| 1. |
Møretrø T,
Hermansen L,
Holck AL,
Sidhu MS,
Rudi K,
Langsrud S,
( 2003 ) Biofilm formation and the presence of the intercellular adhesion locus ica among staphylococci from food and food processing environments. PMID : 12957956 : DOI : 10.1128/aem.69.9.5648-5655.2003 PMC : PMC194930 Abstract >>
In clinical staphylococci, the presence of the ica genes and biofilm formation are considered important for virulence. Biofilm formation may also be of importance for survival and virulence in food-related staphylococci. In the present work, staphylococci from the food industry were found to differ greatly in their abilities to form biofilms on polystyrene. A total of 7 and 21 of 144 food-related strains were found to be strong and weak biofilm formers, respectively. Glucose and sodium chloride stimulated biofilm formation. The biofilm-forming strains belonged to nine different coagulase-negative species of Staphylococcus. The icaA gene of the intercellular adhesion locus was detected by Southern blotting and hybridization in 38 of 67 food-related strains tested. The presence of icaA was positively correlated with strong biofilm formation. The icaA gene was partly sequenced for 22 food-related strains from nine different species of Staphylococcus, and their icaA genes were found to have DNA similarities to previously sequenced icaA genes of 69 to 100%. Northern blot analysis indicated that the expression of the ica genes was higher in strong biofilm formers than that seen with strains not forming biofilms. Biofilm formation on polystyrene was positively correlated with biofilm formation on stainless steel and with resistance to quaternary ammonium compounds, a group of disinfectants.
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2. |
Morikawa K,
Inose Y,
Okamura H,
Maruyama A,
Hayashi H,
Takeyasu K,
Ohta T,
( 2003 ) A new staphylococcal sigma factor in the conserved gene cassette: functional significance and implication for the evolutionary processes. PMID : 12875655 : Abstract >>
Staphylococcus aureus is a major human pathogen and causes a serious hospital infection due to the acquired multidrug resistance. Unlike the well-studied bacteria such as Escherichia coli and Bacillus subtilis, which have seven and 18 sigma factors, respectively, only two sigma factors have been known for S. aureus. We searched for possible sigma factor genes by examining the S. aureus genome with a special attention to the gene arrangement around the sigma factor genes of a close relative, B. subtilis. A new sigma factor gene was identified in Staphylococcus. The gene constituted a conserved gene cluster with other genes including translation- and transcription-related genes. Phylogenetic analysis and comparison of the gene sequences among species indicated that the staphylococcal sigma factor originated from a common ancestor of B. subtilis SigH. An over-expression of this sigma factor in S. aureus resulted in a drastic induction of the expression of the com operons that encode proteins required for the natural genetic competence. We demonstrated that the newly identified staphylococcal sigma factor participated in a regulatory network of transcription that controlled the genetic competence genes. In our phylogenetic tree, the factor was classified as a single group with a common function.
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3. |
Dufour P,
Jarraud S,
Vandenesch F,
Greenland T,
Novick RP,
Bes M,
Etienne J,
Lina G,
( 2002 ) High genetic variability of the agr locus in Staphylococcus species. PMID : 11807079 : DOI : 10.1128/jb.184.4.1180-1186.2002 PMC : PMC134794 Abstract >>
The agr quorum-sensing and signal transduction system was initially described in Staphylococcus aureus, where four distinct allelic variants have been sequenced. Western blotting suggests the presence of homologous loci in many other staphylococci, and this has been confirmed for S. epidermidis and S. lugdunensis. In this study we isolated agr-like loci from a range of staphylococci by using PCR amplification from primers common to the six published agr sequences and bracketing the most variable region, associated with quorum-sensing specificity. Positive amplifications were obtained from 14 of 34 staphylococcal species or subspecies tested. Sequences of the amplicons identified 24 distinct variants which exhibited extensive sequence divergence with only 10% of the nucleotides absolutely conserved on multiple alignment. This variability involved all three open reading frames involved in quorum sensing and signal transduction. However, these variants retained several protein signatures, including the conserved cysteine residue of the autoinducing peptide, with the exception of S. intermedius of pigeon origin, which contained a serine in place of cysteine at this position. We discuss hypotheses on the mode of action and the molecular evolution of the agr locus based on comparisons between the newly determined sequences.
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4. |
Poyart C,
Quesne G,
Boumaila C,
Trieu-Cuot P,
( 2001 ) Rapid and accurate species-level identification of coagulase-negative staphylococci by using the sodA gene as a target. PMID : 11724835 : DOI : 10.1128/JCM.39.12.4296-4301.2001 PMC : PMC88539 Abstract >>
Simple PCR and sequencing assays that utilize a single pair of degenerate primers were used to characterize a 429-bp-long DNA fragment internal (sodA(int)) to the sodA gene encoding the manganese-dependent superoxide dismutase in 40 coagulase-negative staphylococcal (CNS) type strains. The topology of the phylogenetic tree obtained was in general agreement with that which was inferred from an analysis of their 16S rRNA or hsp60 gene sequences. Sequence analysis revealed that the staphylococcal sodA genes exhibit a higher divergence than does the corresponding 16S ribosomal DNA. These results confirm that the sodA gene constitutes a highly discriminative target sequence for differentiating closely related bacterial species. Clinical isolates that could not be identified at the species level by phenotypical tests were identified by use of this database. These results demonstrate the usefulness of this method for rapid and accurate species identification of CNS isolates, although it does not allow discrimination of subspecies. The sodA sequence polymorphisms observed with staphylococcal species offer good opportunities for the development of assays based on DNA chip technologies.
