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

Abstract

Neisseria gonorrhoeae is an urgent public health threat due to rapidly increasing incidence and antibiotic resistance. In contrast with the trend of increasing resistance, clinical isolates that have reverted to susceptibility regularly appear, prompting questions about which pressures compete with antibiotics to shape gonococcal evolution. Here, we used genome-wide association to identify loss-of-function (LOF) mutations in the efflux pump mtrCDE operon as a mechanism of increased antibiotic susceptibility and demonstrate that these mutations are overrepresented in cervical relative to urethral isolates. This enrichment holds true for LOF mutations in another efflux pump, farAB, and in urogenitally-adapted versus typical N. meningitidis, providing evidence for a model in which expression of these pumps in the female urogenital tract incurs a fitness cost for pathogenic Neisseria. Overall, our findings highlight the impact of integrating microbial population genomics with host metadata and demonstrate how host environmental pressures can lead to increased antibiotic susceptibility.

Fig. 1. Population structure and susceptibility profile of N. gonorrhoeae global meta-analysis collection.

A midpoint rooted recombination-corrected maximum likelihood phylogeny of 4852 genomes based on 68697 SNPs (Supplementary Table 1) was annotated with binarized resistance (ciprofloxacin) or decreased susceptibility (azithromycin, ceftriaxone) values. Annotation rings are listed in order of ciprofloxacin, ceftriaxone, and azithromycin from innermost to outermost. For ciprofloxacin, MIC < 1 μg/ml is light purple, and MIC ≥ 1 μg/ml is dark purple. For ceftriaxone, MIC < 0.125 μg/ml is light blue, and MIC ≥ 0.125 μg/ml is dark blue. For azithromycin, MIC ≤ 1 μg/ml is light pink, and MIC > 1 μg/ml is dark pink. Branch length represents total number of substitutions after removal of predicted recombination.

Fig. 2. GWAS identifies a variant mapping to mtrC associated with increased susceptibility.

The Manhattan plot shows negative log₁₀-transformed p-values (calculated using likelihood-ratio tests in the GWAS) for the association of unitigs with MICs to azithromycin (pink, n = 4505), ceftriaxone (blue, n = 4497), and ciprofloxacin (purple, n = 4135). The sign of the GWAS regression coefficient β (with positive indicating an association with increased resistance and negative indicating an association with increased susceptibility) is indicated by an X for β < 0 and a dot for β > 0. Labels indicate known influential resistance determinants, and the mtrC variant associated with increased susceptibility was highlighted in gray. A full list of the annotated significant unitigs for each antibiotic can be found in Supplementary Data 2. Inset: schematic of the mtr genetic regulon including structural genes mtrCDE, the activator mtrA, and the repressor mtrR. The approximate genomic location within mtrC and specific nucleotide change of the mtrC GWAS variant relative to the gonococcal NCCP11945 reference genome (i.e., a two base pair deletion in a ‘GC’ dinucleotide repeat) is shown.

Fig. 3. Gonococcal mtrC, mtrA, and farA LOF mutations are associated with cervical infection.

a Sexual behavior of patients infected with isolates with either intact or LOF alleles of mtrC (left), mtrA (middle), or farA (right). b Site of infection in patients infected with isolates with either intact or LOF alleles of mtrC (left), mtrA (middle), or farA (right). mtrA alleles were predicted as LOF only in the absence of other epistatic Mtr overexpression mutations. Statistical significance between genotype and patient metadata was assessed by two-sided Fisher’s exact test: *p < 0.05, **p < 0.01, and ***p < 0.001. Exact p-values from left to right for analyses in a were 0.04021, 1.81 × 10⁻¹¹, 5.06 × 10⁻¹⁰ and for b were 6.49 × 10⁻⁵, 1.64 × 10⁻¹², 1.78 × 10⁻¹². WSM = women who have sex with men, MSW = men who have sex with women, MSMW = men who have sex with men and women, MSM = men who have sex with men.

Fig. 4. mtrC LOF mutations are enriched in a lineage of ST-11 urogenitally-adapted N. meningitidis.

A core-genome maximum likelihood phylogeny based on 25045 SNPs was estimated of all North American ST-11 N. meningitidis strains from PubMLST (n = 456; accessed 2019–09–03) rooted with meningococcal reference genome MC58. Membership in the ST-11 urogenital clade (dark blue) is defined as in Retchless et al.. Genomes with mtrC LOF mutations are indicated in pink. Branch length represents substitutions per site.