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Institut für Molekulare Infektionsbiologie, Universitt Würzburg, Rntgenring 11, 97070 Würzburg, Germany
Departments of Internal Medicine
Pathology and Microbiology, Nebraska Medical Center, Omaha, Nebraska 68198
Robert-Koch-Institut Bereich Wernigerode, Burgstrae 37, 38843 Wernigerode, Germany
ABSTRACT
Staphylococcus epidermidis is part of the normal microflora of the human skin but is also a leading cause of device-associated infections in critically ill patients. Commensal and clinical S. epidermidis isolates differ in their ability to form biofilms on medical devices; the synthesis of biofilms is mediated by the icaADBC operon. Currently, the epidemiological relatedness between ica-positive and -negative isolates is not known; neither is it known whether the ica genes can spread to biofilm-negative strains through horizontal gene transfer. In this study, multilocus sequence typing (MLST) was employed for the clonal analysis of 118 S. epidermidis ica-positive and -negative strains. MLST revealed that the majority of ica-positive and -negative strains were closely related and formed a single clonal complex. Within this complex one sequence type (ST27) was identified which contained exclusively ica-positive isolates and represented the majority of clinical strains tested. ST27 and related ica-positive clones carried different SCCmec cassettes (conferring methicillin resistance) and the insertion sequence IS256. The findings suggest that the S. epidermidis infections analyzed in this report are mainly caused by a single clone (ST27) which occurs preferentially in hospitals and differs from clones in the community. It is hypothesized that the successful establishment of ST27 within nosocomial environments has been facilitated by the presence of genes encoding biofilm and resistance traits.
INTRODUCTION
Staphylococcus epidermidis is a bacterium that constitutes a major component of the normal skin and mucosal microfloras of humans. It is a leading cause of hospital-acquired infections, mostly associated with the use of medical devices in seriously ill or immunocompromised patients. Staphylococcus epidermidis pathogenesis relies on the ability of the bacteria to form thick multilayered biofilms on a wide variety of polymer and metal surfaces (12). In general, biofilms consist of bacteria embedded into a polysaccharide matrix which protects bacteria against hostile environments, including antibiotics and the action of the host immune system, and therefore are an important factor in the development of chronic and recurrent infections (13, 30). Although the production of staphylococcal biofilm is dependent upon multiple regulatory proteins, an essential factor is the presence and expression of the four-gene icaADBC operon (15). The operon encodes enzymes necessary for production of polysaccharide intercellular adhesin (PIA), which is required for biofilm formation and is involved in hemagglutination and bacterial aggregation (7, 19, 20). In animal models, PIA-negative mutants were significantly less likely to cause catheter-associated infections than their PIA-positive isogenic counterparts (27). Epidemiological studies have clearly shown that the majority of commensal Staphylococcus epidermidis isolates lack the ica operon, whereas clinical strains obtained from device-associated infections harbor the genetic information for biofilm formation in addition to certain insertion sequences (IS256) and genes that mediate methicillin and aminoglycoside resistance (1, 9, 10, 17, 36). Currently, the genetic origin of the ica genes in S. epidermidis is not known, and it is also uncertain how biofilm-forming isolates establish and disseminate within the hospital environment. Occurrence of the ica operon in different S. epidermidis genetic backgrounds would suggest mobility and horizontal transfer of the biofilm-mediating genes. However, it is also conceivable that the ica operon imparted to its host a particular evolutionary advantage which allowed a single S. epidermidis clone to spread within multiple hospital environments. Multilocus sequence typing (MLST) has been used as a tool to study the phylogenetic relationships of a variety of bacterial pathogens, including S. aureus and S. epidermidis (8, 29, 32). It can be used to elucidate evolutionary relationships between strains and to identify ancestral genotypes as well as to predict patterns of divergence within groups of related genotypes. In this report, MLST was used to characterize a collection of 118 S. epidermidis isolates. BURST analysis suggested that both ica-positive and -negative isolates were derived from a common ancestral clone and that the majority of ica-positive isolates shared one specific sequence type (ST). We conclude from these data that the majority of the S. epidermidis infections analyzed in this study are caused by a single sequence type which is characterized by its biofilm-forming capacity and antibiotic resistance traits. It is tempting to speculate that this sequence type might represent a suitable genetic background for the acquisition of these genes, resulting in a well-adapted clone which spreads in hospital environments.
