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.

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.

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.

The complete nucleotide sequence of the 6913 bp plasmid pSTE2 from Staphylococcus lentus, which mediates inducible resistance to tetracyclines, macrolides and lincosamides, was determined. The plasmid was analysed for potential reading frames and structural features to gain insight into its development from potential ancestor plasmids. Plasmid pSTE2 was transformed into Staphylococcus aureus RN4220. Suitable restriction fragments were cloned into E. coli plasmid vectors and sequenced. In vitro susceptibility testing was performed to confirm the resistance phenotype mediated by this plasmid. Plasmid pSTE2 consisted of two parts, each of which corresponded closely to previously identified staphylococcal plasmids. The initial 4439 bp represented a pT181-analogous tet(K)-carrying tetracycline resistance plasmid, whereas the remaining 2474 bp represented a pPV141-related erm(C)-carrying macrolide-lincosamide-streptogramin B resistance plasmid. Both putative parental plasmids harboured the staphylococcal recombination site A (RSA) and the pT181-like plasmid also carried the recombinase gene pre whose product acts at RSA. Analysis of the junctions of the pT181-like and the pPV141-like homologous parts in pSTE2 suggested that plasmid pSTE2 developed from pT181- and pPV141-like ancestor plasmids by cointegrate formation at RSA. Plasmid pSTE2 is the first completely sequenced plasmid from S. lentus and represents the product of an in vivo derived RSA-mediated recombination between two compatible plasmids.

The florfenicol-chloramphenicol exporter gene fexA is part of the novel transposon Tn558 from Staphylococcus lentus. Similarities between Tn558 and Tn554 from Staphylococcus aureus included the arrangement of the transposase genes tnpA to -C and an att554-like target sequence. Circular forms of Tn558 were detected and suggest the functional activity of this transposon.

The Staphylococcus lentus plasmid pSCFS2 carries a novel florfenicol-chloramphenicol resistance gene, designated fexA, encoding a protein of 475 amino acids with 14 transmembrane domains. The FexA protein differs from all previously known proteins involved in the efflux of chloramphenicol and florfenicol. Induction of fexA expression by chloramphenicol and florfenicol occurs via translational attenuation.

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.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important multidrug-resistant pathogens around the world. MRSA is generated when methicillin-susceptible S. aureus (MSSA) exogenously acquires a methicillin resistance gene, mecA, carried by a mobile genetic element, staphylococcal cassette chromosome mec (SCCmec), which is speculated to be transmissible across staphylococcal species. However, the origin/reservoir of the mecA gene has remained unclear. Finding the origin/reservoir of the mecA gene is important for understanding the evolution of MRSA. Moreover, it may contribute to more effective control measures for MRSA. Here we report on one of the animal-related Staphylococcus species, S. fleurettii, as the highly probable origin of the mecA gene. The mecA gene of S. fleurettii was found on the chromosome linked with the essential genes for the growth of staphylococci and was not associated with SCCmec. The mecA locus of the S. fleurettii chromosome has a sequence practically identical to that of the mecA-containing region (?12 kbp long) of SCCmec. Furthermore, by analyzing the corresponding gene loci (over 20 kbp in size) of S. sciuri and S. vitulinus, which evolved from a common ancestor with that of S. fleurettii, the speciation-related mecA gene homologues were identified, indicating that mecA of S. fleurettii descended from its ancestor and was not recently acquired. It is speculated that SCCmec came into form by adopting the S. fleurettii mecA gene and its surrounding chromosomal region. Our finding suggests that SCCmec was generated in Staphylococcus cells living in animals by acquiring the intrinsic mecA region of S. fleurettii, which is a commensal bacterium of animals.

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.

In an evolutionarily conserved gene organization (syntenic region), the sigH gene shares exceptionally low homology among staphylococcal species. We analyzed the "positionally cloned" sigH sequences of 39 staphylococcal species. The topology of the SigH phylogenetic tree was consistent with that of 16S rRNA. Certain clinical isolates were successfully differentiated at the species level with the sigH sequence data set. We propose that the sigH gene is a promising molecular target in genotypic identification because it is highly discriminative in differentiating closely related staphylococcal species.

