| The bacterial antibiotic resistance in aquaculture environments such as Trionyx sinensis farm has been very serious.The antibiotic resistant bacteria（ARBs）and antibiotic resistant genes（ARGs）in T.sinensis farms can be disseminated to the human body through the food chain,which is a serious danger to human health.The purpose of this study is to investigate the bacterial antibiotic resistance of T.sinensis farms in Xiaoshan and Lanxi,Zhejiang Province,and to explore the horizontal dissemination mechanism of plasmid mediated carbapenem resistance gene bla NDM,fitness cost of mainly carrying plasmid and mechanism that cause differences in fitness cost.（1）In order to investigate the bacterial antibiotic resistance of T.sinensis farms in different cities,244 strains of bacteria were isolated from T.sinensis farms in Xiaoshan and Lanxi,Zhejiang Province in November 2020.The identification results of 16S r RNA showed that the flora distribution of the two farms was similar,mainly Enterobacteriaceae.Edwardsiella was the dominant genus of isolates.K-B susceptibility test showed that all isolates were resistant to at least one antibiotic.The resistance rate of these ARBs to penicillins and sulfonamides was more than 70%,and some ARBs are resistant to carbapenems.The detection of ARGs by PCR showed that the carbapenem resistance genes had been disseminated in some farms.Comparing the antibiotic resistance of ARBs from different sample sources,it was found that the sludge of the pond is an important repository of ARBs and ARGs in T.sinensis farms.The bacterial antibiotic resistance of Lanxi farm was more serious than that of Xiaoshan farm,which is mainly reflected in:1)the proportion of 14 to 18 antibiotic resistant strains in Lanxi farm reached 34.7%,while the proportion of this type of strains in Xiaoshan farm was only 10.5%;2)Except that the resistance rate of fosfomycin in Xiaoshan was significantly higher than that in Lanxi,the resistance rate of 16 antibiotics in Lanxi was significantly higher than that in Xiaoshan（P<0.05）;3)Except for the other ARGs detected separately in each region,the detection rate in Lanxi ARBs was higher than that in Xiaoshan ARBs.The comprehensive detection rate of type I integron integrase int I1 was 73.4%,and that of type II integron integrase int I2 was 9.0%.Five types of type I integron gene cassettes and one type of type II integron gene cassettes were amplified.The detected gene cassettes mainly contained aminoglycosides and trimethoprim resistance genes.The comprehensive detection rate of gene cassettes of integron integrase positive bacteria was 15.9%（32/201）.The high detection rate of integron integrase and the low detection rate of gene cassettes indicated that many ARBs have the potential to integrate foreign ARGs.（2）In order to explore the dissemination mechanism of bla NDM in T.sinensis farms,6bla NDM positive ARBs were screened from the ARBs in T.sinensis farms.Through the conjugation transfer experiment,the determination of minimum inhibitory concentration and the enzyme production experiment of improved Carba NP method,it was proved that bla NDMexists on the plasmid,and can disseminate among bacteria.The conjugation transfer frequency of Lanxi bla NDM plasmid was higher than that of Xiaoshan bla NDM plasmid.These results suggested that the horizontal dissemination of bla NDM-carrying plasmid through conjugation transfer may be the main reason for the dissemination of bla NDM in T.sinensis farms.By extracting typical plasmids for high-throughput sequencing and fine annotation analysis,it was found that the Inc C plasmid p210704-NDM-1 isolated from Xiaoshan farm carried 7 classes and 13 kinds of ARGs including bla NDM-1.The locus of bla NDM-1 on this plasmid was in a complex integron structure with IS26 and IS4321.The Inc X3 plasmid p210744-NDM-5isolated from Lanxi farm has a simple structure and only carries two ARGs,ble MBL and bla NDM-5.The high proportion of conjugation transfer region sequence of this plasmid（44.7%）indicated it may be easier to disseminate horizontally through conjugation transfer.The locus of bla NDM-5 on this plasmid was in a structure very similar to the Tn125.p21074-NDM-1,p210744-NDM-5,and the similar gene environment of the bla NDM in other researches showed that the structure of bla NDM--ble MBL-trp F-dsb D is relatively stable in different bla NDM-carrying plasmids.And the mobile genetic elements around bla NDM in plasmids,especially ISAba125 upstream of bla NDMin the sequenced plasmid,was one of the main mobile genetic elements to promote the horizontal dissemination of bla NDM.（3）In order to explore the fitness cost difference of plasmids carrying diverse bla NDMsubtypes and the mechanism involved.The fitness cost of plasmids carrying diverse bla NDMsubtypes was characterized by growth dynamics,plasmid stability,motility experiment and competitive experiment.The results showed that the fitness cost of transconjugant XS210704-EC600 wassignificantly higher than that of transconjugant LX210744-NDM-5.And fitness cost of p2107004-NDM-1 carrying bla NDM-1 was higher than that of p210744-NMD-5 carrying bla NDM-5.RT-q PCR was used to detect the transcription difference of fitness related genes.The possible mechanism causing the high fitness cost of bla NDM-1-carrying plasmid p2107004-NDM-1 was as follows:1)The reduction of the transcription level of fim A,the main gene of pili,led to the decline of the synthesis ability of host pili,which hindered conjugation transfer;2)The reduction of the transcription level of rpo N,the RNA polymeraseσfactor gene regulating flagellum synthesis,leads to the decrease of flagellum synthesis ability of the host,which leads to the decrease of transcription level of flagellum assembly related genes fli A,fli C,fli E,and fli Z.Finally reduces the motility of the host;3)The differential expression of ATP binding protein genes（ugp C,rbs A,and gsi A）and outer membrane pore protein gene omp D affects the host’s energy metabolism;4)The reduction of the transcription level of DNA initial replication gene dna A reduces the efficiency of cell division and hindered growth of the host.In summary,there are differences in bacterial antibiotic resistance in T.sinensis farms in different cities,and the sludge of the pond is the most important repository of ARBs and ARGs in T.sinensis farms.The horizontal dissemination of bla NDM-carrying plasmid through conjugation transfer is the main reason for the wide spread of bla NDM,and ISAba125 in the upstream of bla NDM is one of the main mobile genetic elements to promote the horizontal dissemination of bla NDM.In this study,the Inc X3 bla NDM-5-carrying plasmid has higher widespread risk than the Inc C bla NDM-1-carrying plasmid.The results of this study provided a certain basis for the rational use of antibiotics in T.sinensis farms,and provide a certain theoretical basis for controlling and cutting off the ARBs and ARGs in T.sinensis farms to spread antibiotic resistance through the food chain.It is of great significance to alleviate the problem of bacterial antibiotic resistance and ensure food safety. |
