| Escherichia coli（E.coli）is a kind of conditioned pathogenic bacteria widely existing in nature,which can cause gastrointestinal infection,secondary infection and systemic disease.Avian colibacillosis is one of the most common infectious diseases in large-scale poultry breeding,which is mostly treated and prevented by antibiotics in clinical practice.Polymyxin is considered as the last line of defense in the treatment of multi-drug resistant gram-negative bacteria infection.Irrational use or even abuse of antibiotics in farms leads to the generation of multi-drug resistant strains,which not only cannot effectively control colibacillosis in poultry,but also brings serious food safety problems.Therefore,this study aims to provide data support for poultry industry norms and clinical rational use of antibiotics in the treatment of colibacillosis by studying drug resistance,distribution of virulence genes,serum groups and comparison of pathogenicity of E.coli from white feather broilers,and provide a theoretical basis for the prevention and control of E.coli from poultry.In this study,135 strains of E.coli from white feathered broilers in Shandong Province were selected from the laboratory,which were identified and preserved in 2021,9 strains were from 1-day-old chickens,44 were from unhatched dead embryos,74 were from 34 w apparently healthy chickens,and 8 were from diseased chickens.They were activated in Mac Conkey AGAR medium,and 72 strains of E.coli were randomly selected for serotyping,and 135 strains of E.coli were phylogenetic grouped by 4-fold PCR.A total of 15 antibiotics commonly used in nine categories were selected and 135 strains of E.coli were detected by disk diffusion method and broth dilution method.A total of 27 drug resistance genes in six categories and Class I integrons were detected in 135 E.coli strains.135 strains of E.coli were detected and analyzed with 13 kinds of virulence genes to clarify their drug resistance and virulence gene distribution.It was of concern in this study whether the differences in the serotypes of pathogenic E.coli were directly related to the pathogenicity of the isolates.One enteroinvasive Escherichia coli（21EC01）and one enteropathogenic Escherichia coli（21EC02）were selected to evaluate the pathogenicity of G.mellonella and 3-day-old SPF chickens,respectively.In this study,a total of 3 pathogenic E.coli serotypes were detected in 72 E.coli strains,including 15 enteropathogenic Escherichia coli,1 enteroinvasive Escherichia coli and 7enterotoxigenic Escherichia coli.Phylogenetic cluster results showed that 135 E.coli strains were derived from 8 serogroups A,B1,B2,D,E,F,A or C and CladeⅠ.The results of drug resistance phenotype showed that the multiple drug resistance rate of135 E.coli strains reached 100%,with 56 kinds of drug resistance spectrum.The resistance rate of 135 E.coli strains to penicillin amoxicillin and ampicillin was 96.3%.The drug resistance rates of tetracycline and doxycycline were 98.5%and 99.3%,respectively.The drug resistance rates of cotrimoxazole and sulfamethymidine were 97.0%and 97.8%,respectively.45 strains of polymyxin-resistant E.coli were detected,with a resistance rate of33.3%.The resistance rate of 34 w apparently healthy chicken E.coli was the highest 58.1%（43/74）.Meropenem resistant strains were detected only in E.coli from infected chickens,and the resistance rate was 25%（2/8）.The carrying rates ofβ-lactam bla TEMand bla CTX-Mresistance genes in 135 E.coli strains were 96.3%and 92.6%,respectively.Among the aminoglycosides,the detection rates of aa C2and aa C4 resistance genes in 135 E.coli strains were 31.1%and 68.2%,respectively,and the detection rate of aa C4 in E.coli from unhatched dead embryos was 100%（8/8）.The sul1resistance gene carrying rate of 135 E.coli strains was 17.0%,only the unexhaled dead embryo and 34 w apparently healthy chicken E.coli carried sul1 resistance gene,the carrying rate was 31.8%（14/44）and 12.2%（9/74）,respectively.The detection rate of polymyxin mcr-1 resistance gene in 135 E.coli strains was 3.0%,and only the E.coli from unhatched dead embryos carried the mcr-1 resistance gene,with a carrying rate of 9.1%（4/44）.No mcr-2～mcr-9 resistance genes was detected in 135 E.coli strains.The detection rate of Class I integrons in 135 E.coli strains was 31.1%,and no Class I integrons were detected in the E.coli of diseased chicken.In addition,the analysis of the drug-resistant phenotype and drug-resistant genes of E.coli showed that inβ-lactam,the drug-resistant phenotype of E.coli resistant to ceftriaxone and cefotaxime had extremely significant correlation with the bla CTX-Mgene（P<0.01）.Among aminoglycosides,a significant correlation was found between amikacin resistant E.coli resistance phenotypes and aa C4 gene（P=0.043）.Four virulence genes（irp2,EAST1,fyu A and F17）were detected in 13 virulence genes of135 E.coli strains,and the detection rates were 14.1%,16.3%,14.8%and 4.4%,respectively.No virulence genes of eae A,hly A,Stx1,Stx2,K99,987p,LT,F18 and CS31A were detected in 135 E.coli strains.The detection rates of EAST1 and irp2 virulence factors were the highest,both 37.5%（3/8）.The LD50of enteroinvasive Escherichia coli（21EC01）and enteropathogenic Escherichia coli（21EC02）were 4.2×105CFU and 3.1×104CFU,respectively.The pathogenicity of enteroinvasive Escherichia coli to SPF chickens was weak,and the LD50of enteropathogenic Escherichia coli strain to SPF chickens was 2.7×108CFU.The results showed that the pathogenicity of different E.coli serotypes to different animals was different,which provided a theoretical basis for further study of its pathogenesis.In this study,135 strains of E.coli were found to have serious drug resistance,with multiple drug resistance rate reaching 100%,and resistance rates to penicillin,tetracycline and sulfonamide antibiotics were all over 95%,and 45 strains of polymyxin-resistant E.coli were detected,which should be paid great attention to.This study has guiding significance for broiler breeding standards and rational use of antibiotics in clinical treatment of E.coli. |
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