Antimicrobial resistance and population structure of Staphylococcus aureus recovered from pigs in Belgium

Staphylococcus aureus is a common facultative pathogen that has since long been recognized as a burden in both human and veterinary medicine. S. aureus is well known to be frequently resistant to antimicrobial agents which may lead to complications in the treatment of its infections and increase the cost of treatments. During the last decade, an increasing number of studies reported the presence of methicillin-resistant Staphylococcus aureus (MRSA) in animals. Most studies have focused on the asymptomatic carriage of MRSA among pigs, in which clonal complex (CC) 398 is the dominant lineage. During 2013, a survey was performed in different pig farms randomly selected over Belgium, with the aim of monitoring the current epidemiology and antimicrobial susceptibility of MRSA among asymptomatic pigs. From 328 farms nose swabs were taken from 20 animals and pooled. MRSA was isolated using the standard method proposed by the European Food Safety Authority (EFSA). MRSA identification was performed using the triplex 16S rRNA-mecA-nuc PCR. All isolates were characterised by means of susceptibility testing by a microbroth-dilution method using epidemiological cut-off values (Eucast), SCCmec typing, spa-typing and by the sau1-hsdS1 clonal complex (CC) 398 PCR. CC398 PCR negative isolates were subjected to multi-locus sequence typing (MLST). Selected isolates were subjected to DNA microarray-based typing for detection of resistance and virulence genes. MRSA was detected in 215 farms [65.6% (95% CI: 60.1%-71%)] out of 328 farms sampled. Most isolates (n=207) were positive for the sau1-hsdS1 CC398 PCR. The remaining eight isolates were ST9 (one isolate), ST80 (two isolates), ST239 (one isolate) or ST398 (four isolates) as demonstrated by MLST. A total of 22 different spa types were identified. The spa types t044, t337 and t4150 were found in the ST80, ST9 and ST239 isolates, respectively. Nineteen spa types were found among the CC398 isolates, but most were t011 (n=180, 85%). Regarding to the SCCmec typing, most isolates carried SCCmec V, and less carried SCCmec IV or III. More than 90% of the isolates were epidemiological resistant to tetracycline and trimethoprim and high resistance rates (between 66% and 45%) were also found for ciprofloxacin, clindamycin, erythromycin, kanamycin and gentamicin. Lower epidemiological resistance levels (between 30% and 10%) were detected for streptomycin, fusidic acid, sulfamethoxazole, quinupristin/dalfopristin, tiamulin, rifampicin, chloramphenicol and mupirocin. All isolates were susceptible for vancomycin. More than 90% of the isolates were multi-resistant, and half of them were resistant to at least seven different antibiotics. Microarray analysis showed that most genes were homogeneously distributed among the CC398 isolates. The non-CC398 isolates carried additional virulence genes, as the egc-like cluster with enterotoxins genes (seg, seh, sei, selm, seln, selo, selu). Interestingly, the ST80 strains carried the leukocidin Panton-Valentine (lukPV) and lukED genes. Regarding to antimicrobial resistance genes, all CC398 isolates investigated carried the tetracycline resistance gene tet(M). Most CC398 isolates carried the bla operon (blaZ, blaI, and blaR) encoding for penicillin-ampicillin resistance and the tetracycline resistance gene tet(K). Some CC398 isolates carried genes encoding resistance to the macrolide-lincosamide-streptogramin group [erm(B), erm(C), lnu(A), vga(A)], aminoglycosides (aacA-aphD, aadD, aphA3, sat) and/or chloramphenicol (fexA). One fexA positive isolate was additionally positive for the multi-resistance gene cfr. The MRSA prevalence among pigs in Belgium remains similar to previous studies performed on 2007 and 2009. As has been demonstrated before, the CC398 isolates were highly multi-resistant. However, in this survey there is a larger diversity in spa-types than ever detected before. Moreover, in this survey we have detected the European clone ST80-IV, which corresponded to the main community-acquired (CA-) MRSA clone in Europe. The ST80-IV had the Panton-Valentine leucocidin and had emerged recently as a cause of healthcare-associated infections. The recovery of this CA-MRSA from livestock indicates that one should remain vigilant to the evolution of LA-MRSA CC398