Home / BCRC Content / 10067 / 

Return

  Research Article

The information shown in this page was generated using the cross-referenced linkage within public domain database between their strains and BCRC related strains. Usually the information provided from public domain databases varies with diffent confidences and errors, BCRC provides the related information here at best effort, but BCRC doesn't take the responsibility about the correctness of the information provided here.

1. Willems  RJ, Top  J, Smith  DJ, Roper  DI, North  SE, Woodford  N,     ( 2003 )

Mutations in the DNA mismatch repair proteins MutS and MutL of oxazolidinone-resistant or -susceptible Enterococcus faecium.

Antimicrobial agents and chemotherapy 47 (10)
PMID : 14506009  :   DOI  :   10.1128/aac.47.10.3061-3066.2003     PMC  :   PMC201155    
Abstract >>
Mutations in mutS and mutL, which encode DNA mismatch repair (MMR) proteins, can confer hypermutator phenotypes and may facilitate the emergence of mutational antibiotic resistance in bacteria. Linezolid-resistant enterococci (LRE) rarely emerge during therapy and contain mutations in 23S rRNA genes. As enterococci with defective MMR could be prone to the development of oxazolidinone resistance mutations, we investigated 13 clinical isolates of Enterococcus faecium, including 2 LRE, for mutations in mutSL. A 4,944-bp fragment spanning mutSL was sequenced from two pairs of linezolid-resistant (MICs, 64 micro g/ml) and linezolid-susceptible (MICs, 2 micro g/ml) E. faecium isolates (one pair from Austria and one pair from the United Kingdom) identical by pulsed-field gel electrophoresis. The pairs represented distinct strains in which linezolid resistance had emerged during therapy. The MutSL peptides of all four isolates had amino acid substitutions compared with the sequence of E. faecium strain DO (used for genome sequencing). These were Val352Ile (one pair of isolates only) and Met628Leu in MutS and Leu387Pro, Tyr406Phe, Thr415Ser, Phe427Leu, and Phe565Ile in MutL. The significance of these changes remains unknown; these isolates did not show a demonstrable hypermutator phenotype. The same substitutions were found in two of nine geographically diverse linezolid-susceptible enterococcal isolates; the other seven isolates had MutSL sequences identical to that of strain DO. Multilocus sequence typing revealed that all isolates with alternate MutSL peptides belonged to a distinct lineage of a prevalent E. faecium clonal complex, designated CC17. Further studies are needed to investigate the prevalence of these MutSL mutations and their possible roles in the emergence of E. faecium strains resistant to oxazolidinones and other antibiotic classes.
KeywordMeSH Terms
Mutation
2. Arthur  M, Molinas  C, Courvalin  P,     ( 1992 )

Sequence of the vanY gene required for production of a vancomycin-inducible D,D-carboxypeptidase in Enterococcus faecium BM4147.

Gene 120 (1)
PMID : 1398115  :   DOI  :   10.1016/0378-1119(92)90017-j    
Abstract >>
Cloning and nucleotide sequencing identified the vanY gene as a member of the vancomycin-resistance van gene cluster of enterococcal plasmid, pIP816. The vanY gene was necessary for synthesis of the vancomycin-inducible D,D-carboxypeptidase activity previously proposed to be responsible for glycopeptide resistance. However, this activity was not required for peptidoglycan synthesis in the presence of glycopeptides. The deduced product of vanY did not display significant similarity with other D,D-carboxypeptidases.
KeywordMeSH Terms
3. Handwerger  S, Discotto  L, Thanassi  J, Pucci  MJ,     ( 1992 )

Insertional inactivation of a gene which controls expression of vancomycin resistance on plasmid pHKK100.

FEMS microbiology letters 71 (1)
PMID : 1320585  :   DOI  :   10.1016/0378-1097(92)90533-t    
Abstract >>
Expression of inducible high level vancomycin resistance (Vmr) in enterococci appears to require other plasmid-encoded genes in addition to the previously described structural genes vanA and vanH. Tn917 mutagenesis was used to identify such a region in the Vmr plasmid pHKK100. Insertional inactivation of a 693-bp open reading frame upstream from vanH resulted in complete loss of Vmr. This putative 26,642-Da protein has been designated VanR.
KeywordMeSH Terms
4. Kehoe  LE, Snidwongse  J, Courvalin  P, Rafferty  JB, Murray  IA,     ( 2003 )

Structural basis of Synercid (quinupristin-dalfopristin) resistance in Gram-positive bacterial pathogens.

The Journal of biological chemistry 278 (32)
PMID : 12771141  :   DOI  :   10.1074/jbc.M303766200    
Abstract >>
Synercid, a new semisynthetic streptogramin-derived antibiotic containing dalfopristin and quinupristin, is used in treatment of life-threatening infections caused by glycopeptide-resistant Enterococcus faecium and other bacterial pathogens. However, dissemination of genes encoding virginiamycin acetyltransferases, enzymes that confer resistance to streptogramins, threatens to limit the medical utility of the quinupristin-dalfopristin combination. Here we present structures of virginiamycin acetyltransferase D (VatD) determined at 1.8 A resolution in the absence of ligands, at 2.8 A resolution bound to dalfopristin, and at 3.0 A resolution in the presence of acetyl-coenzyme A. Dalfopristin is bound by VatD in a similar conformation to that described previously for the streptogramin virginiamycin M1. However, specific interactions with the substrate are altered as a consequence of a conformational change in the pyrollidine ring that is propagated to adjacent constituents of the dalfopristin macrocycle. Inactivation of dalfopristin involves acetyl transfer from acetyl-coenzyme A to the sole (O-18) hydroxy group of the antibiotic that lies close to the side chain of the strictly conserved residue, His-82. Replacement of residue 82 by alanine is accompanied by a fall in specific activity of >105-fold, indicating that the imidazole moiety of His-82 is a major determinant of catalytic rate enhancement by VatD. The structure of the VatD-dalfopristin complex can be used to predict positions where further structural modification of the drug might preclude enzyme binding and thereby circumvent Synercid resistance.
KeywordMeSH Terms
Drug Resistance
5. Folli  C, Ramazzina  I, Arcidiaco  P, Stoppini  M, Berni  R,     ( 2003 )

Purification of bacteriocin AS-48 from an Enterococcus faecium strain and analysis of the gene cluster involved in its production.

FEMS microbiology letters 221 (1)
PMID : 12694923  :   DOI  :   10.1016/S0378-1097(03)00176-9    
Abstract >>
The cyclic bacteriocin AS-48 has previously been shown to be produced by Enterococcus faecalis strains. A bacteriocin has been purified from an E. faecium strain (E. faecium 7C5), and it has been found to possess molecular mass, cyclization and amino acid sequence typical of bacteriocin AS-48. In addition to the structural gene as-48A, the sequence analysis of the AS-48 gene cluster present in E. faecium 7C5 has revealed the presence of several putative coding regions presumably involved in bacteriocin production and immunity. The results of DNA hybridization assays have indicated that the AS-48 gene cluster and the gene pd78 are present on the same plasmid, possibly the pPD1 plasmid, in E. faecium 7C5.
KeywordMeSH Terms
Multigene Family
Peptides
6. Teng  F, Kawalec  M, Weinstock  GM, Hryniewicz  W, Murray  BE,     ( 2003 )

An Enterococcus faecium secreted antigen, SagA, exhibits broad-spectrum binding to extracellular matrix proteins and appears essential for E. faecium growth.

Infection and immunity 71 (9)
PMID : 12933846  :   DOI  :   10.1128/iai.71.9.5033-5041.2003     PMC  :   PMC187350    
Abstract >>
A gene encoding a major secreted antigen, SagA, was identified in Enterococcus faecium by screening an E. faecium genomic expression library with sera from patients with E. faecium-associated endocarditis. Recombinant SagA protein showed broad-spectrum binding to extracellular matrix (ECM) proteins, including fibrinogen, collagen type I, collagen type IV, fibronectin, and laminin. A fibrinogen-binding protein, purified from culture supernatants of an E. faecium clinical isolate, was found to match the N-terminal sequence of the predicted SagA protein and to react with the anti-SagA antibody, confirming that it was the SagA protein; this protein appeared as an 80- to 90-kDa smear on a Western blot that was sensitive to proteinase K and resistant to periodate treatment and glycoprotein staining. When overexpressed in E. faecium and Escherichia coli, the native and recombinant SagA proteins formed stable oligomers, apparently via their C-terminal domains. The SagA protein is composed of three domains: (i) a putative coiled-coil N-terminal domain that shows homology to the N-terminal domain of Streptococcus mutans SagA protein (42% similarity), previously shown to be involved in cell wall integrity and cell shape maintenance, and to the P45 protein of Listeria monocytogenes (41% similarity); (ii) a central domain containing direct repeats; and (iii) a C-terminal domain that is similar to that found in various proteins, including P45 (50% similarity) and P60 (52% similarity) of L. monocytogenes. The P45 and P60 proteins both have cell wall hydrolase activity, and the latter has also been shown to be involved in virulence, whereas cell wall hydrolase activity was not detected for SagA protein. The E. faecium sagA gene, like the S. mutans homologue, is located in a cluster of genes encoding proteins that appear to be involved in cell wall metabolism and could not be disrupted unless it was first transcomplemented, suggesting that the sagA gene is essential for E. faecium growth and may be involved in cell wall metabolism. In conclusion, the extracelluar E. faecium SagA protein is apparently essential for growth, shows broad-spectrum binding to ECM proteins, forms oligomers, and is antigenic during infection.
KeywordMeSH Terms
Non-programmatic
7. Werner  G, Willems  RJ, Hildebrandt  B, Klare  I, Witte  W,     ( 2003 )

Influence of transferable genetic determinants on the outcome of typing methods commonly used for Enterococcus faecium.

Journal of clinical microbiology 41 (4)
PMID : 12682136  :   DOI  :   10.1128/jcm.41.4.1499-1506.2003     PMC  :   PMC153884    
Abstract >>
A variety of methods is used for a molecular typing of Enterococcus spp. and related gram-positive bacteria including macrorestriction analysis using pulsed-field gel electrophoresis (PFGE), ribotyping, rapid amplification of polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP). To test the influence of transferable determinants on the outcome of different typing methods commonly used for enterococci, we established a homogenous strain collection of 24 transconjugants resulting from filter matings with antibiotic-resistant Enterococcus faecium. As expected, AFLP, RAPD, and PFGE all identified our model bacteria as strongly related. However, distinct differences in the resolving and discriminatory power of the tested methods could be clearly addressed. In PFGE, 22 of 24 transconjugants possessed less than a three-band difference to the recipient pattern and would be regarded as strongly related. Three different RAPD PCRs were tested; in two reactions, identical patterns for all transconjugants and the recipient were produced. One RAPD PCR produced an identical pattern for 18 transconjugants and the recipient and a clearly different pattern for the remaining 6 transconjugants due to a newly appearing fragment resulting from acquisition of the tetL gene. AFLP clusters all transconjugants into a group of major relatedness. Percent similarities were highly dependent on the method used for calculating the similarity coefficient (curve-based versus band-based similarity coefficient). Fragment patterns of digested plasmids showed the possession of nonidentical plasmids in most transconjugants. PFGE still could be recommended as the method of choice. Nevertheless, the more-modern AFLP approach produces patterns of comparable discriminatory power while possessing some advantages over PFGE (less-time-consuming internal standards). Plasmid fingerprints can be included to subdifferentiate enterococcal isolates possessing identical macrorestriction and PCR typing patterns.
KeywordMeSH Terms
Bacterial Typing Techniques
Conjugation, Genetic
8. Nallapareddy  SR, Weinstock  GM, Murray  BE,     ( 2003 )

Clinical isolates of Enterococcus faecium exhibit strain-specific collagen binding mediated by Acm, a new member of the MSCRAMM family.

Molecular microbiology 47 (6)
PMID : 12622825  :   DOI  :   10.1046/j.1365-2958.2003.03417.x    
Abstract >>
A collagen-binding adhesin of Enterococcus faecium, Acm, was identified. Acm shows 62% similarity to the Staphylococcus aureus collagen adhesin Cna over the entire protein and is more similar to Cna (60% and 75% similarity with Cna A and B domains respectively) than to the Enterococcus faecalis collagen-binding adhesin, Ace, which shares homology with Acm only in the A domain. Despite the detection of acm in 32 out of 32 E. faecium isolates, only 11 of these (all clinical isolates, including four vancomycin-resistant endocarditis isolates and seven other isolates) exhibited binding to collagen type I (CI). Although acm from three CI-binding vancomycin-resistant E. faecium clinical isolates showed 100% identity, analysis of acm genes and their promoter regions from six non-CI-binding strains identified deletions or mutations that introduced stop codons and/or IS elements within the gene or the promoter region in five out of six strains, suggesting that the presence of an intact functional acm gene is necessary for binding of E. faecium strains to CI. Recombinant Acm A domain showed specific and concentration-dependent binding to collagen, and this protein competed with E. faecium binding to immobilized CI. Consistent with the adherence phenotype and sequence data, probing with Acm-specific IgGs purified from anti-recombinant Acm A polyclonal rabbit serum confirmed the surface expression of Acm in three out of three collagen-binding clinical isolates of E. faecium tested, but in none of the strains with a non-functional pseudo acm gene. Introduction of a functional acm gene into two non-CI-binding natural acm mutant strains conferred a CI-binding phenotype, further confirming that native Acm is sufficient for the binding of E. faecium to CI. These results demonstrate that acm, which encodes a potential virulence factor, is functional only in certain infection-derived clinical isolates of E. faecium, and suggest that Acm is the primary adhesin responsible for the ability of E. faecium to bind collagen.
KeywordMeSH Terms
9. Park  SH, Itoh  K, Fujisawa  T,     ( 2003 )

Characteristics and identification of enterocins produced by Enterococcus faecium JCM 5804T.

Journal of applied microbiology 95 (2)
PMID : 12859761  :  
Abstract >>
To screen bacteriocin-producing lactic acid bacteria (LAB) in 52 type and reference strains, which have not previously been studied, with respect to bacteriocins, and to characterize the presence of bacteriocins. Only Enterococcus faecium JCM 5804T showed bacteriocin-like activity. It inhibited the growth of Lactobacillus spp., Enterococcus spp., Clostridium spp., Listeria monocytogenes, and vancomycin resistant Enterococcus (VRE). However, it was not effective against Gram-negative strains, Weisella spp., Leuconostoc spp., Lactococcus spp., or methicillin resistant Staphylococcus aureus (MRSA). The inhibitory activity of Ent. faecium JCM 5804T was inactivated by proteinase K, trypsin, alpha-chymotrypsin, and papain, but not by lysozyme, lipase, catalase, or beta-glucosidase. The inhibitory activity was stable at 100 degrees C for 30 min, and had a pH range from 2 to 10. The molecular weight of the partially purified bacteriocin(s) was approx. 4.5 kDa, according to tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Polymerase chain reaction and direct sequencing methods identified three different types of bacteriocins produced by Ent. faecium JCM 5804T, enterocin A, enterocin B, and enterocin P-like bacteriocin. Enterococcus faecium JCM 5804T produced three different types of bacteriocins, and they inhibited LAB and pathogens. This is the first report of enterocin A, enterocin B, and enterocin P-like bacteriocin, detected in Ent. faecium JCM 5804T among LAB type and reference strains.
KeywordMeSH Terms
10. Grady  R, Hayes  F,     ( 2003 )

Axe-Txe, a broad-spectrum proteic toxin-antitoxin system specified by a multidrug-resistant, clinical isolate of Enterococcus faecium.

Molecular microbiology 47 (5)
PMID : 12603745  :   DOI  :   10.1046/j.1365-2958.2003.03387.x    
Abstract >>
Enterococcal species of bacteria are now acknowledged as leading causes of bacteraemia and other serious nosocomial infections. However, surprisingly little is known about the molecular mechanisms that promote the segregational stability of antibiotic resistance and other plasmids in these bacteria. Plasmid pRUM (24 873 bp) is a multidrug resistance plasmid identified in a clinical isolate of Enterococcus faecium. A novel proteic-based toxin-antitoxin cassette identified on pRUM was demonstrated to be a functional segregational stability module in both its native host and evolutionarily diverse bacterial species. Induced expression of the toxin protein (Txe) of this system resulted in growth inhibition in Escherichia coli. The toxic effect of Txe was alleviated by co-expression of the antitoxin protein, Axe. Homologues of the axe and txe genes are present in the genomes of a diversity of Eubacteria. These homologues (yefM-yoeB) present in the E. coli chromosome function as a toxin-antitoxin mechanism, although the Axe and YefM antitoxin components demonstrate specificity for their cognate toxin proteins in vivo. Axe-Txe is one of the first functional proteic toxin-antitoxin systems to be accurately described for Gram-positive bacteria.
KeywordMeSH Terms
11. Burk  DL, Ghuman  N, Wybenga-Groot  LE, Berghuis  AM,     ( 2003 )

X-ray structure of the AAC(6')-Ii antibiotic resistance enzyme at 1.8 A resolution; examination of oligomeric arrangements in GNAT superfamily members.

Protein science : a publication of the Protein Society 12 (3)
PMID : 12592013  :   DOI  :   10.1110/ps.0233503     PMC  :   PMC2312454    
Abstract >>
The rise of antibiotic resistance as a public health concern has led to increased interest in studying the ways in which bacteria avoid the effects of antibiotics. Enzymatic inactivation by several families of enzymes has been observed to be the predominant mechanism of resistance to aminoglycoside antibiotics such as kanamycin and gentamicin. Despite the importance of acetyltransferases in bacterial resistance to aminoglycoside antibiotics, relatively little is known about their structure and mechanism. Here we report the three-dimensional atomic structure of the aminoglycoside acetyltransferase AAC(6')-Ii in complex with coenzyme A (CoA). This structure unambiguously identifies the physiologically relevant AAC(6')-Ii dimer species, and reveals that the enzyme structure is similar in the AcCoA and CoA bound forms. AAC(6')-Ii is a member of the GCN5-related N-acetyltransferase (GNAT) superfamily of acetyltransferases, a diverse group of enzymes that possess a conserved structural motif, despite low sequence homology. AAC(6')-Ii is also a member of a subset of enzymes in the GNAT superfamily that form multimeric complexes. The dimer arrangements within the multimeric GNAT superfamily members are compared, revealing that AAC(6')-Ii forms a dimer assembly that is different from that observed in the other multimeric GNAT superfamily members. This different assembly may provide insight into the evolutionary processes governing dimer formation.
KeywordMeSH Terms
Drug Resistance, Bacterial
12. Lee  WG, Kim  W,     ( 2003 )

Identification of a novel insertion sequence in vanB2-containing Enterococcus faecium.

Letters in applied microbiology 36 (3)
PMID : 12581381  :  
Abstract >>
The characterization of a novel insertion sequence (IS) in vanB2-containing Enterococcus faecium was conducted. Direct PCR amplification of ORFC region of Tn5382 from DNA extracted from vanB2-containing E. faecium, and sequence analysis were performed. A novel IS was identified. It is 1418 bp in length and contains one putative open reading frame that is similar to transposase. There exists inverted terminal repeats of 12 bp, but direct repeats are not present. According to high similarity to putative transposases of IS3 members, such as, IS150, IS861, IS1077 and IS911, we designated it ISEnfa3. Since ISEnfa3 was detected in all vanB2-containing strains examined so far, it could be used as a tool for epidemiological study.
KeywordMeSH Terms
Genes, Bacterial
13. Depardieu  F, Reynolds  PE, Courvalin  P,     ( 2003 )

VanD-type vancomycin-resistant Enterococcus faecium 10/96A.

Antimicrobial agents and chemotherapy 47 (1)
PMID : 12499162  :   DOI  :   10.1128/aac.47.1.7-18.2003     PMC  :   PMC149003    
Abstract >>
VanD type Enterococcus faecium 10/96A is constitutively resistant to vancomycin and to low levels of teicoplanin by nearly exclusive synthesis of peptidoglycan precursors terminating in D-alanyl-D-lactate (L. M. Dalla Costa, P. E. Reynolds, H. A. Souza, D. C. Souza, M. F. Palepou, and N. Woodford, Antimicrob. Agents Chemother. 44:3444-3446, 2000). A G(184)S mutation adjacent to the serine involved in the binding of D-Ala1 in the D-alanine:D-alanine ligase (Ddl) led to production of an impaired Ddl and accounts for the lack of D-alanyl-D-alanine-containing peptidoglycan precursors. The sequence of the vanD gene cluster revealed eight open reading frames. The organization of this operon, assigned to a chromosomal location, was similar to those in other VanD type strains. The distal part encoded the VanH(D) dehydrogenase, the VanD ligase, and the VanX(D) dipeptidase, which were homologous to the corresponding proteins in VanD-type strains. Upstream from the structural genes for these proteins was the vanY(D) gene; a frameshift mutation in this gene resulted in premature termination of the encoded protein and accounted for the lack of penicillin-susceptible D,D-carboxypeptidase activity. Analysis of the translated sequence downstream from the stop codon, but in a different reading frame because of the frameshift mutation, indicated homology with penicillin binding proteins (PBPs) with a high degree of identity with VanY(D) from VanD-type strains. The 5' end of the gene cluster contained the vanR(D)-vanS(D) genes for a putative two-component regulatory system. Insertion of ISEfa4 in the vanS(D) gene led to constitutive expression of vancomycin resistance. This new insertion belonged to the IS605 family and was composed of two open reading frames encoding putative transposases of two unrelated insertion sequence elements, IS200 and IS1341.
KeywordMeSH Terms
14. Dahl  KH, Røkenes  TP, Lundblad  EW, Sundsfjord  A,     ( 2003 )

Nonconjugative transposition of the vanB-containing Tn5382-like element in Enterococcus faecium.

Antimicrobial agents and chemotherapy 47 (2)
PMID : 12543693  :   DOI  :   10.1128/aac.47.2.786-789.2003     PMC  :   PMC151725    
Abstract >>
The vanB2 operon encoding glycopeptide resistance is an integral part of the putative conjugative transposon Tn5382. Characterization of clinical glycopeptide resistant derivatives from an epidemic ampicillin-resistant Enterococcus faecium strain showed precise chromosomal or plasmid insertions of a vanB2-containing Tn5382-like element. Conjugative transposition of the Tn5382-like element was not demonstrated in retransfer studies.
KeywordMeSH Terms
15. Rice  LB, Carias  L, Rudin  S, Vael  C, Goossens  H, Konstabel  C, Klare  I, Nallapareddy  SR, Huang  W, Murray  BE,     ( 2003 )

A potential virulence gene, hylEfm, predominates in Enterococcus faecium of clinical origin.

The Journal of infectious diseases 187 (3)
PMID : 12552437  :   DOI  :   10.1086/367711    
Abstract >>
An open reading frame (hyl(Efm)) with homologies to previously described hyaluronidase genes has been identified in nonstool isolates of Enterococcus faecium. E. faecium isolates (n=577) from diverse sources were screened for the presence of hyl(Efm) and esp(Efm), a putative virulence gene associated with epidemic E. faecium strains. The presence of esp(Efm) was roughly twice that of hyl(Efm), but both were found primarily in vancomycin-resistant E. faecium isolates in nonstool cultures obtained from patients hospitalized in the United States. These data suggest that specific E. faecium strains may be enriched in determinants that make them more likely to cause clinical infections. Differences in the prevalence of these strains may help explain variations in the clinical importance of multiresistant E. faecium across different continents.
KeywordMeSH Terms
16. Tanimoto  K, Ike  Y,     ( 2002 )

Analysis of the conjugal transfer system of the pheromone-independent highly transferable Enterococcus plasmid pMG1: identification of a tra gene (traA) up-regulated during conjugation.

Journal of bacteriology 184 (20)
PMID : 12270839  :   DOI  :   10.1128/jb.184.20.5800-5804.2002     PMC  :   PMC139602    
Abstract >>
pMG1 (65.1 kbp) is a pheromone-independent Enterococcus faecium conjugative plasmid conferring gentamicin resistance. pMG1 is able to transfer to enterococci at a high frequency in broth mating experiments, and it does not respond to the sex pheromones which are involved in the high-frequency transfer system of some conjugative plasmids in Enterococcus faecalis. To analyze regulation of tra gene expression in pMG1, transcripts of pMG1 were examined during conjugation. RNA samples were prepared from mating mixtures 20, 40, 80, and 160 min after initiation of mating and were subsequently analyzed by Northern hybridization by using a variety of pMG1 DNA fragments as probes. One transcript of gene 71ORF2 increased to the maximal level 20 min after the start of mating. The level of this transcript decreased after 40 min of mating to the same level as the level in the control donor culture. The increase was not observed in cultures of the donor cells or recipient cells. These findings suggested that the increase was a conjugation-specific event. The 71ORF2 gene of pMG1 was disrupted by using the suicide vector pMG226 carrying an erythromycin resistance gene that is expressed in enterococci. The transfer frequencies of mutant plasmid pMG229, which had a disrupted 71ORF2 gene in pMG1, and the parent plasmid pMG1 were 1.6 x 10(-7) and 1.1 x 10(-3) per donor cell, respectively, in broth mating experiments, and the transfer frequencies of pMG229 and pMG1 were 2.7 x 10(-3) and 8.5 x 10(-2) per donor cell, respectively, in filter mating experiments. This indicated that the transfer frequency of plasmid pMG229 was reduced during broth mating and was not altered during filter mating. 71ORF2, which is designated traA, is a gene involved in the tra gene system for conjugation, and the product of this gene is associated with the formation or stabilization of mating aggregates during broth mating.
KeywordMeSH Terms
Conjugation, Genetic
Up-Regulation
17. Burnie  J, Carter  T, Rigg  G, Hodgetts  S, Donohoe  M, Matthews  R,     ( 2002 )

Identification of ABC transporters in vancomycin-resistant Enterococcus faecium as potential targets for antibody therapy.

FEMS immunology and medical microbiology 33 (3)
PMID : 12110480  :   DOI  :   10.1111/j.1574-695X.2002.tb00589.x    
Abstract >>
The occurrence of an outbreak of septicaemias due to vancomycin-resistant Enterococcus faecium (VRE), in Manchester, UK, provided an opportunity to examine the antibody responses in patients infected by the same strain. Immunoblotting sera from 24 cases, six of whom died, showed an immunodominant cluster of antigens at 34, 54 and 97 kDa, with a statistically significant correlate between survival and immunoglobulin G to the 34 and 97 kDa bands (P<0.05). Screening a genomic expression library of VRE with seropositive serum and peritoneal dialysate from a survivor gave a recombinant clone with two contiguous open reading frames, the derived amino acid sequences of which both showed sequence homologue with ABC transporters, with a Walker A and Walker B motif and the signature sequence LSGGQ. The first open reading frame (putative VRE ABC1) showed 57% homologue with YbxA from Bacillus subtilis. A partial sequence (putative VRE ABC2) was also obtained, in the same recombinant clone, of a second ABC transporter with 72% homologue with ybaE from B. subtilis. Affinity selection with the seropositive serum and peritoneal dialysate used to screen the library showed that the eluted antibody bound to the 97, 54, 34 and 30 kDa bands. Direct amino acid sequencing identified this as a possible ABC transporter. Rabbit antiserum against peptides representing Walker A and an area adjacent to the Walker B site cross-reacted with bands at 34, 54, 97, 110 kDa and at 30, 34 and 54 kDa respectively. This therefore appeared to be an immunodominant complex of ABC transporters of which the smallest was the 30 kDa antigen. Epitope mapping of this antigen with seropositive patients' sera delineated three linear epitopes (KVGIV, FGPKNF and RVAI). The Walker A site represented by peptide 1 (GHNGSGKSTLAKTIN), epitope RVAI represented by peptides 2 (MRRVAIAGVLAMPRE) and 3 (ELSGGQMRRVAIAGV), epitope KVGIV represented by peptide 4 (LKPIRKKVGIVFQFP), and recombinant VRE ABC1 and VRE ABC2 expressed in Escherichia coli pBAD were then used to isolate human genetically recombinant antibodies from a phage antibody display library. An assessment of the protective potential of these antibodies was carried out in a mouse model of the infection. This study suggests that an ABC transporter homologue could be a target for antibody therapy against VRE infections.
KeywordMeSH Terms
Vancomycin Resistance
18. Homan  WL, Tribe  D, Poznanski  S, Li  M, Hogg  G, Spalburg  E, Van Embden  JD, Willems  RJ,     ( 2002 )

Multilocus sequence typing scheme for Enterococcus faecium.

Journal of clinical microbiology 40 (6)
PMID : 12037049  :   DOI  :   10.1128/jcm.40.6.1963-1971.2002     PMC  :   PMC130786    
Abstract >>
A multilocus sequence typing (MLST) scheme has been developed for Enterococcus faecium. Internal fragments from seven housekeeping genes of 123 epidemiologically unlinked isolates from humans and livestock and 16 human-derived isolates from several outbreaks in the United States, the United Kingdom, Australia, and The Netherlands were analyzed. A total of 62 sequence types were detected in vancomycin-sensitive E. faecium (VSEF) and vancomycin-resistant E. faecium (VREF) isolates. VSEF isolates were genetically more diverse than VREF isolates. Both VSEF and VREF isolates clustered in host-specific lineages that were similar to the host-specific clustering obtained by amplified fragment length polymorphism analysis. Outbreak isolates from hospitalized humans clustered in a subgroup that was defined by the presence of a unique allele from the housekeeping gene purK and the surface protein gene esp. The MLST results suggest that epidemic lineages of E. faecium emerged recently worldwide, while genetic variation in both VREF and VSEF was created by longer-term recombination. The results show that MLST of E. faecium provides an excellent tool for isolate characterization and long-term epidemiologic analysis.
KeywordMeSH Terms
Alleles
Base Sequence
19. Kawalec  M, Gniadkowski  M, Kedzierska  J, Skotnicki  A, Fiett  J, Hryniewicz  W,     ( 2001 )

Selection of a teicoplanin-resistant Enterococcus faecium mutant during an outbreak caused by vancomycin-resistant enterococci with the vanB phenotype.

Journal of clinical microbiology 39 (12)
PMID : 11724832  :   DOI  :   10.1128/JCM.39.12.4274-4282.2001     PMC  :   PMC88536    
Abstract >>
Vancomycin-resistant enterococci (VRE) have recently become an increasing problem in hospitals in Poland, being responsible for a growing number of nosocomial outbreaks. In this work, we have analyzed the second outbreak of VRE with the VanB phenotype to be identified in the country. It was caused by clonal dissemination of a single strain of vancomycin-resistant Enterococcus faecalis (VRES) and horizontal transmission of vancomycin resistance genes among several vancomycin-resistant Enterococcus faecium (VREM) strains. Two similar restriction fragment length polymorphism types of the vanB gene cluster characterized VRES and VREM isolates, and they both contained the same vanB2 variant of the vanB gene. Two vancomycin-susceptible E. faecium (VSEM) isolates, recovered from the same wards during the outbreak, proved to be related to certain VREM isolates and could represent endemic strains that had acquired vancomycin resistance. One VSEM and four VREM isolates, all identified in the same patient, belonged to a single clone, although they revealed remarkable diversity in terms of susceptibility, PFGE patterns, plasmid content, and number of vanB gene cluster copies. Most probably they reflected the dynamic evolution of an E. faecium strain in the course of infection of a single patient. One of the VREM isolates turned out to be resistant to teicoplanin, which coincided with the use of this antibiotic in the patient's therapy. Its vanB gene variant differed by a single mutation from that found in other isolates; however, it also lacked a large part of the vanB gene cluster, including the regulatory genes vanR(B) and -S(B), and the vancomycin-inducible promoter P(YB). Expression of the resistance genes vanH(B), -B, and -X(B) was constitutive in the mutant, and this phenomenon was responsible for its unusual phenotype.
KeywordMeSH Terms
Disease Outbreaks
20. Oana  K, Okimura  Y, Kawakami  Y, Hayashida  N, Shimosaka  M, Okazaki  M, Hayashi  T, Ohnishi  M,     ( 2002 )

Physical and genetic map of Enterococcus faecium ATCC19434 and demonstration of intra- and interspecific genomic diversity in enterococci.

FEMS microbiology letters 207 (2)
PMID : 11958930  :   DOI  :   10.1111/j.1574-6968.2002.tb11041.x    
Abstract >>
A physical map of the Enterococcus faecium ATCC19434 chromosome was constructed by NotI, I-CeuI and Sse8387I. The chromosome was a circular DNA of 2600 kb in size, and contained six rRNA operons (rrn). The locations and orientations of the six rrn operons and 24 different determinants were mapped. Genomes of three additional E. faecium strains were also analyzed by I-CeuI digestion, and the genome sizes were found to vary from 2550 to 2995 kb. We further investigated the genome sizes and number of rrn operons in four E. faecalis, one E. avium, and one E. durans strains. The genome sizes were larger than E. faecium: 3000-3250 kb in E. faecalis, 3445 kb in E. avium, and 3070 kb in E. durans. E. avium and E. durans contained six rrn operons as in E. faecium, but all the E. faecalis strains possessed four rrn operons.
KeywordMeSH Terms
21. Hasman  H, Aarestrup  FM,     ( 2002 )

tcrB, a gene conferring transferable copper resistance in Enterococcus faecium: occurrence, transferability, and linkage to macrolide and glycopeptide resistance.

Antimicrobial agents and chemotherapy 46 (5)
PMID : 11959576  :   DOI  :   10.1128/aac.46.5.1410-1416.2002     PMC  :   PMC127162    
Abstract >>
A newly discovered gene, designated tcrB, which is located on a conjugative plasmid conferring acquired copper resistance in Enterococcus faecium, was identified in an isolate from a pig. The tcrB gene encodes a putative protein belonging to the CPx-type ATPase family with homology (46%) to the CopB protein from Enterococcus hirae. The tcrB gene was found in E. faecium isolated from pigs (75%), broilers (34%), calves (16%), and humans (10%) but not in isolates from sheep. Resistant isolates, containing the tcrB gene, grew on brain heart infusion agar plates containing up to 28 mM CuSO4 compared to only 4 mM for the susceptible isolates. Copper resistance, and therefore the presence of the tcrB gene, was strongly correlated to macrolide and glycopeptide resistance in isolates from pigs, and the tcrB gene was shown to be located on the same conjugative plasmid as the genes responsible for resistance to these two antimicrobial agents. The frequent occurrence of this new copper resistance gene in isolates from pigs, where copper sulfate is being used in large amounts as feed additive, suggests that the use of copper has selected for resistance.
KeywordMeSH Terms
22. Simjee  S, McDermott  PF, Wagner  DD, White  DG,     ( 2001 )

Variation within the vat(E) allele of Enterococcus faecium isolates from retail poultry samples.

Antimicrobial agents and chemotherapy 45 (10)
PMID : 11557494  :   DOI  :   10.1128/AAC.45.10.2931-2932.2001     PMC  :   PMC90756    
Abstract >>
In a survey of retail meat samples, twelve quinupristin-dalfopristin-resistant (MICs, > or =4 mg/liter) Enterococcus faecium isolates that carried a vat(E) gene were recovered. DNA sequence comparison revealed five new variations in the vat(E) allele among 12 isolates, which were designated vat(E-4) through vat(E-8); two isolates had vat(E-1). There was no correlation between the number of base changes and the quinupristin-dalfopristin MIC.
KeywordMeSH Terms
Acetyltransferases
23. Sugantino  M, Roderick  SL,     ( 2002 )

Crystal structure of Vat(D): an acetyltransferase that inactivates streptogramin group A antibiotics.

Biochemistry 41 (7)
PMID : 11841212  :   DOI  :   10.1021/bi011991b    
Abstract >>
The streptogramin class of antibiotics act to inhibit bacterial protein synthesis, and their semisynthetic derivatives, such as dalfopristin-quinupristin (Synercid), are used to treat serious or life-threatening infections due to multiply antibiotic resistant bacteria. Acquired resistance of the nosocomial pathogen Enterococcus faecium to the group A component of natural and semisynthetic streptogramin mixtures is a prerequisite for the streptogramin resistance phenotype and is mediated by a streptogramin acetyltransferase. The crystal structure of Vat(D), a streptogramin acetyltransferase from a human urinary isolate of E. faecium, has been determined as an apoenzyme and in complex with either acetyl-CoA or virginiamycin M1 and CoA. These structures illustrate the location and arrangement of residues at the active site, and point to His 82 as a residue that may function as a general base. The structural similarity of Vat(D) to the xenobiotic acetyltransferase from Pseudomonas aeruginosa indicates similarities in the catalytic mechanism for these enzymes as well as several shared and distinctive antibiotic binding interactions between these enzymes and their respective substrates. These results reveal the molecular basis for a reaction by which Gram-positive cocci acquire resistance to a last resort antibiotic.
KeywordMeSH Terms
Bacterial Proteins
24. Casadewall  B, Reynolds  PE, Courvalin  P,     ( 2001 )

Regulation of expression of the vanD glycopeptide resistance gene cluster from Enterococcus faecium BM4339.

Journal of bacteriology 183 (11)
PMID : 11344152  :   DOI  :   10.1128/JB.183.11.3436-3446.2001     PMC  :   PMC99642    
Abstract >>
A new open reading frame, encoding a putative integrase-like protein, was detected downstream from the six genes of the vanD glycopeptide resistance cluster in Enterococcus faecium BM4339 (B. Casadewall and P. Courvalin, J. Bacteriol. 181:3644-3648, 1999). In this cluster, genes coding for the VanR(D)-VanS(D) two-component regulatory system were cotranscribed from the P(R(D)) promoter, whereas transcription of the vanY(D), vanH(D), vanD, vanX(D), and intD genes was initiated from the P(Y(D)) promoter located between vanS(D) and vanY(D) (the D subscript indicates that the gene is part of the vanD operon). The VanR(D)-VanS(D) regulatory system is likely to activate transcription of the resistance genes from the promoter P(Y(D)). Glycopeptide-susceptible derivatives of BM4339 were obtained by trans complementation of the frameshift mutation in the ddl gene, restoring functional D-alanine:D-alanine ligase activity in this strain. The glycopeptide-susceptible transformant BM4409, producing only D-alanyl-D-alanine-terminating peptidoglycan precursors, did not express the resistance genes encoding the VanY(D) D,D-carboxypeptidase, the VanH(D) dehydrogenase, the VanD ligase, the VanX(D) D,D-dipeptidase, and also the IntD integrase, although the regulatory region of the vanD cluster was still transcribed. In BM4409, the absence of VanR(D)-VanS(D), apparently dependent, transcription from promoter P(Y(D)) correlated with the lack of D-alanyl-D-lactate-terminating precursors. The vanX(D) gene was transcribed in BM4339, but detectable amounts of VanX(D) D,D-dipeptidase were not synthesized. However, the gene directed synthesis of an active enzyme when cloned on a multicopy plasmid in Escherichia coli, suggesting that the enzyme was unstable in BM4339 or that it had very low activity that was detectable only under conditions of high gene dosage. This activity is not required for glycopeptide resistance in BM4339, since this strain cannot synthesize D-alanyl-D-alanine.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Peptide Synthases
25. Lu  JJ, Perng  CL, Ho  MF, Chiueh  TS, Lee  WH,     ( 2001 )

High prevalence of VanB2 vancomycin-resistant Enterococcus faecium in Taiwan.

Journal of clinical microbiology 39 (6)
PMID : 11376048  :   DOI  :   10.1128/JCM.39.6.2140-2145.2001     PMC  :   PMC88102    
Abstract >>
Thirty-six VanB glycopeptide-resistant Enterococcus faecium isolates were collected from patients in five different hospitals in Taiwan. The vancomycin resistance genes were amplified by the long vanB PCR, which amplifies the 6,373-bp vanB gene cluster including the vanR(B2), vanS(B2), vanY(B2), vanW(B2), vanH(B2), vanB2, and vanX(B2) genes. The deduced amino acid sequences were found to be 95 to 98% homologous to those of the vanB1 gene cluster: VanR(B1), 97%; VanS(B1), 97%; VanY(B1), 96%; VanH(B1), 95%; VanB1, 96%; and VanX(B1), 98%. Restriction enzyme analysis of the long vanB PCR products revealed that all 36 isolates had the same vanB2-specific pattern. DNA sequence analysis of the vanB2 gene, which is a D-Ala-D-Lac ligase gene, revealed that none of the 36 sequences were identical to the previously published vanB2 sequence. Thirty-one isolates had 1 nucleotide different from the published vanB2 sequence. The sequences of the other five isolates differed from the published vanB2 sequence by 2 or 3 nucleotides. Four isolates with a low or moderate resistance to vancomycin (MIC = 4 to 32 microg/ml) were found to have the same leucine-to-methionine change at amino acid position 308 of the vanB2 gene. The genomic DNAs of all 36 isolates were digested with SmaI and then typed by pulsed-field gel electrophoresis (PFGE). Eight different PFGE types (I to VIII) were observed, and type I was found to be prevalent in all hospitals examined in this study. This result suggests that intra- and interhospital dissemination of this E. faecium strain has occurred in Taiwan.
KeywordMeSH Terms
26. Willems  RJ, Homan  W, Top  J, van Santen-Verheuvel  M, Tribe  D, Manzioros  X, Gaillard  C, Vandenbroucke-Grauls  CM, Mascini  EM, van Kregten  E, van Embden  JD, Bonten  MJ,     ( 2001 )

Variant esp gene as a marker of a distinct genetic lineage of vancomycin-resistant Enterococcus faecium spreading in hospitals.

Lancet (London, England) 357 (9259)
PMID : 11265956  :   DOI  :   10.1016/S0140-6736(00)04205-7    
Abstract >>
In the USA, vancomycin-resistant Enterococcus faecium (VREF) is endemic in hospitals, despite lack of carriage among healthy individuals. In Europe, however, hospital outbreaks are rare, but VREF carriage among healthy individuals and livestock is common. We used amplified fragment-length polymorphism analysis to genotype 120 VREF isolates associated with hospital outbreaks and 45 non-epidemic isolates from the USA, Europe, and Australia. We also looked for the esp virulence gene in these isolates and in 98 VREF from animals. A specific E. faecium subpopulation genetically distinct from non-epidemic VREF isolates was found to be the cause of the hospital epidemics in all three continents. This subpopulation contained a variant of the esp gene that was absent in all non-epidemic and animal isolates. Identification of the variant esp gene will be important in guiding infection-control strategies, and the Esp protein could be a new target for antibacterial therapy.
KeywordMeSH Terms
Vancomycin Resistance
27. Lee  WG, Jernigan  JA, Rasheed  JK, Anderson  GJ, Tenover  FC,     ( 2001 )

Possible horizontal transfer of the vanB2 gene among genetically diverse strains of vancomycin-resistant Enterococcus faecium in a Korean hospital.

Journal of clinical microbiology 39 (3)
PMID : 11230450  :   DOI  :   10.1128/JCM.39.3.1165-1168.2001     PMC  :   PMC87896    
Abstract >>
A total of 25 isolates of vanB-containing Enterococcus faecium were recovered from patients in a single Korean hospital over a 20-month period. There were two distinct vanB2 patterns among the 11 pulsed-field gel electrophoresis types; 17 contained the prototype vanB2 and 8 contained a novel vanB2 with a 177-bp deletion in vanY(B). Both vanB2 genes were transmissible in vitro at a mean frequency of 1.1 x 10(-8) transconjugants/donor. These results suggest the horizontal spread of vanB2 is occurring among genetically diverse strains of E. faecium in Korean hospitals.
KeywordMeSH Terms
Gene Transfer, Horizontal
28. Rice  LB, Carias  LL, Hutton-Thomas  R, Sifaoui  F, Gutmann  L, Rudin  SD,     ( 2001 )

Penicillin-binding protein 5 and expression of ampicillin resistance in Enterococcus faecium.

Antimicrobial agents and chemotherapy 45 (5)
PMID : 11302814  :   DOI  :   10.1128/AAC.45.5.1480-1486.2001     PMC  :   PMC90492    
Abstract >>
We report a structural and transcriptional analysis of the pbp5 region of Enterococcus faecium C68. pbp5 exists within a larger operon that includes upstream open reading frames (ORFs) corresponding to previously reported psr (penicillin-binding protein synthesis repressor) and ftsW (whose product is a transmembrane protein that interacts with PBP3 in Escherichia coli septum formation) genes. Hybridization of mRNA from C68, CV133, and four ampicillin-resistant CV133 mutants revealed four distinct transcripts from this region, consisting of (i) E. faecium ftsW (ftsW(Efm)) alone; (ii) psr and pbp5; (iii) pbp5 alone; and (iv) ftsW(Efm), psr, and pbp5. Quantities of the different transcripts varied between strains and did not always correlate with quantities of PBP5 or levels of ampicillin resistance. Since the psr of C68 is presumably nonfunctional due to an insertion of an extra nucleotide in the codon for the 44th amino acid, the region extending from the ftsW(Efm) promoter through the pbp5 gene of C68 was cloned in E. coli to facilitate mutagenesis. The psr ORF was regenerated using site-directed mutagenesis and introduced into E. faecium D344-SRF on conjugative shuttle vector pTCV-lac (pCWR558 [psr ORF interrupted]; pCWR583 [psr ORF intact]). Ampicillin MICs for both D344-SRF(pCWR558) and D344-SRF(pCWR583) were 64 microg/ml. Quantities of pbp5 transcript and protein were similar in strains containing either construct regardless of whether they were grown in the presence or absence of ampicillin, arguing against a role for PSR as a repressor of pbp5 transcription. However, quantities of psr transcript were increased in D344-SRF(pCWR583) compared to D344-SRF(pCWR558), especially after growth in ampicillin; suggesting that PSR acts in some manner to activate its own transcription.
KeywordMeSH Terms
Hexosyltransferases
Membrane Proteins
Peptidyl Transferases
29. Soltani  M, Beighton  D, Philpott-Howard  J, Woodford  N,     ( 2001 )

Identification of vat(E-3), a novel gene encoding resistance to quinupristin-dalfopristin in a strain of Enterococcus faecium from a hospital patient in the United Kingdom.

Antimicrobial agents and chemotherapy 45 (2)
PMID : 11269234  :   DOI  :   10.1128/aac.45.2.645-646.2001     PMC  :   PMC90348    
Abstract >>
N/A
KeywordMeSH Terms
Acetyltransferases
30. Gholizadeh  Y, Prevost  M, Van Bambeke  F, Casadewall  B, Tulkens  PM, Courvalin  P,     ( 2001 )

Sequencing of the ddl gene and modeling of the mutated D-alanine:D-alanine ligase in glycopeptide-dependent strains of Enterococcus faecium.

