Azithromycin susceptibility of Neisseria gonorrhoeae in the USA in 2017: a genomic analysis of surveillance data

Abstract

Background: The number of cases of gonorrhoea in the USA and worldwide caused by Neisseria gonorrhoeae is increasing (555 608 reported US cases in 2017, and 87 million cases worldwide in 2016). Many countries report declining in vitro susceptibility of azithromycin, which is a concern because azithromycin and ceftriaxone are the recommended dual treatment in many countries. We aimed to identify strain types associated with decreased susceptibility to azithromycin.

Methods: We did a genomic analysis of N gonorrhoeae isolates obtained by the US Gonococcal Isolate Surveillance Project. Isolates were whole-genome sequenced based on decreased susceptibility to azithromycin (minimal inhibitory concentration [MIC] ≥2 μg/mL, using agar dilution antibiotic susceptibility testing) and geographical representation. Bioinformatic analyses established genomic diversity, strain population dynamics, and antimicrobial resistance profiles.

Findings: 410 isolates were sorted into more than 20 unique phylogenetic clades. One predominant persistent clade (consisting of 97 isolates) included the most isolates with azithromycin MICs of 2 μg/mL or higher (61 of 97 [63%] vs 59 of 311 [19%]; p<0·0001) and carried a mosaic mtr (multiple transferable resistance) locus (68 of 97 [70%] vs two of 313 [1%]; p<0·0001). Of the remaining 313 isolates, 57 (18%) had decreased susceptibility to azithromycin (MIC ≥4 μg/mL), which was attributed to 23S rRNA variants (56 of 57 [98%]) and formed phylogenetically diverse clades, showing various levels of clonal expansion.

Interpretation: Reduced azithromycin susceptibility was associated with expanding and persistent clades harbouring two well described resistance mechanisms, mosaic mtr locus and 23S rRNA variants. Understanding the role of recombination, particularly within the mtr locus, on the fitness and expansion of strains with decreased susceptibility has important implications for the public health response to minimise gonorrhoea transmission.

Funding: US Centers for Disease Control and Prevention (CDC), CDC Combating Antibiotic Resistant Bacteria initiative, Oak Ridge Institute for Science Education, US Department of Energy/CDC/Emory University, National Institutes of Health, and Biomedical Laboratory Research and Development Service of the US Department of Veterans Affairs.

Figure 1:. Distribution of multilocus sequence types for 410 isolates from the USA in 2017

The 22 most numerous sequence types, which include five or more isolates, are shown according to susceptibility to azithromycin, ceftriaxone, and cefixime. Decreased susceptibility to azithromycin is defined as MIC ≥2 μg/mL. Decreased susceptibility to cefixime is defined as MIC ≥0·250 μg/mL. Decreased susceptibility to ceftriaxone is defined as MIC ≥0·125 μg/mL. MIC=minimum inhibitory concentration.

Figure 2:. Maximum-likelihood core-genome phylogenetic tree of 410 Neisseria gonorrhoeae isolates from the USA in 2017

The reference genome was FA19 (GenBank accession CP012026.1). 19 clades (labelled A–S) are shown, with susceptibility profiles and relevant antibiotic resistance determinants. Variants are described in appendix 1 (pp 19–20). Isolates susceptible to azithromycin are defined as MIC ≤1 μg/mL; decreased susceptibility to azithromycin is categorised with MICs of 2–4 μg/mL, 8 μg/mL, and ≥16 μg/mL. Isolates susceptible to cefixime are defined as MIC ≤0·125 μg/mL, and those with decreased susceptibility are defined as MIC ≥0·250 μg/mL. Isolates susceptible to ceftriaxone are defined as MIC ≤0·060 μg/mL, and those with decreased susceptibility are defined as MIC ≥0·125 μg/mL. Isolates susceptible to ciprofloxacin are defined as MIC ≤0·5 μg/mL, and those with decreased susceptibility are defined as MIC ≥1·0 μg/mL. The antimicrobial resistance determinants 23S rRNA 2611C→T and 2059A→G are categorised by cumulative number of variant rRNA copies present (one to four copies). penA identifies mosaic and non-mosaic penA alleles. Categorisation of isolates by sexual orientation of the individual from whom the isolate was obtained is also shown. MIC=minimum inhibitory concentration. MSW=men who have sex with women. MSM=men who have sex with men. MSMW=men who have sex with men and women.

Figure 3:. Region of the maximum-likelihood core-genome phylogenetic tree representing clade A

Variants are described in appendix 1 (pp 19–20). Aligned antimicrobial determinants emphasise the separation of the parental clade (ST9363) and subclade 9363.1. Isolates susceptible to azithromycin are defined as MIC ≤1 μg/mL; decreased susceptibility to azithromycin is categorised with MICs of 2–4 μg/mL, 8 μg/mL, and ≥16 μg/mL. The antimicrobial resistance determinants 23S rRNA 2611C→T and 2059A→G are categorised by cumulative number of variant rRNA copies present (one to four copies). mtrR locus is described by mtrR mosaic (wild-type non-mosaic or mosaic) and mtrR promoter (C substitution or A deletion in the 13-bp inverted repeat). Amino acid substitutions in MtrR (Ala39Thr, Gly45Asp, and His105Tyr), MtrD (Lys823Glu, representing mtrD mosaicity), and PorB (Gly120Lys and Gly121Asp or Asn) are also shown. mtr operon and porB and penA are defined antibiotic resistance genes for cefixime, ceftriaxone, and penicillin.^, penA identifies mosaic and non-mosaic penA alleles. MIC=minimum inhibitory concentration.

Figure 4:. Schematic of mtr locus of Neisseria gonorrhoeae

Arrows indicate the direction of transcription. The coloured lines above the mtr locus indicate the mosaic region in clade A isolates with mosaic mtrD only (upper line) and in subclade 9363.1 with mosaic mtrR, promoter, and mosaic mtrCDE (lower line).