Isolation And Identification Of Antibiotic-resistant Bacteria In Soil And Water Environment Of Aquaculture And Research On Their Potential Risks

China is a big producer and user of antibiotics.However,the unreasonable application of antibiotics in medical treatment,animal husbandry,aquaculture and other industries leads to the widespread existence of antibiotic-resistant bacteria,which seriously affects human health.In recent years,the research on drug-resistant bacteria has been widespread in the medical industry.However,the research on drug-resistant bacteria in aquaculture,which is closely related to human life,is still scarce.Therefore,it is very necessary to study the number,species and drug resistance of drug-resistant bacteria in soil and water environment of aquaculture industry,and some suggestions can be put forward for the use of antibiotics in aquaculture industry.In this study,the aquaculture soil and water environment samples were taken as the research object.The total number of bacteria and the number of drug-resistant bacteria in the samples were counted by plate colony method,and the comparative analysis was carried out.The isolated drug-resistant bacteria were identified by PCR and 16S r DNA sequencing,and the minimum inhibitory concentration(MICs)of the conditional pathogens for 10 antibiotics such as polymyxin was detected.Through the influence of four efflux pump inhibitors on the resistance of the strains,the existence of efflux pump was deduced,and the resistance mechanism of antibiotic-resistant strains was discussed.To identify whether the strain could transfer the resistance gene level to the standard quality control strain(Escherichia coli ATCC25922DN).Finally,heterologous expression of the efflux pump gene from Pol-G4 strain of Stenotrophomonas sp.and MICs analysis were performed.We discovered the following results through the research of this subject:(1)Based on the total number of colonies and drug-resistant bacteria in the breeding industry of water and soil environment in the order of the detection of the total number of bacteria in the 10~5CFU/m L and above and with plenty of drug-resistant bacteria,is given to illustrate the drug-resistant bacteria breeding development,the important ways to expand and spread in the study sample separation and identification of the 86 strains of drug-resistant bacteria,containing a large number of Cocci,Bacillus,Pseudomonas,etc.Under different screening pressures of antibiotics,the number of drug-resistant bacteria isolated from the samples was significantly less than the total number of bacteria in the samples.Part of the isolated drug-resistant bacteria belonged to opportunistic pathogens.(2)The minimum inhibitory concentration of drug-resistant bacteria to different antibiotics was detected,and it was found that all the isolated drug-resistant bacteria were resistant to more than 3 kinds of antibiotics,belonging to multiple drug-resistant bacteria.In addition,most of the drug-resistant bacteria had strong resistance to ampicillin and amikacin,and the resistance to fluxamicin was significantly lower than other antibiotics tested.(3)In this study,the mechanism of action of antibiotic-resistant bacteria was preliminarily discussed.The experimental results proved that efflux pump inhibitors could indeed reduce the resistance of the strains to an antibiotic,and it was inferred that the isolated antibiotic-resistant bacteria might have related efflux pump.From the genome sequencing results of a few strains,the whole genome contains a large number of antibiotic resistance genes.(4)Some antibiotic-resistant bacteria have potential environmental risks,and some strains can transfer resistance genes to E.coli ATCC 25922DN at the same level.For example,strain MER-M202(Pandoraea Pnomenusa sp.)made antibiotic-sensitive Escherichia coli resistant through conjugation transfer experiment.It was concluded that horizontal gene transfer was an important way to spread the resistance gene in aquaculture and water environment.(5)Recombinant vector was constructed for the predicted target gene of Pol-G4 strain,and the recombinant expression vectors of RS16900,RS17565,RS18690,RS08745,RS06240,RS06780 and RS05865 were successfully constructed.