Protein science : a publication of the Protein Society 10 (4)
PMID : 11274474  :   DOI  :   10.1110/ps.39101     PMC  :   PMC2373979    
Abstract >>
Glycopeptide dependence for growth in enterococci results from mutations in the ddl gene that inactivate the host D-Ala:D-Ala ligase. The strains require glycopeptides as inducers for synthesis of resistance proteins, which allows for the production of peptidoglycan precursors ending in D-Ala-D-Lac instead of D-Ala-D-Ala. The sequences of the ddl gene from nine glycopeptide-dependent Enterococcus faecium clinical isolates were determined. Each one had a mutation consisting either in a 5-bp insertion at position 41 leading to an early stop codon, an in-frame 6-bp deletion causing the loss of two residues (KDVA243-246 to KA), or single base-pair changes resulting in an amino acid substitution (E13 --> G, G99 --> R, V241 --> D, D295 --> G, P313 --> L). The potential consequences of the deletion and point mutations on the 3-D structure of the enzyme were evaluated by comparative molecular modeling of the E. faecium enzyme, using the X-ray structure of the homologous Escherichia coli D-Ala:D-Ala ligase DdlB as a template. All mutated residues were found either to interact directly with one of the substrates of the enzymatic reaction (E13 and D295) or to stabilize the position of critical residues in the active site. Maintenance of the 3-D structure in the vicinity of these mutations in the active site appears critical for D-Ala:D-Ala ligase activity.
KeywordMeSH Terms
Models, Molecular
31. Ke  D, Boissinot  M, Huletsky  A, Picard  FJ, Frenette  J, Ouellette  M, Roy  PH, Bergeron  MG,     ( 2000 )

Evidence for horizontal gene transfer in evolution of elongation factor Tu in enterococci.

Journal of bacteriology 182 (24)
PMID : 11092850  :   DOI  :   10.1128/jb.182.24.6913-6920.2000     PMC  :   PMC94815    
Abstract >>
The elongation factor Tu, encoded by tuf genes, is a GTP binding protein that plays a central role in protein synthesis. One to three tuf genes per genome are present, depending on the bacterial species. Most low-G+C-content gram-positive bacteria carry only one tuf gene. We have designed degenerate PCR primers derived from consensus sequences of the tuf gene to amplify partial tuf sequences from 17 enterococcal species and other phylogenetically related species. The amplified DNA fragments were sequenced either by direct sequencing or by sequencing cloned inserts containing putative amplicons. Two different tuf genes (tufA and tufB) were found in 11 enterococcal species, including Enterococcus avium, Enterococcus casseliflavus, Enterococcus dispar, Enterococcus durans, Enterococcus faecium, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, and Enterococcus raffinosus. For the other six enterococcal species (Enterococcus cecorum, Enterococcus columbae, Enterococcus faecalis, Enterococcus sulfureus, Enterococcus saccharolyticus, and Enterococcus solitarius), only the tufA gene was present. Based on 16S rRNA gene sequence analysis, the 11 species having two tuf genes all have a common ancestor, while the six species having only one copy diverged from the enterococcal lineage before that common ancestor. The presence of one or two copies of the tuf gene in enterococci was confirmed by Southern hybridization. Phylogenetic analysis of tuf sequences demonstrated that the enterococcal tufA gene branches with the Bacillus, Listeria, and Staphylococcus genera, while the enterococcal tufB gene clusters with the genera Streptococcus and Lactococcus. Primary structure analysis showed that four amino acid residues encoded within the sequenced regions are conserved and unique to the enterococcal tufB genes and the tuf genes of streptococci and Lactococcus lactis. The data suggest that an ancestral streptococcus or a streptococcus-related species may have horizontally transferred a tuf gene to the common ancestor of the 11 enterococcal species which now carry two tuf genes.
KeywordMeSH Terms
Evolution, Molecular
Gene Transfer, Horizontal
Genes, Bacterial
32. Singh  KV, Malathum  K, Murray  BE,     ( 2001 )

Disruption of an Enterococcus faecium species-specific gene, a homologue of acquired macrolide resistance genes of staphylococci, is associated with an increase in macrolide susceptibility.

Antimicrobial agents and chemotherapy 45 (1)
PMID : 11120975  :   DOI  :   10.1128/AAC.45.1.263-266.2001     PMC  :   PMC90270    
Abstract >>
The complete sequence (1,479 nucleotides) of msrC, part of which was recently reported by others using a different strain, was determined. This gene was found in 233 of 233 isolates of Enterococcus faecium but in none of 265 other enterococci. Disruption of msrC was associated with a two- to eightfold decrease in MICs of erythromycin azithromycin, tylosin, and quinupristin, suggesting that it may explain in part the apparent greater intrinsic resistance to macrolides of isolates of E. faecium relative to many streptococci. This endogenous, species-specific gene of E. faecium is 53% identical to msr(A), suggesting that it may be a remote progenitor of the acquired macrolide resistance gene found in some isolates of staphylococci.
KeywordMeSH Terms
33. Werner  G, Hildebrandt  B, Klare  I, Witte  W,     ( 2000 )

Linkage of determinants for streptogramin A, macrolide-lincosamide-streptogramin B, and chloramphenicol resistance on a conjugative plasmid in Enterococcus faecium and dissemination of this cluster among streptogramin-resistant enterococci.

International journal of medical microbiology : IJMM 290 (6)
PMID : 11100829  :   DOI  :   10.1016/S1438-4221(00)80020-X    
Abstract >>
A new streptogramin A resistance gene, satG (= vatE), has been recently identified in Enterococcus faecium UW1965 (Werner and Witte 1999. Antimicrob. Agents Chemother. 43: 1813-1814). Further sequence analysis of this plasmid revealed that vatE is in a cluster together with other resistance genes. The identified ORFs were nearly identical with the already known genes ermB and cat. The ermB fragment exhibited more than 99% identity with a resistance region from the streptococcal plasmid pIP501, whereas the cat fragment also contained a truncated rep gene homologue with more than 99% identity to sequences in small staphylococcal plasmids. The cat-rep and the ermB-vatE segments were linked by an IS1216V insertion sequence widely distributed among enterococci. PCR analysis of additional 76 streptogramin-resistant isolates possessing vatE and ermB revealed a linkage of both genes in 45 isolates (59%); 15 of them with a gene arrangement, cat-repU-IS1216V-ermB-vatE, identical to the reference strain UW1965. An identical linkage of IS1216V-ermB-vatE was found among isolates from poultry manure, poultry meat, stool samples of humans, and hospital patients indicating a possible spread of the resistance gene cluster via the food chain to humans.
KeywordMeSH Terms
Macrolides
Multigene Family
34. Goh  SH, Facklam  RR, Chang  M, Hill  JE, Tyrrell  GJ, Burns  EC, Chan  D, He  C, Rahim  T, Shaw  C, Hemmingsen  SM,     ( 2000 )

Identification of Enterococcus species and phenotypically similar Lactococcus and Vagococcus species by reverse checkerboard hybridization to chaperonin 60 gene sequences.

Journal of clinical microbiology 38 (11)
PMID : 11060051  :   PMC  :   PMC87524    
Abstract >>
Data from four recent studies (S. H. Goh et al., J. Clin. Microbiol. 36:2164-2166, 1998; S. H. Goh et al., J. Clin. Microbiol. 34:818-823, 1996; S. H. Goh et al., J. Clin. Microbiol. 35:3116-3121, 1997; A. Y. C. Kwok et al., Int. J. Syst. Bacteriol. 49:1181-1192, 1999) suggest that an approximately 600-bp region of the chaperonin 60 (Cpn60) gene, amplified by PCR with a single pair of degenerate primers, has utility as a potentially universal target for bacterial identification (ID). This Cpn60 gene ID method correctly identified isolates representative of numerous staphylococcal species and Streptococcus iniae, a human and animal pathogen. We report herein that this method enabled us to distinguish clearly between 17 Enterococcus species (Enterococcus asini, Enterococcus rattus, Enterococcus dispar, Enterococcus gallinarum, Enterococcus hirae, Enterococcus durans, Enterococcus cecorum, Enterococcus faecalis, Enterococcus mundtii, Enterococcus casseliflavus, Enterococcus faecium, Enterococcus malodoratus, Enterococcus raffinosus, Enterococcus avium, Enterococcus pseudoavium, Enterococcus new sp. strain Facklam, and Enterococcus saccharolyticus), and Vagococcus fluvialis, Lactococcus lactis, and Lactococcus garvieae. From 123 blind-tested samples, only two discrepancies were observed between the Facklam and Collins phenotyping method (R. R. Facklam and M. D. Collins, J. Clin. Microbiol. 27:731-734, 1989) and the Cpn60 ID method. In each case, the discrepancies were resolved in favor of the Cpn60 ID method. The species distributions of the 123 blind-tested isolates were Enterococcus new sp. strain Facklam (ATCC 700913), 3; E. asini, 1; E. rattus, 4; E. dispar, 2; E. gallinarum, 20; E. hirae, 9; E. durans, 9; E. faecalis, 12; E. mundtii, 3; E. casseliflavus, 8; E. faecium, 25; E. malodoratus, 3; E. raffinosus, 8; E. avium, 4; E. pseudoavium, 1; an unknown Enterococcus clinical isolate, sp. strain R871; Vagococcus fluvialis, 4; Lactococcus garvieae, 3; Lactococcus lactis, 3; Leuconostoc sp., 1; and Pediococcus sp., 1. The Cpn60 gene ID method, coupled with reverse checkerboard hybridization, is an effective method for the identification of Enterococcus and related organisms.
KeywordMeSH Terms
35. Huyton  T, Roper  DI,     ( 2000 )

The molecular basis of vancomycin resistance in clinically relevant Enterococci: crystal structure of D-alanyl-D-lactate ligase (VanA).

Proceedings of the National Academy of Sciences of the United States of America 97 (16)
PMID : 10908650  :   DOI  :   10.1073/pnas.150116497     PMC  :   PMC16797    
Abstract >>
d-alanine-d-lactate ligase from Enterococcus faecium BM4147 is directly responsible for the biosynthesis of alternate cell-wall precursors in bacteria, which are resistant to the glycopeptide antibiotic vancomycin. The crystal structure has been determined with data extending to 2.5-A resolution. This structure shows that the active site has unexpected interactions and is distinct from previous models for d-alanyl-d-lactate ligase mechanistic studies. It appears that the preference of the enzyme for lactate as a ligand over d-alanine could be mediated by electrostatic effects and/or a hydrogen-bonding network, which principally involve His-244. The structure of d-alanyl-d-lactate ligase provides a revised interpretation of the molecular events that lead to vancomycin resistance.
KeywordMeSH Terms
Vancomycin Resistance
36. Myhre  MR, Dahl  KH, Simonsen  GS,     ( 2000 )

Typeability of Tn1546-like elements in vancomycin-resistant enterococci using long-range PCRs and specific analysis of polymorphic regions.

Microbial drug resistance (Larchmont, N.Y.) 6 (1)
PMID : 10868807  :   DOI  :   10.1089/mdr.2000.6.49    
Abstract >>
Molecular subtyping of the VanA-type resistance element Tn1546 in an international collection of 81 genomically diverse vancomycin-resistant enterococci (VRE) from human, animal, and environmental reservoirs was evaluated by restriction analysis of long-range PCR amplicons (PCR-RFLP), single gene PCRs, Southern blot analysis of genomic digests, and partial DNA sequencing. A dominant Tn1546-RFLP in accordance with Enterococcus faecium BM4147 was detected in 43 of the 49 long-range PCR positive strains from ecologically diverse sources in several European countries and the US. Tn1546-like elements from the 32 (40%) long-range PCR negative strains were typed into 17 different groups by single-gene PCRs and Southern blot analysis of the ORF1, ORF2, vanS-vanH, vanX-vanY, and vanZ regions. All these isolates showed deletions in the ORF1 and/or vanZ primer binding regions explaining the failure of long-range PCR amplification. Enlarged vanS-vanH or vanX-vanY fragments were detected in 7 (22%) and 16 (50%) of the long-range PCR negative strains, respectively. The enlarged vanS-vanH regions of five clinical isolates from the US (n = 2), Ireland (n = 2), and Norway (n = 1) contained identical IS1251-like insertions indicating intercontinental spread of the vanA gene cluster. Intergenic vanS-vanH IS1251 insertions have so far not been reported in European studies. Structural rearrangements of Tn1546-like elements may represent single recombination events that can serve as fingerprints in the molecular examination of vanA gene cluster evolution and transmission. The optimal strategy for such analysis has yet to be determined. Two alternative long-range PCRs with subsequent RFLP analysis were successfully used to type the majority of vanA gene clusters in an ecologically and geographically heterogeneous VRE strain collection, but failed to detect and type a group of variant Tn1546-like elements truncated in the left-end ORF1/ORF2 region. Further subtyping of such variants should specifically target the polymorphic vanS-vanH and vanX-vanY regions.
KeywordMeSH Terms
DNA Transposable Elements
37. Ingraham  KA, Iordanescu  S, So  CY, Holmes  DJ, Chalker  AF, Bryant  AP, Brown  JR, Wilding  EI,     ( 2000 )

Identification, evolution, and essentiality of the mevalonate pathway for isopentenyl diphosphate biosynthesis in gram-positive cocci.

Journal of bacteriology 182 (15)
PMID : 10894743  :   DOI  :   10.1128/jb.182.15.4319-4327.2000     PMC  :   PMC101949    
Abstract >>
The mevalonate pathway and the glyceraldehyde 3-phosphate (GAP)-pyruvate pathway are alternative routes for the biosynthesis of the central isoprenoid precursor, isopentenyl diphosphate. Genomic analysis revealed that the staphylococci, streptococci, and enterococci possess genes predicted to encode all of the enzymes of the mevalonate pathway and not the GAP-pyruvate pathway, unlike Bacillus subtilis and most gram-negative bacteria studied, which possess only components of the latter pathway. Phylogenetic and comparative genome analyses suggest that the genes for mevalonate biosynthesis in gram-positive cocci, which are highly divergent from those of mammals, were horizontally transferred from a primitive eukaryotic cell. Enterococci uniquely encode a bifunctional protein predicted to possess both 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and acetyl-CoA acetyltransferase activities. Genetic disruption experiments have shown that five genes encoding proteins involved in this pathway (HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase) are essential for the in vitro growth of Streptococcus pneumoniae under standard conditions. Allelic replacement of the HMG-CoA synthase gene rendered the organism auxotrophic for mevalonate and severely attenuated in a murine respiratory tract infection model. The mevalonate pathway thus represents a potential antibacterial target in the low-G+C gram-positive cocci.
KeywordMeSH Terms
Hemiterpenes
38. Conly  J, Dedier  H, Peters  G, Boyd  DA,     ( 2000 )

Molecular characterization of the vanD gene cluster and a novel insertion element in a vancomycin-resistant enterococcus isolated in Canada.

Journal of clinical microbiology 38 (6)
PMID : 10835012  :   PMC  :   PMC86817    
Abstract >>
A single vanD-containing Enterococcus faecium strain (N97-330) was isolated in Canada. The vanD-containing region was cloned and sequenced. Although the proteins have more than 96% identity to a previously described vanD region in BM4339, the vanS(D) gene contains a frameshift mutation that leads to a predicted truncated protein. Furthermore, sequence analysis of the ddl gene revealed the presence of an IS982-like element (ISEfm1) which interrupted the D-Ala-D-Ala ligase. This suggested the constitutive expression of the vanD operon, which was confirmed. Pulsed-field gel electrophoresis fingerprinting demonstrated that BM4339 was not related to N97-330 (>15 band differences). Both strains contained multiple copies of the IS982-like element.
KeywordMeSH Terms
DNA Transposable Elements
Multigene Family
39. Kao  SJ, You  I, Clewell  DB, Donabedian  SM, Zervos  MJ, Petrin  J, Shaw  KJ, Chow  JW,     ( 2000 )

Detection of the high-level aminoglycoside resistance gene aph(2")-Ib in Enterococcus faecium.

Antimicrobial agents and chemotherapy 44 (10)
PMID : 10991878  :   DOI  :   10.1128/aac.44.10.2876-2879.2000     PMC  :   PMC90169    
Abstract >>
A new high-level gentamicin resistance gene, designated aph(2")-Ib, was cloned from Enterococcus faecium SF11770. The deduced amino acid sequence of the 897-bp open reading frame of aph(2")-Ib shares homology with the aminoglycoside-modifying enzymes AAC(6')-APH(2"), APH(2")-Ic, and APH(2")-Id. The observed phosphotransferase activity is designated APH(2")-Ib.
KeywordMeSH Terms
Bacterial Proteins
40. Lundblad  EW, Rokenes  TP, Dahl  KH,     ( 2000 )

Genetic linkage of the vanB2 gene cluster to Tn5382 in vancomycin-resistant enterococci and characterization of two novel insertion sequences.

Microbiology (Reading, England) 146 (Pt 6) (N/A)
PMID : 10846225  :   DOI  :   10.1099/00221287-146-6-1469    
Abstract >>
VanB-type vancomycin resistance is encoded by the vanB gene cluster, which disseminates by horizontal gene transfer and clonal spread of vancomycin-resistant enterococci (VRE). Genetic linkage of the vanB gene cluster to transposon Tn5382 and the insertion sequences IS16 and IS256-like has previously been shown. In this study linkage of defined vanB gene cluster subtypes to these elements was examined. All the vanB2 subtype strains studied (n=14) revealed co-hybridization of vanB and Tn5382, whereas the strains of vanB1 (n=8) and vanB3 (n=1) subtypes were Tn5382 negative. Conjugative cotransfer of the vanB2 gene cluster and Tn5382 was demonstrated for two strains. DNA sequencing of the vanX(B)-ORFC region in vanB2 strains confirmed that the vanB2 gene cluster is an integral part of Tn5382. No general pattern of linkage was observed with regard to IS16 and IS256-like. Two novel insertion sequences were identified in specific vanB2 subtype strains. (i) A 1611 bp element (ISEnfa110) was detected in the left flank of Tn5382. Its insertion site, lack of terminal inverted and direct repeats, and two conserved motifs in its putative transposase all conform to the conventions of the IS110 family. (ii) A 787 bp element (ISEnfa200) was detected in the vanS(B)-vanY(B) intergenic region. Its ORF encoded a putative protein with 60-70% identity to transposases of the IS200 family. No further copies of ISEnfa110 were found by colony hybridization of 181 enterococcal isolates, whereas ISEnfa200 was found in four additional vanB2 strains from the USA. The five strains had identical ISEnfa200 element insertion sites, and Tn5382 was located downstream from a pbp5 gene conferring high-level ampicillin resistance. These isolates showed related PFGE patterns, suggesting possible clonal spread of a VRE strain harbouring a Tn5382-vanB2-ISEnfa200 element linked to a pbp5 gene conferring ampicillin resistance.
KeywordMeSH Terms
Genes, Bacterial
Multigene Family
41. Peterson  DL, Gleisner  JM, Blakley  RL,     ( 1975 )

The structure of the mutant dihydrofolate reductase from Streptococcus faecium. Amino acid sequence of peptide CNBr 7 and complete sequence of the protein.

The Journal of biological chemistry 250 (13)
PMID : 1097435  :  
Abstract >>
The complete amino acid sequence of the mutant dihydrofolate reductase from Streptococcus faecium var. Durans strain A has been determined by sequence analysis of peptides produced by tryptic, chymotryptic, thermolytic, and mild acid cleavage of the large peptide CNBr 7 and from previously reported studies. The sequence of the S. faecium enzyme is compared to the reported sequence of dihydrofolate reductase from Escherichia coli and the two are shown to contain two domains of substantial homology. One of these domains consists of the NH2-terminal 60 residues and is considered to contribute the dihydrofolate binding site. The second domain probably contains the dinucleotide binding structure. Comparison of the sequences of the dihydrofolate reductases with those of larger dehydrogenases of known structure failed to show any evidence for homology. Considerations of size and predictions of secondary structure also suggest that the second domain in the reductases has no structural similarity to the nucleotide binding site in the larger dehydrogenases. It is concluded that the two reductases are related, although distantly, but that they have evolved from an ancestral protein different from the primitive predecessor of the other oxidoreductases.
KeywordMeSH Terms
42. Jensen  LB, Hammerum  AM, Aarestrup  FM,     ( 2000 )

Linkage of vat(E) and erm(B) in streptogamin-resistant Enterococcus faecium isolates from Europe.

Antimicrobial agents and chemotherapy 44 (8)
PMID : 11023445  :   DOI  :   10.1128/aac.44.8.2231-2232.2000     PMC  :   PMC90050    
Abstract >>
N/A
KeywordMeSH Terms
Bacterial Proteins
43. McGregor  KF, Young  HK,     ( 2000 )

Identification and characterization of vanB2 glycopeptide resistance elements in enterococci isolated in Scotland.

Antimicrobial agents and chemotherapy 44 (9)
PMID : 10952577  :   DOI  :   10.1128/aac.44.9.2341-2348.2000     PMC  :   PMC90067    
Abstract >>
Thirty-two vanB glycopeptide-resistant enterococci (28 Enterococcus faecium and 4 Enterococcus faecalis) were collected from hospitalized patients in Glasgow, Edinburgh, Dundee, and Aberdeen, Scotland, and the vanB element in each was compared to vanB1 of E. faecalis strain ATCC 51299. HhaI digestion of PCR fragments of the vanB ligase gene was used to identify vanB subtypes. All E. faecium isolates were vanB2, and all E. faecalis isolates were vanB1. Restriction fragment length polymorphism analysis of a 5,180-bp vanS(B)-vanX(B) long-PCR fragment of the vanB cluster showed the loss of HaeII restriction sites in vanS(B), vanW, and vanX(B) in strains containing a vanB2 ligase gene. Partial sequences of genes in the vanB2 cluster for two genomically distinct Scottish isolates were >99.8% identical to each other. vanS(B2), vanX(B2), and vanB2 sequences differed at the nucleotide level from those of vanS(B), vanX(B), and vanB by 4.2, 4.6, and 4.8%, respectively. The vanB2 resistance element appears to be widespread among VanB glycopeptide-resistant E. faecium strains isolated in Scottish hospitals.
KeywordMeSH Terms
Glycopeptides
Serine-Type D-Ala-D-Ala Carboxypeptidase
44. Zarazaga  M, Alonso  A, Ruiz-Larrea  F, Portillo  A,     ( 2000 )

Macrolide resistance genes in Enterococcus spp.

Antimicrobial agents and chemotherapy 44 (4)
PMID : 10722498  :   DOI  :   10.1128/aac.44.4.967-971.2000     PMC  :   PMC89799    
Abstract >>
Seventy-eight isolates of different Enterococcus species (E. faecalis, n = 27; E. faecium, n = 23; E. durans, n = 8; E. avium, n = 6; E. hirae, n = 9; E. gallinarum, n = 3; and E. casseliflavus, n = 2) with a variety of erythromycin resistance phenotypes were examined for the presence of macrolide resistance genes (ermA, ermB, ermC, ermTR, mefA/E, and msrA). Positive PCR amplifications of ermB were obtained for 39 of 40 highly erythromycin-resistant Enterococcus isolates (MICs, >128 microg/ml) of different species; the remaining highly resistant E. faecium isolate was positive for PCR amplification of ermA but was negative for PCR amplification of the ermB and ermC genes. For all enterococcal strains for which erythromycin MICs were < or =32 microg/ml PCRs were negative for erm methylase genes. For all E. faecium isolates PCR amplified products of the expected size of 400 bp were obtained when msrA primers were used, with the results being independent of the erythromycin resistance phenotype. All the other enterococcal species gave negative results by msrA PCRs. Sequencing of the msrA PCR products from either erythromycin-susceptible, low-level-resistant, or highly resistant E. faecium strains showed that the amplicons did not correspond to the msrA gene described for Staphylococcus epidermidis but corresponded to a new putative efflux determinant, which showed 62% identity with the msrA gene at the DNA level and 72% similarity at the amino acid level. This new gene was named msrC.
KeywordMeSH Terms
Membrane Transport Proteins
45. Quesnes  G, Poyart  C,     ( 2000 )

Sequencing the gene encoding manganese-dependent superoxide dismutase for rapid species identification of enterococci.

Journal of clinical microbiology 38 (1)
PMID : 10618129  :   PMC  :   PMC88737    
Abstract >>
Simple PCR and sequencing assays that utilize a single pair of degenerate primers were used to characterize a 438-bp-long DNA fragment internal (sodA(int)) to the sodA gene encoding the manganese-dependent superoxide dismutase in 19 enterococcal type strains (Enterococcus avium, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus columbae, Enterococcus dispar, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus flavescens, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, Enterococcus raffinosus, Enterococcus saccharolyticus, Enterococcus seriolicida, Enterococcus solitarius, and Enterococcus sulfureus). Sequence analysis of the sodA(int) fragments enabled reliable identification of 18 enterococcal species, including E. casseliflavus-E. flavescens and E. gallinarum. The sodA(int) fragments of E. casseliflavus and E. flavescens were almost identical (99.5% sequence identity), which suggests that they should be associated in a single species. Our results confirm that the sodA gene constitutes a more discriminative target sequence than 16S rRNA gene in differentiating closely related bacterial species.
KeywordMeSH Terms
Genes, Bacterial
46. Allignet  J, Aubert  S, Haroche  J,     ( 2000 )

satG, conferring resistance to streptogramin A, is widely distributed in Enterococcus faecium strains but not in staphylococci.

Antimicrobial agents and chemotherapy 44 (1)
PMID : 10602747  :   DOI  :   10.1128/aac.44.1.190-191.2000     PMC  :   PMC89652    
Abstract >>
A gene almost identical to satG was isolated from an Enterococcus faecium strain. This gene was transferred to a Staphylococcus aureus recipient strain where it conferred resistance to streptogramin A. satG was found to be widely distributed among E. faecium strains but not detected among staphylococci.
KeywordMeSH Terms
47. Ménard  C, Roy  PH, Martineau  F, Picard  FJ, Ke  D,     ( 1999 )

Development of a PCR assay for rapid detection of enterococci.

Journal of clinical microbiology 37 (11)
PMID : 10523541  :   PMC  :   PMC85677    
Abstract >>
Enterococci are becoming major nosocomial pathogens, and increasing resistance to vancomycin has been well documented. Conventional identification methods, which are based on culturing, require 2 to 3 days to provide results. PCR has provided a means for the culture-independent detection of enterococci in a variety of clinical specimens and is capable of yielding results in just a few hours. However, all PCR-based assays developed so far are species specific only for clinically important enterococci. We have developed a PCR-based assay which allows the detection of enterococci at the genus level by targeting the tuf gene, which encodes elongation factor EF-Tu. Initially, we compared the nucleotide sequences of the tuf gene from several bacterial species (available in public databases) and designed degenerate PCR primers derived from conserved regions. These primers were used to amplify a target region of 803 bp from four enterococcal species (Enterococcus avium, E. faecalis, E. faecium, and E. gallinarum). Subsequently, the complete nucleotide sequences of these amplicons were determined. The analysis of a multiple alignment of these sequences revealed regions conserved among enterococci but distinct from those of other bacteria. PCR primers complementary to these regions allowed amplification of genomic DNAs from 14 of 15 species of enterococci tested (E. solitarius DNA could not be amplified). There was no amplification with a majority of 79 nonenterococcal bacterial species, except for 2 Abiotrophia species and several Listeria species. Furthermore, this assay efficiently amplified all 159 clinical isolates of enterococci tested (61 E. faecium, 77 E. faecalis, 9 E. gallinarum, and 12 E. casseliflavus isolates). Interestingly, the preliminary sequence comparison of the amplicons for four enterococcal species demonstrated that there were some sequence variations which may be used to generate species-specific internal probes. In conclusion, this rapid PCR-based assay is capable of detecting all clinically important enterococci and has potential for use in clinical microbiology laboratories.
KeywordMeSH Terms
48. Witte  W, Werner  G,     ( 1999 )

Characterization of a new enterococcal gene, satG, encoding a putative acetyltransferase conferring resistance to Streptogramin A compounds.

Antimicrobial agents and chemotherapy 43 (7)
PMID : 10438336  :   PMC  :   PMC89374    
Abstract >>
N/A
KeywordMeSH Terms
Genes, Bacterial
49. DelVecchio  VG, McCleskey  FK, Niemeyer  DM, Musso  RE, Liu  R,     ( 1999 )

Cloning of insertion sequence IS1485 from Enterococcus species.

Plasmid 42 (1)
PMID : 10413664  :   DOI  :   10.1006/plas.1999.1409    
Abstract >>
We have cloned and identified an insertion sequence, IS1485, that was present in several members of the genus Enterococcus. IS1485 exists in varying copy numbers with at least 12 copies in E. durans (ATCC 11576), 3 copies in E. faecium (ATCC 19434), and one copy each in E. faecalis (ATCC 19433) and E. avium (ATCC 14024). It was also detected in clinical strains of E. gallinarum, E. casseliflavus, and E. saccharolyticus. IS1485 is 1366 bp in length, it has imperfect terminal inverted repeats with 25 of the terminal 39 residues matched, and it contains three open reading frames exceeding 50 codons, designated orfA, orfB, and orfC. The largest, orfB, was located 36 bp downstream and in the -1 reading frame relative to orfA; orfC is oriented in the opposite direction and overlaps orfA. The genetic organization of IS1485 resembles that of members of the IS3 family of transposable elements. Sequence homology exists with several members of the IS3 family especially with IS199 from Streptococcus mutans.
KeywordMeSH Terms
50. Samore  MH, Tenover  FC, Venkataraman  L, Thauvin-Eliopoulos  C, Clark  NC, Ostrowsky  BE,     ( 1999 )

A cluster of VanD vancomycin-resistant Enterococcus faecium: molecular characterization and clinical epidemiology.

The Journal of infectious diseases 180 (4)
PMID : 10479146  :   DOI  :   10.1086/315030    
Abstract >>
VanD-mediated glycopeptide resistance has been reported for an isolate of Enterococcus faecium, BM4339. Three clinical isolates of vancomycin-resistant E. faecium collected from 3 patients during a 6-week period in 1993 had agar dilution MICs of vancomycin and teicoplanin of 128 and 4 microg/mL, respectively. Polymerase chain reaction (PCR) using degenerate primers complementary to genes encoding d-Ala-d-X ligases yielded a 630-bp product that was similar to the published partial sequence of vanD. By use of inverse PCR, vanD, vanHD, and two partial flanking open-reading frames were sequenced. The deduced amino acid sequence of VanD showed 67% identity with VanA and VanB. vanD appeared to be located on the chromosome and was not transferable to other enterococci. The 3 isolates were indistinguishable by pulsed-field gel electrophoresis and differed from BM4339. No other isolates carrying vanD were found in a subset of 875 recent US isolates of vancomycin-resistant enterococci.
KeywordMeSH Terms
Vancomycin Resistance
51. Draker  K, Wright  GD, Berghuis  AM,     ( 1999 )

Crystal structure of an aminoglycoside 6'-N-acetyltransferase: defining the GCN5-related N-acetyltransferase superfamily fold.

Structure (London, England : 1993) 7 (5)
PMID : 10378269  :  
Abstract >>
The predominant mechanism of antibiotic resistance employed by pathogenic bacteria against the clinically used aminoglycosides is chemical modification of the drug. The detoxification reactions are catalyzed by enzymes that promote either the phosphorylation, adenylation or acetylation of aminoglycosides. Structural studies of these aminoglycoside-modifying enzymes may assist in the development of therapeutic agents that could circumvent antibiotic resistance. In addition, such studies may shed light on the development of antibiotic resistance and the evolution of different enzyme classes. The crystal structure of the aminoglycoside-modifying enzyme aminoglycoside 6'-N-acetyltransferase type li (AAC(6')-li) in complex with the cofactor acetyl coenzyme A has been determined at 2.7 A resolution. The structure establishes that this acetyltransferase belongs to the GCN5-related N-acetyltransferase superfamily, which includes such enzymes as the histone acetyltransferases GCN5 and Hat1. Comparison of the AAC(6')-li structure with the crystal structures of two other members of this superfamily, Serratia marcescens aminoglycoside 3-N-acetyltransferase and yeast histone acetyltransferase Hat1, reveals that of the 84 residues that are structurally similar, only three are conserved and none can be implicated as catalytic residues. Despite the negligible sequence identity, functional studies show that AAC(6')-li possesses protein acetylation activity. Thus, AAC(6')-li is both a structural and functional homolog of the GCN5-related histone acetyltransferases.
KeywordMeSH Terms
DNA-Binding Proteins
Protein Folding
Saccharomyces cerevisiae Proteins
52. Courvalin  P,     ( 1999 )

Characterization of the vanD glycopeptide resistance gene cluster from Enterococcus faecium BM4339.

Journal of bacteriology 181 (12)
PMID : 10368136  :   PMC  :   PMC93839    
Abstract >>
VanD-type resistance to glycopeptides in Enterococcus faecium BM4339 is due to constitutive synthesis of D-alanyl-D-lactate-terminating peptidoglycan precursors (B. P?richon, P. Reynolds, and P. Courvalin, Antimicrob. Agents Chemother. 41:2016-2018, 1997). The sequence of a 5,780-bp fragment was determined and revealed six open reading frames. The 3' distal part encoded the VanHD dehydrogenase, the VanD ligase, and the VanXD DD-dipeptidase, which were highly similar to the corresponding proteins in VanA and VanB types of resistance. The deduced VanYD protein was homologous to penicillin-binding proteins that display DD-carboxypeptidase activity. The 5' end coded for the putative VanRD-VanSD two-component regulatory system. Due to a frameshift mutation in the chromosomal ddl gene, BM4339 produced an impaired D-alanine:D-alanine ligase. However, since expression of the resistance genes is constitutive, growth of E. faecium BM4339 was not dependent on the presence of glycopeptides in the culture medium.
KeywordMeSH Terms
Carboxypeptidases
Membrane Proteins
Multigene Family
Open Reading Frames
53. Holzapfel  WH, Vederas  JC, Quadri  LE,     ( 1999 )

Atypical genetic locus associated with constitutive production of enterocin B by Enterococcus faecium BFE 900.

Applied and environmental microbiology 65 (5)
PMID : 10224016  :   PMC  :   PMC91313    
Abstract >>
A purified bacteriocin produced by Enterococcus faecium BFE 900 isolated from black olives was shown by Edman degradation and mass spectrometric analyses to be identical to enterocin B produced by E. faecium T136 from meat (P. Casaus, T. Nilsen, L. M. Cintas, I. F. Nes, P. E. Hern?ndez, and H. Holo, Microbiology 143:2287-2294, 1997). The structural gene was located on a 2.2-kb HindIII fragment and a 12.0-kb EcoRI chromosomal fragment. The genetic characteristics and production of EntB by E. faecium BFE 900 differed from that described so far by the presence of a conserved sequence like a regulatory box upstream of the EntB gene, and its production was constitutive and not regulated. The 2.2-kb chromosomal fragment contained the hitherto undetected immunity gene for EntB in an atypical orientation that is the reverse of that of the structural gene. Typical transport and other genes associated with bacteriocin production were not detected on the 12.0-kb chromosomal fragment containing the EntB structural gene. This makes the EntB genetic system different from most other bacteriocin systems, where transport and possible regulatory genes are clustered. EntB was subcloned and expressed by the dedicated secretion machinery of Carnobacterium piscicola LV17A. The structural gene was amplified by PCR, fused to the divergicin A signal peptide, and expressed by the general secretory pathway in Enterococcus faecalis ATCC 19433.
KeywordMeSH Terms
Genes, Bacterial
54. Palepou  MF, Woodford  N,     ( 1999 )

Nucleotide sequence of IS1542, an insertion sequence identified within VanA glycopeptide resistance elements of enterococci.

FEMS microbiology letters 173 (2)
PMID : 10227164  :   DOI  :   10.1111/j.1574-6968.1999.tb13523.x    
Abstract >>
IS1542 is an insertion sequence-like element which was originally identified in the orf2-vanR intergenic region of VanA resistance elements of glycopeptide-resistant Enterococcus faecium from hospital patients and from non-human sources in the UK. The nucleotide sequence of IS1542 was determined. It showed 81% homology with IS256 and contained an open reading frame sufficient to encode a 390 amino acid peptide predicted to have 87.2% identity with the transposase of IS256. By PCR, IS1542 was detected in the genomes of 26 of 28 (93%) VanA enterococci isolated from poultry in the UK and Ireland, but in only two of 65 (3%) glycopeptide-sensitive or VanC enterococci from hospital patients in the UK and Brazil. IS1542 may be a useful marker for evolutionary and epidemiological studies of VanA glycopeptide resistance in enterococci.
KeywordMeSH Terms
DNA Transposable Elements
55. Dahl  KH, Sundsfjord  A, Olsvik  O,     ( 1999 )

Heterogeneity in the vanB gene cluster of genomically diverse clinical strains of vancomycin-resistant enterococci.

Antimicrobial agents and chemotherapy 43 (5)
PMID : 10223921  :   PMC  :   PMC89118    
Abstract >>
Molecular analysis of 17 genomically unrelated clinical VanB-type vancomycin-resistant enterococcus isolates from hospital patients in Germany, Norway, Sweden, the United Kingdom and the United States revealed three subtypes of the vanB gene cluster-vanB1, vanB2, and vanB3-which was in accordance with previous subtyping of the ligase gene sequence. There was no correlation between vanB subtype and levels of vancomycin resistance. All strains studied carried a structurally conserved vanB gene cluster as shown by long-range PCR (long PCR) covering 5,959 bp of the published sequence in vanB1 strain V583. Restriction analysis of long PCR amplicons displayed one unique vanB1 pattern and a second vanB2- and vanB3-specific pattern. The vanSB-vanYB intergenic sequences with flanking coding regions were identical within each vanB subtype with one exception. A U.S. vanB2 isolate had a 789-bp enlargement of this region containing a putative open reading frame (ORF) with substantial homology to an ORF in the Clostridium perfringens IS1469 insertion element. The molecular heterogeneity within the vanB gene cluster has implications for the selection of PCR primers, as the primers must ensure detection of all vanB subtypes, and is of importance when considering reservoirs and dissemination of vanB resistance. The molecular identity within the vanB1 and the vanB2 subtype indicates horizontal transmission of both gene clusters between isolates in different geographical areas. Restriction analysis of long PCR vanB amplicons may reveal specific varieties that can be used as epidemiological markers for mobile determinants conferring VanB-type resistance. The finding of three distinct vanB gene clusters should encourage a search for different environmental reservoirs of vanB resistance determinants.
KeywordMeSH Terms
Genome, Bacterial
Multigene Family
56. Fita  I, Rubio  V, Barcelona  B, Uriarte  M, Alzari  PM,     ( 1999 )

Carbamate kinase: New structural machinery for making carbamoyl phosphate, the common precursor of pyrimidines and arginine.

Protein science : a publication of the Protein Society 8 (4)
PMID : 10211841  :   DOI  :   10.1110/ps.8.4.934     PMC  :   PMC2144312    
Abstract >>
The enzymes carbamoyl phosphate synthetase (CPS) and carbamate kinase (CK) make carbamoyl phosphate in the same way: by ATP-phosphorylation of carbamate. The carbamate used by CK is made chemically, whereas CPS itself synthesizes its own carbamate in a process involving the phosphorylation of bicarbonate. Bicarbonate and carbamate are analogs and the phosphorylations are carried out by homologous 40 kDa regions of the 120 kDa CPS polypeptide. CK can also phosphorylate bicarbonate and is a homodimer of a 33 kDa subunit that was believed to resemble the 40 kDa regions of CPS. Such belief is disproven now by the CK structure reported here. The structure does not conform to the biotin carboxylase fold found in the 40 kDa regions of CPS, and presents a new type of fold possibly shared by homologous acylphosphate-making enzymes. A molecular 16-stranded open beta-sheet surrounded by alpha-helices is the hallmark of the CK dimer. Each subunit also contains two smaller sheets and a large crevice found at the location expected for the active center. Intersubunit interactions are very large and involve a central hydrophobic patch and more hydrophilic peripheral contacts. The crevice holds a sulfate that may occupy the site of an ATP phosphate, and is lined by conserved residues. Site-directed mutations tested at two of these residues inactivate the enzyme. These findings support active site location in the crevice. The orientation of the crevices in the dimer precludes their physical cooperation in the catalytic process. Such cooperation is not needed in the CK reaction but is a requirement of the mechanism of CPSs.
KeywordMeSH Terms
57. O'Keeffe  T, Hill  C,     ( 1999 )

Characterization and heterologous expression of the genes encoding enterocin a production, immunity, and regulation in Enterococcus faecium DPC1146.

Applied and environmental microbiology 65 (4)
PMID : 10103244  :   PMC  :   PMC91214    
Abstract >>
Enterocin A is a small, heat-stable, antilisterial bacteriocin produced by Enterococcus faecium DPC1146. The sequence of a 10, 879-bp chromosomal region containing at least 12 open reading frames (ORFs), 7 of which are predicted to play a role in enterocin biosynthesis, is presented. The genes entA, entI, and entF encode the enterocin A prepeptide, the putative immunity protein, and the induction factor prepeptide, respectively. The deduced proteins EntK and EntR resemble the histidine kinase and response regulator proteins of two-component signal transducing systems of the AgrC-AgrA type. The predicted proteins EntT and EntD are homologous to ABC (ATP-binding cassette) transporters and accessory factors, respectively, of several other bacteriocin systems and to proteins implicated in the signal-sequence-independent export of Escherichia coli hemolysin A. Immediately downstream of the entT and entD genes are two ORFs, the product of one of which, ORF4, is very similar to the product of the yteI gene of Bacillus subtilis and to E. coli protease IV, a signal peptide peptidase known to be involved in outer membrane lipoprotein export. Another potential bacteriocin is encoded in the opposite direction to the other genes in the enterocin cluster. This putative bacteriocin-like peptide is similar to LafX, one of the components of the lactacin F complex. A deletion which included one of two direct repeats upstream of the entA gene abolished enterocin A activity, immunity, and ability to induce bacteriocin production. Transposon insertion upstream of the entF gene also had the same effect, but this mutant could be complemented by exogenously supplied induction factor. The putative EntI peptide was shown to be involved in the immunity to enterocin A. Cloning of a 10.5-kb amplicon comprising all predicted ORFs and regulatory regions resulted in heterologous production of enterocin A and induction factor in Enterococcus faecalis, while a four-gene construct (entAITD) under the control of a constitutive promoter resulted in heterologous enterocin A production in both E. faecalis and Lactococcus lactis.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
58. Jalal  S, Wretlind  B,     ( 1999 )

Alterations in GyrA and ParC associated with fluoroquinolone resistance in Enterococcus faecium.

Antimicrobial agents and chemotherapy 43 (4)
PMID : 10103206  :   PMC  :   PMC89232    
Abstract >>
High-level quinolone resistance in Enterococcus faecium was associated with mutations in both gyrA and parC genes in 10 of 11 resistant strains. On low-level resistant strain without such mutations may instead possess an efflux mechanism or alterations in the other subunits of the gyrase or topoisomerase IV genes. These findings are similar to those for other gram-positive bacteria, such as Enterococcus faecalis.
KeywordMeSH Terms
59. Leclercq  R, Stockman  BJ, Farley  KA, Yurek  DA,     ( 1999 )

A new resistance gene, linB, conferring resistance to lincosamides by nucleotidylation in Enterococcus faecium HM1025.

Antimicrobial agents and chemotherapy 43 (4)
PMID : 10103201  :   PMC  :   PMC89227    
Abstract >>
Resistance to lincomycin and clindamycin in the clinical isolate Enterococcus faecium HM1025 is due to a ribosomal methylase encoded by an ermAM-like gene and the plasmid-mediated inactivation of these antibiotics. We have cloned and determined the nucleotide sequence of the gene responsible for the inactivation of lincosamides, linB. This gene encodes a 267-amino-acid lincosamide nucleotidyltransferase. The enzyme catalyzes 3(5'-adenylation) (the adenylation of the hydroxyl group in position 3 of the molecules) of lincomycin and clindamycin. Expression of linB was observed in both Escherichia coli and Staphylococcus aureus. The deduced amino acid sequence of the enzyme did not display any significant homology with staphylococcal nucleotidyltransferases encoded by linA and linA' genes. Sequences homologous to linB were found in 14 other clinical isolates of E. faecium, indicating the spread of the resistance trait in this species.
KeywordMeSH Terms
Macrolides
60. Hendrickx  AP, van Wamel  WJ, Posthuma  G, Bonten  MJ, Willems  RJ,     ( 2007 )

Five genes encoding surface-exposed LPXTG proteins are enriched in hospital-adapted Enterococcus faecium clonal complex 17 isolates.

Journal of bacteriology 189 (22)
PMID : 17873043  :   DOI  :   10.1128/JB.00664-07     PMC  :   PMC2168695    
Abstract >>
Most Enterococcus faecium isolates associated with hospital outbreaks and invasive infections belong to a distinct genetic subpopulation called clonal complex 17 (CC17). It has been postulated that the genetic evolution of CC17 involves the acquisition of various genes involved in antibiotic resistance, metabolic pathways, and virulence. To gain insight into additional genes that may have favored the rapid emergence of this nosocomial pathogen, we aimed to identify surface-exposed LPXTG cell wall-anchored proteins (CWAPs) specifically enriched in CC17 E. faecium. Using PCR and Southern and dot blot hybridizations, 131 E. faecium isolates (40 CC17 and 91 non-CC17) were screened for the presence of 22 putative CWAP genes identified from the E. faecium TX0016 genome. Five genes encoding LPXTG surface proteins were specifically enriched in E. faecium CC17 isolates. These five LPXTG surface protein genes were found in 28 to 40 (70 to 100%) of CC17 and in only 7 to 24 (8 to 26%) of non-CC17 isolates (P < 0.05). Three of these CWAP genes clustered together on the E. faecium TX0016 genome, which may comprise a novel enterococcal pathogenicity island covering E. faecium contig 609. Expression at the mRNA level was demonstrated, and immunotransmission electron microscopy revealed an association of the five LPXTG surface proteins with the cell wall. Minimal spanning tree analysis based on the presence and absence of 22 CWAP genes revealed grouping of all 40 CC17 strains together with 18 hospital-derived but evolutionary unrelated non-CC17 isolates in a distinct CWAP-enriched cluster, suggesting horizontal transfer of CWAP genes and a role of these CWAPs in hospital adaptation.
KeywordMeSH Terms
61. Michel  C, Pelletier  C, Boussaha  M, Douet  DG, Lautraite  A, Tailliez  P,     ( 2007 )

Diversity of lactic acid bacteria associated with fish and the fish farm environment, established by amplified rRNA gene restriction analysis.

