Occurrence Of Antibiotic Resistance And Incidence Of Antimicrobial-resistance Genes And Integrons In Bacteria Isolated From Anguilla Rostrata And Farming Water

Aquaculture has used antimicrobials directly or indirectly,which has promoted the selection of antimicrobial-resistant bacteria.Therefore,Antimicrobials were weaken to prevent and treat bacterial infections.Resistant bacteria from aquatic animals can infect humans through the food chain or directly contact and transfer their resistance genes to human flora or related human pathogens.Thus,the aims of this study were to investigate the composition of drug-resistant bacteria in Anguilla rostrata and aquaculture water,and analyze the characterization of resistance phenotypes,genetypes and integrons of resistant bacteria,then study on the removal of antibiotics resistance by pass-generation test.Drug-resistant bacteria in the samples from intestine,epidermis,gill of Anguilla rostrata and the pond water were screened by five antimicrobials.One hundred and eight resistant strains were isolated and identified by phylogenetic analysis of 16 S rDNA sequences,and were classified into four classed 20 genera,and the high incidence of genera were Aeromonas,Citrobacter,and Acinetobacter.The antimicrobial susceptibility test results of 17 kinds of antimicrobial showed that the frequency of resistance to amoxicillin(90.7%)was high,and so with tetracycline,rifampicin,antifolates,and amphenicols(60%~80%).The percentage of multiple resistance to three or more varieties of antibiotics was 93.5%,and among these multi-resistant isolates two strains were resistant to 13 kinds of antimicrobials and two were resistant to 14 kinds of antimicrobials.The multiple antibiotic resistance(MAR)index of isolates in the water sample were higher than that from intestine,epidermis,gill.Among genera,MAR index of Citrobacter and Klebsiella were high,while that of Acinetobacter was low relatively.Detection of 29 different antimicrobial resistance genes using PCR showed that the blaTEM gene was found in all tested isolates,and the tetC gene and the sulI gene were detected in 96.3%strains,followed by the genes aadA(74.1%)and floR(69.4%).The qnrB gene and the arr2/3gene were observed in about half of the isolates.In addition,all strains carried two resistance genes at least and there was detected the strain Klebsiella pneumoniae C11 at most containing 18 kinds of resistance genes.Class Ⅰ integron were present in 86 of the antibiotic-resistant strains;ten out of 108 isolates carried class Ⅱ integron;while class Ⅲ integron were not detected.The ten strains carried class Ⅱ integron all contained class Ⅰ integron.The variable region of class Ⅰ integron was amplified in74 strains successfully,including 33 strains which carried ‘empty’ class Ⅰ integron and 41 strains that carried gene cassettes.Fourteen types of integron cassette arrays were found among class Ⅰ integron-positive isolates.A new array,dfrB4-catB3-blaOXA-10-aadA1,was discovered in this study.The gene cassette array dfrA12-orfF-aadA2 was found to be the most widely distributed.The main cassettes contained genes encoding resistance to aminoglycosides(aad)and trimethoprim(dfr)in the class Ⅰ integron.All class Ⅱ integron-positive strains had only a single gene cassette array,dfrA1-catB2-sat2-aadA1.After passage culture of the antibiotic-resistant strain T4 under no antimicrobials cultivation condition,the rate of antibiotic-resistant strain was fallen rapidly which indicated the conditions has a major impact on the removal of antibiotics resistance.Ultraviolet irradiation did not affect the antibiotics resistance of strain T4.Under high temperature of 37 ℃ or low concentration of antimicrobials condition,passage culture of the antibiotic-resistant strain still was resistant to antimicrobials basically,that is to these condition could maintain the stability of drug resistance.The existence of SDS went against the removal of antibiotics resistance.The strain removed antibiotics resistance to TET has removed the genes tetD,floR and dfrA12 and class Ⅰ integron,but failed to remove the resistant genes strA/B,aphA1 and blaTEM,while these six genes and class Ⅰ integron were carried by primary strain T4.This study analyzed the diversity and antimicrobial resistance of drug-resistant bacteria isolated from ecological communities of Anguilla rostrata,supplemented the scarce and inadequate investigation of resistant genes and integrons about Anguilla rostrata and explored the influence factors for the removal of antibiotics resistance to explore possible approaches to remove the drug-resistance of bacteria.