| Vibrio parahaemolyticus(VP)is the leading foodborne pathogen in costal regions,and the infection outbreaks caused by eating VP-infected seafood products have ranked first worldwide.With the expansion of aquaculture,the phenomenon of antibiotic resistance due to overuse or misuse of antibiotics has become increasingly alarming.Studying the VP antimicrobial resistance is an urgent scientific problem nowadays.In this study,136 VP isolates(aquatic isolates n=65,clinical isolates n=65,environmental isolates n=6)isolated from 11 different areas of Ningbo in 2012 were detected the antimicrobial resistance profiles of 18different antimicrobials by agar dilution method and identified the relevant antimicrobial resistance genes of antimicrobial resistant isolates by PCR.The results showed that,carbenicillin(CAR)resistance was the most prevalent among aquatic(100.0%),clinical(100.0%)and environmental(100.0%)isolates.Ampicillin(AMP)resistance rate followed,showing a biphasic distribution with 46.1%in aquatic,9.2%in clinical and 83.3%in environmental isolates,respectively.Notably,only aquatic isolates showed resistance to sulphamethoxazole/trimethoprim(SXT),streptomycin(STR),tetracycline(TET)and nalidixic acid(NAL),with a ratio of 7.7%,3.1%,3.1%,1.5%,respectively.No other resistance phenotype was found in VP isolates.Six out of eighteen antimicrobial resistance genes belonging to five classes were present.The detected rate of CARB-17,strA,str B,sul II,gyrA,parC was 100%(136/136),50%(1/2),100%(2/2),60%(3/5),100%(1/1),100%(1/1),respectively.The detected results of virulence genes showed that,in clinical isolates,75.4%contained tdh,3.1%carried trh,7.7%had both genes.Only tdh was detected in aquatic and environmental isolates,with the ratio of 6.2%,83.3%,respectively.All four of T3SS1genes were detected in all isolates,all four of T3SS2?genes were only present in tdh~+/trh~—isolates(100%,58/58),while T3SS2?genes were only present in trh~+isolates(71.4%,5/7).The results above showed that antimicrobial resistance in aquatic isolates was severer than others,while the virulence genes in clinical isolates were the highest.ERIC-PCR molecular typing showed that all the 136 isolates were classified into 9 clusters at a similarity coefficient of 0.66.Aquatic isolates present in all clusters except D.Clinical isolates shared clusters A,B,C,G with aquatic isolates.Environmental isolates co-existed in cluster E with aquatic isolates.In order to identify the impact of residual antimicrobials with low concerntraiton in aquaculture on VP,two isolates ATCC33846 and SJTUF30407 were tested against 18 antimicrobials before and after exposuring to subinhibitory concerntraiton(SIC)of trimethoprim(TMP).The results showed that antimicrobial phenotype of ATCC33846 was unchanged after inducing and subculturing for two weeks.SJTUF30407remained the same antimicrobial phenotype except SXT after exposuring to SIC of TMP and this SXT phenotype was not recoveried after two weeks of subculture.RT-qPCR was used to assess the expression change of efflux transporter genes vmeB,cytoskeleton genes mreB,minE,ftsZ,quorum-sensing regulator genes luxR,biofilm formation genes cpsQ,mfpA and virulence regulator genes fur,rpoS,toxR in SJTUF30407 in response to the SIC of TMP induction.The results showed that ftsZ,mfpA,mreB were found to be down-regulated 2.30,2.35,2.28 times in response to the SIC of TMP,in contrast,toxR was up-regulated 2.16 times.The expression of cpsQ(1.38),fur(1.13),minE(1.86),vmeB(1.27)did not change under SIC of TMP induction.The results suggested that the SIC of TMP could change the expression of genes relevant to morphological structure and virulence. |
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