Applied and environmental microbiology 73 (9)
PMID : 17337536  :   DOI  :   10.1128/AEM.01852-06     PMC  :   PMC1892897    
Abstract >>
Lactic acid bacteria have become a major source of concern for aquaculture in recent decades. In addition to true pathogenic species of worldwide significance, such as Streptococcus iniae and Lactococcus garvieae, several species have been reported to produce occasional fish mortalities in limited geographic areas, and many unidentifiable or ill-defined isolates are regularly isolated from fish or fish products. To clarify the nature and prevalence of different fish-associated bacteria belonging to the lactic acid bacterium group, a collection of 57 isolates of different origins was studied and compared with a set of 22 type strains, using amplified rRNA gene restriction analysis (ARDRA). Twelve distinct clusters were delineated on the basis of ARDRA profiles and were confirmed by sequencing of sodA and 16S rRNA genes. These clusters included the following: Lactococcus raffinolactis, L. garvieae, Lactococcus l., S. iniae, S. dysgalactiae, S. parauberis, S. agalactiae, Carnobacterium spp., the Enterococcus "faecium" group, a heterogeneous Enterococcus-like cluster comprising indiscernible representatives of Vagococcus fluvialis or the recently recognized V. carniphilus, V. salmoninarum, and Aerococcus spp. Interestingly, the L. lactis and L. raffinolactis clusters appeared to include many commensals of fish, so opportunistic infections caused by these species cannot be disregarded. The significance for fish populations and fish food processing of three or four genetic clusters of uncertain or complex definition, namely, Aerococcus and Enterococcus clusters, should be established more accurately.
KeywordMeSH Terms
Aquaculture
Biodiversity
Phylogeny
62. Poeta  P, Costa  D, Igrejas  G, Sáenz  Y, Zarazaga  M, Rodrigues  J, Torres  C,     ( 2007 )

Polymorphisms of the pbp5 gene and correlation with ampicillin resistance in Enterococcus faecium isolates of animal origin.

Journal of medical microbiology 56 (Pt 2)
PMID : 17244806  :   DOI  :   10.1099/jmm.0.46778-0    
Abstract >>
The C-terminal region of the pbp5 gene was sequenced in 11 ampicillin-resistant and 5 ampicillin-susceptible Enterococcus faecium isolates of animal origin, and compared with a pbp5 reference sequence (GenBank accession no. X84860). Eight different pbp5 alleles (designated A-H) were detected when amino acid changes in the region 461-629 were considered. Three of these alleles (A-C) were detected in ampicillin-susceptible isolates (MIC range 1-8 microg ml-1), and included the changes 470H-->Q, 471V-->I, 487Q-->L, 581I-->V, 595E-->A or 622E-->D. The remaining five alleles (D-H) were found in ampicillin-resistant isolates (MIC range 32-256 microg ml-1); three of these alleles (F-H) presented a serine insertion at position 466', in addition to other important amino acid changes (485M-->A, 496N-->K, 499A-->T, 525E-->D, 586V-->L or 629E-->V). The other two alleles presented the amino acid changes 496N-->K and 629E-->V (allele D), and 470H-->Q (allele F). A correlation between deduced amino acid changes in PBP5 and ampicillin MICs was detected in animal E. faecium isolates.
KeywordMeSH Terms
Ampicillin Resistance
Polymorphism, Genetic
63. Sletvold  H, Johnsen  PJ, Simonsen  GS, Aasnaes  B, Sundsfjord  A, Nielsen  KM,     ( 2007 )

Comparative DNA analysis of two vanA plasmids from Enterococcus faecium strains isolated from poultry and a poultry farmer in Norway.

Antimicrobial agents and chemotherapy 51 (2)
PMID : 17116680  :   DOI  :   10.1128/AAC.00557-06     PMC  :   PMC1797720    
Abstract >>
The DNA sequences of two plasmids carrying vanA, pVEF1 (39,626 bp) and pVEF2 (39,714 bp), were determined. Forty-three shared coding sequences were identified, and the only nucleotide difference was an 88-bp indel. A postsegregational killing system was identified. This system possibly explains the persistence of the vanA gene cluster in Norwegian poultry farms.
KeywordMeSH Terms
Plasmids
64. Arlindo  S, Calo  P, Franco  C, Prado  M, Cepeda  A, Barros-Velázquez  J,     ( 2006 )

Single nucleotide polymorphism analysis of the enterocin P structural gene of Enterococcus faecium strains isolated from nonfermented animal foods.

Molecular nutrition & food research 50 (12)
PMID : 17103378  :   DOI  :   10.1002/mnfr.200600178    
Abstract >>
The bacteriocins produced by two lactic acid bacteria isolated from nonfermented fresh meat and fish, respectively, and exhibiting a remarkable antilisterial activity, were characterized. Bacteriocinogenic strains were identified as Enterococcus faecium and the maximum bacteriocin production by both strains was detected in the stationary phase of growth. The activity against Listeria monocytogenes was maintained in pH range of 3-7 and was stable in both strains after heating at 100 or 121 degrees C. The genes coding for enterocin P were detected, isolated, and sequenced in both E. faecium strains. They exhibited DNA/DNA homology in the 87.1-97.2% range with respect to the other four enterocin P genes reported so far. Three single nucleotide polymorphism events, silent at the amino acid level, were detected at nucleotide positions 45 (G/A), 75 (A/G), and 90 (T/C) in E. faecium LHICA 28-4 and may explain the differences reported for those loci in other enterocin P-producing E. faecium strains. This work provides the first description of enterocin P-producing E. faecium strains in nonfermented foodstuffs and, in the case of E. faecium LHICA 51, the first report of an enterocin P-producing strain isolated from fish so far.
KeywordMeSH Terms
Polymorphism, Single Nucleotide
65. Todokoro  D, Tomita  H, Inoue  T, Ike  Y,     ( 2006 )

Genetic analysis of bacteriocin 43 of vancomycin-resistant Enterococcus faecium.

Applied and environmental microbiology 72 (11)
PMID : 17088377  :   DOI  :   10.1128/AEM.00934-06     PMC  :   PMC1636183    
Abstract >>
A total of 636 vancomycin-resistant Enterococcus faecium (VRE) isolates obtained between 1994 and 1999 from the Medical School Hospital of the University of Michigan were tested for bacteriocin production. Of the 277 (44%) bacteriocinogenic strains, 21 were active against E. faecalis, E. faecium, E. hirae, E. durans, and Listeria monocytogenes. Of those 21 strains, a representative bacteriocin of strain VRE82, designated bacteriocin 43, was found to be encoded on mobilizable plasmid pDT1 (6.2 kbp). Nine open reading frames (ORFs), ORF1 to ORF9, were presented on pDT1 and were oriented in the same direction. The bacteriocin 43 locus (bac43) consists of the bacteriocin gene bacA (ORF1) and the immunity gene bacB (ORF2). The deduced bacA product is 74 amino acids in length with a putative signal peptide of 30 amino acids at the N terminus. The bacB gene encodes a deduced 95-amino-acid protein without a signal sequence. The predicted mature BacA protein (44 amino acids) showed sequence homology with the membrane-active class IIa bacteriocins of lactic acid bacteria and showed 86% homology with bacteriocin 31 from E. faecalis YI717 and 98% homology with bacteriocin RC714. Southern analysis with a bac43 probe of each plasmid DNA from the 21 strains showed hybridization to a specific fragment corresponding to the 6.2-kbp EcoRI fragment, suggesting that the strains harbored the pDT1-like plasmid (6.2 kb) which encoded the bacteriocin 43-type bacteriocin. The bac43 determinant was not identified among non-VRE clinical isolates.
KeywordMeSH Terms
Vancomycin Resistance
66. De Kwaadsteniet  M, Fraser  T, Van Reenen  CA, Dicks  LM,     ( 2006 )

Bacteriocin T8, a novel class IIa sec-dependent bacteriocin produced by Enterococcus faecium T8, isolated from vaginal secretions of children infected with human immunodeficiency virus.

Applied and environmental microbiology 72 (7)
PMID : 16820469  :   DOI  :   10.1128/AEM.00436-06     PMC  :   PMC1489345    
Abstract >>
Enterococcus faecium T8, isolated from vaginal secretions of children with human immunodeficiency virus, produces a class IIa sec-dependent bacteriocin that is structurally different from three other class IIa sec-dependent bacteriocins, i.e., enterocin P and an enterocin P-like bacteriocin, produced by Enterococcus faecium, and bacteriocin 31, produced by Enterococcus faecalis, and from a class III bacteriocin produced by E. faecalis. The genes encoding the bacteriocin, immunity protein, mobilization protein, and relaxase nuclease are located on a 7-kb plasmid. Bacteriocin T8 has a molecular mass of 5.1 kDa based on its DNA sequence, similar to the 5.0 kDa recorded for bacteriocin 31 but larger than the 4.6 kDa reported for enterocin P. At the amino acid level, bacteriocin T8 is 69% homologous to bacteriocin 31 and 47% homologous to enterocin P. Bacteriocin T8 is active against E. faecalis isolated from patients diagnosed with vaginosis, against Lactobacillus sakei, and against a Propionibacterium sp. The peptide is heat stable (60 min at 100 degrees C) and remains active in phosphate buffer from pH 4.0 to 10.0. The mode of activity is bactericidal, as determined with E. faecalis.
KeywordMeSH Terms
67. Criado  R, Diep  DB, Aakra  A, Gutiérrez  J, Nes  IF, Hernández  PE, Cintas  LM,     ( 2006 )

Complete sequence of the enterocin Q-encoding plasmid pCIZ2 from the multiple bacteriocin producer Enterococcus faecium L50 and genetic characterization of enterocin Q production and immunity.

Applied and environmental microbiology 72 (10)
PMID : 17021217  :   DOI  :   10.1128/AEM.00859-06     PMC  :   PMC1610292    
Abstract >>
The locations of the genetic determinants for enterocin L50 (EntL50A and EntL50B), enterocin Q (EntQ), and enterocin P (EntP) in the multiple bacteriocin producer Enterococcus faecium strain L50 were determined. These bacteriocin genes occur at different locations; entL50AB (encoding EntL50A and EntL50B) are on the 50-kb plasmid pCIZ1, entqA (encoding EntQ) is on the 7.4-kb plasmid pCIZ2, and entP (encoding EntP) is on the chromosome. The complete nucleotide sequence of pCIZ2 was determined to be 7,383 bp long and contains 10 putative open reading frames (ORFs) organized in three distinct regions. The first region contains three ORFs: entqA preceded by two divergently oriented genes, entqB and entqC. EntqB shows high levels of similarity to bacterial ATP-binding cassette (ABC) transporters, while EntqC displays no significant similarity to any known protein. The second region encompasses four ORFs (orf4 to orf7), and ORF4 and ORF5 display high levels of similarity to mobilization proteins from E. faecium and Enterococcus faecalis. In addition, features resembling a transfer origin region (oriT) were found in the promoter area of orf4. The third region contains three ORFs (orf8 to orf10), and ORF8 and ORF9 exhibit similarity to the replication initiator protein RepE from E. faecalis and to RepB proteins, respectively. To clarify the minimum requirement for EntQ synthesis, we subcloned and heterologously expressed a 2,371-bp fragment from pCIZ2 that encompasses only the entqA, entqB, and entqC genes in Lactobacillus sakei, and we demonstrated that this fragment is sufficient for EntQ production. Moreover, we also obtained experimental results indicating that EntqB is involved in ABC transporter-mediated EntQ secretion, while EntqC confers immunity to this bacteriocin.
KeywordMeSH Terms
68. Roberts  AP, Davis  IJ, Seville  L, Villedieu  A, Mullany  P,     ( 2006 )

Characterization of the ends and target site of a novel tetracycline resistance-encoding conjugative transposon from Enterococcus faecium 664.1H1.

Journal of bacteriology 188 (12)
PMID : 16740942  :   DOI  :   10.1128/JB.00129-06     PMC  :   PMC1482970    
Abstract >>
Enterococcus faecium 664.1H1 is multiply antibiotic resistant and mercury resistant. In this study, the genetic support for the tetracycline resistance of E. faecium 664.1H1 was characterized. The tet(S) gene is responsible for tetracycline resistance, and this gene is located on the chromosome of E. faecium 664.1H1, on a novel conjugative transposon. The element is transferable to Enterococcus faecalis, where it integrates into a specific site. The element was designated EfcTn1. The integrase of EfcTn1 is related to the integrase proteins found on staphylococcal pathogenicity islands. We show that the transposon is flanked by an 18-bp direct repeat, a copy of which is also present at the target site and at the joint of a circular form, and we propose a mechanism of insertion and excision.
KeywordMeSH Terms
Genes, Bacterial
69. Marcobal  A, de las Rivas  B, Muñoz  R,     ( 2006 )

First genetic characterization of a bacterial beta-phenylethylamine biosynthetic enzyme in Enterococcus faecium RM58.

FEMS microbiology letters 258 (1)
PMID : 16630269  :   DOI  :   10.1111/j.1574-6968.2006.00206.x    
Abstract >>
Enterococcus faecium RM58 produces beta-phenylethylamine and tyramine. A gene from Ent. faecium RM58 coding for a 625 amino-acid residues protein that shows 85% identity to Enterococcus faecalis tyrosine decarboxylase has been expressed in Escherichia coli, resulting in L-phenylalanine and L-tyrosine decarboxylase activities. Both activities were lost when a truncated protein lacking 84 amino acids at its C-terminus was expressed in E. coli. This study constitutes the first genetic characterization of a bacterial protein having L-phenylalanine decarboxylase activity and solves a long-standing question regarding the specificity of tyrosine decarboxylases in enterococci.
KeywordMeSH Terms
70. Ruiz-Barba  JL, Floriano  B, Maldonado-Barragán  A, Jiménez-Díaz  R,     ( 2007 )

Molecular analysis of the 21-kb bacteriocin-encoding plasmid pEF1 from Enterococcus faecium 6T1a.

Plasmid 57 (2)
PMID : 16893567  :   DOI  :   10.1016/j.plasmid.2006.06.003    
Abstract >>
The complete 21,344-bp DNA sequence of the bacteriocin-encoding plasmid pEF1 from Enterococcus faecium 6T1a was determined. Thirty-four putative open reading frames which could code for proteins longer than 42 amino acids were found. Those included the structural genes encoding for the previously described bacteriocins enterocin I and J (also named as enterocins L50A and L50B). After comparison to sequences in public databases, analysis of the gene organization of pEF1 suggests a modular structure with three different functional domains: the replication region, the bacteriocin region and the mobilization plus UV-resistance region. This genetic mosaic structure most probably evolved through recombination events promoted by transposable elements. The hypothesis that the bacteriocin cluster on pEF1 could act as a functional plasmid stabilization module in E. faecium 6T1a is discussed.
KeywordMeSH Terms
Sequence Analysis, DNA
71. Hummel  A, Holzapfel  WH, Franz  CM,     ( 2007 )

Characterisation and transfer of antibiotic resistance genes from enterococci isolated from food.

Systematic and applied microbiology 30 (1)
PMID : 16563685  :   DOI  :   10.1016/j.syapm.2006.02.004    
Abstract >>
The genetic determinants responsible for the resistances against the antibiotics tetracycline [tet(M), tet(O), tet(S), tet(K) and tet(L)], erythromycin (ermA,B,C; mefA,E; msrA/B; and ereA,B) and chloramphenicol (cat) of 38 antibiotic-resistant Enterococcus faecium and Enterococcus faecalis strains from food were characterised. In addition, the transferability of resistance genes was also assessed using filter mating assays. The tet(L) determinant was the most commonly detected among tetracycline-resistant enterococci (94% of the strains), followed by the tet(M) gene, which occurred in 63.0% of the strains. Tet(K) occurred in 56.0% of the resistant strains, while genes for tet(O) and tet(S) could not be detected. The integrase gene of the Tn916-1545 family of transposons was present in 81.3% of the tetracycline resistant strains, indicating that resistance genes might be transferable by transposons. All chloramphenicol-resistant strains carried a cat gene. 81.8% of the erythromycin-resistant strains carried the ermB gene. Two (9.5%) of the 21 erythromycin-resistant strains, which did not contain ermA,B,C, ereA,B and mphA genes harboured the msrC gene encoding an erythromycin efflux pump, which was confirmed by sequencing the PCR amplicon. In addition, all E. faecium strains contained the msrC gene, but none of the E. faecalis strains. Transfer of the genetic determinants for antibiotic resistance could only be demonstrated in one filter mating experiment, where both the tet(M) and tet(L) genes were transferred from E. faecalis FAIR-E 315 to the E. faecalis OG1X recipient strain. Our results show the presence of various types of resistance genes as well as transposon integrase genes associated with transferable resistances in enterococci, indicating a potential for gene transfer in the food environment.
KeywordMeSH Terms
Food Microbiology
Genes, Bacterial
72. Inoue  T, Tomita  H, Ike  Y,     ( 2006 )

Bac 32, a novel bacteriocin widely disseminated among clinical isolates of Enterococcus faecium.

Antimicrobial agents and chemotherapy 50 (4)
PMID : 16569830  :   DOI  :   10.1128/AAC.50.4.1202-1212.2006     PMC  :   PMC1426941    
Abstract >>
A total of 636 vancomycin-resistant Enterococcus faecium (VRE) isolates that had been obtained between 1994 and 1999 from the Medical School Hospital of the University of Michigan, Ann Arbor, were tested for bacteriocin production. Two hundred seventy-seven (44%) of the strains were bacteriocinogenic; and 193 of these exhibited activity against Enterococcus faecium, Enterococcus hirae, and Enterococcus durans. Strain VRE200 harbors the highly efficient conjugative gentamicin resistance plasmid pG200 (70 kb) and bacteriocin plasmid pTI1 (12.5 kb). The bacteriocin encoded on pTI1 was designated bacteriocin 32 (Bac 32). Bacteriocin 32 was active against E. faecium, E. hirae, and E. durans but showed no activity against Listeria monocytogenes. The Bac 32 genetic locus consists of a bacteriocin gene (bacA) and an immunity gene (bacB). Neither of these genes showed significant homology to any known bacteriocin determinants. The deduced bacA product is 89 amino acids in length, with a putative signal peptide of 19 amino acids at the N terminus. The bacB gene encodes a deduced 55-amino-acid protein without a signal sequence. One hundred eighty-nine strains (97.9%) of the 193 strains with activity against the 3 test enterococcal strains gave rise to the expected specific PCR product with a primer specific for bacA, indicating that there is a high incidence of Bac 32 production among VRE clinical isolates. Data from Southern analyses of plasmid DNA from 189 of the Bac 32-producing strains with a plasmid pTI1-specific probe suggested that 137 (72.5%) of the strains harbored a pTI1-type plasmid. Bac 32 or Bac 32-type bacteriocin activity and the determinant genes were also identified in 22 (39.3%) of a total of 56 vancomycin-sensitive E. faecium clinical isolates, which suggests that this bacteriocin is widely disseminated among E. faecium strains.
KeywordMeSH Terms
73. Agersø  Y, Pedersen  AG, Aarestrup  FM,     ( 2006 )

Identification of Tn5397-like and Tn916-like transposons and diversity of the tetracycline resistance gene tet(M) in enterococci from humans, pigs and poultry.

The Journal of antimicrobial chemotherapy 57 (5)
PMID : 16565159  :   DOI  :   10.1093/jac/dkl069    
Abstract >>
To analyse the sequence diversity of the tetracycline resistance gene tet(M) and its location on mobile elements in Enterococcus faecium and Enterococcus faecalis from humans, pigs and poultry in Denmark. A total of 76 isolates were screened for Tn916/Tn1545-like and Tn5397-like transposons using PCR. tet(M) was sequenced in 15 of the isolates and compared with tet(M) sequences submitted to GenBank (phylogenetic analysis and signs of recombination). Plasmids were extracted, filter-mating experiments were performed and Tn5397-like transposons were further characterized in selected isolates. In 8 of 13 isolates of E. faecium from broilers, tet(M) was present on Tn5397-like transposons, whereas tet(M) was predominantly associated with Tn916/Tn1545-like transposons in E. faecium from pigs and humans, as well as in E. faecalis from humans, pigs and broilers (50 of 63 isolates). The tet(M) genes were divided into three major subgroups according to the phylogenetic analysis. Subgroup I consisted of tet(M) from Clostridium difficile and E. faecium associated with Tn5397-like elements, subgroup II consisted of tet(M) located on Tn916/Tn1545 family transposons and subgroup III consisted of tet(M) associated with composite elements containing several resistance genes. We found evidence of recombination both within and between these groups. Moreover, we identified an E. faecium isolate with both Tn916/Tn1545-like and Tn5397-like elements. This study showed that enterococci contain diverse tet(M) genes present on different mobile elements, which may suggest that enterococci play an important role in the evolution and horizontal spread of mobile elements carrying tet(M). This is the first report of Tn5397-like elements in enterococci.
KeywordMeSH Terms
DNA Transposable Elements
Genetic Variation
74. Jung  WK, Hong  SK, Lim  JY, Lim  SK, Kwon  NH, Kim  JM, Koo  HC, Kim  SH, Seo  KS, Ike  Y, Tanimoto  K, Park  YH,     ( 2006 )

Phenotypic and genetic characterization of vancomycin-resistant enterococci from hospitalized humans and from poultry in Korea.

FEMS microbiology letters 260 (2)
PMID : 16842344  :   DOI  :   10.1111/j.1574-6968.2006.00311.x    
Abstract >>
Vancomycin resistant enterococci (VRE) isolates from humans (23 isolates) and poultry (20 isolates) were characterized by antibiotic susceptibility, vancomycin resistance transferability, pulsed-field gel electrophoresis (PFGE), and structural analysis of Tn1546-like elements. VRE isolates from humans and poultry showed different resistance patterns, transferability, and transfer rate. In addition to these phenotypic differences between humans and poultry VRE, PFGE and the structure of Tn1546-like elements were also distinct. Most poultry isolates (16/20) were identical to the prototype vanA transposon, Tn1546, while most human isolates (21/23) had multiple integrations of insertion sequence. The transmission of VRE and vancomycin resistance determinant between humans and poultry could not be demonstrated in this study.
KeywordMeSH Terms
Hospitalization
Vancomycin Resistance
75. Launay  A, Ballard  SA, Johnson  PD, Grayson  ML, Lambert  T,     ( 2006 )

Transfer of vancomycin resistance transposon Tn1549 from Clostridium symbiosum to Enterococcus spp. in the gut of gnotobiotic mice.

Antimicrobial agents and chemotherapy 50 (3)
PMID : 16495268  :   DOI  :   10.1128/AAC.50.3.1054-1062.2006     PMC  :   PMC1426432    
Abstract >>
The vancomycin resistance vanB2 gene cluster is disseminated worldwide and has been found in phylogenetically remote bacterial genera. The vanB2 operon is part of conjugative transposons Tn1549/Tn5382, but conjugative transposition of these elements has not been demonstrated. We have obtained transfer of a Tn1549-like element (referred to herein as "Tn1549-like") from Clostridium symbiosum MLG101 to Enterococcus faecium 64/3 and Enterococcus faecalis JH2-2 in the digestive tract of gnotobiotic mice and to E. faecium 64/3 in vitro. Retransfer of Tn1549-like from an E. faecium transconjugant also containing Tn916 to E. faecium BM77 was obtained in vitro, albeit at a very low frequency. Transfer efficiency was found to be both donor and recipient dependent. Pulsed-field gel electrophoresis analysis of total SmaI-digested DNA of 48 transconjugants indicated in 27 instances the acquisition of ca. 34 kb of DNA. Two transconjugants harbored two copies of the transposon. Sequencing of the flanking regions of Tn1549-like in 48 transconjugants revealed 29 integration events in 26 loci in the E. faecium genome, and two hot spots for insertion were identified. Integration of the transposon was associated with the acquisition of 5 (n = 18) or 6 (n = 7) bp of donor DNA or with 5-bp duplications of target DNA in the remaining transconjugants. These data demonstrate functionality of the Tn1549-like element and attest that the transfer of the vanB operon between enterococci and human commensal anaerobes occurs in the intestinal environment.
KeywordMeSH Terms
DNA Transposable Elements
Germ-Free Life
76. Davis  IJ, Roberts  AP, Ready  D, Richards  H, Wilson  M, Mullany  P,     ( 2005 )

Linkage of a novel mercury resistance operon with streptomycin resistance on a conjugative plasmid in Enterococcus faecium.

Plasmid 54 (1)
PMID : 15907536  :   DOI  :   10.1016/j.plasmid.2004.10.004    
Abstract >>
It has been shown that the mercury in dental amalgam and other environmental sources can select for mercury resistant bacteria and that this can lead to an increase in resistance to antibiotics. To understand more about this linkage we have investigated the genetic basis for mercury and antibiotic resistance in a variety of oral bacteria. In this study we have cloned and sequenced the mer operon from an Enterococcus faecium strain which was resistant to mercury, tetracycline, and streptomycin. This strain was isolated, in a previous investigation, from a cynomolgus monkey post-installation of amalgam fillings. The mer operon was contained within a putative transposon (Tnmer1) of the ISL3 family. This element was located on a streptomycin resistant plasmid, pPPM1000, which shares homology with pRE25.
KeywordMeSH Terms
Operon
77. Qu  TT, Chen  YG, Yu  YS, Wei  ZQ, Zhou  ZH, Li  LJ,     ( 2006 )

Genotypic diversity and epidemiology of high-level gentamicin resistant Enterococcus in a Chinese hospital.

The Journal of infection 52 (2)
PMID : 15904965  :   DOI  :   10.1016/j.jinf.2005.02.030    
Abstract >>
To investigate the antibiotics resistance of Enterococcus, the aminoglycoside-modifying enzymes (AME) and homology of high-level gentamicin resistant (HLGR) Enterococcus in clinical specimens for the implementation of effective infection control measures. The resistance of 13 antimicrobial agents was determined by Kirby-Bauer (K-B) or agar dilution method. And the HLGR and high-level streptomycin resistant (HLSR) isolates were screened by agar screen. Production of beta-lactamases was tested by the nitrocefin disc method. The aminoglycoside-modifying enzyme genes were detected by polymerase chain reaction (PCR). Pulsed-field gel electrophoresis (PFGE) was used to analyze the homology of HLGR isolates from in-patients. No isolates resistant to linezolid, vancomycin and teicoplanin were found. Ampicillin-resistant isolates did not produce beta-lactamases and 68 HLGR isolates were screened at the rate of 64.2%. The positive rate of aac(6')-Ie-aph(2'')-Ia was 86.8% and 3 isolates had the new AME gene designated aph(2'')-Ie mostly similar to aph(2'')-Id. Among 51 HLGR isolates from in-patients, PFGE grouped 17 Enterococcus faecalis (E. faecalis) isolates into 4 clusters (A-D), and 33 Enterococcus faecium (E. faecium) isolates into 8 clusters (A-H), of which the A cluster is the main. HLGR has become the important antibiotic resistance pathogen causing nocosomial infection. And the aac(6')-Ie-aph(2'')-Ia gene was the main aminoglycoside-modifying enzyme gene leading to HLGR.
KeywordMeSH Terms
78. Top  J, Butaye  P, Haesebrouck  F, Willems  R, Decostere  A, De Leener  E, Martel  A, De Graef  EM,     ( 2005 )

Molecular analysis of human, porcine, and poultry Enterococcus faecium isolates and their erm(B) genes.

Applied and environmental microbiology 71 (5)
PMID : 15870371  :   DOI  :   10.1128/AEM.71.5.2766-2770.2005     PMC  :   PMC1087536    
Abstract >>
Fifty-nine erm(B)-positive Enterococcus faecium strains isolated from pigs, broilers, and humans were typed using multilocus sequence typing (MLST), and the coding sequence of the erm(B) gene was determined. Identical erm(B) gene sequences were detected in genetically unrelated isolates. Furthermore, genetically indistinguishable strains were found to contain different erm(B) alleles. This may suggest that horizontal exchange of the erm(B) gene between animal and human E. faecium strains or the existence of a common reservoir of erm(B) genes might be more important than direct transmission of resistant strains.
KeywordMeSH Terms
Genes, Bacterial
79. Naser  S, Thompson  FL, Hoste  B, Gevers  D, Vandemeulebroecke  K, Cleenwerck  I, Thompson  CC, Vancanneyt  M, Swings  J,     ( 2005 )

Phylogeny and identification of Enterococci by atpA gene sequence analysis.

Journal of clinical microbiology 43 (5)
PMID : 15872246  :   DOI  :   10.1128/JCM.43.5.2224-2230.2005     PMC  :   PMC1153757    
Abstract >>
The relatedness among 91 Enterococcus strains representing all validly described species was investigated by comparing a 1,102-bp fragment of atpA, the gene encoding the alpha subunit of ATP synthase. The relationships observed were in agreement with the phylogeny inferred from 16S rRNA gene sequence analysis. However, atpA gene sequences were much more discriminatory than 16S rRNA for species differentiation. All species were differentiated on the basis of atpA sequences with, at a maximum, 92% similarity. Six members of the Enterococcus faecium species group (E. faecium, E. hirae, E. durans, E. villorum, E. mundtii, and E. ratti) showed > 99% 16S rRNA gene sequence similarity, but the highest value of atpA gene sequence similarity was only 89.9%. The intraspecies atpA sequence similarities for all species except E. faecium strains varied from 98.6 to 100%; the E. faecium strains had a lower atpA sequence similarity of 96.3%. Our data clearly show that atpA provides an alternative tool for the phylogenetic study and identification of enterococci.
KeywordMeSH Terms
80. Ballard  SA, Pertile  KK, Lim  M, Johnson  PD, Grayson  ML,     ( 2005 )

Molecular characterization of vanB elements in naturally occurring gut anaerobes.

Antimicrobial agents and chemotherapy 49 (5)
PMID : 15855482  :   DOI  :   10.1128/AAC.49.5.1688-1694.2005     PMC  :   PMC1087664    
Abstract >>
Previously, we reported the isolation of 10 vancomycin-resistant gram-positive anaerobic bacilli carrying the vanB ligase gene from nine hemodialysis patients (S. A. Ballard et al., Antimicrob. Agents Chemother. 49:77-81, 2005; T. P. Stinear et al., Lancet 357:855-856, 2001). In the present study, the molecular and evolutionary relationship of the vanB resistance element within these 10 anaerobes and two vancomycin-resistant Enterococcus faecium strains were examined. PCR analysis and nucleotide sequencing demonstrated that all 12 isolates carried the vanB operon associated with an element identical to Tn1549 and Tn5382 of Enterococcus. Restriction fragment length polymorphism analysis of the vanB operon in these isolates revealed two distinct patterns, and sequencing showed that minor base differences existed. PCR amplification of the joint region of a circular intermediate was demonstrated in nine of these organisms, a finding indicative of an ability to excise and circularize, an intermediate step in transposition and conjugative transfer. Southern hybridization with a vanB-vanX(B) probe suggests that there is one insert of the transposon in all isolates. Sequence analysis of the integration site revealed distinct sequences: the Tn1549/5382 element within E. faecium was inserted within the host chromosome, whereas nucleotide sequences surrounding the Tn1549/5382 element in the 10 anaerobes showed no significant homology to sequences in the GenBank database. We demonstrate considerable similarity between the Tn1549/5382 element identified in 10 anaerobe isolates with that found in enterococci. The homology and potential to transpose suggest a recent horizontal transfer event may have occurred. However, the original direction of transposition and the mechanism involved remains unknown.
KeywordMeSH Terms
81. Kang  JH, Lee  MS,     ( 2005 )

Characterization of a bacteriocin produced by Enterococcus faecium GM-1 isolated from an infant.

Journal of applied microbiology 98 (5)
PMID : 15836487  :   DOI  :   10.1111/j.1365-2672.2005.02556.x    
Abstract >>
To partially characterize the bacteriocin produced by the GM-1 strain of Enterococcus faecium, isolated from the faeces of a newborn human infant. The bacteriocin produced by E. faecium GM-1 showed a broad spectrum of activity against indicator strains of Escherichia coli, Staphylococcus aureus, Vibrio spp., Salmonella typhimurium, Listeria monocytogenes, Lactobacillus acidophilus, and Streptococcus thermophilus. Treatment of the GM-1 bacteriocin with proteolytic enzymes reduced its inhibitory activities. The bacteriocin was stable at 100 degrees C for 20 min and displayed inhibitory activity at neutral pH. The optimal production of bacteriocin from E. faecium GM-1 was obtained when the culture conditions were pH 6.0-6.5 and 35-40 degrees C. The inhibitory activity of the bacteriocin was not substantially changed by the use of different carbon sources in the media, except when galactose was substituted for glucose. The use of a sole nitrogen source caused a decrease in inhibitory activity. A bacteriocin gene similar to enterocin P was identified from the total DNA of E. faecium GM-1 by PCR and direct sequencing methods. E. faecium GM-1, which was isolated from the faeces of a newborn baby, produces an enterocin P-like bacteriocin with inhibitory activity against Gram-positive and Gram-negative bacteria, including food-borne pathogens. E. faecium GM-1, isolated from infant faeces, produces a new bacteriocin that is similar to enterocin P. This bacteriocin is heat stable and has a broad antibacterial spectrum that includes both Gram-positive and Gram-negative bacteria.
KeywordMeSH Terms
82. Rice  LB, Carias  LL, Marshall  S, Rudin  SD, Hutton-Thomas  R,     ( 2005 )

Tn5386, a novel Tn916-like mobile element in Enterococcus faecium D344R that interacts with Tn916 to yield a large genomic deletion.

Journal of bacteriology 187 (19)
PMID : 16166528  :   DOI  :   10.1128/JB.187.19.6668-6677.2005     PMC  :   PMC1251567    
Abstract >>
We describe Tn5386, a novel ca.-29-kb Tn916-like mobile element discovered to occur in ampicillin-resistant, Tn916-containing Enterococcus faecium D344R. PCR amplification experiments after overnight growth with or without tetracycline revealed "joint" regions of circularized Tn5386 composed of 6-bp sequences linking different transposon termini. In one case (no tetracycline), the termini were consistent with those derived by target site analysis of the integrated element. In the other case, the termini were virtually identical in distance from the integrase binding regions, as seen with Tn916. These data are consistent with a model in which one PCR product results from the action of Tn5386 integrase, whereas the other results from the action of the Tn916 integrase on Tn5386. Spontaneous conversion of D344R to an ampicillin-susceptible phenotype (D344SRF) was associated with a 178-kb deletion extending from the left end of Tn5386 to the left end of Tn916. Examination of the Tn5386 junction after the large deletion event suggests that the deletion resulted from an interaction between the nonintegrase ends of Tn5386 and Tn916. The terminus of Tn5386 identified in this reaction suggested that it may have resulted from the activity of the Tn916 integrase (Int(Tn916)). The "joint" of the circular element resulting from this excision was amplifiable from D344R, the sequence of which revealed a heteroduplex consistent with Int(Tn916)-mediated excision. In contrast, Tn5386 joints amplified from ampicillin-susceptible D344SRF revealed ends consistent with Tn5386 integrase activity, reflecting the absence of Tn916 from this strain. Tn5386 represents a new member of the Tn916 transposon family. Our data suggest that excision of Tn5386 can be catalyzed by the Tn916 integrase and that large genomic deletions may result from the interaction between these heterologous elements.
KeywordMeSH Terms
Gene Deletion
Genome, Bacterial
83. Tomita  H, Ike  Y,     ( 2005 )

Genetic analysis of transfer-related regions of the vancomycin resistance Enterococcus conjugative plasmid pHTbeta: identification of oriT and a putative relaxase gene.

Journal of bacteriology 187 (22)
PMID : 16267297  :   DOI  :   10.1128/JB.187.22.7727-7737.2005     PMC  :   PMC1280310    
Abstract >>
The pHT plasmids pHTalpha (65.9 kbp), pHTbeta (63.7 kbp), and pHTgamma (66.5 kbp) are highly conjugative pheromone-independent pMG1-like plasmids that carry Tn1546-like transposons encoding vancomycin resistance. pHTbeta is the prototype plasmid, and the pHTalpha and pHTgamma plasmids are derivatives of the insertion into pHTbeta of an IS232-like (2.2 kbp) element and a group II intron (2.8 kbp), respectively. The complete nucleotide sequence of the pHTbeta plasmid was determined and, with the exception of the Tn1546-like insertion (10,851 bp), was found to be 52,890 bp. Sixty-one open reading frames (ORFs) having the same transcript orientation were identified. A homology search revealed that 22 of the pHTbeta (pHT) plasmid ORFs showed similarities to the ORFs identified on the pXO2 plasmid (96.2 kbp), which is the virulence plasmid essential for capsule formation by Bacillus anthracis; however, the functions of most of the ORFs remain unknown. Most other ORFs did not show any significant homology to reported genes for which functions have been analyzed. To investigate the highly efficient transfer mechanism of the pHT plasmid, mutations with 174 unique insertions of transposon Tn917-lac insertion mutants of pHTbeta were obtained. Of the 174 derivatives, 92 showed decrease or loss in transfer frequency, and 74 showed normal transfer frequency and LacZ expression. Eight derivatives showed normal transfer and no LacZ expression. Inserts within the 174 derivatives were mapped to 124 different sites on pHTbeta. The Tn917-lac insertions which resulted in altered transfer frequency mapped to three separate regions designated I, II, and III, which were separated by segments in which insertions of Tn917-lac did not affect transfer. There was no region homologous to the previously reported oriT sequences in the pHT plasmid. The oriT was cloned by selection for the ability to mobilize the vector plasmid pAM401. The oriT region resided in a noncoding region (192 bp) between ORF31 and ORF32 and contained three direct repeat sequences and two inverted repeat sequences. ORF34, encoding a 506-amino-acid protein which was located downstream of the oriT region, contains the three conserved motifs (I to III) of the DNA relaxase/nickase of mobile plasmids. The transfer abilities of the Tn917-lac-insertion mutants of ORF34 or a mutant of ORF34 with an in-frame motif III deletion were completely abolished. The sequence of the oriT region and the deduced relaxase/nickase protein of ORF34 showed no significant similarity to the oriT and relaxase/nickase of other conjugative plasmids, respectively. The putative relaxase/nickase protein of ORF34 could be classified as a new member of the MOB(MG) family.
KeywordMeSH Terms
84. Mahbub Alam  M, Kobayashi  N, Ishino  M, Sumi  A, Kobayashi  K, Uehara  N, Watanabe  N,     ( 2005 )

Detection of a novel aph(2") allele (aph[2"]-Ie) conferring high-level gentamicin resistance and a spectinomycin resistance gene ant(9)-Ia (aad 9) in clinical isolates of enterococci.

Microbial drug resistance (Larchmont, N.Y.) 11 (3)
PMID : 16201926  :   DOI  :   10.1089/mdr.2005.11.239    
Abstract >>
Aminoglycoside-modifying enzymes (AMEs) are major factors that confer aminoglycoside resistance to enterococci. In an epidemiologic study on distribution of 12 AME genes in 534 recent clinical strains isolated from a Japanese hospital, two uncommon AME genes, ant(9)-Ia and a novel aph(2") allele, aph(2")-Ie, were detected. ant(9)-Ia had been reported only in Staphylococcus aureus and encodes spectinomycin adenylyltransferase ANT(9)-I, which confers resistance to spectinomycin. The ant(9)-Ia gene was detected in three strains, a single strain each of Enterococcus faecalis, E. faecium, and E. avium. Nucleotide sequences of ant(9)-Ia from these three enterococcal species were identical to that reported for S. aureus and considered to be located on Tn 554. The new aph(2") allele, designated aph(2")-Ie, was identified in three E. faecium strains. The aph(2")-Ie allele was genetically close to aph(2")-Id reported in E. casseliflavus (93.7% amino acid sequence identity; 96.3% similarity), while distant from aph(2")-Ia, aph(2")-Ib, or aph(2")-Ic (26.3-29.5% amino acid sequence identity). Sequence divergence between APH(2")-Id and APH(2")-Ie was mostly located in amino-terminal half. In contrast, sequences corresponding to the three motifs required for aminoglycoside phosphotransferase were conserved except for a single amino acid. Three E. faecium strains having aph(2")-Ie showed high-level resistance to gentamicin and streptomycin, but not to kanamycin, dibekacin, and tobramycin, unlike enzyme specificity described for aph(2")-Id in E. casseliflavus. Such a difference in resistance phenotype was suggested to be related to amino acid sequence divergence between APH(2")-Id and APH(2")-Ie.
KeywordMeSH Terms
85. Jung  WK, Hong  SK, Koo  HC, Kwon  NH, Park  YH,     ( 2005 )

Nucleotide sequence of IS1678, an insertion sequence in the vanA cluster of enterococci.

Antimicrobial agents and chemotherapy 49 (4)
PMID : 15793169  :   DOI  :   10.1128/AAC.49.4.1666-1667.2005     PMC  :   PMC1068654    
Abstract >>
N/A
KeywordMeSH Terms
Base Sequence
DNA Transposable Elements
Multigene Family
86. Kim  SW, Jeong  EJ, Kang  HS, Tak  JI, Bang  WY, Heo  JB, Jeong  JY, Yoon  GM, Kang  HY, Bahk  JD,     ( 2006 )

Role of RepB in the replication of plasmid pJB01 isolated from Enterococcus faecium JC1.

Plasmid 55 (2)
PMID : 16188315  :   DOI  :   10.1016/j.plasmid.2005.08.002    
Abstract >>
The plasmid pJB01 (GenBank Accession No. AY425961) isolated from the pathogenic bacterium, Enterococcus faecium JC1, is 2235 base pairs in length and consists of a putative double-strand origin (dso), a single-strand origin, a counter-transcribed RNA, and three open reading frames. A comparison of a few replication factors and motifs, bind and nic regions, for replication initiation on the nucleotide sequence level revealed that it belongs to the pMV158 family among RC-replicating plasmids. A runoff DNA synthesis assay demonstrated that nicking occurred between G525 and A526, which is located on the internal loop of a putative secondary structure in the dso. Unlike all the other plasmids of the pMV158 family having two or three direct repeats, pJB01 has three non-tandem direct repeats of 5'-CAACAAA-3' separated by four nucleotides, as the RepB-binding site in the dso. Moreover, the nick site on the internal loop is located at 77 nucleotides upstream from the RepB-binding region. Irrespective of the structural difference of direct repeats from other members of the pMV158 family, we think, it is still a new member of this plasmid family. The introduction of mutations in conserved regions of RepB confirmed that RepB N-moiety is important for nicking/nick-closing activity. Within N-moiety, especially all of the motif R-III, the Y100 in R-IV and Y116 in R-V residues, played particularly critical roles in this activity, however, for its binding, both of the N- and C-moieties of RepB were needed.
KeywordMeSH Terms
87. Johnsen  L, Dalhus  B, Leiros  I, Nissen-Meyer  J,     ( 2005 )

1.6-Angstroms crystal structure of EntA-im. A bacterial immunity protein conferring immunity to the antimicrobial activity of the pediocin-like bacteriocin enterocin A.

The Journal of biological chemistry 280 (19)
PMID : 15753083  :   DOI  :   10.1074/jbc.M501386200    
Abstract >>
Many Gram-positive bacteria produce ribosomally synthesized antimicrobial peptides, often termed bacteriocins. Genes encoding pediocin-like bacteriocins are generally cotranscribed with or in close vicinity to a gene encoding a cognate immunity protein that protects the bacteriocin-producer from their own bacteriocin. We present the first crystal structure of a pediocin-like immunity protein, EntA-im, conferring immunity to the bacteriocin enterocin A. Determination of the structure of this 103-amino acid protein revealed that it folds into an antiparallel four-helix bundle with a flexible C-terminal part. The fact that the immunity protein conferring immunity to carnobacteriocin B2 also consists of a four-helix bundle (Sprules, T., Kawulka, K. E., and Vederas, J. C. (2004) Biochemistry 43, 11740-11749) strongly indicates that this is a conserved structural motif in all pediocin-like immunity proteins. The C-terminal half of the immunity protein contains a region that recognizes the C-terminal half of the cognate bacteriocin, and the flexibility in the C-terminal end of the immunity protein might thus be an important characteristic that enables the immunity protein to interact with its cognate bacteriocin. By homology modeling of three other pediocin-like immunity proteins and calculation of the surface charge distribution for EntA-im and the three structure models, different charge distributions were observed. The differences in the latter part of helix 3, the beginning of helix 4, and the loop connecting these helices might also be of importance in determining the specificity.
KeywordMeSH Terms
88. Burk  DL, Xiong  B, Breitbach  C, Berghuis  AM,     ( 2005 )

Structures of aminoglycoside acetyltransferase AAC(6')-Ii in a novel crystal form: structural and normal-mode analyses.

Acta crystallographica. Section D, Biological crystallography 61 (Pt 9)
PMID : 16131761  :   DOI  :   10.1107/S0907444905021487    
Abstract >>
The aminoglycoside-modifying enzyme aminoglycoside 6'-N-acetyltransferase type Ii [AAC(6')-Ii] has been crystallized with its cofactor coenzyme A in space group C222(1), with unit-cell parameters a = 71.5, b = 127.4, c = 76.9 A and one physiologically relevant dimer species per asymmetric unit. The space group previously observed for this complex was P2(1)2(1)2(1), with two dimers per asymmetric unit. By comparing the six available protomer structures of the AAC(6')-Ii-CoA complex, it has been possible to identify regions of plasticity within the protein. Normal-mode analysis of this complex suggests that this plasticity is not an artefact of crystal-packing forces, but that the region of the protomer that displays multiple conformations is intrinsically flexible. It is conjectured that the flexibility is relevant for the cooperative activity observed for the enzyme.
KeywordMeSH Terms
89. Lu  JJ, Chang  TY, Perng  CL, Lee  SY,     ( 2005 )

The vanB2 gene cluster of the majority of vancomycin-resistant Enterococcus faecium isolates from Taiwan is associated with the pbp5 gene and is carried by Tn5382 containing a novel insertion sequence.

Antimicrobial agents and chemotherapy 49 (9)
PMID : 16127076  :   DOI  :   10.1128/AAC.49.9.3937-3939.2005     PMC  :   PMC1195393    
Abstract >>
Thirty-two vanB2 Enterococcus faecium isolates were found to harbor Tn5382. Twenty-four isolates had a 1,419-bp sequence inserted within the open reading frame (ORF) C of Tn5382. This 1,419-bp sequence contained a 638-bp ORF with a 72% amino acid sequence homology with the transposase gene of IS150. Thirty isolates had the pbp5 gene linked to Tn5382.
KeywordMeSH Terms
90. Hasman  H,     ( 2005 )

The tcrB gene is part of the tcrYAZB operon conferring copper resistance in Enterococcus faecium and Enterococcus faecalis.

