Cross-Border Dissemination of Methicillin-Resistant Staphylococcus aureus, Euregio Meuse-Rhin Region

MRSA clones were associated with hospital-associated clonal complexes and with Panton-Valentine leukocidin–positive community-associated MRSA.

tal-isolated MRSA (23.6%, 13.8%, and 0.6% in Belgium, Germany, and the Netherlands, respectively) (2). Consequently, cross-border transfer of patients may affect the dissemination and prevalence of MRSA, particularly when patients are transferred from countries with a relatively high prevalence to a country with a low prevalence.
A study of MRSA isolates from the EMR between December 1999 and February 2004 showed that isolates from clonal complex (CC) 5 and CC 8, which harbor the resistance elements staphylococcal cassette chromosome mec (SCCmec) types I-IV, had been disseminated in the EMR (2). Our aim was to investigate the emergence, dissemination, and diversity of MRSA clones in the EMR during a 10-month period in 2005 and 2006 and to compare the results with those of the previous study. We used sequencing of the short sequence repeat (SSR) region of the S. aureus protein A gene (spa typing), multilocus sequence typing (MLST), and SCCmec typing by PCR to investigate the genetic background of all MRSA isolates. The spa locus was typed to provide more detailed information about prevalent MRSA clones in the EMR, especially because the previous study used only MLST analyses on a small subset of isolates (2). Finally, because an increase of Panton-Valentine leukocidin (PVL)-positive MRSA isolates in the Netherlands has recently been observed (3), we investigated the possible spread of PVL-positive MRSA clones into hospitals in the EMR, as well as the prevalence of the virulence factors collagen adhesion (CNA) and toxic shock syndrome toxin-1 (TSST-1).

MRSA Isolates
We investigated 257 MRSA isolates, cultured during July 2005-April 2006 from 8 geographically closely related hospitals in the EMR. The hospitals included 1 hospital in Belgium (General Hospital Vesalius, Tongeren, 355 beds), 2 hospitals in Germany (General Hospital Dūren, 521 beds, and Marien Hospital, Aachen, 321 beds), and 5 hospitals in the Netherlands (Atrium Medical Center, Heerlen, 811 beds; Orbis Medical and Care Center, Sittard, 578 beds; Laurentius Hospital, Roermond, a 458-bed general hospital; University Hospital Maastricht, a tertiary hospital, 680 beds; and VieCuri Medical Center, Venlo, a 554-bed general hospital). The 257 MRSA isolates comprised 44 from Belgium, 92 from Germany, and 121 from the Netherlands. Isolates from the Belgian and German hospitals were from patients with MRSA infection; Dutch isolates were from patients carrying MRSA who were admitted to the Dutch hospitals. All isolates were identifi ed as S. aureus by Gram stain, catalase, and coagulase testing. The presence of the mecA gene was determined as described previously (2).

Antimicrobial Drug Susceptibility Testing
The susceptibility pattern of the MRSA isolates was determined according to the guidelines of the Clinical and Laboratory Standards Institute (4). Susceptibility to the following antimicrobial agents was determined as MIC: cefaclor, cefuroxime, clindamycin, ciprofl oxacin, clarithromycin, gentamicin, linezolid, moxifl oxacin, oxacillin, penicillin, rifampin, teicoplanin, tetracycline, trimethoprim/sulfamethoxazole, and vancomycin. The susceptibility to fucidic acid and mupirocin (Rosco, Taastrup, Denmark) was determined by using the disk-diffusion method (5,6). MRSA isolates resistant to clarithromycin were tested for inducible clindamycin resistance by using the D-test (7).

Typing Methods
SCCmec typing was performed as described by Oliveira et al. (8) with the modifi cation described previously (2). SCCmec type I elements that lack locus A (pls region) are indistinguishable (9) from SCCmec type IV elements when the method of Oliveira et al. is used (8). Furthermore, locus D (dcs region) is detected in both SCCmec types IV and VI (10). Therefore, SCCmec elements that were typed as SCCmec type IV using the method of Oliveira et al. (8) were further analyzed for presence of the ccrAB gene. SCCmec elements that could not be typed with the method of Oliveira et al (8) were further analyzed by using the methods of Ito et al. (11) and Zhang et al. (12).
Real-time amplifi cation of the spa gene was performed as described previously, followed by sequencing of the SSR region (13). The spa types were clustered into spa-CCs using the algorithm Based Upon Repeat Pattern (BURP) with the Ridom StaphType version 1.4 software package (www. ridom.de). Because spa typing, together with the algorithm BURP, yields results concordant with typing results obtained by MLST and pulsed-fi eld gel electrophoresis (13), the associated CCs, as determined with MLST, were allocated through the Ridom SpaServer (http://spaserver.ridom. de). To confi rm the association between MLST and spa typing, in combination with BURP, MLST was performed on a representative set of 12 strains of each major spa type and spa-CC (2).The presence of CNA, PVL, and TSST-1 was determined with real-time PCR assays (14,15).