MATERIALS AND METHODS
Bacterial isolates. A total of 118 S. epidermidis strains, 62 ica-positive and 56 ica-negative isolates, were analyzed in this study. The collection consisted of 53 isolates of commensal origin and 65 clinical isolates collected between 1991 and 2004 in six different areas of Germany (89 isolates), one hospital in Ireland (1 isolate), one hospital in Norway (8 isolates), and five different areas in North America (18 isolates) (Table 1). S. epidermidis ATCC12228 and S. epidermidis RP62A, two reference strains of which the full-genome nucleotide sequences have recently been published, were also included in the study (11, 34). Clinical strains originated from blood cultures of patients with venous access (n = 31), prosthetic and native valve endocarditis (n = 3 and n = 5, respectively), cerebrospinal fluid (n = 1), and catheter-related urinary tract infections (n = 25). Commensal isolates were obtained from nasal swabs of healthy volunteers (n = 41); 11 commensal isolates were taken from nasal swabs of health care workers. Detailed strain information and references are available on the www.mlst.net site in the S. epidermidis database. Isolates were identified to the species level by biochemical characterization using the API-20-Staph system (bioMerieux, Marcy l'Etoile, France). Isolates that exhibited ambiguous results by this method were retested by amplification and direct nucleotide sequencing of PCR fragments coding for 16S rRNA genes using the primers 5'-TACGGCTACCTTGTTACGACTT-3' and 5'-GAGTTTGATCCTGGCTCA-3' (18).
ica-, mecA-, and IS256-specific PCRs and Southern hybridizations. Isolation of chromosomal DNA, restriction enzyme cleavage, and Southern blotting were done as described previously (36). The ica operon and IS256 were detected by Southern hybridization. For this purpose icaA- and IS256-specific gene probes were amplified using the primers IcaA-forward (5'-GAC CTC GAA GTC AAT AGA GGT-3') and IcaA-reverse (5'-CCC AGT ATA ACG TTG GAT ACC-3') and the primers IS256-forward (5'-TGA AAA GCG AAG AGA TTC AAA GC-3') and IS256-reverse (5'-ATG TAG GTC CAT AAG AAC GGC-3'), respectively, with S. epidermidis RP62A chromosomal DNA as the template and conditions as described previously (17, 37). Bacterial strains were tested for the presence of the mecA gene by PCR using the primers 5'-AAA ATC GAT GGT AAA GGT TGG C-3' and 5'-AGT TCT GCA GTA CCG GAT TTG C-3' (accession no. X52593) and the following conditions: 1 min at 95°C, 30 s at 52°C, and 45 s at 72°C for 30 cycles. The resulting 532-bp PCR fragment was visualized by agarose gel electrophoresis and ethidium bromide staining. S. epidermidis RP62A and S. carnosus TM300 served as positive and negative controls, respectively.
Determination of SCCmec types. SCCmec typing was performed by a multiplex PCR approach with subsequent visualization of the amplified DNA fragment patterns by agarose gel electrophoresis and ethidium bromide staining (24). To distinguish between SCCmec types I to IV, primers and conditions identical to those described by Oliveira et al. (24) were used, with modifications of primers PlsF (5'-GGG GTG GTT AAT GGT ATG AAT AAA-3') and PlsR (5'-CGG AAT GTT GCT CTT GGT TGT GCG TTT TC-3') (23).
