Azithromycin Resistance through Interspecific Acquisition of an Epistasis-Dependent Efflux Pump Component and Transcriptional Regulator in Neisseria gonorrhoeae

Abstract

Mosaic interspecifically acquired alleles of the multiple transferable resistance (mtr) efflux pump operon correlate with increased resistance to azithromycin in Neisseria gonorrhoeae in epidemiological studies. However, whether and how these alleles cause resistance is unclear. Here, we use population genomics, transformations, and transcriptional analyses to dissect the relationship between variant mtr alleles and azithromycin resistance. We find that the locus encompassing the mtrR transcriptional repressor and the mtrCDE pump is a hot spot of interspecific recombination introducing alleles from Neisseria meningitidis and Neisseria lactamica into N. gonorrhoeae, with multiple rare haplotypes in linkage disequilibrium at mtrD and the mtr promoter region. Transformations demonstrate that resistance to azithromycin, as well as to other antimicrobial compounds such as polymyxin B and crystal violet, is mediated through epistasis between these two loci and that the full-length mosaic mtrD allele is required. Gene expression profiling reveals the mechanism of resistance in mosaics couples novel mtrD alleles with promoter mutations that increase expression of the pump. Overall, our results demonstrate that epistatic interactions at mtr gained from multiple neisserial species has contributed to increased gonococcal resistance to diverse antimicrobial agents.IMPORTANCENeisseria gonorrhoeae is the sexually transmitted bacterial pathogen responsible for more than 100 million cases of gonorrhea worldwide each year. The incidence of resistance to the macrolide azithromycin has increased in the past decade; however, a large proportion of the genetic basis of resistance remains unexplained. This study is the first to conclusively demonstrate the acquisition of macrolide resistance through mtr alleles from other Neisseria species, demonstrating that commensal Neisseria bacteria are a reservoir for antibiotic resistance to macrolides, extending the role of interspecies mosaicism in resistance beyond what has been previously described for cephalosporins. Ultimately, our results emphasize that future fine-mapping of genome-wide interspecies mosaicism may be valuable in understanding the pathways to antimicrobial resistance. Our results also have implications for diagnostics and public health surveillance and control, as they can be used to inform the development of sequence-based tools to monitor and control the spread of antibiotic-resistant gonorrhea.

Keywords: Neisseria gonorrhoeae; antibiotic resistance; efflux pump; epistasis; gonorrhea; macrolide.

FIG 1

Horizontal gene transfer (HGT) of mtr introduces novel adaptive genetic variation into Neisseria gonorrhoeae. (A) The FA1090 genomic position of the mtrRCDE locus is presented, with the locations of DNA uptake sequences (DUS) displayed (black circles). These DUS sequences are shared with closely related Neisseria species and increase the frequency of DNA uptake and recombination (58). The U.S. gonococcal population (n = 1,102 isolates) shows patterns of elevated allelic diversity as measured by π and elevated SNP densities as predicted by Gubbins (52) across mtrRCDE. Depressed values of Tajima’s D (Tajima’s D < 0) indicate an excess frequency of rare mutations across this region. The average values for these parameters are plotted for mtrD (blue dashed lines) and the genome-wide average (gray dashed lines). (B) The highest allelic diversity within mtrRCDE was found at mtrD and (C) an excess of rare alleles was detected across mtrRCDE (Tajima’s D < 0) compared to the rest of the genome. The values that are significantly different are indicated by a bar and asterisks as follows: *, P less than or equal to 0.05; **, P less than or equal to 0.001; ***, P less than or equal to 0.0001. Values that are not significantly different (N.S.) are indicated. mtrp, mtr promoter. (D) Depressed genealogical sorting index (gsi) values compared to those of a 25-kb mtr flanking region indicate extensive admixture of alleles between N. gonorrhoeae and other Neisseria across all mtr loci. Signatures of high allelic diversity, an excess of rare mutations, and interspecies admixture taken altogether suggest the recent introduction of novel genetic variation into this region. (E) Linkage disequilibrium was also observed, with the highest linkage measured by r² occurring within mtrD and the mtr promoter regions. (F) r² values plotted across mtrCDE in reference to their position in strain FA1090 are represented from 0 (yellow for low linkage) to 1 (blue for high linkage). Positions of the Correia element insertion (CE), A-to-C transversion in the mtrR promoter inverted repeat (C), and glycine to aspartic acid MtrR amino acid substitution (G45D) are indicated. Large tracts of high linkage suggest recent recombination of alleles from another source or maintenance of particular haplotypes due to selection. LD, linkage disequilibrium.

