ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 2010, p. 4589–4595 0066-4804/10/$12.00 doi:10.1128/AAC.00470-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 54, No. 11
Diversity of Staphylococcal Cassette Chromosome mec Elements in Predominant Methicillin-Resistant Staphylococcus aureus Clones in a Small Geographic Area䌤 Patrick Basset,* Laurence Senn, Vale´rie Vogel, Giorgio Zanetti, and Dominique S. Blanc Hospital Preventive Medicine Service, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland Received 7 April 2010/Returned for modification 13 July 2010/Accepted 6 August 2010
Recent population genetic studies suggest that staphylococcal cassette chromosome mec (SCCmec) was acquired much more frequently than previously thought. In the present study, we aimed to investigate the diversity of SCCmec elements in a local methicillin-resistant Staphylococcus aureus (MRSA) population. Each MRSA isolate (one per patient) recovered in the Vaud canton of Switzerland from January 2005 to December 2008 was analyzed by the double-locus sequence typing (DLST) method and SCCmec typing. DLST analysis indicated that 1,884/2,036 isolates (92.5%) belong to four predominant clones. As expected from the local spread of a clone, most isolates within clones harbored an identical SCCmec type. However, three to seven SCCmec types have been recovered in every predominant DLST clone, suggesting that some of these elements might have been acquired locally. This pattern could also be explained by distinct importations of related isolates into the study region. The addition of a third highly variable locus to further increase the discriminatory power of typing as well as epidemiological data suggested that most ambiguous situations were explained by the second hypothesis. In conclusion, our study showed that even if the acquisition of new SCCmec elements at a local level likely occurs, it does not explain all the diversity observed in the study region. cus sequence typing (MLST) data have revealed isolates with identical sequence types (STs) but with different SCCmec types. These studies demonstrated that the cassette has been acquired on multiple occasions (at least 20 times) during the evolution of S. aureus (17, 24, 30). However, the multiple acquisitions of SCCmec elements at the local level compared to international spread remain debated. The sampling of identical clones in different countries suggested that a single SCCmec acquisition was usually followed by clonal spread. However, methicillin-sensitive S. aureus (MSSA) strains genetically identical to the predominant MRSA strains have been observed at the local level (16, 18), suggesting local acquisitions of SCCmec. Recently, a study based on single-nucleotide polymorphism (SNP) discovery in a worldwide collection of ST 5 showed a close association between phylogenetic lineages and geography (26). These data indicate that the geographical spread of MRSA over long distances is a rare event compared with the frequency of local SCCmec acquisition. However, this hypothesis was never carefully investigated in a large and local population. A previous study by Nu ¨bel et al. (26) also highlighted the importance of using typing tools with high discriminatory power when investigating the epidemiology and microevolution of MRSA clones. For this reason, we recently developed the double-locus sequence typing (DLST) method based on the analysis of approximately 500 bp of the highly variable clfB and spa genes. This method has a high discriminatory power comparable to that of pulsed-field gel electrophoresis (PFGE) (23) and a good congruence with MLST results (3), and it allows the study of local epidemiological collections of isolates as well as the microevolution of clones (4, 27). Molecular epidemiological surveillance of MRSA in Western Switzerland revealed the successive emergence of four lineages (6). DLST analysis showed that isolates of these
Methicillin-resistant Staphylococcus aureus (MRSA) is currently one of the most commonly identified antibiotic-resistant nosocomial pathogens worldwide. Numerous studies found only one or a few predominant clones in places where a high proportion of MRSA isolates among S. aureus strains was encountered (1, 2, 11, 12). These clones were found not only in the same region but also in distant countries. For example, the molecular epidemiology of more than 3,000 MRSA isolates from Southern Europe, the United States, and South America showed that nearly 70% of them belonged to five major pandemic clones (1, 28, 29). Each of these clones has been hypothesized to be particularly transmissible and/or well adapted to the hospital environment (5, 10). Appropriate control of the important MRSA problem requires a thorough understanding of the processes underlying the emergence and spread of MRSA clones. The key step for the emergence of MRSA clones is the acquisition of staphylococcal cassette chromosome mec (SCCmec) (21), which carries the mecA gene, responsible for methicillin resistance (25). The major role of SCCmec is illustrated by the relationship between the different waves of MRSA and evolutionary changes in SCCmec (7). Although SCCmec typing is essential for the characterization of MRSA clones, a rationalized nomenclature for SCCmec was proposed only recently (8, 20). At least eight main types (types I to VIII) as well as several subtypes of SCCmec elements have been reported so far for MRSA strains (20). Population genetic studies based on the analysis of multilo* Corresponding author. Mailing address: Service of Hospital Preventive Medicine, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne, Switzerland. Phone: 41 21 314 40 61. Fax: 41 21 314 02 62. E-mail:
[email protected]. 䌤 Published ahead of print on 16 August 2010. 4589
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FIG. 1. DLST SLV clustering using eBurst on 2,036 MRSA isolates recovered in the Vaud canton (Switzerland) between 2005 and 2008. Each circle represents one DLST type, and the diameter of the circles reflects the frequency of that type (i.e., number of isolates). Linked DLST types differ at one of the two loci (clfB or spa), and each color represents an SCCmec type.
