Azithromycin resistance through interspecific acquisition of an epistasis dependent efflux pump component and transcriptional regulator in Neisseria gonorrhoeae

ABSTRACT Mosaic interspecifically acquired alleles of the multiple transferable resistance (mtr) efflux pump operon correlate with reduced susceptibility to azithromycin in Neisseria gonorrhoeae 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 mtr alleles and azithromycin resistance. We find that the locus encompassing the mtrR transcriptional repressor and the mtrCDE pump is a hotspot of interspecific recombination introducing alleles from N. meningitidis and N. lactamica into N. gonorrhoeae, with multiple rare haplotypes in linkage disequilibrium at mtrD and the mtr promoter region. Transformations demonstrated that resistance is mediated through epistasis between these two loci and that the full length of the mosaic mtrD allele is required. Gene expression profiling revealed the mechanism of resistance in mosaics couples the novel mtrDalleles with promoter mutations enhancing expression of the pump. Overall, our results demonstrate that epistatic interactions at mtr gained from multiple Neisseria has contributed to azithromycin resistance in the gonococcal population. AUTHOR SUMMARYNeisseria gonorrhoeae is the sexually transmitted bacterial pathogen responsible for over 100 million cases of gonorrhea worldwide each year. The incidence of reduced susceptibility to the macrolide class antibiotic azithromycin has increased in the past decade; however, a large proportion of the genetic basis of resistance to this drug remains unexplained. Recently, resistance has been shown to be highly associated with mosaic alleles of the multiple transferable resistance (mtr) efflux pump, which have been gained via horizontal gene exchange with other Neisseria. However, if and how these alleles caused resistance was unknown. Here, we demonstrate that resistance has been gained through epistasis between mtrD and the mtr promoter region using evidence from both population genomics and experimental genetic manipulation. Epistasis also acts within the mtrD locus alone, requiring the full length of the gene for phenotypic resistance. Transcriptomic profiling indicates that the mechanism of resistance in mosaics is likely derived from both structural changes to mtrD, coupled with promoter mutations that result in regulatory changes to mtrCDE.