The extended-spectrum cephalosporin ceftriaxone (CRO) is the recommended first-line treatment of Neisseria gonorrhoeae infections in several countries. However, the emergence of CRO-resistant N. gonorrhoeae strains has been reported in this decade.1 Thus, alternative therapeutic methods besides CRO monotherapy have been proposed; for example, dual antimicrobial therapies such as the combination of azithromycin (AZM) and CRO. In particular, AZM and CRO and dual therapy have been used after CRO monotherapy treatment failure for gonococcal pharyngeal infection.1 However, N. gonorrhoeae strains with high-level AZM resistance (HL-AziR) have been isolated worldwide,2–4 with sustained transmission of HL-AziR gonococcal strains in some regions.4–8 Moreover, HL-AziR gonococcal strains with reduced CRO susceptibility have been reported,7 and both CRO- and AZM-resistant strains have been reported from Australia and the United Kingdom.9,10
Just one strain, FC488, has been confirmed as HL-AziR in Japan11; thus, information on these resistant strains is limited. Recently, we identified another HL-AziR gonococcal strain, GU20180115-5. Herein, we describe the first 2 HL-AziR gonococcal strains, FC488 and GU20180115-5, isolated in Japan. The relationship between the HL-AziR gonococcal strains isolated in Japan and in other regions was investigated.
MATERIALS AND METHODS
Our ongoing surveillance started in 2015 as a research project under the Japan Agency for Medical Research and Development (JP16fk0108214 and JP18fk0108062). The surveillance system and the results of this project have been partially reported.11 During this surveillance (2015–2018), 3495 N. gonorrhoeae strains have been isolated in Japan, and we found the 2 HL-AziR gonococcal strains FC488 isolated in Osaka (population of 8.8 million people) in 2015 and GU20180115-5 isolated in Tokyo (14 million people) in 2018. FC488 and GU20180115-5 were isolated from men in their 40s and 30s showing symptoms of urethritis. However, there was no other information on sexual contacts or sexual orientation of the patients. These 2 strains have been deposited in the Gifu University Center for Conservation of Microbial Genetic Resource, Organization for Research and Community Development. Strains FC488 and GU20180115-5 are registered as GTC_21798 and GTC_21915, respectively.
Antimicrobial susceptibility tests were performed using an agar dilution method in accordance with the Clinical and Laboratory Standards Institute protocol.12
Genomic DNA was extracted with a QIAamp DNA Mini Kit (QIAGEN, Venlo, the Netherlands). Genomic DNA libraries were prepared using a Nextera XT DNA Sample Prep Kit (Illumina, San Diego, CA). The pooled libraries were analyzed by multiplexed paired-end sequencing (300-mer × 2) using a MiSeq (Illumina). The short reads were assembled using SPAdes v.3.11.1 software with the “–careful” option.13 The draft genomes were analyzed by multilocus sequence typing (MLST), N. gonorrhoeae multiantigen sequence typing (NG-MAST), and N. gonorrhoeae sequence typing for antimicrobial resistance, using the PubMLST database (https://pubmlst.org/neisseria/). The phylogenetic relationships of the AZM resistant N. gonorrhoeae strains were inferred by mapping-based analysis. Core genome single-nucleotide polymorphisms (SNPs) were identified with BactSNP v.1.1.0 software14 using the N. gonorrhoeae WHO F strain genome (GenBank accession no. LT591897) as the reference strain. Repetitive regions longer than 50 bp detected by MUMmer v.3.225915 as previously described16 and recombinogenic regions detected by Gubbins17 were removed from further analysis. The resulting concatenated SNP sequences were used for further analyses. Phylogenetic relationships were determined by reconstructing a phylogenetic tree using the maximum likelihood method with IQ-TREE software and 1000 ultrafast bootstrap replicates.18 The bacterial population structure was analyzed using the hierBAPS Bayesian-based clustering algorithm to assign lineages.19
The minimum inhibitory concentrations (MIC) of AZM, CRO, cefixime, penicillin G, ciprofloxacin, and spectinomycin were 512, 0.016, 0.008, 0.5, 8, and 16 mg/L against N. gonorrhoeae strain FC488 and 1024, 0.016, 0.016, 1, 8, and 16 mg/L against N. gonorrhoeae strain GU20180115-5. Both strains were resistant to AZM and ciprofloxacin.