clones are comprised within four clusters of related strains, each consisting of a founder (DLST types 1-1, 2-2, 3-3, and 4-4) and surrounded by related strains (single-locus variants [SLVs]) (4). The local spread of several clones represents a promising opportunity to understand the patterns of acquisition of SCCmec elements. Consequently, we aimed in the present study to investigate the diversity of SCCmec elements in this local population with the hypothesis that the occurrence of multiple elements in isolates of a disseminating clone is an indication of local acquisitions. Identification of the changes and genetic relationships among SCCmec on both the local and global scales is of primary importance to understand how and why new MRSA clones emerge. MATERIALS AND METHODS Setting and bacterial isolates. Medical laboratories and infection control programs of the canton of Vaud, Switzerland, report all MRSA isolates (from infected or colonized inpatients or outpatients) to our reference laboratory (University Hospital of Lausanne). All isolates recovered between January 2005 and December 2008 were identified at the species level and checked for methicillin resistance (disk diffusion antibiogram with oxacillin and/or agglutination test for the detection of the PBP2⬘ protein [Slidex MRSA detection; bioMe´rieux]). One isolate per patient was saved by the reference laboratory. For each patient, demographic data were collected (e.g., name, age, and sex), as was the date and site of MRSA isolation. In addition, a susceptibility profile encompassing eight antibiotics was performed by using the Kirby-Bauer method according to CLSI guidelines (9). Typing methods. DLST was performed on each isolate according to a method previously described (23). In short, this method is based on the sequencing of two highly polymorphic adhesin genes (i.e., clfB and spa). For each gene, an arbitrary number is given to each allele, and the combination of both clfB and spa alleles constitutes the DLST type.
To increase the discriminatory power of the DLST method, a third polymorphic marker (i.e., clfA) was further analyzed, as described previously (23), on a subsample of isolates of the four main clones. This subsample contained all the isolates with an SCCmec type different from the majority as well as isolates randomly drawn from each of the four main clones. The number of isolates analyzed from each clone was proportional to the frequency of that clone. SCCmec typing. The SCCmec element of each isolate was determined with the first two multiplex PCRs (M-PCRs) according to a scheme reported previously by Kondo et al. (22). In addition, SCCmec subtypes were also determined for a subsample of 396 isolates with multiplex PCRs 3 and 4 of the scheme reported previously by Kondo et al. (22). This subsample was randomly selected from among all isolates. Clone definition. Whereas DLST markers seem highly stable during local epidemiological investigations, they could undergo changes over a long period. Thus, we also considered related genotypes in our definition of clones. As the reconstruction of genetic relatedness is highly problematic with sequences containing repeats, we used an approach similar to the analysis of MLST data with the BURST (based upon related sequence type) algorithm. We considered only allelic data, and relatedness was defined by clustering all DLST types sharing one of the two alleles (single-locus variant [SLV]). With this approach, a clone was defined by the cluster of SLV DLST types and its founder. This analysis was performed by using eBurst (http://eburst.mlst.net/). The same SLV definition was applied to the analysis of the subsample of isolates for which the clfA gene was also sequenced.
