| In recent years,the increasingly antimicrobial resistance of gram-negative bacteria,particularly Enterobacteriaceae,has become a serious public health issue worldwide.Resistance genes spread mainly by plasmids among bacteria from various sources,such as humans and animals,and pose a threat to clinic therapy and public health.IncHI2 plasmid is one of the most popular plasmids among Enterobacteriaceae,and is frequently involved in multiple resistance genes.The transmission of resistance genes mediated by IncHI2 plasmids is one of global concern.In this study,we investigated the prevalence of IncHI2 plasmids and resistance genes located on IncHI2 plasmids in Escherichia coli from different sources（patients,food-producing animals,and food）in 2011-2014 in Guangdong Province,and complete sequences of IncHI2 plasimds,in order to provide theoretical basis for slowing down the spread of antimicrobial resistance.A total of 1844 E.coli isolated from different sources in Guangdong Province from 2011to 2014 were detected IncHI2 replicon by PCR-based replicon typing（PBRT）.The results showed that the prevalence of IncHI2-plasmids in 1844 E.coli isolates was 2.6%（n=48）,the chicken isolates（4.3%）showed highest prevalence,followed by food isolates（2.9%）and porcine isolates（2.2%）,human isolates showed lowest IncHI2 prevalence（1.6%）.A total of40 transconjugants/transformants were successfully obtained.S1-PFGE showed that all transconjugants/transformants carried single plasmid with size ranging from150 kb to400kb,they were further analyzed by plasmid double locus sequence typing（pDLST）,mainly IncHI2-ST3（n=32）,followed by IncHI2-ST1（n=3）and IncHI2-ST4（n=1）,and the remainingfourIncHI2plasmidswerenottypable.Inadditional,40transconjugants/transformants were further detected other replicons by PBRT,17transconjugants/transformants also contained other replicons,including IncFII（n=9）,IncN（n=6）,and IncFII-IncN（n=2）.Five IncHI2-positive original isolates were randomly selected to calculate conjugative transfer frequency,the results showed IncHI2-plasmids to E.coli C600 at frequencies of 10-5 to 10-3 cells per recipient cell.Agardilutionmethod wasusedto test the suceptibility of40transconjugants/transformants against 19 antimicrobial agents.The results showed that most isolates were highly resistant to ampicillin（97.5%）,ceftiofur（70%）,chloramphenicol（80%）,florfenicol（70%）and cotrimoxazole（72.5%）.Furthermore,the resistance rates of colistin and fosfomycin were 25%and 35%,respectively.On the other hand,39transconjugants/transformants were resistant to more than three classes of antibiotics,and 22strains（55%）were resistant against eight to 11 agents.Forty transconjugants/transformants were further determined antimicrobial resistance genes by PCR.Among them,30,32,14 and eight isolates carried blaCTX-M,floR,fosA3,and mcr-1.The most predominant CTX-M-encoding gene was blaCTX-M-65（n=11）,followed by blaCTX-M-14（n=9）.Prevalence of blaCTX-M among isolates from animal-derived food（56.5%）and humans（62.5%）were significantly lower than those of chickens and pigs（100%）.In this study,bla CTX-M-floR were most commonly co-transferred（n=24）.Similarly,bla CTX-M-fosA3,blaCTX-M-oqxAB,blaCTX-M-aac（6’）-Ib-cr and blaCTX-M-oqxAB-floR were co-transfered in 11,nine,six,and six,respectively.Additionally,eight strains carried mcr-1,blaCTX-M and floR.IncHI2-ST3 plasmids from pig（SHP45）,human（LDHF400）and food（YJMC8）were sequenced by PacBio.The results showed that the sizes were 251493 bp,249152 bp,249153bp respectively with GC content 46.53%,46.51%,and 46.51%.These three plasmids contained a typical IncHI2-type backbone,which encodes genes for plasmid replication,conjugative transfer,maintenance,and stability.The complete sequences of pHNLDF400and pHNYJC8 were almost identical,with only 2-bp base differences.The backbone of plasmid pHNSHP45-2 was identical to pHNLDF400 and pHNYJC8,except the module ISApl1-mcr-1 was inserted into the backbone of pHNSHP45-2.Furthermore,all three plasmids contained numerous resistant genes,including oqxAB,sul1,ΔaadA2,dfrA12,aphA1,sul3,aadA1a,cmlA1,aadA2,floR,sul2,aph（4）-Ia and aac（3）-IV.On the other hand,pHNLDF400 and pHNYJC8 contained the|aac（6’）-Ib-cr|blaOXA-1|catB3|arr3|qacEΔ1|sul|cassette array,whereas pHNSHP45-2 harboured a segment containing blaCTX-M-14 and fosA3.It is speculated that these plasmids may be derived from the same IncHI2 plasmid through stepwise acquisition and recombination by integron and insertion sequence,such as IS26 and ISApl1,carrying drug-resistant genes.In conclusion,similar multi-resistant IncHI2 plasmids were distributed in E.coli from different sources（food,animals and clinical humans）,and mediated resistance genes such as mcr-1,bla CTX-M,and fosA3 disseminated among humans,animals,and food.IncHI2 plasmids acquiring or losing mobile elements,such as integron and insertion sequence,throughout evolution and eventually spread further among E.coli from various resources.The horizontal transmission of IncHI2 plasmids,as carriers of multiple resistance genes,among E.coli in animals,animal-derived food,and humans,is not only a challenge to animal husbandry industry,but also poses a serious threat to human therapy and public health. |
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