| Escherichia coli is the most common conditional pathogen in aquaculture environments which can cause infections of varying degrees in livestock.Antibiotics are widely used in aquaculture as therapeutic drugs and growth promoters,making more and more bacteria resistant,even full-resistant bacteria.In addition,these drug resistance genes can be transmitted between humans and animals by the food chain and environment,posing a huge threat to aquaculture and human health.Since the widespread use of extended-spectrum ?-lactam antibiotics,more and more bacteria have developed resistance.Extended-spectrum?-lactamase(ESBL)has strong enzymatic activity whcih can hydrolyze cephalosporin,penicillin and monocyclic ?-lactam antibiotic aztreonam.blaCTXM is one of the most important genotypes of ESBL.The aim of this study was to investigate the drug resistance status and major drug resistance genes of E.coli in cultured environments,and to analyze the gene structure of plasmids of multidrug resistant bacteria.Inour previous study,we collected a large number of strains in the farm mainly in the samples of feces,urine,soil and surrounding water environment,and identified by 16S.Finally,206 strains of Escherichia coli,48 strains of Proteus mirabilis and 32 strains of Providence were obtained.The KB-method was used to detect the resistant phenotype of E.coli.The results showed that E.coli in livingstock farm had higher resistance rate to tetracycline and cefazolin.The rate of resistance to tigecycline and polymyxin is the lowest.The established multiplex PCR detection technology was used to detect the drug resistance genotypes of drug-resistant bacteria which showed that six extended-spectrum ?-lactamase resistance genes were detected in all drug-resistant strains.The detection rate from high to low was blaTEM?blaCTX-M?blaSHV?blaOAX-1?blaKPC and blaNDM.Three corresponding resistance genes were also detected in the quinolone-resistant bacteria,and the detection rates were qnrD,qnrB,and qnrA from high to low.Four resistant genes were detected in macrolide-resistant bacteria,and the detection rates were mphE,mphA,ermA and mphB ranged from high to low.We finally selected two multidrug-resistant strains for further study:E.coli 1709008 and 1710032.Activities of class carbapenemase in bacterial cell extracts were determined by novel CarbaNP tests.Bacterial antimicrobial susceptibies were tested by VITEK 2.Plasmid conjugal transfer experiments were carried out with the rifampin-resistant E.coli EC600.Plasmid DNA was isolated from E.coli tarnsconjugant using a Qiagen Large Construct Kit,and then sequenced with a mate-pair library with average insert size of 5,000 bp through an illumine MiSeq sequencer.The results showed that strain 1709008 was able to produce carbapenemase type A,and 1710032 was able to produce carbapenemase type B.Conjutransfer experiments showed that the resistance of these two strains could mobile,and the transconjugant strains were resistant to penicillins,cephalosporins and monocyclic ?-lactam antibiotics.The two-generation sequenced data were spliced to obtain two multi-drug resistant plasmids p1709008-CTXM and p1710032-CTXM,respectively.Both of the resistant plasmids belong to the IncFII type plasmid containing the repA2-6-1-4 structure responsible for encoding the replication initiation protein.Both p 1709008-CTXM and p 1710032-CTXM contain the blaCTXM gene,and both are mediated by the mobile original ISEcpl-blaCTX-M-55-orf477.ISEcp1 not only enables the overall movement of the structure,but also provides a strong promoter to promote the expression of blaCTX-M-55.In addition,both plasmids contain type 1 integron In54 to provide the resistant methicone resistance gene dfrA17,the sulfa resistance gene sull.The macrolide resistance gene mphA was introduced by the mobile original IS26-mph(A)-mrx-mphR(A)-IS6100 unit.The tetracycline resistance gene tetA is provided by the transposon Tn1721.In general,both plasmids have a complex chimeric structure,not only the maintenance region responsible for plasmid stability,the binding transfer region responsible for horizontal transfer of the plasmid,but also the exogenous insertion containing a large number of drug resistance genes and mobile elements.This study confirms that it is precisely because of the presence of these resistant plasmids that bacteria develop resistance and that this resistance can be transferred between bacteria by horizontal transfer of plasmids. |
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