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5. |
Drancourt M,
Raoult D,
( 2002 ) rpoB gene sequence-based identification of Staphylococcus species. PMID : 11923353 : DOI : 10.1128/jcm.40.4.1333-1338.2002 PMC : PMC140360 Abstract >>
The complete sequence of rpoB, the gene encoding the beta subunit of RNA polymerase was determined for Staphylococcus saccharolyticus, Staphylococcus lugdunensis, S taphylococcus caprae, and Staphylococcus intermedius and partial sequences were obtained for an additional 27 Staphylococcus species. The complete rpoB sequences varied in length from 3,452 to 3,845 bp and had a 36.8 to 39.2% GC content. The partial sequences had 71.6 to 93.6% interspecies homology and exhibited a 0.08 to 0.8% intraspecific divergence. With a few exceptions, the phylogenetic relationships inferred from the partial rpoB sequences were in agreement with those previously derived from DNA-DNA hybridization studies and analyses of 16S ribosomal DNA gene sequences and partial HSP60 gene sequences. The staphylococcal rpoB sequence database we established enabled us to develop a molecular method for identifying Staphylococcus isolates by PCR followed by direct sequencing of the 751-bp amplicon. In blind tests, this method correctly identified 10 Staphylococcus isolates, and no positive results were obtained with 10 non-Staphylococcus gram-positive and gram-negative bacterial isolates. We propose partial sequencing of the rpoB gene as a new tool for the accurate identification of Staphylococcus isolates.
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6. |
Martineau F,
Picard FJ,
Ke D,
Paradis S,
Roy PH,
Ouellette M,
Bergeron MG,
( 2001 ) Development of a PCR assay for identification of staphylococci at genus and species levels. PMID : 11427566 : DOI : 10.1128/JCM.39.7.2541-2547.2001 PMC : PMC88182 Abstract >>
We have developed a PCR-based assay which allows the detection of staphylococci at the genus level by targeting the tuf gene, which encodes the elongation factor Tu. Degenerate PCR primers derived from consensus regions of several tuf genes were used to amplify a target region of 884 bp from 11 representative staphylococcal species. Subsequently, the entire nucleotide sequence of these amplicons was determined. The analysis of a multiple alignment of these sequences revealed regions conserved among staphylococci but distinct from those of other gram-positive bacteria genetically related to staphylococci. PCR primers complementary to these regions could amplify specifically and efficiently a DNA fragment of 370 bp for all of 27 different staphylococcal species tested. There was no amplification with genomic DNA prepared from 53 nonstaphylococcal species tested to verify the specificity of the assay (20 gram positive and 33 gram negative). Furthermore, this assay amplified efficiently all 27 American Type Culture Collection (ATCC) staphylococcal reference strains as well as 307 clinical isolates of staphylococci from the Qu?bec City region. Analysis of the multiple sequence alignment for the 884-bp fragment for the 11 staphylococcal species as well as comparison of the sequences for the 370-bp amplicon from five unrelated ATCC and clinical strains for each of the species S. aureus, S. epidermidis, S. haemolyticus, S. hominis, and S. saprophyticus demonstrated sufficient interspecies polymorphism to generate genus- and species-specific capture probes. This sequence information allowed the development of Staphylococcus-specific and species-specific (targeting S. aureus, S. epidermidis, S. haemolyticus, S. hominis, or S. saprophyticus) capture probes hybridizing to the 370-bp amplicon. In conclusion, this PCR assay is suitable for detection of staphylococci at both genus and species levels.
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7. |
Linde HJ,
Schmidt M,
Fuchs E,
Reischl U,
Niller HH,
Lehn N,
( 2001 ) In vitro activities of six quinolones and mechanisms of resistance in Staphylococcus aureus and coagulase-negative staphylococci. PMID : 11302827 : DOI : 10.1128/AAC.45.5.1553-1557.2001 PMC : PMC90505 Abstract >>
Of 94 clinical isolates of Staphylococcus aureus (n = 51) and coagulase-negative staphylococci (CNS) (n = 43), mutations in the quinolone resistance-determining region of topoisomerases GrlA, GrlB, GyrA, and GyrB together with MICs of six quinolones were analyzed. Amino acid substitutions at identical residues (GrlA residues 80 and 84; GyrA residues 84 and 88) were found in S. aureus and CNS. Active efflux, as suggested by blocking by reserpine, contributed substantially to the resistance phenotype in some strains. Among ciprofloxacin, clinafloxacin, levofloxacin, nalidixic acid, trovafloxacin, and sparfloxacin, a 0.5-microg/ml concentration of sparfloxacin discriminated best between strains with two or three mutations and those with no mutations.
