Whole-genome Sequencing And Comparative Genomic Analysis Of Three Pig Associated Methicillin-resistant Staphylococcus Aureus

Pig associated methicillin-resistant Staphylococcus aureus(MRSA)has become the focus of global public health issues as a pathogen of serious harm to aquaculture and biological food safety.From 2012 to 2017,we isolated 75 MRSA strains from farm,slaughterhouse and market in Guangdong Province.To study the carrying status of drug resistance genes,virulence genes and the genetic variation pattern of pig-associated MRSA,ST9-MRSA isolate HP,ST239-MRSA isolate M48,ST1516-MRSA isolate M51 were selected.By focusing on the genome characteristics of these three MRSA isolates and combining with the genome information of 86 S.aureus isolates in NCBI database,the information of potential pathogenicity,evolutionary relationship and strategy of the isolates were obtained.The results are as follows:1.The sequencing results showed that chromosome of isolates HP,M48 and M51 were2,850,509 bp,3,050,015 bp,2,786,527 bp,respectively.And the GC contents were all 32.8%.The total number of open reading fragments were 2,663,2,894 and 2,642,respectively.Polysaccharide lyase was only recognized in isolates HP and M48.2.According to the results of the phylogenetic tree,36 isolates of 14 ST types from 12 different countries and regions gathered in branch I;53 isolates of 14 ST types from 13 different countries and regions gathered in branch II.The isolates were widely distributed and did not show obvious regional correlation.Virulence genes such as hlg A,hlg B,map,fnb B and resistance genes such as gyr A and gyr B were widely presented in human and pig isolates,while ant(1)-Ia,aph(1')-IIIa,erm A,erm C and isd D,luk F-PV,luk S-PV were mostly present in human isolates.The tet M gene only existed in 17 human isolates,14 of which were MRSA isolates.3.SNP(Single nucleotide polymorphisms)and MLST(Multilocus sequence typing)clustering results showed that HP was associated with three ST9 pig isolates;M48 was associated with ST239 human isolates form Korea and Canada.M51 and three ST8 human isolates form the USA were closely related.BRIG analysis displayed that the main differences were concentrated in the accessory genomes such as SCCmec,genomic islands and prophages.The results of Mauve analysis also displayed that many genomic rearrangements including translocation and inversion among isolates in each group.Pan-genome analysis showed that the specific gene content of HP,M48,and M51 were 10.9%,16.3%,and 13.4%.the unique genes of HP and M48 were 60% and 70%,respectively.4.46 different drug resistance genes and 72 different virulence genes have been identified.Gene esa C and esx B of the T7 SS system were lost in strain M48.Genomic islands HPGI1,HPGI2,M48GI2 and M51GI1 contained IS insertion sequences including IS256,and aminoglycoside resistance gene was in downstream of IS256 such as aph(2'')-Ia,aac(6')/aph(2''),aph(3')-IIIa and ant(6).All isolates carried CRISPR system without cas gene,their ability of obtain exogenous genes was stronger than other isolates.