J Med Microbiol 55 (2006), 1675-1683; DOI: 10.1099/jmm.0.46700-0
© 2006 Society for General Microbiology
ISSN 1473-5644
Identification of different clonal complexes and diverse amino acid substitutions in penicillin-binding protein 2 (PBP2) associated with borderline oxacillin resistance in Canadian Staphylococcus aureus isolates
Jeya Nadarajah1,
Mark J. S. Lee1,
Lisa Louie1,
Latha Jacob1,
Andrew E. Simor1,
Marie Louie2 and
Martin J. McGavin1
1 University of Toronto Department of Laboratory Medicine and Pathobiology, and Sunnybrook and Women's College Health Sciences Centre, Department of Microbiology, Toronto, ON, Canada
2 University of Calgary, Calgary, AB, Canada
Correspondence
Martin J. McGavin
martin.mcgavin{at}sri.utoronto.ca
Received 26 April 2006
Accepted 8 August 2006
Borderline oxacillin-resistant Staphylococcus aureus (BORSA) exhibit oxacillin MIC values of 1–8 µg ml–1, but lack mecA, which encodes the low-affinity penicillin-binding protein (PBP)2a. The relationship of the BORSA phenotype with specific genetic backgrounds was assessed, as well as amino acid sequence variation in the normal PBP2. Among 38 BORSA, 26 had a common PFGE profile of genomic DNA, and were multilocus sequence type (ST)25. The other isolates were genetically diverse. Complete pbp2 sequences were determined for three BORSA, corresponding to ST25, ST1 and ST47, which were selected on the basis of lacking blaZ-encoded ß-lactamase. The essential transpeptidase-domain-encoding segment of pbp2 was also sequenced from seven additional ST25 isolates. Amino acid substitutions occurred in the transpeptidase domain of all BORSA, irrespective of clonal type. A Gln629
Pro substitution was common to all ST25 BORSA, but most could be distinguished from one another by additional unique substitutions in the transpeptidase domain. The ST1 and ST47 isolates also possessed unique substitutions in the transpeptidase domain. Plasmid-mediated expression of pbp2 from an ST25 or ST1 isolate in S. aureus RN6390 increased its oxacillin MIC from 0.25 to 4 µg ml–1, while pbp2 from a susceptible strain, ATCC 25923, had no effect. Therefore, different amino acid substitutions in PBP2 of diverse BORSA lineages contribute to borderline resistance. The predominant ST25 lineage was not related to any of the five clonal complexes that contain meticillin-resistant S. aureus (MRSA), suggesting that ST25 cannot readily acquire mecA-mediated resistance.
Abbreviations: BORSA, borderline oxacillin-resistant S. aureus; CC, clonal complex; MLST, multilocus sequence typing; MRSA, meticillin-resistant S. aureus; MSSA, meticillin-susceptible S. aureus; PBP, penicillin-binding protein; ST, sequence type.
The GenBank/EMBL/DDBJ accession numbers of pbp2 sequences determined from strains MA15 and MSH12 in this study are AF540019 and AF540020, respectively. Accession numbers AF540021–AF540028 were assigned for the respective partial nucleotide sequences of pbp2 from additional BORSA isolates MA4, MA6, MA8, MA9, MA13, MA14, MSH7 and OGH2.
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INTRODUCTION
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Staphylococcus aureus is a leading cause of nosocomial infections, and is distinguished by its ability to cause infection in virtually every tissue and organ system of the body (Archer, 1998). Therefore, rapid eradication of S. aureus is of paramount importance in the treatment and control of nosocomial infections. However, the introduction of penicillin into clinical practice was quickly followed by the appearance of ß-lactamase-mediated resistance. The introduction of ß-lactamase-resistant semi-synthetic forms of penicillin was also quickly followed by the acquisition of an alternative penicillin-binding protein (PBP), PBP2a, encoded by mecA on the staphylococcal cassette chromosome SCCmec (Hiramatsu et al., 2001; Ito et al., 2001). Meticillin-resistant S. aureus (MRSA) are resistant to all members of the ß-lactam family, due to the low affinity of PBP2a for ß-lactams (Hartman & Tomasz, 1984). As endemic hospital-associated MRSA are typically resistant to multiple antimicrobial agents, an increased emphasis has been placed on their rapid identification, both for determining an appropriate antimicrobial regimen, and to enable appropriate infection control measures (Louie et al., 2000; van Griethuysen et al., 2001; Yamazumi et al., 2001). A by-product of the quest for more sensitive diagnostic techniques has been the identification of strains with an intermediate level of resistance, known as borderline oxacillin-resistant S. aureus (BORSA) (Varaldo, 1993; Varaldo et al., 1993).
