Study On O-serogroup,Virulence Factor And Drug Resistance Of Non-O157 Shiga Toxin-producing Escherichia Coli From Cattle In Xinjiang

Objective:To systematically study the O-serogroup,virulence genes and drug resistance of non-O157Shiga toxin-producing Escherichia coli from Xinjiang cattle,and preliminary explore whether non-O157STEC virulence genes and drug resistance genes co-transfer.Methods:A total of 127 cattle non-O157 STEC strains collected from Changji,Yili,Urumqi,Bole and Shihezi in Xinjiang from 2015 to 2019 were selected as the research object.(1)Multiplex PCR(E.coli O-genotyping PCR)was used to screen non-O157 STEC O serogroups.(2)The non-O157 STEC phylogenetic grouping of Escherichia coli was carried out by multiplex PCR,and the Shiga toxin gene subtypes(stx_1a,stx_1c,stx_1d and stx_2a²g)and the hemolysin gene ehx A were detected,and ehx A positive strains were tested on 5%sheep blood plates Of hemolysis.(3)The K-B method was used to conduct drug susceptibility tests on non-O157 STEC of 18 antimicrobial drugs,and conduct a conjugation test on drug-resistant strains to verify whether the drug-resistant genes can undergo horizontal transfer.(4)The multi-drug resistant strains were sequenced to annotate drug resistance genes and virulence genes.Results:(1)A total of 19 O-serogroups,including O145,O22,O179,O8,O29,O81,O88,O113,O130,O28ac,O116,O119,O21,O48,O140,O147,O183,O110 and O159,were detected in 127 Xinjiang cattle non-O157 STEC strains.(2)The phylogenetic grouping results showed that the cattle source non-O157 STEC was dominated by group A(116/127,91.39%),followed by group B1(9/127,7.09%).The detection rates of stx_1a,stx_2a,stx_2c and ehx A in 127 non-O157 STEC strains were 96.85%(123/127),78.74%(100/127),1.57%(2/127)and 93.70%(119/127),respectively.A total of 8 virulence gene profiles are carried,mainly stx_1a/stx_2a/ehx A(54.33%,69/127).41 ehx A positive strains showed hemolytic activity on sheep red blood cells.(3)There are 8 resistant strains in 127 non-O157 STEC strains.They are all multi-drug resistant phenotypes,resistant to 4 to 13 antimicrobial drugs,including?-lactams,tetracyclines,sulfonamides,amido alcohols and aminoglycosides,And there are TET-SXT-CHL-STR resistance combination in the drug resistance spectrum.The 8 non-O157 STECs all carry the tet A gene and Inc A/C plasmid.The conjugative transfer test showed that 8 resistant bacteria can transfer all or part of the resistant phenotype to E.coli J53.The tet A gene and Inc A/C plasmid carried by 8 resistant bacteria can be transferred to E.coli J53.The rate is 100%.Among them,6 strains transferred stx_1a,stx_2a and ehx A to E.coli J53,with a transfer rate of75.00%.(4)The whole genome framework map sequencing annotated 70 kinds of drug resistance genes and141 kinds of virulence genes.Conclusion:The distribution of non-O157 STEC O-serotype in Xinjiang cattle was diversified,mainly O145,O22 and O179.There were 8 virulence gene profiles,and the dominant pattern was stx_1a/stx_2a/ehx A.Co-transfer of non-O157 STEC drug resistance gene and virulence gene was found for the first time.In this study,the serogroups,virulence factors and drug resistance of non-O157 STEC from Xinjiang cattle were systematically evaluated.As a reservoir of drug resistance and virulence genes,non-O157 STEC from cattle is a potential threat to public health security in Xinjiang.