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8. |
Av-Gay Y,
Dovichi NJ,
Chow AW,
Bay SJ,
Su SC,
Reynolds RP,
( 1999 ) Species identification and phylogenetic relationships based on partial HSP60 gene sequences within the genus Staphylococcus. PMID : 10425778 : DOI : 10.1099/00207713-49-3-1181 Abstract >>
The phylogenetic relationships among 36 validly described species or subspecies within the genus Staphylococcus were investigated by cloning and sequencing their 60 kDa heat-shock protein (HSP60) genes using a set of universal degenerate HSP60 PCR primers. The cloned partial HSP60 DNA sequences from nine Staphylococcus aureus strains were highly conserved (97-100% DNA sequence similarity; mean 98%), indicating that the HSP60 gene of multiple isolates within the same species have little microheterogeneity. At the subspecies level, DNA sequence similarity among members of S. aureus, Staphylococcus schleiferi, Staphylococcus cohnii and Staphylococcus capitis ranged from 91 to 98%. At the interspecies level, sequence similarity among 23 distinct species of staphylococci ranged from 74 to 93% (mean 82%). By comparison, the highest sequence similarity of Bacillus subtilis and Escherichia coli with members within the genus Staphylococcus was only 70 and 59%, respectively. Importantly, phylogenetic analysis based on the neighbour-joining distance method revealed remarkable concordance between the tree derived from partial HSP60 gene sequences and that based on genomic DNA-DNA hybridization, while 16S rRNA gene sequences correlated less well. The results demonstrate that DNA sequences from the highly conserved and ubiquitous HSP60 gene offer a convenient and accurate tool for species-specific identification and phylogenetic analysis of staphylococci.
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9. |
Fitzgibbon JE,
Nahvi MD,
John JF,
( 1999 ) Topoisomerase sequences of coagulase-negative staphylococcal isolates resistant to ciprofloxacin or trovafloxacin. PMID : 10390214 : PMC : PMC89335 Abstract >>
Coagulase-negative staphylococcal isolates (n = 188) were screened for susceptibility to oxacillin, ciprofloxacin, and trovafloxacin, a new fluoroquinolone. At an oxacillin concentration of >/=4 microg/ml, 43% were methicillin resistant; of these, 70% were ciprofloxacin resistant (MIC, >/=4 microg/ml). Of the methicillin-resistant, ciprofloxacin-resistant isolates, 46% were susceptible to /=8 microg/ml) and increased trovafloxacin MICs (0.25 to 2 microg/ml) could be conferred by the combined presence of single mutations in each gyrA and grlA gene. Trovafloxacin MICs of >/=8 microg/ml also occurred, but these required an additional mutation in grlA.
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10. |
Layer F,
Ghebremedhin B,
König W,
König B,
( 2007 ) Differentiation of Staphylococcus spp. by terminal-restriction fragment length polymorphism analysis of glyceraldehyde-3-phosphate dehydrogenase-encoding gene. PMID : 17681623 : DOI : 10.1016/j.mimet.2007.06.015 Abstract >>
Classical phenotypic and biochemical testing do not lead to correct identification of the distinct Staphylococcus species. Therefore, the aim of our study was to develop a method for the reliable and accurate determination of distinct Staphylococcus species. In the present study, the 931-934-bp partial sequences of the glyceraldehyde-3-phosphate dehydrogenase-encoding (gap) gene of 28 validly described Staphylococcus species were amplified and sequenced. By using the respective sequence information we performed a terminal-restriction fragment length polymorphism (T-RFLP) analysis. For T-RFLP the partial gap gene was amplified with double-fluorescently labelled primers and digested with the restriction enzymes DdeI, BspHI and TaqI. Distinctive T-RFLP patterns were rendered by the use of capillary electrophoresis with laser-induced fluorescence detection. This molecular method allowed us to identify all 28 Staphylococcus species with high specificity. This was validated by analysis of 34 Staphylococcus epidermidis and 28 Staphylococcus haemolyticus isolates. These results demonstrate the feasibility and applicability of the T-RFLP method based on the partial gap gene sequences for rapid and accurate species identification.