Although BORSA infections can be treated with ß-lactams (Varaldo, 1993), outbreaks continue to occur (Balslev et al., 2005); BORSA have been associated with both community-acquired and nosocomial infections in multiple institutions, and have been isolated from various infection sites, including skin and surgical wounds (Kernodle et al., 1998; Massanari et al., 1988; Suggs et al., 1999). Although BORSA do not exhibit a single trait that differentiates them from fully susceptible or resistant strains, many share a common 94/96 phage type, a high degree of genetic relatedness, and a 17.2 kb plasmid that confers hyper-production of ß-lactamase (Barg et al., 1991; Massidda et al., 1996; McMurray et al., 1990). This last feature has been proposed as a factor that contributes to the BORSA phenotype (Barg et al., 1991; McDougal & Thornsberry, 1986), as well as a novel meticillinase (Keseru et al., 2005; Massidda et al., 1992). Others have noted that spontaneous amino acid substitutions in PBP2 of S. aureus can be induced by selection for growth in the presence of ceftizoxime (Leski & Tomasz, 2005), and different substitutions in PBP2 have been noted in one study conducted with a single clinical BORSA strain, CDC-6 (Hackbarth et al., 1995).
Although these archetypal BORSA share a high degree of genetic relatedness, several issues are unresolved, including their association with the overall population structure of S. aureus, which is predominantly clonal (Feil et al., 2003, 2004; Feil & Enright, 2004), the extent to which amino acid substitutions in PBP2 from the BORSA strain CDC-6 are shared by other related BORSA, and the presence of amino acid substitutions in PBP2 from BORSA strains that do not conform to the prototypic 94/96 phage type strains represented by CDC-6. In this respect, diverse amino acid substitutions within the equivalent of PBP2 in Streptococcus pneumoniae also promote reduced susceptibility to ß-lactams (Asahi et al., 1999; Mouz et al., 1999). However, the genetic competence of Strep. pneumoniae and its capacity for interspecies recombination have contributed to a mosaic structure of PBP in penicillin-resistant strains (Dessen et al., 2001; Laible et al., 1991), whereas S. aureus is not noted for genetic competence (Tortosa & Dubnau, 1999), and is more likely to alter gene function by point mutation than genetic recombination (Feil et al., 2003, 2004; Feil & Enright, 2004). Herein, we report on the clonal associations of genetically diverse BORSA, an analysis of amino acid substitutions in their PBP2s, and the contribution of the latter to the BORSA phenotype.
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METHODS
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Bacterial strains, plasmids and growth media.
Regional healthcare centres from across Canada were requested to submit S. aureus strains that exhibited an oxacillin MIC 
1 and 
8 µg ml–1, but were mecA negative. All strains that satisfied these criteria were confirmed as S. aureus which lacked mecA, using a multiplex PCR assay that detected both the nuclease gene (specific for S. aureus) and mecA, with detection of bacterial 16S rRNA as an internal control, as described previously (Louie et al., 2002). Control organisms used with each batch of samples tested included S. aureus ATCC 43300 (MRSA) and meticillin-susceptible S. aureus (MSSA) ATCC 29213. By these criteria, 38 BORSA were collected over a period of 8 years (1990–1998). The isolates were from different geographic locations in Quebec, Ontario and Manitoba, and deemed to be epidemiologically unrelated, as evident either from the identification of distinct genetic backgrounds or from the existence of strains of a similar genetic background that originated from different locations or non-overlapping time spans.
Confirmed BORSA were subjected to antibiotic-susceptibility testing, using cation-adjusted Mueller–Hinton broth supplemented with 2 % NaCl, unless otherwise indicated, according to standard guidelines (National Committee for Clinical Laboratory Standards, 2003). Phage typing and PFGE of SmaI-digested genomic DNA were conducted as described previously (Simor et al., 2001, 2002). Additional strains and plasmids used in this study are described in Table 1
. S. aureus was grown at 37 °C in brain heart infusion (BHI) broth (Difco). Escherichia coli was grown at 37 °C in Luria–Bertani medium (Difco). Media were supplemented with chloramphenicol (10 µg ml–1) or ampicillin (50 µg ml–1), where appropriate.
