| 1. |
Povelainen M,
Eneyskaya EV,
Kulminskaya AA,
Ivanen DR,
Kalkkinen N,
Neustroev KN,
Miasnikov AN,
( 2003 ) Biochemical and genetic characterization of a novel enzyme of pentitol metabolism: D-arabitol-phosphate dehydrogenase. PMID : 12467497 : DOI : 10.1042/BJ20021096 PMC : PMC1223252 Abstract >>
An enzyme with a specificity that has not been described previously, D-arabitol-phosphate dehydrogenase (APDH), has been purified from cell lysate of Enterococcus avium. SDS/PAGE indicated that the enzyme had a molecular mass of 41+/-2 kDa, whereas a molecular mass of 160+/-5 kDa was observed under non-denaturing conditions, implying that the APDH may exist as a tetramer with identical subunits. Purified APDH was found to have a narrow substrate specificity, converting only D-arabitol 1-phosphate and D-arabitol 5-phosphate into xylulose 5-phosphate and ribulose 5-phosphate, respectively, in the oxidative reaction. Both NAD(+) and NADP(+) were accepted as cofactors. Based on the partial protein sequences, the APDH gene was cloned. Homology comparisons place APDH within the medium-range dehydrogenase family. Unlike most members of this family, APDH requires Mn(2+) but no Zn(2+) for enzymic activity. The DNA sequence surrounding the gene suggests that it belongs to an operon that also contains several components of phosphotransferase system. Both biochemical evidence and protein sequence homology comparisons indicate that similar enzymes are widespread among the Gram-positive bacteria. Their apparent biological role is to participate in arabitol catabolism via the 'arabitol phosphate route', similar to the ribitol and xylitol catabolic routes described previously.
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2. |
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. 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.
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3. |
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. 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.
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4. |
Ménard C,
Roy PH,
Martineau F,
Picard FJ,
Ke D,
( 1999 ) Development of a PCR assay for rapid detection of enterococci. 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.
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5. |
Quesnes G,
Poyart C,
( 2000 ) Sequencing the gene encoding manganese-dependent superoxide dismutase for rapid species identification of enterococci. 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.
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6. |
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. 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.
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7. |
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. 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.
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8. |
Hill JE,
Penny SL,
Crowell KG,
Goh SH,
Hemmingsen SM,
( 2004 ) cpnDB: a chaperonin sequence database. 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.
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9. |
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. 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.
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10. |
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. 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.
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11. |
Tamura T,
Noda M,
Ozaki M,
Maruyama M,
Matoba Y,
Kumagai T,
Sugiyama M,
( 2010 ) Establishment of an efficient fermentation system of gamma-aminobutyric acid by a lactic acid bacterium, Enterococcus avium G-15, isolated from carrot leaves. PMID : 20930374 : DOI : 10.1248/bpb.33.1673 Abstract >>
In the present study, we successfully isolated a carrot leaf-derived lactic acid bacterium that produces gamma-aminobutyric acid (GABA) from monosodium L-glutamate (L-MSG) at a hyper conversion rate. The GABA-producing bacterium, identified as Enterococcus (E.) avium G-15, produced 115.7��6.4 g/l GABA at a conversion rate of 86.0��5.0% from the added L-MSG under the optimum culture condition by a continuous L-MSG feeding method using a jar-fermentor, suggesting that the bacterium displays a great potential ability for the commercial-level fermentation production of GABA. Using the reverse transcription polymerase chain reaction (RT-PCR) method, we analyzed the expression of genes for the GABA transporter and glutamate decarboxylase, designated gadT and gadG, respectively, which were cloned from the E. avium G-15 chromosome. Both genes were expressed even without the added L-MSG, but their expression was enhanced by the addition of L-MSG.
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12. |
Birri DJ,
Brede DA,
Forberg T,
Holo H,
Nes IF,
( 2010 ) Molecular and genetic characterization of a novel bacteriocin locus in Enterococcus avium isolates from infants. PMID : 19933345 : DOI : 10.1128/AEM.01597-09 PMC : PMC2805230 Abstract >>
Enterococci are among the most common human intestinal lactic acid bacteria, and they are known to produce bacteriocins. In this study, fecal enterococci were isolated from infants and screened for bacteriocin production. Bacteriocin-producing Enterococcus avium isolates were obtained, and a new pediocin-like bacteriocin was purified and characterized. This bacteriocin, termed avicin A, was found to be produced by isolates from two healthy infants. It was purified to homogeneity from culture supernatant by ion-exchange and reversed-phase chromatography, and part of its amino acid sequence was obtained. The sequence of a 7-kb DNA fragment of a bacteriocin locus was determined by PCR and DNA sequencing. The bacteriocin locus was organized into four operon-like structures consisting of (i) the structural genes encoding avicin A and its immunity protein, (ii) a divergicin-like bacteriocin (avicin B) gene, (iii) an ABC bacteriocin transporter gene and two regulatory genes (histamine protein kinase- and response regulator-encoding genes), and (iv) induction peptide pheromone- and transport accessory protein-encoding genes. It was shown that the production of avicin A was regulated by the peptide pheromone-inducible regulatory system. Avicin A shows very high levels of similarity to mundticin KS and enterocin CRL35. This bacteriocin showed strong antimicrobial activity against many species of Gram-positive bacteria, including the food-borne pathogen Listeria monocytogenes. The avicin A locus is the first bacteriocin locus identified in E. avium to be characterized at the molecular level.
