Constrained evolutionary paths to macrolide resistance in aNeisseriacommensal converge on ribosomal genes through sequence duplication

Abstract

Neisseria commensals are an indisputable source of resistance for their pathogenic relatives; however, the evolutionary paths commensal species take to reduced susceptibility in this genus have been relatively underexplored. Here, we leverage in vitro selection as a powerful screen to identify the genetic adaptations that produce azithromycin resistance (≤ 2 μg/mL) in the Neisseria commensal, N. elongata. Across multiple lineages (n=7/16), we find mutations encoding resistance converge on the gene encoding the 50S ribosomal L34 protein (rpmH) and the intergenic region proximal to the 30S ribosomal S3 protein (rpsC) through duplication events. Importantly, one of the laboratory evolved mutations in rpmH is identical, and two nearly identical, to those recently reported to confer high-level resistance to azithromycin in N. gonorrhoeae. Transformations into the ancestral N. elongata lineage confirmed the causality of both rpmH and rpsC mutations. Though most lineages inheriting duplications suffered in vitro fitness costs, one variant showed no growth defect, suggesting the possibility that it may be sustained in natural populations. Finally, we assessed the potential of horizontal transfer of derived resistance mutations into multiple strains of N. gonorrhoeae. Though we were unable to transform N. gonorrhoeae in this case, studies like this will be critical for predicting commensal alleles that are at risk of rapid dissemination into pathogen populations.

Doxycycline PEP can induce doxycycline resistance in Klebsiella pneumoniae in a Galleria mellonella model of PEP

“…After overnight incubation at 36°C at 5 (v/v)% CO2, the MIC was noted. A standard loopful of culture (5 mm) was taken from the margin of growth from the most resistant colonies, following the protocol of Wadsworth et al (Balduck et al, 2022;Raisman et al, 2022). This growth was then suspended in PBS and re-inoculated on a fresh BD BBLTM Chocolate II agar plate (GC Choc) and a new doxycycline E-test was placed.…”