Antimicrobial Drug Susceptibility Patterns
All 257 MRSA isolates were resistant to the β-lactam antimicrobial agents cefaclor, cefuroxime, oxacillin, and penicillin and were susceptible to linezolid, teicoplanin, and vancomycin. Most isolates were also resistant to ciprofl oxacin (84%) and moxifl oxacin (82%). The Dutch MRSA isolates were more often susceptible to ciprofl oxacin and moxifl oxacin than were the Belgian and German isolates (Table 1) (p<0.05). Furthermore, 78% of the MRSA isolates were resistant to clarithromycin, and 62%, to clindamycin. Susceptibility for clarithromycin and clindamycin differed by country (Table 1). A total of 41 MRSA isolates (19 from Belgium, 5 from Germany, and 17 from the Netherlands) was resistant to clarithromycin and susceptible to clindamycin. The D-test showed that 31 (76%) of these 41 MRSA isolates had the inducible clindamycin resistant phenotype, including 15 from Belgium, 5 from Germany, and 11 from the Netherlands.

Distribution of MRSA Clones
SCCmec type IV was predominant in MRSA isolates from Belgium (77%), whereas MRSA isolates from Germany harbored mainly SCCmec type II (82%). MRSA isolates from the Dutch region harbored both SCCmec type II and IV (27% and 65%, respectively). Although 25 (10%) of the 257 MRSA isolates harbored an SCCmec element that could not be typed with the method of Oliveira et al. (8), they could be typed with the other methods. Seven MRSA isolates from Belgium harbored a SCCmec type III element that lacked Tn554, which is usually characteristic for SCCmec type III. From the German region, 1 isolate that had a nontypeable SCCmec element harbored ccrC, locus E, and Tn554. The method of Zhang et al. (12) classifi ed this element as SCCmec type III. In the Netherlands, 17 MRSA isolates contained a nontypeable SCCmec element as defi ned by Oliveira et al. (8). Ten of these were classifi ed as SCCmec type IV, lacking locus D. The remaining 7 harbored ccrC, characteristic for SCCmec type V, and were classifi ed as such with the method of Zhang et al. (12).
The ST5-MRSA-II (New York/Japan) clone was found mainly in Germany and the Netherlands, and the ST45-MRSA-IV (Berlin) clone was found in Belgium and the Netherlands. Furthermore, the ST5-MRSA-IV (Pediatric) clone was found among the Dutch isolates. The MRSA isolates classifi ed as CC30 (ST30-MRSA-IV and ST36-MRSA-II) were found only in the Netherlands. Most of the ST8-MRSA-IV (UK EMRSA-2/6) isolates were found in the Netherlands. Furthermore, several ST398-MRSA-IV and ST398-MRSA-V isolates were found in certain Dutch hospitals (Figure; Table 3).

Prevalence of Virulence Factors
Eleven (5%)  Nine (4%) of the 257 MRSA isolates were positive for the tst gene, 4 isolates were classifi ed as ST22-MRSA-IV, 3 as ST36-MRSA-II, 1 as ST30-MRSA-IV, and 1 could not be classifi ed as an MRSA clone (spa type t779). All isolates were from the Netherlands and were positive for the cna gene; none harbored PVL.
Ninety-fi ve (37%) of the 257 MRSA isolates were positive for the cna gene (34 from Belgium, 9 from Germany, and 52 from the Netherlands). All MRSA isolates from CC30, CC45, and ST398 harbored the cna gene. Furthermore, 1 isolate from CC5, 1 from CC80, 6 classifi ed as singletons (associated with ST22 and ST89), and 2 excluded from the BURP analyses were positive for the cna gene.