Microbiology (Reading, England) 151 (Pt 9)
PMID : 16151212  :   DOI  :   10.1099/mic.0.28109-0    
Abstract >>
The plasmid-localized tcrB (transferable copper-resistance gene B) gene from Enterococcus faecium was identified to be part of an operon called the tcrYAZB operon, which has a genetic organization similar to the copYZAB copper-homeostasis gene cluster from Enterococcus hirae. Putative promoter (P(tcr))- and repressor-binding sites highly similar to the E. hirae cop-promoter region were identified upstream of the tcrYAZB genes. The P(tcr) promoter was cloned in both the absence and the presence of the proximal repressor-encoding tcrY gene into a promoter-probe vector. Induction of the promoter was shown in liquid growth medium containing increasing concentrations of copper sulphate. To determine the growth advantage conferred by the tcrYAZB genes in a copper environment, a tcr-deletion mutant was isolated, and its growth was compared with that of its copper-resistant ancestor (strain A17sv1) in sublethal concentrations of copper sulphate. A competition assay using these two isogenic strains showed that copper sulphate concentrations of 3 mmol l(-1) and above are sufficient to select for copper resistance.
KeywordMeSH Terms
91. Ballard  SA, Grabsch  EA, Johnson  PD, Grayson  ML,     ( 2005 )

Comparison of three PCR primer sets for identification of vanB gene carriage in feces and correlation with carriage of vancomycin-resistant enterococci: interference by vanB-containing anaerobic bacilli.

Antimicrobial agents and chemotherapy 49 (1)
PMID : 15616278  :   DOI  :   10.1128/AAC.49.1.77-81.2005     PMC  :   PMC538908    
Abstract >>
We assessed the sensitivities and specificities of three previously described PCR primers on enrichment broth cultures of feces for the accurate detection of fecal carriage of vancomycin-resistant enterococci (VRE). In addition, we investigated specimens that were vanB PCR positive but VRE culture negative for the presence of other vanB-containing pathogens. Feces from 59 patients (12 patients carrying vanB Enterococcus faecium strains and 47 patients negative for VRE carriage) were cultured for 36 h in aerobic brain heart infusion (BHI) broth, anaerobic BHI (AnO(2)BHI) broth, or aerobic Enterococcosel (EC) broth. DNA was extracted from the cultures and tested for the presence of vanB by using the PCR primers of Dutka-Malen et al. (S. Dutka-Malen, S. Evers, and P. Courvalin, J. Clin. Microbiol. 33:24-27, 1995), Bell et al. (J. M. Bell, J. C. Paton, and J. Turnidge, J. Clin. Microbiol. 36:2187-2190, 1998), and Stinear et al. (T. P. Stinear, D. C. Olden, P. D. R. Johnson, J. K. Davies, and M. L. Grayson, Lancet 357:855-856, 2001). The sensitivity (specificity) of PCR compared with the results of culture on BHI, AnO(2)BHI, and EC broths were 67% (96%), 50% (94%), and 17% (100%), respectively, with the primers of Dutka-Malen et al.; 92% (60%), 92% (45%), and 92% (83%), respectively, with the primers of Bell et al.; and 92% (49%), 92% (43%), and 100% (51%) respectively, with the primers of Stinear et al. The primers of both Bell et al. and Stinear et al. were significantly more sensitive than those of Dutka-Malen et al. in EC broth (P = 0.001 and P < 0.001, respectively). The poor specificities for all primer pairs were due in part to the isolation and identification of six anaerobic gram-positive bacilli, Clostridium hathewayi (n = 3), a Clostridium innocuum-like organism (n = 1), Clostridium bolteae (n = 1), and Ruminococcus lactaris-like (n = 1), from five fecal specimens that were vanB positive but VRE culture negative. All six organisms were demonstrated to contain a vanB gene identical to that of VRE. VanB-containing bowel anaerobes may result in false-positive interpretation of PCR-positive fecal enrichment cultures as VRE, regardless of the primers and protocols used.
KeywordMeSH Terms
DNA Primers
92. Tsai  JC, Hsueh  PR, Lin  HM, Chang  HJ, Ho  SW, Teng  LJ,     ( 2005 )

Identification of clinically relevant enterococcus species by direct sequencing of groES and spacer region.

Journal of clinical microbiology 43 (1)
PMID : 15634977  :   DOI  :   10.1128/JCM.43.1.235-241.2005     PMC  :   PMC540105    
Abstract >>
We determined the groESL sequences (groES, groEL, and the intergenic spacer) of 10 clinically relevant Enterococcus species and evaluated the feasibility of identifying Enterococcus species on the basis of these sequences. Seven common clinical Enterococcus species, E. faecalis, E. faecium, E. casseliflavus, E. gallinarum, E. avium, E. raffinosus, and E. hirae, and three less common Enterococcus species, E. cecorum, E. durans, and E. mundtii, were examined in this study. We found that the groES genes of these enterococcal species are identical in length (285 nucleotides) and contain an unusual putative start codon, GTG. The lengths and sequences of the intergenic regions (spacers between the groES and groEL genes) are quite variable (17 to 57 bp in length) among Enterococcus species but are conserved in strains within each species, with only a few exceptions. Considerable variation of groES or groEL sequences was also observed. The evolutionary trees of groES or groEL sequences revealed similarities among Enterococcus species. However, the overall intraspecies variation of groES was less than that of groEL. The high interspecies variation and low intraspecies variation indicate that the groES and spacer sequences are more useful than groEL for identification of clinically relevant Enterococcus species. The sequences of these two genetic traits, groES and spacer, can be determined by a single PCR and direct sequencing and may provide important information for the differentiation of closely related species of Enterococcus.
KeywordMeSH Terms
Bacterial Typing Techniques
Sequence Analysis, DNA
93. Tang  XB, Si  SY, Zhang  YQ,     ( 2004 )

Identification of a new peptide deformylase gene from enterococcus faecium and establishment of a new screening model targeted on PDF for novel antibiotics.

Biomedical and environmental sciences : BES 17 (3)
PMID : 15602833  :  
Abstract >>
To identify a new peptide deformylase (PDF) gene (Genebank Accession AY238515) from Enterococcus faecium and to establish a new screening model targeted on PDF. A new PDF gene was identified by BLAST analysis and PCR and was subsequently over-expressed in the prokaryotic expression host E. coli B121(DE3). Over-expressed protein was purified for enzymatic assay by metal affinity chromatography and a new screening model was established for novel antibiotics. A new PDF gene of Enterococcus faecium was identified successfully. Ten positive samples were picked up from 8000 compound library and the microbial fermentation broth samples. A new PDF of gene Enterococcus faecium was first identified and the model had a high efficacy. Positive samples screened may be antibacterial agents of broad spectrum.
KeywordMeSH Terms
Drug Evaluation, Preclinical
94. Arthur  M, Molinas  C, Courvalin  P,     ( 1992 )

The VanS-VanR two-component regulatory system controls synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147.

Journal of bacteriology 174 (8)
PMID : 1556077  :   DOI  :   10.1128/jb.174.8.2582-2591.1992     PMC  :   PMC205897    
Abstract >>
Plasmid pIP816 of Enterococcus faecium BM4147 confers inducible resistance to vancomycin and encodes the VanH dehydrogenase and the VanA ligase for synthesis of depsipeptide-containing peptidoglycan precursors which bind the antibiotic with reduced affinity. We have characterized a cluster of five genes of pIP816 sufficient for peptidoglycan synthesis in the presence of vancomycin. The distal part of the van cluster encodes VanH, VanA, and a third enzyme, VanX, all of which are necessary for resistance. Synthesis of these enzymes was regulated at the transcriptional level by the VanS-VanR two-component regulatory system encoded by the proximal part of the cluster. VanR was a transcriptional activator related to response regulators of the OmpR subclass. VanS stimulated VanR-dependent transcription and was related to membrane-associated histidine protein kinases which control the level of phosphorylation of response regulators. Analysis of transcriptional fusions with a reporter gene and RNA mapping indicated that the VanR-VanS two-component regulatory system activates a promoter used for cotranscription of the vanH, vanA, and vanX resistance genes.
KeywordMeSH Terms
Bacterial Proteins
Drug Resistance, Microbial
Genes, Bacterial
95. Kim  SH, Lee  DG, Yoo  JH, Choi  SM, Choi  JH, Shin  WS, Lee  K, Yong  D, Lee  WG, Youn  BS, Kang  MW,     ( 2004 )

DD1.5k, the gene preferentially expressed in bloodstream isolates of vancomycin-resistant Enterococcus faecium.

Journal of microbiology (Seoul, Korea) 42 (2)
PMID : 15357309  :  
Abstract >>
Vancomycin-resistant Enterococcus faecium (VREFM) is becoming a threatening pathogen. We identified a gene called DD1.5K by differential display-PCR, which was preferentially expressed in the bloodstream isolates of VREFM. Due to its amino acid similarity to transfer complex protein, trsE, and tissue-specific expression, this gene may be involved in virulence of VREFM.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
96. Boyd  DA, Kibsey  P, Roscoe  D, Mulvey  MR,     ( 2004 )

Enterococcus faecium N03-0072 carries a new VanD-type vancomycin resistance determinant: characterization of the VanD5 operon.

The Journal of antimicrobial chemotherapy 54 (3)
PMID : 15308604  :   DOI  :   10.1093/jac/dkh391    
Abstract >>
To genotypically characterize the vancomycin resistance mechanism of Enterococcus faecium N03-0072, which was negative by PCR for the currently known van genotypes. PCR was used to amplify the entire vancomycin resistance operon and the complete nucleotide sequence was determined by dideoxy cycle sequencing. Analysis revealed a VanD-type operon with 94% nucleotide identity to the VanD4 operon and 90% nucleotide identity to the VanD1/D3 operons. A set of universal primers was designed in order to identify all current vanD variants by PCR. E. faecium N03-0072 carries a new VanD-type operon, designated VanD5.
KeywordMeSH Terms
97. Hill  JE, Penny  SL, Crowell  KG, Goh  SH, Hemmingsen  SM,     ( 2004 )

cpnDB: a chaperonin sequence database.

Genome research 14 (8)
PMID : 15289485  :   DOI  :   10.1101/gr.2649204     PMC  :   PMC509277    
Abstract >>
Type I chaperonins are molecular chaperones present in virtually all bacteria, some archaea and the plastids and mitochondria of eukaryotes. Sequences of cpn60 genes, encoding 60-kDa chaperonin protein subunits (CPN60, also known as GroEL or HSP60), are useful for phylogenetic studies and as targets for detection and identification of organisms. Conveniently, a 549-567-bp segment of the cpn60 coding region can be amplified with universal PCR primers. Here, we introduce cpnDB, a curated collection of cpn60 sequence data collected from public databases or generated by a network of collaborators exploiting the cpn60 target in clinical, phylogenetic, and microbial ecology studies. The growing database currently contains approximately 2000 records covering over 240 genera of bacteria, eukaryotes, and archaea. The database also contains over 60 sequences for the archaeal Type II chaperonin (thermosome, a homolog of eukaryotic cytoplasmic chaperonin) from 19 archaeal genera. As the largest curated collection of sequences available for a protein-encoding gene, cpnDB provides a resource for researchers interested in exploiting the power of cpn60 as a diagnostic or as a target for phylogenetic or microbial ecology studies, as well as those interested in broader subjects such as lateral gene transfer and codon usage. We built cpnDB from open source tools and it is available at http://cpndb.cbr.nrc.ca.
KeywordMeSH Terms
98. Moy  TI, Mylonakis  E, Calderwood  SB, Ausubel  FM,     ( 2004 )

Cytotoxicity of hydrogen peroxide produced by Enterococcus faecium.

Infection and immunity 72 (8)
PMID : 15271910  :   DOI  :   10.1128/IAI.72.8.4512-4520.2004     PMC  :   PMC470665    
Abstract >>
Although the opportunistic bacterial pathogen Enterococcus faecium is a leading source of nosocomial infections, it appears to lack many of the overt virulence factors produced by other bacterial pathogens, and the underlying mechanism of pathogenesis is not clear. Using E. faecium-mediated killing of the nematode worm Caenorhabditis elegans as an indicator of toxicity, we determined that E. faecium produces hydrogen peroxide at levels that cause cellular damage. We identified E. faecium transposon insertion mutants with altered C. elegans killing activity, and these mutants were altered in hydrogen peroxide production. Mutation of an NADH oxidase-encoding gene eliminated nearly all NADH oxidase activity and reduced hydrogen peroxide production. Mutation of an NADH peroxidase-encoding gene resulted in the enhanced accumulation of hydrogen peroxide. E. faecium is able to produce hydrogen peroxide by using glycerol-3-phosphate oxidase, and addition of glycerol to the culture medium enhanced the killing of C. elegans. Conversely, addition of glucose, which leads to the down-regulation of glycerol metabolism, prevented both C. elegans killing and hydrogen peroxide production. Lastly, detoxification of hydrogen peroxide either by exogenously added catalase or by a C. elegans transgenic strain overproducing catalase prevented E. faecium-mediated killing. These results suggest that hydrogen peroxide produced by E. faecium has cytotoxic effects and highlight the utility of C. elegans pathogenicity models for identifying bacterial virulence factors.
KeywordMeSH Terms
99. Wright  GD, Molinas  C, Arthur  M, Courvalin  P, Walsh  CT,     ( 1992 )

Characterization of vanY, a DD-carboxypeptidase from vancomycin-resistant Enterococcus faecium BM4147.

Antimicrobial agents and chemotherapy 36 (7)
PMID : 1510448  :   DOI  :   10.1128/aac.36.7.1514     PMC  :   PMC191613    
Abstract >>
VanY is a protein with a molecular mass of 34.8 kDa encoded by vanY, a member of the high-level vancomycin resistance gene cluster found on plasmid pIP816 in Enterococcus faecium BM4147. Extracts from Escherichia coli JM83 bearing plasmid pAT383, which contains the vanY gene, were examined for enzymatic hydrolysis of peptidoglycan precursors. VanY was associated with the cell membranes and cleaved the C-terminal D-alanine residue of UDP-muramyl-pentapeptide but did not display transpeptidase or beta-lactamase activities. The DD-carboxypeptidase activity was not inhibited by beta-lactam antibiotics. VanY released the C-terminal D-hydroxy acid from depsipeptides produced by the vancomycin resistance protein VanA. These results demonstrate that VanY should contribute in vivo to the hydrolysis of both the D-alanyl-D-alanine- and the depsipeptide-containing peptidoglycan precursors.
KeywordMeSH Terms
100. Handwerger  S, Pucci  MJ, Volk  KJ, Liu  J, Lee  MS,     ( 1992 )

The cytoplasmic peptidoglycan precursor of vancomycin-resistant Enterococcus faecalis terminates in lactate.

Journal of bacteriology 174 (18)
PMID : 1522072  :   DOI  :   10.1128/jb.174.18.5982-5984.1992     PMC  :   PMC207137    
Abstract >>
Vancomycin resistance plasmids in enterococci carry the genes vanH and vanA, which encode enzymes catalyzing, respectively, the reduction of 2-keto acids to 2-D-hydroxy acids and the addition of D-hydroxy acids to D-alanine. It has therefore been postulated that resistant cells produce peptidoglycan precursors that terminate in the depsipeptide D-alanine-2-D-hydroxy acid rather than the dipeptide D-alanine-D-alanine, thus preventing vancomycin binding (M. Arthur, C. Molinas, T. D. H. Bugg, G. D. Wright, C. T. Walsh, and P. Courvalin, Antimicrob. Agents Chemother. 36:867-869, 1992). In the present work, a cytoplasmic peptidoglycan precursor was isolated from vancomycin-resistant Enterococcus faecalis and analyzed by mass spectrometry, which suggested the structure UDP-N-acetyl-muramyl-L-Ala-D-Glu-L-Lys-D-Ala-D-lactate.
KeywordMeSH Terms
101. Lorenzo-Díaz  F, Delgado  T, Reyes-Darias  JA, Flores  C, Méndez-Alvarez  S, Villar  J, Sierra  A, Claverie-Martín  F,     ( 2004 )

Characterization of the first VanB vancomycin-resistant Enterococcus faecium isolated in a Spanish hospital.

Current microbiology 48 (3)
PMID : 15057465  :   DOI  :   10.1007/s00284-003-4125-2    
Abstract >>
We report the detection and characterization of the first vancomycin-resistant VanB-type Enterococcus faecium to be isolated in a Spanish hospital. Sequence analysis of the vanB gene showed that this isolate belonged to subtype vanB2. Moreover, PCR amplification analysis indicated that the vanB gene cluster was linked to a Tn5382-like transposon.
KeywordMeSH Terms
Vancomycin Resistance
102. Drancourt  M, Roux  V, Fournier  PE, Raoult  D,     ( 2004 )

rpoB gene sequence-based identification of aerobic Gram-positive cocci of the genera Streptococcus, Enterococcus, Gemella, Abiotrophia, and Granulicatella.

Journal of clinical microbiology 42 (2)
PMID : 14766807  :   DOI  :   10.1128/jcm.42.2.497-504.2004     PMC  :   PMC344509    
Abstract >>
We developed a new molecular tool based on rpoB gene (encoding the beta subunit of RNA polymerase) sequencing to identify streptococci. We first sequenced the complete rpoB gene for Streptococcus anginosus, S. equinus, and Abiotrophia defectiva. Sequences were aligned with these of S. pyogenes, S. agalactiae, and S. pneumoniae available in GenBank. Using an in-house analysis program (SVARAP), we identified a 740-bp variable region surrounded by conserved, 20-bp zones and, by using these conserved zones as PCR primer targets, we amplified and sequenced this variable region in an additional 30 Streptococcus, Enterococcus, Gemella, Granulicatella, and Abiotrophia species. This region exhibited 71.2 to 99.3% interspecies homology. We therefore applied our identification system by PCR amplification and sequencing to a collection of 102 streptococci and 60 bacterial isolates belonging to other genera. Amplicons were obtained in streptococci and Bacillus cereus, and sequencing allowed us to make a correct identification of streptococci. Molecular signatures were determined for the discrimination of closely related species within the S. pneumoniae-S. oralis-S. mitis group and the S. agalactiae-S. difficile group. These signatures allowed us to design a S. pneumoniae-specific PCR and sequencing primer pair.
KeywordMeSH Terms
103. Leavis  H, Top  J, Shankar  N, Borgen  K, Bonten  M, van Embden  J, Willems  RJ,     ( 2004 )

A novel putative enterococcal pathogenicity island linked to the esp virulence gene of Enterococcus faecium and associated with epidemicity.

Journal of bacteriology 186 (3)
PMID : 14729692  :   DOI  :   10.1128/jb.186.3.672-682.2004     PMC  :   PMC321477    
Abstract >>
Enterococcus faecalis harbors a virulence-associated surface protein encoded by the esp gene. This gene has been shown to be part of a 150-kb putative pathogenicity island. A gene similar to esp has recently been found in Enterococcus faecium isolates recovered from hospitalized patients. In the present study we analyzed the polymorphism in the esp gene of E. faecium, and we investigated the association of esp with neighboring chromosomal genes. The esp gene showed considerable sequence heterogeneity in the regions encoding the nonrepeat N- and C-terminal domains of the Esp protein as well as differences in the number of repeats. DNA sequencing of chromosomal regions flanking the esp gene of E. faecium revealed seven open reading frames, representing putative genes implicated in virulence, regulation of transcription, and antibiotic resistance. These flanking regions were invariably associated with the presence or absence of the esp gene in E. faecium, indicating that esp in E. faecium is part of a distinct genetic element. Because of the presence of virulence genes in this gene cluster, the lower G+C content relative to that of the genome, and the presence of esp in E. faecium isolates associated with nosocomial outbreaks and clinically documented infections, we conclude that this genetic element constitutes a putative pathogenicity island, the first one described in E. faecium. Except for the presence of esp and araC, this pathogenicity island is completely different from the esp-containing pathogenicity island previously disclosed in E. faecalis.
KeywordMeSH Terms
104. Gueneau de Novoa  P, Williams  KP,     ( 2004 )

The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts.

Nucleic acids research 32 (Database issue)
PMID : 14681369  :   DOI  :   10.1093/nar/gkh102     PMC  :   PMC308836    
Abstract >>
tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.
KeywordMeSH Terms
Databases, Nucleic Acid
Evolution, Molecular
Internet
105. Tomita  H, Tanimoto  K, Hayakawa  S, Morinaga  K, Ezaki  K, Oshima  H, Ike  Y,     ( 2003 )

Highly conjugative pMG1-like plasmids carrying Tn1546-like transposons that encode vancomycin resistance in Enterococcus faecium.

Journal of bacteriology 185 (23)
PMID : 14617670  :   DOI  :   10.1128/jb.185.23.7024-7028.2003     PMC  :   PMC262697    
Abstract >>
A total of 12 VanA-type vancomycin-resistant enterococci, consisting of 10 Enterococcus faecium isolates and two Enterococcus avium isolates, were examined in detail. The vancomycin resistance conjugative plasmids pHTalpha (65.9 kbp), pHTbeta (63.7 kbp), and pHTgamma (66.5 kbp) were isolated from each of three different E. faecium strains. The plasmids transferred highly efficiently between enterococcus strains during broth mating and were homologous with pMG1 (Gm(r); 65.1 kb).
KeywordMeSH Terms
106. Depardieu  F, Courvalin  P, Msadek  T,     ( 2003 )

A six amino acid deletion, partially overlapping the VanSB G2 ATP-binding motif, leads to constitutive glycopeptide resistance in VanB-type Enterococcus faecium.

Molecular microbiology 50 (3)
PMID : 14617162  :   DOI  :   10.1046/j.1365-2958.2003.03771.x    
Abstract >>
Enterococcus faecium clinical isolate BM4524, resistant to vancomycin and susceptible to teicoplanin, harboured a chromosomal vanB cluster, including the vanSB/vanRB two-component system regulatory genes. Enterococcus faecium strain BM4525, isolated two weeks later from the same patient, was resistant to high levels of both glycopeptides. The ddl gene of BM4525 had a 2 bp insertion leading to an impaired d-alanine:d-alanine ligase. Sequencing of the vanB operon in BM4525 also revealed an 18 bp deletion in the vanSB gene designated vanSBDelta. The resulting six amino acid deletion partially overlapped the G2 ATP-binding domain of the VanSBDelta histidine kinase leading to constitutive expression of the resistance genes. Sequence analysis indicated that the deletion occurred between two tandemly arranged heptanucleotide direct repeats, separated by 11 base-pairs. The VanSB, VanSBDelta and VanRB proteins were overproduced in Escherichia coli and purified. In vitro autophosphorylation of the VanSB and VanSBDelta histidine kinases and phosphotransfer to the VanRB response regulator did not differ significantly. However, VanSBDelta was deficient in VanRB phosphatase activity leading to accumulation of phosphorylated VanRB. Increased glycopeptide resistance in E. faecium BM4525 was therefore a result of the lack of production of d-alanyl-d-alanine ending pentapeptide and to constitutive synthesis of d-alanyl-d-lactate terminating peptidoglycan precursors, following loss of d-alanine:d-alanine ligase and of VanSB phosphatase activity respectively. We suggest that the heptanucleotide direct repeat in vanSB may favour the appearance of high level constitutively expressed vancomycin resistance through a 'slippage' type of genetic rearrangement in VanB-type strains.
KeywordMeSH Terms
Bacterial Proteins
Sequence Deletion
107. Lebreton  F, Depardieu  F, Bourdon  N, Fines-Guyon  M, Berger  P, Camiade  S, Leclercq  R, Courvalin  P, Cattoir  V,     ( 2011 )

D-Ala-d-Ser VanN-type transferable vancomycin resistance in Enterococcus faecium.

Antimicrobial agents and chemotherapy 55 (10)
PMID : 21807981  :   DOI  :   10.1128/AAC.00714-11     PMC  :   PMC3187002    
Abstract >>
Enterococcus faecium UCN71, isolated from a blood culture, was resistant to low levels of vancomycin (MIC, 16 �gg/ml) but susceptible to teicoplanin (MIC, 0.5 �gg/ml). No amplification was observed with primers specific for the previously described glycopeptide resistance ligase genes, but a PCR product corresponding to a gene called vanN was obtained using degenerate primers and was sequenced. The deduced VanN protein was related (65% identity) to the d-alanine:d-serine VanL ligase. The organization of the vanN gene cluster, determined using degenerate primers and by thermal asymmetric interlaced (TAIL)-PCR, was similar to that of the vanC operons. A single promoter upstream from the resistance operon was identified by rapid amplification of cDNA ends (RACE)-PCR. The presence of peptidoglycan precursors ending in d-serine and d,d-peptidase activities in the absence of vancomycin indicated constitutive expression of the resistance operon. VanN-type resistance was transferable by conjugation to E. faecium. This is the first report of transferable d-Ala-d-Ser-type resistance in E. faecium.
KeywordMeSH Terms
108. López  M, Kadlec  K, Schwarz  S, Torres  C,     ( 2012 )

First detection of the staphylococcal trimethoprim resistance gene dfrK and the dfrK-carrying transposon Tn559 in enterococci.

Microbial drug resistance (Larchmont, N.Y.) 18 (1)
PMID : 21718151  :   DOI  :   10.1089/mdr.2011.0073    
Abstract >>
The trimethoprim resistance gene dfrK has been recently described in Staphylococcus aureus, but so far has not been found in other bacteria. A total of 166 enterococci of different species (E. faecium, E. faecalis, E. hirae, E. durans, E. gallinarum, and E. casseliflavus) and origins (food, clinical diseases in humans, healthy humans or animals, and sewage) were studied for their susceptibility to trimethoprim as determined by agar dilution (European Committee on Antimicrobial Susceptibility Testing) and the presence of (a) the dfrK gene and its genetic environment and (b) other dfr genes. The dfrK gene was detected in 49% of the enterococci (64% and 42% of isolates with minimum inhibitory concentrations of ?2 mg/L or ?1 mg/L, respectively). The tet(L)-dfrK linkage was detected in 21% of dfrK-positive enterococci. The chromosomal location of the dfrK gene was identified in one E. faecium isolate in which the dfrK was not linked to tet(L) gene but was part of a Tn559 element, which was integrated in the chromosomal radC gene. This Tn559 element was also found in 14 additional isolates. All combinations of dfr genes were detected among the isolates tested (dfrK, dfrG, dfrF, dfrK+dfrG, dfrK+dfrF, dfrF+dfrG, and dfrF+dfrG+dfrK). The gene dfrK gene was found together with other dfr genes in 58% of the tested enterococci. This study suggested an exchange of the trimethoprim resistance gene dfrK between enterococci and staphylococci, as previously observed for the trimethoprim resistance gene dfrG.
KeywordMeSH Terms
DNA Transposable Elements
109. Li  X, Alvarez  V, Harper  WJ, Wang  HH,     ( 2011 )

Persistent, toxin-antitoxin system-independent, tetracycline resistance-encoding plasmid from a dairy Enterococcus faecium isolate.

Applied and environmental microbiology 77 (20)
PMID : 21784909  :   DOI  :   10.1128/AEM.05168-11     PMC  :   PMC3194845    
Abstract >>
A tetracycline-resistant (Tet(r)) dairy Enterococcus faecium isolate designated M7M2 was found to carry both tet(M) and tet(L) genes on a 19.6-kb plasmid. After consecutive transfer in the absence of tetracycline, the resistance-encoding plasmid persisted in 99% of the progenies. DNA sequence analysis revealed that the 19.6-kb plasmid contained 28 open reading frames (ORFs), including a tet(M)-tet(L)-mob gene cluster, as well as a 10.6-kb backbone highly homologous (99.9%) to the reported plasmid pRE25, but without an identified toxin-antitoxin (TA) plasmid stabilization system. The derived backbone plasmid without the Tet(r) determinants exhibited a 100% retention rate in the presence of acridine orange, suggesting the presence of a TA-independent plasmid stabilization mechanism, with its impact on the persistence of a broad spectrum of resistance-encoding traits still to be elucidated. The tet(M)-tet(L) gene cluster from M7M2 was functional and transmissible and led to acquired resistance in Enterococcus faecalis OG1RF by electroporation and in Streptococcus mutans UA159 by natural transformation. Southern hybridization showed that both the tet(M) and tet(L) genes were integrated into the chromosome of S. mutans UA159, while the whole plasmid was transferred to and retained in E. faecalis OG1RF. Quantitative real-time reverse transcription-PCR (RT-PCR) indicated tetracycline-induced transcription of both the tet(M) and tet(L) genes of pM7M2. The results indicated that multiple mechanisms might have contributed to the persistence of antibiotic resistance-encoding genes and that the plasmids pM7M2, pIP816, and pRE25 are likely correlated evolutionarily.
KeywordMeSH Terms
Plasmids
Tetracycline Resistance
110. Capozzi  V, Ladero  V, Beneduce  L, Fernández  M, Alvarez  MA, Benoit  B, Laurent  B, Grieco  F, Spano  G,     ( 2011 )

Isolation and characterization of tyramine-producing Enterococcus faecium strains from red wine.

Food microbiology 28 (3)
PMID : 21356448  :   DOI  :   10.1016/j.fm.2010.10.005    
Abstract >>
Enterococcus faecium strains were isolated from red wines undergoing malolactic fermentation and identified by comparison of their 16S rDNA gene sequences with those included in the GenEMBL Databases. The tyrosine decarboxylase gene was identified in all the strains analysed by PCR using gene-specific primers and the ability to produce tyramine in a synthetic media was analysed by RP-HPLC. Survival of an E. faecium strain was also evaluated in microvinification assays using two different musts with different ethanol concentrations (10% and 12% (v/v)). Tyramine production was monitored during the vinification trials. Our results suggest that E. faecium strains isolated from wine are able to produce tyramine and tolerate wine conditions following a pre-acidic stress.
KeywordMeSH Terms
Enterococcus faecium
111. Yamashita  H, Tomita  H, Inoue  T, Ike  Y,     ( 2011 )

Genetic organization and mode of action of a novel bacteriocin, bacteriocin 51: determinant of VanA-type vancomycin-resistant Enterococcus faecium.

Antimicrobial agents and chemotherapy 55 (9)
PMID : 21709077  :   DOI  :   10.1128/AAC.01274-10     PMC  :   PMC3165305    
Abstract >>
Bacteriocin 51 (Bac 51) is encoded on the mobile plasmid pHY (6,037 bp), which was isolated from vancomycin-resistant Enterococcus faecium VRE38. Bacteriocin 51 is active against E. faecium, E. hirae, and E. durans. Sequence analysis of pHY showed that it encodes nine open reading frames (ORFs) from ORF1 to ORF9 (in that order). Genetic analysis suggested that ORF1 and ORF2, which were designated bacA and bacB, respectively, are the bacteriocin and immunity genes. bacA encodes a 144-amino-acid protein. The deduced BacA protein has a typical signal sequence at its amino terminus, and a potential signal peptidase-processing site corresponding to the V-E-A sequence is located between the 37th and 39th amino acids. The predicted mature BacA protein consists of 105 amino acids. A potential promoter sequence was identified upstream of the start codon. bacB encodes a 55-amino-acid protein. No obvious promoter or terminator sequence was identified between bacA and bacB. Northern blot analysis of bacA and bacB with a bacA RNA probe produced a transcript of approximately 700 nucleotides, which corresponded to the combined nucleotide sizes of bacA and bacB, indicating that transcription was initiated from the promoter upstream of bacA, continued through bacB, and was terminated at the terminator downstream of bacB. The transcription start site was determined to be the T nucleotide located 6 nucleotides downstream from the -10 promoter sequence. These results indicate that bacA and bacB constitute an operon and that bacA is the bacteriocin structural gene while bacB is the immunity gene. The purified C-terminally His tagged BacA protein of Bac 51 showed bacteriostatic activity against the indicator strain. The purified C-terminally His tagged BacA protein of Bac 32 (whose mature BacA protein has 54 amino acids) and the culture filtrates of the Bac 31- and Bac 43-producing E. faecalis strain FA2-2 showed bactericidal activity. Bac 31 and Bac 43 are pore-forming bacteriocins, unlike the newly characterized bacteriocin Bac 51.
KeywordMeSH Terms
112. Muñoz-Atienza  E, Landeta  G, de las Rivas  B, Gómez-Sala  B, Muñoz  R, Hernández  PE, Cintas  LM, Herranz  C,     ( 2011 )

Phenotypic and genetic evaluations of biogenic amine production by lactic acid bacteria isolated from fish and fish products.

International journal of food microbiology 146 (2)
PMID : 21411165  :   DOI  :   10.1016/j.ijfoodmicro.2011.02.024    
Abstract >>
In this work, biogenic amine production (histamine, tyramine and putrescine) by a collection of 74 lactic acid bacteria of aquatic origin has been investigated by means of amino acid decarboxylation by growth on decarboxylase differential medium, biogenic amine detection by thin-layer chromatography (TLC) and decarboxylase gene detection by PCR. None of the evaluated strains showed neither production of histamine and putrescine, nor presence of the genetic determinants encoding the corresponding decarboxylase activities. However, the tyrosine decarboxylase gene (tdc) was present in all the enterococcal strains, and tyramine production was detected by TLC in all of them but Enterococcus faecium BCS59 and MV5. Analysis of the tyrosine decarboxylase operon of these strains revealed the presence of an insertion sequence upstream tdc that could be responsible for their lack of tyrosine decarboxylase activity.
KeywordMeSH Terms
113. Rahkila  R, Johansson  P, Säde  E, Björkroth  J,     ( 2011 )

Identification of enterococci from broiler products and a broiler processing plant and description of Enterococcus viikkiensis sp. nov.

Applied and environmental microbiology 77 (4)
PMID : 21183650  :   DOI  :   10.1128/AEM.02412-10     PMC  :   PMC3067211    
Abstract >>
In two previous studies dealing with lactic acid bacteria (LAB) from modified-atmosphere-packaged (MAP) broiler products and a broiler processing plant, several isolates remained unidentified. According to 16S rRNA gene sequence analysis, 36 isolates were assigned to the genus Enterococcus. Numerical analysis of combined HindIII and EcoRI ribopatterns of these isolates resulted in species-specific clusters that were congruent with the clusters obtained by both DNA-directed RNA polymerase subunit A (rpoA) and phenylalanyl-tRNA synthetase �\ chain (pheS) housekeeping gene analyses. In the analyses, a group of five isolates distinct from any known enterococcal species clustered together. The five isolates were positioned in the Enterococcus avium group, with E. devriesei being the closest phylogenetic neighbor. The DNA-DNA hybridization levels with E. devriesei ranged from 28.8 to 54.3% and indicated that these strains represented a novel species. The name Enterococcus viikkiensis sp. nov. is proposed, with strain DSM 24043(T) (LMG 26075(T)) being the type strain. Our study demonstrated that the identification of enterococci within the E. avium phylogenetic group demands polyphasic taxonomic approaches. The rpoA and pheS gene similarities (99.0 to 99.2% and 94.3 to 95.4%, respectively) between E. viikkiensis and its closest phylogenetic neighbor, E. devriesei, were higher than those previously reported within the enterococci. In addition, the phenotypic profiles of the species in the E. avium group were also highly similar, and some traits were found to be misleading for enterococci, such as E. viikkiensis does not grow at 45�XC. The numerical analysis of combined HindIII and EcoRI ribopatterns was of considerable assistance in distinguishing enterococcal species within the E. avium group.
KeywordMeSH Terms
Bacterial Typing Techniques
Meat-Packing Industry
114. Kerbauy  G, Perugini  MR, Yamauchi  LM, Yamada-Ogatta  SF,     ( 2011 )

Vancomycin-dependent Enterococcus faecium vanA: characterization of the first case isolated in a university hospital in Brazil.

Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas 44 (3)
PMID : 21271180  :   DOI  :   10.1590/s0100-879x2011007500006    
Abstract >>
In this study, we report the characterization of a strain of Enterococcus faecium vanA, which grows only in the presence of vancomycin (VDEfm-UEL). The bacterium was isolated from the feces of a female patient who had undergone surgical treatment of Reinke's edema and was receiving intravenous vancomycin therapy for infection with methicillin/oxacillin-resistant Staphylococcus aureus, a postoperative complication. Antimicrobial dependence was further confirmed by the vancomycin E-test. VDEfm-UEL was also shown to be resistant to ampicillin, ciprofloxacin, chloramphenicol, erythromycin, levofloxacin, penicillin, rifampicin, and teicoplanin. The putative virulence genes efaA, gelE and esp were detected by PCR. The ddl gene from VDEfm-UEL was cloned and sequenced. Vancomycin dependence seems to be associated with the insertion of a nucleotide in that sequence, which results in a frame-shift mutation, introducing a premature stop codon. This is the first report of vancomycin-dependent E. faecium isolation in a university hospital in Brazil.
KeywordMeSH Terms
115. Galimand  M, Schmitt  E, Panvert  M, Desmolaize  B, Douthwaite  S, Mechulam  Y, Courvalin  P,     ( 2011 )

Intrinsic resistance to aminoglycosides in Enterococcus faecium is conferred by the 16S rRNA m5C1404-specific methyltransferase EfmM.

RNA (New York, N.Y.) 17 (2)
PMID : 21159796  :   DOI  :   10.1261/rna.2233511     PMC  :   PMC3022275    
Abstract >>
Aminoglycosides are ribosome-targeting antibiotics and a major drug group of choice in the treatment of serious enterococcal infections. Here we show that aminoglycoside resistance in Enterococcus faecium strain CIP 54-32 is conferred by the chromosomal gene efmM, encoding the E. faecium methyltransferase, as well as by the previously characterized aac(6')-Ii that encodes a 6'-N-aminoglycoside acetyltransferase. Inactivation of efmM in E. faecium increases susceptibility to the aminoglycosides kanamycin and tobramycin, and, conversely, expression of a recombinant version of efmM in Escherichia coli confers resistance to these drugs. The EfmM protein shows significant sequence similarity to E. coli RsmF (previously called YebU), which is a 5-methylcytidine (m?C) methyltransferase modifying 16S rRNA nucleotide C1407. The target for EfmM is shown by mass spectrometry to be a neighboring 16S rRNA nucleotide at C1404. EfmM uses the methyl group donor S-adenosyl-L-methionine to catalyze formation of m?C1404 on the 30S ribosomal subunit, whereas naked 16S rRNA and the 70S ribosome are not substrates. Addition of the 5-methyl to C1404 sterically hinders aminoglycoside binding. Crystallographic structure determination of EfmM at 2.28 ? resolution reveals an N-terminal domain connected to a central methyltransferase domain that is linked by a flexible lysine-rich region to two C-terminal subdomains. Mutagenesis of the methyltransferase domain established that two cysteines at specific tertiary locations are required for catalysis. The tertiary structure of EfmM is highly similar to that of RsmF, consistent with m?C formation at adjacent sites on the 30S subunit, while distinctive structural features account for the enzymes' respective specificities for nucleotides C1404 and C1407.
KeywordMeSH Terms
116. Halvorsen  EM, Williams  JJ, Bhimani  AJ, Billings  EA, Hergenrother  PJ,     ( 2011 )

Txe, an endoribonuclease of the enterococcal Axe-Txe toxin-antitoxin system, cleaves mRNA and inhibits protein synthesis.

Microbiology (Reading, England) 157 (Pt 2)
PMID : 21030436  :   DOI  :   10.1099/mic.0.045492-0     PMC  :   PMC3090131    
Abstract >>
The axe-txe operon encodes a toxin-antitoxin (TA) pair, Axe-Txe, that was initially identified on the multidrug-resistance plasmid pRUM in Enterococcus faecium. In Escherichia coli, expression of the Txe toxin is known to inhibit cell growth, and co-expression of the antitoxin, Axe, counteracts the toxic effect of Txe. Here, we report the nucleotide sequence of pS177, a 39 kb multidrug-resistant plasmid isolated from vancomycin-resistant Ent. faecium, which harbours the axe-txe operon and the vanA gene cluster. RT-PCR analysis revealed that the axe-txe transcript is produced by strain S177 as well as by other vancomycin-resistant enteroccoci. Moreover, we determine the mechanism by which the Txe protein exerts its toxic activity. Txe inhibits protein synthesis in E. coli without affecting DNA or RNA synthesis, and inhibits protein synthesis in a cell-free system. Using in vivo primer extension analysis, we demonstrate that Txe preferentially cleaves single-stranded mRNA at the first base after an AUG start codon. We conclude that Txe is an endoribonuclease which cleaves mRNA and inhibits protein synthesis.
KeywordMeSH Terms
117. Yohda  M, Okada  H, Kumagai  H,     ( 1991 )

Molecular cloning and nucleotide sequencing of the aspartate racemase gene from lactic acid bacteria Streptococcus thermophilus.

Biochimica et biophysica acta 1089 (2)
PMID : 2054383  :   DOI  :   10.1016/0167-4781(91)90013-c    
Abstract >>
The gene coding aspartate racemase (EC 5.1.1.13) was cloned from the lactic acid bacteria Streptococcus thermophilus IAM10064 and expressed efficiently in Escherichia coli. The 2.1 kilobase pairs long full length clone had an open reading frame of 729 nucleotides coding for 243 amino acids. The calculated molecular weight of 27,945 agreed well with the apparent molecular weight of 28,000 found in sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of the aspartate racemase purified from S. thermophilus. The N-terminal amino acid sequence from the purified protein exactly matches the derived sequence. In addition, the amino acid composition compiled from the derived sequence is very similar to that obtained from the purified recombinant protein. No significantly homologous proteins were found in a protein sequence data bank. Even the homology scores with alanine racemases of Salmonella typhimurium and Bacillus stearothermophilus were low. Aspartate racemase was overproduced in Escherichia coli NM522 with plasmid pAG6-2-7, which was constructed from two copies of the gene linked with a tac promoter and plasmid vector pUC18. The amount of aspartate racemase increases with the growth of E. coli and almost no degradation of the enzyme was observed. The maximum amount of the produced enzyme reached approx. 20% of the total protein of E. coli.
KeywordMeSH Terms
118. Laverde Gomez  JA, van Schaik  W, Freitas  AR, Coque  TM, Weaver  KE, Francia  MV, Witte  W, Werner  G,     ( 2011 )

A multiresistance megaplasmid pLG1 bearing a hylEfm genomic island in hospital Enterococcus faecium isolates.

International journal of medical microbiology : IJMM 301 (2)
PMID : 20951641  :   DOI  :   10.1016/j.ijmm.2010.08.015    
Abstract >>
Enterococcus faecium is considered to be a nosocomial pathogen with increasing medical importance. The putative virulence factor, hyl(Efm), encoding a putative hyaluronidase, is enriched among the hospital-associated polyclonal subpopulation of E. faecium.. The hyl(Efm) gene is described to be part of a genomic island and was recently identified to be plasmid-located. Here, we present a description of the structure, localization, and distribution of the putative pathogenicity factor hyl(Efm) and its putative island among 39 clinical isolates and elucidate the composition and host range of pLG1, a hyl(Efm) multiresistance plasmid of approximately 281.02kb. The hyl(Efm) gene was located within a 17,824-bp element highly similar to the putative genomic island (GI) structure that had been previously described. This genomic region was conserved among 39 hyl(Efm)-positive strains with variation in a specific region downstream of hyl(Efm) in 18 strains. The putative hyl(Efm) was located on large plasmids (150-350kb) in 37 strains. pLG1 could be horizontally transferred into four different E. faecium recipient strains (n=4) but not into E. faecalis (n=3). Sequencing of pLG1 resolved putative plasmid replication, conjugation, and maintenance determinants as well as a pilin gene cluster, carbon uptake and utilization genes, heavy metal and antibiotic resistance clusters. The hyl(Efm) transferable plasmid pLG1 bears additional putative pathogenicity factors and antibiotic resistance genes. These findings suggest horizontal gene transfer of virulence factors and antibiotic resistance gene clusters by a single genetic event (conjugative transfer) which might be triggered by heavy antibiotic use common in health care units where E. faecium is increasingly prevalent.
KeywordMeSH Terms
Genomic Islands
119. Amachawadi  RG, Shelton  NW, Jacob  ME, Shi  X, Narayanan  SK, Zurek  L, Dritz  SS, Nelssen  JL, Tokach  MD, Nagaraja  TG,     ( 2010 )

Occurrence of tcrB, a transferable copper resistance gene, in fecal enterococci of swine.

Foodborne pathogens and disease 7 (9)
PMID : 20500052  :   DOI  :   10.1089/fpd.2010.0540    
Abstract >>
High concentration of copper, fed as copper sulfate, is often used to increase growth rates in swine. Bacteria exposed to copper may acquire resistance, and in Enterococcus faecium and Enterococcus faecalis, a plasmid-borne transferable copper resistance (tcrB) gene that confers copper resistance has been reported. Our objectives were to determine the occurrence of tcrB in fecal enterococci from weaned piglets fed diets with a normal supplemental level (16.5 ppm) or an elevated supplemental level (125 ppm) of copper and to determine the association of tcrB with copper, erythromycin, and vancomycin resistance. A total of 323 enterococcal isolates were examined and 15 (4.6%) isolates (14 E. faecium and 1 E. faecalis) were positive for tcrB. Fifteen tcrB-positive and 15 randomly chosen tcrB-negative isolates from piglets fed the normal supplemental level of copper were tested for erm(B), tet(M), vanA, and vanB genes and susceptibilities to copper, erythromycin, tetracyclines, and vancomycin. All tcrB-positive and -negative isolates contained erm(B) and tet(M), but not vanA and vanB. The mean minimum inhibitory concentration of copper for tcrB-positive (21.1 mM) was higher (p < 0.001) compared with tcrB-negative isolates (6.1 mM). All isolates were resistant to erythromycin and tetracyclines and susceptible to vancomycin. The transferability of the tcrB gene from tcrB-positive strains to tcrB-negative strains was demonstrated by conjugation. The potential link between tcrB and antibiotic resistance genes and the propensity of enterococci to transfer tcrB to other strains raises the possibility that copper supplementation may exert selection pressure for antibiotic-resistant enterococci. This study is the first report on the occurrence of the tcrB gene in enterococci isolated from swine in the United States.
KeywordMeSH Terms
120. Hu  CB, Malaphan  W, Zendo  T, Nakayama  J, Sonomoto  K,     ( 2010 )

Enterocin X, a novel two-peptide bacteriocin from Enterococcus faecium KU-B5, has an antibacterial spectrum entirely different from those of its component peptides.

Applied and environmental microbiology 76 (13)
PMID : 20418437  :   DOI  :   10.1128/AEM.02264-09     PMC  :   PMC2897431    
Abstract >>
Enterocin X, composed of two antibacterial peptides (Xalpha and Xbeta), is a novel class IIb bacteriocin from Enterococcus faecium KU-B5. When combined, Xalpha and Xbeta display variably enhanced or reduced antibacterial activity toward a panel of indicators compared to each peptide individually. In E. faecium strains that produce enterocins A and B, such as KU-B5, only one additional bacteriocin had previously been known.
KeywordMeSH Terms
Anti-Bacterial Agents
121. López  M, Sáenz  Y, Alvarez-Martínez  MJ, Marco  F, Robredo  B, Rojo-Bezares  B, Ruiz-Larrea  F, Zarazaga  M, Torres  C,     ( 2010 )

Tn1546 structures and multilocus sequence typing of vanA-containing enterococci of animal, human and food origin.

