| Antibiotics applied in animals,humans,and agriculture systems are directly entering the water sources and environment through community or hospital waste,wastewater,and sewage,which are playing key roles in the proliferation of antibiotics resistant bacteria.At present,these Antibiotic-resistant bacteria are posing a serious threat to global health and the economy.Baoxiang River located in Kunming,Yunnan Province,China,which is commonly polluted with urban and agricultural pollution.Therefore,the current study was designed to investigate the prevalence of drug resistance Enterobacteriaceae bacteria in the Baoxiang river,which provided a scientific basis for the rational use of drugs,environment,and control of drug-resistant bacteria and drug-resistant genes in Kunming city.In this study,51water samples or soil samples were collected from 11 areas around the Baoxiang River Basin,from July to August 2020.951 Enterobacteriaceae isolates were isolated and purified,including 699 water isolates and 252 soil isolates.The drug resistance characteristics of Enterobacteriaceae bacteria were investigated for antimicrobial susceptibility tests,plasmid transformation tests and Bacterial genome sequence technology.The main research results of current study are as follows:（1）In the antimicrobial susceptibility tests（Agar dilution）of 951 Enterobacteriaceae isolates,the results showed that 23.7%of 166 water samples isolates were sensitive to all tested antiiotics.The prevalence of drug-resistant isolates was high in rural areas compared to urban area.The prevalence of all 15 tested antibiotic resistance isolates among urban samples was 21（21/144,14.6%）.Among urban samples,150（150/699,40.5%）isolates were sensitive to all tested antibiotics.The prevalence of 8 antibiotic-resistant isolates was 34（34/185,18.4%）,which accounted for the largest proportion of multidrug resistance in urban and Dianchi regions,respectively.The prevalence of drug resistance of soil bacteria was similar to that of water samples,with 15 antibiotic resistance in rural areas（38/98,38.8%）and in urban areas（24/154,15.6%）.（2）The homology of 60 multidrug-resistant Enterococci isolates was studied showed that Klebsiella pneumoniae was the main bacterium isolated in rural areas,while E.coli was the main bacterium isolated in urban areas and Dianchi Lake.After MCR-1 harboring plasmid transformation,susceptible E.coli ATCC25922 and DH5αreceptor cells exhibited similar resistance phenotypes and identical resistant genotypes(bla TEM,bla CTX-M,bla SHV,and MCR-1)to the donor bacteria（Klebsiella pneumoniae S12111）.Further,PCR results showed that the detection rate of bla TEMresistance gene was the highest（56/60,93.3%）followed by bla SHV,bla CTX-Mand MCR-1（20/60,33.3%）.（3）Bacterial genome scanning sequencing was used to analyze multidrug-resistant Klebsiella pneumoniae S12111 carrying MCR-1-resistant plasmids.The results showed that the total genome length of Klebsiella pneumoniae S12111 was 5,453,542 bp,carrying 361 resistance genes,607 virulence genes,and 1,234 pathogenic genes.There are a large number of genes involved in metabolic activities.MLST typing showed that Klebsiella pneumoniae S12111belonged to ST292 and maybe the main source of the CC292 clone group.To sum up,the multi-drug resistance of water and soil Enterobacteriaceae isolates is mainly sensitive.Klebsiella pneumoniae S12111 carrying the MCR-1 resistance gene plasmid can spread across species and lead to the emergence of drug-resistant phenotypes and genotypes of sensitive strains.A strain of Klebsiella pneumoniae S12111 carrying the MCR-1 resistance gene plasmid was found for the first time in a rural water source,belonging to ST292 and possibly the main source of CC292.It warned us that continuous monitoring of multidrug-resistant Enterobacteriaceae bacteria,which is of great significance to protect the ecological security in the Baoxiang River Basin. |
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