Discussion
Because cross-border healthcare is an issue in the EMR, and the prevalence of MRSA differs among the countries forming the EMR, studying the possible emergence, spread, and diversity of MRSA clones within and among these countries is important (2). In addition to MRSA clones from CC5 and CC8, found previously in the EMR, we observed MRSA isolates from CC30 and CC45. Furthermore, the Dutch isolates had a more heterogeneous genetic background than did MRSA isolates from Belgium and Germany. The prevalence of PVL-positive MRSA isolates, belonging to ST1, 8, 30, 80 and 89, was higher than that found in the previous study (5% vs. 1.3%) (2). The antimicrobial susceptibility of the MRSA isolates depends on the S. aureus lineage. The observation that the Dutch MRSA isolates were more often susceptible to ciprofl oxacin and moxifl oxacin than were isolates from Belgium and Germany can be explained by the fact that the isolates associated with ST5-MRSA-IV, ST22-MRSA-IV, and ST30-MRSA-IV, which were susceptible to ciprofl oxacin and moxifl oxacin, were mainly observed in the Netherlands. Although ST22-MRSA-IV is commonly susceptible to tetracycline, the ST22-MRSA-IV isolates in this study were resistant to tetracycline (16). S. aureus can harbor resistance genes on mobile genetic elements on the genome, such as Tn554, as well as on plasmids, and these can be exchanged among S. aureus lineages, possibly because of antimicrobial drug pressure (17).
Primarily because of the Dutch "search-and-destroy" policy, isolates derived from colonized persons were available from the Netherlands, whereas isolates from Belgium and Germany were derived from infections. However, nasal carriers are at increased risk of acquiring MRSA infection (18). Consequently, not preventing the spread of MRSA among nasal carriers could lead to MRSA infection among these persons. Furthermore, the molecular epidemiology of MRSA can vary widely among hospitals. In the Dutch hospitals of the EMR, MRSA clones in each hospital were diverse, whereas in the Belgian hospital and 2 German hospitals, 1 MRSA clone predominated, showing that the number of hospitals is unlikely to have biased the results of our study.
Most of the MRSA isolates from Belgium were associated with the Berlin clone (ST45-MRSA-IV). This clone has previously been found in Belgium, Germany, and the Netherlands (19). Most of the MRSA isolates from Germany were associated with the New York/Japan clone (ST5-MRSA-II), previously found in Belgium and Germany (2,19). Most of the Dutch MRSA isolates belonged to 5 MRSA clones (Table 3). Twenty-fi ve percent of the Dutch isolates were associated with the New York/Japan clone (ST5-MRSA-II), which has not been previously found in the Netherlands. The Pediatric clone (ST5-MRSA-IV), which represented 14% of the Dutch isolates, has been found in Belgium but not in the Netherlands (20,21). The Berlin clone (ST45-MRSA-IV), comprising 21% of the Dutch isolates, and the UK EMRSA-2/-6 clone (ST8-MR-SA-IV), comprising 16% of the Dutch isolates, have been described in all 3 EMR countries (19,20). In addition, some less prevalent MRSA clones were observed. Four tst-positive MRSA isolates were associated with the UK EMRSA-15 clone (ST22-MRSA-IV), previously found in Belgium and Germany but not in the Netherlands (19,20). Three Dutch MRSA isolates (spa type t012), harboring SCCmec type II, were associated with the CC30 lineage. These isolates might be derived from the UK EMRSA-16 (ST36-MRSA-II) clone (spa type t018) because spa types t012 and t018 differ in 1 spa repeat (r24) and are thus related. Furthermore, both clones harbor the cna and tst genes (22,23). The highly endemic UK EMRSA-16 clone has not been observed before in the Netherlands, although this clone has previously been found in Belgium (24). Seven and 5 of the Dutch MRSA isolates were associated with ST398-MRSA- IV and ST398-MRSA-V, respectively, MRSA clones usually observed in pigs and among screening samples from pig farmers (25). The ST398 clone is now observed among screening samples of veterinarians from many countries in Europe, including Belgium and Germany (26). However, ST398 also has been isolated from several forms of human infections in Germany (27). The ST398 isolates from our study were positive for the cna gene, suggesting a higher virulence than that of the CNA-negative German ST398 MRSA isolates (27). One Dutch MRSA strain was associated with the ST30-MRSA-IV clone, previously reported in Belgium, Germany, and France (20,21,28). The more diverse genetic background among MRSA isolates in the Dutch part of the EMR and the close cooperation of hospitals in the EMR might suggest that importation of MRSA from Belgium and Germany has occurred through crossborder healthcare (Table 4) (2). Other, less likely, explanations for the diversity of MRSA clones in the Netherlands are the spread of MRSA from countries other than Belgium or Germany (19) and the emergence of new MRSA clones in vivo through transfer of the SCCmec element from methicillin-resistant coagulase-negative staphylococci to methicillin-sensitive S. aureus strains (29). We could not determine the SCCmec type for 10% of the MRSA isolates by using the method of Oliveira et al. (8). This percentage was similar to that found in other studies (30,31) but higher than the 3% previously found in the EMR (2). The relatively large number of nontypeable SCCmec types found in this study, probably formed by homologous recombination among SCCmec elements, supports the need for a new system for SCCmec typing and nomenclature (19).