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11. |
Lüthje P,
von Köckritz-Blickwede M,
Schwarz S,
( 2007 ) Identification and characterization of nine novel types of small staphylococcal plasmids carrying the lincosamide nucleotidyltransferase gene lnu(A). PMID : 17329268 : DOI : 10.1093/jac/dkm008 Abstract >>
To date, very little is known about lincosamide resistance plasmids in staphylococci with only a single lnu(A)-carrying staphylococcal plasmid having been sequenced completely. The aim of this study was to characterize small lnu(A)-carrying plasmids isolated from bovine coagulase-negative staphylococci (CoNS). Nine CoNS isolates with MICs of the lincosamide pirlimycin of 1-4 mg/L were tested for the presence of the lnu(A) gene. Its location was determined by Southern-blot hybridization. The lnu(A)-carrying plasmids were transformed into Staphylococcus aureus RN4220 and compared by restriction mapping and subsequent sequencing. Selected plasmids were investigated for their copy number and their lnu(A) gene expression via RT real-time PCR. The lnu(A) gene was detected on plasmids in all isolates. Sequence analysis revealed that these plasmids carried a rep gene, coding for the replication initiator protein, and the resistance gene lnu(A), coding for a lincosamide nucleotidyltransferase. While the Lnu(A) proteins were closely related (91.3-100% amino acid identity), the Rep proteins differed distinctly (27.4-100% amino acid identity), but showed similarity (81.4-98.5%) to Rep proteins of other small staphylococcal resistance plasmids. Sequence features of rolling-circle plasmids, such as the single-strand (ssoA) and double-strand (dso) origins of replication, were identified. For two plasmid types detected, the lincosamide resistance level varied with regard to the amounts of lnu(A) transcripts detected. Structurally different lnu(A)-carrying plasmids were detected in various CoNS species. The detection of the same lnu(A) gene in different plasmid backbones suggested the exchange of the gene via interplasmid recombinational events.
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12. |
Layer F,
Ghebremedhin B,
Moder KA,
König W,
König B,
( 2006 ) Comparative study using various methods for identification of Staphylococcus species in clinical specimens. PMID : 16891498 : DOI : 10.1128/JCM.00226-06 PMC : PMC1594629 Abstract >>
Coagulase-negative staphylococci (CNS) play a predominant role in nosocomial infections. Rapid, reliable identification of these organisms is essential for accurate diagnosis and prompt effective treatment of these infections. Quite recently, the VITEK 2 g-positive (gram-positive [GP]) identification card (bioM?rieux) has been redesigned for greater accuracy in the identification of gram-positive cocci. We compared the BD Phoenix (Becton Dickinson) and VITEK 2 (bioM?rieux) automated microbiology systems, using their respective update version cards, and the API ID32 STAPH test. The glyceraldehyde-3-phosphate dehydrogenase (gap) gene-based T-RFLP (terminal restriction fragment length polymorphism) method was used for verifying the results. In total, 86 clinical isolates of CNS and 27 reference strains were analyzed. The results show that for identification of CNS, the automated identification methods using the newest VITEK 2 and BD Phoenix identification cards are comparable. However, API ID32 STAPH revealed more correct results compared to both automated microbiology systems. Despite the increased performance of the phenotypic automated identification systems compared to the former versions, molecular methods, e.g., the gap-based T-RFLP method, still show superior accuracy in identifying Staphylococcus species other than Staphylococcus aureus.
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13. |
Corbière Morot-Bizot S,
Leroy S,
Talon R,
( 2006 ) Staphylococcal community of a small unit manufacturing traditional dry fermented sausages. PMID : 16488037 : DOI : 10.1016/j.ijfoodmicro.2005.12.006 Abstract >>
The level and the diversity of the staphylococcal community occurring in the environment and meat products of a small unit manufacturing traditional dry fermented sausages were investigated at two seasons: winter and spring. Gram-positive cocci were enumerated and a collection of 412 Staphylococcus isolates was made. Multiplex PCR, pulse-field gel electrophoresis (PFGE) and sequencing of the sodA gene were used to identify and characterize the isolates. High counts of Staphylococcus were found in final traditional sausages, reaching about 6 log CFU/g in winter and about 8 log CFU/g in spring. In the environment, the counts varied from 2 log to 7 log/100 cm(2), the higher colonisation being observed on the surface of the drying and cold rooms, cutting tables and the butcher's block. The combination of the three methods allowed the identification of seven species of Staphylococcus in spring and five in winter. S. equorum and S. succinus dominated both in environment and in meat products, 49% and 33% of the isolates, respectively. The other identified species were in decreasing order S. saprophyticus (6%), S. xylosus (5%), S. carnosus (5%), S. simulans (1%) and S. warneri (1%). The two species S. xylosus and S. carnosus were sporadically isolated during the spring. PFGE allowed the assignment of S. equorum to eight pulsotypes showing a wide diversity among this species. But the entire environment and the meat products were dominated by one pulsotype. For S. succinus, three pulsotypes were found with one dominant mainly isolated during the spring sampling. This study highlighted the diversity of staphylococci isolated in the environment and the meat products of a small processing unit manufacturing traditional dry fermented sausages. The S. equorum and S. succinus species rarely described in meat products and never in the environment had great capacity to colonise the entire small processing unit and the meat products.