The Journal of antimicrobial chemotherapy 65 (8)
PMID : 20519356  :   DOI  :   10.1093/jac/dkq192    
Abstract >>
To characterize the Tn1546 structure and to perform the genetic typing of 51 PFGE-unrelated vanA-containing enterococci of different origin (clinical, food and faecal samples of healthy humans and healthy poultry). Tn1546 structure was characterized by a PCR primer walking strategy and sequencing. Multilocus sequence typing (MLST) was performed for Enterococcus faecium and Enterococcus faecalis strains, and esp and hyl genes were detected by PCR. Nine different Tn1546 structures were identified in the studied strains. Type I was the most prevalent structure (75%) (identical to GenBank M97297). Two new Tn1546 structures were identified (in three clinical and animal strains), containing two new insertion sequences (ISs; ISEfa11 disrupting vanS and ISEfa10 disrupting orf1). An additional new Tn1546 structure was found in one animal strain, containing ISEf1 interrupting vanY and IS1542 in the orf2-vanR region. A high diversity of sequence types (STs) was detected among clinical (6 ST/18 strains) and non-clinical E. faecium strains (18 ST/24 strains). STs associated with clonal complexes CC17 and CC9 were mainly detected among clinical and non-clinical E. faecium strains, respectively. Seven new STs were identified in non-clinical strains. The esp and hyl genes were only found among clinical E. faecium strains. A moderate variability in Tn1546 structure has been detected among unrelated vanA-containing enterococci of different origins, showing three new structures including two new ISs. A high diversity of STs was detected among E. faecium strains, especially among non-clinical strains, and new STs have been identified.
KeywordMeSH Terms
DNA Transposable Elements
Food Microbiology
122. Bjørkeng  EK, Tessema  GT, Lundblad  EW, Butaye  P, Willems  R, Sollid  JE, Sundsfjord  A, Hegstad  K,     ( 2010 )

ccrABEnt serine recombinase genes are widely distributed in the Enterococcus faecium and Enterococcus casseliflavus species groups and are expressed in E. faecium.

Microbiology (Reading, England) 156 (Pt 12)
PMID : 20817645  :   DOI  :   10.1099/mic.0.041491-0     PMC  :   PMC3068701    
Abstract >>
The presence, distribution and expression of cassette chromosome recombinase (ccr) genes, which are homologous to the staphylococcal ccrAB genes and are designated ccrAB(Ent) genes, were examined in enterococcal isolates (n=421) representing 13 different species. A total of 118 (28 %) isolates were positive for ccrAB(Ent) genes by PCR, and a number of these were confirmed by Southern hybridization with a ccrA(Ent) probe (n=76) and partial DNA sequencing of ccrA(Ent) and ccrB(Ent) genes (n=38). ccrAB(Ent) genes were present in Enterococcus faecium (58/216, 27 %), Enterococcus durans (31/38, 82 %), Enterococcus hirae (27/52, 50 %), Enterococcus casseliflavus (1/4, 25 %) and Enterococcus gallinarum (1/2, 50 %). In the eight other species tested, including Enterococcus faecalis (n=94), ccrAB(Ent) genes were not found. Thirty-eight sequenced ccrAB(Ent) genes from five different enterococcal species showed 94-100 % nucleotide sequence identity and linkage PCRs showed heterogeneity in the ccrAB(Ent) flanking chromosomal genes. Expression analysis of ccrAB(Ent) genes from the E. faecium DO strain showed constitutive expression as a bicistronic mRNA. The ccrAB(Ent) mRNA levels were lower during log phase than stationary phase in relation to total mRNA. Multilocus sequence typing was performed on 39 isolates. ccrAB(Ent) genes were detected in both hospital-related (10/29, 34 %) and non-hospital (4/10, 40 %) strains of E. faecium. Various sequence types were represented by both ccrAB(Ent) positive and negative isolates, suggesting acquisition or loss of ccrAB(Ent) in E. faecium. In summary, ccrAB(Ent) genes, potentially involved in genome plasticity, are expressed in E. faecium and are widely distributed in the E. faecium and E. casseliflavus species groups.
KeywordMeSH Terms
123. Kadlec  K, Schwarz  S,     ( 2010 )

Identification of the novel dfrK-carrying transposon Tn559 in a porcine methicillin-susceptible Staphylococcus aureus ST398 strain.

Antimicrobial agents and chemotherapy 54 (8)
PMID : 20498309  :   DOI  :   10.1128/AAC.00464-10     PMC  :   PMC2916295    
Abstract >>
The trimethoprim resistance gene dfrK was found to be part of the novel Tn554-related transposon Tn559 integrated in the chromosomal radC gene of a porcine methicillin-susceptible Staphylococcus aureus ST398 strain. While Tn559 and Tn554 had similar arrangements of the transposase genes tnpA, tnpB, and tnpC, the Tn554-associated resistance genes erm(A) and spc were replaced by dfrK in Tn559. Circular forms of Tn559 were detected and suggest the functional activity of this transposon.
KeywordMeSH Terms
124. Jung  YH, Shin  ES, Kim  O, Yoo  JS, Lee  KM, Yoo  JI, Chung  GT, Lee  YS,     ( 2010 )

Characterization of two newly identified genes, vgaD and vatH, [corrected] conferring resistance to streptogramin A in Enterococcus faecium.

Antimicrobial agents and chemotherapy 54 (11)
PMID : 20713681  :   DOI  :   10.1128/AAC.00798-09     PMC  :   PMC2976166    
Abstract >>
We characterized two new streptogramin A resistance genes from quinupristin-dalfopristin-resistant Enterococcus faecium JS79, which was selected from 79 E. faecium isolates lacking known genes encoding streptogramin A acetyltransferase. A 5,650-bp fragment of HindIII-digested plasmid DNA from E. faecium JS79 was cloned and sequenced. The fragment contained two open reading frames carrying resistance genes related to streptogramin A, namely, genes for an acetyltransferase and an ATP efflux pump. The first open reading frame comprised 648 bp encoding 216 amino acids with a predicted left-handed parallel �]-helix domain structure; this new gene was designated vatH. [corrected] The second open reading frame consisted of 1,575 bp encoding 525 amino acids with two predicted ATPase binding cassette transporters comprised of Walker A, Walker B, and LSSG motifs; this gene was designated vgaD. vgaD is located 65 bp upstream from vatH, [corrected] was detected together with vatH [corrected] in 12 of 179 quinupristin-dalfopristin-resistant E. faecium isolates, and was located on the same plasmid. Also, the 5.6-kb HindIII-digested fragment which was observed in JS79 was detected in nine vgaD- and vatH-containing [corrected] E. faecium isolates by Southern hybridization. Therefore, it was expected that these two genes were strongly correlated with each other and that they may be composed of a transposon. Importantly, vgaD is the first identified ABC transporter conferring resistance to streptogramin A in E. faecium. Pulsed-field gel electrophoresis patterns and sequence types of vgaD- and vatH-containing [corrected] E. faecium isolates differed for isolates from humans and nonhumans.
KeywordMeSH Terms
125. Xu  X, Lin  D, Yan  G, Ye  X, Wu  S, Guo  Y, Zhu  D, Hu  F, Zhang  Y, Wang  F, Jacoby  GA, Wang  M,     ( 2010 )

vanM, a new glycopeptide resistance gene cluster found in Enterococcus faecium.

Antimicrobial agents and chemotherapy 54 (11)
PMID : 20733041  :   DOI  :   10.1128/AAC.01710-09     PMC  :   PMC2976141    
Abstract >>
Since glycopeptide-resistant enterococci (GRE) were reported in 1988, they have appeared in hospitals worldwide. Seven van gene cluster types (vanA, vanB, vanC, vanD, vanE, vanG, and vanL) are currently known. We investigated a clinical strain of Enterococcus faecium Efm-HS0661 that was isolated in 2006 from an inpatient with intra-abdominal infection in Shanghai. It was resistant to most antimicrobials, including vancomycin (MIC, >256 �gg/ml) and teicoplanin (MIC, 96 �gg/ml). Glycopeptide resistance could be transferred to E. faecium BM4105RF by conjugation. The donor and its transconjugant were negative by PCR for the known van genes. By cloning and primer walk sequencing, we discovered a novel van gene cluster, designated vanM. The vanM ligase gene was 1,032-bp in length and encoded a 343-amino-acid protein that shared 79.9, 70.8, 66.3, and 78.8% amino acid identity with VanA, VanB, VanD, and VanF, respectively. Although the vanM DNA sequence was closest to vanA, the organization of the vanM gene cluster was most similar to that of vanD. Upstream from the vanM cluster was an IS1216-like element, which may play a role in the dissemination of this resistance determinant. Liquid chromatography-mass spectrometry analysis of peptidoglycan precursors extracted from the VanM-type strain Efm-HS0661 treated with vancomycin or teicoplanin revealed a modified precursor (UDP-N-acetylmuramic acid [MurNAc]-tetrapeptide-D-Lac), indicating that VanM, like VanA, confers glycopeptide resistance by the inducible synthesis of precursor ending in D-Ala-D-Lac.
KeywordMeSH Terms
126. Freitas  AR, Tedim  AP, Novais  C, Ruiz-Garbajosa  P, Werner  G, Laverde-Gomez  JA, Cantón  R, Peixe  L, Baquero  F, Coque  TM,     ( 2010 )

Global spread of the hyl(Efm) colonization-virulence gene in megaplasmids of the Enterococcus faecium CC17 polyclonal subcluster.

Antimicrobial agents and chemotherapy 54 (6)
PMID : 20385861  :   DOI  :   10.1128/AAC.00134-10     PMC  :   PMC2876360    
Abstract >>
Enterococcus faecium has increasingly been reported as a nosocomial pathogen since the early 1990s, presumptively associated with the expansion of a human-associated Enterococcus faecium polyclonal subcluster known as clonal complex 17 (CC17) that has progressively acquired different antibiotic resistance (ampicillin and vancomycin) and virulence (esp(Efm), hyl(Efm), and fms) traits. We analyzed the presence and the location of a putative glycoside hydrolase hyl(Efm) gene among E. faecium strains obtained from hospitalized patients (255 patients; outbreak, bacteremic, and/or disseminated isolates from 23 countries and five continents; 1986 to 2009) and from nonclinical origins (isolates obtained from healthy humans [25 isolates], poultry [30], swine [90], and the environment [55]; 1999 to 2007). Clonal relatedness was established by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Plasmid analysis included determination of content and size (S1-PFGE), transferability (filter mating), screening of Rep initiator proteins (PCR), and location of vanA, vanB, ermB, and hyl(Efm) genes (S1/I-CeuI hybridization). Most E. faecium isolates contained large plasmids (>150 kb) and showed variable contents of van, hyl(Efm), or esp(Efm). The hyl(Efm) gene was associated with megaplasmids (170 to 375 kb) of worldwide spread (ST16, ST17, and ST18) or locally predominant (ST192, ST203, ST280, and ST412) ampicillin-resistant CC17 clones collected in the five continents since the early 1990s. All but one hyl(Efm)-positive isolate belonged to the CC17 polyclonal subcluster. The presence of hyl(Efm) megaplasmids among CC17 from Europe, Australia, Asia, and Africa since at least the mid-1990s was documented. This study further demonstrates the pandemic expansion of particular CC17 clones before acquisition of vancomycin resistance and putative virulence traits and describes the presence of megaplasmids in most of the contemporary E. faecium isolates with different origins.
KeywordMeSH Terms
Genes, Bacterial
127. Morar  M, Bhullar  K, Hughes  DW, Junop  M, Wright  GD,     ( 2009 )

Structure and mechanism of the lincosamide antibiotic adenylyltransferase LinB.

Structure (London, England : 1993) 17 (12)
PMID : 20004168  :   DOI  :   10.1016/j.str.2009.10.013    
Abstract >>
Lincosamides make up an important class of antibiotics used against a wide range of pathogens, including methicillin-resistant Staphylococcus aureus. Predictably, lincosamide-resistant microorganisms have emerged with antibiotic modification as one of their major resistance strategies. Inactivating enzymes LinB/A catalyze adenylylation of the drug; however, little is known about their mechanistic and structural properties. We determined two X-ray structures of LinB: ternary substrate- and binary product-bound complexes. Structural and kinetic characterization of LinB, mutagenesis, solvent isotope effect, and product inhibition studies are consistent with a mechanism involving direct in-line nucleotidyl transfer. The characterization of LinB enabled its classification as a member of a nucleotidyltransferase superfamily, along with nucleotide polymerases and aminoglycoside nucleotidyltransferases, and this relationship offers further support for the LinB mechanism. The LinB structure provides an evolutionary link to ancient nucleotide polymerases and suggests that, like protein kinases and acetyltransferases, these are proto-resistance elements from which drug resistance can evolve.
KeywordMeSH Terms
128. Sacco  E, Hugonnet  JE, Josseaume  N, Cremniter  J, Dubost  L, Marie  A, Patin  D, Blanot  D, Rice  LB, Mainardi  JL, Arthur  M,     ( 2010 )

Activation of the L,D-transpeptidation peptidoglycan cross-linking pathway by a metallo-D,D-carboxypeptidase in Enterococcus faecium.

Molecular microbiology 75 (4)
PMID : 20025663  :   DOI  :   10.1111/j.1365-2958.2009.07014.x    
Abstract >>
Bypass of the penicillin-binding proteins by an L,D-transpeptidase (Ldt(fm)) confers cross-resistance to beta-lactam and glycopeptide antibiotics in mutants of Enterococcus faecium selected in vitro. Ldt(fm) is produced by the parental strain D344S although it insignificantly contributes to peptidoglycan cross-linking as pentapeptide stems cannot be used as acyl donors by this enzyme. Here we show that production of the tetrapeptide substrate of Ldt(fm) is controlled by a two-component regulatory system (DdcRS) and a metallo-D,D-carboxypeptidase (DdcY). The locus was silent in D344S and its activation was due to amino acid substitutions in DdcS or DdcR that led to production of DdcY and hydrolysis of the C-terminal D-Ala residue of the cytoplasmic peptidoglycan precursor UDP-MurNAc-pentapeptide. The T(161)A and T(161)M substitutions affected a position of DdcS known to be essential for the phosphatase activity of related sensor kinases. Complete elimination of UDP-MurNAc-pentapeptide, which was required specifically for resistance to glycopeptides, involved substitutions in DdcY that increased the catalytic efficiency of the enzyme (E(127)K) and affected its interaction with the cell envelope (I(14)N). The ddc locus displays striking similarities with portions of the van vancomycin resistance gene clusters, suggesting possible routes of emergence of cross-resistance to glycopeptides and beta-lactams in natural conditions.
KeywordMeSH Terms
129. Qu  TT, Zhang  JL, Zhou  ZH, Wei  ZQ, Yu  YS, Chen  YG, Li  LJ,     ( 2009 )

Heteroresistance to teicoplanin in Enterococcus faecium harboring the vanA gene.

Journal of clinical microbiology 47 (12)
PMID : 19846656  :   DOI  :   10.1128/JCM.01802-09     PMC  :   PMC2786671    
Abstract >>
N/A
KeywordMeSH Terms
Drug Resistance, Multiple, Bacterial
130. Macwana  SR, Punj  S, Cooper  J, Schwenk  E, John  GH,     ( 2010 )

Identification and isolation of an azoreductase from Enterococcus faecium.

Current issues in molecular biology 12 (1)
PMID : 19741272  :  
Abstract >>
Azo dyes are commonly used in many commercial industries. Some of the azo dyes can produce carcinogenic compounds after being metabolized by azoreductase. Several human intestinal microbiota possess azoreductase activity which plays an important role in the toxicity and mutagenicity of these azo dye compounds. The acpD gene product (AzoEf1) responsible for the azoreductase activity of Enterococcus faecium, an intestinal bacterium, was heterologously expressed, purified and characterized. The protein sequence shares 67% identity with the azoreductase from Enterococcus faecalis, AzoA. Although AzoEf1 possesses many commonalities with AzoA, there are differences in coenzyme preference, residues associated with FMN binding, substrate specificity, and specific activity. AzoEf1 utilized both NADH and NADPH for the reduction of azo dyes, and it contains a leucyl residue at position 104 and threonyl residue at position 19 which differ from AzoA at the active site. Its specific activity was 5095 M/min/mg and its catalytic efficiency for Methyl red reduction was lower than AzoA.
KeywordMeSH Terms
131. Vermette  CJ, Russell  AH, Desai  AR, Hill  JE,     ( 2010 )

Resolution of phenotypically distinct strains of Enterococcus spp. in a complex microbial community using cpn60 universal target sequencing.

Microbial ecology 59 (1)
PMID : 19844647  :   DOI  :   10.1007/s00248-009-9601-1    
Abstract >>
Characterization of complex microbial communities is frequently based on the examination of polymerase chain reaction amplified sequences from a single phylogenetic marker, usually the 16S rRNA gene. However, this commonly used target often does not offer robust resolution of species or sub-species and is thus not a sufficiently informative target for understanding microbial population dynamics occurring at the strain level. We have used the cpn60 universal target sequence to characterize Enterococcus isolates from feces of growing pigs and have shown that sub-species groups, not detected using 16S rRNA sequences, can be resolved. Furthermore, groups resolved by cpn60-based phylogenetic analysis have distinct phenotypes. We report changes in the structure and function of Enterococcus communities in pig feces sampled from individual animals at three times, from suckling through to maturity. Enterococcus faecalis was largely replaced by Enterococcus hirae between suckling and 9 weeks of age, and a shift from one sub-species group of E. hirae to another was observed in all animals between 9 and 15 weeks. Conversely, E. faecalis strains remained consistent throughout the study period. Our results demonstrate that cpn60 sequences can be used to detect strain level changes in Enterococcus populations during succession in the fecal microbiota of growing pigs.
KeywordMeSH Terms
132. Young  PG, Walanj  R, Lakshmi  V, Byrnes  LJ, Metcalf  P, Baker  EN, Vakulenko  SB, Smith  CA,     ( 2009 )

The crystal structures of substrate and nucleotide complexes of Enterococcus faecium aminoglycoside-2''-phosphotransferase-IIa [APH(2'')-IIa] provide insights into substrate selectivity in the APH(2'') subfamily.

Journal of bacteriology 191 (13)
PMID : 19429619  :   DOI  :   10.1128/JB.00149-09     PMC  :   PMC2698469    
Abstract >>
Aminoglycoside-2''-phosphotransferase-IIa [APH(2'')-IIa] is one of a number of homologous bacterial enzymes responsible for the deactivation of the aminoglycoside family of antibiotics and is thus a major component in bacterial resistance to these compounds. APH(2'')-IIa produces resistance to several clinically important aminoglycosides (including kanamycin and gentamicin) in both gram-positive and gram-negative bacteria, most notably in Enterococcus species. We have determined the structures of two complexes of APH(2'')-IIa, the binary gentamicin complex and a ternary complex containing adenosine-5'-(beta,gamma-methylene)triphosphate (AMPPCP) and streptomycin. This is the first crystal structure of a member of the APH(2'') family of aminoglycoside phosphotransferases. The structure of the gentamicin-APH(2'')-IIa complex was solved by multiwavelength anomalous diffraction methods from a single selenomethionine-substituted crystal and was refined to a crystallographic R factor of 0.210 (R(free), 0.271) at a resolution of 2.5 A. The structure of the AMPPCP-streptomycin complex was solved by molecular replacement using the gentamicin-APH(2'')-IIa complex as the starting model. The enzyme has a two-domain structure with the substrate binding site located in a cleft in the C-terminal domain. Gentamicin binding is facilitated by a number of conserved acidic residues lining the binding cleft, with the A and B rings of the substrate forming the majority of the interactions. The inhibitor streptomycin, although binding in the same pocket as gentamicin, is orientated such that no potential phosphorylation sites are adjacent to the catalytic aspartate residue. The binding of gentamicin and streptomycin provides structural insights into the substrate selectivity of the APH(2'') subfamily of aminoglycoside phosphotransferases, specifically, the selectivity between the 4,6-disubstituted and the 4,5-disubstituted aminoglycosides.
KeywordMeSH Terms
133. Bugg  TD, Wright  GD, Dutka-Malen  S, Arthur  M, Courvalin  P, Walsh  CT,     ( 1991 )

Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA.

Biochemistry 30 (43)
PMID : 1931965  :   DOI  :   10.1021/bi00107a007    
Abstract >>
Vancomycin resistance in Enterococcus faecium BM4147 is mediated by vancomycin resistance proteins VanA and VanH. VanA is a D-alanine:D-alanine ligase of altered substrate specificity [Bugg, T. D. H., Dutka-Malen, S., Arthur, M., Courvalin, P., & Walsh, C. T. (1991) Biochemistry 30, 2017-2021], while the sequence of VanH is related to those of alpha-keto acid dehydrogenases [Arthur, M., Molinas, C., Dutka-Malen, S., & Courvalin, P. (1991) Gene (submitted)]. We report purification of VanH to homogeneity, characterization as a D-specific alpha-keto acid dehydrogenase, and comparison with D-lactate dehydrogenases from Leuconostoc mesenteroides and Lactobacillus leichmanii. VanA was found to catalyze ester bond formation between D-alanine and the D-hydroxy acid products of VanH, the best substrate being D-2-hydroxybutyrate (Km = 0.60 mM). The VanA product D-alanyl-D-2-hydroxybutyrate could then be incorporated into the UDPMurNAc-pentapeptide peptidoglycan precursor by D-Ala-D-Ala adding enzyme from Escherichia coli or by crude extract from E. faecium BM4147. The vancomycin binding constant of a synthetic modified peptidoglycan analogue N-acetyl-D-alanyl-D-2-hydroxybutyrate (Kd greater than 73 mM) was greater than 1000-fold higher than the binding constant for N-acetyl-D-alanyl-D-alanine (Kd = 54 microM), partly due to the disruption of a hydrogen bond in the vancomycin-target complex, thus providing a molecular rationale for high-level vancomycin resistance.
KeywordMeSH Terms
Carbon-Oxygen Ligases
134. Nishioka  T, Ogawa  W, Kuroda  T, Katsu  T, Tsuchiya  T,     ( 2009 )

Gene cloning and characterization of EfmA, a multidrug efflux pump, from Enterococcus faecium.

Biological & pharmaceutical bulletin 32 (3)
PMID : 19252300  :   DOI  :   10.1248/bpb.32.483    
Abstract >>
A DNA fragment responsible for resistance to antimicrobial agents was cloned from chromosomal DNA of Enterococcus faecium FN-1, a clinically isolated strain. Escherichia coli KAM32, a drug-hypersusceptible mutant, was used as a host for gene cloning. Cells of E. coli KAM32 harboring a recombinant plasmid (pTFM8) carrying the DNA fragment became resistant to fluoroquinolones, macrolides, ethidium bromide, 4',6-diamidino-2-phenylindole (DAPI) and tetraphenylphosphonium chloride (TPPCl). Three complete open reading frames (ORFs) were found in the DNA insert of pTFM8, and the deduced amino acid sequences of one of the ORFs showed high similarity to Mdt(A) from Lactococcus lactis. Mdt(A) is a multidrug efflux pump belonging to a major facilitator superfamily. We designated the ORF efmA. E. coli KAM32 cells harboring the efmA showed energy-dependent efflux of DAPI and TPP(+). We also observed norfloxacin/H(+) antiport due to EfmA. The mRNA expression of efmA was observed in E. faecium FN-1 grown without any exogenously added antimicrobial agents. Thus, we conclude that efmA is constitutively expressed under laboratory growth conditions and would contribute to intrinsic resistance against multiple antimicrobial agents in E. faecium FN-1.
KeywordMeSH Terms
Drug Resistance, Multiple, Bacterial
135. San Millan  A, Depardieu  F, Godreuil  S, Courvalin  P,     ( 2009 )

VanB-type Enterococcus faecium clinical isolate successively inducibly resistant to, dependent on, and constitutively resistant to vancomycin.

Antimicrobial agents and chemotherapy 53 (5)
PMID : 19273676  :   DOI  :   10.1128/AAC.00034-09     PMC  :   PMC2681533    
Abstract >>
Three Enterococcus faecium strains isolated successively from the same patient, vancomycin-resistant strain BM4659, vancomycin-dependent strain BM4660, and vancomycin-revertant strain BM4661, were indistinguishable by pulsed-field gel electrophoresis and harbored plasmid pIP846, which confers VanB-type resistance. The vancomycin dependence of strain BM4660 was due to mutation P(175)L, which suppressed the activity of the host Ddl D-Ala:D-Ala ligase. Reversion to resistance in strain BM4661 was due to a G-to-C transversion in the transcription terminator of the vanRS(B) operon that lowered the free energy of pairing from -13.08 to -6.65 kcal/mol, leading to low-level constitutive expression of the resistance genes from the P(RB) promoter, as indicated by analysis of peptidoglycan precursors and of VanX(B) D,D-dipeptidase activity. Transcription of the resistance genes, studied by Northern hybridization and reverse transcription, initiated from the P(YB) resistance promoter, was inducible in strains BM4659 and BM4660, whereas it started from the P(RB) regulatory promoter in strain BM4661, where it was superinducible. Strain BM4661 provides the first example of reversion to vancomycin resistance of a VanB-type dependent strain not due to a compensatory mutation in the ddl or vanS(B) gene. Instead, a mutation in the transcription terminator of the regulatory genes resulted in transcriptional readthrough of the resistance genes from the P(RB) promoter in the absence of vancomycin.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Vancomycin Resistance
136. Izquierdo  E, Cai  Y, Marchioni  E, Ennahar  S,     ( 2009 )

Genetic identification of the bacteriocins produced by Enterococcus faecium IT62 and evidence that bacteriocin 32 is identical to enterocin IT.

Antimicrobial agents and chemotherapy 53 (5)
PMID : 19273675  :   DOI  :   10.1128/AAC.00052-09     PMC  :   PMC2681497    
Abstract >>
Enterococcus faecium IT62, a strain isolated from ryegrass in Japan, produces three bacteriocins (enterocins L50A, L50B, and IT) that have been previously purified and the primary structures of which have been determined by amino acid sequencing (E. Izquierdo, A. Bednarczyk, C. Schaeffer, Y. Cai, E. Marchioni, A. Van Dorsselaer, and S. Ennahar, Antimicrob. Agents Chemother., 52:1917-1923, 2008). Genetic analysis showed that the bacteriocins of E. faecium IT62 are plasmid encoded, but with the structural genes specifying enterocin L50A and enterocin L50B being carried by a plasmid (pTAB1) that is separate from the one (pTIT1) carrying the structural gene of enterocin IT. Sequencing analysis of a 1,475-bp region from pTAB1 identified two consecutive open reading frames corresponding, with the exception of 2 bp, to the genes entL50A and entL50B, encoding EntL50A and EntL50B, respectively. Both bacteriocins are synthesized without N-terminal leader sequences. Genetic analysis of a sequenced 1,380-bp pTIT1 fragment showed that the genes entIT and entIM, encoding enterocin IT and its immunity protein, respectively, were both found in E. faecium VRE200 for bacteriocin 32. Enterocin IT, a 6,390-Da peptide made up of 54 amino acids, has been previously shown to be identical to the C-terminal part of bacteriocin 32, a 7,998-Da bacteriocin produced by E. faecium VRE200 whose structure was deduced from its structural gene (T. Inoue, H. Tomita, and Y. Ike, Antimicrob. Agents Chemother., 50:1202-1212, 2006). By combining the biochemical and genetic data on enterocin IT, it was concluded that bacteriocin 32 is in fact identical to enterocin IT, both being encoded by the same plasmid-borne gene, and that the N-terminal leader peptide for this bacteriocin is 35 amino acids long and not 19 amino acids long as previously reported.
KeywordMeSH Terms
Bacteriocins
137. Damborg  P, Top  J, Hendrickx  AP, Dawson  S, Willems  RJ, Guardabassi  L,     ( 2009 )

Dogs are a reservoir of ampicillin-resistant Enterococcus faecium lineages associated with human infections.

Applied and environmental microbiology 75 (8)
PMID : 19233953  :   DOI  :   10.1128/AEM.02035-08     PMC  :   PMC2675212    
Abstract >>
Ampicillin resistance is a marker for hospital-associated Enterococcus faecium. Feces from 208 dogs were selectively screened for the occurrence of ampicillin-resistant E. faecium (AREF). AREF was detected in 42 (23%) of 183 dogs screened in a cross-sectional study in the United Kingdom and in 19 (76%) of 25 dogs studied longitudinally in Denmark. AREF carriage was intermittent in all dogs studied longitudinally. Multilocus sequence typing of 63 canine AREF isolates revealed the presence of 13 distinct sequence types. Approximately 76% of the isolates belonged to hospital-adapted clonal complex 17 (CC17), including those of sequence types ST-78 and ST-192, which are widespread in European and Asian hospitals. Longitudinal screening of 18 healthy humans living in contact with 13 of the dogs under study resulted in the identification of a single, intermittent CC17 carrier. This person carried one of the sequence types (ST-78) recovered from his dog. Based on PCR and Southern hybridization analyses, the putative virulence gene cluster from orf903 to orf907 was widespread in canine AREF isolates (present in 97%), whereas orf2351 (present in 26% of isolates) and orf2430 (present in 31%) were strongly associated with CC17-related sequence types (P<0.05). Surprisingly, esp and hyl were not detected in any of the isolates. The antimicrobial resistance profiles of canine AREF isolates generally differed from those previously described for clinical human isolates. The results indicate that dogs are frequent carriers of CC17-related lineages and may play a role in the spread of this nosocomial pathogen. The distinctive virulence and antimicrobial resistance profiles observed among canine AREF isolates raise interesting questions about the origin and evolution of the strains causing human infections.
KeywordMeSH Terms
Ampicillin Resistance
Bacterial Typing Techniques
Disease Reservoirs
138. Depardieu  F, Foucault  ML, Bell  J, Dubouix  A, Guibert  M, Lavigne  JP, Levast  M, Courvalin  P,     ( 2009 )

New combinations of mutations in VanD-Type vancomycin-resistant Enterococcus faecium, Enterococcus faecalis, and Enterococcus avium strains.

Antimicrobial agents and chemotherapy 53 (5)
PMID : 19258279  :   DOI  :   10.1128/AAC.01348-08     PMC  :   PMC2681503    
Abstract >>
We studied the clinical isolates Enterococcus faecium NEF1, resistant to high levels of vancomycin (MIC, 512 microg/ml) and teicoplanin (MIC, 64 microg/ml); Enterococcus faecium BM4653 and BM4656 and Enterococcus avium BM4655, resistant to moderate levels of vancomycin (MIC, 32 microg/ml) and to low levels of teicoplanin (MIC, 4 microg/ml); and Enterococcus faecalis BM4654, moderately resistant to vancomycin (MIC, 16 microg/ml) but susceptible to teicoplanin (MIC, 0.5 microg/ml). The strains were distinct, were constitutively resistant via the synthesis of peptidoglycan precursors ending in D-alanyl-D-lactate, and harbored a chromosomal vanD gene cluster that was not transferable. New mutations were found in conserved domains of VanS(D): at T(170)I near the phosphorylation site in NEF1, at V(67)A at the membrane surface in BM4653, at G(340)S in the G2 ATP-binding domain in BM4655, in the F domain in BM4656 (a 6-bp insertion), and in the G1 and G2 domains of BM4654 (three mutations). The mutations resulted in constitutivity, presumably through the loss of the phosphatase activity of the sensor. The chromosomal Ddl D-Ala:D-Ala ligase had an IS19 copy in NEF1, a mutation in the serine (S(185)F) or near the arginine (T(289)P) involved in D-Ala1 binding in BM4653 or BM4655, respectively, and a mutation next to the lysine (P(180)S) involved in D-Ala2 binding in BM4654, leading to the production of an impaired enzyme. In BM4653 vanY(D), a new insertion sequence, ISEfa9, belonging to the IS3 family, resulted in the absence of D,D-carboxypeptidase activity. Strain BM4656 had a functional D-Ala:D-Ala ligase, associated with high levels of both VanX(D) and VanY(D) activities, and is the first example of a VanD-type strain with a functional Ddl enzyme. Study of these five clinical isolates, displaying various assortments of mutations, confirms that all VanD-type strains isolated so far have undergone mutations in the vanS(D) or vanR(D) gene, leading to constitutive resistance, but that the Ddl host ligase is not always impaired. Based on sequence differences, the vanD gene clusters could be assigned to two subtypes: vanD-1 and vanD-4.
KeywordMeSH Terms
Mutation
139. Nam  KH, Kim  KH, Kim  EE, Hwang  KY,     ( 2009 )

Crystal structure of an EfPDF complex with Met-Ala-Ser based on crystallographic packing.

Biochemical and biophysical research communications 381 (4)
PMID : 19249287  :   DOI  :   10.1016/j.bbrc.2009.02.113    
Abstract >>
PDF (peptide deformylase) plays a critical role in the production of mature proteins by removing the N-formyl polypeptide of nascent proteins in the prokaryote cell system. This protein is essential for bacterial growth, making it an attractive target for the design of new antibiotics. Accordingly, PDF has been evaluated as a drug target; however, architectural mechanism studies of PDF have not yet fully elucidated its molecular function. We recently reported the crystal structure of PDF produced by Enterococcus faecium [K.H. Nam, J.I. Ham, A. Priyadarshi, E.E. Kim, N. Chung, K.Y. Hwang, "Insight into the antibacterial drug design and architectural mechanism of peptide recognition from the E. faecium peptide deformylase structure", Proteins 74 (2009) 261-265]. Here, we present the crystal structure of the EfPDF complex with MAS (Met-Ser-Ala), thereby not only delineating the architectural mechanism for the recognition of mimic-peptides by N-terminal cleaved expression peptide, but also suggesting possible targets for rational design of antibacterial drugs. In addition to their implications for drug design, these structural studies will facilitate elucidation of the architectural mechanism responsible for the peptide recognition of PDF.
KeywordMeSH Terms
140. Okada  H, Yohda  M, Giga-Hama  Y, Ueno  Y, Ohdo  S, Kumagai  H,     ( 1991 )

Distribution and purification of aspartate racemase in lactic acid bacteria.

Biochimica et biophysica acta 1078 (3)
PMID : 1907199  :   DOI  :   10.1016/0167-4838(91)90159-w    
Abstract >>
The distribution of aspartate racemase (EC 5.1.1.13) in various kinds of bacteria demonstrated that the enzyme occurs in lactic acid bacteria, such as Streptococcus species and Lactobacillus species. The enzyme from Streptococcus thermophilus IAM10064 was more thermostable than that from Streptococcus lactis IAM1198 which contained the enzyme most abundantly among the lactic acid bacteria we examined here. We purified the enzyme about 3400-fold to homogeneity from cell-free extract of S. thermophilus, which is composed of two identical subunits with a molecular weight of 28,000 as a homodimer. The enzyme utilizes specifically aspartate as a substrate, but not alanine and glutamate. Maximal reaction velocity was observed at 37 degrees C and around pH 8.0. The sequence of the NH2-terminal amino acids of the enzyme was determined to be Met-Glu-Asn-Phe-Phe-Ser-Ile-Leu-Gly-XXX-Met-Gly-Thr-Met-Ala-Thr-Glu-Ser- Phe-.
KeywordMeSH Terms
141. Arthur  M, Molinas  C, Dutka-Malen  S, Courvalin  P,     ( 1991 )

Structural relationship between the vancomycin resistance protein VanH and 2-hydroxycarboxylic acid dehydrogenases.

Gene 103 (1)
PMID : 1908809  :   DOI  :   10.1016/0378-1119(91)90405-z    
Abstract >>
Sequencing upstream from the vanA gene of enterococcal plasmid pIP816 that confers Vm resistance revealed the presence of an ORF which could code for a protein of 322 aa designated VanH. Extensive aa similarity was detected between VanH and 2-hydroxycarboxylic acid dehydrogenase. We discuss possible roles for VanH in the synthesis of a novel type of peptidoglycan precursors with lower affinity for Vm.
KeywordMeSH Terms
142. Valdezate  S, Labayru  C, Navarro  A, Mantecón  MA, Ortega  M, Coque  TM, García  M, Saéz-Nieto  JA,     ( 2009 )

Large clonal outbreak of multidrug-resistant CC17 ST17 Enterococcus faecium containing Tn5382 in a Spanish hospital.

The Journal of antimicrobial chemotherapy 63 (1)
PMID : 19001448  :   DOI  :   10.1093/jac/dkn449    
Abstract >>
A large clonal outbreak of multidrug-resistant CC17 ST17 Enterococcus faecium containing Tn5382 in a hospital in the north of Spain is described. We characterized vancomycin-resistant E. faecium isolates from 10 infected and 40 colonized inpatients from a single hospital by PFGE, multiple-locus variable-number tandem-repeat analysis (MLVA) and multilocus sequence typing (MLST). Genes encoding antibiotic resistance (ampicillin, aminoglycosides, macrolides, quinupristin/dalfopristin, quinolones, tetracycline) and putative virulence traits were analysed. All isolates showed highly similar PFGE profiles and were assigned to the type MT1 by MLVA and to ST17 (CC17) by MLST. The Tn5382 type identified in all isolates was linked to pbp5 and contained a 5 bp deletion and 10 point mutations within the intergenic vanS(B)-vanY(B) region. Other resistance genes identified were erm(B), mef(E), tet(M), ant(6')-Ia, aph(3')-IIIa and aac(6')-Ie-aph(2'')-Ia. All isolates carried the unexpressed tet(M) gene. The high level of ciprofloxacin resistance was attributable to the first described Gly-61 and Ile-80 mutations in ParC and the Tyr-83 or Arg-83 mutations in GyrA. All isolates contained esp. The presence of hyl was variable. A large clonal outbreak caused by multidrug-resistant CC17 E. faecium containing pbp5-Tn5382 is described. The persistence of this clone, which has been recovered from both hospital and community settings since 2005, and the possibility of transferring this Tn5382 to other epidemic ampicillin-resistant clonal types currently circulating in Spain might contribute to increasing the prevalence of vancomycin-resistant enterococci in our area. This study constitutes the first description of mef(E) in E. faecium.
KeywordMeSH Terms
DNA Transposable Elements
Drug Resistance, Multiple, Bacterial
143. Sletvold  H, Johnsen  PJ, Hamre  I, Simonsen  GS, Sundsfjord  A, Nielsen  KM,     ( 2008 )

Complete sequence of Enterococcus faecium pVEF3 and the detection of an omega-epsilon-zeta toxin-antitoxin module and an ABC transporter.

Plasmid 60 (1)
PMID : 18511120  :   DOI  :   10.1016/j.plasmid.2008.04.002    
Abstract >>
Glycopeptide resistant Enterococcus faecium (GREF) persists on Norwegian poultry farms despite the ban on the growth promoter avoparcin. The biological basis for long-term persistence of avoparcin resistance is not fully understood. This study presents the complete DNA sequence of the E. faecium R-plasmid pVEF3 and functional studies of some plasmid-encoded traits (a toxin-antitoxin (TA) system and an ABC transporter) that may be of importance for plasmid persistence. The pVEF3 (63.1 kbp), isolated from an E. faecium strain of poultry origin sampled in Norway in 1999, has 71 coding sequences including the vanA avoparcin/vancomycin resistance encoding gene cluster. pVEF3 encodes the TA system omega-epsilon-zeta, and plasmid stability tests and transcription analysis show that omega-epsilon-zeta is functional in Enterococcus faecalis OGIX, although with decreasing effect over time. The predicted ABC transporter was not found to confer reduced susceptibility to any of the 28 substances tested. The TA system identified in the pVEF-type plasmids may contribute to vanA plasmid persistence on Norwegian poultry farms. However, size and compositional heterogeneity among E. faecium vanA plasmids suggest that additional plasmid maintenance systems in combination with host specific factors and frequent horizontal gene transfer and rearrangement causes the observed plasmid composition and distribution patterns.
KeywordMeSH Terms
144. Hendrickx  AP, Bonten  MJ, van Luit-Asbroek  M, Schapendonk  CM, Kragten  AH, Willems  RJ,     ( 2008 )

Expression of two distinct types of pili by a hospital-acquired Enterococcus faecium isolate.

Microbiology (Reading, England) 154 (Pt 10)
PMID : 18832326  :   DOI  :   10.1099/mic.0.2008/020891-0    
Abstract >>
Surface filamentous structures designated pili, and implicated in virulence, have been found on the surfaces of several Gram-positive pathogens. This work describes the conditional expression of two phenotypically distinct pilus-like structures, designated PilA and PilB, on the surface of a hospital-adapted Enterococcus faecium bloodstream isolate. E. faecium is an emerging Gram-positive opportunistic pathogen that can cause severe disease, particularly in immunocompromised patients. Expression of PilA- and PilB-type pili was analysed during different phases of growth in broth culture. During growth, PilA and PilB pilin subunits were expressed around the cross-wall in early-exponential-phase cells. Polymerization and migration of short PilB-type pili towards the poles occurred in cells from the exponential phase and long polymerized pili were expressed at the poles of cells grown to stationary phase. In contrast, PilA-type pili were not expressed in broth culture, but only when cells were grown on solid media. Furthermore, surface expression of the PilA- and PilB-type pili was regulated in a temperature-dependent manner, as polymerization of two distinct types of pili at the surface only occurred when cells were grown at 37 degrees C; no pili were observed on cells grown at 21 degrees C. Hospital-aquired E. faecium isolates were specifically enriched in pilin gene clusters, suggesting that conditional expression of pili may contribute to E. faecium pathogenesis.
KeywordMeSH Terms
145. Nam  KH, Ham  JI, Priyadarshi  A, Kim  EE, Chung  N, Hwang  KY,     ( 2009 )

Insight into the antibacterial drug design and architectural mechanism of peptide recognition from the E. faecium peptide deformylase structure.

Proteins 74 (1)
PMID : 18831047  :   DOI  :   10.1002/prot.22257    
Abstract >>
N/A
KeywordMeSH Terms
146. Garcia-Migura  L, Hasman  H, Jensen  LB,     ( 2009 )

Presence of pRI1: a small cryptic mobilizable plasmid isolated from Enterococcus faecium of human and animal origin.

Current microbiology 58 (2)
PMID : 18830745  :   DOI  :   10.1007/s00284-008-9266-x    
Abstract >>
This study focused on the molecular characterization of a small cryptic, mobilizable plasmid (6038 bp) sequenced from an E. faecium 9631160-1 of poultry origin. Sequence analysis of pRI1 revealed seven open reading frames. pRI1 contained an IS 100% identical to ISEfa4. This insertion element disrupted a putative mobilization gene (mobA) which presented 99% similarity to the one described in plasmid pJS42 (NC_010291). pRI1 harbored a cluster of four coding sequences which exhibited a homology to those described in contig 658 (from nucleotide 8940 to nt 10515) of E. faecium DO. In addition, a rep almost identical to the repA from the pEFNP1 and pKQ10 plasmids from E. faecium was also identified. Presence of the pRI1 replication initiation gene (rep) was analyzed in a panel of 159 E. faecium isolates of human and animal origin from different European countries, of which 60 tested positive for the presence of pRI1-rep. Conjugation experiments verified transfer of the pRI1 together with conjugative plasmids harboring resistance to vancomycin and streptogramin. The presence of pRI1 in enterococcal isolates geographically separated and from different origin demonstrates the ability of enterococci to acquire and transfer mobile genetic elements, emphasizing the need for further studies to reveal the meaning and role that these cryptic plasmids play in nature.
KeywordMeSH Terms
Gene Transfer, Horizontal
147. Sung  K, Khan  SA, Nawaz  MS,     ( 2008 )

Genetic diversity of Tn1546-like elements in clinical isolates of vancomycin-resistant enterococci.

International journal of antimicrobial agents 31 (6)
PMID : 18462926  :   DOI  :   10.1016/j.ijantimicag.2008.01.030    
Abstract >>
We have investigated the genetic diversity of Tn1546 among 17 vancomycin-resistant enterococci (VRE) isolates of Enterococcus faecium. Most of these multidrug-resistant strains harboured plasmids of 2 kb to >300 kb in size. The vancomycin resistance marker vanA was located on both the plasmid and the chromosomal DNA. VRE isolates 18 and 22 failed to amplify the orf1-IR(R) and orf2-IR(R) but contained the orf1 and orf2. VRE3 failed to amplify the orf1, orf2, vanR and vanS, but still yielded a larger than expected (4.4 kb vs. 2.3 kb) vanSH amplicon. VRE9, 10, 21 and 22 also yielded larger (5.5 kb) vanSH amplicons; all others yielded 4.0 kb vanSH amplicons. Sequence analysis of the vanSH amplicons from VRE9, 10, 21 and 22 revealed the presence of IS1251 between the vanS and vanH genes in these isolates. The observed vanSH amplicon from VRE3 contained orf31, orf30 and orf29 of the plasmid pRUM followed by the vanHAXYZ region of Tn1546. Translocation of Tn1546 to a pRUM-like plasmid in VRE3 resulted in the loss of its orf1, orf2, vanR and vanS elements and a loss of the orf32 of pRUM, leading to a unique structural arrangement of vanA elements that is hitherto unknown.
KeywordMeSH Terms
148. Svetoch  EA, Eruslanov  BV, Perelygin  VV, Mitsevich  EV, Mitsevich  IP, Borzenkov  VN, Levchuk  VP, Svetoch  OE, Kovalev  YN, Stepanshin  YG, Siragusa  GR, Seal  BS, Stern  NJ,     ( 2008 )

Diverse antimicrobial killing by Enterococcus faecium E 50-52 bacteriocin.

Journal of agricultural and food chemistry 56 (6)
PMID : 18293921  :   DOI  :   10.1021/jf073284g    
Abstract >>
An effective bacteriocin was identified and characterized. Lactic acid bacteria were screened against Campylobacter jejuni. One bacteriocin producer, Enterococcus faecium (NRRL B-30746), was studied. The isolate was grown, and the bacteriocin was purified to single-band homogeneity. Biochemical traits indicated that the peptide was a Class IIa bacteriocin, and it was named E 50-52. The bacteriocin had a molecular weight of 3339.7 and an isoelectric point of 8.0. The minimal inhibitory concentrations of E 50-52 against C. jejuni, Yersinia spp., Salmonella spp., Escherichia coli O157:H7, Shigella dysenteriae, Morganella morganii, Staphylococcus spp., and Listeria spp. ranged from 0.025 to 32 microg/mL. In therapeutic broiler trials, oral treatment with E 50-52 reduced both C. jejuni and Salmonella enteritidis by more than 100,000-fold in the ceca, and systemic S. enteritidis was reduced in the liver and spleen. The wide range of antibacterial activity of bacteriocin E 50-52 against pathogens provides a promising alternative to antibiotics.
KeywordMeSH Terms
149. Chan  YY, Abd Nasir  MH, Yahaya  MA, Salleh  NM, Md Dan  AD, Musa  AM, Ravichandran  M,     ( 2008 )

Low prevalence of vancomycin- and bifunctional aminoglycoside-resistant enterococci isolated from poultry farms in Malaysia.

