Molecular Epide Miology Of ESBLs And Mcr-1 Gene Among Enterobacteriaceae Isolated From Retail Vegetables

In recent years,the increase in antimicrobial resistance has become a major worldwide public health problem,especiallythe emerging of extended-spectrum-β-lactamase（ESBLs）that confer bacterial resistance to the third and four generation cephalosporins.In this context,colistin is frequently used clinically as the last resort antibiotic against ESBL-producingespeciallycarbapenemase-producinggram-negativebacteria infections.Therefore,the recent identification of new plasmid-mediated resistance gene mcr-1 has become a research hotspot.In this study,we investigated the molecular epidemiology of Enterobacteriaceae producing ESBLs and/or mcr-1 that was isolated from vegetables,aiming to provide a theoretical basis to prevent further spread of antimicrobial resistance.A total of 708 vegetable samples,including 168 tomatoes,201cucumbers,166 carrots and 173 Lettuceswere collected from retail market in Guangzhou between May 2015 to August 2016.150Escherichia coli isolates were randomly collected fromagar plates without antibiotics and 202 ESBLs-producing Enterobacteriaceae isolates,including 142klebsiellapneumoniaisolates,32E.coliisolates,8Enterobacter cloacaeisolates,7Citrobacter isolates and 5 ornithinolytica isolateswere obtained from CHROM agar plates.Minimum inhibitory concentration（MIC）of these isolateswere determined by the agar dilution method.O f the 202 ESBLs-producing Enterobacteriaceae collected,all showed resistance toampicillin and cefotaxime,over70%were resistant to cefquinome,tetracycline,doxycycline,fosfomycin and sulfamethoxazole,about 65%were resistant to Olaquindox,about 50%were resistant to streptomycin,chloramphenicol and florfenicol.A total of 187（92.57%）ESBLs-producing Enterobacteriaceae were positive forbla_CTX-M.The dominant prevalence genetype was CTX-M-14（n=56,29.95%）,CTX-M-3（n=43,22.99%）and CTX-M-15（n=30,16.04%）.A total of six（2.97%）ESBLs-producingEnterobacteriaceaeisolates,including four E.coli and two Raoultella ornithinolytica isolates,and three（2.0%）randomly isolatedE.coli were positive for mcr-1.Conjugation experiments were performed on these nine mcr-1-carring isolates.It showed that the mcr-1gene from five E.coli were successfully transferred by conjugation.S1 pulsed-field gel electrophoresis（PFGE）and Southern blotting indicated that the mcr-1 was located on chromosome（two conjugation-failed E.coli isolates）and on plasmid（two R.ornithinolytica isolates）.Replicon typing was conducted for the 5 transconjugants.The results showed that two were Inc HI2 plasmids,two were IncI2plasmids,and one was Inc X4 plasmid.The floR,fosA3 and oqx AB genes were detected in transconjugants carrying IncHI2 plasmids,which demonstrated thatfloR fosA3 and oqx AB were usually co-transferred with mcr-1 gene by Inc HI2 plasmids.The sequence types（STs）of the seven mcr-1-positive E.coli isolates were also determined by multilocus sequence typing（MLST）.The result showed that they belonged to six different sequence types,including ST48,ST69,ST156,ST206,ST795 and ST2505.The results demonstrated that the prevalence of ESBLs-positiveEnterobacteriaceaein vegetable was pretty high.The mainβ-lactamase gene groups were bla_{CT X-M-14},bla_CTX-M-3andbla_CTX-M-15.Themcr-1 gene was primarily disseminated in vegetable Enterobacteriaceae through Inc HI2,Inc I2 and IncX4 plasmids.Moreover,other resistance genes,like flo R,fosA3 andoqxAB,were also co-transferred with mcr-1 via IncHI2 plasmids.So we should pay more attention to antibiotics resistance gene contamination in vegetables,and prevent fresh vegetables from becoming a possible route for the spread of antibiotic resistance.