MLST. Internal PCR fragments of seven housekeeping genes were amplified using previously described primers (32): for carbamate kinase (arcC), arcC-F (5'-TGT GAT GAG CAC GCT ACC GTT AG-3') and arcC-R (5'-TCC AAG TAA ACC CAT CGG TC TG-3'); for shikimate 5-dehydrogenase (aroE), aroE-F (5'-CAT TGG ATT ACC TCT TTG TTC AGC-3') and aroE-R (5'-CAA GCG AAA TCT GTT GGG G-3'); for glycerol kinase (glpK), glpK-F (5'-CATCACCACGGTCAAAACATGC-3') and glpK-R (5'-CAG GTC GTC CAA TCT ATC ACG C-3'); for guanylate kinase (gmk), gmk-F (5'-TCG ATT CTT AGC GAG TTC AAC C-3') and gmk-R (5'-CCT TCA GGT GTT GGA AAG GG-3'); for phosphate acetyltransferase (pta), pta-F (5'-TAC TGC ATC GTA TCC ACC TAA ACG-3') and pta-R (5'-TGG TGC TGC ACA TTC TAC TGG AG-3'); for triosephosphate isomerase (tpiA), tpiA-F (5'-CCA CCA TAT TGA ATA CGT GTA GCG-3') and tpiA-R (5'-GCT TAC TTT GAA GAA AGC GGT G-3'); and for acetyl coenzyme A acetyltransferase (yqi), yqi-F (5'-TGC TGG ACG GAG TTG TGC TAA C-3') and yqi-R (5'-ATC CTG CTC GTA TTG CTG CG-3'). Nucleotide sequences were determined for both strands by direct, automated sequencing of the PCR products by use of an ABI Prism 310 DNA sequencer (Applied Biosystems, Darmstadt, Germany) or a LiCor model 4000L DNA sequencer (LiCor Biosciences, Lincoln, NE). Nucleotide sequences were compared to known alleles for each locus via the MLST website (http://www.mlst.net). For each isolate a seven-digit allelic profile was established which defined a sequence type (ST). Clonal analysis of the STs was performed using BURST, a Web-implemented clustering algorithm (http://www.mlst.net), which divides MLST data sets into groups of related isolates and predicts the founding genotype of each clonal complex (28). STs with at least five of seven identical alleles were defined as a clonal group.
Recombination analysis. Recombination rates were estimated by pairwise comparison of the nucleotide sequence of each single-locus variant (SLV; i.e., an ST which differs from another ST in only one locus and remains unchanged in the other six) with that of its predicted clonal ancestor as described previously (6). Briefly, BURST was used to identify SLVs and their associated ancestor STs. The nucleotide sequences of the various alleles were compared to each other using Sequence Output, a Macintosh program available from the MLST website, which allows for displaying polymorphic sites. An allele was determined to have resulted from point mutation when two criteria were fulfilled: first, when the allele differed only in one nucleotide site from its putative ancestor allele, and second, when the allele was unique within the data set of this study and within the data deposited in the mlst.net database. Recombination was determined to have occurred when an allele differed in more than one nucleotide site or when the allele varied in a single site but corresponded to a known allele found elsewhere in the database.
RESULTS
MLST profiles and ica presence in clinical and commensal isolates. MLST revealed a total of 28 different sequence types (STs) among the 118 S. epidermidis isolates (Table 1). Sequence types ST2 to ST29 corresponded to known allelic profiles in the mlst.net S. epidermidis database, whereas ST33 to ST56 represented novel sequence types. Entire nucleotide sequences of all alleles are available via the mlst.net database. The majority of the clinical strains (58 of 65; 89.2%) were ica positive (Fig. 1A) and displayed nine different STs (Fig. 1B). In this respect, ST27 was a remarkable clone. It harbored almost half of all clinical isolates tested (32 of 65; 49.2%) and the bulk of the ica-positive isolates (35 of 62; 56.4%) (Fig. 1B). Among commensal strains of healthy volunteers, the ica operon was found in only 4 of 41 isolates (9.7%); three of these belonged to ST27 and one to ST12. Commensal strains from hospital staff, however, carried the ica operon in 8 of 11 isolates (72.7%). These isolates were grouped into ST2 (n = 3), ST7 (n = 1), ST12 (n = 1), ST18 (n = 1), ST33 (n = 1), and ST34 (n = 1).
Clonal relationship of the STs. BURST was used to divide the 118 isolates into clonal complexes by comparison of their allelic profiles (Fig. 2). This analysis indicated that the majority of isolates are closely related to each other and form a single major group or clonal complex (Fig. 2). ST2, which was the sequence type that generated the most abundant single-locus variants (SLVs), was considered the common ancestor clone. A total of 30 isolates, both ica positives and negatives (7 versus 23), belonged to ST2. ST27, the clone that represented the majority of the ica-positive isolates (56.4%) and 49.2% of the clinical isolates (Fig. 1), is a single-locus variant of ST2 differing only in the arcC allele. Interestingly, this clone is a source of isolates which carry, concomitantly with the ica operon, genes that mediate methicillin resistance (mecA) and the insertion sequence IS256 (see below).
