Bacterial Genome-wide Association Study Of Drug Resistance Genes In Gram-negative Bacteria From Different Sources

The excessive abuse of antibiotics in the livestock and poultry breeding industry has caused most drugs to be excreted in the environment in the form of secondary metabolites or progenitors with feces or urine.The farmland and water sources around the living environment of humans and captive livestock and poultry are seriously affected.The content of antibiotics in the medium is significantly increased,which speeds up the production of drug-resistant microorganisms in the environment.Bacterial resistance genes and drug-resistant strains can be widely spread and spread through animals,humans and the environment,posing a serious threat to global public health.The World Health Organization lists Carbapenems-resistant and extended-spectrum ?-lactamase-producing Acinetobacter baumannii and Enterobacteriaceae strains as Class I focus.The current research on the spread and transmission mechanism of multidrug-resistant Gram-negative bacteria is mainly focused on human clinical and captive livestock and poultry.The public health safety risks that may be caused by drug-resistant bacteria carried by wild animals are easily overlooked.How multidrug-resistant Gram-negative bacteria play a role in epidemiology is poorly understood.Therefore,we aim at multi-source gram-negative bacteria with wild animals and humans as the research object,carry out drug-resistant phenotype and Bacterial Genome-wide Association Study(BGWAS)research,and systematically analyze multi-drug-resistant gram-negative bacteria The genetic evolution mechanism of drug-resistant genes and the ecological mechanism of epidemics are of great significance for clarifying the spread of drug-resistant genes and drug-resistant strains.In this study,215 strains of Enterobacteriaceae and Acinetobacter were isolated and identified from fresh stool samples from 57 different species.Based on the results of the drug susceptibility test,81 gram-negative bacteria were selected for whole-genome sequencing and whole-genome association analysis.Among 77 Enterobacteriaceae strains,80.36%(45/56)Klebsiella sp.,84.62%(11/13)Proteus sp.,100%(5/5)E.coli,Morganella morganii,Citrobacter werkmanii and Serratia marcescens(1 strain each)showed high tolerance to ampicillin,and most strains also had strong resistance to cephalosporins,sulfamethoxazole/trimethoprim,and ciprofloxacin.Studies have shown that the genomic DNA of K.pneumoniae,P.mirabilis,E.coli,Citrobacter werkmanii,Morganella morganii and Acinetobacter baumannii isolates mainly carry the extended-spectrum ?-lactamase gene,and part of the Proteus sp.chromosome carries Metal-?-Lactamases.The lactamase gene blaNDM-1.It was also found that sulfonamides,fluoroquinolones,tetracyclines and trimethoprim resistance genes were widely distributed on the chromosomes and plasmids of all strains.Further studies have confirmed that there are a large number of insert sequences,transposons and integron structures related to gene movement on the chromosome or plasmid of the strain,which together mediate the transfer of drug resistance.When a strain carries a drug-resistant plasmid,the drug-resistant phenotype is mostly conferred by the drug-resistant plasmid,but when the strain does not carry a drug-resistant plasmid,the drug-resistant gene carried by the strain's genome is more closely related to the drug-resistant phenotype.Research work on the K.pneumoniae M297-1 isolate from Zhengzhou Zoo red kangaroo samples confirmed that the ST290 K.pneumoniae isolate M297-1 genome Chr-M297-1,IncFIB/IncFII type multireplicon plasmid pM297-1.1,The IncFII/IncQ1 multireplicon plasmid pM297-1.2 carries a variety of drug resistance genes and is associated with the structure of movable elements to jointly promote the spread of drug resistance genes between bacteria.Plasmid conjugation experiments confirmed that plasmids pM297-1.1 and pM297-1.2 can mediate the conjugation and transfer of M297-1 and E.coli J53 AZr,and promote the spread of drug-resistant genes.Taking 56 isolates of Klebsiella sp.as the main research object,the internal relationship between the antibiotic resistance phenotype of the bacteria and the multireplicon plasmid carried by the host Klebsiellal sp.was systematically analyzed.The results showed that multireplicon plasmids have stronger drug resistance gene carrying capacity than non-multireplicon plasmids,and can endow strains with multiple drug-resistant phenotypes.Further analysis of 28 multireplicon plasmids found that they have no obvious preference for the host strain sequence type(ST),but the analysis confirmed that K.pneumoniae may be the best adaptive host for multireplicon plasmids.In summary,this study found that most of the strains carrying multireplicon plasmids originated from wild animal samples,suggesting that the habitat of wild animals may be contaminated by antibiotics,and wild animals will become multi-drug resistant bacteria and multireplicon resistant plasmids.As an important preservation host,wild animal host bacteria carrying multireplicon plasmids have stronger transmission advantages and may play an important role in the spread of drug-resistant bacteria in Southeast Asia.