Plasmid isolation and Southern hybridization.
Plasmid isolation from either E. coli or S. aureus was performed using reagents provided with the GenElute plasmid kit (Sigma), using a lysostaphin treatment step for S. aureus, as necessary. Southern hybridization of HindIII-digested genomic DNA was performed as described previously (Rice et al., 2001), using the Amersham ECL Direct chemiluminescence labelling system. A 316 bp probe representing the 3' end of the S. aureus blaZ-coding sequence was generated by PCR with the forward and reverse primers 5'-TTCAACACCTGCTGCTTTC-3' and 5'-CACTCTTGGCGGTTTCAC-3', using genomic DNA of S. aureus ATCC 29213 as template.
Cloning and sequencing of pbp2.
The pbp2 gene is preceded by prfA (Pinho et al., 1998), encoding a putative DNA recombination and repair function, and the two genes can be transcribed independently or as a polycistronic RNA originating from the prfA promoter. To ensure optimal expression, PCR of genomic DNA was employed to amplify a 3.2 kb fragment, containing pfrApbp2, using forward and reverse primers 5'-cgggatccCACATACTTGTACTTGCCTC-3' and 5'-cccgagctcTTTATGTTGAGTGGA-3', respectively. A 1.28 kb fragment spanning the transpeptidase domain (amino acids 278–701) of pbp2 was amplified with primers 5'-cgggatccGCGAACTTAGTAAATCGTACTCCTG-3' and 5'-gggctgcagCTGCTGTTGCTGATGTGTC-3'. Underlined lower-case nucleotides represent added BamHI, PstI or SacI restriction enzyme sites required for cloning. PCR was performed with AmpliTaq Gold (Roche), and the products were cloned into E. coli INV
F', using the pCR2.1 vector and protocols provided with the Original TA Cloning system (Invitrogen). Plasmid DNA was extracted from selected clones, and sequenced by the University of Toronto Hospital for Sick Children DNA sequencing facility. The 3.2 kb pfrApbp2 fragments were excised from pCR2.1 with BamHI and SacI, ligated with plasmid pRN5548 (Novick et al., 1993), and electroporated into S. aureus RN4220 and RN6390 as detailed in Table 1
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Nucleotide sequence analyses and multilocus sequence typing (MLST).
Sequences obtained from this study were compared to pbp2 from S. aureus strains for which genome sequences are available, and other strains for which pbp2 sequences have been published or deposited in GenBank, as detailed in Table 2. The accession numbers of pbp2 sequences determined from strains MA15 and MSH12 in this study are AF540019 and AF540020, respectively. Accession numbers AF540021–AF540028 were assigned for the respective partial nucleotide sequences of pbp2 from additional BORSA isolates MA4, MA6, MA8, MA9, MA13, MA14, MSH7 and OGH2.
MLST was conducted as described for S. aureus (Enright et al., 2000) using seven gene loci: arcC, aroE, glpF, gmk, pta, tpi and yqi. Each PCR amplicon was sequenced using the forward and reverse primers that were employed for amplification. Automated data analyses and assignment of STs were conducted using the programs provided by the http://saureus.mlst.net/ site, hosted at Imperial College, London, and funded by the Wellcome Trust.
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RESULTS AND DISCUSSION
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Genotypic and phenotypic characterization of BORSA
Genetic diversity among 38 BORSA was assessed by phage typing and PFGE of SmaI-digested genomic DNA using established protocols (Simor et al., 2001, 2002). The plasmid profile of each BORSA isolate was also assessed by agarose gel electrophoresis. The details of representative isolates are presented in Table 3. The majority of BORSA (n=26) possessed an identical ‘type A’ PFGE profile of SmaI-digested genomic DNA, and the 94/96 phage type (group 5) that was previously noted as a common feature of BORSA (Kernodle et al., 1998). Most type A BORSA (n=20) possessed blaZ on a 17.2 kb plasmid, as previously noted for BORSA of the 94/96 phage type (Massidda et al., 1996; McMurray et al., 1990). However, six BORSA did not possess a plasmid. Southern hybridization of HindIII-digested genomic DNA with a probe specific for blaZ revealed that five of these carried a genomic copy of blaZ (data not shown), while a single strain MSH12 did not have blaZ (Table 3). MLST revealed that MSH12 and two other type A BORSA (MA8 and MA14) were ST25 (Table 3). The 12 BORSA that were not PFGE type A had diverse phage types and eight different PFGE patterns (B–I); they included strains MA15 and MA6, corresponding to ST1 and ST47, respectively, which also did not harbour blaZ (Table 3). These two strains, together with MSH12, exhibited MIC values of 1–2 µg ml–1 for penicillin, and 4–8 µg ml–1 for oxacillin (Table 3). Other isolates that possessed blaZ maintained MIC values of 2–8 µg ml–1 for oxacillin, whereas the MIC for penicillin was greater than 64 µg ml–1 (Table 3).
