| With the widespread use of antibiotics,antibiotic resistance（AMR）has become one of the important factors threatening human health.Migratory birds play an important epidemiological role in the spread of drug-resistant bacteria,and are transporters and repositories of drug-resistant bacteria.At present,studies have confirmed that migratory birds can carry and transmit drug resistance genes during migration.Escherichia coli is an important conditional pathogen and the best model for studying the spread of drug resistance.At present,some scholars have studied the drug resistance and antibiotic resistance gene（ARGs）of E.coli carried by migratory birds in some areas of China,but the comprehensive analysis of drug resistance and pathogenicity of E.coli from migratory birds in China has not been reported.In order to better understand the drug resistance and pathogenicity of bacteria from migratory birds in China,samples of migratory bird feces and other from six provinces in China were collected,including Ciconiiformes,Anseriformes,Gruiformes and Charadriiformes.The samples were separated and transmitted by selective culture medium.E.coli isolates and their drug-resistant phenotypes were identified by specific 16s r DNA identification,BD PhoenixTM-100system automatic microbial identification/drug sensitivity system.According to the results of drug susceptibility identification,PCR and other methods were used to further detect antibiotic resistance genes such as beta-lactams,tetracyclines and sulfonamides,integrase genes and virulence genes.The identified strains of drug-resistant E.coli were subjected to E-test,and the results were evaluated according to the Clinical and Laboratory Standards Institute（version 2019）.Finally,multiple drug-resistant bacteria and E.coli carrying pathogenic genes were selected for next-generation sequencing.The sequencing results were analyzed by genomics,and the drug resistance gene,virulence gene,serotype,plasmid type and MLST typing of E.coli were further analyzed,and the drug resistance,pathogenicity and molecular epidemiological association between them were analyzed comprehensively.The results show that:In this study,556 strains of E.coli（34.0%）were isolated from 1636 faeces,throat swabs and anal swabs of migratory birds from Liaoning Province,Inner Mongolia Autonomous region,Ningxia Hui Autonomous region,Jiangxi Province,Guangdong Province and Guangxi Zhuang Autonomous region.Drug susceptibility identification results:there were155 strains（27.9%）of drug-resistant E.coli,which were resistant to 16 kinds of antibiotics,including tetracycline（25.5%）,ampicillin（17.4%）,trimethoprim/sulfamethoxazole（14.7%）,piperacillin（13.7%）,chloramphenicol（12.4%）,ciprofloxacin（4.0%）,levofloxacin（3.8%）,ampicillin/sulbactam（3.4%）,gentamicin（3.2%）,cefazolin（2.9%）,cefotaxime（1.8%）,cefepime（1.6%）,aztreonam（1.1%）,polymyxin（0.9%）,amoxicillin/clavulanic（0.4%）and amikacin（0.2%）.All were sensitive to moxifloxacin,imipenem,meropenem,ceftazidime and piperacillin/tazobactam.Among the 155 strains of drug-resistant E.coli,11 strains were ESBLs-producing E.coli,accounting for 7.1%,and 91 strains of multi-drug-resistant bacteria,accounting for 58.7%,were mainly resistant to 3 kinds（31.9%）and 4 kinds（29.7%）.The drug resistance and virulence genes of 155 drug-resistant E.coli were identified.Detection results of drug resistance genes:the detection rates of beta-lactam resistance gene bla CTX-Mand bla TEM-1were 6.5%and 31.0%,respectively;the detection rates of tetracycline resistance gene tet（A）,tet（B）and tet（M）were 80.6%,9.0%and 5.2%,respectively;the detection rates of sul1,sul2 and sul3 were 6.5%,47.1%and 9.7%,respectively;the detection rates of chloramphenicol resistance gene flo R and cml A were 39.4%and 43.9%,respectively.Among the 10 drug resistance genes,tet（A）（80.6%）and cml A（43.9%）were the dominant genes.The results of virulence gene identification were:the detection rate of EAST-1 was14.6%,and the detection rate of eae was 2.9%.In addition,85 strains（54.8%）were detected to carry the integrase gene int I1,of which 74 strains were multi-drug resistant.This result suggests that integron plays an important role in mediating the spread of drug resistance.The PCR amplification results of drug resistance genes and virulence genes of 107strains of E.coli screened were supplemented by genomic analysis.The results of genomics analysis showed that among the O serotypes,O8（11.2%）and O103（7.5%）were the dominant serotypes,and the results of other serotypes were scattered.Among the H serotypes,H7（15.9%）and H8（10.3%）were the predominant serotypes.The results of multi-locus sequence typing showed that there were as many as 55 strains in MLST type,and 8 strains were not aligned to MLST type.The dominant types were ST2077（8.4%）and ST48（7.5%）.The results of plasmid typing showed that the plasmid type of the multi-strains was mainly Inc FIB（54.2%）,and all 107 strains carried 1 to 6 types of plasmids.The analysis of resistance genes and virulence genes showed that:quinolone resistance gene qnr S1（48.6%）,folic acid metabolism pathway inhibitor gene dfr A14（47.7%）and aminoglycoside resistance gene aph（3’’）-Ib（45.8%）had a higher carrier rate;Two strains of E.coli carried Shiga toxins stx2,and were identified as enterohemorrhagic Escherichia coli because they also carried the virulence gene eae.We also found a mcr-10 positive strain,which is the first time that mcr-10gene has been detected in E.coli isolates from migratory birds in my country.To sum up,this study carried out the isolation and identification,drug susceptibility testing,drug resistance gene screening and genomic analysis of migratory bird E.coli from six provinces across the country.Through this article,we understand the drug resistance and pathogenicity levels of E.coli from migratory birds in my country,which further confirms that migratory birds are potential communicators of antibiotic resistance.This paper also provides a data basis for the research on drug resistance and pathogenicity of bacteria from wild animals in my country. |
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