International journal of food microbiology 122 (1��2��)
PMID : 18187222  :   DOI  :   10.1016/j.ijfoodmicro.2007.11.063    
Abstract >>
A total of 225 samples from poultry farms and the surrounding environment were screened for vancomycin-resistant enterococci (VRE) and bifunctional aminoglycoside-resistant enterococci using conventional microbiological tests and a nanoplex polymerase chain reaction (PCR) assay. Three (1.3%) of the samples were found to contain vancomycin-resistant isolates (MIC>256 microg/mL) that had a vanA genotype. The three vanA positive VRE isolates were identified as different species. Only one isolate (Enterococcus faecium F 4/13_54) was sensitive to teicoplanin (MIC<0. 12-0.35 microg/mL); the other two VRE (E. faecalis A 21_35 and E. gallinarum F 5/10_1) were resistant to teicoplanin (MIC 3.6-->16 microg/mL). The vanC genotype was observed in nine (4%) of the samples collected. High-level gentamicin-resistant (HLGR) enterococci (with MIC ranging between 100 and 500 microg/mL) were detected in 44 samples. However, only 40 of these were found to possess the aac(6')-aph(2'') gene. The overall prevalence of VRE among the samples from the poultry farms and environment was 5.3%, but the prevalence of the clinically significant vanA VRE was 1.3%, and the prevalence of bifunctional aminoglycoside-resistant enterococci was slightly higher, at 19.5%.
KeywordMeSH Terms
Vancomycin Resistance
150. Derome  A, Hoischen  C, Bussiek  M, Grady  R, Adamczyk  M, Kedzierska  B, Diekmann  S, Barill?  D, Hayes  F,     ( 2008 )

Centromere anatomy in the multidrug-resistant pathogen Enterococcus faecium.

Proceedings of the National Academy of Sciences of the United States of America 105 (6)
PMID : 18245388  :   DOI  :   10.1073/pnas.0704681105     PMC  :   PMC2538891    
Abstract >>
Multidrug-resistant variants of the opportunistic human pathogen Enterococcus have recently emerged as leading agents of nosocomial infection. The acquisition of plasmid-borne resistance genes is a driving force in antibiotic-resistance evolution in enterococci. The segregation locus of a high-level gentamicin-resistance plasmid, pGENT, in Enterococcus faecium was identified and dissected. This locus includes overlapping genes encoding PrgP, a member of the ParA superfamily of segregation proteins, and PrgO, a site-specific DNA binding homodimer that recognizes the cenE centromere upstream of prgPO. The centromere has a distinctive organization comprising three subsites, CESII separates CESI and CESIII, each of which harbors seven TATA boxes spaced by half-helical turns. PrgO independently binds both CESI and CESIII, but with different affinities. The topography of the complex was probed by atomic force microscopy, revealing discrete PrgO foci positioned asymmetrically at the CESI and CESIII subsites. Bending analysis demonstrated that cenE is intrinsically curved. The organization of the cenE site and of certain other plasmid centromeres mirrors that of yeast centromeres, which may reflect a common architectural requirement during assembly of the mitotic apparatus in yeast and bacteria. Moreover, segregation modules homologous to that of pGENT are widely disseminated on vancomycin and other resistance plasmids in enterococci. An improved understanding of segrosome assembly may highlight new interventions geared toward combating antibiotic resistance in these insidious pathogens.
KeywordMeSH Terms
Centromere
151. Qu  TT, Chen  YG, Yu  YS, Lv  HX, Dong  XQ, Xiao  Z, Gu  HQ, Li  LJ,     ( 2007 )

Molecular characterization of vancomycin-resistant enterococci in Hangzhou, China.

The Journal of antimicrobial chemotherapy 60 (6)
PMID : 17951264  :   DOI  :   10.1093/jac/dkm399    
Abstract >>
N/A
KeywordMeSH Terms
DNA Transposable Elements
Vancomycin Resistance
152. Li  S, Zhang  Z, Mi  ZH,     ( 2007 )

Vancomycin-resistant enterococci in a Chinese hospital.

Current microbiology 55 (2)
PMID : 17619103  :   DOI  :   10.1007/s00284-005-0484-1    
Abstract >>
In investigating the prevalence of vancomycin-resistant enterococci (VREs) in a hospital in China, 338 enterococci were detected by using multiplex polymerase chain reaction. Eleven VREs were found, including one VanA E. faecalis, one VanB E. faecium, three VanB E. faecalis, five VanC(1) E. gallinarum, and one VanC(2) E. flavescens. VITEK 2, microbroth dilution, and E-test were used to determine the susceptibilities of the VREs to certain antimicrobial agents; multiple-drug resistance of the 11 VREs was distinct. Compared with phenotypic methods, multiplex PCR rapidly detected 11 VREs and provided van genotype information. This is the first study that sequenced van genes of VRE isolates in China to date and found type VanB in China. Because of the relatively low isolating rate, early detection of VRE is vital.
KeywordMeSH Terms
153. Mareková  M, Lauková  A, Skaugen  M, Nes  I,     ( 2007 )

Isolation and characterization of a new bacteriocin, termed enterocin M, produced by environmental isolate Enterococcus faecium AL41.

Journal of industrial microbiology & biotechnology 34 (8)
PMID : 17551760  :   DOI  :   10.1007/s10295-007-0226-4    
Abstract >>
The new bacteriocin, termed enterocin M, produced by Enterococcus faecium AL 41 showed a wide spectrum of inhibitory activity against the indicator organisms from different sources. It was purified by (NH4)2SO4 precipitation, cation-exchange chromatography and reverse phase chromatography (FPLC). The purified peptide was sequenced by N-terminal amino acid Edman degradation and a mass spectrometry analysis was performed. By combining the data obtained from amino acid sequence (39 N-terminal amino acid residues was determined) and the molecular weight (determined to be 4628 Da) it was concluded that the purified enterocin M is a new bacteriocin, which is very similar to enterocin P. However, its molecular weight is different from enterocin P (4701.25). Of the first 39 N-terminal residues of enterocin M, valine was found in position 20 and a lysine in position 35, while enterocin P has tryptophane residues in these positions.
KeywordMeSH Terms
154. Fürst  P, Mösch  HU, Solioz  M,     ( 1989 )

A protein of unusual composition from Enterococcus faecium.

Nucleic acids research 17 (16)
PMID : 2780297  :   DOI  :   10.1093/nar/17.16.6724     PMC  :   PMC318365    
Abstract >>
N/A
KeywordMeSH Terms
Genes
Genes, Bacterial
155. Pinholt  M, Gumpert  H, Bayliss  S, Nielsen  JB, Vorobieva  V, Pedersen  M, Feil  E, Worning  P, Westh  H,     ( 2017 )

Genomic analysis of 495 vancomycin-resistant Enterococcus faecium reveals broad dissemination of a vanA plasmid in more than 19 clones from Copenhagen, Denmark.

The Journal of antimicrobial chemotherapy 72 (1)
PMID : 27605596  :   DOI  :   10.1093/jac/dkw360    
Abstract >>
From 2012 to 2014, there has been a huge increase in vancomycin-resistant (vanA) Enterococcus faecium (VREfm) in Copenhagen, Denmark, with 602 patients infected or colonized with VREfm in 2014 compared with just 22 in 2012. The objective of this study was to describe the genetic epidemiology of VREfm to assess the contribution of clonal spread and horizontal transfer of the vanA transposon (Tn1546) and plasmid in the dissemination of VREfm in hospitals. VREfm from Copenhagen, Denmark (2012-14) were whole-genome sequenced. The clonal structure was determined and the structure of Tn1546-like transposons was characterized. One VREfm isolate belonging to the largest clonal group was sequenced using long-read technology to close a 37 kb vanA plasmid. Phylogeny revealed a polyclonal structure where 495 VREfm isolates were divided into 13 main groups and 7 small groups. The majority of the isolates were located in three groups (n = 44, 100 and 218) and clonal spread of VREfm between wards and hospitals was identified. Five Tn1546-like transposon types were identified. A dominant truncated transposon (type 4, 92%) was spread across all but one VREfm group. The closed vanA plasmid was highly covered by reads from isolates containing the type 4 transposon. This study suggests that it was the dissemination of the type 4 Tn1546-like transposon and plasmid via horizontal transfer to multiple populations of E. faecium, followed by clonal spread of new VREfm clones, that contributed to the increase in and diversity of VREfm in Danish hospitals.
KeywordMeSH Terms
Genetic Variation
156. Sinel  C, Cosquer  T, Auzou  M, Goux  D, Giard  JC, Cattoir  V,     ( 2016 )

Sequential steps of daptomycin resistance in Enterococcus faecium and reversion to hypersusceptibility through IS-mediated inactivation of the liaFSR operon.

The Journal of antimicrobial chemotherapy 71 (10)
PMID : 27353469  :   DOI  :   10.1093/jac/dkw229    
Abstract >>
To improve understanding of mechanisms of daptomycin resistance and to dissect the genetic basis of reversion to daptomycin hypersusceptibility in Enterococcus faecium. Daptomycin-resistant mutants (Mut4, Mut8, Mut16, Mut32, Mut64 and Mut128 with MICs from 4 to 128 mg/L) were obtained in vitro from E. faecium strain Aus0004 (MIC at 2 mg/L). The entire genome sequences of Mut64 and Mut128 were determined as well as those of liaFSR and cls genes for other mutants and corresponding revertants (named Rev4 to Rev128). The study of daptomycin resistance stability was performed without any selective pressure. The expression of liaF, liaS and liaR genes was quantified by quantitative RT-PCR. By comparative genomic analysis, substitutions Asn13Ser in cls and Gly92Asp in liaS were identified in Mut64 and Mut128. Only the liaS mutation was found in Mut16 and Mut32 while Mut4 and Mut8 were devoid of any mutation. After 15 days, all mutants except Mut4 reverted to daptomycin hypersusceptibility (MICs from 0.12 to 0.25 mg/L). In all revertants (except Rev4 and Rev8), an IS was found in the liaFSR operon with a dramatic decrease of its expression: IS66 in the promoter region of liaF (Rev16 and Rev64), IS30 in liaR (Rev32) and IS982 in liaF (Rev128). We demonstrated the stepwise and sequential acquisition of mutations in liaS and in cls leading to daptomycin resistance in E. faecium, and the instability of daptomycin resistance as well as the role of liaFSR inactivation in reversion to daptomycin hypersusceptibility.
KeywordMeSH Terms
DNA Transposable Elements
Operon
157. Boyd  DA, Lalancette  C, Lévesque  S, Golding  GR,     ( 2016 )

Characterization of a genomic island harbouring a new vanD allele from Enterococcus faecium N15-508 isolated in Canada.

The Journal of antimicrobial chemotherapy 71 (7)
PMID : 27084917  :   DOI  :   10.1093/jac/dkw063    
Abstract >>
N/A
KeywordMeSH Terms
Alleles
Genomic Islands
158. Rizzotti  L, Rossi  F, Torriani  S,     ( 2016 )

Biocide and antibiotic resistance of Enterococcus faecalis and Enterococcus faecium isolated from the swine meat chain.

Food microbiology 60 (N/A)
PMID : 27554158  :   DOI  :   10.1016/j.fm.2016.07.009    
Abstract >>
In this study nine strains of Enterococcus faecalis and 12 strains of Enterococcus faecium, isolated from different sample types in the swine meat chain and previously characterized for the presence of antibiotic resistance genes, were examined for phenotypic tolerance to seven biocides (chlorexidine, benzalkonium chloride, triclosan, sodium hypochlorite, 2-propanol, formaldehyde and hydrogen peroxide) and resistance to nine antibiotics (ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol). Moreover, the presence of efflux system encoding genes qacA/B, qacC, qacE, qacE�G1, emeA, and stress response genes, sigV and gsp65, involved in the tolerance to biocides, was analysed. Most strains were not tolerant to the biocides, but showed minimum inhibitory concentrations (MICs) higher than the recommended cut-off values for all the antibiotics tested, except for vancomycin and chloramphenicol. Only weak correlations, if any, were found between biocide and antibiotic resistance data. One E. faecalis strain was tolerant to triclosan and one E. faecium strain, with higher tolerance to chlorexidine than the other strains tested, was found to carry a qacA/B gene. Our results indicated that phenotypic resistance to antibiotics is very frequent in enterococcal isolates from the swine meat chain, but phenotypic tolerance to biocides is not common. On the other hand, the gene qacA/B was found for the first time in the species E. faecium, an indication of the necessity to adopt measures suitable to control the spread of biocide resistance determinants among enterococci.
KeywordMeSH Terms
Biocide tolerance
Enterococci
Stress response genes
qac genes
Drug Resistance, Multiple, Bacterial
159. Molale  LG, Bezuidenhout  CC,     ( 2016 )

Virulence determinants and production of extracellular enzymes in Enterococcus spp. from surface water sources.

Water science and technology : a journal of the International Association on Water Pollution Research 73 (8)
PMID : 27120635  :   DOI  :   10.2166/wst.2016.015    
Abstract >>
Virulence factors in Enterococcus may be indicative of potential pathogenicity. The aim of this study was to determine the relationship between the presence of clinically relevant virulence genes, in Enterococcus spp. from environmental water, and their in vitro expression. One hundred and twenty-four Enterococcus isolates (seven species), from five surface water systems in the North West Province, South Africa, were screened for the presence of asa1, cylA, esp, gelE and hyl using polymerase chain reaction. The expression of cylA, hyl and gelE was determined by phenotypic assessments. Sixty-five percent of the isolates were positive for one virulence gene and 13% for two or more. Most frequently detected genes were gelE (32%) and cylA (28%). Enterococcal surface protein was absent in all isolates screened. The presence of virulence genes was correlated with their extracellular enzyme production. The results show that a large percentage of these environmental Enterococcus spp. possess virulence factors that could be expressed in vitro. This is a cause for concern and could have implications for individuals using this water for recreational and cultural purposes. Further investigation is required into the sources of these potential pathogenic Enterococcus isolates and measures to minimize their presence in water sources.
KeywordMeSH Terms
Water Microbiology
160. Guzmán Prieto  AM, Urbanus  RT, Zhang  X, Bierschenk  D, Koekman  CA, van Luit-Asbroek  M, Ouwerkerk  JP, Pape  M, Paganelli  FL, Wobser  D, Huebner  J, Hendrickx  AP, Bonten  MJ, Willems  RJ, van Schaik  W,     ( 2015 )

The N-terminal domain of the thermo-regulated surface protein PrpA of Enterococcus faecium binds to fibrinogen, fibronectin and platelets.

Scientific reports 5 (N/A)
PMID : 26675410  :   DOI  :   10.1038/srep18255     PMC  :   PMC4682149    
Abstract >>
Enterococcus faecium is a commensal of the mammalian gastrointestinal tract, but is also found in non-enteric environments where it can grow between 10 �XC and 45 �XC. E. faecium has recently emerged as a multi-drug resistant nosocomial pathogen. We hypothesized that genes involved in the colonization and infection of mammals exhibit temperature-regulated expression control and we therefore performed a transcriptome analysis of the clinical isolate E. faecium E1162, during mid-exponential growth at 25 �XC and 37 �XC. One of the genes that exhibited differential expression between 25 �XC and 37 �XC, was predicted to encode a peptidoglycan-anchored surface protein. The N-terminal domain of this protein is unique to E. faecium and closely related enterococci, while the C-terminal domain is homologous to the Streptococcus agalactiae surface protein BibA. This region of the protein contains proline-rich repeats, leading us to name the protein PrpA for proline-rich protein A. We found that PrpA is a surface-exposed protein which is most abundant during exponential growth at 37 �XC in E. faecium E1162. The heterologously expressed and purified N-terminal domain of PrpA was able to bind to the extracellular matrix proteins fibrinogen and fibronectin. In addition, the N-terminal domain of PrpA interacted with both non-activated and activated platelets.
KeywordMeSH Terms
161. Brenciani  A, Morroni  G, Vincenzi  C, Manso  E, Mingoia  M, Giovanetti  E, Varaldo  PE,     ( 2016 )

Detection in Italy of two clinical Enterococcus faecium isolates carrying both the oxazolidinone and phenicol resistance gene optrA and a silent multiresistance gene cfr.

The Journal of antimicrobial chemotherapy 71 (4)
PMID : 26702919  :   DOI  :   10.1093/jac/dkv438    
Abstract >>
N/A
KeywordMeSH Terms
Drug Resistance, Multiple, Bacterial
Genes, Bacterial
162. Baettig  OM, Shi  K, Yachnin  BJ, Burk  DL, Berghuis  AM,     ( 2016 )

Comprehensive characterization of ligand-induced plasticity changes in a dimeric enzyme.

The FEBS journal 283 (16)
PMID : 27333541  :   DOI  :   10.1111/febs.13788     PMC  :   PMC5053276    
Abstract >>
An enzyme's inherent structural plasticity is frequently associated with substrate binding, yet detailed structural characterization of flexible proteins remains challenging. This study employs complementary biophysical methods to characterize the partially unfolded structure of substrate-free AAC(6')-Ii, an N-acetyltransferase of the GCN5-related N-acetyltransferase (GNAT) superfamily implicated in conferring broad-spectrum aminoglycoside resistance on Enterococcus faecium. The X-ray crystal structure of AAC(6')-Ii is analyzed to identify relative motions of the structural elements that constitute the dimeric enzyme. Comparison with the previously elucidated crystal structure of AAC(6')-Ii with acetyl coenzyme A (AcCoA) reveals conformational changes that occur upon substrate binding. Our understanding of the enzyme's structural plasticity is further refined with small-angle X-ray scattering and circular dichroism analyses, which together reveal how flexible structural elements impact dimerization and substrate binding. These results clarify the extent of unfolding that AAC(6')-Ii undergoes in the absence of AcCoA and provide a structural connection to previously observed allosteric cooperativity of this enzyme. Structural data are available in the PDB database under the accession number 5E96.
KeywordMeSH Terms
antibiotic resistance
ligand-induced plasticity
protein flexibility
allosteric cooperativity
aminoglycoside 6′-N-acetyltransferase type-Ii
163. Perez  RH, Ishibashi  N, Inoue  T, Himeno  K, Masuda  Y, Sawa  N, Zendo  T, Wilaipun  P, Leelawatcharamas  V, Nakayama  J, Sonomoto  K,     ( 2016 )

Functional Analysis of Genes Involved in the Biosynthesis of Enterocin NKR-5-3B, a Novel Circular Bacteriocin.

Journal of bacteriology 198 (2)
PMID : 26503847  :   DOI  :   10.1128/JB.00692-15     PMC  :   PMC4751792    
Abstract >>
A putative biosynthetic gene cluster of the enterocin NKR-5-3B (Ent53B), a novel circular bacteriocin, was analyzed by sequencing the flanking regions around enkB, the Ent53B structural gene, using a fosmid library. A region approximately 9 kb in length was obtained, and the enkB1, enkB2, enkB3, and enkB4 genes, encoding putative biosynthetic proteins involved in the production, maturation, and secretion of Ent53B, were identified. We also determined the identity of proteins mediating self-immunity against the effects of Ent53B. Heterologous expression systems in various heterologous hosts, such as Enterococcus faecalis and Lactococcus lactis strains, were successfully established. The production and secretion of the mature Ent53B required the cooperative functions of five genes. Ent53B was produced only by those heterologous hosts that expressed protein products of the enkB, enkB1, enkB2, enkB3, and enkB4 genes. Moreover, self-immunity against the antimicrobial action of Ent53B was conferred by at least two independent mechanisms. Heterologous hosts harboring the intact enkB4 gene and/or a combination of intact enkB1 and enkB3 genes were immune to the inhibitory action of Ent53B. In addition to their potential application as food preservatives, circular bacteriocins are now considered possible alternatives to therapeutic antibiotics due to the exceptional stability conferred by their circular structure. The successful practical application of circular bacteriocins will become possible only if the molecular details of their biosynthesis are fully understood. The results of the present study offer a new perspective on the possible mechanism of circular bacteriocin biosynthesis. In addition, since some enterococcal strains are associated with pathogenicity, virulence, and drug resistance, the establishment of the first multigenus host heterologous production of Ent53B has very high practical significance, as it widens the scope of possible Ent53B applications.
KeywordMeSH Terms
164. Boyd  DA, Lévesque  S, Picard  AC, Golding  GR,     ( 2015 )

Vancomycin-resistant Enterococcus faecium harbouring vanN in Canada: a case and complete sequence of pEfm12493 harbouring the vanN operon.

The Journal of antimicrobial chemotherapy 70 (7)
PMID : 25754999  :   DOI  :   10.1093/jac/dkv057    
Abstract >>
N/A
KeywordMeSH Terms
Enterococcus faecium
d-Ala-d-Ser ligase
vancomycin resistance
Genes, Bacterial
Plasmids
Vancomycin Resistance
165. Bhatt  P, Sahni  AK, Praharaj  AK, Grover  N, Kumar  M, Chaudhari  CN, Khajuria  A,     ( 2015 )

Detection of glycopeptide resistance genes in enterococci by multiplex PCR.

Medical journal, Armed Forces India 71 (1)
PMID : 25609863  :   DOI  :   10.1016/j.mjafi.2014.03.005     PMC  :   PMC4297841    
Abstract >>
Vancomycin Resistant Enterococci (VRE) are a major cause of nosocomial infections. There are various phenotypic and genotypic methods of detection of glycopeptide resistance in enterococci. This study utilizes multiplex PCR for reliable detection of various glycopeptides resistance genes in VRE. This study was conducted to detect and to assess the prevalence of vancomycin resistance among enterococci isolates. From October 2011 to June 2013, a total of 96 non-repetitive isolates of enterococci from various clinical samples were analyzed. VRE were identified by Kirby Bauer disc diffusion method with Clinical and Laboratory Standards Institute (CLSI) guidelines. Minimum inhibitory concentration (MIC) of all isolates for vancomycin and teicoplanin was determined by E-test. Multiplex PCR was carried out for all enterococci isolates using six sets of primers. Out of 96 isolates, 14 (14.6%) were found to be resistant to vancomycin by vancomycin E-test method (MIC ?32 �gg/ml). Out of these 14 isolates, 13 were also resistant to teicoplanin (MIC ?16 �gg/ml). VanA gene was detected in all the 14 isolates by Multiplex PCR. One of the PCR amplicons was sent for sequencing and the sequence received was submitted in the GenBank (GenBank accession no. KF181100). Prevalence of VRE in this study was 14.6%. Multiplex PCR is a robust, sensitive and specific technique, which can be used for rapid detection of various glycopeptide resistance genes. Rapid identification of patients infected or colonized with VRE is essential for implementation of appropriate control measures to prevent their spread.
KeywordMeSH Terms
Clinical and Laboratory Standards Institute
GenBank
Minimum inhibitory concentration
Multiplex PCR
Vancomycin resistant enterococci
166. Paganelli  FL, de Been  M, Braat  JC, Hoogenboezem  T, Vink  C, Bayjanov  J, Rogers  MR, Huebner  J, Bonten  MJ, Willems  RJ, Leavis  HL,     ( 2015 )

Distinct SagA from Hospital-Associated Clade A1 Enterococcus faecium Strains Contributes to Biofilm Formation.

Applied and environmental microbiology 81 (19)
PMID : 26209668  :   DOI  :   10.1128/AEM.01716-15     PMC  :   PMC4561713    
Abstract >>
Enterococcus faecium is an important nosocomial pathogen causing biofilm-mediated infections. Elucidation of E. faecium biofilm pathogenesis is pivotal for the development of new strategies to treat these infections. In several bacteria, extracellular DNA (eDNA) and proteins act as matrix components contributing to biofilm development. In this study, we investigated biofilm formation capacity and the roles of eDNA and secreted proteins for 83 E. faecium strains with different phylogenetic origins that clustered in clade A1 and clade B. Although there was no significant difference in biofilm formation between E. faecium strains from these two clades, the addition of DNase I or proteinase K to biofilms demonstrated that eDNA is essential for biofilm formation in most E. faecium strains, whereas proteolysis impacted primarily biofilms of E. faecium clade A1 strains. Secreted antigen A (SagA) was the most abundant protein in biofilms from E. faecium clade A1 and B strains, although its localization differed between the two groups. sagA was present in all sequenced E. faecium strains, with a consistent difference in the repeat region between the clades, which correlated with the susceptibility of biofilms to proteinase K. This indicates an association between the SagA variable repeat profile and the localization and contribution of SagA in E. faecium biofilms.
KeywordMeSH Terms
Biofilms
167. Beukers  AG, Zaheer  R, Cook  SR, Stanford  K, Chaves  AV, Ward  MP, McAllister  TA,     ( 2015 )

Effect of in-feed administration and withdrawal of tylosin phosphate on antibiotic resistance in enterococci isolated from feedlot steers.

Frontiers in microbiology 6 (N/A)
PMID : 26074889  :   DOI  :   10.3389/fmicb.2015.00483     PMC  :   PMC4444845    
Abstract >>
Tylosin phosphate is a macrolide commonly administered to cattle in North America for the control of liver abscesses. This study investigated the effect of in-feed administration of tylosin phosphate to cattle at subtherapeutic levels and its subsequent withdrawal on macrolide resistance using enterococci as an indicator bacterium. Fecal samples were collected from steers that received no antibiotics and steers administered tylosin phosphate (11 ppm) in-feed for 197 days and withdrawn 28 days before slaughter. Enterococcus species isolated from fecal samples were identified through sequencing the groES-EL intergenic spacer region and subject to antimicrobial susceptibility testing, identification of resistance determinants and pulsed-field gel electrophoresis profiling. Tylosin increased (P < 0.05) the proportion of ery(R) and tyl(R) enterococci within the population. Just prior to its removal, the proportion of ery(R) and tyl(R) resistant enterococci began decreasing and continued to decrease after tylosin was withdrawn from the diet until there was no difference (P > 0.05) between treatments on d 225. This suggests that antibiotic withdrawal prior to slaughter contributes to a reduction in the proportion of macrolide resistant enterococci entering the food chain. Among the 504 enterococci isolates characterized, Enterococcus hirae was found to predominate (n = 431), followed by Enterococcus villorum (n = 32), Enterococcus faecium (n = 21), Enterococcus durans (n = 7), Enterococcus casseliflavus (n = 4), Enterococcus mundtii (n = 4), Enterococcus gallinarum (n = 3), Enterococcus faecalis (n = 1), and Enterococcus thailandicus (n = 1). The diversity of enterococci was greater in steers at arrival than at exit from the feedlot. Erythromycin resistant isolates harbored the erm(B) and/or msrC gene. Similar PFGE profiles of ery(R) E. hirae pre- and post-antibiotic treatment suggest that increased abundance of ery(R) enterococci after administration of tylosin phosphate reflects selection for strains that were already present within the gastrointestinal tract of cattle at arrival.
KeywordMeSH Terms
antimicrobial resistance
beef cattle
enterococci
erythromycin
subtherapeutic macrolides
tylosin
antimicrobial resistance
beef cattle
enterococci
erythromycin
subtherapeutic macrolides
tylosin
168. Murphree  CA, Heist  EP, Moe  LA,     ( 2014 )

Antibiotic resistance among cultured bacterial isolates from bioethanol fermentation facilities across the United States.

Current microbiology 69 (3)
PMID : 24748439  :   DOI  :   10.1007/s00284-014-0583-y    
Abstract >>
Bacterial contamination of fuel ethanol fermentations by lactic acid bacteria (LAB) can have crippling effects on bioethanol production. Producers have had success controlling bacterial growth through prophylactic addition of antibiotics to fermentors, yet concerns have arisen about antibiotic resistance among the LAB. Here, we report on mechanisms used by 32 LAB isolates from eight different US bioethanol facilities to persist under conditions of antibiotic stress. Minimum inhibitory concentration assays with penicillin, erythromycin, and virginiamycin revealed broad resistance to each of the antibiotics as well as high levels of resistance to individual antibiotics. Phenotypic assays revealed that antibiotic inactivation mechanisms contributed to the high levels of individual resistances among the isolates, especially to erythromycin and virginiamycin, yet none of the isolates appeared to use a �]-lactamase. Biofilm formation was noted among the majority of the isolates and may contribute to persistence under low levels of antibiotics. Nearly all of the isolates carried at least one canonical antibiotic resistance gene and many carried more than one. The erythromycin ribosomal methyltransferase (erm) gene class was found in 19 of 32 isolates, yet a number of these isolates exhibit little to no resistance to erythromycin. The erm genes were present in 15 isolates that encoded more than one antibiotic resistance mechanism, suggestive of potential genetic linkages.
KeywordMeSH Terms
Drug Resistance, Bacterial
Industrial Microbiology
169. Sadowy  E, Luczkiewicz  A,     ( 2014 )

Drug-resistant and hospital-associated Enterococcus faecium from wastewater, riverine estuary and anthropogenically impacted marine catchment basin.

BMC microbiology 14 (N/A)
PMID : 24629030  :   DOI  :   10.1186/1471-2180-14-66     PMC  :   PMC4004213    
Abstract >>
Enterococci, ubiquitous colonizers of humans and other animals, play an increasingly important role in health-care associated infections (HAIs). It is believed that the recent evolution of two clinically relevant species, Enterococcus faecalis and Enterococcus faecium occurred in a big part in a hospital environment, leading to formation of high-risk enterococcal clonal complexes (HiRECCs), which combine multidrug resistance with increased pathogenicity and epidemicity. The aim of this study was to establish the species composition in wastewater, its marine recipient as well as a river estuary and to investigate the antimicrobial susceptibility of collected isolates. Molecular methods were additionally applied to test the presence of HiRRECC-related E. faecium. Two wastewater treatment plants (WWTPs), their marine outfalls and Vistula river that influence significantly the quality of waters in Gulf of Gdansk were sampled to investigate the presence of Enterococcus spp. Four-hundred-twenty-eight isolates were obtained, including E. faecium (244 isolates, 57.0%), E. hirae (113 isolates, 26.4%) and E. faecalis (63 isolates, 14.7%); other species (E. gallinarum/casseliflavus, E. durans and E. avium) accounted for 1.9%. Antimicrobial susceptibility testing revealed the presence of isolates resistant to erythromycin, tetracycline, amipicillin, fluoroquinolones and aminoglycosides (high-level resistance), especially among E. faecium, where such isolates were usually characterized by multilocus sequence types associated with nosocomial lineages 17, 18 and 78 of this species representing HiRECC, formerly called CC17. These isolates not only carried several resistance determinants but were also enriched in genes encoding pathogenicity factors (Esp, pili) and genes associated with mobile genetic elements (MGE), a feature also typical for nosocomial HiRECC. Our data show that WWTPs constitute an important source of enterococcal strains carrying antimicrobial resistance determinants, often associated with the presence of MGE, for the recipient water environment, thus increasing a pool of such genes for other organisms. The presence of HiRECCs in wastewaters and marine/river environment may indicate that adaptations gained in hospitals may be also beneficial for survival of such clones in other settings. There is an obvious need to monitor the release and spread of such strains in order to elucidate better ways to curb their dissemination.
KeywordMeSH Terms
Drug Resistance, Bacterial
170. Pietta  E, Montealegre  MC, Roh  JH, Cocconcelli  PS, Murray  BE,     ( 2014 )

Enterococcus faecium PBP5-S/R, the missing link between PBP5-S and PBP5-R.

Antimicrobial agents and chemotherapy 58 (11)
PMID : 25182648  :   DOI  :   10.1128/AAC.03648-14     PMC  :   PMC4249377    
Abstract >>
During a study to investigate the evolution of ampicillin resistance in Enterococcus faecium, we observed that a number of E. faecium strains, mainly from the recently described subclade A2, showed PBP5 sequences in between PBP5-S and PBP5-R. These hybrid PBP5-S/R patterns reveal a progression of amino acid changes from the S form to the R form of this protein; however, these changes do not strictly correlate with changes in ampicillin MICs.
KeywordMeSH Terms
171. Spitaels  F, Wieme  AD, Janssens  M, Aerts  M, Daniel  HM, Van Landschoot  A, De Vuyst  L, Vandamme  P,     ( 2014 )

The microbial diversity of traditional spontaneously fermented lambic beer.

PloS one 9 (4)
PMID : 24748344  :   DOI  :   10.1371/journal.pone.0095384     PMC  :   PMC3991685    
Abstract >>
Lambic sour beers are the products of a spontaneous fermentation that lasts for one to three years before bottling. The present study determined the microbiota involved in the fermentation of lambic beers by sampling two fermentation batches during two years in the most traditional lambic brewery of Belgium, using culture-dependent and culture-independent methods. From 14 samples per fermentation, over 2000 bacterial and yeast isolates were obtained and identified. Although minor variations in the microbiota between casks and batches and a considerable species diversity were found, a characteristic microbial succession was identified. This succession started with a dominance of Enterobacteriaceae in the first month, which were replaced at 2 months by Pediococcus damnosus and Saccharomyces spp., the latter being replaced by Dekkera bruxellensis at 6 months fermentation duration.
KeywordMeSH Terms
Beer
Fermentation
Microbiota
172. Wang  XM, Li  XS, Wang  YB, Wei  FS, Zhang  SM, Shang  YH, Du  XD,     ( 2015 )

Characterization of a multidrug resistance plasmid from Enterococcus faecium that harbours a mobilized bcrABDR locus.

The Journal of antimicrobial chemotherapy 70 (2)
PMID : 25324422  :   DOI  :   10.1093/jac/dku416    
Abstract >>
N/A
KeywordMeSH Terms
E. faecium
ISEnfa1
bacitracin resistance
Drug Resistance, Multiple, Bacterial
173. Li  XS, Dong  WC, Wang  XM, Hu  GZ, Wang  YB, Cai  BY, Wu  CM, Wang  Y, Du  XD,     ( 2014 )

Presence and genetic environment of pleuromutilin-lincosamide-streptogramin A resistance gene lsa(E) in enterococci of human and swine origin.

The Journal of antimicrobial chemotherapy 69 (5)
PMID : 24379302  :   DOI  :   10.1093/jac/dkt502    
Abstract >>
N/A
KeywordMeSH Terms
PLSA resistance
Enterococcus faecalis
Enterococcus faecium
Drug Resistance, Bacterial
174. Woegerbauer  M, Zeinzinger  J, Springer  B, Hufnagl  P, Indra  A, Korschineck  I, Hofrichter  J, Kopacka  I, Fuchs  R, Steinwider  J, Fuchs  K, Nielsen  KM, Allerberger  F,     ( 2014 )

Prevalence of the aminoglycoside phosphotransferase genes aph(3')-IIIa and aph(3')-IIa in Escherichia coli, Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica and Staphylococcus aureus isolates in Austria.

Journal of medical microbiology 63 (Pt 2)
PMID : 24194558  :   DOI  :   10.1099/jmm.0.065789-0    
Abstract >>
The aminoglycoside phosphotransferase aph(3')-IIa primarily inactivates kanamycin and neomycin, whilst aph(3')-IIIa also inactivates amikacin. The aim of this study was to determine the frequency of both resistance genes in major human pathogens to obtain their baseline prevalence in the gene pool of these bacterial populations in Austria. In total, 10 541 Escherichia coli, Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica and Staphylococcus aureus isolates were collected representatively without selection bias between 2008 and 2011. Isolates were analysed by aph(3')-IIIa/nptIII- and aph(3')-IIa/nptII-specific TaqMan real-time PCR. For positive strains, MICs using Etests were performed and resistance gene sequences were determined. The overall prevalence of aph(3')-IIIa/nptIII was 1.62 % (95 % confidence interval: 1.38-1.88 %). In Escherichia coli, enterococci, Staphylococcus aureus, P. aeruginosa and Salmonella spp., the aph(3')-IIIa/nptIII prevalence was 0.47 % (0-1.47 %), 37.53 % (32.84-42.40 %), 2.90 % (1.51-5.02 %), 0 % (0-0.32 %) and 0 % (0-0.037 %), respectively. Eleven of a total of 169 carriers showed single-nucleotide polymorphisms in the resistance allele. The overall prevalence of aph(3')-IIa/nptII was 0.0096 % (0-0.046 %). Escherichia coli (0-0.70 %), enterococci (0-0.75 %), Staphylococcus aureus (0-0.73 %) and P. aeruginosa (0-0.32 %) did not carry aph(3')-IIa. A single Salmonella isolate was positive, resulting in an aph(3')-IIa prevalence of 0.013 % (0-0.058 %). aph(3')-IIIa/nptIII carriers were moderately prevalent in the strains tested except for in enterococci, which appeared to be an important reservoir for aph(3')-IIIa. aph(3')-IIa/nptII genes were detected at clinically irrelevant frequencies and played no significant role in the aminoglycoside resistance gene pool during the observation period.
KeywordMeSH Terms
Drug Resistance, Bacterial
175. Padmasini  E, Padmaraj  R, Ramesh  SS,     ( N/A )

Detection of Vancomycin resistant Enterococci with vanA genotype in clinical isolates from a tertiary care centre.

Indian journal of medical microbiology 32 (1)
PMID : 24399401  :   DOI  :   10.4103/0255-0857.124339    
Abstract >>
N/A
KeywordMeSH Terms
Vancomycin Resistance
176. Santona  A, Paglietti  B, Al-Qahtani  AA, Bohol  MF, Senok  A, Deligios  M, Rubino  S, Al-Ahdal  MN,     ( 2014 )

Novel type of VanB2 teicoplanin-resistant hospital-associated Enterococcus faecium.

International journal of antimicrobial agents 44 (2)
PMID : 25059441  :   DOI  :   10.1016/j.ijantimicag.2014.05.005    
Abstract >>
Seven high-risk clones of vancomycin-resistant Enterococcus faecium (VREF) belonging to clonal complex 17 were identified using multilocus sequence typing (MLST) among clinical isolates from Saudi Arabia. Among these isolates, a new hospital-associated sequence type (ST795), VanB(2)-type teicoplanin-resistant strain was detected. Its unusual phenotype resulted from a new combination of mutations in the ddl, vanS and vanW genes, which confirmed the trend of evolution in VanB-type resistance. Furthermore, characteristics of adaptation and persistence in the hospital environment of ST795 were emphasised by the presence of genes and clusters recognised to be specific for hospital-associated VREF.
KeywordMeSH Terms
Enterococcus faecium
Hospital-associated
MLST
Teicoplanin resistance mechanism
Drug Resistance, Bacterial
177. Szakacs  TA, Kalan  L, McConnell  MJ, Eshaghi  A, Shahinas  D, McGeer  A, Wright  GD, Low  DE, Patel  SN,     ( 2014 )

Outbreak of vancomycin-susceptible Enterococcus faecium containing the wild-type vanA gene.

Journal of clinical microbiology 52 (5)
PMID : 24523464  :   DOI  :   10.1128/JCM.03563-13     PMC  :   PMC3993680    
Abstract >>
Accurate detection of vancomycin-resistant enterococci (VRE) is essential in preventing transmission in health care settings. Chromogenic media are widely used for screening VRE because of fast turnaround times (TAT) and high sensitivity. We report an outbreak of Enterococcus faecium bearing vanA yet susceptible to vancomycin (vancomycin-variable Enterococcus [VVE]). Between October 2009 to March 2011, clinical and screening specimens (n=14,747) were screened for VRE using VRE-selective medium and/or PCR. VVE isolates were genotyped to determine relatedness. Plasmids from these isolates were characterized by sequencing. Overall, 52 VVE isolates were identified, comprising 15% of all VRE isolates identified. Isolates demonstrated growth on Brilliance VRE agar (Oxoid) at 24 h of incubation but did not grow on brain heart infusion agar with 6 �gg/ml vancomycin (Oxoid) or bile esculin azide agar with 6 �gg/ml vancomycin (Oxoid) and were susceptible to vancomycin. Genotyping of 20 randomly selected VVE isolates revealed that 15/20 were identical, while 5 were highly related. PCR of the VVE transposon confirmed the presence of vanHAXY gene cluster; however, vanS (sensor) and vanR (regulator) genes were absent. The outbreak was controlled through routine infection control measures. We report an emergence of a fit strain of E. faecium containing vanA yet susceptible to vancomycin. Whether this new strain represents VRE has yet to be determined; however, unique testing procedures are required for reliable identification of VVE.
KeywordMeSH Terms
Disease Outbreaks
178. Frolkova  P, Ghosh  A, Svec  P, Zurek  L, Literak  I,     ( 2012 )

Use of the manganese-dependent superoxide dismutase gene sodA for rapid identification of recently described enterococcal species.

Folia microbiologica 57 (5)
PMID : 22570141  :   DOI  :   10.1007/s12223-012-0115-8    
Abstract >>
N/A
KeywordMeSH Terms
179. Miyanaga  A, Fujisawa  S, Furukawa  N, Arai  K, Nakajima  M, Taguchi  H,     ( 2013 )

The crystal structure of D-mandelate dehydrogenase reveals its distinct substrate and coenzyme recognition mechanisms from those of 2-ketopantoate reductase.

Biochemical and biophysical research communications 439 (1)
PMID : 23954635  :   DOI  :   10.1016/j.bbrc.2013.08.019    
Abstract >>
D-Mandelate dehydrogenases (D-ManDHs), belonging to a new d-2-hydroxyacid dehydrogenase family, catalyze the conversion between benzoylformate and d-mandelate using NAD as a coenzyme. We determined the first D-ManDH structure, that of ManDH2 from Enterococcus faecalis IAM10071. The overall structure showed ManDH2 has a similar fold to 2-ketopantoate reductase (KPR), which catalyzes the conversion of 2-ketopantoate to d-pantoate using NADP as a coenzyme. They share conserved catalytic residues, indicating ManDH2 has the same reaction mechanism as KPR. However, ManDH2 exhibits significant structural variations in the coenzyme and substrate binding sites compared to KPR. These structural observations could explain their different coenzyme and substrate specificities.
KeywordMeSH Terms
2-Ketopantoate reductase
2-ketopantoate reductase from Escherichia coli
CENDH
Coenzyme specificity
Crystal structure
EcKPR
KPR
ManDH2
N-(1-d-carboxyethyl)-l-norvaline dehydrogenase from Arthrobacter sp.
Substrate specificity
d-2-HydDH
d-2-hydroxyacid dehydrogenase
d-ManDH
d-Mandelate dehydrogenase
d-mandelate dehydrogenase from Enterococcus faecalis IAM10071
rmsd
root mean square deviation.
180. Wang  R, Kan  B, Li  J, Zhang  L,     ( 2012 )

Antibiotic resistance of Vibrio cholerae O1 El Tor strains from the seventh pandemic in China, 1961-2010.

International journal of antimicrobial agents 40 (4)
PMID : 22867881  :   DOI  :   10.1016/j.ijantimicag.2012.06.010    
Abstract >>
Antibiotic resistance is observed with increasing frequency among epidemic Vibrio cholerae strains in some countries. In this study, the antibiotic resistance profiles of V. cholerae O1 El Tor strains isolated in China from 1961 to 2010 were analysed. The frequency of antibiotic resistance among the seventh pandemic El Tor isolates from China remained low, except for resistance to nalidixic acid (45.9%), tetracycline (11%) and trimethoprim/sulfamethoxazole (38.5%). All test strains in the first multiyear epidemic in the 1960s were sensitive to all test antibiotics, whereas strains from the 1990s and later showed a rapid increase in the prevalence of resistance. The class I integron was present primarily among strains isolated between 1993 and 1998, and the prevalence of the SXT element was much greater among strains isolated after 1993. This study determined the regional resistance characteristics of epidemic clones in China and serves as a warning of the rapid dissemination of resistance in the past 20 years.
KeywordMeSH Terms
Drug Resistance, Bacterial
Pandemics
181. Xu  X, Chen  C, Lin  D, Guo  Q, Hu  F, Zhu  D, Li  G, Wang  M,     ( 2013 )

The fosfomycin resistance gene fosB3 is located on a transferable, extrachromosomal circular intermediate in clinical Enterococcus faecium isolates.

PloS one 8 (10)
PMID : 24205114  :   DOI  :   10.1371/journal.pone.0078106     PMC  :   PMC3812183    
Abstract >>
Some VanM-type vancomycin-resistant Enterococcus faecium isolates from China are also resistant to fosfomycin. To investigate the mechanism of fosfomycin resistance in these clinical isolates, antimicrobial susceptibility testing, filter-mating, Illumina/Solexa sequencing, inverse PCR and fosfomycin resistance gene cloning were performed. Three E. faecium clinical isolates were highly resistant to fosfomycin and vancomycin with minimal inhibitory concentrations (MICs) >1024 ?g/ml and >256 ?g/ml, respectively. The fosfomycin and vancomycin resistance of these strains could be co-transferred by conjugation. They carried a fosfomycin resistance gene fosB encoding a protein differing by one or two amino acids from FosB, which is encoded on staphylococcal plasmids. Accordingly, the gene was designated fosB3. The fosB3 gene was cloned into pMD19-T, and transformed into E. coli DH5�\. The fosfomycin MIC for transformants with fosB3 was 750-fold higher than transformants without fosB3. The fosB3 gene could be transferred by an extrachromosomal circular intermediate. The results indicate that the fosB3 gene is transferable, can mediate high level fosfomycin resistance in both Gram-positive and Gram-negative bacteria, and can be located on a circular intermediate.
KeywordMeSH Terms
182. Isnard  C, Malbruny  B, Leclercq  R, Cattoir  V,     ( 2013 )

Genetic basis for in vitro and in vivo resistance to lincosamides, streptogramins A, and pleuromutilins (LSAP phenotype) in Enterococcus faecium.