Detection of the ica genes in an unrelated genetic background. BURST analysis identified four additional STs which were unrelated to the major clonal group (Fig. 2). Each of these STs was represented by one isolate. ST33 and ST35 shared five identical alleles and were therefore regarded as a minor group (Table 1 and Fig. 2). ST40 and ST54 are singletons and are unrelated to each other and to any other ST identified in this study. Interestingly, ST33 represented an ica-positive strain, indicating that the ica operon can also be present in S. epidermidis lineages that differ from the ST2-derived clonal complex.
SCCmec typing and IS256 detection. In previous studies we have shown a correlation between the presence of the ica operon, methicillin resistance, and detection of the insertion sequence IS256 in clinical S. epidermidis isolates (17). Methicillin resistance in staphylococci is mediated by the mecA gene, which is located on a large, mobile DNA element termed SCCmec (16). Four major SCCmec types have been identified for which mobility and repeated transfer into different genetic backgrounds of S. aureus and coagulase-negative staphylococci has been demonstrated (5, 8, 32). IS256 is an insertion sequence element which is part of the aminoglycoside resistance-mediating transposon Tn4001 but also occurs independently in multiple copies in the genomes of staphylococci and enterococci. IS256 has the capacity to influence expression of the ica operon and subsequent biofilm formation by reversible insertion into the ica operon and its regulatory genes as well as by chromosomal rearrangements (2, 14, 35, 37). To investigate whether SCCmec cassettes and IS256 occur in specific STs, all isolates were tested for the presence of these genes. Of the 118 S. epidermidis strains tested, 43 (37 clinical isolates and 6 commensal isolates) were mecA positive. Four of the commensal isolates originated from hospital staff members. SCCmecII was detected in 13 strains, SCCmecIII in 14 strains, and SCCmecIV in 13 strains. Three mecA-positive isolates were nontypeable, and none of the strains carried an SCCmecI cassette (Table 1). ST27, the most prevalent ica-positive clone, contained 27 (62.8%) of the 43 methicillin-resistant isolates. Four different SCCmec cassettes were found in these 27 mecA-positive isolates: SCCmecII (n = 12), SCCmecIII (n = 12), SCCmecIV (n = 1), and SCCmec NT (n = 2). IS256 was detected in 45 of 118 S. epidermidis strains: 40 clinical strains and 5 commensals. Four of the five IS256-positive commensals were obtained from health care workers. IS256-carrying strains were detected in nine STs which contained both ica-positive and SCCmec-positive isolates, and ST27 again represented the clone containing the most IS256-positive isolates (28 of 45; 62.2%). Taken together, these data suggest that SCCmec and IS256 occur jointly in specific S. epidermidis clones which harbor the ica operon and occur preferentially in the hospital environment.
Geographic distribution of ica-positive clones. Table 1 summarizes the properties of each ST with respect to ica and IS256 presence, including SCCmec typing as well as its geographic distribution. ica-positive STs were preferentially found in medical facilities. Interestingly, these clones were not restricted to a specific geographic area. ica-positive ST27 isolates were identified in hospitals from four regions in Germany, in one hospital in Norway, and at four locations in the United States. Except for ST12, ST28, ST33, and ST51, which represent only one or two isolates, respectively, all ica-positive STs were detected in more than one hospital (Table 1).
Estimates of recombination rates. To address the question of whether S. epidermidis strains diverge by point mutation or by recombination, a pairwise comparison of single-locus variants (SLV) was done by analysis of their polymorphic sites (data not shown). An allele was considered as likely to have arisen by point mutation when (i) it differed only in one nucleotide from the corresponding ancestor allele and (ii) the allele was unique in the data set and the database on the S. epidermidis mlst.net site. In contrast, recombination was assumed when the allele differed in more than one nucleotide and/or was already present in the MLST database (6). Table 2 shows that 12 of 15 SLVs likely evolved by recombination and only 3 of 15 by point mutation, suggesting that recombination is the major mechanism for divergence of S. epidermidis strains.
