Revisiting the potential impact of doxycycline post-exposure prophylaxis on the selection of doxycycline resistance in Neisseria commensals

Doxycycline post-exposure prophylaxis (doxy-PEP) is a strategy to reduce bacterial sexually transmitted infections. However, the impact of doxy-PEP on resistance emergence is as of yet unclear. Commensal Neisseria are known reservoirs of resistance for gonococci through horizontal gene transfer (HGT), and are more likely to experience bystander selection from doxy-PEP as they are universally carried. The consequences of doxycycline selection on commensal Neisseria will be critical to investigate to understand possible resistance mechanisms that may be transferred to an important human pathogen. Here, collection of commensals from human hosts demonstrated 46% of isolates carry doxycycline resistance; and doxycycline resistance was significantly greater in participants self-reporting doxycycline use in the past 6 months. High-level doxycycline resistance (> 8 µg/mL) was always associated with the ribosomal protection protein (tetM) and pConj. In vitro selection of Neisseria commensals (N. cinerea, N. canis, N. elongata, and N. subflava) resulted in 12 of 16 lineages evolving doxycycline resistance (> 1 µg/mL). An A46T substitution in the repressor of the Mtr efflux pump (MtrR) and a V57M substitution in the 30 ribosomal protein S10 were associated with elevated MICs. Mutations in ribosomal components also emerged (i.e., 16 S rRNA G1057C, RplX A14T). We find the MtrR 46T, RpsJ 57M, and RplX 14T in natural commensal populations. In vitro co-evolution of N. gonorrhoeae with Neisseria commensals demonstrated rapid transfer of the pConj plasmid to N. subflava and N. cinerea, and pbla to N. cinerea. This work underscores the importance of commensal Neisseria as reservoirs of doxycycline resistance, and demonstrates a link between doxycycline use and the emergence of resistance. Though novel chromosomal resistance mutations are nominated herein, resistance emergence in natural commensal populations appears to be mainly associated with acquisition of the tetM gene. A secondary danger to pConj acquisition, is spread of pbla and β-lactam resistance, which we demonstrate here in vitro. Ultimately, characterizing the contemporary prevalence of doxycycline resistance, and underlying resistance mechanisms, in commensal communities may help us to predict the long-term impact of doxy-PEP on Neisseria, and the likelihood of transferring resistance across species' boundaries.