Based on these data, we conclude that hyperproduction of ß-lactamase is not necessary to promote a BORSA phenotype. Although we cannot exclude the possibility of a novel meticillinase that has been described elsewhere (Keseru et al., 2005; Massidda et al., 1992), such an enzyme would have to exhibit little or no activity towards penicillin to account for the low penicillin MIC values that were noted in the few BORSA that lack blaZ.
Analysis of amino acid substitutions in PBP2
PBP2 is a 727 amino acid protein, with an N-terminal transglycosylase domain spanning amino acids 76–244, and a C-terminal transpeptidase domain spanning amino acids 360–653 (Fig. 1) that is essential for peptidoglycan cross-linking (Pinho et al., 2001a,b), and is inhibited by the ß-lactam family of antibiotics. PBPs also have conserved penicillin-binding motifs (Frere & Joris, 1985; Ghuysen, 1991, 1994), which in PBP2 of S. aureus consist of 398SSLK, 454SFN and 583KTG (Fig. 1), with Ser398 being the primary active site serine. We anticipated that if variation in PBP2 was responsible for the BORSA phenotype, we would detect amino acid substitutions in the transpeptidase domain of BORSA strains, but not in known PBP2 sequences from MSSA or MRSA.
We first used the BLAST program (www.ncbi.nlm.nih.gov/blast) to assess variation among known PBP2 sequences obtained from S. aureus genomes or GenBank depositions, as listed in Table 2. Three strains (COL, USA300 and NCTC 8325-4) had identical sequences, relative to which several others had a limited number of amino acid substitutions, in the N-terminal transglycosylase domain (Thr113Ile; Tyr197Cys), in a spacer segment that separates the transglycosylase and transpeptidase domain (Pro285Ala; Ala315Glu), or close to the C-terminus (Ser707Leu) (Fig. 1, Table 2). Strains ATCC 9144 and ATCC 6538P possessed one or two substitutions in the transpeptidase domain, but are both MSSA (Fuller et al., 2005; Hackbarth et al., 1995). The bovine strain RF122, which also harboured a unique substitution in the transpeptidase domain, is described as being penicillin sensitive (Fitzgerald et al., 2000), and is also assumed to be MSSA. Therefore, among S. aureus strains that do not exhibit a BORSA phenotype, amino acid substitutions in the transpeptidase domain of PBP2 are unusual, and do not promote resistance.
We then sequenced pbp2 from the ST1 BORSA strain MA15 and the ST25 BORSA strain MSH12 for comparative purposes. Surprisingly, PBP2 from the ST1 BORSA strain MA15 harboured a single Asp606Val substitution in its transpeptidase domain, at the same site as Asp606Ala in the oxacillin-susceptible strain ATCC 6538P (Table 4). The ST25 BORSA strain MSH12 possessed four substitutions in its transpeptidase domain (Ala405Val; Met580Thr; Gln629Pro; Asn650Asp), all of them different from that of the ST1 strain (Table 4). As the ST1 and ST25 BORSA did not have any common substitutions, we sequenced a pbp2 segment encoding amino acids 278–701 of PBP2, from seven additional ST25 BORSA and the ST47 strain MA6. Each of the ST25 isolates had the Gln629Pro substitution that was present in MSH12, and previously noted in the prototypic BORSA strain CDC-6 (Hackbarth et al., 1995). This defining feature of ST25 BORSA is the only substitution in strains MA8 and MA9 (Table 4). The other ST25 strains, MSH12, MA4, MA13, MA14, MSH7 and OGH2, harboured three, one, one, four, two and one additional substitutions, respectively, that were unique to each isolate. The ST47 isolate MA6 had a single Thr586Ile substitution in the transpeptidase domain.