DISCUSSION
Prevalence of ST27 and other ica-positive clones in hospital settings. Employing a recently established MLST scheme, this study demonstrated that the majority of the S. epidermidis infections analyzed in this report are caused by a single clone (i.e., ST27) whose presence is widespread within hospital settings in Germany. Interestingly, this sequence type was also detected in a Norwegian hospital and in medical facilities in the United States. In addition to ST27, 11 other ica-positive clones with similar properties were identified. The presence of these clones in the hospital environment is in agreement with a recent MLST study on the distribution of SCCmec cassettes in clinical S. epidermidis isolates (32). Wisplinghoff and coworkers analyzed a North American collection of 67 methicillin-resistant and -susceptible S. epidermidis isolates from blood cultures and prosthetic valve endocarditis. Nine of the 32 MLST profiles identified in the SCCmec study coincide with ica-positive STs identified in our report, i.e., ST2, ST5, ST7, ST8, ST12, ST18, ST27, ST28, and ST29. Moreover, there was good agreement with respect to the SCCmec types identified in the STs of both reports. At present we do not know whether the isolates analyzed by Wisplinghoff et al. are also ica and IS256 positive, but their origination from device-associated infections makes it very likely that they are also biofilm formers.
ST27 and other ica-positive clones were rarely found outside of medical facilities, suggesting that they are highly adapted to the hospital environment and differ from commensal S. epidermidis in the community. It is conceivable that patients who are admitted to a hospital are soon colonized by these biofilm-forming, multiresistant S. epidermidis isolates and that this newly acquired endogenous microflora represents the origin for a later infection. This hypothesis is supported by a recent ecological study in bone marrow transplantation patients demonstrating that these individuals carry ica-, mecA-, and IS256-positive S. epidermidis isolates as commensals (26). Colonization of hospital staff by ica-positive S. epidermidis isolates in our report highlights again the presence of such strains in the hospital environment. However, larger studies are necessary to elucidate whether or not the personnel might function as transmitters of biofilm-forming strains to patients. Moreover, it will be interesting to elucidate the possible impact of other virulence-associated factors such as the phenol-soluble modulins or other biofilm-mediating genes such as aap and bhp in these clones (4, 25, 33).
Origin and attributes of ST27 isolates. In addition to ica, the majority of ST27 isolates carry different SCCmec cassettes and multiple copies of the insertion sequence IS256. It is unknown why ST27 has become the dominant ST within nosocomial environments in Germany; however, the answer may be related to the increased reliance on implanted medical devices within immunocompromised patients. These devices represent a suitable habitat for colonization, and the extended use of antibiotics and disinfectants generates a highly selective pressure for the generation of resistant variants. Occupation of this niche requires specific prerequisites which are obviously met by ST27. The ica operon enables the bacteria to stick to polymer and metal surfaces, and the ability to gain resistance determinants helps ST27 isolates withstand hostile external conditions. The presence of different SCCmec cassettes in ST27 suggests repeated, independent transfer of SCCmec DNA into this genetic background and is an indication of the capacity of ST27 to exchange genetic material. The question of whether or not the ica genes are also subject to horizontal gene transfer must remain open at this stage of experimental work. However, the detection of the ica operon in an unrelated S. epidermidis background in our study and the identification of ica homologues in pathogenic Escherichia coli and a broad range of other bacterial species support the hypothesis of mobility and lateral transfer of biofilm-encoding genes (31). The recent publication of the genome sequences of a methicillin-resistant ica-positive S. epidermidis clinical strain and an ica-negative commensal isolate revealed that most of the genetic variations between the two genomes are attributed to genomic islands, phages, and integrated plasmids (11, 34). In this regard, it was concluded that lateral gene transfer between staphylococci and other low-GC gram-positive bacteria is common and contributes considerably to resistance and virulence development of S. epidermidis (11). Interestingly, we observed in studies of the spontaneous emergence of biofilm-negative variants that DNA encompassing the ica operon is unstable and can be readily deleted from the S. epidermidis genome (P. D. Fey and W. Ziebuhr, unpublished data). Instability of genetic material is often an indication of mobility, and in this respect it is also conceivable that the ica operon represents mobile DNA that has been lost in the commensal S. epidermidis isolates. Another finding of our study is that the generation of SLVs within S. epidermidis seems to occur generally by recombination rather than by point mutation. This is in contrast to what has been described in S. aureus studies, where clonal complexes were shown to expand primarily by point mutation (6). The molecular background for this difference between the two species remains to be elucidated, but it might reflect again the capacity of S. epidermidis to acquire and incorporate foreign DNA.
