Increased power from bacterial genome-wide association conditional on known effects identifiesNeisseria gonorrhoeaemacrolide resistance mutations in the 50S ribosomal protein L4

Abstract

19The emergence of resistance to azithromycin complicates treatment of N. gonorrhoeae, the 20 etiologic agent of gonorrhea. Population genomic analyses of clinical isolates have 21 demonstrated that some azithromycin resistance remains unexplained after accounting for the 22 contributions of known resistance mutations in the 23S rRNA and the MtrCDE efflux pump. 23Bacterial genome-wide association studies (GWAS) offer a promising approach for identifying 24 novel resistance genes but must adequately address the challenge of controlling for genetic 25 confounders while maintaining power to detect variants with lower effect sizes. Compared to a 26 standard univariate GWAS, conducting GWAS conditioned on known resistance mutations with 27 high effect sizes substantially reduced the number of variants that reached genome-wide 28 significance and identified a G70D mutation in the 50S ribosomal protein L4 (encoded by the 29 gene rplD) as significantly associated with increased azithromycin minimum inhibitory 30 concentrations (β = 1.03, 95% CI [0.76, 1.30]). The role and prevalence of these rplD mutations 31 in conferring macrolide resistance in N. gonorrhoeae had been unclear. Here, we experimentally 32 confirmed our GWAS results, identified other resistance-associated mutations in RplD, and 33 showed that in total these RplD binding site mutations are prevalent (present in 5.42% of 4850 34 isolates) and geographically and temporally widespread (identified in 21/65 countries across two 35 decades). Overall, our findings demonstrate the utility of conditional associations for improving 36 the performance of microbial GWAS and advance our understanding of the genetic basis of 37 macrolide resistance in a prevalent multidrug-resistant pathogen.

Molecular pathways to high-level azithromycin resistance in Neisseria gonorrhoeae

“…However, a recent bacterial genome-wide association study identified the G70D mutation in the 50S ribosomal protein L4 as being significantly associated with increased azithromycin MICs in clinical isolates of N. gonorrhoeae. 32 The rplD G70D mutation was present in 231 out of 4850 isolates (4.76%) and an additional 34 isolates contained other mutations at amino acid positions 68, 69 and 70. These other putative L4 binding site mutations were associated with significantly higher azithromycin MICs compared to both L4 G70D and L4 wild-type strains.…”

Efflux Pump Antibiotic Binding Site Mutations Are Associated with Azithromycin Nonsusceptibility in Clinical Neisseria gonorrhoeae Isolates

“…The isolate with an MtrD R714H mutation and the lowest observed azithromycin MIC of 0.19 μg/ml did not have an mtrR promoter mutation, consistent with epistasis across the mtrRCDE operon ( 4 ). Contributions from ribosomal mutations can also synergistically increase macrolide resistance: the isolate with an MtrD K823E substitution and the highest observed azithromycin MIC of 8.0 μg/ml contained an RplD G70S mutation previously implicated in macrolide resistance ( 5 ). Seven MtrD isolates also had mosaic penA XXXIV alleles conferring cephalosporin reduced susceptibility, indicating a potential route to dual therapy resistance.…”

Adaptation to the cervical environment is associated with increased antibiotic susceptibility in Neisseria gonorrhoeae

“…MICs were log 2transformed for use as continuous outcome variables in linear modeling and GWAS. We modeled transformed MICs using a panel of known resistance markers 11,12 and included the recently characterized mosaic mtrCDE alleles 5 and rplD G70D substitution 78 To visualize the continuous MICs using thresholds as on Fig. 1, we binarized MICs using the CLSI resistance breakpoint for ciprofloxacin, the CLSI nonsusceptibility breakpoint for azithromycin, and the CDC GISP surveillance breakpoint for ceftriaxone.…”