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Antimicrobial resistance in Neisseria gonorrhoeae is a global health concern. Strains from two internationally circulating sequence types, ST-7363 and ST-1901, have acquired resistance to treatment with third-generation cephalosporins mainly due to the emergence of mosaic penA alleles. These two STs were first detected in Japan; however, when and how the mosaic penA alleles emerged and spread to other countries remains unknown. Here, we addressed the evolution of penA alleles by obtaining complete genomes from three Japanese ST-1901 clinical isolates harboring mosaic penA allele 34 (penA-34) dating from 2005 and generating a phylogenetic representation of 1,075 strains sampled from 37 countries. We also sequenced the genomes of 103 Japanese ST-7363 N. gonorrhoeae isolates from 1996-2005 and reconstructed a phylogeny including 88 previously sequenced genomes. Based on an estimate of the time of emergence of ST-1901 harboring mosaic penA-34 and ST-7363 harboring mosaic penA-10, and >300 additional genome sequences of Japanese strains representing multiple STs isolated in 1996-2015, we suggest that penA-34 in ST-1901 was generated from penA-10 via recombination with another Neisseria species, followed by a second recombination event with a gonococcal strain harboring wildtype penA-1. Following the acquisition of penA-10 in ST-7363, a dominant sub-lineage rapidly acquired fluoroquinolone resistance mutations at GyrA 95 and ParC 87-88, possibly due to independent mutations rather than horizontal gene transfer. Literature data suggest the emergence of these resistance determinants may reflect selection from the standard treatment regimens in Japan at that time. Our findings highlight how recombination and antibiotic use across and within Neisseria species intersect in driving the emergence and spread of drug-resistant gonorrhea.Author summaryAntimicrobial resistance is recognized as one of the greatest threats to human health, and Neisseria gonorrhoeae resistance is classified as one of the most urgent. The two major internationally spreading lineages resistant. to first line drugs likely originated in Japan, but when and how their genetic resistance determinants emerged remain unknown. In this study, we conducted an evolutionary analysis using clinical N. gonorrhoeae isolates from 37 countries, including a historical collection of Japanese isolates, to investigate the emergence of resistance in each of the two major lineages. We showed that the penA allele responsible for resistance to cephalosporins, the first-line treatment for gonorrhea, was possibly generated by two recombination events, one from another Neisseria species and one from another N. gonorrhoeae lineage. We also showed that mutations responsible for resistance to a previously widely used antibiotic treatment occurred twice independently in one of the two major lineages. The emergence of the genetic resistance determinants potentially reflects selection from the standard treatment regimen at that time. Our findings highlight how recombination (horizontal gene transfer) and antibiotic use across and within a bacterial species intersect in driving the emergence and spread of antimicrobial resistance genes and mutations.

Background Neisseria gonorrhoeae strains with resistance to extended-spectrum cephalosporins (ESCs), last options for first-line monotherapy of gonorrhoea, likely emerged and initially disseminated in Japan, followed by international transmission. In recent years, multi-locus sequence typing (MLST) ST1901 and N. gonorrhoeae multiantigen sequence typing (NG-MAST) ST1407 isolates with the mosaic penicillin-binding protein (PBP) 2 XXXIV have accounted for most ESC resistance globally. Our aim was to elucidate the initial emergence and transmission of ESC-resistant strains by detailed examination of N. gonorrhoeae isolates from 1995 to 2005 in Kanagawa, Japan. Methods N. gonorrhoeae isolates were examined phenotypically (n = 690) and genetically (n = 372) by agar dilution method (cefixime, ceftriaxone and ciprofloxacin), penA gene sequencing, MLST and NG-MAST. Results Already in 1995, one cefixime-resistant (CFM-R) isolate was found, which is the first CFM-R isolate described globally. After 1996, the prevalence of CFM-R and CFM-decreased susceptibility (CFM-DS) isolates significantly increased, with the peak resistance level in 2002 (57.1 % CFM-R). In 1997–2002, the CFM-R MLST ST7363 strain type with the mosaic PBP 2 X was predominant among CFM-R/DS isolates. The first CFM-R/DS MLST ST1901 clone(s), which became the predominant CFM-R/DS strain type(s) already in 2003–2005, possessed the mosaic PBP 2 X, which was possibly originally transferred from the MLST ST7363 strains, and subsequently acquired the mosaic PBP 2 XXXIV. The first MLST ST1901 and NG-MAST ST1407 isolate was identified in Kanagawa already in 2003. Conclusions The two main internationally spread cefixime-resistant gonococcal clones, MLST ST7363 and ST1901 (NG-MAST ST1407 most frequent internationally) that also have shown their capacity to develop high-level ceftriaxone resistance (superbugs H041 and F89), likely emerged, evolved and started to disseminate in the metropolitan area, including Kanagawa, in Japan, which was followed by global transmission.