Introduction Reduced susceptibility to azithromycin (Azi^RS) is increasing in frequency in Neisseria gonorrhoeae. We have shown that non-gonococcal alleles at the mtr operon, encoding the MtrCDE efflux pump and the transcription repressor, are associated with Azi^RS and sought to define further their role in resistance.

Methods We transformed a susceptible strain (MIC <0.125 µg/mL) using gDNA from gonococcal donors carrying mosaic mtr loci and selected for Azi^RS (MIC ≥2 µg/mL) transformants. We sequenced the transformants’ genomes to define the transformed DNA, compared growth rates of parent and transformant strains, described the phylogenetic distribution of mtrR, mtrC, mtrD, and mtrE alleles across Neisserial species, and defined the sequence diversity and π at these loci.

Results Transformation studies confirm mosaic mtr alleles cause Azi^RS. Phylogenetic patterns support frequent Azi^RS-associated recombination of mtrR, mtrC, and mtrD between N. gonorrhoeae and other Neisseria. Conversely, 16 S and mtrE displayed almost exclusive species-based clade topology and no recombination events associated with Azi^RS. Within gonococcal populations, reduced nucleotide diversity at mtrE (n=1102, π=0.0054) compared to mtrRCD (π=0.014) suggests either equal rates of interspecific recombination across the mtr operon with increased levels of purifying selection acting to purge the introduction of novel alleles at mtrE, or a ‘hotspot’ of interspecific recombination at mtrRCD. Recombination of mosaic mtr alleles into novel gonococcal genomic backgrounds does not deleteriously affect growth rate in vitro, raising questions regarding the associated fitness cost.

Conclusion This work supports that Neisseria serve as a reservoir of gonococcal Azi^RS through interchange of mtr alleles. The genomic epidemiological evidence of multiple acquisitions of these alleles underscores the importance of screening for mtr mosaics to prevent outbreaks of Azi^RS.