Genomic Characterization of Neisseria gonorrhoeae Strains from 2016 U.S. Sentinel Surveillance Displaying Reduced Susceptibility to Azithromycin

In 2016, the proportion of Neisseria gonorrhoeae isolates with reduced susceptibility to azithromycin rose to 3.6%. A phylogenetic analysis of 334 N. gonorrhoeae isolates collected in 2016 revealed a single, geographically diverse lineage of isolates with MICs of 2 to 16 μg/ml that carried a mosaic-like mtr locus, whereas the majority of isolates with MICs of ≥16 μg/ml appeared sporadically and carried 23S rRNA mutations.

ABSTRACT

In 2016, the proportion of Neisseria gonorrhoeae isolates with reduced susceptibility to azithromycin rose to 3.6%. A phylogenetic analysis of 334 N. gonorrhoeae isolates collected in 2016 revealed a single, geographically diverse lineage of isolates with MICs of 2 to 16 μg/ml that carried a mosaic-like mtr locus, whereas the majority of isolates with MICs of ≥16 μg/ml appeared sporadically and carried 23S rRNA mutations. Continued molecular surveillance of N. gonorrhoeae isolates will identify new resistance mechanisms.

TEXT

Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea, has consistently developed resistance to each recommended antibiotic, resulting in its designation as an urgent threat by the Centers for Disease Control and Prevention (CDC) and as a high-priority antibiotic-resistant pathogen by the World Health Organization (1, 2). Increasing numbers of N. gonorrhoeae isolates with reduced susceptibility to azithromycin (AZM) are reported in the United States and internationally, including in China (3), Canada (4), and Europe (5). In the United States, surveillance efforts through the Gonococcal Isolate Surveillance Project (GISP) indicated an increased incidence of reduced susceptibility to AZM from 0.2% in 2012 to 3.6% in 2016 (6). Given this sharp increase, we focused on understanding the genetics of all the isolates from 2016 with reduced susceptibility to AZM, defined as an MIC of ≥2 μg/ml. In a previous report, 117 isolates from 2016 with an AZM MIC of ≥2 μg/ml were genetically analyzed (7). In this report, we expand this analysis to 177 isolates, which represents 95% of all such isolates from 2016.

Isolates with an AZM MIC of <16 μg/ml cluster into a single, diverse clade sharing a common genetic mechanism.

In 2016, 95% (177/186) of all gonococcal isolates collected by GISP with an AZM MIC of ≥2 μg/ml as determined by agar dilution were available for whole-genome sequencing (WGS) on an Illumina MiSeq (8). We investigated the relatedness of the strains by performing sequence typing using MLST and NG-MAST and a core genome single nucleotide polymorphism (SNP) analysis with recombination filtering followed by generation of a maximum-likelihood phylogenetic tree (Fig. 1). We also selected a stratified random sample of 10 isolates from each GISP sentinel site from 2016, of which 157 isolates were available for sequencing and contained mostly susceptible isolates. The distribution of AZM MICs in the sampled susceptible isolates was similar to that of all susceptible isolates in GISP, suggesting that these isolates reflect the circulating U.S. AZM-susceptible gonorrhea population (compare reference 6 and Fig. S1 in the supplemental material). We also investigated alterations in the 23S rRNA alleles or the multiple transferable resistance regulator (mtrR) locus, which contribute to reduced susceptibility to AZM (Tables 1 and 2) using a custom pipeline written in Python (9, 10). A total of 13 clades containing three or more isolates were identified using Bayesian analysis of population structure (BAPS) (11). We identified one large clade of highly related isolates with high nodal support (clade A [n = 119]; within-clade difference, 87 ± 58 SNPs) (Fig. 1), which is a subset of BAPS cluster 4. Whereas the most common sequence type (ST) in clade A was ST9363 (100/119), others, including ST11422 and ST8134, were present. Clade A contained 39 total NG-MAST STs, with ST3935 (21/119) being the most common, followed by ST8241 (19/119) and ST12302 (12/119). Clade A is distinguished from the rest of the cluster by the presence of a mosaic-like mtrR locus, which contributes to elevated macrolide MICs by increasing expression of the MtrCDE efflux pump (12). Chi-square analysis showed that a mosaic-like mtrR locus or an A-to-C transversion was highly associated with an MIC range of 2 to 4 μg/ml (χ² = 204.71, P < 0.0001; or χ² = 174.98, P < 0.0001, respectively) (Table 1). The success of the organisms in clade A, as suggested by the presence of similar isolates in a genomic analysis of GISP isolates from 2000 to 2013 (13) and whose presence has been continuous since 2014 (7), suggests that the mosaic-like mtrR locus contributes to their ability to survive and be transmitted. In support of this hypothesis, the transformation of a susceptible strain with a mosaic-like mtr locus is sufficient to increase the AZM MIC of the recipient strain (12, 14). Across the data set, the most predominant MLST type was ST9363 (n = 130) (Fig. 1), followed by ST1584 (n = 23), ST11422 (n = 12), ST1893 (n = 11), and ST1579 (n = 10). Some STs, e.g., ST9363, were found across the country, whereas others were more restricted (e.g., ST7371 or ST8154 on the East Coast). For NG-MAST, ST3935 was the most common (n = 23), followed by ST8241 (n = 19), ST7638 (n = 15), and ST12302 (n = 12) (for new NG-MAST STs, see Fig. S3 and Supplementary Methods in the supplemental material).

