Diversity Of Mcr-1-Carrying Enterobacteriaceae In Chicken Gut

Colistin is a polymyxin antibiotic widely used in animal production and human disease treatment.The resistance mechanisms of colistin were recognized as chromosomally mediated and could not transmissible,until the first description of the plasmid-mediated colistin resistance gene mcr-1.Since then,increasingly follow-up reports have proved a worldwide spread of this gene,and it had emerged in humans,animals,environments and food.Furthermore,the emergence of mcr-2 to mcr-5 and numerous of mutants had to attract our attention to mcr-like genes,as well as the monitoring of colistin resistance.As we know,traditional methods of antibiotic resistance epidemic research were usually performed by selecting a single clone for further study.However,this single clone per sample method was likely to underestimating species diversity and transmission events.Therefore,a new method was used in this study to explore the different possibilities in the same sample and provide a new idea for the future research.Nine mcr-1-positive cecum swabs from randomly selected sick chickens were collected from three animal hospitals located in Heilongjiang,Anhui and Guangdong province in China.For each sample,a dilution gradient was selected on which growth was sparse enough to enable 50 single isolates in a plate.All single clones of three parallel experiments were picked and purified for further analysis.A total of 1094 colistin resistance isolates were obtained from nine samples,including 99 natural resistance isolates?M.morganii,P.vulgaris/penneri,P.mirabilis,P.stuartii and S.marcescens?and 995 acquired resistance isolates?E.coli,E.fergusonii,K.pneumoniae,K.quasipneumoniae and E.cloacae?,among which,mcr-1-positive rate was 88.4%?968/1094?,including 962 E.coli,2 E.fergusonii,2K.quasipneumoniae and 2 K.pneumonia.Notably,it's the first report of mcr-1-positive E.fergusonii and K.quasipneumoniae.Additionally,mcr-1 and mcr-3 genes were detected in a single E.coli isolate.The results of MIC showed that mcr-1-positive isolates were resistant to TET?98.2%?,S/T?92.7%?and FFC?91.7%?,more than 98%of these isolates showed multidrug resistance.Transconjugants were mainly resistant to FFC?32.3%?,OLA?27.1%?and FOS?22.9%?.The mcr-1-positive isolates carried multiple AMR genes,mainly including floR?91.5%?,bla_CTX-M?71.2%?,oqxAB?62.7%?.The mainly co-transfer genes in transconjugants were bla_CTX-M?32.3%?,floR?28.1%?and fosA3?24.0%?.Mutations of amino acid at pmrA and pmrB were found in mcr-like-negative non-natural colistin resistance isolates of MIC>2?2 E.coli and14 K.pneumonia?.102 mcr-1-positive E.coli isolates from nine samples were divided into 46 clusters by PFGE,and various clusters were obtained from each sample.Totally,59 mcr-1-positive isolates were selected for WGS,and 41 sequence types?STs?were identified.ST10 was the most prevalent ST in mcr-1-positive E.coli isolates,which was identified in all three animal hospitals.ST48 and ST1485 were relatively popular.The mcr-1 genes were located on the chromosome and IncI2,IncX4,IncHI2,p0111 and IncHI1 type plasmids through analysis whole genome sequencing.And the diversity existed between the same types of plasmid.The genetic environment of mcr-1 was also diversity,consisted of multiple insertion sequences?ISApl1,IS1,IS629 and IS26?.This study clarified the abundant diversity of mcr-1-positive Enterobacteriaceae isolates in four-dimension,chicken from species,relations of clone,plasmid types and genetic environment,and provided a methodological reference for the study of resistance diversity of bacteria in the future.