RESULTS Local DLST analysis. Between January 2005 and December 2008, a total of 248 DLST genotypes were observed among the 2,036 isolates (one per patient) recovered in the Vaud canton of Switzerland (Fig. 1). Cluster analysis revealed that the majority (92.5%) of these isolates belonged to the four predominant clones corresponding to the founders and single-locus
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TABLE 1. SCCmec types observed among all strains and in the four major DLST clones of Western Switzerland No. of strains of SCCmec type I
II
IIImercury
IV
V
VI
Ua
Total no. of strains
1-1 2-2 3-3 4-4 Other
0 9 1 192 1
0 1,282 2 2 4
0 0 0 0 23
61 18 237 3 92
2 10 0 2 24
0 2 2 0 0
1 2 46 10 8
64 1,323 288 209 152
All
203
1,290
23
411
38
4
67
2,036
DLST clone
a
Unusual SCCmec variants.
variants of DLST genotypes 1-1 (n ⫽ 64; 3.1%), 2-2 (n ⫽ 1,323; 65.0%), 3-3 (n ⫽ 288; 14.1%), and 4-4 (n ⫽ 209; 10.3%). In addition, 14 smaller clusters, composed of 2 to 10 DLST types, and 25 isolated DLST types were observed. SCCmec analysis. The analysis of the SCCmec types of the 2,036 isolates with the first two PCRs described previously by Kondo (22) showed the presence of the six main types (Fig. 1 and Table 1): 203 (10.0%) of SCCmec type I, 1,290 (63.4%) of SCCmec type II, 23 (1.1%) of SCCmec type IIImercury (IIImerc), 411 (20.2%) of SCCmec type IV, 38 (1.9%) of SCCmec type V, and 4 (0.2%) of SCCmec type VI. In addition, 67 isolates (3.3%) showed an atypical PCR pattern with the scheme of Kondo et al., representing eight different SCCmec variants (Table 2). The majority of isolates in each of the four main clones harbored an identical SCCmec type: SCCmec type IV in 61 (95%) isolates of DLST clone 1-1, SCCmec type II in 1,282 (97%) isolates of clone 2-2, SCCmec type IV in 237 (82%) isolates of clone 3-3, and SCCmec type I in 192 (92%) isolates of clone 4-4. Interestingly, SCCmec types different from the predominant one were present in each of the four predominant clones (Table 1): SCCmec type V plus one atypical variant in clone 1-1; SCCmec types I, IV, V, and VI plus two atypical variants in clone 2-2; SCCmec types I, II, and VI plus three atypical variants in clone 3-3; and SCCmec types II, IV, and V plus one atypical variant in clone 4-4. The SCCmec elements of 396 isolates were further subtyped based on differences of the J1 region (M-PCRs 3 and 4 [22]). This analysis revealed that the majority of isolates of SCCmec
TABLE 2. Atypical SCCmec patterns observed with the first two PCRsa Variant
No. of isolatesb
mec class(es)c
U1 U2 U3 U4 U5 U6
1 2 11 1 1 6
B and C B B C A B
2 1 2 1 2 2
U7 U8
1 44
? ?
2 2
a b c
ccr type(s)
and and and and and
2 4 5 4 5
PCRs described previously by Kondo et al. (22). Number of isolates with this SCCmec pattern. ?, unknown bands.