The 7 Belgian MRSA isolates with the nontypeable SCCmec type III element were associated with CC5 and had the related spa types t045 and t1107 (http://spaserver. ridom.de). Although SCCmec type III usually is found in the CC8 genetic background, such as in the ST239-MRSA-III clone, an MRSA associated with CC5 (spa type t045) and harboring SCCmec type III recently was observed in Belgium (32). This might suggest that a new MRSA clone, ST5-MRSA-III, is beginning to emerge in Belgium.
The nontypeable SCCmec element of the German MRSA isolate harbored locus E and ccrC, specifi c for SCCmec type V (2), and Tn554, normally carried by SCCmec type II, III, and SCCmercury. Zhang et al. (12) classifi ed this element as SCCmec type III, but the SCCmec type III-specifi c primers used by this method are situated near locus E on SCCmercury (33), indicating that this element could be a SCCmercury element containing mecA. Further investigation is needed into the structure of this element.
Previous studies have shown that MRSA isolates classifi ed as community-associated usually harbor either SCCmec Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15  type IV or V, and often PVL, but may differ in their genetic backgrounds (CC1, CC8, CC30, CC59 and CC80) (34). In the EMR, 5% of the MRSA isolates were positive for PVL, which is higher than the previously reported 1.3% (2). Thus, PVL-positive MRSA isolates with a heterogeneous genetic background are emerging in the EMR. PVL-positive MRSA isolates associated with ST8-MRSA-IV, ST30-MRSA-IV, and ST80-MRSA-IV have been isolated in the Netherlands (3,35). In the present study, 2 of the PVL-positive MRSA isolates harbored SCCmec type V. The different genetic background of these isolates, i.e., ST89 and ST772, a single-locus variant of ST1 at the pta locus, might suggest that SCCmec type V was introduced on different occasions into different S. aureus lineages. A PVL-positive ST772-MRSA-V has been observed in Germany (36). One of the PVL-positive isolates harbored SCCmec type I, and such isolates with a ST30 and ST37 genetic background have been described in the Netherlands (3). Although a recent study suggested that CNA and PVL combined contribute to virulence, only 6 of the 11 PVL-positive MRSA isolates from the EMR harbored the cna gene (37). Further studies are needed to investigate the contribution of the combination of CNA and PVL to virulence.
The genetic background of 1 PVL-positive ST45-MRSA-IV isolate from Belgium was similar to that of the Berlin clone. Hitherto, only PVL-negative isolates with this background have been found in EMR countries (19,20). PVL-positive MRSA isolates, associated with the major CA-MRSA clones (ST8-MRSA-IV, ST30-MRSA-IV, and ST80-MRSA-IV) have been reported from Belgium (38). Because PVL is situated on a phage, the genes encoding PVL might have been transferred to S. aureus with a CC45 genetic background (34).
Our study found a PVL-positive MRSA isolate from Germany with spa type t042 (spa repeat pattern r26r23r12r34r34r33r34). This spa type is strongly related to spa types t044 and t131 (spa repeat patterns r07r23r12r34r34r33r34 and r07r23r12r34r33r34, respectively), which are usually associated with the CA-MRSA ST80-MRSA-IV clone found in Germany (34).
The cna gene has been previously observed among MRSA isolates from CC22, CC30, and CC45 (23,29). Therefore, the presence of the can gene might, together with spa typing, be used as a marker for different genetic backgrounds.
MRSA clones associated with the hospital associated-MRSA CCs 5,8,22,30, and 45, the PVL-positive CA-MRSA CCs 1, 8, 30, 80, and 89, as well as MRSA related to pigs (ST389-MRSA-IV/V) were observed in the EMR. Dissemination of these clones is possible because of the introduction of new MRSA clones associated with travel; with patients who have previously been admitted to a hospital abroad (cross-border healthcare); or with other high-risk patients, such as pig-farmers and their families. Therefore, a cross-border search-and-contain policy may help control the further spread of MRSA and reduce the fi nancial cost to hospitals, nursing homes, and the community in the EMR.