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14. |
Tormo MA,
Knecht E,
Götz F,
Lasa I,
Penadés JR,
( 2005 ) Bap-dependent biofilm formation by pathogenic species of Staphylococcus: evidence of horizontal gene transfer? PMID : 16000737 : DOI : 10.1099/mic.0.27865-0 Abstract >>
The biofilm-associated protein (Bap) is a surface protein implicated in biofilm formation by Staphylococcus aureus isolated from chronic mastitis infections. The bap gene is carried in a putative composite transposon inserted in SaPIbov2, a mobile staphylococcal pathogenicity island. In this study, bap orthologue genes from several staphylococcal species, including Staphylococcus epidermidis, Staphylococcus chromogenes, Staphylococcus xylosus, Staphylococcus simulans and Staphylococcus hyicus, were identified, cloned and sequenced. Sequence analysis comparison of the bap gene from these species revealed a very high sequence similarity, suggesting the horizontal gene transfer of SaPIbov2 amongst them. However, sequence analyses of the flanking region revealed that the bap gene of these species was not contained in the SaPIbov2 pathogenicity island. Although they did not contain the icaADBC operon, all the coagulase-negative staphylococcal isolates harbouring bap were strong biofilm producers. Disruption of the bap gene in S. epidermidis abolished its capacity to form a biofilm, whereas heterologous complementation of a biofilm-negative strain of S. aureus with the Bap protein from S. epidermidis bestowed the capacity to form a biofilm on a polystyrene surface. Altogether, these results demonstrate that Bap orthologues from coagulase-negative staphylococci induce an alternative mechanism of biofilm formation that is independent of the PIA/PNAG exopolysaccharide.
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15. |
Ceotto H,
Holo H,
da Costa KF,
Nascimento Jdos S,
Salehian Z,
Nes IF,
Bastos Mdo C,
( 2010 ) Nukacin 3299, a lantibiotic produced by Staphylococcus simulans 3299 identical to nukacin ISK-1. PMID : 20627619 : DOI : 10.1016/j.vetmic.2010.04.032 Abstract >>
Nukacin 3299 (formerly designated simulancin 3299), produced by a Staphylococcus simulans strain involved in bovine mastitis in Brazil, is the first peptide bacteriocin described in this staphylococcal species. With the intent to elucidate some aspects of its biology, nukacin 3299 was purified and characterized. The mass of the purified bacteriocin was shown to be 2957.3 Da, and the peptide N-terminal amino acids (KKKSGVI) were identified by Edman degradation. The nukacin 3299 structural gene, nukA, was detected by PCR and DNA sequencing, showing that this bacteriocin is identical to nukacin ISK-1, a 27-amino acid type-A (II) lantibiotic produced by Staphylococcus warneri ISK-1, isolated from a "nukadoko", in Japan. The genes involved in nukacin 3299 biosynthesis are located on plasmid pRJ97 (>27 kb). They have an organization similar to that of the nukacin ISK-1 gene cluster, excepted for the presence of an IS257/431 element (791 bp) present between the orf1 and nukA genes of the nukacin 3299 gene cluster. The presence of this insertion sequence is expected to affect the expression of orf1, whose function is presently unknown. Nukacin 3299 proved to be sensitive to proteolytic enzymes and relatively stable at different temperatures and between pH 3.0-9.0. Nukacin 3299 exhibited activity towards staphylococcal strains involved in bovine mastitis, showing a potential application on mastitis control, a disease with great economic impact.
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16. |
Bergeron M,
Dauwalder O,
Gouy M,
Freydiere AM,
Bes M,
Meugnier H,
Benito Y,
Etienne J,
Lina G,
Vandenesch F,
Boisset S,
( 2011 ) Species identification of staphylococci by amplification and sequencing of the tuf gene compared to the gap gene and by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. PMID : 20967479 : DOI : 10.1007/s10096-010-1091-z Abstract >>
Staphylococcal species, notably, coagulase-negative staphylococci (CoNS), are frequently misidentified using phenotypic methods. The partial nucleotide sequences of the tuf and gap genes were determined in 47 reference strains to assess their suitability, practicability, and discriminatory power as target molecules for staphylococcal identification. The partial tuf gene sequence was selected and further assessed with a collection of 186 strains, including 35 species and subspecies. Then, to evaluate the efficacy of this genotyping method versus the technology of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), the 186 strains were identified using MALDI-TOF-MS (Axima? Shimadzu) coupled to the SARAMIS? database (AnagnosTec). The French National Reference Center for Staphylococci identification method was used as a reference. One hundred and eighty-four strains (98.9%) were correctly identified by tuf gene sequencing. Only one strain was misidentified and one was unidentified. MALDI-TOF-MS identified correctly 138 isolates (74.2%). Four strains were misidentified, 39 were unidentified, five were identified at the group (hominis/warneri) level, and one strain was identified at the genus level. These results confirm the value of MALDI-TOF-MS identification for common species in clinical laboratory practice and the value of the partial tuf gene sequence for the identification of all staphylococcal species as required in a reference laboratory.
