Evolutionary paths to macrolide resistance in a Neisseria commensal converge on ribosomal genes through short sequence duplications

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 that reduce susceptibility to azithromycin converge on the locus encoding the 50S ribosomal L34 protein (rpmH) and the intergenic region proximal to the 30S ribosomal S3 protein (rpsC) through short tandem duplication events. Interestingly, one of the laboratory evolved mutations in rpmH is identical (7LKRTYQ12), and two nearly identical, to those recently reported to contribute to high-level azithromycin resistance 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. Ultimately, studies like this will be critical for predicting commensal alleles that could rapidly disseminate into pathogen populations via allelic exchange across recombinogenic microbial genera.

Tolerance to Ceftriaxone in Neisseria gonorrhoeae: Rapid Induction in WHO P Reference Strain and Detection in Clinical Isolates

“…We used a protocol based on the work of Raisman et al 2022. In their study, they were able to induce resistance to azithromycin in N. elongata by performing crossover E-tests [ 40 ]. In contrast, Levin-Reisman et al 2017 induced AMR via continuing the cyclical intermittent antibiotic exposure set-up for up to 17 cycles [ 8 , 40 ].…”

“…In their study, they were able to induce resistance to azithromycin in N. elongata by performing crossover E-tests [ 40 ]. In contrast, Levin-Reisman et al 2017 induced AMR via continuing the cyclical intermittent antibiotic exposure set-up for up to 17 cycles [ 8 , 40 ]. An additional important consideration is that gonococcal resistance to CRO is more difficult to induce in vitro compared to AZM and ampicillin [ 41 , 42 , 43 ].…”

“…To analyse whether tolerance accelerates the development of CRO resistance, selective pressure was applied on tolerant colonies obtained through cyclic CRO exposure of WHO P reference strain (n = 13) and non-tolerant sensitive strains (n = 4), in parallel, using a modified cross-plating protocol of Raisman et al 2022 [ 40 ].…”

Canary in the Coal Mine: How Resistance Surveillance in Commensals Could Help Curb the Spread of AMR in Pathogenic Neisseria

“…Additionally, in vitro evolution experiments can reveal the spontaneous mutations caused by DNA replication and repair errors which increase mean fitness in new selective environments ( 148 ), and are quick and easy to implement in the research laboratory due to the short generation times of Neisseria (~60 min). Using this approach, multiple tandem duplications in the locus encoding the 50S ribosomal L34 protein ( rpmH ) and the intergenic region proximal to the 30S ribosomal S3 protein ( rpsC ) were identified to increase resistance (≥2 μg/mL) to the macrolide antibiotic azithromycin in the commensal N. elongata in 20 days (or approximately 480 generations) ( 149 ). A similar experiment also described an identical 7LKRTYQ12 sequence duplication in rpmH as a transient steppingstone to high level azithromycin resistance in N. gonorrhoeae ( 150 ).…”

Pre-exposure to azithromycin enhances gonococcal resilience to subsequent ciprofloxacin exposure: an in vitro study

“…Recent studies have suggested that azithromycin may promote antimicrobial resistance to other classes of antimicrobials via inducing mutations that act as stepping-stones to antimicrobial resistance. [4][5][6] In vitro, culture experiments with Mycobacterium smegmatis have found that antimicrobial-induced mutations in ribosomal proteins reduce susceptibility to various antimicrobials in a stepping-stone manner. 4 Ciprofloxacin, for example, first selects for mutations in four ribosomal proteins.…”