DLST type(s)
3-3 47-46 4-36, 93-85 247-46 2-2 1-52, 99-89, 10-348, 90-3, 375-41 190-353 3-3, 3-33, 3-281, 17-3, 132-3, 190-173
types I (50/53), II (251/252), and IIImerc (4/4) had a single J1 subtype (Table 3). No amplification was observed for the remaining isolates of these types. In contrast, isolates that carried type IV had three J1 subtypes (subtypes IV.1 [19/80], IV.3 [19/80], and IV.4 [8/80]) as well as a significant proportion of undetermined subtypes (34/80). The diversity of subtypes in SCCmec type IV is likely explained in part by its occurrence in several DLST clones (e.g., DLST clones 1-1 and 3-3 as well as several other low-frequency clones) (Table 1 and Fig. 1). Antibiotic susceptibility. Minority SCCmec elements were correlated with a change in antibiotic susceptibility in two predominant clones (Table 4). The isolates of clone 2-2 SCCmec type I differed from the isolates of the predominant clone 2-2 SCCmec type II mainly in their susceptibility to ciprofloxacin, erythromycin, and fusidic acid, whereas the isolates of clone 2-2 SCCmec type V differed in their susceptibility to erythromycin and gentamicin. The isolates of clone 4-4 SCCmec pattern U3 differed from the isolates of the predominant clone 4-4 SCCmec type I in their susceptibility to clindamycin and gentamicin. Addition of the clfA marker. A total of 341 isolates from the four major clones and distributed in 84 DLST types were further analyzed with the clfA gene. This increased the discriminatory power, since 106 types were obtained with this analysis. As expected, most isolates clustered into the four newly defined clones (clones 1-1-1, 2-2-2, 3-3-3, and 4-4-4, with the clfA allele in the third position) (Fig. 2). The addition of the clfA allele segregated only eight types (11/341 isolates; 3.2%) from the four major clones, suggesting that they did not belong to the local disseminating clones (Fig. 2). Several isolates, classified primarily into DLST clones 2-2 and 4-4, shared clfA allele 19 and were clustered together. Interestingly, none of these x-x-19 isolates did carry SCCmec types I and II, which were predominant in clones 2-2 and 4-4, respectively. SCCmec types different from the predominant one were still observed for most of the four newly defined clones (types IV and V plus one atypical variant in clone 1-1-1, types II and IV plus one atypical variant in clone 2-2-2, and types I, II, IV, and VI plus three atypical variants in clone 3-3-3), suggesting that these clones acquired new SCCmec elements during their spread at the local level. Epidemiological data. To better understand the presence of SCCmec elements different than the predominant one within the four newly defined clones, we looked for the origins of patients carrying these elements (Table 5). Interestingly, all the patients (10/10) that carried clone 2-2 with SCCmec type V
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ANTIMICROB. AGENTS CHEMOTHER. TABLE 3. SCCmec subtypes observed among a subsample of 396 isolates No. of samples of SCCmec type
Clone
I
1-1 2-2 3-3 4-4 Other All a
II
V
Total no. of samples
0 2 0 1 4 7
11 259 38 53 36 396
IV III.1merc
Ia
I.1
IIa
II.1
0 2 0 1 0 3
0 0 0 50 0 50
0 1 0 0 0 1
0 251 0 0 0 251
0 0 0 0 4 4
IVa
IV.1
IV.3
IV.4
2 0 23 0 9 34
1 2 6 1 10 19
0 1 9 0 9 19
8 0 0 0 0 8
Undetermined subtypes.
