Molecular pathways to high-level azithromycin resistance in Neisseria gonorrhoeae

Abstract

Background The prevalence of azithromycin resistance in Neisseria gonorrhoeae is increasing in numerous populations worldwide. Objectives To characterize the genetic pathways leading to high-level azithromycin resistance. Methods A customized morbidostat was used to subject two N. gonorrhoeae reference strains (WHO-F and WHO-X) to dynamically sustained azithromycin pressure. We tracked stepwise evolution of resistance by whole genome sequencing. Results Within 26 days, all cultures evolved high-level azithromycin resistance. Typically, the first step towards resistance was found in transitory mutations in genes rplD, rplV and rpmH (encoding the ribosomal proteins L4, L22 and L34 respectively), followed by mutations in the MtrCDE-encoded efflux pump and the 23S rRNA gene. Low- to high-level resistance was associated with mutations in the ribosomal proteins and MtrCDE efflux pump. However, high-level resistance was consistently associated with mutations in the 23S ribosomal RNA, mainly the well-known A2059G and C2611T mutations, but also at position A2058G. Conclusions This study enabled us to track previously reported mutations and identify novel mutations in ribosomal proteins (L4, L22 and L34) that may play a role in the genesis of azithromycin resistance in N. gonorrhoeae.

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

“… Evolved in-frame sequence duplications in Nel lineages introduce 8SVTKRKRT15, 7LKRTYQ12, and 8HIMKRTYQ15 insertions, which are identical to (7LKRTYQ12) and nearly identical to those recently discovered to confer high-level macrolide resistance in Ngo [ 32 ]. …”

“…The evolved L34 mutations uncovered within this study in Nel are identical or nearly identical to those recently reported to have emerged in an azithromycin-selection study in Ngo. The L34 duplications 7PSVTKRKR14, 7PSVTNTYQP14, and 7LKRTYQ12 were found to contribute to high levels of resistance to azithromycin in multiple Ngo strains [ 32 ]; and occurred in similar or the same locations as the Nel duplications 8SVTKRKRT15, 8HIMKRTYQ15, and 7LKRTYQ12 respectively ( Fig 3 ). Preserved identity and location of rpmH mutations across Neisseria species suggests that this is a conserved mechanism of macrolide resistance across the genus.…”

“…Laboratory evolution experiments reveal the spontaneous mutations caused by DNA replication and repair errors which increase mean fitness in new selective environments [ 30 ], and are ideal to elucidate the mechanisms underlying novel adaptations in bacteria due to their short generation times. In vitro selection has previously been used successfully to nominate the mutations underlying ceftriaxone and azithromycin reduced susceptibility in Ngo [ 31 , 32 ], and thus is a promising tool for the discovery of undescribed resistance mechanisms in Neisseria commensals.…”

Sub-Inhibitory Concentrations of Chlorhexidine Induce Resistance to Chlorhexidine and Decrease Antibiotic Susceptibility in Neisseria gonorrhoeae

“…The morbidostat was built following the detailed instructions by Toprak et al, modified for N. gonorrhoeae according to Toprak et al (2013), Verhoeven et al (2019). The experiment was carried out exactly as previously reported in the context of a N. gonorrhoeae morbidostat experiment selecting for azithromycin resistance in our laboratory (Laumen et al, 2021). In brief, N. gonorrhoeae cultures were put under sustained Listerine R (n = 6) and chlorhexidine pressure (n = 5), whereas two negative control cultures where exclusively exposed to gonococcal (GC) broth supplemented with 1% IsoVitaleX (BD BBLTM) and vancomycin, colistin, nystatin and trimethoprim selective supplement (VCNT), hereafter called GC medium.…”

Association between quinolone use in food animals and gonococcal resistance to ciprofloxacin: an ecological study

“…Growth rates were measured in standard 15ml vials in the NGmorbidostat whose construction and optimization for the culture of N. gonorrhoeae has been described elsewhere [33, 34]. The vials were continuously stirred at a speed of 200 rotations per minute (rpm) via a magnetic stirrer.…”