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Table 4. Comparison of amino acid substitutions in the transpeptidase domain of known PBP2 sequences compared to BORSA
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This substitution is of interest because it is adjacent to the 583KTG penicillin-binding motif, altering the normal 583KTGT586 sequence to 583KTGI, and replaces a small polar amino acid with one with a bulky hydrophobic side chain. When the PBP2 sequence of S. aureus is aligned with that of the PBP 2x protein of Strep. pneumoniae, the 583KTGT sequence in PBP2 is synonymous with the similar 547KSGT550 penicillin-binding motif in PBP 2x (data not shown). Of particular interest, both laboratory-induced and clinical Strep. pneumoniae isolates that exhibit resistance to the extended-spectrum ß-lactam antibiotic cefotaxime harbour a Thr550Ala substitution (547KSGA550) adjacent to this motif (Grebe & Hakenbeck, 1996). Curiously, this substitution promotes cefotaxime resistance, but results in loss of cross-resistance to oxacillin or benzyl penicillin (Grebe & Hakenbeck, 1996). Similarly, it has been noted that a spontaneously selected Pro458Leu substitution adjacent to the 454SFN penicillin-binding motif of PBP2 from S. aureus promotes reduced affinity for the ß-lactam ceftizoxime without affecting the binding of other ß-lactam antibiotics (Leski & Tomasz, 2005). Therefore, it is evident that a single amino acid substitution can exert a complex phenotype with respect to susceptibility or resistance towards different ß-lactam antibiotics. This may explain why BORSA strains MA15 and MSH12, which lack blaZ, exhibit MIC values for oxacillin that are higher than those determined for penicillin.
Expression of BORSA PBP2 confers a borderline-resistant phenotype upon S. aureus RN6390
When PCR products containing the pfrApbp2 genes from the MSSA strain ATCC 25923, and BORSA strains MA15 and MSH12, were cloned into pRN5548 for expression in S. aureus RN6390, increased expression of an approximately 81 kDa protein corresponding to the expected size of PBP2 was observed, but not in RN6390 harbouring pRN5548 alone (Fig. 2). Although each RN6390 derivative exhibited increased PBP2 expression, the MIC for oxacillin was not altered in RN6390 carrying pb_AT derived from the meticillin-sensitive strain ATCC 25923, whereas S. aureus RN6390 harbouring pRN_pbMS or pRN_pbMA plasmids had MICs that were comparable to those of the parent BORSA strains MSH12 and MA15, respectively (Table 5). The pbp2 genes from strains MA15 and MSH12 were selected for expression in S. aureus RN6390 because these strains lacked blaZ-encoded ß-lactamase as a potential contributor to the BORSA phenotype. From these data, we conclude that the BORSA pbp2 gene alone is sufficient to confer a borderline-resistance phenotype. As the ST1 strain MA15 possessed just one amino acid substitution, Asp606Val, in the transpeptidase domain of its PBP2, and expression of this PBP2 in S. aureus RN6390 promoted an oxacillin MIC of 4 µg ml–1 (Table 5), our findings strongly implicate a causative role for this substitution in promoting a BORSA phenotype. Although the Gln629Pro substitution appears to be a common feature of all ST25 BORSA isolates, we cannot exclude the possibility that this substitution is a characteristic feature of the ST25 genetic background, irrespective of BORSA phenotype. Future studies will focus on assessing the influence of this and other substitutions that we have identified as specific mediators of a BORSA phenotype.
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Fig. 2. SDS-PAGE of total cell lysate protein (10 µg) derived from S. aureus RN6390 harbouring plasmids pRN_pbMA (lane 1), pRN_pbMS (lane 2), pRN_pbAT (lane 3), pRN5548 (lane 4) or RN6390 alone (lane 5). The black arrow points to a  82 kDa protein corresponding to the expected size of PBP2, which is expressed at a higher level due to plasmids pRN_pbMA, pRN_pbMS and pRN_pbAT, but not with vector alone (lane 4) or in RN6390 without vector (lane 5).
