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.

Keywords: Neisseria; commensals; horizontal gene transfer; in vitro evolution.

Figure 1.

In vitro selection of four commensal Neisseria species with ciprofloxacin. In brief, four experimental replicates of each species were passaged for 20 days on selective gradients created with Etest strips. Cells for each passage were selected by sweeping the zone of inhibition (ZOI) and a 1 cm band in the bacterial lawn surrounding the ZOI. MIC trajectories are shown for (A) N. cinerea, (B) N. elongata, (C) N. canis, and (D) N. subflava. Expanded scales are shown in panels E-G for N. cinerea, N. elongata, and N. canis respectively. (H) CDs identity of in vitro derived mutations for drug-selected lineages. The percentage of mutational hits within a given gene is displayed as a heatmap, with brighter blue coloration indicating more frequent occurrence of a mutation within a CDS in replicate evolved lineages for each species.

Figure 2.

(A) UPGMA phylogeny of gyrA of 2235 PubMLST Neisseria isolates. The ciprofloxacin reduced susceptibility-associated mutation GyrA 91I circulates within commensal Neisseria populations, however has not been transferred to Neisseria gonorrhoeae. The coloring in the inner ring represents the Neisseria species, followed by amino acid identity at GyrA positions 89, 91, and 95 respectively. The derived 91I mutation was found in N. lactamica, N. subflava, and N. polysaccharea (n=70 or ~ 3% of isolates). (B) UPGMA phylogeny of the gyrB locus for 2302 PubMLST Neisseria isolates. GyrB does not show variation in the zoliflodacin reduced susceptibility mutation (D429V) or derived P739H mutation in commensal populations. The coloring in the inner ring represents the Neisseria species, followed by amino acid identity at GyrB positions 429 and 739 respectively.

Figure 3.

(A) UPGMA phylogeny of the parC locus for 2256 PubMLST Neisseria isolates. ParC 86N, 87R/N, and 91K mutations associated with ciprofloxacin resistance in gonococci were limited to the gonococcal clade and were not present in the commensal Neisseria; however, a single gonococcal isolate inherited the experimentally-derived ParC G85C substitution that emerged in N. canis post-selection. The coloring in the inner ring represents the Neisseria species, followed by amino acid identity at ParC positions 86, 87, and 91 and respectively. The subpanel highlights 3 gonococcal isolates with commensal-like parC alleles. (B) UPGMA phylogeny of the parE locus for 2287 PubMLST Neisseria isolates. The coloring in the inner ring represents the Neisseria species, followed by amino acid identity at ParE positions 320 and 410 respectively. The subpanel highlights 4 gonococcal isolates with commensal-like parE alleles.