Mechanism Of Quinolone Resistance Antibiotics In Riemerella Anatipestifer

Riemerella anatipestifer(RA)is a bacterial infectious disease pathogen that can infect domestic ducks,geese,turkeys,and various other domestic and wild birds,causing huge economic losses to the breeding industry.Quinolone is one of the most widely used drugs in clinic.In recent years,the resistance to quinolone of RA is serious.In this study,the resistance mechanism of RA to quinolones was explored based on the detection of drug resistance phenotype of RA isolates.The main results are as follows: 1.Detection of quinolone resistance phenotype of Riemerella anatipestiferFor a total of 162 isolates of RA,the MICs were determined for four quinolone antimicrobial agents: the first generation quinolone drug nalidixic acid,the third generation quinolone ciprofloxacin,enrofloxacin and ofloxacin.Compared with the isolates before 1979,the resistance of the isolates after 1979 to three fluoroquinolones increased significantly.137 RA strains isolated from 2013 to 2018 from 48 farms were selected for quinolone resistance analysis.The MICs showed that the resistance rates of 137 clinical isolates to the four quinolones were 100%(137/137),97.8%(134/137),99.3%(136/137)and 97.8%(134/137),respectively.2.The resistance mutation of DNA helicase and ? topoisomerase in Riemerella anatipestifer.We compared the gyrA,gyrB,parC,and parE genes of the strains for 37 whole-genome sequences.A region with a high mutation probability that included a large number of mutations was selected as the target region for PCR amplification.The above gene fragments of 125 remaining strains were amplified.The mutation and amino acid substitution of each strain were aligned and analyzed.The results showed that the high frequency mutation sites were the amino acid position 83 in GyrA,including two mutation types S83 R and S83 I,with a total mutation frequency of 93.2%.In addition,in GyrA,amino acid 465 has the mutation type C465 R with mutation frequency of 42.6%.In ParC,amino acid 586 contains either V586 T or V586 A mutation types,with a total mutation frequency of 41.4%.In addition,the mutation frequencies of amino acids V799 A and 811 I811 V in ParC are 72.2% and 67.3%,respectively.In ParE,the mutation frequencies of V357 I,H358Y,R541 K,D564K were 80.9%,80.9%,82.1% and 44.4%,respectively.Quinolone-resistant mutants were obtained by in vitro induction mutation technique.Site-directed mutagenesis and natural transformation techniques were used to construct in vivo mutants and overexpression strains of RA.The results showed that only S83 R mutants were isolated from the induced mutants.The MICs of nalidixic acid have a 4-8-fold increase and that of three fluoroquinolone drugs have a 512-1024-fold increase.Only the MICs of ATCC 11845 gyrA(S83I)and ATCC 11845 gyr A(S83I)+ parE(V357I+H358Y+R541K)strains against three fluoroquinolone antibiotics have a 4-8-fold increase,but the MICs of nalidixic acid were not significantly changed.The MICs of the overexpressed strains were not significantly changed compared with their parents.3.Study on the efflux of Riemerella anatipestifer on quinolones.Two efflux pump inhibitors,carbonyl cyanide m-chlorophenylhydrazone(CCCP)and verapamil(VP),were used to test the efflux pump inhibition of 24 genome sequencing RA strains.Compared with the MIC results without efflux pump inhibitors,the susceptibility of RA RCAD0131,RCAD0181 and RCAD0377 strains to three fluoroquinolones decreased significantly.Quinolone resistance-related efflux pumps are involved in the resistance mechanism of RA to quinolones.In conclusion,this study examined the resistance of RA to quinolones,and demonstrated that target mutations and quinolone-related efflux pumps mediated the resistance of RA to quinolones.