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Analysis of clonal associations among BORSA
Prior to our study, it was known that most BORSA shared a number of common properties, including a high degree of genetic relatedness, the 94/96 phage type, and a 17.2 kb plasmid that encodes blaZ (Barg et al., 1991; Massidda et al., 1996; McMurray et al., 1990). Our study has established that these isolates are of the ST25 genetic lineage, but also that the BORSA phenotype is not restricted to this lineage. In this context, our data allow for the first time a comparison of the genetic backgrounds of different BORSA strains in relation to the eight major clonal complexes (CCs) that define the population structure of S. aureus (Feil et al., 2003). Strain MA15 was identified as ST1, which is the founding member of the CC1. Other ST1 strains within the CC1 complex include the high-virulence community-acquired MRSA strain MW2, and community-acquired meticillin-susceptible strain MSSA476 (Holden et al., 2004), but these strains do not have the Asp606Val substitution that we identified in PBP2 of strain MA15. Other ST1 isolates in the S. aureus MLST database (http://saureus.mlst.net/) include MSSA nasal-carriage isolates, endemic hospital-associated MSSA, and community-acquired MSSA and MRSA, originating from Canada, Denmark, Australia and the USA. As strains of this genetic background have a proven ability to acquire mecA-mediated resistance, there is presumably no selection pressure for maintenance of the Asp606Val substitution in the ST1 background.
Similarly, the ST47 group, to which BORSA strain MA6 was assigned, is a single-locus variant of ST45, which defines CC45 (Feil et al., 2003), and includes the Berlin strain of epidemic MRSA (Robinson & Enright, 2004). The nine ST47 isolates in the S. aureus MLST database are exclusively meticillin susceptible, but are annotated as the progenitor of the ST45 Berlin strain of epidemic MRSA. Therefore, this genetic background can also readily acquire mecA-mediated resistance, which would eliminate any positive selection for maintenance of the single Thr586Ile substitution that we have noted in the transpeptidase domain of this strain.
The ST25 clone to which the majority of BORSA belong is the founding member of CC25. There are presently 34 ST25 isolates in the MLST database; these are exclusively meticillin susceptible, and do not appear to have spawned any successful variants, because reducing the stringency of comparison to find one-, two-, three-, or four-locus variants identifies 17 additional isolates represented by 14 STs. As these STs are not in any of the five major CCs to which MRSA belong (Enright et al., 2002), it appears that members of CC25 cannot readily acquire mecA-mediated resistance. For this reason, we propose that there is a positive selection for stable maintenance of the diverse amino acid substitutions that we have noted in nosocomial isolates belonging to the ST25 genetic background. Intriguingly, a recent study that employed MLST to assess genetic variation among 258 bovine-associated S. aureus isolates from the USA, Chile and the UK (Smith et al., 2005) revealed that ST25 was one of two predominant STs, ST25 (n=73) and ST124 (n=86), which collectively formed >60 % of bovine isolates. The ST25 strain was the most widely disseminated, being found in the UK, and in 27 of 43 herds that were tested in the USA, and was also significantly more likely to be isolated from teat skin than milk.
Potentially, the prevalence of ST25 S. aureus as a bovine-colonizing strain could be related to their propensity to acquire amino acid substitutions in PBP2, and the management of dairy herds (Chriel & Dietz, 2003), in which antibiotics can be administered during ‘dry’ or non-lactating periods, as a prophylaxis to prevent the incidence of mastitis during future lactation (Bradley & Green, 2004). Such practices could select for ST25 BORSA as a predominant colonizing strain of dairy cattle, and provide a stable reservoir for maintenance of the BORSA phenotype and transmission to humans.
An interesting feature of the ST25 BORSA for which PBP2 sequences were determined is the remarkable variation, apart from Gln629Pro, in the other amino acid substitutions that are present. Studies of S. aureus population structure, combined with genome-sequencing projects, strongly support the close association of specific substitutions with strains of a common genetic background. The ST1 strains MW2 and MSSA476 were isolated on different continents (Holden et al., 2004; Lindsay & Holden, 2004), and harbour a common Pro285Ala substitution, while the closely related MRSA strains N315 and Mu50 isolated in Japan in 1982 and 1997, respectively (Enright et al., 2002; Kuroda et al., 2001), share a single Tyr197Cys substitution (Table 2). It is therefore remarkable that most of the ST25 BORSA exhibit unique strain-specific substitutions in their transpeptidase domains (Table 4). The occurrence of eight substitutions in the partial PBP2 sequence of strain MA14 may indicate a high rate of point mutation within the ST25 group. Alternatively, or in addition, these may be compensatory mutations that arise in response to antibiotic exposure.
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ACKNOWLEDGEMENTS
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J. N. and M. L. were the recipients of scholarship awards from the National Sciences and Engineering Research Council of Canada.
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