ABSTRACTSince its discovery in the early 2000s, methicillin-resistantStaphylococcus aureus(MRSA) clonal complex 398 (CC398) has become a rapidly emerging cause of human infections, most often associated with livestock exposure. We applied whole-genome sequence typing to characterize a diverse collection of CC398 isolates (n= 89), including MRSA and methicillin-susceptibleS. aureus(MSSA) from animals and humans spanning 19 countries and four continents. We identified 4,238 single nucleotide polymorphisms (SNPs) among the 89 core genomes. Minimal homoplasy (consistency index = 0.9591) was detected among parsimony-informative SNPs, allowing for the generation of a highly accurate phylogenetic reconstruction of the CC398 clonal lineage. Phylogenetic analyses revealed that MSSA from humans formed the most ancestral clades. The most derived lineages were composed predominantly of livestock-associated MRSA possessing three different staphylococcal cassette chromosomemecelement (SCCmec) types (IV, V, and VII-like) including nine subtypes. The human-associated isolates from the basal clades carried phages encoding human innate immune modulators that were largely missing among the livestock-associated isolates. Our results strongly suggest that livestock-associated MRSA CC398 originated in humans as MSSA. The lineage appears to have undergone a rapid radiation in conjunction with the jump from humans to livestock, where it subsequently acquired tetracycline and methicillin resistance. Further analyses are required to estimate the number of independent genetic events leading to the methicillin-resistant sublineages, but the diversity of SCCmecsubtypes is suggestive of strong and diverse antimicrobial selection associated with food animal production.IMPORTANCEModern food animal production is characterized by densely concentrated animals and routine antibiotic use, which may facilitate the emergence of novel antibiotic-resistant zoonotic pathogens. Our findings strongly support the idea that livestock-associated MRSA CC398 originated as MSSA in humans. The jump of CC398 from humans to livestock was accompanied by the loss of phage-carried human virulence genes, which likely attenuated its zoonotic potential, but it was also accompanied by the acquisition of tetracycline and methicillin resistance. Our findings exemplify a bidirectional zoonotic exchange and underscore the potential public health risks of widespread antibiotic use in food animal production.

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Please cite this article as: Cavaco, L.M., Hasman, H., Aarestrup, F.M., on behalf of the MRSA collaborating group (MRSA-CG), Zinc resistance of Staphylococcus aureus of animal origin is strongly associated with methicillin resistance, Veterinary Microbiology (2010Microbiology ( ), doi:10.1016Microbiology ( /j.vetmic.2011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The test population consisted of 476 porcine MRSA isolates from ten European 30 countries, 18 porcine MRSA isolates from Canada and seven MRSA from China, 92 31 MRSA and 60 methicillin-susceptible S. aureus (MSSA) isolates from veal calves in the 32Netherlands and 88 porcine MSSA isolates from four European countries. Most porcine 33 MRSA (n=454) and all bovine MRSA belonged to clonal complex (CC) 398 whereas 37 34 of the pig MRSA from Europe and the seven Chinese isolates belonged to other CCs 35 and 3 isolates were not classified into a CC. 36All isolates were tested for susceptibility to zinc chloride and copper sulphate using 37 agar dilution and tested by PCR for the czrC gene encoding zinc resistance. 38Phenotypic zinc resistance (MIC>2mM) was observed in 74% (n=324) and 42% (n=39) 39 of European MRSA CC398 from pigs and veal calves respectively, and in 44% of the 40 Canadian isolates (n=8), but not among the Chinese isolates. Almost all (99%) zinc-41 resistant MRSA carried czrC. Of the 37 European non-CC398 MRSA, 62% were 42 resistant to zinc, but only 46% of them carried czrC,. The MICs of the MSSA isolates to 43 zinc chloride ranged from 1 to 4 mM and none carried czrC. The MICs of copper 44 sulphate were neither associated with methicillin resistance nor with the detection of 45 czrC. 46This study showed that zinc resistance and the czrC gene is widespread among 47 CC398 MRSA isolates. This suggests that the use of zinc in feed might have 48 contributed to the emergence of MRSA. 49 50

S taphylococcus aureus is a major pathogen causing considerable human morbidity and mortality worldwide, and it is a leading cause of infections of some economically important livestock species and, as a prominent bacterial cause of contagious bovine mastitis, a major economic burden for the dairy cattle industry (1). Methicillin-resistant S. aureus (MRSA) lineages are causes of health care-and community-associated infections, which are a major burden of disease on a global scale (2). In the last decade, MRSA sequence type 398 (ST398) has found an ecological niche in the pig, cattle, and poultry industries, although other MRSA lineages (e.g., ST1, ST5, ST9, ST97, ST130, and ST433) have been identified in farmed animals worldwide (3) All these lineages are currently termed "livestock-associated MRSA" (LA-MRSA).The MRSA clonal complex 97 (CC97) lineage has been reported as the second most prevalent MRSA lineage in the Italian pig industry, where spa types t1730 and t4795, both belonging to ST97, were estimated to have colonized 11% of finishing holdings (4). In Italy, in the last 5 years, the CC97 MRSA lineage has also been increasingly detected in cattle primary production, either from mastitis in dairy cattle or from bulk tank milk (5-7). Occasionally, CC97 MRSA isolates have been detected in cattle in Germany and Spain.CC97 is one of the major S. aureus clonal complexes in bovines (8), and recently, a livestock origin of the human pandemic CC97 MRSA strains has been demonstrated, resulting in two emergent human epidemic CC97 community-acquired/associated MRSA (CA-MRSA) clones (8).The aim of this study was to determine the genetic relatedness of MRSA and methicillin-susceptible S. aureus (MSSA) CC97 isolates from pigs and dairy cattle in Italy in the last few years (2008 to 2012) for epidemiological and risk assessment purposes.

Methicillin-resistant Staphylococcus aureus (MRSA) Sequence Type (ST)1, Clonal Complex(CC)1, SCCmec V is one of the major Livestock-Associated (LA-) lineages in pig farming industry in Italy and is associated with pigs in other European countries. Recently, it has been increasingly detected in Italian dairy cattle herds. The aim of this study was to analyse the differences between ST1 MRSA and methicillin-susceptible S. aureus (MSSA) from cattle and pig herds in Italy and Europe and human isolates. Sixty-tree animal isolates from different holdings and 20 human isolates were characterized by pulsed-field gel electrophoresis (PFGE), spa-typing, SCCmec typing, and by micro-array analysis for several virulence, antimicrobial resistance, and strain/host-specific marker genes. Three major PFGE clusters were detected. The bovine isolates shared a high (≥90% to 100%) similarity with human isolates and carried the same SCCmec type IVa. They often showed genetic features typical of human adaptation or present in human-associated CC1: Immune evasion cluster (IEC) genes sak and scn, or sea; sat and aphA3-mediated aminoglycoside resistance. Contrary, typical markers of porcine origin in Italy and Spain, like erm(A) mediated macrolide-lincosamide-streptograminB, and of vga(A)-mediated pleuromutilin resistance were always absent in human and bovine isolates. Most of ST(CC)1 MRSA from dairy cattle were multidrug-resistant and contained virulence and immunomodulatory genes associated with full capability of colonizing humans. As such, these strains may represent a greater human hazard than the porcine strains. The zoonotic capacity of CC1 LA-MRSA from livestock must be taken seriously and measures should be implemented at farm-level to prevent spill-over.