Epidemiological Characteristics And Phylogenetic Analysis Of Pig Associated Methicillin-resistant Staphylococcus Aureus

Staphylococcus aureus is a commensal opportunistic pathogen present in both healthy and diseased humans and animals.Methicillin-resistant S.aureus(MRSA)evolved from methicillin-susceptible S.aureus(MSSA)by acquisition of mecA.In 2017,World Health Organization published a catalogue of 12 families of bacteria that pose the greatest threat to human health and stratified MRSA into the priority tier-High.Livestock is an important reservoir for MRSA,especially pigs.As the biggest swine production nation,the high prevalence of MRSA in pigs was observed,but the intensive investigation was still absence and the phylogenetics of pig associated MRSA in China were still unclear.The aims of this study were to investigate the prevalence of pig associated MRSA and its molecular characteristics by collecting samples from different regions,determine the phylogenetics of pig associated MRSA in China by whole genome sequences analyses,and provide scientific basis for both risk assessment and containment of LA-MRSA.2,420 non-duplicate samples were collected from pigs at swine farms and slaughterhouses in Henan,Shandong,Shanghai,Ningxia during 2014.MRSA isolates were identified in 270(11.2%)samples,and the prevalence of MRSA differed significantly between different regions.The isolates were characterized by antimicrobial susceptibility testing,resistance genes screening,pulsed-field gel electrophoresis(PFGE),multilocus sequence typing(MLST)and spa typing.All isolates were non-susceptible to oxacillin,cefoxitin,clindamycin,chloramphenicol,florfenicol,ciprofloxacin,and valnemulin.High rates of resistance were also observed for tetracycline,erythromycin,Quinupristin/dalfopristin,and gentamicin.Nine rifampicin-non-susceptible isolates and three linezolid-non-susceptible isolates were detected.mecA(β-lactams resistance gene)and lsa(E)gene(pleuromutilin-lincosamide-streptogramin A resistance gene)were identified in all MRSA isolates,fexA(phenicol resistance gene)in 99%isolates,and cfr(linezolid-non-susceptible)in three isolates.Molecular typing results showed that all MRSA isolates belonged to the clonal complex 9 and spa type t899,and clonal transmission appeared among different regions.Rifampicin-non-susceptible MRSA from pigs were include in a further study to investigate the molecular basis of rifampicin resistance.The results showed that rifampicin-non-susceptible rate of MRSA from pigs reached 2.0%-3.2%during 2013-2014,and the molecular typing of the isolates underlined the clonal transmission of the rifampicin resistant MRSA among different regions.Mutations in the rpoB gene were response for the rifampicin resistance,and multisite mutations conferred high-level rifampicin-resistant.481His→Asn was the most common mutation followed by 471 Asp→Tyr.94 pig associated MRSA collected from different regions during 2009-2014 were selected for second generation(94)and third generation(2)whole genome sequencing.The two complete genome sequences of the pig associated MRSA showed extremely high similarity with only few regions deletion and translocation,which mainly associated with mobile genetic elements.one of the complete genome contained the pseudo SCCmec(cpSCCmec).The phylogenetic tree were constructed based on the single nucleotide polymorphisms(SNPs).The results showed that the successful pig associated MRSA in different regions evolved from the same ancestor,and developed before 2009.The analyses of the accessory genome indicated that most of the pig associated MRSA contained SCCmec XII,and φSCCmec appeared in 8 isolates as well.Some of the resistant genes,such as mecA、aadE、spw、lnu(B)、lsa(E)、tet(L)、dfrG、blaZ、aadD、fexA,were common in pig associated MRSA in China,and some of showed a rising trend,such as aacA-aphD、erm(C).Horizontal gene transfer among MRSA isolates intensified the multi-resistant situation.In conclusion,pig associated MRSA predominant clone ST9-t899 in different regions may evolved from the same ancestor,which developed before 2009 and cross transformed among different regions in China.The pig associated MRSA from different regions showed resistance to a wide range of antibiotics with homogeneous antimicrobial resistance pheno-and genotypes.Horizontal gene transfer among MRSA isolates intensified the multi-resistant situation.Where are the successful pig associated clones from?What kind of role is φSCCmec playing during the evolutionary process of pig associated MRSA?Further study is still needed.