Dual roles of genes required for intrinsic resistance to clarithromycin in evasion of killing by serum complement in Haemophilus influenzae.

Macrolide antibiotics are commonly prescribed to treat Haemophilus influenzae respiratory tract infections. Studies have primarily focused on emerging H. influenzae strains with acquired macrolide resistance, while the bacterium's intrinsic resistance to antibiotics has been underexamined. Here, we used a genome-wide approach of transposon insertion-site sequencing to screen an H. influenzae mutant library grown in sub-inhibitory doses of the macrolide antibiotic clarithromycin (CLR) to identify 33 genes involved in intrinsic CLR resistance. Almost half of these genes are also needed for survival in the mouse lung. We focused on candidate genes necessary for both intrinsic macrolide resistance and lung survival. Two of these genes affect the outer-membrane composition of H. influenzae, orfH and omp26. Deletions of these genes in Rd and nontypeable H. influenzae clinical isolates, Hi375 and NT127, conferred sensitivity to CLR and polymyxin B and increased membrane permeability to ethidium bromide (EtBr). The omp26 mutant was sensitive to killing by human serum. Deletions of orfH or omp26 in an acrR mutant strain overexpressing a multidrug efflux pump abrogated resistance of the acrR mutant to CLR and restored permeability to EtBr. Thus, deletion of these genes not only mitigates the effects of an acquired resistance mechanism but also remarkably overrides it. Complementation of these deletion mutations restored CLR resistance and decreased permeability to EtBr. Our results indicate that the subset of genes with dual roles in intrinsic resistance and host lung survival may provide potential novel combination antimicrobial therapeutic targets.