The determinants of AZM resistance were further analyzed. The target alleles for antimicrobial resistance in strains FC488 and GU20180115-5 were identified from the whole-genome sequencing (WGS) data using the sequence of strain FA1090 as the reference sequence. Both strains contained an A2143G mutation, corresponding to the A2059G mutation in the Escherichia coli genome, in loop V in all 4 23S rDNA genes in the N. gonorrhoeae genome. This mutation resulted in resistance to HL-AziR gonococcal strains.20 An analysis of mtrR showed that both FC488 and GU20180115-5 strains carried an adenine deletion in the 13-bp inverted repeat in the mtrR promoter region and a G45D mutation in the mtrR coding sequence; these were associated with increased macrolide resistance.21
To investigate the genetic relationship among HL-AziR strains, we used the WGS data of the FC488 and GU20180115-5 strains and those of the 116 publicly available HL-AziR strains in the BioProject database (Supplementary Table S1, https://links.lww.com/OLQ/A562). Through Bayesian analysis of the core genome SNPs in these strains, their population structure was reconstructed and 5 clades were identified (Fig. 1, Supplementary Table S1, https://links.lww.com/OLQ/A562), with strains FC488 and GU20180115-5 clustered in clade A (n = 9). The phylogenetic analysis indicated that clade A can be subdivided into clades A1 and A2 (Fig. 1). Clade A2 contained the CRO- and AZM-resistant strains isolated in the United Kingdom and Australia,9,10 and clade A1 contained the strains isolated in China and Japan, except strain 101259. Because clade A2 strains may have been introduced from Asia,10,22 clade A may be an Asia-derived HL-AziR phylogenetic branch.
The molecular typing of clade A strains is shown in Figure 1. N. gonorrhoeae sequence typing for antimicrobial resistance23 showed that both strains isolated in Japan, and all clade A1 strains were type 202 and clade A2 strains were type 996. Type 996 strains are the same as type 202 strains except for the penA allele, which is penA-60.001 in type 996 strains and penA-2.002 in type 202 strains. Multilocus sequence typing showed that clade A strains were ST12039, except strain FC488 that was ST10899 (a single-locus pdhC-variant of ST12039), consistent with the phylogenetic analysis results. N. gonorrhoeae multiantigen sequence typing showed that clade A1 strains, including the HL-AziR FC488 strain, were NG-MAST 1866. On the contrary, the HL-AziR GU20180115-5 strains were ST16497, in which there is a single-nucleotide substitution in the porB allele of the ST1866 strains. Clade A2 strains were phylogenetically different from clade A1 strains.
The HL-AziR gonococcal strains in MLST12039 (clade A) are spreading globally.4,8–10 Some MLST12039 strains carrying penA 60.001, with CRO and AZM resistance, have been reported in Australia and the United Kingdom.9,10 Ceftriaxone and AZM double-resistant strains can result from the acquisition of the penA 60.001 allele from clade A2 strains by transformation. The spread of these double-resistant strains is a global concern and requires enhanced surveillance of their antimicrobial susceptibilities. The development of a simple assay system, which can detect multiple HL-AziR alleles simultaneously, would be useful for on-time monitoring of the occurrence of this kind of resistant gonococcal strains.
The HL-AziR gonococcal strains typed as MLST12039 and NG-MAST16497 were isolated in Taiwan4 and Japan. In addition, strains that were a combination of MLST10899 and NG-MAST1866 have been reported in Taiwan, China,4,8 and Japan. N. gonorrhoeae multiantigen sequence typing 1866 HL-AziR strains have spread in eastern Asia.3,4,8,24 Several reports have shown that the HL-AziR N. gonorrhoeae strains in China are NG-MAST1866.3,8,24 Another ST10133 strain, related to NG-MAST1866, has a different porB allele but the same tbpB allele as clade A1 strains: this strain was isolated in Australia, but its source has been suggested to be China or Hong Kong.25 Because no other NG-MAST1866 or NG-MAST16497 strain was found in Japan before the emergence of FC488 and GU20180115-5,26 these 2 strains may have been from regions in eastern Asia other than Japan, as in the case of NG-MAST 10133 strain.25
We report here the first HL-AziR gonococcal strains isolated in Japan. Fortunately, there has been no further spread of these strains in Japan, so their emergence may be only a sporadic event in Japan. However, HL-AziR N. gonorrhoeae strains that are clade A1 strains (NG-MAST1866, NG-MAST 16497) have been frequently observed in eastern Asia.4,8,24 The spread of these strains both inside and outside of Asia needs to be monitored by both WGS data analysis of isolated strains and enhanced surveillance of their antibiotic resistance.
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