It is tempting to speculate that ST27 isolates represent an ideal genetic background for biofilm and resistance genes, resulting in well-adapted strains which are then selected in the hospital environment. The presence of multiple copies of IS256 in the ST27 genome might support this adaptation process by an ongoing generation of novel phenotypic and genotypic variants. Thus, involvement of IS256 in genome flexibility has been shown for the modulation of biofilm, resistance, and global regulatory gene expression (2, 3, 21, 22, 35, 37). Therefore, the combination of biofilm formation, antibiotic resistance, and genetic flexibility may explain why ST27 has become the dominant ST within medical facilities. The data of this report provide novel insights into the population structure of S. epidermidis and may suggest new infection control measures for the management of these infections in the future.
ACKNOWLEDGMENTS
We are grateful to our colleagues who provided us with Staphylococcus epidermidis isolates: K. Naber, R. Marre, E. Straube, D. Mack, F. Gtz, and W. Thomas (Germany); P. Meyer (Norway); V. D. Fowler, G. L. Archer, and R. J. Sheretz (United States); and J. P. O'Gara (Ireland). We thank M. C. Enright and S. O'Hanlon (University of Bath, Bath, United Kingdom) for data entry into the mlst.net database.
The work of S.K. and W.Z. was supported by the Deutsche Forschungsgemeinschaft SFB479 and the Federal Ministry of Education and Research (BMBF03F0401C) and that of K.O. by the Deutsche Forschungsgemeinschaft SFB630. P.D.F. was supported by grant 5RO1 AI49311 from the National Institute of Allergy and Infectious Diseases.
REFERENCES
Cho, S. H., K. Naber, J. Hacker, and W. Ziebuhr. 2002. Detection of the icaADBC gene cluster and biofilm formation in Staphylococcus epidermidis isolates from catheter-related urinary tract infections. Int. J. Antimicrob. Agents 19:570-575.
Conlon, K. M., H. Humphreys, and J. P. O'Gara. 2004. Inactivations of rsbU and sarA by IS256 represent novel mechanisms of biofilm phenotypic variation in Staphylococcus epidermidis. J. Bacteriol. 186:6208-6219.
Couto, I., S. W. Wu, A. Tomasz, and H. de Lencastre. 2003. Development of methicillin resistance in clinical isolates of Staphylococcus sciuri by transcriptional activation of the mecA homologue. J. Bacteriol. 185:645-653.
Cucarella, C., C. Solano, J. Valle, B. Amorena, I. Lasa, and J. R. Penades. 2001. Bap, a Staphylococcus aureus surface protein involved in biofilm formation. J. Bacteriol. 183:2888-2896.
Enright, M. C., D. A. Robinson, G. Randle, E. J. Feil, H. Grundmann, and B. G. Spratt. 2002. The evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA). Proc. Natl. Acad. Sci. USA 99:7687-7692.
Feil, E. J., J. E. Cooper, H. Grundmann, D. A. Robinson, M. C. Enright, T. Berendt, S. J. Peacock, J. M. Smith, M. Murphy, B. G. Spratt, C. E. Moore, and N. P. Day. 2003. How clonal is Staphylococcus aureus J. Bacteriol. 185:3307-3316.
Fey, P. D., J. S. Ulphani, F. Gtz, C. Heilmann, D. Mack, and M. E. Rupp. 1999. Characterization of the relationship between polysaccharide intercellular adhesin and hemagglutination in Staphylococcus epidermidis. J. Infect. Dis. 179:1561-1564.
Fitzgerald, J. R., D. E. Sturdevant, S. M. Mackie, S. R. Gill, and J. M. Musser. 2001. Evolutionary genomics of Staphylococcus aureus: insights into the origin of methicillin-resistant strains and the toxic shock syndrome epidemic. Proc. Natl. Acad. Sci. USA 98:8821-8826.
Frebourg, N. B., S. Lefebvre, S. Baert, and J. F. Lemeland. 2000. PCR-based assay for discrimination between invasive and contaminating Staphylococcus epidermidis strains. J. Clin. Microbiol. 38:877-880.
