Ceftriaxone-resistant, multidrug-resistant Neisseria gonorrhoeae with a novel mosaic penA-237.001 gene, France, June 2022

We report a ceftriaxone-resistant, multidrug-resistant urogenital gonorrhoea case in a heterosexual woman in France, June 2022. The woman was successfully treated with azithromycin 2 g. She had unprotected sex with her regular partner, who developed urethritis following travel to Vietnam and Switzerland. Whole genome sequencing of the gonococcal isolate (F92) identified MLST ST1901, NG-STAR CC-199, and the novel mosaic penA-237.001, which caused ceftriaxone resistance. penA-237.001 is 98.7% identical to penA-60.001, reported in various ceftriaxone-resistant strains, including the internationally spreading FC428 clone.

We report a ceftriaxone-resistant, multidrug-resistant urogenital gonorrhoea case in a heterosexual woman in France, June 2022. The woman was successfully treated with azithromycin 2 g. She had unprotected sex with her regular partner, who developed urethritis following travel to Vietnam and Switzerland. Whole genome sequencing of the gonococcal isolate (F92) identified MLST ST1901, NG-STAR CC-199, and the novel mosaic penA-237.001, which caused ceftriaxone resistance. penA-237.001 is 98.7% identical to penA-60.001, reported in various ceftriaxone-resistant strains, including the internationally spreading FC428 clone.
The emergence of ceftriaxone resistance in Neisseria gonorrhoeae (NG) has become a relevant concern worldwide. Very few new antibiotics are available for treatment of such cases. Here, we describe a case of local transmission in France following possible importation of a ceftriaxone-resistant Asian multidrugresistant (MDR)-NG by the partner of the heterosexual case.

Clinical case description and diagnosis
In May 2022, a heterosexual woman in her late 40s with vaginal discharge consulted her general practitioner. Three days before symptom onset, she had vaginal unprotected sexual intercourse with her regular male partner, who had recently returned from business trips to Vietnam and Switzerland. The partner had urethritis symptoms, but he was not available for medical examination. The woman was screened (only vaginal swab) for Neisseria gonorrhoeae with a nucleic acid amplification test (NAAT) (Allplex STI Essential Assay Q, Seegene) and N. gonorrhoeae (NG) culture. The pathogen was detected by NAAT but viral culture was negative. Serological tests for HIV, hepatitis B virus/ hepatitis C virus infections and syphilis were negative. The woman was empirically treated with a single intramuscular dose of ceftriaxone 1 g and, within one week, symptoms were resolved and NAAT was negative (test of cure was negative 7 days post-treatment). However, approximately 6 weeks later, in June 2022, the woman returned with vaginal discharge, after having sexual intercourse only with her partner. The NAAT and culture of vaginal swabs were NG-positive. Extragenital sites were not screened as the woman reported no oral/anal sex. Previous histories of STIs in the woman and her partner were not known.
Antimicrobial susceptibility testing was performed on the vaginal sample. No empirical treatment was given before antimicrobial susceptibility testing results were available. Results revealed that the isolate (referred to as F92) was resistant to ceftriaxone and susceptible to azithromycin. Thus, the general practitioner did not administer ceftriaxone and, accordingly, only a single oral dose of azithromycin 2 g was given, which successfully treated the infection. Tests of cure performed 2 weeks post-treatment, including NAATs on vaginal, anorectal and oropharyngeal swabs and culture, were negative.

Antimicrobial susceptibility testing
The F92 isolate was cultured from a vaginal swab on PolyViteX agar (bioMérieux) in a humid 5% CO 2enriched atmosphere for 24 h at + 36 ± 1°C. Species verification was performed by matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry (Vitek MS, BioMérieux). Minimum inhibitory concentrations (MICs) were determined by Etest (BioMérieux) for 12 antimicrobial drugs, and interpreted using European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations [1], and by agar dilution for novel antimicrobials zoliflodacin [2], gepotidacin [3], and lefamulin [4]. The F92 isolate was resistant to ceftriaxone and other extended-spectrum cephalosporins (ESCs), benzylpenicillin, ciprofloxacin, and tetracycline, but susceptible to azithromycin and spectinomycin. The MICs of gentamicin, rifampicin and ertapenem as well as zoliflodacin, gepotidacin and lefamulin were considered wild-type according to previous publications and because no known antimicrobial resistance determinants for these antimicrobials were detected (Table).

