Amino acid substitutions in the region between RpoB clusters II and III on rifampin susceptibility in Haemophilus influenzae.

Abstract

Rifampin is a potent chemoprophylactic antibiotic for Haemophilus influenzae infection, and the resistance rate in H. influenzae is low. In this study, we assessed rifampin resistance-related genetic variations in H. influenzae. Rifampin susceptibility testing and whole-genome sequencing were performed in 51 H. influenzae isolates. Variations associated with rifampin resistance were identified using Fisher's exact tests. Functional assays were performed to evaluate the effect of RpoB substitutions on rifampin susceptibility. Using the genome of the Rd KW20 H. influenzae strain as the reference, we detected 40 genetic variations in rpoB, which resulted in 39 deduced amino acid substitutions among the isolates. Isolate A0586 was resistant to rifampin, with a minimum inhibitory concentration (MIC) = 8μg/mL. Phylogenetic analyses revealed that the RpoB sequence of isolate A0586 was distinct from other isolates. Five substitutions, including H526N located in cluster I and L623F, R628C, L645F, and L672F in the region between clusters II and III, were unique to isolate A0586. In two rifampin-susceptible H. influenzae isolates, RpoB-H526N alone and in combination with RpoB-L672F increased the MICs of rifampin to 4 and 8μg/mL, respectively. RpoB-L672F did not affect cell growth and transcription in H. influenzae isolates. No amino acid substitutions in the AcrAB-TolC efflux pump or outer membrane proteins were found to be associated with rifampin resistance in H. influenzae. Our findings indicate that L672F substitution in the region between RpoB clusters II and III has an aggravating effect on rifampin resistance in H. influenzae.