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17. |
Capurro A,
Artursson K,
Waller KP,
Bengtsson B,
Ericsson-Unnerstad H,
Aspán A,
( 2009 ) Comparison of a commercialized phenotyping system, antimicrobial susceptibility testing, and tuf gene sequence-based genotyping for species-level identification of coagulase-negative staphylococci isolated from cases of bovine mastitis. PMID : 18930604 : DOI : 10.1016/j.vetmic.2008.08.028 Abstract >>
In order to evaluate the usefulness of some phenotypic and genotypic methods for species identification of coagulase-negative staphylococci (CNS), isolates were obtained from bovine cases of clinical and sub-clinical mastitis from different geographical areas in Sweden. By using the Staph-Zym test, antimicrobial susceptibility testing, and sequencing of part of the CNS tuf gene and, when needed, part of the 16S rRNA gene we characterized 82 clinical isolates and 24 reference strains of 18 different species of staphylococci. The genotypic methods identified nine different species of CNS among the 82 milk isolates. A comparison with results obtained by tuf gene sequencing showed that Staph-Zym correctly identified CNS reference strains to species level more often than bovine milk CNS isolates (83% and 61%, respectively). In addition, tests supplementary to the Staph-Zym were frequently needed in both groups of isolates (50% of reference strains and 33% of milk isolates) to obtain an identification of the strain. It is notable that Staph-Zym judged two isolates as CNS, although they belonged to other species, could not give a species name in 11% of the bovine CNS isolates, and gave 28% of the isolates an incorrect species name. The present study indicates that the studied phenotypic methods are unreliable for identification of CNS from bovine intra-mammary infections.
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18. |
Naushad S,
Barkema HW,
Luby C,
Condas LA,
Nobrega DB,
Carson DA,
De Buck J,
( 2016 ) Comprehensive Phylogenetic Analysis of Bovine Non-aureus Staphylococci Species Based on Whole-Genome Sequencing. PMID : 28066335 : DOI : 10.3389/fmicb.2016.01990 PMC : PMC5168469 Abstract >>
Non-aureus staphylococci (NAS), a heterogeneous group of a large number of species and subspecies, are the most frequently isolated pathogens from intramammary infections in dairy cattle. Phylogenetic relationships among bovine NAS species are controversial and have mostly been determined based on single-gene trees. Herein, we analyzed phylogeny of bovine NAS species using whole-genome sequencing (WGS) of 441 distinct isolates. In addition, evolutionary relationships among bovine NAS were estimated from multilocus data of 16S rRNA, hsp60, rpoB, sodA, and tuf genes and sequences from these and numerous other single genes/proteins. All phylogenies were created with FastTree, Maximum-Likelihood, Maximum-Parsimony, and Neighbor-Joining methods. Regardless of methodology, WGS-trees clearly separated bovine NAS species into five monophyletic coherent clades. Furthermore, there were consistent interspecies relationships within clades in all WGS phylogenetic reconstructions. Except for the Maximum-Parsimony tree, multilocus data analysis similarly produced five clades. There were large variations in determining clades and interspecies relationships in single gene/protein trees, under different methods of tree constructions, highlighting limitations of using single genes for determining bovine NAS phylogeny. However, based on WGS data, we established a robust phylogeny of bovine NAS species, unaffected by method or model of evolutionary reconstructions. Therefore, it is now possible to determine associations between phylogeny and many biological traits, such as virulence, antimicrobial resistance, environmental niche, geographical distribution, and host specificity.
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19. |
Marty E,
Buchs J,
Eugster-Meier E,
Lacroix C,
Meile L,
( 2012 ) Identification of staphylococci and dominant lactic acid bacteria in spontaneously fermented Swiss meat products using PCR-RFLP. PMID : 22202869 : DOI : 10.1016/j.fm.2011.09.011 Abstract >>
Pathogenic, spoilage, and technologically important microorganisms were monitored in 21 spontaneously fermented Swiss meat products manufactured with meat from wildlife or animals grown in natural habitat. Thereby, PCR-restriction fragment length polymorphism (RFLP) on rpoB and 16S rRNA gene sequences provided a powerful tool for fast and accurate identification of the main microbial population. Lactobacillus sakei and Lactobacillus curvatus dominated in fermented meat products followed by Staphylococcus species, which constituted 88.2% of all Gram-positive, catalase-positive cocci (GCC(+)) with cell counts varying from 2.6 to 7.0 log cfu/g during maturation. Staphylococcus equorum was prevalent in frequency and cell counts during maturation (18.0%; 5.0-7.3 log cfu/g) and in the end products (28.4%; 1.8-6.2 log cfu/g) implicating a new presumptive starter species for meat fermentation. Nine out of 14 end products indicated safety risks to consumers due to the high incidence of Staphylococcus saprophyticus or Staphylococcus epidermidis combined with cell counts of 7.4 and 4.9 log cfu/g, respectively. This fact was supported by the detection of Staphylococcus aureus and Enterobacteriaceae in ready-to-eat products strongly exceeding the tolerable limit of 2 log cfu/g. Spontaneously fermented meat products produced from wildlife or animals grown in natural habitats not only gave rise to hygienic and safety concerns but also provided new presumptive starter strains.
