Genomic surveillance of Neisseria gonorrhoeae to investigate the distribution and evolution of antimicrobial-resistance determinants and lineages

Abstract

The first extensively drug resistant (XDR) Neisseria gonorrhoeae strain with high resistance to the extended-spectrum cephalosporin ceftriaxone was identified in 2009 in Japan, but no other strain with this antimicrobial-resistance profile has been reported since. However, surveillance to date has been based on phenotypic methods and sequence typing, not genome sequencing. Therefore, little is known about the local population structure at the genomic level, and how resistance determinants and lineages are distributed and evolve. We analysed the whole-genome sequence data and the antimicrobial-susceptibility testing results of 204 strains sampled in a region where the first XDR ceftriaxone-resistant N. gonorrhoeae was isolated, complemented with 67 additional genomes from other time frames and locations within Japan. Strains resistant to ceftriaxone were not found, but we discovered a sequence type (ST)7363 sub-lineage susceptible to ceftriaxone and cefixime in which the mosaic penA allele responsible for reduced susceptibility had reverted to a susceptible allele by recombination. Approximately 85 % of isolates showed resistance to fluoroquinolones (ciprofloxacin) explained by linked amino acid substitutions at positions 91 and 95 of GyrA with 99 % sensitivity and 100 % specificity. Approximately 10 % showed resistance to macrolides (azithromycin), for which genetic determinants are less clear. Furthermore, we revealed different evolutionary paths of the two major lineages: single acquisition of penA X in the ST7363-associated lineage, followed by multiple independent acquisitions of the penA X and XXXIV in the ST1901-associated lineage. Our study provides a detailed picture of the distribution of resistance determinants and disentangles the evolution of the two major lineages spreading worldwide.

Keywords: Neisseria gonorrhoeae; antimicrobial resistance; cephalosporin; coalescent; fluoroquinolone; genomic epidemiology; macrolide; phylogeny; recombination; surveillance.

Fig. 1.

Whole-genome sequence phylogeny, resistance patterns of the antimicrobials and genetic polymorphisms. Left: a clonal phylogeny with corrected branch lengths to account for homologous recombination. The blue vertical line represents the notable sub-lineage in ST7363, whereas the blue arrow represents the outgroup of the sub-lineage. The red arrow indicates strain H041 (WHO X). Heatmap: column 1, ST: the two major STs (ST7363 and ST1901), single-locus variants of the former (ST7356, ST1932) and the latter (ST7360 and ST1579), and a new ST (double-locus variant of ST1901) are coloured. Columns 2 and 3: susceptible/resistant (S/R) categories according to the EUCAST MIC breakpoint of cefixime (CFM) and ceftriaxone (CRO). The presence or absence of genetic features is shown in columns 4–14, 16–20, 22–26 as marked: grey indicates absence, other colours indicate presence. Column 4: any mosaic penA allele. Columns 5–10: a specific mosaic penA allele. Column 11: penA V. Columns 12–14: nonsynonymous amino acid changes from wild-types in PorB. Column 15: susceptible/intermediate/resistant (S/I/R) categories according to the EUCAST MIC breakpoints of ciprofloxacin (CIP). Columns 16–17: nonsynonymous amino acid changes from wild-type in GyrA. Columns 18–20: nonsynonymous amino acid changes from wild-type in ParC. Column 21: susceptible/intermediate/resistant (S/I/R) categories according to the EUCAST MIC breakpoints of azithromycin (AZI). Column 22: the adenosine deletion in the mtrR promoter. Columns 23–24: the mutations in the 23S rRNA gene. Columns 25–26: plasmid-encoded bla_TEM and tetM. The blue rectangles indicate notable sub-lineages in ST7363 described in the text.

Fig. 2.

Gene tree of penA, resistance patterns of cefixime and ceftriaxone, and penA polymorphisms. Heatmap: column 1, ST: the ST of the genomes harbouring the penA alleles. The two major STs (ST7363 and ST1901), single-locus variants of the former (ST7356, ST1932) and the latter (ST7360 and ST1579), and a new ST (double-locus variant of ST1901) are coloured. Columns 2–3: susceptible/resistant (S/R) categories according to the EUCAST MIC breakpoint of cefixime (CFM) and ceftriaxone (CRO). The presence or absence of genetic features is shown in columns 4–11 as marked: grey indicates absence, other colours indicate presence. Column 4: any mosaic penA allele. Columns 5–10: a specific mosaic penA allele. Column 11: penA V. The inset on the right is a schematic representation illustrating PBP2 amino acid sequences encoded by the penA alleles. The sequence blocks shaded with the same colour represent identical amino acid sequences. The susceptible penA allele reverted by recombination is composed of penA X (first half, white) and penA V (second half, yellow-green).

Fig. 3.

Time-resolved phylogeny and penA polymorphisms. All nodes and branches with posterior support values with 0.9 are coloured in purple. The cyan branches indicate acquisition of penA X or XXXIV, whereas the yellow branch indicates loss of penA X by recombination. The cyan, yellow and green circles indicate nodes with 95 % HPD described in the text. Heatmap: column 1, ST: the ST of the genomes harbouring the penA alleles. The two major STs (ST7363 and ST1901), single-locus variants of the former (ST7356, ST1932) and the latter (ST7360 and ST1579), and a new ST (double-locus variant of ST1901) are coloured. Columns 2–3: susceptible/resistant (S/R) categories according to the EUCAST MIC breakpoint of cefixime (CFM) and ceftriaxone (CRO). The presence or absence of genetic features is shown in columns 4–9 as marked: grey indicates absence, other colours indicate presence. Column 4: any mosaic penA allele. Columns 5–7: penA H041-type, X and variants, and XXXIV and variants. Column 8: penA V. Column 9: variants of penA V: light and dark yellow-green indicate 4 and 5 amino acid variants, respectively. (a) ST7363 and its single locus variants. The blue rectangle indicates the notable sub-lineage sharing the susceptible penA allele reverted by recombination. (b) ST1901-associated lineage. The yellow-green arrow indicates an exceptional strain isolated in 2015 that contained the ancestral susceptible penA V variant.