Screening Of Multidrug Resistant Bacteria And Study On The Mechanism Of Drug Resistance Gene Transfer In Sewage Treatment Plant

Antibiotics are substances with sterilization,bacteriostasis and growth promoting effects.They are widely used in the fields of medical treatment and animal husbandry.The use of antibiotics in China is often in the forefront of the world,but the actual utilization rate of antibiotics is limited.Some antibiotics enter the environment with the life activities of human and animals,causing selective pressure,making microorganisms in the environment resistant to drugs and leading to the enrichment of drug-resistant genes.Drug resistance genes can be replicated and transferred,threatening human health and environmental safety.In order to analyze the conjugation and transfer mechanism of drug-resistant genes in multidrug-resistant bacteria in sewage plants in northern China,multidrug-resistant bacteria capable of conjugation were screened from a sewage plant in Harbin.Through drug sensitivity test and whole genome sequencing,the biological characteristics of the strains were comprehensively analyzed,and the mechanism of drug-resistant gene transfer was further explored from the genomic level.To provide theoretical basis and data support for the treatment of microbial drug resistance in the future.The main results are as follows:（1）A total of 29 drug-resistant bacteria were screened from the sewage plant,of which 25 were multi drug-resistant bacteria,8 were able to tolerate all the antibiotics selected in this experiment,and most of them were highly resistant to sulfonamides and macrolides.The multi drug resistant bacteria that can undergo conjugation transfer are named C1 and Z1,and the zygote is named CZ.Through morphological observation,physiological and biochemical experiments and molecular biology identification,C1 is Escherichia coli,Z1 and CZ are Proteus mirabilis.The 16S rRNA sequences of Z1 and CZ strains reached 99.65% repeatability.（2）The growth curves of the three strains were measured.The results showed that the growth laws of the three strains were similar,but the growth rates were slightly different.The results showed that the MIC of C1 strain to 4 antibiotics were TET > NOR > CIP = ERM,Z1 strain to 6 antibiotics were SMX = SMZ > ERM >TET = DOX > AZI,and CZ strain to 8 antibiotics were SMX = SMZ > TET = ERM >DOX = AZI > NOR > CIP.The MIC of the strain to sulfa antibiotics was the highest.（3）The results showed that erythromycin had little effect on the growth of strain C1;Tetracycline,ciprofloxacin and norfloxacin delayed the growth of C1 strain and decreased the biomass.The growth trend of Z1 strain under the action of sulfonamides was basically the same as that of the control group,but the final number of bacteria was lower than that of the control group;At 36 h,the biomass of the strain under the action of some concentrations of tetracycline and macrolide antibiotics was higher than that of the control group.The growth of CZ strain under the action of sulfonamides and quinolones was basically the same as that of the control group;Under the action of tetracycline antibiotics,the growth of the strain showed an obvious delay period,and the effect of doxycycline was greater than that of tetracycline;Erythromycin and azithromycin belong to macrolide antibiotics,but their effects on the growth of CZ strain are different.（4）The whole genome sequencing of three strains showed that C1 strain had a circular chromosome with the size of 4782114 bp and two circular plasmids with the size of 60306 bp and 92065 bp respectively.Z1 and CZ strains each had a circular chromosome with the size of 4039185 bp and 4063678 bp respectively,and no plasmid was detected.A total of 3161 genes were annotated in the go database for strain C1,2547 genes for Z1 strain and 2571 genes for CZ strain.After KEGG annotation,the genes of the three strains were divided into six functional categories,of which the most related genes were annotated to the metabolic pathway.（5）Based on the CARD database,the args of three strains were annotated.A total of 129 drug resistance genes were detected in C1 strain,and 115 drug resistance genes were detected in Z1 strain and CZ strain.According to the prediction of gene Island,there are 18 gene islands in C1 strain,8 gene islands in Z1 strain and 9 gene islands in CZ strain.C1 strain can predict 13 protophages,Z1 and CZ strains can predict 5 and 6 protophages respectively.Some protophages carry args,which may lead to drug resistance transfer.