Study On The Characteristics Of Conjugative Transfer Of β-Lactamase Resistance Genes In Cefotaxime-Resistant Bacteria And The Effect Of Chlorination On Them

With the widespread use of third-generation cephalosporin antibiotics（such as cefotaxime）,antibiotic resistance bacteria has rapidly emerged,and the spread ofβ-lactamase resistance genes has also attracted more and more attention.At present,there are few reports on the problem of the transmission ofβ-lactamase resistance genes against cefotaxime-resistant bacteria.Therefore,this study took the cefotaxime-resistant strains in the main landscape waters of Xi’an as the research object,investigated the conjugative transfer characteristics that could was observed in cefotaxime-resistant strains under natural conditions,and explore the effect of chlorination on the conjugative transfer ofβ-lactamase resistance genes,which could reveal the conjugative transfer characteristics of cefotaxime-resistant bacteria in urban landscape waters,and provide scientific basis for the spread control of antibiotic-resistant bacteria in urban landscape waters.The conjugation experiments with 216 cefotaxime-resistant strains isolated from five urban landscape waters in Xi’an city as candidate donor strains and Escherichia coli NK5449 as the recipient bacteria were conducted to determine the conjugative transfer frequency.PCR was used to analyze the distribution of threeβ-lactamase resistance genes(bla _CTX-M,bla _TEM,bla _SHV)and class I integron integrase gene（int I）on the plasmid of the donor strains.Using RT-PCR technology,mRNA expression of outer membrane genes（omp A,omp C,omp F）and conjugative transfer-related genes（trbC,tra A,trb Bp,tra F,trf Ap,tra J,kor A,kor B,trb A）were detected in these donor strains.Based on this,a donor strain with the highest frequency of conjugative transfer was selected to investigate the change of conjugative transfer frequency after chlorination.Real-time fluorescence quantitative RT-PCR technology was used to further study the effect of chlorination on gene mRNA expression.The results showed that conjugative transfer was observed in a total of 12cefotaxime-resistant strains,which were identified as Escherichia coli,Aeromonas veronii and Bacillus cereus,respectivley.The conjugative transfer frequency ranged from 3.95×10^-8to 3.07×10^-3.The detection rates of threeβ-lactamase resistance genes bla _CTX-M,bla _TEMand bla _SHVon the plasmids of the 12 donor strains were 58.33%,58.33%and 8.33%,as well as class I integron were 100%.Outer membrane protein genes（omp A,omp C,omp F）,sex pilus-coding gene（trbC）and mating pair formation gene（trb Bp）showed active mRNA expression in these donor strains.Escherichia coli L414 is the donor strain with the highest frequency of conjugative transfer in this study.Using Na Cl O solution with an effective chlorine concentration of10 mg/L for 30 min,the number of Escherichia coli L414 was reduced from 1.49×10⁸CFU/m L to 2.67×10³CFU/m L,and the frequency of conjugative transfer was reduced from 2.09×10^-3to 5.19×10^-6.Chlorinatio can promote or inhibit the mRNA expression of bacterial outer membrane protein genes（omp A,omp C,omp F）and sex pili-encoding gene（trbC）,and the type of action is related to the effective chlorine concentration.Under the chlorination,the frequency of conjugative transfer between bacteria showed a significant positive correlation with the number of donor bacteria（p<0.05）,while there was a significant negative correlation with the mRNA expression levels of outer membrane protein genes（omp A and omp C）（p<0.05）.