Galdbart, J. O., J. Allignet, H. S. Tung, C. Ryden, and N. El Solh. 2000. Screening for Staphylococcus epidermidis markers discriminating between skin-flora strains and those responsible for infections of joint prostheses. J. Infect. Dis. 182:351-355.
Gill, S. R., D. E. Fouts, G. L. Archer, E. F. Mongodin, R. T. Deboy, J. Ravel, I. T. Paulsen, J. F. Kolonay, L. Brinkac, M. Beanan, R. J. Dodson, S. C. Daugherty, R. Madupu, S. V. Angiuoli, A. S. Durkin, D. H. Haft, J. Vamathevan, H. Khouri, T. Utterback, C. Lee, G. Dimitrov, L. Jiang, H. Qin, J. Weidman, K. Tran, K. Kang, I. R. Hance, K. E. Nelson, and C. M. Fraser. 2005. Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain. J. Bacteriol. 187:2426-2438.
Gtz, F. 2002. Staphylococcus and biofilms. Mol. Microbiol. 43:1367-1378.
Hall-Stoodley, L., J. W. Costerton, and P. Stoodley. 2004. Bacterial biofilms: from the natural environment to infectious diseases. Nat. Rev. Microbiol. 2:95-108.
Handke, L. D., K. M. Conlon, S. R. Slater, S. Elbaruni, F. Fitzpatrick, H. Humphreys, W. P. Giles, M. E. Rupp, P. D. Fey, and J. P. O'Gara. 2004. Genetic and phenotypic analysis of biofilm phenotypic variation in multiple Staphylococcus epidermidis isolates. J. Med. Microbiol. 53:367-374.
Heilmann, C., O. Schweitzer, C. Gerke, N. Vanittanakom, D. Mack, and F. Gtz. 1996. Molecular basis of intercellular adhesion in the biofilm-forming Staphylococcus epidermidis. Mol. Microbiol. 20:1083-1091.
Ito, T., K. Okuma, X. X. Ma, H. Yuzawa, and K. Hiramatsu. 2003. Insights on antibiotic resistance of Staphylococcus aureus from its whole genome: genomic island SCC. Drug Resist. Updates 6:41-52.
Kozitskaya, S., S. H. Cho, K. Dietrich, R. Marre, K. Naber, and W. Ziebuhr. 2004. The bacterial insertion sequence element IS256 occurs preferentially in nosocomial Staphylococcus epidermidis isolates: association with biofilm formation and resistance to aminoglycosides. Infect. Immun. 72:1210-1215.
Lane, D. J. 1991. 16S/23S rRNA sequencing, p. 115-175. In E. Stackebrandt and M. Goodfellow (ed.), Nucleic acids techniques in bacterial systematics. John Wiley & Sons, New York, N.Y.
Mack, D., W. Fischer, A. Krokotsch, K. Leopold, R. Hartmann, H. Egge, and R. Laufs. 1996. The intercellular adhesin involved in biofilm accumulation of Staphylococcus epidermidis is a linear beta-1,6-linked glucosaminoglycan: purification and structural analysis. J. Bacteriol. 178:175-183.
Mack, D., J. Riedewald, H. Rohde, T. Magnus, H. H. Feucht, H. A. Elsner, R. Laufs, and M. E. Rupp. 1999. Essential functional role of the polysaccharide intercellular adhesin of Staphylococcus epidermidis in hemagglutination. Infect. Immun. 67:1004-1008.
Maki, H., N. McCallum, M. Bischoff, A. Wada, and B. Berger-Bchi. 2004. tcaA inactivation increases glycopeptide resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 48:1953-1959.
Maki, H., and K. Murakami. 1997. Formation of potent hybrid promoters of the mutant llm gene by IS256 transposition in methicillin-resistant Staphylococcus aureus. J. Bacteriol. 179:6944-6948.
O'Brien, F. G., T. T. Lim, F. N. Chong, G. W. Coombs, M. C. Enright, D. A. Robinson, A. Monk, B. Said-Salim, B. N. Kreiswirth, and W. B. Grubb. 2004. Diversity among community isolates of methicillin-resistant Staphylococcus aureus in Australia. J. Clin. Microbiol. 42:3185-3190.