Antimicrobial agents and chemotherapy 57 (9)
PMID : 23836170  :   DOI  :   10.1128/AAC.01030-13     PMC  :   PMC3754343    
Abstract >>
As opposed to Enterococcus faecalis, which is intrinsically resistant to lincosamides, streptogramins A, and pleuromutilins (LSAP phenotype) by production of the ABC protein Lsa(A), Enterococcus faecium is naturally susceptible. Since this phenotype may be selected for in vivo by quinupristin-dalfopristin (Q-D), the aim of this study was to investigate the molecular mechanism of acquired LSAP resistance in E. faecium. Six LSAP-resistant in vitro mutants of E. faecium HM1070 as well as three different pairs of clinical isolates (pre- and postexposure to Q-D) were studied. The full genome sequence of an in vitro mutant (E. faecium UCN90B) was determined by using 454 sequencing technology and was compared with that of the parental strain. Single-nucleotide replacement was carried out to confirm the role of this mutation. By comparative genomic analysis, a point mutation was found within a 1,503-bp gene coding for an ABC homologue showing 66% amino acid identity with Lsa(A). This mutation (C1349T) led to an amino acid substitution (Thr450Ile). An identical mutation was identified in all in vitro and in vivo resistant strains but was not present in susceptible strains. The wild-type allele was named eat(A) (for Enterococcus ABC transporter), and its mutated allelic variant was named eat(A)v. The introduction of eat(A)v from UCN90B into HM1070 conferred the LSAP phenotype, whereas that of eat(A) from HM1070 into UCN90B restored susceptibility entirely. This is the first description of the molecular mechanism of acquired LSAP resistance in E. faecium. Characterization of the biochemical mechanism of resistance and the physiological role of this ABC protein need further investigations.
KeywordMeSH Terms
183. Cattoir  V, Leclercq  R, Dhalluin  A, Willems  RJ, Verneuil  N, Zhang  X, Giard  JC, Le Bras  F, Torelli  R, Posteraro  B, Sanguinetti  M,     ( 2012 )

AsrR is an oxidative stress sensing regulator modulating Enterococcus faecium opportunistic traits, antimicrobial resistance, and pathogenicity.

PLoS pathogens 8 (8)
PMID : 22876178  :   DOI  :   10.1371/journal.ppat.1002834     PMC  :   PMC3410868    
Abstract >>
Oxidative stress serves as an important host/environmental signal that triggers a wide range of responses in microorganisms. Here, we identified an oxidative stress sensor and response regulator in the important multidrug-resistant nosocomial pathogen Enterococcus faecium belonging to the MarR family and called AsrR (antibiotic and stress response regulator). The AsrR regulator used cysteine oxidation to sense the hydrogen peroxide which results in its dissociation to promoter DNA. Transcriptome analysis showed that the AsrR regulon was composed of 181 genes, including representing functionally diverse groups involved in pathogenesis, antibiotic and antimicrobial peptide resistance, oxidative stress, and adaptive responses. Consistent with the upregulated expression of the pbp5 gene, encoding a low-affinity penicillin-binding protein, the asrR null mutant was found to be more resistant to �]-lactam antibiotics. Deletion of asrR markedly decreased the bactericidal activity of ampicillin and vancomycin, which are both commonly used to treat infections due to enterococci, and also led to over-expression of two major adhesins, acm and ecbA, which resulted in enhanced in vitro adhesion to human intestinal cells. Additional pathogenic traits were also reinforced in the asrR null mutant including greater capacity than the parental strain to form biofilm in vitro and greater persistance in Galleria mellonella colonization and mouse systemic infection models. Despite overexpression of oxidative stress-response genes, deletion of asrR was associated with a decreased oxidative stress resistance in vitro, which correlated with a reduced resistance to phagocytic killing by murine macrophages. Interestingly, both strains showed similar amounts of intracellular reactive oxygen species. Finally, we observed a mutator phenotype and enhanced DNA transfer frequencies in the asrR deleted strain. These data indicate that AsrR plays a major role in antimicrobial resistance and adaptation for survival within the host, thereby contributes importantly to the opportunistic traits of E. faecium.
KeywordMeSH Terms
beta-Lactam Resistance
184. Chang  SY, Chen  YS, Pan  SF, Lee  YS, Chang  CH, Chang  CH, Yu  B, Wu  HC,     ( 2013 )

Enterocin TW21, a novel bacteriocin from dochi-isolated Enterococcus faecium D081821.

Journal of applied microbiology 115 (3)
PMID : 23725102  :   DOI  :   10.1111/jam.12265    
Abstract >>
Purification and characterization of a novel bacteriocin produced by strain Enterococcus faecium D081821. Enterococcus faecium D081821, isolated from the traditional Taiwanese fermented food dochi (fermented black beans), was previously found to produce a bacteriocin against Listeria monocytogenes and some Gram-positive bacteria. This bacteriocin, termed enterocin TW21, was purified from culture supernatant by ammonium sulfate precipitation, Sep-Pak C18 cartridge, ion-exchange and gel filtration chromatography. Mass spectrometry analysis showed the mass of the peptide to be approximately 5300�P6 Da. The N-terminal amino acid sequencing yielded a partial sequence NH2 -ATYYGNGVYxNTQK by Edman degradation, and it contains the consensus class IIa bacteriocin motif YGNGV in the N-terminal region. The open reading frame (ORF) encoding the bacteriocin was identified from the draft genome sequence of Enterococcus faecium D081821, and sequence analysis of this peptide indicated that enterocin TW21 is a novel bacteriocin. Enterococcus faecium D081821 produced a bacteriocin named enterocin TW21, the molecular weight and amino acid sequence both revealed it to be a novel bacteriocin. A new member of class IIa bacteriocin was identified. This bacteriocin shows great inhibitory ability against L. monocytogenes and could be applied as a natural food preservative.
KeywordMeSH Terms
Enterococcus faecium
dochi
fermented black beans
lactic acid bacteria
novel bacteriocin
185. Bjørkeng  EK, Hjerde  E, Pedersen  T, Sundsfjord  A, Hegstad  K,     ( 2013 )

ICESluvan, a 94-kilobase mosaic integrative conjugative element conferring interspecies transfer of VanB-type glycopeptide resistance, a novel bacitracin resistance locus, and a toxin-antitoxin stabilization system.

Journal of bacteriology 195 (23)
PMID : 24078615  :   DOI  :   10.1128/JB.02165-12     PMC  :   PMC3837959    
Abstract >>
A 94-kb integrative conjugative element (ICESluvan) transferable to Enterococcus faecium and Enterococcus faecalis from an animal isolate of Streptococcus lutetiensis consists of a mosaic of genetic fragments from different Gram-positive bacteria. A variant of ICESluvan was confirmed in S. lutetiensis from a patient. A complete Tn5382/Tn1549 with a vanB2 operon is integrated into a streptococcal ICESde3396-like region harboring a putative bacteriophage exclusion system, a putative agglutinin receptor precursor, and key components of a type IV secretion system. Moreover, ICESluvan encodes a putative MobC family mobilization protein and a relaxase and, thus, in total has all genetic components essential for conjugative transfer. A 9-kb element within Tn5382/Tn1549 encodes, among others, putative proteins similar to the TnpX site-specific recombinase in Faecalibacterium and VanZ in Paenibacillus, which may contribute to the detected low-level teicoplanin resistance. Furthermore, ICESluvan encodes a novel bacitracin resistance locus that is associated with reduced susceptibility to bacitracin when transferred to E. faecium. The expression of a streptococcal pezAT toxin-antitoxin-encoding operon of ICESluvan in S. lutetiensis, E. faecium, and E. faecalis was confirmed by reverse transcription (RT)-PCR, indicating an active toxin-antitoxin system which may contribute to stabilizing ICESluvan within new hosts. Junction PCR and DNA sequencing confirmed that ICESluvan excised to form a circular intermediate in S. lutetiensis, E. faecalis, and E. faecium. Transfer between E. faecalis cells was observed in the presence of helper plasmid pIP964. Sequence analysis of the original S. lutetiensis donor and enterococcal transconjugants showed that ICESluvan integrates in a site-specific manner into the C-terminal end of the chromosomal tRNA methyltransferase gene rumA.
KeywordMeSH Terms
186. Amachawadi  RG, Scott  HM, Alvarado  CA, Mainini  TR, Vinasco  J, Drouillard  JS, Nagaraja  TG,     ( 2013 )

Occurrence of the transferable copper resistance gene tcrB among fecal enterococci of U.S. feedlot cattle fed copper-supplemented diets.

Applied and environmental microbiology 79 (14)
PMID : 23666328  :   DOI  :   10.1128/AEM.00503-13     PMC  :   PMC3697488    
Abstract >>
Copper, an essential micronutrient, is supplemented in the diet at elevated levels to reduce morbidity and mortality and to promote growth in feedlot cattle. Gut bacteria exposed to copper can acquire resistance, which among enterococci is conferred by a transferable copper resistance gene (tcrB) borne on a plasmid. The present study was undertaken to investigate whether the feeding of copper at levels sufficient to promote growth increases the prevalence of the tcrB gene among the fecal enterococci of feedlot cattle. The study was performed with 261 crossbred yearling heifers housed in 24 pens, with pens assigned randomly to a 2��2 factorial arrangement of treatments consisting of dietary copper and a commercial linseed meal-based energy protein supplement. A total of 22 isolates, each identified as Enterococcus faecium, were positive for tcrB with an overall prevalence of 3.8% (22/576). The prevalence was higher among the cattle fed diets supplemented with copper (6.9%) compared to normal copper levels (0.7%). The tcrB-positive isolates always contained both erm(B) and tet(M) genes. Median copper MICs for tcrB-positive and tcrB-negative enterococci were 22 and 4 mM, respectively. The transferability of the tcrB gene was demonstrated via a filter-mating assay. Multilocus variable number tandem repeat analysis revealed a genetically diverse population of enterococci. The finding of a strong association between the copper resistance gene and other antibiotic (tetracycline and tylosin) resistance determinants is significant because enterococci remain potential pathogens and have the propensity to transfer resistance genes to other bacteria in the gut.
KeywordMeSH Terms
Drug Resistance, Bacterial
187. Sonomoto  K, Nakayama  J, Leelawatcharamas  V, Wilaipun  P, Zendo  T, Perez  RH, Masuda  Y,     ( 2012 )

Purification and characterization of multiple bacteriocins and an inducing peptide produced by Enterococcus faecium NKR-5-3 from Thai fermented fish.

Bioscience, biotechnology, and biochemistry 76 (5)
PMID : 22738965  :   DOI  :   10.1271/bbb.110972    
Abstract >>
Enterocins NKR-5-3A, B, C, and D were purified from the culture supernatant of Enterococcus faecium NKR-5-3 and characterized. Among the four purified peptides, enterocin NKR-5-3A (5242.3 Da) was identical to brochocin A, produced by Brochothrix campestris ATCC 43754, in mature peptides, and its putative synergistic peptide, enterocin NKR-5-3Z, was found to be encoded in ent53Z downstream of ent53A, encoding enterocin NKR-5-3A. Enterocin NKR-5-3B (6316.4 Da) showed a broad antimicrobial spectrum, and enterocin NKR-5-3C (4512.8 Da) showed high activity against Listeria. Enterocin NKR-5-3D (2843.5 Da), showing high homology to an inducing peptide produced by Lactobacillus sakei 5, induced the production of the enterocins. The enterocins showed different antimicrobial spectra and intensities. E. faecium NKR-5-3 concomitantly produced enterocins NKR-5-3A, B, C, and D which probably belong to different classes of bacteriocins. Furthermore, NKR-5-3 production was induced by enterocin NKR-5-3D.
KeywordMeSH Terms
188. Dutka-Malen  S, Courvalin  P, Arthur  M,     ( 1990 )

The VANA glycopeptide resistance protein is related to D-alanyl-D-alanine ligase cell wall biosynthesis enzymes.

Molecular & general genetics : MGG 224 (3)
PMID : 2266943  :   DOI  :   10.1007/bf00262430    
Abstract >>
Inducible resistance to the glycopeptide antibiotics vancomycin and teicoplanin is mediated by plasmid pIP816 in Enterococcus faecium strain BM4147. Vancomycin induced the synthesis of a ca. 40 kDa membrane-associated protein designated VANA. The resistance protein was partially purified and its N-terminal sequence was determined. A 1761 bp DNA restriction fragment of pIP816 was cloned into Escherichia coli and sequenced. When expressed in E. coli, this fragment encoded a ca. 40 kDa protein that comigrated with VANA from enterococcal membrane fractions. The ATG translation initiation codon for VANA specified the methionine present at the N-terminus of the protein indicating the absence of signal peptide processing. The amino acid sequence deduced from the sequence of the vanA gene consisted of 343 amino acids giving a protein with a calculated Mr of 37,400. VANA was structurally related to the D-alanyl-D-alanine (D-ala-D-ala) ligases of Salmonella typhimurium (36% amino acid identity) and of E. coli (28%). The vanA gene was able to transcomplement an E. coli mutant with thermosensitive D-ala-D-ala ligase activity. Thus, the inducible resistance protein VANA was structurally and functionally related to cytoplasmic enzymes that synthesize the target of glycopeptide antibiotics. Based on these observations we discuss the possibility that resistance is due to modification of the glycopeptide target.
KeywordMeSH Terms
Carbon-Oxygen Ligases
Drug Resistance, Microbial
Genes, Bacterial
189. Sonomoto  K, Masuda  Y, Leelawatcharamas  V, Yoneyama  F, Ishibashi  N, Wilaipun  P,     ( 2012 )

Identification of enterocin NKR-5-3C, a novel class IIa bacteriocin produced by a multiple bacteriocin producer, Enterococcus faecium NKR-5-3.

Bioscience, biotechnology, and biochemistry 76 (6)
PMID : 22790957  :   DOI  :   10.1271/bbb.120089    
Abstract >>
The structure of enterocin NKR-5-3C, an anti-listerial bacteriocin produced by a multiple bacteriocin producer, Enterococcus faecium NKR-5-3, was determined. Enterocin NKR-5-3C is a novel class IIa bacteriocin that possesses an YGNGL motif sequence and two disulfide bridges in its structure. It is encoded on gene ent53C together with an 18-amino-acid-residue double glycine leader peptide.
KeywordMeSH Terms
Genes, Bacterial
190. Pedersen  T, Sundsfjord  A, Lester  CH, Simonsen  GS, Hegstad  K, Aasnaes  B, Hammerum  AM,     ( 2012 )

Increased high-level gentamicin resistance in invasive Enterococcus faecium is associated with aac(6')Ie-aph(2?)Ia-encoding transferable megaplasmids hosted by major hospital-adapted lineages.

FEMS immunology and medical microbiology 66 (2)
PMID : 22672387  :   DOI  :   10.1111/j.1574-695X.2012.00997.x    
Abstract >>
Gentamicin is important in synergistic bactericidal therapy with cell wall agents for severe enterococcal infections. During 2003-2008, a 10-fold increase in the prevalence of high-level gentamicin resistance (HLGR), to above 50%, in blood culture isolates of Enterococcus faecium, was reported by the Norwegian Surveillance System for Antimicrobial Resistance. A representative national collection of invasive E. faecium isolates (n = 99) from 2008 was examined by a multilevel approach. Genotyping revealed a polyclonal population dominated by major hospital-associated lineages (mainly ST203, ST17, ST18, ST202 and ST192). The presence of aac(6')-Ie-aph(2?)-Ia, encoding the bi-functional aminoglycoside-modifying enzyme, was found in 98% of HLGR isolates (56/57). Furthermore, a significantly higher prevalence of potential virulence genes, toxin-antitoxin loci as well as pRE25 and pRUM type replicons was demonstrated in isolates belonging to major hospital-associated lineages compared to other sequence types. Megaplasmids of pLG1 replicon type (200-330 kb) were present in 90% of the isolates. Co-hybridization analyses revealed genetic linkage of aac(6')-Ie-aph(2?)-Ia to this replicon type. Transfer of HLGR-encoding plasmids was restricted to E. faecium. In conclusion, the increased prevalence of HLGR in invasive E. faecium in Norway is associated with hospital-adapted genetic lineages carrying aac(6')-Ie-aph(2?)-Ia-encoding transferable megaplasmids of the pLG1 replicon type.
KeywordMeSH Terms
Drug Resistance, Bacterial
Plasmids
191. Valdezate  S, Miranda  C, Navarro  A, Freitas  AR, Cabrera  JJ, Carrasco  G, Coque  TM, Jiménez-Romano  E, Saéz-Nieto  JA,     ( 2012 )

Clonal outbreak of ST17 multidrug-resistant Enterococcus faecium harbouring an Inc18-like::Tn1546 plasmid in a haemo-oncology ward of a Spanish hospital.

The Journal of antimicrobial chemotherapy 67 (4)
PMID : 22228676  :   DOI  :   10.1093/jac/dkr545    
Abstract >>
To report a clonal outbreak of ST17 vancomycin-resistant Enterococcus faecium (VREfm) carrying Tn1546 (vanA) in a haemo-oncology ward of a tertiary teaching hospital in the south of Spain (January-September 2009). Twenty-two VREfm strains from 13 patients were characterized by PFGE, multiple-locus variable-number tandem-repeat analysis (MLVA) and multilocus sequence typing (MLST). Genes encoding antibiotic resistance and putative virulence traits and the Tn1546 backbone were investigated by PCR. Plasmid characterization included determination of size (S1-PFGE) and replication modules (PCR, hybridization and sequencing). Patient clinical records were analysed retrospectively. A single ST17 E. faecium clone (MT-7 MLVA type) carrying esp and hyl plus a 30 kb Inc18-like::Tn1546 (IS1216) plasmid was identified. Ampicillin resistance was linked to PBP5 showing mutations at positions 24, 27, 34, 66, 68, 85, 100, 144, 172, 177, 204, 216, 324, 462, 466', 470, 485, 496, 499, 525, 546, 558, 582, 586, 629, 632, 642 and 667. Other resistance genes identified were erm(B), ant(6')-Ia and aph(3')-IIIa. Fluoroquinolone resistance was attributable to ParC (Arg-61 �� Gly and Ser-80 �� Arg) and GyrA (Ser-83 �� Arg) mutations. A nosocomial outbreak caused by an ST17 (CC17) E. faecium clone harbouring Esp and Hyl and a 30 kb Inc18-like::Tn1546 plasmid among haemo-oncology patients is reported. The failure of early infection control practices indicates an undetected reservoir and the ability of this strain to persist over long periods. The potential spread of epidemic clones and broad host plasmids carrying vancomycin resistance in Spain is of concern since it might contribute towards a higher rate of VREfm infection.
KeywordMeSH Terms
Disease Outbreaks
DNA Transposable Elements
Plasmids
192. Liu  H, Wang  Y, Wu  C, Schwarz  S, Shen  Z, Jeon  B, Ding  S, Zhang  Q, Shen  J,     ( 2012 )

A novel phenicol exporter gene, fexB, found in enterococci of animal origin.

The Journal of antimicrobial chemotherapy 67 (2)
PMID : 22096043  :   DOI  :   10.1093/jac/dkr481    
Abstract >>
To investigate two porcine Enterococcus isolates for the genetic basis of phenicol resistance and to determine the location and the genetic environment of the novel resistance gene. A total of 391 isolates with reduced florfenicol susceptibility (MIC ? 16 mg/L), obtained from 557 nasal swabs of individual pigs, were screened by PCR for the known florfenicol resistance genes. Isolates that were negative in these PCRs were analysed for their species assignment and antimicrobial susceptibility. Plasmids were extracted and subjected to transformation and conjugation assays. Restriction fragments of the phenicol resistance plasmids were cloned and sequenced. The sequences obtained were analysed and compared with sequences deposited in the databases. The two isolates, Enterococcus faecium EFM-1 and Enterococcus hirae EH-1, exhibited MICs of chloramphenicol and florfenicol of 64 mg/L and carried a new phenicol resistance gene, designated fexB. This gene codes for a phenicol exporter of 469 amino acids organized in 14 transmembrane domains. The fexB gene was located on the 35 kb pEFM-1 from E. faecium and on the 25.3 kb pEH-1 from E. hirae, respectively. Both plasmids were non-conjugative. The fexB gene was found to be embedded in virtually the same genetic environment of 14.8 kb in both plasmids. To the best of our knowledge, this is the first report of the new florfenicol exporter gene fexB. Based on its plasmid location, horizontal transfer from the enterococci to other bacteria is possible.
KeywordMeSH Terms
193.     ( 1997 )

Enterocin B, a new bacteriocin from Enterococcus faecium T136 which can act synergistically with enterocin A.

Microbiology (Reading, England) 143 (Pt 7) (N/A)
PMID : 9245817  :   DOI  :   10.1099/00221287-143-7-2287    
Abstract >>
The strain Enterococcus faecium T136 produces two bacteriocins, enterocin A, a member of the pediocin family of bacteriocins, and a new bacteriocin termed enterocin B. The N-terminal amino acid sequences of enterocins A and B were determined, and the gene encoding enterocin B was sequenced. The primary translation product was a 71 aa peptide containing a leader peptide of the double-glycine type which is cleaved off to give mature enterocin B of 53 aa. Enterocin B does not belong to the pediocin family of bacteriocins and shows strong homology to carnobacteriocin A. However, sequence similarities in their leader peptides and C-termini suggest that enterocin B and carnobacteriocin A are related to bacteriocins of the pediocin family. Enterocins A and B had only slightly different inhibitory spectra, and both were active against a wide range of Gram-positive bacteria, including listeriae, staphylococci and most lactic acid bacteria tested. Both had bactericidal activities, but survival at a frequency of 10(-4)-10(-2) was observed when sensitive cultures were exposed to either bacteriocin. The number of survivors was drastically reduced when a mixture of the two bacteriocins was added to the cells.
KeywordMeSH Terms
Genes, Bacterial
194.     ( 1997 )

Biochemical and genetic characterization of enterocin P, a novel sec-dependent bacteriocin from Enterococcus faecium P13 with a broad antimicrobial spectrum.

Applied and environmental microbiology 63 (11)
PMID : 9361419  :   PMC  :   PMC168752    
Abstract >>
Enterocin P is a new bacteriocin produced by Enterococcus faecium P13 isolated from a Spanish dry-fermented sausage. Enterocin P inhibited most of tested spoilage and food-borne gram-positive pathogenic bacteria, such as Listeria monocytogenes, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum. Enterocin P is produced during growth in MRS broth from 16 to 45 degrees C; it is heat resistant (60 min at 100 degrees C; 15 min at 121 degrees C) and can withstand exposure to pH between 2.0 and 11.0, freeze-thawing, lyophilization, and long-term storage at 4 and -20 degrees C. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation, gel filtration, cation-exchange, hydrophobic-interaction, and reverse-phase liquid chromatography. The sequence of 43 amino acids of the N terminus was obtained by Edman degradation. DNA sequencing analysis of a 755-bp region revealed the presence of two consecutive open reading frames (ORFs). The first ORF encodes a 71-amino-acid protein containing a hydrophobic N-terminal sec-dependent leader sequence of 27 amino acids followed by the amino acid sequence corresponding to the purified and sequenced enterocin P. The bacteriocin is apparently synthesized as a prepeptide that is cleaved immediately after the Val-Asp-Ala residues (positions -3 to -1), resulting in the mature bacteriocin consisting of 44 amino acids, and with a theoretical molecular weight of 4,493. A second ORF, encoding a putative immunity protein composed of 88 amino acids with a calculated molecular weight of 9,886, was found immediately downstream of the enterocin P structural gene. Enterocin P shows a strong antilisterial activity and has the consensus sequence found in the pediocin-like bacteriocins; however, enterocin P is processed and secreted by the sec-dependent pathway.
KeywordMeSH Terms
195.     ( 1996 )

Structure of the low-affinity penicillin-binding protein 5 PBP5fm in wild-type and highly penicillin-resistant strains of Enterococcus faecium.

Journal of bacteriology 178 (16)
PMID : 8759860  :   DOI  :   10.1128/jb.178.16.4948-4957.1996     PMC  :   PMC178279    
Abstract >>
Among its penicillin-binding proteins (PBPs), Enterococcus faecium possesses a low-affinity PBP5, PBP5fm, which is the main target involved in beta-lactam resistance. A 7.7-kb EcoRI chromosomal fragment of E. faecium D63r containing the pbp5fm gene was cloned and sequenced. Two open reading frames (ORFs) were found. A 2,037-bp ORF encoded the deduced 73.8-kDa PBP5fm, the amino acid sequences of which were, respectively, 99.8, 78.5, and 62% homologous to those of the low-affinity plasmid-encoded PBP3r of Enterococcus hirae S185r and the chromosome-encoded PBP5 of E. hirae R40 and Enterococcus faecalis 56R. A second 597-bp ORF, designated psrfm, was found 2.3 kb upstream of pbp5fm. It appeared to be 285 bp shorter than and 74% homologous with the regulatory gene psr of E. hirae ATCC 9790. Different clinical isolates of E. faecium, for which a wide range of benzylpenicillin MICs were observed, showed that the increases in MICs were related to two mechanisms. For some strains of intermediate resistance (MICs of 16 to 64 micrograms/ml), the increased level of resistance could be explained by the presence of larger quantities of PBP5fm which had an affinity for benzylpenicillin (second-order rate constant of protein acylation [k+2/K] values of 17 to 25 M(-1) s(-1)) that remained unchanged. For the two most highly resistant strains, EFM-1 (MIC, 90 micrograms/ml) and H80721 (MIC, 512 micrograms/ml), the resistance was related to different amino acid substitutions yielding very-low-affinity PBP5fm variants (k+2/K < or = 1.5 M(-1) s(-1)) which were synthesized in small quantities. More specifically, it appeared, with a three-dimensional model of the C-terminal domain of PBP5fm, that the substitutions of Met-485, located in the third position after the conserved SDN triad, by Thr in EFM-1 and by Ala in H80721 were the most likely cause of the decreasing affinity of PBP5fm observed in these strains.
KeywordMeSH Terms
Bacterial Proteins
Hexosyltransferases
Penicillin Resistance
Peptidyl Transferases
196.     ( 1996 )

Mobilization of vancomycin resistance by transposon-mediated fusion of a VanA plasmid with an Enterococcus faecium sex pheromone-response plasmid.

Gene 171 (1)
PMID : 8675038  :   DOI  :   10.1016/0378-1119(96)00022-4    
Abstract >>
A striking feature of recent outbreaks of vancomycin-resistant (VmR) enterococci is the apparent horizontal dissemination of resistance determinants. The plasmids pHKK702 and pHKK703 from Enterococcus faecium clinical isolate R7 have been implicated in the conjugal transfer of VmR. pHKK702 is a 41-kb plasmid that contains an element indistinguishable from the glycopeptide-resistance transposon Tn1546. pHKK703 is an approx. 55-kb putative sex pheromone-response plasmid that is required for conjugative mobilization of pHKK702. During experiments in which strain R7 was used as a donor, a highly conjugative VmR transconjugant was isolated that formed constitutive cellular aggregates. Restriction analyses and DNA hybridizations revealed that the transconjugant harbored a single plasmid of approx. 92 kb and this plasmid (pHKK701) was composed of DNA from both pHKK702 and pHKK703. Results from DNA sequence analyses showed that a 39-kb composite transposon (Tn5506) from pHKK702 had inserted into pHKK703. The left end of Tn5506 contained a single insertion sequence (IS) element, IS1216V2, whereas the right end was composed of a tandem IS structure consisting of the novel 1065-bp IS1252 nested within an IS1216V1 element. Transposition of Tn5506 from pHKK702 to pHKK703 created an 8-bp target sequence duplication at the site of insertion and interrupted an ORF (ORFX) that was 91% identical to that of prgX, a gene proposed to negatively regulate sex pheromone response of the E.faecalis plasmid, pCF10. We propose that the interruption of ORFX by Tn5506 led to the constitutive cellular aggregation phenotype and thereby enhanced the efficiency with which VmR was transferred. Similar IS1216V-mediated transposition events may contribute to the horizontal spread of glycopeptide resistance among enterococci in nature.
KeywordMeSH Terms
197.     ( 1996 )

Characterization and sequence analysis of a stable cryptic plasmid from Enterococcus faecium 226 and development of a stable cloning vector.

Applied and environmental microbiology 62 (4)
PMID : 8919818  :   PMC  :   PMC167923    
Abstract >>
A small cryptic plasmid, pMBB1, isolated from Enterococcus faecium 226 was characterized. The plasmid contained an extremely stable replicon which has limited homology to the lactococcal plasmid pCI305. Sequence analysis of the replicon detected one open reading frame of 822 bp capable of encoding a 32-kDa protein. No detectable single-stranded intermediates were found for the replicon, suggesting that pMBB1 may be included in the same family as pCI305, although pCI305 exhibits a more narrow host range. A small stably maintained vector able to replicate in a variety of lactic acid bacteria, containing a large multiple cloning region, was constructed by using the pMBB1 replicon.
KeywordMeSH Terms
198.     ( 1997 )

The VanS sensor negatively controls VanR-mediated transcriptional activation of glycopeptide resistance genes of Tn1546 and related elements in the absence of induction.

Journal of bacteriology 179 (1)
PMID : 8981985  :   DOI  :   10.1128/jb.179.1.97-106.1997     PMC  :   PMC178666    
Abstract >>
Transposon Tn1546 from Enterococcus faecium BM4147 encodes a histidine protein kinase (VanS) and a response regulator (VanR) that regulate transcription of the vanHAX operon encoding a dehydrogenase (VanH), a ligase (VanA), and a D,D-dipeptidase (VanX). These last three enzymes confer resistance to glycopeptide antibiotics by production of peptidoglycan precursors ending in the depsipeptide D-alanyl-D-lactate. Transcription of vanS and the role of VanS in the regulation of the vanHAX operon were analyzed by inserting a cat reporter gene into vanS. Transcription of cat and vanX was inducible by glycopeptides in partial diploids harboring vanS and vanS(omega)cat but was constitutive in strains containing only vanS(omega)cat. Promoters P(R) and P(H), located upstream from vanR and vanH, respectively, were cloned into a promoter probing vector to study transactivation by chromosomally encoded VanR and VanS. The promoters were inactive in the absence of vanR and vanS, inducible by glycopeptides in the presence of both genes, and constitutively activated by VanR in the absence of VanS. Thus, induction of the vanHAX operon involves an amplification loop resulting from binding of phospho-VanR to the P(R) promoter and increased transcription of the vanR and vanS genes. Full activation of P(R) and P(H) by VanR was observed in the absence of VanS, indicating that the sensor negatively controls VanR in the absence of glycopeptides, presumably by dephosphorylation. Activation of the VanR response regulator in the absence of VanS may involve autophosphorylation of VanR with acetyl phosphate or phosphorylation by a heterologous histidine protein kinase.
KeywordMeSH Terms
Glycopeptides
Serine-Type D-Ala-D-Ala Carboxypeptidase
199.     ( 1996 )

Biochemical and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins.

Applied and environmental microbiology 62 (5)
PMID : 8633865  :   PMC  :   PMC167941    
Abstract >>
A new bacteriocin has been isolated from an Enterococcus faecium strain. The bacteriocin, termed enterocin A, was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and mass spectrometry analysis. By combining the data obtained from amino acid and DNA sequencing, the primary structure of enterocin A was determined. It consists of 47 amino acid residues, and the molecular weight was calculated to be 4,829, assuming that the four cysteine residues form intramolecular disulfide bridges. This molecular weight was confirmed by mass spectrometry analysis. The amino acid sequence of enterocin A shared significant homology with a group of bacteriocins (now termed pediocin-like bacteriocins) isolated from a variety of lactic acid-producing bacteria, which include members of the genera Lactobacillus, Pediococcus, Leuconostoc, and Carnobacterium. Sequencing of the structural gene of enterocin A, which is located on the bacterial chromosome, revealed an N-terminal leader sequence of 18 amino acid residues, which was removed during the maturation process. The enterocin A leader belongs to the double-glycine leaders which are found among most other small nonlantibiotic bacteriocins, some lantibiotics, and colicin V. Downstream of the enterocin A gene was located a second open reading frame, encoding a putative protein of 103 amino acid residues. This gene may encode the immunity factor of enterocin A, and it shares 40% identity with a similar open reading frame in the operon of leucocin AUL 187, another pediocin-like bacteriocin.
KeywordMeSH Terms
200.     ( 1996 )

Evolution of structure and substrate specificity in D-alanine:D-alanine ligases and related enzymes.

Journal of molecular evolution 42 (6)
PMID : 8662022  :  
Abstract >>
The D-alanine:D-alanine-ligase-related enzymes can have three preferential substrate specificities. Usually, these enzymes synthesize D-alanyl-D-alanine. In vancomycin-resistant Gram-positive bacteria, structurally related enzymes synthesize D-alanyl-D-lactate or d-alanyl-d-serine. The sequence of internal fragments of eight structural d-alanine:d-alanine ligase genes from enterococci has been determined. Alignment of the deduced amino acid sequences with those of other related enzymes from Gram-negative and Gram-positive bacteria revealed the presence of four distinct sequence patterns in the putative substrate-binding sites, each correlating with specificity to a particular substrate (D-alanine:D-lactate ligases exhibited two patterns). Phylogenetic analysis showed different clusters. The enterococcal subtree was largely superimposable on that derived from 16S rRNA sequences. In lactic acid bacteria, structural divergence due to differences in substrate specificity was observed. Glycopeptide resistance proteins VanA and VanB, the VanC-type ligases, and DdlA and DdlB from enteric bacteria and Haemophilus influenzae constituted separate clusters.
KeywordMeSH Terms
Evolution, Molecular
201.     ( 1993 )

Characterization of Tn1546, a Tn3-related transposon conferring glycopeptide resistance by synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147.

Journal of bacteriology 175 (1)
PMID : 8380148  :   DOI  :   10.1128/jb.175.1.117-127.1993     PMC  :   PMC196104    
Abstract >>
Sequence determination of the flanking regions of the vancomycin resistance van gene cluster carried by pIP816 in Enterococcus faecium BM4147 revealed similarity to transposons of the Tn3 family. Imperfect inverted repeats (36 of 38 bp) delineated a 10,851-bp element designated Tn1546. The 4-kb region located upstream from the vanR gene contained two open reading frames (ORF) transcribed in opposite directions. The deduced amino acid sequence of ORF1 (988 residues) displayed, respectively, 56 and 42% identity to those of the transposases of Tn4430 from Bacillus thuringiensis and of Tn917 from Enterococcus faecalis. The product of ORF2 (191 residues) was related to the resolvase of Tn917 (33% amino acid identity) and to the Res protein (48%) of plasmid pIP404 from Clostridium perfringens. Tn1546 transposed consecutively in Escherichia coli from plasmid pUC18 into pOX38 and from pOX38 into various sites of pBR329. Transposition was replicative, led to the formation of cointegrates, and produced a 5-bp duplication at the target site. Southern hybridization and DNA amplification revealed the presence of Tn1546-related elements in enterococci highly resistant to glycopeptides. Analysis of sequences surrounding these elements indicated that transposition plays a role in dissemination of the van gene cluster among replicons of human clinical isolates of E. faecium.
KeywordMeSH Terms
202.     ( 1995 )

Identification of chromosomal mobile element conferring high-level vancomycin resistance in Enterococcus faecium.

Antimicrobial agents and chemotherapy 39 (11)
PMID : 8585724  :   DOI  :   10.1128/aac.39.11.2446     PMC  :   PMC162963    
Abstract >>
A clinical isolate of Enterococcus faecium that contains a chromosomally encoded vanA gene cluster, Tn1546::IS1251, transferred vancomycin resistance to the plasmid-free strain Enterococcus faecalis JH2-2 during filter matings. Hybridization of a vanHAXY probe to SmaI restriction-digested genomic DNA separated by pulsed-field gel electrophoresis showed that the vanA gene cluster was located on a 40-kb fragment in the original donor strain and on fragments of different sizes (150 to 450 kb) in the transconjugants. No hybridization to vanA gene cluster probes was obtained with plasmid DNA preparations from the donor or transconjugants. These results suggested that in each case, the van genes had integrated into the recipient chromosome. The transconjugants in turn could act as donors of vancomycin resistance, and resistance was transferable to a Rec- recipient. The results of restriction analyses and DNA hybridizations of genomic DNA from the donor and transconjugants were consistent with the transfer of a mobile element that includes the 12.3-kb Tn1546::IS1251 gene cluster and at least 13 kb of additional DNA. This element has been tentatively designated Tn5482. DNA sequence analysis of a fragment predicted to contain the left end of Tn5482 revealed two insertion sequence-like elements: IS1216V and an apparently truncated IS3-like element. Restriction mapping and DNA hybridization patterns of the van gene clusters of three additional clinical isolates from New York City showed an element similar to Tn5482. Transfer of Tn5482 and related elements may be involved in dissemination of vancomycin resistance.
KeywordMeSH Terms
DNA Transposable Elements
203.     ( 1994 )

Crystallization, characterization and preliminary crystallographic studies of carbamate kinase of Streptococcus faecium.

Journal of molecular biology 235 (4)
PMID : 8308897  :   DOI  :   10.1006/jmbi.1994.1088    
Abstract >>
Crystals of carbamate kinase (E.C.2.7.2.2) suitable for high resolution studies have been obtained, using the hanging drop vapour diffusion technique, with polyethylene glycol 8000 and NaCl as precipitants at pH 6.5 and a temperature of 4 degrees C. Crystals of about 0.3 mm x 0.2 mm x 0.2 mm in size diffract to at least 3.2 A resolution and are stable to X-radiation for more than ten hours. The space group is P2(1)2(1)2(1), with unit cell dimensions a = 84.5 A, b = 99.6 A, c = 173.3 A. Density packing considerations are consistent with the presence of four to five monomers (M(r) of the monomer = 33,000) in the asymmetric unit, two dimers or even a tetramer being favoured by the results of cross-linking experiments of the enzyme in solution.
KeywordMeSH Terms
204.     ( 1996 )

Purification and N-terminal amino acid sequence of Enterocin CRL 35, a 'pediocin-like' bacteriocin produced by Enterococcus faecium CRL 35.

Letters in applied microbiology 22 (6)
PMID : 8695065  :  
Abstract >>
Enterocin CRL 35, a bacteriocin produced by Enterococcus faecium CRL 35 that inhibits food-borne pathogens, was purified by precipitation with (NH4)2SO4, gel filtration, ion exchange and reverse phase chromatography. The partial N-terminal amino acid sequence indicated a strong homology with other 'pediocin-like bacteriocins' previously described.
KeywordMeSH Terms
205.     ( 1993 )

Identification of the satA gene encoding a streptogramin A acetyltransferase in Enterococcus faecium BM4145.

Antimicrobial agents and chemotherapy 37 (10)
PMID : 8257133  :   DOI  :   10.1128/aac.37.10.2119     PMC  :   PMC192238    
Abstract >>
Enterococcus faecium BM4145, a clinical isolate from urine, was resistant to streptogramin group A antibiotics by inactivation. The strain harbored a plasmid containing a gene, satA, responsible for this resistance; this gene was cloned and sequenced. It encoded SatA, a protein deduced to be 23,634 Da in mass and homologous with a new family of chloramphenicol acetyltransferases described in Agrobacterium tumefaciens, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The similarity of SatA to other acetyltransferases, LacA (thiogalactoside acetyltransferase) and CysE (serine acetyltransferase) from E. coli, and to two putative acetyltransferases, NodL from Rhizobium leguminosarum and Urf1 from E. coli, was also observed in a region considered to be the enzyme's active site. Acetylation experiments indicated that acetyl coenzyme A was necessary for SatA activity and that a single acetylated derivative of pristinamycin IIA was produced. Other members of the streptogramin A group such as virginiamycin M and RP54476 were also substrates for the enzyme. We conclude that resistance to the streptogramin A group of antibiotics in E. faecium BM4145 is due to acetylation by an enzyme related to the novel chloramphenicol acetyltransferase family.
KeywordMeSH Terms
206.     ( 1993 )

Characterization of the chromosomal aac(6')-Ii gene specific for Enterococcus faecium.

Antimicrobial agents and chemotherapy 37 (9)
PMID : 8239603  :   DOI  :   10.1128/aac.37.9.1896     PMC  :   PMC188089    
Abstract >>
Chromosomal gene aac(6')-Ii of Enterococcus faecium CIP 54-32, encoding a 6'-N-aminoglycoside acetyltransferase was characterized. The gene was identified as a coding sequence of 549 bp corresponding to a protein with a calculated mass of 20,666 Da. Analysis of the sequence of the deduced protein suggested that it was the second member of a subfamily of AAC(6')-I enzymes. Insertional inactivation of aac(6')-Ii led to aminoglycoside susceptibility of CIP 54-32, suggesting that this gene plays a role in resistance to AAC(6')-I substrates. The gene was detected by DNA hybridization in all 26 strains of E. faecium tested but not in 44 other enterococci of 13 species. These data suggest that the aac(6')-Ii gene is species specific and may be used to identify E. faecium.
KeywordMeSH Terms
Chromosomes, Bacterial
207.     ( 1996 )

A novel tetracycline-resistant determinant, tet(U), is encoded on the plasmid pKq10 in Enterococcus faecium.

Plasmid 35 (2)
PMID : 8700968  :   DOI  :   10.1006/plas.1996.0009    
Abstract >>
Nine tetracycline (Tc)-resistant clinical isolates of Enterococcus faecium were screened for plasmid content using agarose gel electrophoresis. pKQ10, a 1.9-kb plasmid carrying a novel Tc resistance determinant, was isolated from one of the isolates. The nucleotide sequence of this plasmid revealed an open reading frame corresponding to an 11.8-kDa protein and containing 105 amino acid residues. There was some limited similarity between this protein and tet(M), tet(O), tet(Q), tet(S), tetB(P), and otr(A), which overlapped, but did not include, the consensus GTP-binding sequences. The low-level, Tc-resistant determinant of pKQ10, named tet(U), does not appear to correspond to any other known Tc resistance determinant.
KeywordMeSH Terms
Plasmids
208. Reynolds  PE, Depardieu  F, Dutka-Malen  S, Arthur  M, Courvalin  P,     ( 1994 )

Glycopeptide resistance mediated by enterococcal transposon Tn1546 requires production of VanX for hydrolysis of D-alanyl-D-alanine.

Molecular microbiology 13 (6)
PMID : 7854121  :   DOI  :   10.1111/j.1365-2958.1994.tb00497.x    
Abstract >>
Cloning and nucleotide sequencing indicated that transposon Tn1546 from Enterococcus faecium BM4147 encodes a 23,365 Da protein, VanX, required for glycopeptide resistance. The vanX gene was located downstream from genes encoding the VanA ligase and the VanH dehydrogenase which synthesize the depsipeptide D-alanyl-D-lactate (D-Ala-D-Lac). In the presence of ramoplanin, an Enterococcus faecalis JH2-2 derivative producing VanH, VanA and VanX accumulated mainly UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Lac (pentadepsipeptide) and small amounts of UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala (pentapeptide) in the ratio 49:1. Insertional inactivation of vanX led to increased synthesis of pentapeptide with a resulting change in the ratio of pentadepsipeptide: pentapeptide to less than 1:1. Expression of vanX in E. faecalis and Escherichia coli resulted in production of a D,D-dipeptidase that hydrolysed D-Ala-D-Ala. Pentadepsipeptide, pentapeptide and D-Ala-D-Lac were not substrates for the enzyme. These results establish that VanX is required for production of a D,D-dipeptidase that hydrolyses D-Ala-D-Ala, thereby preventing pentapeptide synthesis and subsequent binding of glycopeptides to D-Ala-D-Ala-containing peptidoglycan precursors at the cell surface.
KeywordMeSH Terms
Carbon-Oxygen Ligases
Gene Expression Regulation, Bacterial
Genes, Bacterial
Serine-Type D-Ala-D-Ala Carboxypeptidase
209. Handwerger  S, Skoble  J, Discotto  LF, Pucci  MJ,     ( 1995 )

Heterogeneity of the vanA gene cluster in clinical isolates of enterococci from the northeastern United States.

Antimicrobial agents and chemotherapy 39 (2)
PMID : 7726499  :   DOI  :   10.1128/aac.39.2.362     PMC  :   PMC162544    
Abstract >>
In several strains of Enterococcus faecium isolated in Europe, the cluster of genes encoding high-level resistance to vancomycin (VanA phenotype) resides on a 10.85-kb transposon, Tn1546, or closely related elements. To determine whether Tn1546 was conserved in recent enterococcal isolates from the northeastern United States, seven strains were compared by restriction mapping and DNA hybridization with probes from within the van cluster. Two of the seven strains contained intact Tn1546-like sequences; however, in five of the strains, the organization of the van cluster differed from that of Tn1546. Three of the five strains with variations harbored a novel DNA segment within the van gene cluster. This 1,496-bp segment was similar to IS1165 of Leuconostoc mesenteroides and IS1181 of Staphylococcus aureus and was flanked by 24- and 23-bp imperfect inverted repeats and 8-bp direct repeats. On the basis of these findings, we propose that this element comprises a novel insertion-like sequence, IS1251. Multiple copies of IS1251 were also present at other sites in both resistant and susceptible clinical isolates. Our findings suggest that the van cluster in recent isolates from the northeastern United States differs from that present in the early European VanA phenotype strains.
KeywordMeSH Terms
Carbon-Oxygen Ligases
Genes, Bacterial
Multigene Family
210. Wu  Z, Wright  GD, Walsh  CT,     ( 1995 )

Overexpression, purification, and characterization of VanX, a D-, D-dipeptidase which is essential for vancomycin resistance in Enterococcus faecium BM4147.

Biochemistry 34 (8)
PMID : 7873524  :   DOI  :   10.1021/bi00008a008    
Abstract >>
Vancomycin resistance in Enterococcus faecium requires five genes: vanR, vanS, vanH, vanA, and vanX. The functions and mechanism of four gene products have been known, with VanR/S for signal transduction and transcriptional regulation and VanH/A to synthesize D-Ala-D-lactate. But the function of the fifth gene product, VanX, has been unknown until very recently, when Reynolds and colleagues discovered D-, D-dipeptidase activity in crude extracts of a VanX overproducer [Reynolds, P. E., et al. (1994) Mol. Microbiol. 13, 1065-1070]. We report here the expression of VanX in Escherichia coli and its purification to homogeneity. VanX has been characterized as a metal-activated D-, D-dipeptidase with an optimal pH range of 7-9. The kcat and Km of D-Ala-D-Ala in the absence of divalent metal are determined to be 4.7 s-1 and 1 mM, respectively. However, in the presence of metal cations, kcat can be as high as 788 s-1. VanX is unable to hydrolyze D-Ala-D-lactate, the substituted moiety in the peptidoglycan that leads to vancomycin resistance, not only because of low binding affinity (Ki estimated at 242 mM) but also due to a kcat less than 0.005 s-1. The more than 10(5)-fold differential in catalytic efficiency of VanX for hydrolysis of D-Ala-D-Ala vs D-Ala-D-lactate leaves D-Ala-D-lactate intact for subsequent incorporation into peptidoglycan.(ABSTRACT TRUNCATED AT 250 WORDS)
KeywordMeSH Terms
Serine-Type D-Ala-D-Ala Carboxypeptidase
211. Arthur  M, Depardieu  F, Molinas  C, Reynolds  P, Courvalin  P,     ( 1995 )

The vanZ gene of Tn1546 from Enterococcus faecium BM4147 confers resistance to teicoplanin.