originated from the Balkans, supporting the hypothesis of distant importation rather than a local acquisition of SCCmec type V by clone 2-2. In contrast, most patients (34/43 patients) with clone 3-3 with the atypical SCCmec pattern U8 were hosted in two geographically isolated nursing homes. Similarly, most patients (7/10) carrying clone 4-4 with the unusual SCCmec pattern U3 were living in the same district. DISCUSSION SCCmec elements may carry various resistance genes (13), which highlights the need for an understanding of the patterns of acquisition and transfer of this element. Our analysis of the diversity of SCCmec elements in a local population of Western Switzerland revealed the presence of six main SCCmec types as well as several atypical SCCmec variants. As expected, the majority of isolates within a clone carried an identical cassette (Fig. 1). This pattern supports the local spread of the major
clones of Western Switzerland. Interestingly, a significant proportion of isolates in the four major DLST clones nevertheless carried an SCCmec element different from the majority (Table 1). Several SCCmec types were even found in a single DLST type. The Swiss DLST clones were shown previously to be related to international clones (4); thus, both local acquisition and/or importation from beyond the study region might explain the diversity of SCCmec types in our local population. Although unambiguously discriminating between the two hypotheses is difficult, several elements add useful information. To help distinguish between local and international clones, we further increased the discriminatory power of DLST by sequencing a third locus in all isolates with incongruent SCCmec elements as well as in a subsample of the four major clones. Interestingly, this analysis showed that most isolates with incongruent SCCmec types of clones 2-2 and 4-4 form a cluster with one clfA allele 19 in common (Fig. 2). These two
TABLE 4. Antibiotic susceptibilities of major clones % of isolates susceptible to: Clone
SCCmec type
No. of isolates
Clindamycin
Ciprofloxacin
Ceftriaxone
Erythromycin
Fusidic acid
Gentamicin
Rifampin
Trimethoprimsulfamethoxazole
1-1
IV V U
61 2 1
98 100 100
26 100 0
2 0 0
98 100 100
83 100 0
98 100 100
100 100 100
100 100 100
2-2
II I IV V VI U3 U5
1,282 9 18 10 2 1 1
38 89 56 89 100 100 100
0 89 28 78 0 0 0
0 11 0 11 0 0 0
1 67 39 89 100 100 0
98 0 100 100 100 100 100
100 100 94 0 100 100 100
100 100 94 100 100 100 100
100 100 100 100 100 100 100
3-3
IV I II VI U1 U6 U8
237 1 2 2 1 2 44
79 0 100 100 100 50 95
4 0 0 100 0 0 0
0 0 0 0 0 0 2
73 0 100 100 100 50 98
96 0 100 0 100 50 100
97 100 100 100 100 100 95
100 100 100 100 100 100 100
100 100 100 100 100 100 100
4-4
I II IV V U3
192 2 3 2 10
1 0 33 100 100
0 0 100 100 0
0 0 0 0 0
1 0 33 100 0
99 100 100 50 100
3 100 100 100 100
96 100 100 100 100
100 100 100 100 100
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FIG. 2. SLV clustering using eBurst on 341 MRSA isolates analyzed with the two genes of DLST (i.e., clfB and spa) as well as with clfA. Each circle represents one type, and the diameter of the circles reflects the frequency of that type (i.e., number of isolates). Linked types differ at one of the three loci (clfB, spa, or clfA), and each color represents an SCCmec type.
clones are known to be closely related since both carry isolates of ST 5 and ST 105 and belong to CC5. In addition, clfA allele 19 has been shown to be found in different lineages of ST 5 (our unpublished data), suggesting that the cluster defined by this allele is ancestral and that the incongruent elements were
acquired before the local spread of clones 2-2 and 4-4. Nevertheless, although we used three highly variable markers, several SCCmec elements were still present in three of the four main clones, supporting the hypothesis that some of these elements have been acquired during the local spread of the clones.
TABLE 5. Epidemiological origin of patients with incongruent SCCmec types Clone
DLST plus clfA type(s) (no. of isolates)
SCCmec type
Epidemiological origin (no. of isolates with origin/total no. of isolates)
1-1
1-37-48 (1), 317-37-48 (1) 1-52-1 (1)
V U6
2-2