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13. |
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. 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.
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14. |
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. 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.
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15. |
( 1996 ) Evolution of structure and substrate specificity in D-alanine:D-alanine ligases and related enzymes. 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.
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16. |
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. 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.
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17. |
Yu T,
Li L,
Zhao Q,
Wang P,
Zuo X,
( 2019 ) Complete genome sequence of bile-isolated Enterococcus avium strain 352. PMID : 31049078 : DOI : 10.1186/s13099-019-0294-9 PMC : PMC6480912 Abstract >>
Enterococcus avium is a Gram-positive pathogenic bacterium belonging to the family Enterobacteriaceae. E. avium can cause bacteremia, peritonitis, and intracranial suppurative infection. However, the mechanism of its pathogenesis and its adaptation to a special niche is still unclear. In this study, the E. avium strain 352 was isolated from human bile and whole genome sequencing was performed. The E. avium strain 352 consists of a circular 4,794,392 bp chromosome as well as an 87,705 bp plasmid. The GC content of the chromosome is 38.98%. There are 4905 and 99 protein coding sequences in the chromosome and the plasmid, respectively. The genome of the E. avium strain 352 contains number of genes reported to be associated with bile adaption, including bsh, sbcC, mutS, nifI, galU, and hupB. There are also several virulence-associated genes including esp, fss1, fss3, ecbA, bsh, lap, clpC, clpE, and clpP. This study demonstrates the presence of various virulence factors of the E. avium strain 352, which has the potential to cause infections. Moreover, the genes involved in bile adaption might contribute to its ability to live in bile. Further comparative genomic studies would help to elucidate the evolution of pathogenesis of E. avium.
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18. |
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. 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.
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19. |
Cai J,
Schwarz S,
Chi D,
Wang Z,
Zhang R,
Wang Y,
( 2019 ) Faecal carriage of optrA-positive enterococci in asymptomatic healthy humans in Hangzhou, China. PMID : 30076974 : DOI : 10.1016/j.cmi.2018.07.025 Abstract >>
To investigate the faecal carriage of optrA-positive enterococci among asymptomatic healthy humans in Hangzhou, China, and to characterize the genetic context of optrA. A total of 3458 stool samples from healthy individuals were collected and cultured on a selective medium containing 10 mg/L florfenicol and resulting enterococci were screened for the presence of optrA by PCR. OptrA variants were determined by amino acid sequence comparison with the original OptrA from Enterococcus faecalis E349. Whole genome sequencing and PCR mapping were performed to obtain and analyse the genetic environment of optrA. Similar optrA carriage rates (?3.5%) were detected in samples from adults (55/1558) and children (66/1900). Linezolid resistance rates for E. faecalis, Enterococcus faecium and other Enterococcus species were 58.5% (38/65), 42.3% (11/26) and 0% (0/31), respectively. Nineteen OptrA variants exhibiting different linezolid MICs were identified. Isolates carrying wild-type OptrA and variants RDK, KLDP, KD, D, RDKP, and EDP generally demonstrated linezolid MICs ?8 mg/L. The OptrA variants, with fexA upstream and erm(A) downstream, were flanked by IS1216E at one or both ends. The fexA-optrA(wild-type) was located downstream of a Tn554 transposon, and was inserted into the radC gene. The EDM variant was detected in 31/73 enterococci with linezolid MICs ?4 mg/L. Despite the variable genetic context, Tn558-araC-optrA(EDM)-erm(A)-met was the most common gene array. This study revealed a correlation between linezolid MIC, genetic context and OptrA variant. Intestinal colonization of healthy individuals by optrA-positive enterococci is a concern, and active epidemiological surveillance of optrA is warranted.
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20. |
( 2012 ) Use of tuf as a target for sequence-based identification of Gram-positive cocci of the genus Enterococcus, Streptococcus, coagulase-negative Staphylococcus, and Lactococcus. PMID : 23181410 : DOI : 10.1186/1476-0711-11-31 PMC : PMC3533577 Abstract >>
Accurate identification of isolates belonging to genus Enterococcus, Streptococcus, coagulase-negative Staphylococcus, and Lactococcus at the species level is necessary to provide a better understanding of their pathogenic potential, to aid in making clinical decisions, and to conduct epidemiologic investigations,especially when large blind samples must be analyzed. It is useful to simultaneously identify species in different genera using a single primer pair. We developed a primer pair based on the tuf gene (encoding elongation factor) sequence to identify 56 Gram-positive cocci isolates. The target sequences were amplified from all 56 samples. The sequencing results and the phylogenetic tree derived from the partial tuf gene sequences identified the isolates as three enterococcal species, two lactococcal species, two staphylococcal species, and six streptococcal species, as well as eight isolates that were novel species of the genus Streptococcus. Partial gene sequence analysis of the sodA, dnaK, and 16S RNA genes confirmed the results obtained by tuf gene sequencing. Based on the uniform amplification of the tuf gene from all samples and the ability to identify all isolates at both the genus and species levels, we conclude that the primer pair developed in this research provides a powerful tool for identifying these organisms in clinical laboratories where large blind samples are used.
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