Oliveira, D. C., and H. de Lencastre. 2002. Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 46:2155-2161.
Rohde, H., C. Burdelski, K. Bartscht, M. Hussain, F. Buck, M. A. Horstkotte, J. K. Knobloch, C. Heilmann, M. Herrmann, and D. Mack. 2005. Induction of Staphylococcus epidermidis biofilm formation via proteolytic processing of the accumulation-associated protein by staphylococcal and host proteases. Mol. Microbiol. 55:1883-1895.
Rohde, H., M. Kalitzky, N. Kroger, S. Scherpe, M. A. Horstkotte, J. K. Knobloch, A. R. Zander, and D. Mack. 2004. Detection of virulence-associated genes not useful for discriminating between invasive and commensal Staphylococcus epidermidis strains from a bone marrow transplant unit. J. Clin. Microbiol. 42:5614-5619.
Rupp, M. E., P. D. Fey, C. Heilmann, and F. Gotz. 2001. Characterization of the importance of Staphylococcus epidermidis autolysin and polysaccharide intercellular adhesin in the pathogenesis of intravascular catheter-associated infection in a rat model. J. Infect. Dis. 183:1038-1042.
Smith, J. M., N. H. Smith, M. O'Rourke, and B. G. Spratt. 1993. How clonal are bacteria Proc. Natl. Acad. Sci. USA 90:4384-4388.
Urwin, R., and M. C. Maiden. 2003. Multi-locus sequence typing: a tool for global epidemiology. Trends Microbiol. 11:479-487.
Vuong, C., J. M. Voyich, E. R. Fischer, K. R. Braughton, A. R. Whitney, F. R. DeLeo, and M. Otto. 2004. Polysaccharide intercellular adhesin (PIA) protects Staphylococcus epidermidis against major components of the human innate immune system. Cell Microbiol. 6:269-275.
Wang, X., J. F. Preston III, and T. Romeo. 2004. The pgaABCD locus of Escherichia coli promotes the synthesis of a polysaccharide adhesin required for biofilm formation. J. Bacteriol. 186:2724-2734.
Wisplinghoff, H., A. E. Rosato, M. C. Enright, M. Noto, W. Craig, and G. L. Archer. 2003. Related clones containing SCCmec type IV predominate among clinically significant Staphylococcus epidermidis isolates. Antimicrob. Agents Chemother. 47:3574-3579.
Yao, Y., D. E. Sturdevant, and M. Otto. 2005. Genomewide analysis of gene expression in Staphylococcus epidermidis biofilms: insights into the pathophysiology of S. epidermidis biofilms and the role of phenol-soluble modulins in formation of biofilms. J. Infect. Dis. 191:289-298.
Zhang, Y. Q., S. X. Ren, H. L. Li, Y. X. Wang, G. Fu, J. Yang, Z. Q. Qin, Y. G. Miao, W. Y. Wang, R. S. Chen, Y. Shen, Z. Chen, Z. H. Yuan, G. P. Zhao, D. Qu, A. Danchin, and Y. M. Wen. 2003. Genome-based analysis of virulence genes in a non-biofilm-forming Staphylococcus epidermidis strain (ATCC 12228). Mol. Microbiol. 49:1577-1593.
Ziebuhr, W., K. Dietrich, M. Trautmann, and M. Wilhelm. 2000. Chromosomal rearrangements affecting biofilm production and antibiotic resistance in a Staphylococcus epidermidis strain causing shunt-associated ventriculitis. Int. J. Med. Microbiol. 290:115-120.
Ziebuhr, W., C. Heilmann, F. Gtz, P. Meyer, K. Wilms, E. Straube, and J. Hacker. 1997. Detection of the intercellular adhesion gene cluster (ica) and phase variation in Staphylococcus epidermidis blood culture strains and mucosal isolates. Infect. Immun. 65:890-896.
Ziebuhr, W., V. Krimmer, S. Rachid, I. Loessner, F. Gtz, and J. Hacker. 1999. A novel mechanism of phase variation of virulence in Staphylococcus epidermidis: evidence for control of the polysaccharide intercellular adhesin synthesis by alternating insertion and excision of the insertion sequence element IS256. Mol. Microbiol. 32:345-356.