111-2-2 (1), 372-2-19 (1), 136-2-19 (1), 119-2-19 (3), 119-2-69 (2), 61-65-19 (1) 2-2-2 (4), 2-2-19 (1), 2-2-65 (1), 2-23-19 (1), 2-64-34 (2), 2-255-65 (2), 2-286-72 (1), 2-290-71 (1), 125-2-19 (1), 93-85-19 (1), 370-257-19 (1), 4-2-19 (2) 2-2-19 (9), 102-2-19 (1) 42-163-68 (2) 93-85-19 (1) 2-2-2 (1)
I IV V VI U3 U5
Balkan (10/10)
3-3-3 (25), 3-3-22 (4), 3-33-3 (10), 3-21-3 (1), 3-281-3 (1), 17-3-3 (1), 132-3-3 (1) 3-3-3 (1) 3-3-3 (2) 57-34-3 (2) 322-3-3 (1) 10-348-3 (1), 90-3-3 (1)
U8 I II VI U1 U6
Nursing homes (34/43)
4-286-4 (1), 4-2-2 (1) 271-64-67 (1), 4-338-19 (1), 4-407-19 (1) 4-36-19 (10) 4-64-19 (1), 314-64-19 (1)
II IV U3 V
3-3
4-4
Nursing home (2/2) Nursing home (1/1) Nursing home (1/2)
Eastern Lausanne (7/10) Nursing home (1/2)
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The occurrence of the same DLST allele in isolates with different SCCmec elements may be explained by convergent evolution (i.e., homoplasy). Identical DLST alleles have been identified in isolates recovered several years apart in geographically distant countries like Switzerland and Japan (3). However, there is almost no DLST allele sharing among clonal complexes (3), and a high frequency of homoplasy during local and recent expansions of clones would be surprising considering the high variability of the three genes analyzed in this study. Moreover, Harris et al. (19) previously showed low levels of homoplasy during the expansion of a young clone. Therefore, it is unlikely that most of the SCCmec incongruence is explained by homoplasy. Analysis of the epidemiological relationships among patients carrying SCCmec types different from the predominant one may help us to understand the origin of these elements (Table 5). The international origin of the genotype DLST 2-2 SCCmec type V was clearly identified since all carriers of this strain originated from the Balkans. In contrast, patients that carried clone DLST 4-36 SCCmec variant U3 or DLST 3-3 SCCmec variant U8 were elderly people living in the same district or in the same nursing homes. Although it is difficult to rule out distant importations of the index cases, the presence of uncommon SCCmec types in people with probably few recent travel opportunities suggests a local origin of these clones. Eight atypical SCCmec variants were not typeable with the system described previously by Kondo et al. (22). Most of these elements had a single mec class but carried two distinct ccr regions, suggesting that they were a mix of two elements. Similar SCCmec variants were previously observed for S. aureus (15) and are common in other staphylococcal species such as Staphylococcus epidermidis or S. haemolyticus (31). The low frequency of these elements in the worldwide S. aureus population could be explained by either (i) a low frequency of acquisition of these elements or (ii) difficulties to disseminate worldwide because of lower fitness and/or random processes. The presence of several atypical SCCmec elements in our local population supports the second hypothesis. If SCCmec elements are acquired locally, we would expect to find a correspondence between the local MSSA and MRSA clones. MSSA strains genetically related to major MRSA clones were previously observed at the local level (16, 18). Unfortunately, few data are available regarding the genetic diversity of MSSA isolates in Western Switzerland. Two recent studies of MSSA isolates from Western Switzerland showed strains genetically related to the major MRSA clones (32; our unpublished data). However, these strains were not predominant in the MSSA population, suggesting that the frequent emergence of incongruent isolates from a local MSSA clone is unlikely. Nevertheless, to definitely address the issue of local acquisition, it is necessary to know the clear phylogenetic relationship among isolates. Within clones, this can be obtained only by the sequencing of a large fraction of the core genome in numerous isolates. Although changes in SCCmec elements in clones 2-2 and 4-4 were sometimes correlated with changes in antibiotic susceptibility (Table 4), we have not been able to establish a causal link between these two characteristics. Erythromycin resistance is caused by the ermA gene, located on transposon Tn554, which is generally found in SCCmec types II and III.
