Azithromycin Resistance through Interspecific Acquisition of an Epistasis-Dependent Efflux Pump Component and Transcriptional Regulator in Neisseria gonorrhoeae

Mosaic interspecifically acquired alleles of the multiple transferable resistance ( ) efflux pump operon correlate with increased resistance to azithromycin in in epidemiological studies. However, whether and how these alleles cause resistance is unclear. Here, we use population genomics, transformations, and transcriptional analyses to dissect the relationship between variant alleles and azithromycin resistance. We find that the locus encompassing the transcriptional repressor and the pump is a hot spot of interspecific recombination introducing alleles from and into , with multiple rare haplotypes in linkage disequilibrium at and the promoter region. Transformations demonstrate that resistance to azithromycin, as well as to other antimicrobial compounds such as polymyxin B and crystal violet, is mediated through epistasis between these two loci and that the full-length mosaic allele is required. Gene expression profiling reveals the mechanism of resistance in mosaics couples novel alleles with promoter mutations that increase expression of the pump. Overall, our results demonstrate that epistatic interactions at gained from multiple neisserial species has contributed to increased gonococcal resistance to diverse antimicrobial agents. is the sexually transmitted bacterial pathogen responsible for more than 100 million cases of gonorrhea worldwide each year. The incidence of resistance to the macrolide azithromycin has increased in the past decade; however, a large proportion of the genetic basis of resistance remains unexplained. This study is the first to conclusively demonstrate the acquisition of macrolide resistance through alleles from other species, demonstrating that commensal bacteria are a reservoir for antibiotic resistance to macrolides, extending the role of interspecies mosaicism in resistance beyond what has been previously described for cephalosporins. Ultimately, our results emphasize that future fine-mapping of genome-wide interspecies mosaicism may be valuable in understanding the pathways to antimicrobial resistance. Our results also have implications for diagnostics and public health surveillance and control, as they can be used to inform the development of sequence-based tools to monitor and control the spread of antibiotic-resistant gonorrhea.