FIG 1.

Maximum-likelihood phylogeny of Neisseria gonorrhoeae isolates collected in 2016, including all available isolates exhibiting AZM MICs of ≥2 μg/ml in GISP (n = 117). Lineage characterized by reduced susceptibility to AZM (2 to16 μg/ml) is highlighted in red (clade A). Rings from inside to outside are BAPS clusters, AZM MIC values, HHS regions, 23S rRNA C2611T, 23S rRNA A2059G, mosaic-like mtrR presence, and mtrR promoter mutations.

TABLE 1.

Distribution of mtrR mutations

AZM MIC (μg/ml)	Distribution (no. isolates) of:
	mtrR locus		mtrR promoterc			MtrR
	Wild type	Mosaic	Wild type	Del A	C SNP	39A	39T	44H	44R	45D	45G	47L	47A	Full length	Premature stop
<2.0	157a	0	119a	38	0	100a	57	26	131b	48	109a	157	0	28	129
2.0–4.0	29	113a	24	4	114a	134a	8	6	136b	0	142a	142	0	19	123
8.0–16.0	25	6	12	13	6	20	11	4	27	8	23	31	0	0	31
>16	4	0	0	4	0	4	0	0	4	0	4	4	0	0	4

^aP < 0.0001.

^bP < 0.005.

^cDel A, deletion of an adenine in the inverted repeat of the mtrR promoter; C SNP, A-to-C transversion in the inverted repeat of the mtrR promoter.

TABLE 2.

Distribution of 23S rRNA mutant alleles

AZM MIC (μg/ml)	Distribution (no. isolates) of:
	C2611T				A2059G
	Wild type	2 mutated loci	3 mutated loci	4 mutated loci	Wild type	4 mutated loci
<2.0	156a	1	0	0	157	0
2.0–4.0	126a	0	1	15b	142	0
8.0–16.0	6	0	1	24a	30	1
>16.0	4	0	0	0	0	4

^aP < 0.0001.

^bNot significant.

Isolates with an AZM MIC of ≥16 μg/ml appear sporadically and contain mutations in the 23S rRNA genes.

Twenty isolates had an AZM MIC of ≥16 μg/ml as determined by agar dilution antimicrobial susceptibility testing. Those were further tested by Etest (bioMérieux), and 4 had an AZM MIC of ≥256 μg/ml. Those isolates were represented by ST1579 (n = 4), ST1901 (n = 4), ST7371 (n = 6), ST9363 (n = 5), and ST7822 (n = 1). The four isolates with MICs of ≥256 μg/ml were part of a previously described cluster of isolates with high-level AZM resistance and reduced susceptibility to ceftriaxone, which was successfully contained (15). Isolates with AZM MICs of ≥16 μg/ml were found throughout the tree and grouped into small subclusters (e.g., BAPS cluster 5 contained three subclusters) (Fig. 1). These isolates all contained mutations in either position 2611 or 2059 (E. coli numbering) of the 23S rRNA, which is the target of macrolides (Fig. 1 and Table 2). In N. gonorrhoeae, there are four copies of the 23S rRNA gene in the genome. Four copies of the 23S rRNA allele carrying the C2611T alteration were associated with an MIC range of 8 to 16 μg/ml (χ² = 129.65; P < 0.0001) (Table 2). Isolates with four alleles carrying the A2059G alteration were found to have an AZM MIC of ≥256 μg/ml (Fig. 1 and Table 2). When found in clinical isolates or generated by site-directed mutagenesis in a susceptible strain, the A2059G alteration leads to an AZM MIC of ≥256 μg/ml and, potentially, treatment failure (9, 16, 17).

In contrast to the widespread presence of isolates containing the mosaic-like mtrR locus, isolates with the C2611T alteration appear more sporadically. This is consistent with a hypothesis that the 23S rRNA mutations occur spontaneously and carry a fitness cost. The largest clade of these contains 15 isolates (MLST ST1584 and NG-MAST ST7638) and is found only in HHS regions 9 and 10. Many of these smaller clades were geographically isolated (Fig. 1), further suggesting spontaneous mutation followed by clonal expansion.

WGS for N. gonorrhoeae surveillance in 2016 did not include routine selection of enough susceptible isolates to assess when the 23S rRNA mutations arose in the U.S. N. gonorrhoeae population and whether certain strain types are more prone to developing these mutations. Defined selection criteria for routine WGS of GISP isolates were established in late 2017 and include subsets of both susceptible and resistant isolates. These sequences will provide a valuable resource for tracking of known strains with reduced susceptibility to AZM and detection of emerging strains that appear in the United States.