Re-visiting the potential impact of doxycycline post-exposure prophylaxis (doxy-PEP) on the selection of doxycycline resistance in commensals.

Doxycycline post-exposure prophylaxis (doxy-PEP) is a preventative strategy demonstrated to reduce bacterial sexually transmitted infections in high-risk populations. However, the impact of doxy-PEP on antibiotic resistance acquisition in key members of our microbiomes, is as of yet unclear. For example, commensal are known reservoirs of resistance for gonococci through horizontal gene transfer (HGT), and are more likely to experience bystander selection due to doxy-PEP as they are universally carried. Thus, the consequences of doxycycline selection on commensal will be critical to investigate to understand possible resistance mechanisms that may be transferred to an important human pathogen. Here, we use antibiotic gradients to evolve four commensals ( n=4 per species) across a 20-day time course; and use whole genome sequencing to nominate derived mutations. After selection, 12 of 16 replicates evolved doxycycline resistance (> 1 μg/mL). Across resistant lineages: An A46T substitution in the repressor of the Mtr efflux pump (MtrR) and a V57M substitution in the 30 ribosomal protein S10 were clearly associated with elevated MICs. Additional mutations in ribosomal components also emerged in strains with high MICs (i.e., , RplX A14T). We find the MtrR 46T, RpsJ 57M, and RplX 14T circulating in natural commensal populations. Furthermore, co-evolution of with commensals demonstrated rapid transfer of the pConj plasmid to and , and p to . Finally, collection of novel commensals from human hosts reveals 46% of isolates carrying 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 carriage of the ribosomal protection protein ( and pConj. 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 , and the likelihood of transferring particular genotypes across species' boundaries.