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20. |
Zong Z,
Peng C,
Lü X,
( 2011 ) Diversity of SCCmec elements in methicillin-resistant coagulase-negative staphylococci clinical isolates. PMID : 21637845 : DOI : 10.1371/journal.pone.0020191 PMC : PMC3102682 Abstract >>
Methicillin-resistant coagulase-negative staphylococci (MR-CoNS) are opportunistic pathogens and serve as a large reservoir of staphylococcal cassette chromosome mec (SCCmec). Characterization of SCCmec in MR-CoNS can generate useful information on the mobilization and evolution of this element. Non-repetitive MR-CoNS clinical isolates (n = 84; 39 S. epidermidis, 19 S. haemolyticus, 9 S. hominis, 6 S. capitis, 4 S. warneri, 2 S. cohnii, 2 S. saprophyticus, 1 S. kloosii, 1 S. simulans and 1 S. massiliensis) were collected. All isolates could grow on plates with 4 mg/L cefoxitin and all had mecA as detected by PCR. Strain typing using RAPD and ERIC-PCR revealed that almost all isolates were of different strains. SCCmec typing was performed using multiplex PCR published previously. For isolates in which SCCmec could not be typed, the mec complex classes were determined by additional PCR and the ccr genes were amplified with published or newly-designed primers and then sequenced. SCCmec types were assigned for 63 isolates by multiplex PCR and were assigned for 14 other isolates by PCR targeting mec and ccr. Among 77 isolates with determined SCCmec types, 54 had a single type, including type III (n = 19), IV (n = 14), V (n = 10), II (n = 2), I (n = 1), VIII (n = 1) and five unnamed types (n = 7), while 23 isolates had two types, III+V (n = 12), II+V (n = 8), II+IV (n = 2) or IV+V (n = 1). The five unnamed types were assigned UT1 (class A mec, ccrA1/ccrB4), UT2 (class C1 mec, ccrA4/ccrB4), UT3 (class A mec, ccrA5/ccrB3), UT4 (class C2 mec, ccrA2/ccrB2 plus ccrC1) and UT5 (class A mec, ccrA1/ccrB1 plus ccrC1). SCCmec types III, IV and V were prevalent in MR-CoNS and many isolates could harbor more than one type. Several new types of SCCmec were identified, highlighting the great genetic diversity and the need of developing classification schemes for SCCmec in MR-CoNS.
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21. |
( 1997 ) Studies on prolysostaphin processing and characterization of the lysostaphin immunity factor (Lif) of Staphylococcus simulans biovar staphylolyticus. PMID : 9106216 : DOI : 10.1046/j.1365-2958.1997.2911657.x Abstract >>
Lysostaphin is an extracellular glycylglycine endopeptidase produced by Staphylococcus simulans biovar staphylolyticus ATCC1362 that lyses staphylococcal cells by hydrolysing the polyglycine interpeptide bridges of the peptidoglycan. Renewed analysis of the sequence of the lysostaphin gene (lss), and the sequencing of the amino-terminus of purified prolysostaphin and of mature lysostaphin revealed that lysostaphin is organized as a preproprotein of 493 amino acids (aa), with a signal peptide consisting of 36 aa, a propeptide of 211 aa from which 195 aa are organized in 15 tandem repeats of 13 aa length, and a mature protein of 246 aa. Prolysostaphin is processed in the culture supernatant of S. simulans biovar staphylolyticus by an extracellular cysteine protease. Although prolysostaphin was staphylolytically active, the mature lysostaphin was about 4.5-fold more active. The controlled expression in Staphylococcus carnosus of lss and lss with deletions in the prepropeptide region indicated that the tandem repeats of the propeptide are not necessary for protein export or activation of Lss, but keep Lss in a less active state. Intracellularly expressed pro- and mature lysostaphin exert staphylolytic activity in cell-free extracts, but do not affect growth of the corresponding clones. We characterized a lysostaphin immunity factor gene (lif) which is located in the opposite direction to lss. The expression of lif in S. carnosus led to an increase in the serine/glycine ratio of the interpeptide bridges of peptidoglycan from 2 to 35%, suggesting that lysostaphin immunity depends on serine incorporation into the interpeptide bridge. If, in addition to lif, lss is co-expressed the serine/glycine ratio is further increased to 58%, suggesting that Lss selects for optimal serine incorporation. Lif shows similarity to FemA and FemB proteins, which are involved in the biosynthesis of the glycine interpeptide bridge of staphylococcal peptidoglycan. In contrast to that of Lif, the production of FemA and FemB in S. carnosus does not cause lysostaphin immunity. The putative tRNASer gene located downstream of lss had no recognizable influence on lysostaphin immunity. lss and lif are flanked by insertion sequences, suggesting that S. simulans biovar staphylolyticus received lif and lss by horizontal gene transfer.
