Preliminary Study On Resistance Mechanisms To Rifampin And Fitness Of Rpo B Mutation In Riemerella Anatipestifer

Riemerella anatipestifer?RA?is a bacterial pathogen that can infect ducks,geese,turkeys,and other poultry.In recent years,drug resistance of rifampin has become increasingly serious in RA.The main research contents and results of the rifampin resistance mechanism and effects on the survival and fitness of rpoB gene mutations in RA are as follows:1.Study of rifampin resistance mechanisms in RAThe rifampin MIC was determined in 110 RA,and 89%?98/110?of them was resistant to rifampin,and the MIC value is between 0.5?g/mL²56?g/mL.The correlation between the 18 RA rpoB sequences and MICs was analyzed,and site-directed mutagenesis and natural transformation techniques were used to construct overexpression strains for verification.The results showed that mutations in five amino acid sites of RA rpoB gene?V382I,H491N,R494K,G502K,and S539Y?were related to rifampin resistance.The RA-CH-1 and RCAD0147 strains had identical rpoB mutations,but rifampin MIC of RCAD0147 is eight times higher than that of RA-CH-1,32?g/mL and 256?g/mL respectively.Using the efflux pump inhibitors CCCP and VP,it was found that there was drug efflux pump related to rifampin resistance in RCAD0147.It cooperated with rpoB mutation to increase the resistance of RCAD0147 to rifampin.2.Study of the fitness cost of rpoB mutations in RAFifteen types of rpoB mutations were isolated by using spontaneous mutation experiments,in which R494K was identical to the type of mutation detected in the isolates.The frequency of rpoB gene mutation was calculated to be 10^-8 by flat colony counting method.The 98.8%?247/250?of the rpoB mutations obtained from mutant strains were located in cluster I of rifampin resistance-determining region of rpoB gene.With the exception of D481Y,I537N and S539F,the remaining mutants reached to at least 64?g/mL for rifampin MIC.The H491D and 484::TAA mutant strains showed delayed growth.In vitro competitive experiments showed that the fitness of the mutants was reduced,and the H491D and 484::TAA mutants were most affected?the relative fitness was0.60 and 0.63,respectively?.Finally,the colonization abilities and sensitivities of the R494K and H491D mutants were investigated.The results showed that the sensitivity of the two mutants to H₂O₂ and SNP increased.In vivo colonization experiments showed that the number of live colonies colonized by the two mutants in the trachea and brain was lower than that of the parental strain within 24 hours.This study showed that the single mutants of the rpoB gene in rifampin-resistant RA led to fitness cost and the strains fitness weakened.