In vitro evolution of ciprofloxacin resistance in Neisseria commensals and derived mutation population dynamics in natural Neisseria populations.

Abstract

Commensal Neisseria are members of a healthy human oropharyngeal microbiome; however, they also serve as a reservoir of antimicrobial resistance for their pathogenic relatives. Despite their known importance as sources of novel genetic variation for pathogens, we still do not understand the full suite of resistance mutations commensal species can harbor. Here, we use in vitro selection to assess the mutations that emerge in response to ciprofloxacin selection in commensal Neisseria by passaging 4 replicates of 4 different species in the presence of a selective antibiotic gradient for 20 days; then categorized derived mutations with whole genome sequencing. 10/16 selected cells lines across the 4 species evolved ciprofloxacin resistance (≥ 1 ug/ml); with resistance-contributing mutations primarily emerging in DNA gyrase subunit A and B (gyrA and gyrB), topoisomerase IV subunits C and E (parC and parE), and the multiple transferable efflux pump repressor (mtrR). Of note, these derived mutations appeared in the same loci responsible for ciprofloxacin reduced susceptibility in the pathogenic Neisseria, suggesting conserved mechanisms of resistance across the genus. Additionally, we tested for zoliflodacin cross-resistance in evolved strain lines and found 6 lineages with elevated zoliflodacin minimum inhibitory concentrations. Finally, to interrogate the likelihood of experimentally derived mutations emerging and contributing to resistance in natural Neisseria, we used a population-based approach and identified GyrA 91I as a substitution circulating within commensal Neisseria populations and ParC 85C in a single gonococcal isolate. Small clusters of gonococcal isolates had commensal-like alleles at parC and parE, indicating recent cross-species recombination events.