Molecular investigation
Genomic DNA from F92 was isolated using QIAsymphony (QIAGEN) with the DSP DNA Mini Kit (QIAGEN). Whole genome sequencing (WGS) and bioinformatic analysis were performed as described [5,6], and sequencing reads are available at DNA Data Bank of Japan (DDBJ)/European Nucleotide Archive (ENA)/GenBank (Accession number: JAPIVM000000000).

Discussion
The resistance to ESCs in NG has been low in France (0.8% and 0.2% of cefixime-resistant isolates in 2019 and 2021, respectively) [15] and Europe (0.8% of cefixime-resistant isolates in 2019) [16], since the decline of the widespread MLST ST1901, NG-MAST ST1407 clone [17]. Furthermore, the high-level ceftriaxoneresistant F89 isolate, which emerged in this genomic background, was reported only in France and Spain [14] and its spread was likely limited by a decreased biofitness [18]. The ceftriaxone-resistant F90 and F91 isolates, associated with the internationally spreading FC428 clone containing penA-60 [5,[7][8][9][10][11][12], appeared in France in 2017 and 2019, respectively [5,7]. Notably, the patient with F91 was infected by her husband, who acquired the infection from a sex worker in Cambodia. FC428 has an adequate biofitness [10]. To our knowledge, the F91 has not yet spread further within France.
The assumed importation of MDR-NG to Europe from Asia, where ESC resistance has been reported to be high and increasing [11,13,19], is worrying. The described isolate is ceftriaxone-resistant because of the mosaic penA-237.001, which closely resembles penA-60.001. The genome of this MLST ST1901 strain is genomically distant from the cefixime-resistant MLST ST1901 clone that has caused ESC-resistance -especially in Europe -for over a decade [17]. However, it is similar to recently described Asian ceftriaxonesusceptible isolates carrying a non-mosaic penA-5.002 [20]. Accordingly, the F92 isolate with a novel mosaic penA-237.001 causing ceftriaxone resistance is unique and it may result from a horizontal gene transfer of penA-237.001 from commensal Neisseria species to a NG MLST ST1901 strain with penA-5.002 in Asia. A limitation of our report is that no details were available from the partner, and no strain was available for analysis.

Conclusions
The international emergence and transmission of NG isolates with resistance to ceftriaxone, which is the last remaining option for first-line gonorrhoea treatment, is a public health concern. Increased awareness The F92 isolate from France (red arrow) is compared with other ceftriaxone-resistant gonococcal strains (blue arrows and circles), among publicly available gonococcal genomes (n = 36,310) downloaded from the European Nucleotide Archive using the search term 'Taxon:485' (on 4 August 2022). The internationally spreading ceftriaxone-resistant FC428 clade (including isolates F90 and F91) [5,[7][8][9][10][11][12], the closely related UK722 isolate [12], the first described ceftriaxone-resistant extensively drug-resistant WHO X [21] and WHO Y (F89) [14] strains, and the first two strains with ceftriaxone resistance combined with high-level azithromycin resistance, i.e. AT159 [6] and WHO Q [22], are shown.

Figure 2
Phylogeny of the most closely related publicly available Neisseria gonorrhoeae genome sequences (n = 52) and the ceftriaxone-resistant, multidrug-resistant F92 gonococcal isolate from France, June 2022
of sporadic ceftriaxone-resistant cases and improved prevention, early diagnosis (including contact tracing and treatment of index case and contact) and surveillance of antimicrobial resistance, including test of cure and genome sequencing, are crucial to improve prevention, early diagnosis and surveillance. We show that the new antimicrobials zoliflodacin, gepotidacin and lefamulin all have a high in vitro activity also against the sporadic ceftriaxone-resistant strains. Indeed, novel effective and affordable antimicrobials for treatment of gonorrhoea and, ultimately, an effective gonococcal vaccine are needed.

Ethical statement
Ethical approval for the study was not necessary. Data were obtained from routine antimicrobial surveillance (standard care) and are published with a high level of anonymisation. The patient gave consent to the publication of this case report.

Funding statement
This study received financial support from the French National Public Health Agency (Saint-Maurice, France) via the French Reference Centre for Bacterial STIs, and from the Örebro County Council Research Committee and the Foundation for Medical Research at Örebro University Hospital, Sweden.