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( 1996 ) HSP60 gene sequences as universal targets for microbial species identification: studies with coagulase-negative staphylococci. PMID : 8815090 : PMC : PMC228899 Abstract >>
A set of universal degenerate primers which amplified, by PCR, a 600-bp oligomer encoding a portion of the 60-kDa heat shock protein (HSP60) of both Staphylococcus aureus and Staphylococcus epidermidis were developed. However, when used as a DNA probe, the 600-bp PCR product generated from S. epidermidis failed to cross-hybridize under high-stringency conditions with the genomic DNA of S. aureus and vice versa. To investigate whether species-specific sequences might exist within the highly conserved HSP60 genes among different staphylococci, digoxigenin-labelled HSP60 probes generated by the degenerate HSP60 primers were prepared from the six most commonly isolated Staphylococcus species (S. aureus 8325-4, S. epidermidis 9759, S. haemolyticus ATCC 29970, S. schleiferi ATCC 43808, S. saprophyticus KL122, and S. lugdunensis CRSN 850412). These probes were used for dot blot hybridization with genomic DNA of 58 reference and clinical isolates of Staphylococcus and non-Staphylococcus species. These six Staphylococcus species HSP60 probes correctly identified the entire set of staphylococcal isolates. The species specificity of these HSP60 probes was further demonstrated by dot blot hybridization with PCR-amplified DNA from mixed cultures of different Staphylococcus species and by the partial DNA sequences of these probes. In addition, sequence homology searches of the NCBI BLAST databases with these partial HSP60 DNA sequences yielded the highest matching scores for both S. epidermidis and S. aureus with the corresponding species-specified probes. Finally, the HSP60 degenerate primers were shown to amplify an anticipated 600-bp PCR product from all 29 Staphylococcus species and from all but 2 of 30 other microbial species, including various gram-positive and gram-negative bacteria, mycobacteria, and fungi. These preliminary data suggest the presence of species-specific sequence variation within the highly conserved HSP60 genes of staphylococci. Further work is required to determine whether these degenerate HSP60 primers may be exploited for species-specific microbic identification and phylogenetic investigation of staphylococci and perhaps other microorganisms in general.
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Recsei PA,
Gruss AD,
Novick RP,
( 1987 ) Cloning, sequence, and expression of the lysostaphin gene from Staphylococcus simulans. PMID : 3547405 : DOI : 10.1073/pnas.84.5.1127 PMC : PMC304379 Abstract >>
A 1.5-kilobase-pair fragment of DNA that contains the lysostaphin gene from Staphylococcus simulans and its flanking sequences has been cloned and completely sequenced. The gene encodes a preproenzyme of Mr 42,000. The NH2-terminal sequence of the preproenzyme is composed of a signal peptide followed by seven tandem repeats of a 13-amino acid sequence. Conversion of prolysostaphin to the mature enzyme occurs extracellularly in cultures of S. simulans and involves removal of the NH2-terminal portion of the proenzyme that contains the tandem repeats. The high degree of homology of the repeats suggests that they have arisen by duplication of a 39-base-pair sequence of DNA. In S. simulans, the lysostaphin gene is present on a large beta-lactamase plasmid.
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( 1998 ) Regulation of agr-dependent virulence genes in Staphylococcus aureus by RNAIII from coagulase-negative staphylococci. PMID : 9620969 : PMC : PMC107820 Abstract >>
Many of the genes coding for extracellular toxins, enzymes, and cell surface proteins in Staphylococcus aureus are regulated by a 510-nucleotide (nt) RNA molecule, RNAIII. Transcription of genes encoding secreted toxins and enzymes, including hla (alpha-toxin), saeB (enterotoxin B), tst (toxic shock syndrome toxin 1), and ssp (serine protease), is stimulated, while transcription of genes encoding cell surface proteins, like spa (protein A) and fnb (fibronectin binding proteins), is repressed. Besides being a regulator, RNAIII is also an mRNA coding for staphylococcal delta-lysin. We have identified RNAIII homologs in three different coagulase-negative staphylococci (CoNS), i.e., Staphylococcus epidermidis, Staphylococcus simulans, and Staphylococcus warneri. RNAIII from these CoNS turned out to be very similar to that of S. aureus and contained open reading frames encoding delta-lysin homologs. Though a number of big insertions and/or deletions have occurred, mainly in the 5' half of the molecules, the sequences show a high degree of identity, especially in the first 50 and last 150 nt. The CoNS RNAIII had the ability to completely repress transcription of protein A in an RNAIII-deficient S. aureus mutant and the ability to stimulate transcription of the alpha-toxin and serine protease genes. However, the stimulatory effect was impaired compared to that of S. aureus RNAIII, suggesting that these regulatory functions are independent. By creating S. epidermidis-S. aureus RNAIII hybrids, we could also show that both the 5' and 3' halves of the RNAIII molecule are involved in the transcriptional regulation of alpha-toxin and serine protease mRNAs in S. aureus.
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( 1998 ) Molecular characterization of the erythromycin resistance plasmid pPV142 from Staphylococcus simulans. PMID : 9742700 : DOI : 10.1111/j.1574-6968.1998.tb13158.x Abstract >>
The 2.5-kb erythromycin resistance (EmR) plasmid pPV142 of Staphylococcus simulans 13044 was isolated and characterized. Sequence analysis identified ORF1 and ORF2 encoding a 158-residue replication protein (Rep142) and a 244-residue erythromycin resistance protein (Erm, rRNA adenine N-6-methyltransferase), respectively. Structural analysis and Southern hybridization showed that the rep and ermM genes in pPV142 shared homology with the EmR plasmid pPV141 (2.4 kb) of S. chromogenes 3688 and other EmR plasmids known to exist in staphylococci and bacilli. Based on the presence of a 61-bp repeat upstream of the ermM gene, pPV142 is apparently a unique member of the pSN2 family of EmR plasmid able to express erythromycin resistance constitutively.
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