Study On The In Vitro Fitness Cost Of Antibiotic Resistance-associated Class Ⅱ Integron Of Escherichia Coli From Penaeus Vannamei

As a mobile genetic element,integrons play an important role in the horizontal spread of bacterial resistance genes and the emergence of multi-drug resistant bacteria.In recent years,class I and class Ⅱ integrons have been frequently detected in aquatic products such as Penaeus vannamei.In addition,class Ⅱ integrons have received increasing attention due to their special structure and unclear transmission mechanism that are different from class I integrons.However,the fitness cost of class Ⅱ integrons in related host bacteria is unknown,which severely restricts the understanding of its transmission mechanism.Therefore,in this study,the multi-drug resistant Escherichia coli with class Ⅱ integrons selected from P.vannamei collected from the farmer’s market were used as the material,and the construction of genetic engineering bacteria,the competition test between the strains,and flow cytometry were used for the study of in vitro fitness cost of each important element of class Ⅱ integron in E.coli.The results showed that:(1)A total of 181 strains of E.coli were isolated from 450 samples of P.vannamei collected from a farmer’s market in Zhejiang province,of which 13 strains carrying Ⅱ integron,and positive rate was 7.18%.Class Ⅱ integron positive isolates were multiple antibiotic resistant,and multiple drug resistance coefficient were all greater than 0.2,and resistance pattern were diverse.In addition,13 of class Ⅱ integron gene structures were analyzed.It was found that class Ⅱ integrase were all functional defect type(int I2*).Among them,12 class Ⅱ integron gene structures contained typical gene cassette array dfr A1-sat2-aad A1,and only one class Ⅱ integron gene structure contained the atypical gene cassette array dfr A1-cat B2-sat2-aad A1.(2)Taking the selected typical class Ⅱ integron int I2*-dfr A1-sat2-aad A1 sequence as the template,gene recombination technology was used to construct 13 test strains with different integrases,Pc promoters and gene cassettes.The drug susceptibility test results showed that the antibiotic-resistant gene cassettes of the test strains all expressed the corresponding antibiotic resistance.(3)The bacterial in vitro competition test and flow cytometry analysis were obtained that the cost of class Ⅱ integron structure increased with the increase of strength of the catalytic activity of class Ⅱ integrase.At the same time,the cost increased with the increase of strength of Pc promoter,and the fitness cost of Pc2 A was slightly greater than that of Pc2 B.In addition,the cost of the class Ⅱ integron structure increased with the increase of the number of antibiotic-resistant gene cassettes in the array,and the fitness cost of a single antibiotic-gene cassette was relatively small.Finally,the antibiotic subinhibitory concentration was conducive to the maintenance and enrichment of E.coli carrying typical class Ⅱ integron,which could effectively cope with the external antibiotic pressure,thus promoting the fixation and transmission of the integrons among bacteria.The completion of this project lays a research foundation for further revealing the mechanism of integron-mediated horizontal transmission of antibiotic resistance genes,and provides a theoretical basis for the risk assessment and prevention and control technology of food-borne pathogens.