Rolling the evolutionary dice: Neisseria commensals as proxies for elucidating the underpinnings of antibiotic resistance mechanisms and evolution in human pathogens

Abstract

Species within the genus Neisseria are adept at sharing adaptive allelic variation, with commensal species repeatedly transferring resistance to their pathogenic relative Neisseria gonorrhoeae . However, resistance in commensals is infrequently characterized, limiting our ability to predict novel and potentially transferable resistance mechanisms that ultimately may become important clinically. Unique evolutionary starting places of each Neisseria species will have distinct genomic backgrounds, which may ultimately control the fate of evolving populations in response to selection as epistatic and additive interactions coerce lineages along divergent evolutionary trajectories. Alternatively, similar genetic content present across species due to shared ancestry may constrain existing adaptive solutions. Thus, identifying the paths to resistance across commensals may aid in characterizing the Neisseria resistome—or the reservoir of alleles within the genus as well as its depth. Here, we use in vitro evolution of four commensal species to investigate the potential and repeatability of resistance evolution to two antimicrobials, the macrolide azithromycin and the β-lactam penicillin. After 20 days of selection, commensals evolved resistance to penicillin and azithromycin in 11/16 and 12/16 cases, respectively. Almost all cases of resistance emergence converged on mutations within ribosomal components or the mtrRCDE efflux pump for azithromycin-based selection and mtrRCDE , penA , and rpoB for penicillin selection, thus supporting constrained adaptive solutions despite divergent evolutionary starting points across the genus for these particular drugs. Though drug-selected loci were limited, we do identify novel resistance-imparting mutations. Continuing to explore paths to resistance across different experimental conditions and genomic backgrounds, which could shunt evolution down alternative evolutionary trajectories, will ultimately flesh out the full Neisseria resistome. IMPORTANCE Neisseria gonorrhoeae is a global threat to public health due to its rapid acquisition of antibiotic resistance to all first-line treatments. Recent work has documented that alleles acquired from close commensal relatives have played a large role in the emergence of resistance to macrolides and beta-lactams within gonococcal populations. However, commensals have been relatively underexplored for the resistance genotypes they may harbor. This leaves a gap in our understanding of resistance that could be rapidly acquired by the gonococcus through a known highway of horizontal gene exchange. Here, we characterize resistance mechanisms that can emerge in commensal Neisseria populations via in vitro selection to multiple antimicrobials and begin to define the number of paths to resistance. This study, and other similar works, may ultimately aid both surveillance efforts and clinical diagnostic development by nominating novel and conserved resistance mechanisms that may be at risk of rapid dissemination to pathogen populations.

In vitroevolution of ciprofloxacin resistance inNeisseriacommensals and derived mutation population dynamics in naturalNeisseriapopulations

“…Thus, characterizing the mechanisms that impart ciprofloxacin resistance in Neisseria commensals using in vitro selection may highlight important mutations to consider for gonococcal surveillance programs and may also illuminate novel resistance-conferring mutations that can arise in commensal genomic backgrounds. Previous work using in vitro evolution has pointed to conserved mechanisms of resistance across the genus for azithromycin and penicillin-based selection 36,37,42 , here we expand our enquiry to ciprofloxacin for several Neissiera commensals, and interrogate both the identity and diversity of paths to resistance across different genomic backgrounds.…”

“…Reads shorter than 36 bp, and those missing a mate, were removed from subsequent analysis as well. The reference genomes were assembled and annotated previously 36,37,42 , and the read libraries were mapped back to these reference assemblies using Bowtie2 v.2.2.4 using the "end-to-end" and "verysensitive" options 45 . Ultimately, Pilon v.1.16 46 identified any derived mutations; and custom scripts were used to determine if these mutations were located in any previously annotated genomic features.…”

“…Selected lineages were whole genome sequenced (WGS) and aligned to the draft assemblies reported previously 36,37,42 to nominate derived polymorphisms. Mutations shared with control strains (with no drug selection) were omitted from subsequent analyses.…”

“…Here, we passage four species of commensals using a selective gradient of ciprofloxacin for 20 days, and characterize derived mutations postselection. As in our prior work 36,37 , we query the identity and number of paths to reduced susceptibility, and compare and contrast these paths across different commensal genomic backgrounds. We also test for the potential for the emergence of zoliflodacin cross-resistance.…”