Global Emergence and Dissemination of Neisseria gonorrhoeae ST-9363 Isolates with Reduced Susceptibility to Azithromycin

Abstract

Neisseria gonorrhoeae multilocus sequence type (ST) 9363 core-genogroup isolates have been associated with reduced azithromycin susceptibility (AZMrs) and show evidence of clonal expansion in the United States. Here, we analyze a global collection of ST-9363 core-genogroup genomes to shed light on the emergence and dissemination of this strain. The global population structure of ST-9363 core-genogroup falls into three lineages: Basal, European, and North American; with 32 clades within all lineages. Although, ST-9363 core-genogroup is inferred to have originated from Asia in the mid-19th century; we estimate the three modern lineages emerged from Europe in the late 1970s to early 1980s. The European lineage appears to have emerged and expanded from around 1986 to 1998, spreading into North America and Oceania in the mid-2000s with multiple introductions, along with multiple secondary reintroductions into Europe. Our results suggest two separate acquisition events of mosaic mtrR and mtrR promoter alleles: first during 2009-2011 and again during the 2012-2013 time, facilitating the clonal expansion of this core-genogroup with AZMrs in the United States. By tracking phylodynamic evolutionary trajectories of clades that share distinct demography as well as population-based genomic statistics, we demonstrate how recombination and selective pressures in the mtrCDE efflux operon granted a fitness advantage to establish ST-9363 as a successful gonococcal lineage in the United States and elsewhere. Although it is difficult to pinpoint the exact timing and emergence of this young core-genogroup, it remains critically important to continue monitoring it, as it could acquire additional resistance markers.

Keywords: Neisseria gonorrhoeae; ST-9363; antibiotic resistance; azithromycin; evolution; phylogeography; recombination; timed phylogeny.

Fig. 1.

Whole-genome MCC dated phylogeny reconstructed using BEAST and annotated with TreeStructure clades, MLSTs, geographical locations, and MICs of various antibiotics along with the penA allele profile of all the 1978 ST-9363 core-genogroup gonococcal isolates included in this study. White/blank regions in the figure indicate that the data are not available for the corresponding isolate on the phylogenetic tree. *Azithromycin MIC nonsusceptible ≥1 are shown for the Oceania isolates as the exact MICs were not publicly available and in Williamson et al. (2019) all Oceania isolates with MICs ≥1 μg/ml were considered as resistant to AZM, which does not conform to CLSI breakpoint. HL-AZI-R, high-level azithromycin resistance.

Fig. 2.

Whole-genome MCC dated phylogeny reconstructed using BEAST and annotated with TreeStructure clades, continents, AZM MICS, and the known resistance conferring genetic variants for AZI and CIP of all the 1978 ST-9363 core-genogroup gonococcal isolates included in this study. White/blank regions in the figure indicate that the data are not available for the corresponding isolate on the phylogenetic tree. *Azithromycin MIC nonsusceptible ≥1 are shown for the Oceania isolates as the exact MICs were not publicly available and in Williamson et al. (2019) all Oceania isolates with MICs ≥1 μg/ml were considered as resistant to AZM, which does not conform to CLSI breakpoint. HL-AZI-R, high-level azithromycin resistance.

Fig. 3.

Ancestral state reconstruction based on the MCC dated phylogeny reconstructed using BEAST with geographical locations (Continents) as discrete traits. Each node indicates the estimated posterior probabilities of each of the discrete traits—Europe, North American, and Oceania as pie charts.

Fig. 4.

—(a) Whole-genome dated phylogeny of clade 4 isolates (North American lineage) along with the known resistance conferring genetic variants for AZI and CIP of all the clade 4 ST-9363 core-genogroup gonococcal isolates. (b) Demographic history of clade 4. Time is measured in years and 0 indicates the year 2018 (x axis) and the EPS is scaled to the number of generations per year. (c) Bar diagram showing Tajima’s D statistics estimated for the whole genome, the entire mtr locus genes, mtrD gene and the mtrR and promoter region for the clade 4 isolates. (d) Estimated LD measured by r² for the entire mtr locus, mtrD gene and the mtrR and promoter region for the clade 4 isolates. Both Tajima’s D and r² were estimated over a 100-bp sliding window on the entire whole-genome alignment containing only the clade 4 isolates using the R package called PopGenome. *Azithromycin MIC nonsusceptible ≥1 are shown for the Oceania isolates as the exact MICs were not publicly available and in Williamson et al. (2019) all Oceania isolates with MICs ≥1 μg/ml were considered as resistant to AZM, which does not conform to CLSI breakpoint. HL-AZI-R, high-level azithromycin resistance.