Increased power from conditional bacterial genome-wide association identifies macrolide resistance mutations in Neisseria gonorrhoeae

Abstract

The emergence of resistance to azithromycin complicates treatment of Neisseria gonorrhoeae, the etiologic agent of gonorrhea. Substantial azithromycin resistance remains unexplained after accounting for known resistance mutations. Bacterial genome-wide association studies (GWAS) can identify novel resistance genes but must control for genetic confounders while maintaining power. Here, we show that compared to single-locus GWAS, conducting GWAS conditioned on known resistance mutations reduces the number of false positives and identifies a G70D mutation in the RplD 50S ribosomal protein L4 as significantly associated with increased azithromycin resistance (p-value = 1.08 × 10−11). We experimentally confirm our GWAS results and demonstrate that RplD G70D and other macrolide binding site mutations are prevalent (present in 5.42% of 4850 isolates) and widespread (identified in 21/65 countries across two decades). Overall, our findings demonstrate the utility of conditional associations for improving the performance of microbial GWAS and advance our understanding of the genetic basis of macrolide resistance.

Emergence of a Neisseria gonorrhoeae clone with reduced cephalosporin susceptibility between 2014 and 2019 in Amsterdam, The Netherlands, revealed by genomic population analysis

“…Annotation of resistance genes ( penA, porB, ponA, gyrA, parC , 23S rRNA, mtrA/R/C/D/E, rplD, rplV, rpmH, rpoB, rpoD ) was done using either the allele annotation available in the PubMLST database or by manually aligning allele sequences from PubMLST and identifying previously reported resistance mutations or mosaicism. 9 , 26–29 Raw reads were mapped against all 23S rRNA reference sequences in the PubMLST database to identify heterogeneous A2058/2059G/C2611T mutations in the four different alleles using Ariba v2.14.4. 30 Snakemake v5.6.0 was used for workflow management.…”

A community-driven resource for genomic epidemiology and antimicrobial resistance prediction of Neisseria gonorrhoeae at Pathogenwatch

“…Recently, epistatic interactions between a mosaic mtr promoter region and a mosaic mtrD gene have also been reported to increase the expression of this pump, contributing to macrolide resistance [23,24]. Mutations in rplD have also been associated with reduced susceptibility to this antibiotic [25], and contrarily, loss-offunction mutations in mtrC have been linked to increased susceptibility to several antibiotics including azithromycin [26]. Thus, we can relatively confidently predict decreased susceptibility or resistance to an antimicrobial using the current known genetic mechanisms; however, phenotypic testing is still necessary to detect resistant cases caused by unknown or novel mechanisms.…”

Evidence of Horizontal Gene Transfer of 50S Ribosomal Genes rplB, rplD, and rplY in Neisseria gonorrhoeae

“…The above well-characterized resistance-associated mutations (RAMs) are, however, unable to explain all the variation in gonococcal susceptibility to macrolides ( Ma et al, 2020 ). A point mutation in the 50S ribosomal protein, L4 (G70D), has been implicated in macrolide resistance in N. gonorrhoeae and other bacteria ( Gregory and Dahlberg, 1999 ; Tait-Kamradt et al, 2000 ; Ma et al, 2020 ). Recently, a genome-wide association study conditioned on the known resistance mechanisms, with experimental validation, confirmed that the G70D mutation resulted in reduced susceptibility to azithromycin.…”

“…Recently, a genome-wide association study conditioned on the known resistance mechanisms, with experimental validation, confirmed that the G70D mutation resulted in reduced susceptibility to azithromycin. In addition, the study identified other L4 mutations at amino acid positions 68 (G68D, G68C), 69 (T69I), and 70 (G70S, G70A, G70R, and G70duplication) associated with reduced azithromycin susceptibility ( Ma et al, 2020 ). These mutations are all situated at the end of the L4 loop close to the macrolide binding site ( Gregory and Dahlberg, 1999 ; Tait-Kamradt et al, 2000 ; Ma et al, 2020 ).…”