FIG 2

Divergent mtrD and mtr promoter haplotypes are associated with increases in azithromycin MIC. (A) A maximum likelihood whole-genome-sequence phylogeny of 1,102 Neisseria gonorrhoeae isolates based on single-nucleotide polymorphisms generated from mapping to the FA1090 reference genome (8). The inner annotation ring shows azithromycin MICs on a continuous scale (red shading). The next annotation ring indicates isolates with at least two copies of the C2611T 23S rRNA mutation (dark blue) or isolates with four copies of the A2059G 23S rRNA mutation (magenta). The next annotation ring shows isolates that were identified as interspecies mosaics based on their sequence at mtrR by Grad et al. (8) (dark green). The outermost annotation ring shows isolates identified as mtrD mosaics in this study (teal). Tree scales or bars, 0.01 or 0.1 nucleotide substitutions per position. (B) The entire gonococcal population has a distribution of azithromycin MIC values which fall both above and below the defined resistance threshold (MIC ≥ 1 µg/ml). (C) A maximum likelihood phylogeny built on mtrD alignments shows 109 isolates with full-length mosaic alleles that are highly divergent from the wild-type gonococcal mtrD allele. (D) These 109 isolates are associated with elevated azithromycin MICs (MICs ≥ 0.25 µg/ml). (E) A maximum likelihood phylogeny built on the mtr promoter region shows that all 29 of the mtr promoter mosaics previously described by Grad et al. (8) also have inherited mosaic mtrD. (F) Isolates with both full-length mosaic mtrD and a mosaic mtr promoter region alleles have higher azithromycin MICs (MICs ≥ 1 µg/ml) than those isolates with mosaic mtrD alone.

FIG 3

Unguided genomic DNA transformations demonstrate that the mosaic mtrRCDE region causes increased azithromycin resistance. The 28Bl azithromycin-susceptible recipient strain was transformed with gDNA from mosaic mtr donor strains and selected on plates containing 0.38 to 1 µg/ml azithromycin. Genomic sequencing revealed the boundaries of recombined DNA that azithromycin-resistant transformants had inherited from mosaic donor strains (red bars) in the 28Bl background, as identified by SNP homology using the FA1090 reference genome as a scaffold. The only genomic region that was consistently inherited from donor strains in the transformant cell lines encompassed mtrRCDE for both the N. meningitidis (GCGS0276) and N. lactamica-like (GCGS0402 and GCGS0834) mtr mosaics. Average in vitro recombination tract lengths were 9.5 ± 1.4 kb for strain GCGS0276 (n = 4), 24.3 ± 3.6 kb for GCGS0834 (n = 9), and 31.4 ± 6.0 kb for GCGS0402 (n = 8). The locations of all DNA uptake sequences (DUS) (black circles), as well as the mtr locus (white box), are mapped in reference to the FA1090 strain.

FIG 4

Epistatic interactions between multiple domains of mtrD contribute to elevated azithromycin MICs. (A) GCGS0276 mtrD in the 28Bl background elevated the azithromycin MIC from 0.125 µg/ml to 0.5 µg/ml (red). (B) Primer pairs designed to amplify ~300-bp fragments over the length of mtrD (see Table S4) resulted in no observed transformants on 0.38-µg/ml azithromycin selection plates, suggesting that multiple mutations across mtrD are needed for resistance. (C and D) To determine the regions that contributed to resistance, multiple fragment sizes were constructed by holding the rightmost forward primer (black) constant while adding different reverse primers (gray) (C) and by holding the leftmost reverse primer (black) constant while adding forward primers (gray) to separate reactions (D). The red lines in panels C and D indicate the PCR products that generated transformants on azithromycin selection plates. (E) At a minimum, SNPs at base pair positions 18 to 356 coupled with SNPs at positions 2356 to 2724 (indicated by yellow background and an asterisk) were required to raise the MIC from the 0.125 µg/ml of the recipient 28Bl strain to 0.5 µg/ml, though we are unable to exclude the possibility that additional SNPs between these two regions are also involved.

