Study On Antibiotics Inducing Horizontal Gene Transfer Of β-Lactam Resistance At Sublethal Concentrations

The induction of sublethal antibiotics in the environment promotes the development of resistant mutations of microbiota and horizontal transfer of antibiotic resistance genes（ARGs）,which stimulates the appearance and propagation of resistant bacteria in the environment.Among them,the spread of resistance toβ-lactam antibiotics has attracted the most attention.Extended-spectrumβ-lactamase（ESBLs）and Klebsiella pneumoniae Carbapenemase（KPC）are critical methodologies for bacteria to gain resistance toβ-lactam antibiotics.By studying the occurrence of ESBL genes in a river-reservoir system located in the middle and lower reaches of the Yangtze River,the team found that antibiotics and mobile genetic elements（MGEs）had a significant impact on the distribution of ESBL genes:according to results from variation partitioning and Procrustes analysis,they both had extremely significant correlations to the distribution of ESBL genes and were capable to explain a total of 65.10%of the variance.However,co-occurrence network analysis showed that transformation was not the key method of horizontal transfer of ESBL genes.The ESBL genes are encoded on plasmids,so it was speculated that conjugal transfer was an essential way for ESBL genes to undergo HGT.Streaking was operated to isolate P.aeruginosa and E.coli strains probably capable to composite ESBL and KPC simultaneously from the wastewater of major hospitals in Singapore,as well as verified genus using standard 16S rDNA sequencing;Vitek^?2 was used to verify ESBL and KPC synthesis;and quantitative PCR was used to confirm ESBL and KPC genes are tagged.P.aeruginosa and E.coli strains verified were used as donors of ESBL and KPC-encoding plasmids.gfp-tagged E.coli SCC1 strains resistant to chloramphenicol was chosen as recipients.Response surface analysis and Taguchi design were used to analyze the induction of conjugal transfer under single-exposure and co-exposure of tetracycline（TC）,sulfamethoxazole（SMZ）and ceftazidime（CAZ）at sublethal concentrations.It was found that the ESBL plasmid could be conjugal transferred from P.aeruginosa and E.coli strains to the recipient E.coli SCC1 strains at an average frequency of 0.0015 and 0.0042 respectively without stress from inducing antibiotic,showing low fitness cost and higher conjugal frequency between E.coli strains under exposure of sub-MIC antibiotics.Significant conjugation between E.coli strains occurred under single-exposure or co-exposure of TC less than 0.03 mg·L^-1 and CAZ less than 0.002 mg·L^-1,inhibited by sub-MIC level of TC.Conjugation between P.aeruginosa and E.coli strains was stimulated under exposure of TC and CAZ with concentrations 5times larger than MIC,while no significant induction was detected from sub-MIC antibiotics.