Gene 154 (1)
PMID : 7867956  :   DOI  :   10.1016/0378-1119(94)00851-i    
Abstract >>
A five-gene cluster from Tn1546 confers resistance to the glycopeptide antibiotics vancomycin (Vm) and teicoplanin (Te) by synthesis of pentadepsipeptide peptidoglycan precursors terminating in D-lactate, which replaces D-alanine in the same position of precursors utilized by susceptible enterococci. Cloning and nucleotide sequencing indicated that Tn1546 contains an additional gene, designated vanZ, which confers low-level Te resistance, in the absence of the genes required for pentadepsipeptide synthesis. Analysis of cytoplasmic peptidoglycan precursors, accumulated in the presence of ramoplanin, showed that VanZ-mediated Te resistance does not involve incorporation of a substituent of D-alanine into the precursors.
KeywordMeSH Terms
Genes, Bacterial
212. Poyart  C, Berche  P, Trieu-Cuot  P,     ( 1995 )

Characterization of superoxide dismutase genes from gram-positive bacteria by polymerase chain reaction using degenerate primers.

FEMS microbiology letters 131 (1)
PMID : 7557308  :   DOI  :   10.1016/0378-1097(95)00232-t    
Abstract >>
An internal fragment representing approximately 85% of sod genes from seven Gram-positive bacteria was amplified by using degenerate primers in a polymerase chain reaction assay. The DNA sequences of sod polymerase chain reaction products from Clostridium perfringens, Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes were determined. Comparisons of their deduced amino acid sequences with those of the corresponding regions of the SOD proteins from Bacillus stearothermophilus, Listeria monocytogenes, and Streptococcus mutans revealed strong relatedness. Phylogenetic analysis of SOD peptides showed that members of the genera Streptococcus and those of the genera Enterococcus constitute two well-supported monophyletic groups. The method described in this study provides a means for easy recovery of sod genes and the construction of sod mutants of various Gram-positive pathogens.
KeywordMeSH Terms
213. Miceli de Farias  F, Silva Francisco  M, Nascimento de Sousa Santos  I, Salustiano Marques-Bastos  SL, Lemos Miguel  MA, Mattos Albano  R, de Freire Bastos  MDC,     ( 2019 )

Draft genome sequence of Enterococcus faecium E86, a strain producing broad-spectrum antimicrobial peptides: Description of a novel bacteriocin immunity protein and a novel sequence type.

Journal of global antimicrobial resistance 17 (N/A)
PMID : 31005734  :   DOI  :   10.1016/j.jgar.2019.04.004    
Abstract >>
The aim of this study was to report the draft genome sequence of the bacteriocinogenic strain Enterococcus faecium E86. Bacteriocins are prokaryotic peptides or proteins with antimicrobial activity. The genome information may contribute to the identification of enterocins produced by this strain that exhibit inhibitory activity against the foodborne pathogen Listeria monocytogenes and vancomycin-resistant enterococci (VRE) involved in human infections, among other bacterial genera and species. An Illumina MiSeq platform was used for genome sequencing. De novo assembly of 5 735 838 paired-end reads was done using the A5-miseq pipeline, yielding >300-fold average genome coverage. Genome annotation was performed by the RAST server, and mining of the bacteriocinogenic gene clusters was done using the BAGEL3 and antiSMASH v.4 platforms. The total scaffold size was determined to be 2 689 107 bp, approximately 2.7 Mbp, featuring a G + C content of 38.1%. The genome contains 2858 coding sequences and 74 RNA genes. Genome analyses revealed the presence of: 30 genes involved in drug resistance; 2 bacteriocinogenic gene clusters (for enterocin P and enterocin TW21); EntiTW21, a novel bacteriocin immunity protein and a novel multilocus sequence type (ST1500). This work highlights the potential biotechnological application of this strain for the production of enterocin P, a bacteriocin that can be employed in the food industry as a biopreservative against L. monocytogenes and as an alternative to classical antibiotics against VRE.
KeywordMeSH Terms
Enterocin
Enterococcus faecium
Illumina MiSeq
MLST
VRE
Vancomycin-resistant enterococci
214. Rushton-Green  R, Darnell  RL, Taiaroa  G, Carter  GP, Cook  GM, Morgan  XC,     ( 2019 )

Agricultural Origins of a Highly Persistent Lineage of Vancomycin-Resistant Enterococcus faecalis in New Zealand.

Applied and environmental microbiology 85 (13)
PMID : 31028029  :   DOI  :   10.1128/AEM.00137-19     PMC  :   PMC6581176    
Abstract >>
Enterococcus faecalis and Enterococcus faecium are human and animal gut commensals. Vancomycin-resistant enterococci (VRE) are important opportunistic pathogens with limited treatment options. Historically, the glycopeptide antibiotics vancomycin and avoparcin selected for the emergence of vancomycin resistance in human and animal isolates, respectively, resulting in global cessation of avoparcin use between 1997 and 2000. To better understand human- and animal-associated VRE strains in the postavoparcin era, we sequenced the genomes of 231 VRE isolates from New Zealand (NZ; 75 human clinical, 156 poultry) cultured between 1998 and 2009. E. faecium lineages and their antibiotic resistance carriage patterns strictly delineated between agricultural and human reservoirs, with bacitracin resistance ubiquitous in poultry but absent in clinical E. faecium strains. In contrast, one E. faecalis lineage (ST108) predominated in both poultry and human isolates in the 3 years following avoparcin discontinuation. Both phylogenetic and antimicrobial susceptibility (i.e., ubiquitous bacitracin resistance in both poultry and clinical ST108 isolates) analyses suggest an agricultural origin for the ST108 lineage. VRE isolate resistomes were carried on multiple, heterogeneous plasmids. In some isolate genomes, bacitracin, erythromycin, and vancomycin resistance elements were colocalized, indicating multiple potentially linked selection mechanisms.IMPORTANCE Historical antimicrobial use in NZ agriculture has driven the evolution of ST108, a VRE lineage carrying a range of clinically relevant antimicrobial resistances. The persistence of this lineage in NZ for over a decade indicates that coselection may be an important stabilizing mechanism for its persistence.
KeywordMeSH Terms
Enterococcus
VRE
antimicrobial
bacitracin
faecalis
faecium
genomics
phylogeny
vancomycin
Enterococcus
VRE
antimicrobial
bacitracin
faecalis
faecium
genomics
phylogeny
vancomycin
215. Moon  TM, D'Andréa  ?D, Lee  CW, Soares  A, Jakoncic  J, Desbonnet  C, Garcia-Solache  M, Rice  LB, Page  R, Peti  W,     ( 2018 )

The structures of penicillin-binding protein 4 (PBP4) and PBP5 from Enterococci provide structural insights into �]-lactam resistance.

The Journal of biological chemistry 293 (48)
PMID : 30355734  :   DOI  :   10.1074/jbc.RA118.006052     PMC  :   PMC6290140    
Abstract >>
The final steps of cell-wall biosynthesis in bacteria are carried out by penicillin-binding proteins (PBPs), whose transpeptidase domains form the cross-links in peptidoglycan chains that define the bacterial cell wall. These enzymes are the targets of �]-lactam antibiotics, as their inhibition reduces the structural integrity of the cell wall. Bacterial resistance to antibiotics is a rapidly growing concern; however, the structural underpinnings of PBP-derived antibiotic resistance are poorly understood. PBP4 and PBP5 are low-affinity, class B transpeptidases that confer antibiotic resistance to Enterococcus faecalis and Enterococcus faecium, respectively. Here, we report the crystal structures of PBP4 (1.8 ?) and PBP5 (2.7 ?) in their apo and acyl-enzyme complexes with the �]-lactams benzylpenicillin, imipenem, and ceftaroline. We found that, although these three �]-lactams adopt geometries similar to those observed in other class B PBP structures, there are small, but significant, differences that likely decrease antibiotic efficacy. Further, we also discovered that the N-terminal domain extensions in this class of PBPs undergo large rigid-body rotations without impacting the structure of the catalytic transpeptidase domain. Together, our findings are defining the subtle functional and structural differences in the Enterococcus PBPs that allow them to support transpeptidase activity while also conferring bacterial resistance to antibiotics that function as substrate mimics.
KeywordMeSH Terms
ESKAPE pathogen
Enterococcus
antibiotic action
antibiotic resistance
crystal structure
enzyme structure
penicillin-binding proteins
transpeptidase
β-lactam
ESKAPE pathogen
Enterococcus
antibiotic action
antibiotic resistance
crystal structure
enzyme structure
penicillin-binding proteins
transpeptidase
β-lactam
beta-Lactam Resistance
216. Hung  WW, Chen  YH, Tseng  SP, Jao  YT, Teng  LJ, Hung  WC,     ( 2019 )

Using groEL as the target for identification of Enterococcus faecium clades and 7 clinically relevant Enterococcus species.

Journal of microbiology, immunology, and infection = Wei mian yu gan ran za zhi 52 (2)
PMID : 30473144  :   DOI  :   10.1016/j.jmii.2018.10.008    
Abstract >>
Accurate identification is important for effective treatment because Enterococcus species have talents to cope with various antibiotics either by intrinsic resistance or by acquisition of mobile genetic elements. The groEL gene is a permissive target in identification of bacteria. We aimed to develop simple assays based on groEL for identification of enterococci. We continued our previous work and determined groEL gene sequences of Enterococcus species isolated from clinical specimens. Phylogenetic analysis based on groEL revealed that each strain clustered well with their reference strains (bootstrap value 100%), in which Enterococcusfaecium and Enterococcusgallinarum could be split into two clades. The divergence of E. faecium was coincident with hospital-associated clade, known as clade A, and community-associated clade, known as clade B. A PCR-restriction fragment length polymorphism (PCR-RFLP) assay was therefore designed to differentiate the two E. faecium clades, based on the specific RsaI cutting sites present in the two clades. To differentiate 7 clinical relevant Enterococcus species, the multiplex PCR assay was designed to identify Enterococcusavium, Enterococcuscasseliflavus, Enterococcusfaecalis, E. faecium, E. gallinarum, Enterococcushirae and Enterococcusraffinosus. Specificity was tested with other Enterococcus species including Enterococcuscecorum, Enterococcusdurans and Enterococcusmundtii. None of these bacterial species generated products of similar size to those of the seven Enterococcus species. The simple PCR-RFLP and multiplex PCR assays on the basis of groEL gene provided an alternative way to identify Enterococcus species.
KeywordMeSH Terms
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Phylogeny
217. Zhou  W, Gao  S, Xu  H, Zhang  Z, Chen  F, Shen  H, Zhang  C,     ( 2019 )

Distribution of the optrA gene in Enterococcus isolates at a tertiary care hospital in China.

Journal of global antimicrobial resistance 17 (N/A)
PMID : 30641287  :   DOI  :   10.1016/j.jgar.2019.01.001    
Abstract >>
Linezolid-resistant Enterococcus have spread worldwide. This study investigated the prevalence of linezolid-non-susceptible Enterococcus (LNSE) and the potential mechanism and molecular epidemiology of LNSE isolates from Nanjing, China. Linezolid susceptibility of 2555 Enterococcus was retrospectively determined by Etest. Vancomycin and teicoplanin MICs were determined for LNSE by Etest. PCR and DNA sequencing were used to investigate the potential molecular mechanism. Clonal relatedness between LNSE isolates was analysed by MLST. WGS was also performed. A total of 27 Enterococcus isolates (24 Enterococcus faecalis, 3 Enterococcus faecium) with linezolid MICs of 4-48�gg/mL were identified, among which 20 E. faecalis and 3 E. faecium were positive for optrA. No mutations were found in genes encoding domain V of 23S rRNA or ribosomal proteins L3/L4; the cfr gene was not found. The 24 linezolid-non-susceptible E. faecalis were classified into eight STs (ST16, ST480, ST476, ST631, ST585, ST428, ST25 and ST689). The three linezolid-non-susceptible E. faecium were classified as ST17, ST400 and ST195. Comparison of the deduced OptrA amino acid sequences of the 23 optrA-positive isolates by PCR-based sequencing and WGS with that of the original OptrA from E. faecalis E349 revealed seven variants (KD, EDP, EDM, D, EDD, RDK and DP) in 16 isolates, with no mutations in the remaining 7 isolates. optrA was found downstream of fexA by searching the pE349 sequence based on WGS data. Emergence of LNSE with optrA-mediated resistance and clonal dissemination of ST16 E. faecalis in our hospital may pose a potential public-health threat.
KeywordMeSH Terms
Cfr gene
Enterococcus
Linezolid
Non-susceptible
OptrA
Resistance
WGS
Cfr gene
Enterococcus
Linezolid
Non-susceptible
OptrA
Resistance
WGS
218. Friedrich  K, Brombach  M, Pon  CL,     ( 1988 )

Identification, cloning and sequence of the Streptococcus faecium infB (translational initiation factor IF2) gene.

Molecular & general genetics : MGG 214 (3)
PMID : 3063954  :   DOI  :   10.1007/bf00330501    
Abstract >>
The structural gene for translational initiation factor IF2 (infB) from Streptococcus faecium was identified by cross-hybridization with DNA probes derived from the corresponding gene of Bacillus stearothermophilus. The entire infB gene (ca. 2.8 kb) was cloned and sequenced. The amino acid sequence deduced from the nucleotide sequence shows that S. faecium initiation factor IF2 (785 amino acids, Mr 86,415) displays extensive homology (ca. 69% and 53%) with the region comprising three-quarters of the molecule from the carboxy-terminus of B. stearothermophilus and Escherichia coli IF2, respectively. The region comprising one-quarter of the molecule from the amino-terminus, on the other hand, does not display any significant homology.
KeywordMeSH Terms
Cloning, Molecular
Genes, Bacterial
219. van Kessel  SP, Frye  AK, El-Gendy  AO, Castejon  M, Keshavarzian  A, van Dijk  G, El Aidy  S,     ( 2019 )

Gut bacterial tyrosine decarboxylases restrict levels of levodopa in the treatment of Parkinson's disease.

Nature communications 10 (1)
PMID : 30659181  :   DOI  :   10.1038/s41467-019-08294-y     PMC  :   PMC6338741    
Abstract >>
Human gut microbiota senses its environment and responds by releasing metabolites, some of which are key regulators of human health and disease. In this study, we characterize gut-associated bacteria in their ability to decarboxylate levodopa to dopamine via tyrosine decarboxylases. Bacterial tyrosine decarboxylases efficiently convert levodopa to dopamine, even in the presence of tyrosine, a competitive substrate, or inhibitors of human decarboxylase. In situ levels of levodopa are compromised by high abundance of gut bacterial tyrosine decarboxylase in patients with Parkinson's disease. Finally, the higher relative abundance of bacterial tyrosine decarboxylases at the site of levodopa absorption, proximal small intestine, had a significant impact on levels of levodopa in the plasma of rats. Our results highlight the role of microbial metabolism in drug availability, and specifically, that abundance of bacterial tyrosine decarboxylase in the proximal small intestine can explain the increased dosage regimen of levodopa treatment in Parkinson's disease patients.
KeywordMeSH Terms
220. Melegh  S, Nyul  A, Kovács  K, Kovács  T, Ghidán  ?, Dombrádi  Z, Szabó  J, Berta  B, Lesinszki  V, Pászti  J, Tóth  ?, Mestyán  G,     ( 2018 )

Dissemination of VanA-Type Enterococcus faecium Isolates in Hungary.

Microbial drug resistance (Larchmont, N.Y.) 24 (9)
PMID : 29750597  :   DOI  :   10.1089/mdr.2017.0296    
Abstract >>
Although vanA carrying Enterococcus faecium human clinical isolates have been rarely found in Hungary before 2012, they have been detected in continuously increasing numbers since then. To identify factors associated with their dissemination, we investigated the clonal relatedness and plasmids of 30 vanA carrying E. faecium isolates originating from different Hungarian healthcare institutions from 2012 to 2014. Molecular typing of the isolates (n = 30) was performed with pulsed-field gel electrophoresis (PFGE), multilocus sequence typing, Tn1546 polymerase chain reaction mapping, plasmid restriction fragment length polymorphism analysis, and sequencing. A single Tn1546 variant was detected in all of the isolates. It harbored IS1251 in the vanS-vanH intergenic region, had an entire deletion of the transposase gene and a partial deletion of the resolvase gene, and was located on a pRUM-like plasmid. Based on PFGE, the isolates could be grouped into 13 pulsotypes. Representative strains of these pulsotypes belonged to ST17, ST18, ST80, ST117, and ST203, which are known to be part of the hospital-adapted clades. The increase in the number of vanA carrying E. faecium clinical isolates in Hungary could be explained by the dissemination of pRUM-like vancomycin resistance plasmids in hospital-adapted clonal lineages.
KeywordMeSH Terms
Enterococcus faecium
molecular epidemiology
vancomycin resistance
221. Miller  OK, Banfield  MJ, Schwarz-Linek  U,     ( 2018 )

A new structural class of bacterial thioester domains reveals a slipknot topology.

Protein science : a publication of the Protein Society 27 (9)
PMID : 30052296  :   DOI  :   10.1002/pro.3478     PMC  :   PMC6194298    
Abstract >>
An increasing number of surface-associated proteins identified in Gram-positive bacteria are characterized by intramolecular cross-links in structurally conserved thioester, isopeptide, and ester domains (TIE proteins). Two classes of thioester domains (TEDs) have been predicted based on sequence with, to date, only representatives of Class I structurally characterized. Here, we present crystal structures of three Class II TEDs from Bacillus anthracis, vancomycin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococcus faecium. These proteins are structurally distinct from Class I TEDs due to a �]-sandwich domain that is inserted into the conserved TED fold to form a slipknot structure. Further, the B. anthracis TED domain is presented in the context of a full-length sortase-anchored protein structure (BaTIE). This provides insight into the three-dimensional arrangement of TIE proteins, which emerge as very abundant putative adhesins of Gram-positive bacteria.
KeywordMeSH Terms
Bacillus anthracis
Enterococcus faecium
Staphylococcus aureus
TIE proteins
bacterial surface proteins
crystal structures
thioester domains
Bacillus anthracis
Enterococcus faecium
Staphylococcus aureus
TIE proteins
bacterial surface proteins
crystal structures
thioester domains
222. Strateva  T, Sirakov  I, Dimov  S, Trifonova  A, Savov  E, Mitov  I,     ( 2018 )

Clonal spread of vanA Enterococcus faecium sequence type 203 in Bulgarian hospitals.

Infectious diseases (London, England) 50 (9)
PMID : 29558857  :   DOI  :   10.1080/23744235.2018.1453946    
Abstract >>
N/A
KeywordMeSH Terms
Phylogeny
223. Morroni  G, Brenciani  A, Antonelli  A, D'Andrea  MM, Di Pilato  V, Fioriti  S, Mingoia  M, Vignaroli  C, Cirioni  O, Biavasco  F, Varaldo  PE, Rossolini  GM, Giovanetti  E,     ( 2018 )

Characterization of a Multiresistance Plasmid Carrying the optrA and cfr Resistance Genes From an Enterococcus faecium Clinical Isolate.

Frontiers in microbiology 9 (N/A)
PMID : 30271398  :   DOI  :   10.3389/fmicb.2018.02189     PMC  :   PMC6142821    
Abstract >>
Enterococcus faecium E35048, a bloodstream isolate from Italy, was the first strain where the oxazolidinone resistance gene optrA was detected outside China. The strain was also positive for the oxazolidinone resistance gene cfr. WGS analysis revealed that the two genes were linked (23.1 kb apart), being co-carried by a 41,816-bp plasmid that was named pE35048-oc. This plasmid also carried the macrolide resistance gene erm(B) and a backbone related to that of the well-known Enterococcus faecalis plasmid pRE25 (identity 96%, coverage 65%). The optrA gene context was original, optrA being part of a composite transposon, named Tn6628, which was integrated into the gene encoding for the �a toxin protein (orf19 of pRE25). The cfr gene was flanked by two ISEnfa5 insertion sequences and the element was inserted into an lnu(E) gene. Both optrA and cfr contexts were excisable. pE35048-oc could not be transferred to enterococcal recipients by conjugation or transformation. A plasmid-cured derivative of E. faecium E35048 was obtained following growth at 42�XC, and the complete loss of pE35048-oc was confirmed by WGS. pE35048-oc exhibited some similarity but also notable differences from pEF12-0805, a recently described enterococcal plasmid from human E. faecium also co-carrying optrA and cfr; conversely it was completely unrelated to other optrA- and cfr-carrying plasmids from Staphylococcus sciuri. The optrA-cfr linkage is a matter of concern since it could herald the possibility of a co-spread of the two genes, both involved in resistance to last resort agents such as the oxazolidinones.
KeywordMeSH Terms
Enterococcus faecium
cfr gene
multiresistance plasmid
optrA gene
oxazolidinone resistance
224. Leinweber  H, Alotaibi  SMI, Overballe-Petersen  S, Hansen  F, Hasman  H, Bortolaia  V, Hammerum  AM, Ingmer  H,     ( 2018 )

Vancomycin resistance in Enterococcus faecium isolated from Danish chicken meat is located on a pVEF4-like plasmid persisting in poultry for 18 years.

International journal of antimicrobial agents 52 (2)
PMID : 29621590  :   DOI  :   10.1016/j.ijantimicag.2018.03.019    
Abstract >>
The occurrence of vancomycin-resistant Enterococcus faecium (VREfm) in food is relevant to public health as foodborne VREfm may colonize the gut of consumers and transfer vancomycin resistance genes to the indigenous gut microbiota. Therefore, we determined occurrence and elucidated genetic traits of VREfm in Danish retail chicken meat. Three out of 40 samples (7.5%) from two slaughterhouses yielded VREfm (vancomycin MIC > 32 mg/L). This is the first report of VREfm in Danish retail poultry meat since 2010 (DANMAP). All three VREfm belonged to the sequence type ST32, cluster type CT1068. Using whole genome sequencing, we detected transposon Tn1546 harbouring the vanA operon encoding vancomycin resistance. The vanA operon was located on a 43.4 kb plasmid highly similar (99.9% identity across 97.5% of the sequence) to pVEF4, which was observed in VREfm in Norwegian poultry in 1998 and in Danish poultry in 2010. The remarkable persistence of a pVEF4-like plasmid in enterococcal populations may be explained by the presence of two independent plasmid stability systems, the �s/�`/�a toxin-antitoxin system and the prgOPN gene cluster. Filter mating experiments showed that the pVEF4-like plasmid could transfer between E. faecium strains in vitro and that transfer occurred concomitantly with a larger, co-residing plasmid. The data presented here indicate that poultry meat constitutes a reservoir of VREfm and further investigations are needed to assess the risk of foodborne transmission to humans.
KeywordMeSH Terms
Enterococcus faecium
Retail chicken meat
ST32 (CT1068)
VRE
pVEF4
vanA
Gene Expression Regulation, Bacterial
225.     ( 2013 )

High abundance and diversity of antimicrobial resistance determinants among early vancomycin-resistant Enterococcus faecium in Poland.

European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology 32 (9)
PMID : 23558365  :   DOI  :   10.1007/s10096-013-1868-y    
Abstract >>
The purpose of this study was to investigate the clonal structure, antimicrobial resistance phenotypes and their determinants among early vancomycin-resistant Enterococcus faecium (VREm) isolates in Poland. Two hundred and eighty-one VREm isolates collected between 1997 and 2005 were studied. VREm isolates were characterised by multilocus sequence typing (MLST). The presence of antimicrobial resistance determinants, transposon-specific genes, IS16 and esp Efm was checked by polymerase chain reaction (PCR). Ciprofloxacin and ampicillin resistance determinants were investigated by sequencing. Two hundred and twenty-two (79 %) and 59 (21 %) VREm isolates were vanA- and vanB-positive, respectively. Among 135 representative isolates, MLST yielded 33 different sequence types (STs), of which 29 were characteristic of hospital-associated E. faecium; 128 (94.8 %) and 123 (91.1 %) isolates harboured the IS16 and esp Efm genes, and all 135 isolates were resistant to ciprofloxacin and ampicillin. Resistance to tetracycline (71.1 % isolates) was mostly associated with tetM (75.0 %) and the concomitant presence of the Tn916 integrase gene. High-level resistance to streptomycin (93.3 % of isolates) and high-level resistance to gentamicin (94.1 % of isolates) were due to ant(6')-Ia and aac(6')-Ie-aph(2?) genes, respectively, the latter of which is known to be located on various Tn4001-type transposons. Fifteen combinations of mutations in the quinolone-determining regions of GyrA and ParC were identified, including changes not previously reported, such as S83F and A84P in GyrA. Twenty-three variants of the penicillin-binding protein PBP5 occurred in the studied group, and novel insertions at amino acid positions 433 and 568 were identified. This analysis revealed the predominance of hospital-associated strains of E. faecium, carrying an abundant and divergent range of resistance determinants among early VREm isolates in Poland.
KeywordMeSH Terms
226. Shin  E, Mduma  S, Keyyu  J, Fyumagwa  R, Lee  Y,     ( 2017 )

An Investigation of Enterococcus Species Isolated from the African Buffalo (Syncerus caffer) in Serengeti National Park, Tanzania.

Microbes and environments 32 (4)
PMID : 29081464  :   DOI  :   10.1264/jsme2.ME17025     PMC  :   PMC5745028    
Abstract >>
We isolated Enterococcus species that colonized in the African buffalo (Syncerus caffer) in order to investigate their genetic relatedness and antimicrobial susceptibility. A total of 219 isolates were obtained and a 16S rRNA gene sequence analysis showed they were classified into Enterococcus avium, E. casseliflavus, E. faecalis, E. faecium, E. hirae, or E. mundtii. Multilocus sequence typing of E. faecalis and E. faecium isolates indicated that some of the isolates showed an evolutionary distance that was far from the primary founders. The antimicrobial susceptibility of the enterococcal isolates suggested that the significant transmission of antimicrobial resistance via human intervention had not yet occurred.
KeywordMeSH Terms
African buffalo
Enterococcus species
MLST
antimicrobial susceptibility
phylogeny
African buffalo
Enterococcus species
MLST
antimicrobial susceptibility
phylogeny
227. Sun  L, Zhang  P, Qu  T, Chen  Y, Hua  X, Shi  K, Yu  Y,     ( 2017 )

Identification of Novel Conjugative Plasmids with Multiple Copies of fosB that Confer High-Level Fosfomycin Resistance to Vancomycin-Resistant Enterococci.

Frontiers in microbiology 8 (N/A)
PMID : 28861056  :   DOI  :   10.3389/fmicb.2017.01541     PMC  :   PMC5559704    
Abstract >>
To further characterize the fosB-carrying plasmids of 19 vancomycin-resistant enterococci, the complete sequences of the fosB- and vanA-containing plasmids of Enterococcus faecium (pEMA120) and E. avium (pEA19081) were obtained by single-molecule, real-time sequencing. We found that these two plasmids are essentially identical (99.99% nucleotide sequence identity), which proved the possibility of interspecies transmission. Comparative analysis of the plasmids revealed that the backbone of pEMA120 is 99% similar to a conjugative fosB-negative E. faecium plasmid, pZB18. There is a traE disrupted in the transfer region of pEMA120, in comparison to pZB18 with an intact traE. The difference of their transfer frequencies between pEMA120 and pZB18 suggests this interruption of traE might affect conjugative transfer. Two copies of the fosB gene linked to a tnpA gene, forming an ISL3-like transposon, were found at separate locations within pEMA120, which had not been reported previously. These two fosB-carrying transposons were confirmed to form circular intermediates by inverse PCR. The hybridization of plasmid DNA digested by BsaI, having restriction site within the fosB sequence, demonstrated that the presence of multiple copies of fosB per plasmid is common. The total copy number of the fosB gene as revealed by qRT-PCR did not correlate with fosfomycin MICs or growth rates at sub-MICs of fosfomycin in different transconjugants. From susceptibility tests, the fosB gene, regardless of the copy number, conferred high fosfomycin MICs that ranged from 16384 to 65536 �gg/ml. This first complete nucleotide sequence of a plasmid carrying two copies of fosB in VRE suggests that the fosB gene can transfer to multiple loci of plasmids by the ISL3 family transposase TnpA, possibly in the form of circular intermediates, leading to the dissemination of high fosfomycin resistance in VRE.
KeywordMeSH Terms
copy number
fosB
fosfomycin resistance
single-molecule real-time sequencing
vancomycin resistant enterococci
copy number
fosB
fosfomycin resistance
single-molecule real-time sequencing
vancomycin resistant enterococci
copy number
fosB
fosfomycin resistance
single-molecule real-time sequencing
vancomycin resistant enterococci
228.     ( 2013 )

Characterization of Enterococcus faecium with macrolide resistance and reduced susceptibility to quinupristin/dalfopristin in a Japanese hospital: detection of extensive diversity in erm(B)-regulator regions.

Microbial drug resistance (Larchmont, N.Y.) 19 (4)
PMID : 23442208  :   DOI  :   10.1089/mdr.2012.0176    
Abstract >>
Cross-resistance to macrolide, lincosamide, and streptogramin B (MLSB) antibiotics is mainly mediated by the erm (erythromycin ribosome methylation) genes that encode 23S rRNA methylases in enterococi, and various mechanisms are involved in the streptogramin B resistance. Prevalence of MLSB resistance and its genetic mechanisms were analyzed for a total of 159 strains of Enterococcus faecium isolated from clinical specimens in a university hospital in Japan from 1997 to 2006. Resistance to erythromycin (EM) and clindamycin was detected in 88.1% and 89.9% of all the strains examined, respectively, and expression of resistance was totally constitutive. Although none of the strain was resistant to quinupristin/dalfopristin (Q/D), 28 strains (17.6%) showed intermediate resistance to Q/D (MIC: 2 �gg/ml). The erm(B) gene was detected in 139 strains (87.4%), and msrC was found in all the strains examined, whereas no other known MLSB resistance genes were identified. The erm(B) regulator region (RR) containing a coding region of the leader peptide was classified into 13 genetic variations (L1-L3, M, S1-S7, D, and R genotypes) in 56 strains. However, no relatedness was identified between the erm(B) RR genotype and EM resistance, or reduced susceptibility to Q/D, although most of Q/D-intermediate strains were assigned to the L1, L2, and S1 genotypes. Q/D-intermediate strains were classified into five multiple-locus variable-number tandem-repeat analysis (MLVA) types, including four types of clonal complex (CC)-C1, five sequence types (STs), including four STs of CC-17, and several resistance gene/virulence factor profiles. The present study revealed the occurrence of Q/D-intermediate E. faecium, which are composed of heterogeneous strains in Japan, and more genetic diversity in the erm(B) RRs than those reported previously.
KeywordMeSH Terms
229.     ( 2013 )

Detection of mobile genetic elements in pediocin PA-1 like producing lactic acid bacteria.

Journal of basic microbiology 53 (7)
PMID : 22915312  :   DOI  :   10.1002/jobm.201200079    
Abstract >>
To evaluate the presence of mobile genetic elements (MGEs) in intergeneric and interspecific pediocin producing lactic acid bacteria (LAB) the flanking regions of the pediocin PA-1/AcH (pediocin PA-1) operon was characterized. In Enterococcus faecium Acr4 and Lactobacillus plantarum Acr2 a variation in the amplicon size in the downstream region of the operon was identified, suggesting a deletion in this region. Beyond that, in pediocin PA-1 encoding plasmids MGEs such as ISLpl1 and mobilization regions were detected by Southern hybridization analysis. Phylogenetic analyses of the E. faecium Acr4 ISLpl1 gene sequence suggested the gene transfer from lactobacilli in the environment. The tyrosine recombinase detected in pediocin plasmids of Pediococcus acidilactici H and K7 indicate a possible transfer of the entire operon among LAB. Since these elements are known to be associated with transfer of genes linked to the bacteriocin production, antibiotic resistance, and sugar utilization, we suggest similar mechanism for natural spread of pediocin PA-1 operon among different bacterial species.
KeywordMeSH Terms
ISlpl1
Lactic acid bacteria
Mobile genetic elements
Pediocin PA-1
Plasmids
Interspersed Repetitive Sequences
230.     ( 2012 )

Identification of VanN-type vancomycin resistance in an Enterococcus faecium isolate from chicken meat in Japan.

Antimicrobial agents and chemotherapy 56 (12)
PMID : 23006756  :   DOI  :   10.1128/AAC.00747-12     PMC  :   PMC3497174    
Abstract >>
Five VanN-type vancomycin-resistant Enterococcus faecium strains were isolated from a sample of domestic chicken meat in Japan. All isolates showed low-level resistance to vancomycin (MIC, 12 mg/liter) and had the same pulsed-field gel electrophoresis profile. The vancomycin resistance was encoded on a large plasmid (160 kbp) and was expressed constitutively. The VanN-type resistance operon was identical to the first resistance operon to be reported, with the exception of a 1-bp deletion in vanT(N) and a 1-bp substitution in vanS(N).
KeywordMeSH Terms
231.     ( 1998 )

Differences in the occurrence of two base pair variants of Tn1546 from vancomycin-resistant enterococci from humans, pigs, and poultry.

Antimicrobial agents and chemotherapy 42 (9)
PMID : 9736587  :   PMC  :   PMC105857    
Abstract >>
N/A
KeywordMeSH Terms
DNA Transposable Elements
232.     ( 2012 )

Different genetic supports for the tet(S) gene in Enterococci.

Antimicrobial agents and chemotherapy 56 (11)
PMID : 22908170  :   DOI  :   10.1128/AAC.00758-12     PMC  :   PMC3486547    
Abstract >>
The diversity of tet(S) genetic contexts of 13 enterococci from human, animal, and environmental samples from different geographical areas is reported. The tet(S) gene was linked to either CTn6000 variants of chromosomal location or composite platforms flanked by IS1216 located on plasmids (?40 to 115 kb). The comparative analysis of all tet(S) genetic elements available in the GenBank databases suggests that CTn6000 might be the origin of a variety of tet(S)-carrying platforms that were mobilized to different plasmids.
KeywordMeSH Terms
DNA Transposable Elements
Genes, Bacterial
Plasmids
233.     ( 1998 )

Genetic linkage and cotransfer of a novel, vanB-containing transposon (Tn5382) and a low-affinity penicillin-binding protein 5 gene in a clinical vancomycin-resistant Enterococcus faecium isolate.

Journal of bacteriology 180 (17)
PMID : 9721279  :   PMC  :   PMC107451    
Abstract >>
Mechanisms for the intercellular transfer of VanB-type vancomycin resistance determinants and for the almost universal association of these determinants with those for high-level ampicillin resistance remain poorly defined. We report the discovery of Tn5382, a ca. 27-kb putative transposon encoding VanB-type glycopeptide resistance in Enterococcus faecium. Open reading frames internal to the right end of Tn5382 and downstream of the vanXB dipeptidase gene exhibit significant homology to genes encoding the excisase and integrase of conjugative transposon Tn916. The ends of Tn5382 are also homologous to the ends of Tn916, especially in regions bound by the integrase enzyme. PCR amplification experiments indicate that Tn5382 excises to form a circular intermediate in E. faecium. Integration of Tn5382 in the chromosome of E. faecium C68 has occurred 113 bp downstream of the stop codon for the pbp5 gene, which encodes high-level ampicillin resistance in this clinical isolate. Transfer of vancomycin, ampicillin, and tetracycline resistance from C68 to an E. faecium recipient strain occurs at low frequency in vitro and is associated with acquisition of a 130- to 160-kb segment of DNA that contains Tn5382, the pbp5 gene, and its putative repressor gene, psr. The interenterococcal transfer of this large chromosomal element appears to be the primary mechanism for vanB operon spread in northeast Ohio. These results expand the known family of Tn916-related transposons, suggest a mechanism for vanB operon entry into and dissemination among enterococci, and provide an explanation for the nearly universal association of vancomycin and high-level ampicillin resistance in clinical E. faecium strains.
KeywordMeSH Terms
DNA Transposable Elements
Genetic Linkage
Hexosyltransferases
Peptidyl Transferases
234.     ( 1998 )

In vivo testing of an Enterococcus faecalis efaA mutant and use of efaA homologs for species identification.

FEMS immunology and medical microbiology 21 (4)
PMID : 9753005  :   DOI  :   10.1111/j.1574-695X.1998.tb01180.x    
Abstract >>
Disruption of the previously described efaA (from Enterococcus faecalis antigen A) gene was generated in E. faecalis strain OG1RF and loss of an 37-kDa immunoreactive band from the mutant was demonstrated in Western blots. In a mouse peritonitis model, mice infected with the efaAfs (fs=from Enterococcus faecalis) mutant showed more prolonged survival than mice infected with the parent strain OG1RF. These results suggest that efaAfs encodes a function important for infection of mice by enterococci. An efaA-like gene was also identified in E. faecium DNA libraries and its deduced amino acid sequence showed 73% similarity to EfaA of E. faecalis and 42-63% similarities to a group of streptococcal virulence and adhesion associated proteins that are components of ATP-binding cassette transport systems. Intragenic probes representing efaAfs, recAfs, efaAfm (fm=from E. faecium) and gyrAfm were tested for their ability to identify E. faecalis and E. faecium using colony lysates of 133 enterococci and one Streptococcus sp. Probes of E. faecium and E. faecalis origin hybridized to all isolates of E. faecium and E. faecalis, respectively, regardless of their clinical source but not to any of 29 other enterococci. These results suggest that the use of gene probes may prove helpful in identification of isolates of E. faecium and E. faecalis.
KeywordMeSH Terms
Mutation
235.     ( 1998 )

The structure of VanX reveals a novel amino-dipeptidase involved in mediating transposon-based vancomycin resistance.

Molecular cell 2 (1)
PMID : 9702193  :  
Abstract >>
VanX is a zinc-dependent D-alanyl-D-alanine dipeptidase that is a critical component in a system that mediates transposon-based vancomycin resistance in enterococci. It is also a key drug target in circumventing clinical vancomycin resistance. The structure of VanX from E. faecium has been solved by X-ray crystallography and reveals a Zn(2+)-dipeptidase with a unique overall fold and a well-defined active site confined within a cavity of limited size. The crystal structures of VanX, the VanX:D-alanyl-D-alanine complex, the VanX:D-alanine complex, and VanX in complex with phosphonate and phosphinate transition-state analog inhibitors, are also presented at high resolution. Structural homology searches of known structures revealed that the fold of VanX is similar to those of two proteins: the N-terminal fragment of murine Sonic hedgehog and the Zn(2+)-dependent N-acyl-D-alanyl-D-alanine carboxypeptidase of S. albus G.
KeywordMeSH Terms
Drug Resistance, Microbial
Protein Conformation
Serine-Type D-Ala-D-Ala Carboxypeptidase
Trans-Activators
236.     ( 1998 )

Internal size variations in Tn1546-like elements due to the presence of IS1216V.

FEMS microbiology letters 169 (2)
PMID : 9868780  :   DOI  :   10.1111/j.1574-6968.1998.tb13339.x    
Abstract >>
In this study, internal size variations in the VanA gene cluster Tn1546, encoding resistance to glycopeptides, is described. Studies of previously uncharacterized size variations of an internal region, encoding the vanX and vanY genes of Tn1546, revealed that these variations were due to the presence of the IS sequence, IS1216V. This IS sequence has previously been found integrated in Tn1546. Integration of the IS1216V element created both deletions and a duplication in a non-essential region of Tn1546. In several isolates, the entire vanY gene was deleted, proving that this gene is non-essential for vancomycin resistance.
KeywordMeSH Terms
Genes, Bacterial
237.     ( 1998 )

Purification and genetic characterization of enterocin I from Enterococcus faecium 6T1a, a novel antilisterial plasmid-encoded bacteriocin which does not belong to the pediocin family of bacteriocins.

Applied and environmental microbiology 64 (12)
PMID : 9835578  :   PMC  :   PMC90938    
Abstract >>
Enterocin I (ENTI) is a novel bacteriocin produced by Enterococcus faecium 6T1a, a strain originally isolated from a Spanish-style green olive fermentation. The bacteriocin is active against many olive spoilage and food-borne gram-positive pathogenic bacteria, including clostridia, propionibacteria, and Listeria monocytogenes. ENTI was purified to homogeneity by ammonium sulfate precipitation, binding to an SP-Sepharose fast-flow column, and phenyl-Sepharose CL-4B and C2/C18 reverse-phase chromatography. The purification procedure resulted in a final yield of 954% and a 170,000-fold increase in specific activity. The primary structure of ENTI was determined by amino acid and nucleotide sequencing. ENTI consists of 44 amino acids and does not show significant sequence similarity with any other previously described bacteriocin. Sequencing of the entI structural gene, which is located on the 23-kb plasmid pEF1 of E. faecium 6T1a, revealed the absence of a leader peptide at the N-terminal region of the gene product. A second open reading frame, ORF2, located downstream of entI, encodes a putative protein that is 72.7% identical to ENTI. entI and ORF2 appear to be cotranscribed, yielding an mRNA of ca. 0.35 kb. A gene encoding immunity to ENTI was not identified. However, curing experiments demonstrated that both enterocin production and immunity are conferred by pEF1.
KeywordMeSH Terms
238.     ( 1998 )

Carbamate kinase from Enterococcus faecalis and Enterococcus faecium--cloning of the genes, studies on the enzyme expressed in Escherichia coli, and sequence similarity with N-acetyl-L-glutamate kinase.

European journal of biochemistry 253 (1)
PMID : 9578487  :   DOI  :   10.1046/j.1432-1327.1998.2530280.x    
Abstract >>
Carbamate kinase (CK) catalyzes the reversible reaction NH2COO- + ATP <--> NHCOOPO3(2-) + ADP, serving to synthesize ATP from carbamoyl phosphate in those microorganisms that derive energy from anaerobic arginine degradation via the arginine dihydrolase pathway. We report here the cloning and sequencing of the CK gene from Enterococcus faecalis and Enterococcus faecium and we demonstrate that the amino acid sequence of CK is identical in the two species. The enzyme, expressed and isolated from Escherichia coli using simple purification procedures, was used to generate crystals suitable for X-ray studies and to investigate the utilization by CK of bicarbonate and other carbamate analogs. CK had a bicarbonate-dependent ATPase activity and, therefore, is able to synthesize carboxyphosphate, an unstable compound that is an intermediate in the reactions catalyzed by carbamoyl-phosphate synthetase (CPS) and by biotin carboxylase. Other functional similarities with CPS include the utilization of acetate by CK with a similarly high Km and the similar Km values of CK for carbamate and of CPS for bicarbonate. Enterococcal CK was inhibited by adenosine(5')pentaphospho(5')adenosine (Ap5A) and Ap6A and, less powerfully, by Ap4A, whereas Ap3A is essentially non-inhibitory. Thus, inhibition by Ap5A seems not to be a valid criterion to differentiate between CK and CPS, for the two enzymes can be inhibited by Ap5A. All these results support the relatedness of CK and CPS. Finally, we used limited proteolysis: (a) to localize the epitopes for monoclonal antibodies obtained against CK; (b) to demonstrate the importance of the C-terminus for enzyme activity; and (c) to show that Arg158 is highly exposed and may be essential for activity. Comparison of the sequence of CK with known protein sequences demonstrates considerable similarity of CK with bacterial N-acetylglutamate kinases, strongly suggesting that these two enzymes may share a similar structure and the same catalytic mechanism.
KeywordMeSH Terms
Genes, Bacterial
239.     ( 1998 )

Enterocins L50A and L50B, two novel bacteriocins from Enterococcus faecium L50, are related to staphylococcal hemolysins.

Journal of bacteriology 180 (8)
PMID : 9555877  :   PMC  :   PMC107121    
Abstract >>
Enterocin L50 (EntL50), initially referred to as pediocin L50 (L. M. Cintas, J. M. Rodr?guez, M. F. Fern?ndez, K. Sletten, I. F. Nes, P. E. Hern?ndez, and H. Holo, Appl. Environ. Microbiol. 61:2643-2648, 1995), is a plasmid-encoded broad-spectrum bacteriocin produced by Enterococcus faecium L50. It has previously been purified from the culture supernatant and partly sequenced by Edman degradation. In the present work, the nucleotide sequence of the EntL50 locus was determined, and several putative open reading frames (ORFs) were identified. Unexpectedly, two ORFs were found to encode EntL50-like peptides. These peptides, termed enterocin L50A (EntL50A) and enterocin L50B (EntL50B), have 72% sequence identity and consist of 44 and 43 amino acids, respectively. Interestingly, a comparison of the deduced sequences of EntL50A and EntL50B with the corresponding sequences obtained by Edman degradation shows that these bacteriocins, in contrast to other peptide bacteriocins, are secreted without an N-terminal leader sequence or signal peptide. Expression in vivo and in vitro transcription/translation experiments demonstrated that entL50A and entL50B are the only genes required to obtain antimicrobial activity, strongly indicating that their bacteriocin products are not posttranslationally modified. Both bacteriocins possess antimicrobial activity on their own, with EntL50A being the most active. In addition, when the two bacteriocins were combined, a considerable synergism was observed, especially with some indicator strains. Even though the enterocins in some respects are similar to class II bacteriocins, several conserved features common to class II bacteriocins are absent from the EntL50 system. The enterocins have more in common with members of a small group of cytolytic peptides secreted by certain staphylococci. We therefore propose that the enterocins L50A and L50B and the staphylococcal cytolysins together constitute a new family of peptide toxins, unrelated to class II bacteriocins, which possess bactericidal and/or hemolytic activity.
KeywordMeSH Terms
240.     ( 1998 )

DNA sequence variation within vanA, vanB, vanC-1, and vanC-2/3 genes of clinical Enterococcus isolates.

Antimicrobial agents and chemotherapy 42 (1)
PMID : 9449290  :   PMC  :   PMC105485    
Abstract >>
We studied the DNA sequence variation of van genes of 34 isolates of Enterococcus spp. The isolates containing the vanB gene exhibited between 0 and 41 base pair changes per 801 bp studied when the vanB sequences were compared to that of the reference strain Enterococcus faecalis V583. The isolates carrying the vanC-2 gene exhibited between 0 and 23 base pair changes per 346 bp studied when the vanC-2 sequences were compared to that of the reference strain E. casseliflavus ATCC 25788. Little variation was noted in the vanA and vanC-1 genes.
KeywordMeSH Terms

331, Shih-Pin Rd., Hsinchu 30062, Taiwan

Phone: +886-3-5223191

E-mail: bcrcweb@firdi.org.tw

web maintainance: +886-3-5223191 ext 593

Copyright © 2018.BCRC All rights reserved.The duplication or use of information and data such as texts or images or any linkage the website at the "bcrc.firdi.org.tw" is only permitted with the indication of the source or with prior approval by the BCRC(Bioresource Collection and Research Center).