Molecular Biological Characterization Of Non-O157 Shiga Toxin-producing Escherichia Coli From Cattle,Sheep And Camels In Xinjiang

Objectives:In order to investigate the distribution of phylogenetic clustering,serotype,virulence genes,drug resistance and genetic diversity of non-O157 Shiga toxin-producing Escherichia coli（STEC）from cattle,sheep and camel in Xinjiang,to provide data support for the monitoring of animal STEC resistance and molecular epidemiology.Methods:（1）A total of 2 312 feces,rectal and carcass swabs of cattle,sheep and camels were collected from livestock farms,slaughterhouses and markets in Urumqi,Altay,Yili,Kumul,Bayinguolin,Aksu,Kashgar and Hotan of Xinjiang in 2018 to 2019,of which 1 628 were from cattle,542 from sheep and 142from camels were isolated and identified for non-O157 STEC.The isolates were screened and isolated using Eosin-methylene Blue Medium and CHROMagar ^TMSTEC base,and STEC was identified with specific primers.The STEC isolates were phylogenetic and clustered by multiplex PCR.（2）The virulence genes stx₁,stx₂（including subtypes）,eae A,tir,esp B,saa,iha,ecp A,ehx A,kat P,esp P,sub AB and cdt C of STEC from cattle,sheep and camel were detected by PCR,and O-serogroup were detected.（3）The resistance phenotypes of the isolates was determined by using a Kirby-Bauer disk diffusion method and some key isolates were classified by pulse field gel electrophoresis to explore the genetic relationship between different sources of non-O157 STEC.Result:（1）The 94 strains of STEC were isolated and identified from 2 312 samples,including 57 strains from cattle,25 strains from sheep and 12 strains from camels.46.8%（44/94）only carried stx₁,6.4%（6/94）only carried stx₂,and 46.8%（44/94）carried stx₁+stx₂.The results of phylogenetic analysis showed that 94non-O157 STEC strains could be divided into three groups,with B1 group as the main group（83/94,88.3%）,E group（7/94,7.4%）and A group（4/94,4.3%）as the second group.（2）The detection of Shiga toxin subtype showed that the detection rates of stx_1a,stx_1c,stx_2aand stx_2din 94 STEC strains were 83%,60.6%,21.3%and 50%respectively.stx_1a+stx_2dwas the main source of cattle and stx_1a+stx_1cwas the main source of sheep and camel.The 9 virulence genes were detected,including eae A（9.6%）,tir（9.6%）,esp B（11.7%）,saa（41.5%）,iha（88.3%）,ecp A（96.8%）,ehx A（80.9%）,kat P（22.3%）and sub AB（44.7%）.There were 20 kinds of virulence spectrum,and the most abundant one was from cattle（14/20,70%）.Contained 9serum groups,including O146（n=14）,O22（n=7）,O3（n=4）,O168（n=4）,O8（n=3）,O167（n=2）,O88（n=1）,O112Ab（n=1）and O147（n=1）.（3）Among 94 STEC strains,14 strains（14.9%）were drug-resistant,and the drug resistance rates of STEC isolates to ceftazidime,tetracycline,cefotaxime,ampicillin and aztreonam ranged from 3.2%to 5.3%,to cotrimoxazole,cefepime,piperacillin-tazobactam,ampicillin-sulbactam,amoxicillin-clavulanate and polymyxin B ranged from 1.1%to 2.1%.The STEC strains from cattle,sheep and human diarrhea were in the same cluster according to the principle that the similarity of node connection to a single isolate or isolate group was greater than 80%.Conclusion:Cattle,sheep and camels are all the reservoirs of non-O157 STEC,and carry abundant virulence factors and multiple serum groups,which are at risk of infecting humans and should be prevented and controlled during slaughter and processing.