Staphylococci are frequently isolated from food processing environments, and it has been speculated whether survival after cleaning and disinfection with benzalkonium chloride (BC)-containing disinfectants is due to biofilm formation, matrix composition, or BC e?ux mechanisms. Out of 35 food associated staphylococci, eight produced biofilm in a microtiter plate assay and were identified as Staphylococcus capitis (2), S. cohnii, S. epidermidis, S. lentus (2), and S. saprophyticus (2). The eight biofilm producing strains were characterized using whole genome sequencing. Three of these strains contained the ica operon responsible for production of a polysaccharide matrix, and formed a biofilm which was detached upon exposure to the polysaccharide degrading enzyme Dispersin B, but not Proteinase K or trypsin. These strains were more tolerant to the lethal effect of BC both in suspension and biofilm than the remaining five biofilm producing strains. The five BC susceptible strains were characterized by lack of the ica operon, and their biofilms were detached by Proteinase K or trypsin, but not Dispersin B, indicating that proteins were major structural components of their biofilm matrix. Several novel cell wall anchored repeat domain proteins with domain structures similar to that of MSCRAMM adhesins were identified in the genomes of these strains, potentially representing novel mechanisms of ica-independent biofilm accumulation. Biofilms from all strains showed similar levels of detachment after exposure to alkaline chlorine, which is used for cleaning in the food industry. Strains with qac genes encoding BC e?ux pumps could grow at higher concentrations of BC than strains without these genes, but no differences were observed at biocidal concentrations. In conclusion, the biofilm matrix of food associated staphylococci varies with respect to protein or polysaccharide nature, and this may affect the sensitivity toward a commonly used disinfectant.

The multiresistance gene cfr was identified for the first time in streptococci, namely, in porcine Streptococcus suis isolate S10. The cfr gene was detected on the ~100-kb plasmid pStrcfr, where it was bracketed by two copies of the novel insertion sequence ISEnfa5, located in the same orientation. The detection of a cfr- and ISEnfa5-containing amplicon by inverse PCR suggests that ISEnfa5 may play a role in the dissemination of cfr.

The present study focussed on the analysis of the genetic environment of the multi-resistance gene cfr detected among 21, mostly methicillin-resistant, coagulase-negative Staphylococcus (CoNS) isolates obtained from chickens, ducks and pigs in China. It included sequencing of the regions up- and downstream of the cfr gene on various plasmid types in 13 isolates, such as pSS-02 and pSS-02-like (n=7), pSS-03-like (n=1), pJP1-like (n=3), pSS-04 (n=1) and pJP2 (n=1). This analysis revealed that insertion sequences (IS21-558, IS256, IS257, or IS1216E) and other resistance genes (aacA-aphD and aadD for aminoglycoside resistance, ble for bleomycin resistance, fosD for fosfomycin resistance, erm(B) and erm(C) for macrolide-lincosamide-streptogramin B resistance, or fexA for phenicol resistance) coexisted on the respective plasmids. In the chromosomal copies of cfr identified in eight S. lentus isolates, the cfr gene was found to be bracketed by insertion sequences, such as IS256 or ISEnfa5. Stability tests confirmed that all chromosomal cfr-containing regions could be looped out via IS-mediated recombination. The observations made in this study extend the rather rudimentary knowledge about the genetic environment of cfr in staphylococci from chickens and ducks and confirmed that insertion sequences play an important role in the dissemination of cfr, not only among different types of plasmids, but also for the integration in the chromosomal DNA.

The search for a specific rRNA methylase motif led to the identification of the new macrolide, lincosamide, and streptogramin B resistance gene erm(43) in Staphylococcus lentus. An inducible resistance phenotype was demonstrated by cloning and expressing erm(43) and its regulatory region in Staphylococcus aureus. The erm(43) gene was detected in two different DNA fragments, of 6,230 bp and 1,559 bp, that were each integrated at the same location in the chromosome in several S. lentus isolates of human, dog, and chicken origin.

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.

The 8.0-kb macrolide-lincosamide-streptogramin B resistance plasmid pSES20 from Staphylococcus lentus harbored part of a Tn917-like transposon including the left terminal repeat, a gene almost identical to ermB, and its regulatory region, as well as the internal direct repeat. Homology between pSES20 and Tn917 ended at a sequence closely related to those of the resolution sites of Tn917 and Tn552 and staphylococcal recombination sites.