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Therefore, the change in erythromycin susceptibility is likely explained by changes in SCCmec elements. In contrast, ciprofloxacin resistance is most often chromosomal and not associated with the SCCmec element (14). Similarly, most isolates resistant to gentamicin, clindamycin, and fusidic acid carried SCCmec type I or V, which are known to cause only -lactam antibiotic resistance (13). These data suggest that the resistance patterns to these antibiotics are not associated with changes in SCCmec elements but that the different SCCmec elements found in clones 2-2 and 4-4 are likely associated with different closely related genetic backgrounds. In conclusion, our analysis of the diversity of SCCmec elements in a local population of MRSA with a highly discriminatory method showed the presence of the six main SCCmec types as well as several atypical SCCmec variants. Most isolates within clones carried an identical cassette, supporting the local spread of these clones. Nevertheless, a significant proportion of isolates from the predominant clones had an SCCmec element different from the majority. Although it is difficult to discriminate between the hypotheses of local acquisition and importation from beyond the study region, our analyses suggest that even if the acquisition of new SCCmec elements at a local level likely occurs, it does not explain all the diversity observed at the local level. ACKNOWLEDGMENTS We are grateful to all laboratories and infection control programs in the Vaud canton of Switzerland that provided us with MRSA isolates and to Caroline Choulat for technical assistance. We also thank three anonymous reviewers for the helpful comments. REFERENCES 1. Aires de Sousa, M., and H. de Lencastre. 2004. Bridges from hospitals to the laboratory: genetic portraits of methicillin-resistant Staphylococcus aureus clones. FEMS Immunol. Med. Microbiol. 40:101–111. 2. Aparicio, P., J. Richardson, S. Martin, A. Vindel, R. R. Marples, and B. D. Cookson. 1992. An epidemic methicillin-resistant strain of Staphylococcus aureus in Spain. Epidemiol. Infect. 108:287–298. 3. Basset, P., N. B. Hammer, G. Kuhn, V. Vogel, O. Sakwinska, and D. S. Blanc. 2009. Staphylococcus aureus clfB and spa alleles of the repeat regions are segregated into major phylogenetic lineages. Infect. Genet. Evol. 9:941–947. doi:10.1016/j.meegid.2009.06.015. 4. Basset, P., L. Senn, G. Prod’hom, J. Bille, P. Francioli, G. Zanetti, and D. S. Blanc. 2010. Usefulness of double locus sequence typing (DLST) for regional and international epidemiological surveillance of methicillin-resistant Staphylococcus aureus. Clin. Microbiol. Infect. 16:1289–1296. 5. Blanc, D. S., C. Petignat, P. Moreillon, J. Entenza, M. C. Eisenring, H. Kleiber, A. Wenger, N. Troillet, C. H. Blanc, and P. Francioli. 1999. Unusual spread of a penicillin-susceptible methicillin-resistant Staphylococcus aureus clone in a geographic area of low incidence. Clin. Infect. Dis. 29:1512–1518. 6. Blanc, D. S., C. Petignat, A. Wenger, G. Kuhn, Y. Vallet, D. Fracheboud, S. Trachsel, A. Reymond, N. Troillet, H. H. Siegrist, S. Oeuvray, M. Bes, J. Etienne, J. Bille, P. Francioli, and G. Zanetti. 2007. Changing molecular epidemiology of methicillin-resistant Staphylococcus aureus in a small geographic area over an eight-year period. J. Clin. Microbiol. 45:3729–3736. 7. Chambers, H. F., and F. R. DeLeo. 2009. Waves of resistance: Staphylococcus aureus in the antibiotic era. Nat. Rev. Microbiol. 7:629–641. doi:10.1038/ nrmicro2200. 8. Chongtrakool, P., T. Ito, X. X. Ma, Y. Kondo, S. Trakulsomboon, C. Tiensasitorn, M. Jamklang, T. Chavalit, J. H. Song, and K. Hiramatsu. 2006. Staphylococcal cassette chromosome mec (SCCmec) typing of methicillinresistant Staphylococcus aureus strains isolated in 11 Asian countries: a proposal for a new nomenclature for SCCmec elements. Antimicrob. Agents Chemother. 50:1001–1012. 9. Clinical and Laboratory Standards Institute. 2010. Performance standards for antimicrobial susceptibility testing. Twentieth informational supplement. Clinical and Laboratory Standards Institute, Wayne, PA. 10. Cookson, B. D., and I. Phillips. 1988. Epidemic methicillin-resistant Staphylococcus aureus. J. Antimicrob. Chemother. 21(Suppl. C):57–65. 11. de Lencastre, H., E. P. Severina, H. Milch, M. Konkoly Thege, and A. Tomasz. 1997. Wide geographic distribution of a unique methicillin-resistant
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