FIG 5

Alignment of wild-type and mosaic Neisseria gonorrhoeae MtrD with N. lactamica and N. meningitidis reference sequences. Amino acid alignment of MtrD sequences from N. gonorrhoeae FA1090 (AE004969.1), N. lactamica 020-06 (FN995097.1), and N. meningitidis NZ-05/33 (CP002424.1) with those of the gonococcal isolates used in this study are shown. The minor variants for each amino acid position are shown in red on a pink background. Though most of the amino acid changes in isolates with mosaic MtrDs are homologous to those found in either the chosen N. lactamica and N. meningitidis references, some amino acid changes are unique and thus have been either horizontally acquired from another source or represent de novo mutations. Overlaid on the alignment are the secondary structure elements of the protein: β pleated sheets (blue arrows); α-helices (red spirals); and transmembrane helices (TM) (green boxes) (40). Domains of MtrD are annotated and include the MtrE docking domain (DN and DC) and the MtrD pore domain (PN1, PN2, PC1, and PC2) (40). Epistatically interacting domains of MtrD that enhanced azithromycin MIC in mosaic isolates as deduced from transformation experiments are shown on yellow background.

FIG 6

Epistasis between mtrD and the mtr promoter region yields increased azithromycin resistance in mosaic isolates. (A) While transformation of the entire GCGS0276 mtrRCDE region perfectly reconstructed the donor strain MIC of 1 µg/ml in the 28Bl background (yellow line), GCGS0276 mtrD was the only region within mtrRCDE that could independently increase resistance to azithromycin (from 0.125 to 0.5 µg/ml [red line]). This suggested that epistasis across loci contributed to resistance in mtr mosaics. (B) To determine the additional locus needed to produce the high-level resistance seen in mtr mosaics, PCR products were designed to amplify mtrD in addition to other loci within mtrRCDE. Here, we found that the addition of the mtr promoter to constructs containing mtrD was sufficient to raise the MIC of 28Bl to the GCGS0276 phenotype of 1 µg/ml.

FIG 7

Alignment of wild-type and mosaic Neisseria gonorrhoeae mtr promoters with N. lactamica and N. meningitidis references. Nucleotide alignment of the mtr promoter region from N. gonorrhoeae FA1090 (AE004969.1), N. lactamica 020-06 (FN995097.1), and N. meningitidis NZ-05/33 (CP002424.1) with the gonococcal isolates used in this study. The minor allele variants for each position are shown in red on a pink background. Sequence features include the interleaved mtrCDE and mtrR promoters (yellow background), MtrR binding site (maroon bars), inverted repeat in the mtrR promoter (black bar), 153-bp Correia element insertion (red arrows), and integration host factor (IHF) binding site (blue bar) (21, 29, 30, 33). The C-to-T transition 120 bp upstream of the mtrC start codon (mtr₁₂₀ [purple background]) has been demonstrated to increase expression of the efflux pump components and enhance resistance to various substrates of the pump (29); however, no T variants were detected at this site. Deletion of a single A (A deletion) in the mtrR promoter inverted repeated (blue background) has also been shown to increase expression of the efflux pump and enhance mtr-mediated resistance (30); no A-deletion variants were present. Interestingly, the N. lactamica reference, N. meningitidis reference, and all 29 isolates with mosaic mtr promoter regions had a C transversion in the second position within this repeat (blue background).

FIG 8

The N. meningitidis-like GCGS0276 mosaic mtr promoter sequence upregulates expression of mtr efflux pump component mRNAs. The 28Bl recipient strain (green), 28Bl transformants with mosaic GCGS0276 mtrD (blue), and 28Bl transformants with mosaic GCGS0276 mtrRCDE (teal) were exposed to sub-MIC (0.125 µg/ml) concentrations of azithromycin (AZI) for 120 min and subjected to whole-transcriptome sequencing before and after the addition of the antimicrobial. The 28BlΔGCGS0276-mtrD strain was then used as a reference to calculate the relative expression of transcripts for other strains by condition by gene. In both the presence and absence of drug, transformants with the presence of a mosaic mtr promoter region yielded significantly upregulated pump component mRNAs (false-discovery rate [FDR] of <0.0001), while transformants that inherited mosaic mtrD alleles did not significantly alter mtrCDE regulation from the 28Bl recipient strain levels. Values that are significantly different are indicated by asterisks as follows: *, P less than or equal to 0.05; *** P less than or equal to 0.0001.