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John C. Cartee1, Thitima Cherdtrakulkiat12, Sandeep J. Joseph, Rossaphorn Kittiyaowamarn, Natnaree Girdthep, Pongsathorn Sangprasert, Porntip Paopang, Thidathip Wongsurawat, Piroon Jenjaroenpun, Perapon Nitayanon, Rebekah Frankson, Silvina Masciotra, Teodora Wi, Ismael Maatouk, Ellen N. Kersh, Andrey S. Borisov, and Chanwit Tribuddharat
Author affiliation: US Centers for Disease Control and Prevention, Atlanta, Georgia, USA (J.C. Cartee, T. Cherdtrakulkiat, S.J. Joseph, R. Frankson, S. Masciotra, E.N. Kersh, A.S. Borisov); Thailand Ministry of Public Health–US Centers for Disease Control and Prevention Collaboration, Nonthaburi, Thailand (T. Cherdtrakulkiat, S. Masciotra, A.S. Borisov); Ministry of Public Health, Nonthaburi (R. Kittiyaowamarn, N. Girdthep, P. Sangprasert, P. Paopang); Mahidol University, Bangkok, Thailand (T. Wongsurawat, P. Jenjaroenpun, P. Nitayanon, C. Tribuddharat); World Health Organization, Geneva, Switzerland (T. Wi, I. Maatouk)
Neisseria meningitidis is known to cause severe invasive infections, such as meningitis, but asymptomatic N. meningitidis nasopharyngeal colonization occurs in ≈10% of the human population (1). N. meningitidis–associated urethritis cases also have been reported, including a cluster caused by nongroupable clonal complex 11 (CC11) N. meningitidis detected in the United States in 2015 (2,3). That US clade has since expanded globally, and N. meningitidis urethritis cases have been found in the United Kingdom, Japan, and Vietnam (4–6). Phylogenetic analysis revealed that the globally expanding clade has formed a distinct branch within the CC11 lineage, designated as the N. meningitidis urethritis clade (NmUC) (3,6,7). Further genomic characterization of NmUC revealed integration of multiple Neisseria gonorrhoeae genomic regions into genomes of N. meningitidis isolates via recombination, and those recombinations are thought to increase the ability of N. meningitidis to colonize the urethra (3,6,7).
Antimicrobial resistance (AMR) is not yet well established in NmUC (3), but recombination between N. meningitidis and N. gonorrhoeae is a concern because AMR is prevalent in N. gonorrhoeae (5,8). In a 2019–2020 outbreak of N. meningitidis–associated urethritis in Vietnam, isolates from NmUC had elevated MICs to ciprofloxacin (5). The isolates harbored common mutations found in N. gonorrhoeae that confer resistance to ciprofloxacin (9), which is concerning because ciprofloxacin is commonly used for prophylaxis against invasive meningococcal diseases (8). We analyzed isolates collected from a cluster of N. meningitidis–associated urethritis among men in Thailand to assess AMR and urethral adaptation.
In 2015, Thailand began surveillance for N. gonorrhoeae urethritis as part of the World Health Organization Enhanced Gonococcal Antimicrobial Surveillance Programme (EGASP; https://www.who.int/initiatives/gonococcal-antimicrobial-surveillance-programme). Through EGASP surveillance, 31 urethritis-causing N. meningitidis isolates were collected from men in Thailand during 2017–2023 (10). All cases were successfully treated with 250 mg or 500 mg intramuscular ceftriaxone in accordance with national guidelines for male patients with urethral symptoms and gram-negative intracellular diplococci. The national reference laboratory (accredited according to ISO 15189:2012) confirmed N. meningitidis in isolates by using culture and biochemical characteristics of Neisseria spp. bacteria.
We selected 16 isolates for further investigation, ensuring representation from the earliest detected case to the most recent case (Appendix). We combined whole-genome long-read sequencing using a PromethION P2i with an R10.4.1 flow cell (both Oxford Nanopore Technologies, https://nanoporetech.com) and short-read sequencing using NovaSeq 6000 (Illumina Inc., https://www.illumina.com) and 150-bp paired-end reads for hybrid genome assembly according to standard protocols (11). We used PubMLST (https://pubmlst.org) to determine the specific gene alleles and genotype of the isolates (12).
Multilocus sequence typing showed that 15 of 16 isolates belonged to CC11 and 1 to sequence type 35 clonal complex (CC35). Within the CC11 isolates, 3 isolates (NM13, NM14, and NM15) collected during April–June 2022 from men who had sex with women were separated by an average of 13 pairwise single-nucleotide polymorphisms (SNPs), representing a potential transmission cluster or locally circulating strain.
Figure 1
Figure 1. Maximum-likelihood phylogenetic tree from investigation of antimicrobial-resistant clonal complex 11 Neisseria meningitidis–associated urethritis cluster, Thailand. The tree is based on core-genome single-nucleotide polymorphisms of 259 clonal complex 11 …
To clarify the global placement of the CC11 urethral N. meningitidis isolates from Thailand, we performed core-genome SNP phylogenetic analysis on the 15 CC11 isolates from Thailand and 241 previously reported global NmUC isolates (Figure 1). Most (14/15) isolates from Thailand formed a monophyletic clade with the newly emerging NmUC-B subclade (6). NmUC-B is diverging from the original US NmUC, and isolates from NmUC-B were collected in Europe and Asia during 2019–2023. Of note, we found the first N. meningitidis isolate (NM1) collected from Thailand’s EGASP activities in 2017 formed an outgroup for the entire NmUC CC11 and is distantly related to the rest of the isolates from Thailand that clustered in the NmUC-B subclade.
Figure 2
Figure 2. Maximum-likelihood phylogenetic tree of clonal complex 35 Neisseria meningitidis isolates used in investigation of antimicrobial-resistant clonal complex 11 N. meningitidis–associated urethritis cluster, Thailand. The tree is…
We genotyped 1 isolate (NM4) as CC35 and found it was distantly related to the CC11 isolates. We conducted a phylogenetic analysis of that CC35 isolate and 63 CC35 isolates collected during 2017–2024 from Africa, Asia Pacific, Europe, and the United States (Figure 2). The other 63 isolates were collected from multiple disease types, including conjunctivitis, invasive infections, meningitidis, and septicemia, and from asymptomatic carriers. Isolate NM4 was the only isolate from a urethritis case and was most closely related (141 SNPs difference) to an isolate collected in Germany in 2017 from an invasive infection case.
We conducted antimicrobial susceptibility testing for azithromycin, cefixime, ceftriaxone, ciprofloxacin, and gentamicin on the 16 selected isolates by using ETEST Antibiotic Susceptibility Testing Reagent Strips (bioMérieux, https://www.biomerieux.com). We determined categorical MICs for N. meningitidis by following published guidelines (13). Most (15/16) isolates were susceptible to all antimicrobial drugs (Table). Three isolates from the NmUC-B subclade had an elevated MIC or resistance to ciprofloxacin: MIC for isolate NM8 was 0.125 µg/mL, for NM10 was 1.5 µg/mL, and for NM16 was 0.38 µg/mL. Upon further genomic analysis into AMR marker determinants, NM10 carried dual mutations, T91F and D95A, in the gyrA allele and NM16 carried the gyrA T91I mutation. Those gryA variants have been shown to reduce susceptibility to ciprofloxacin in both N. gonorrhoeae and N. meningitidis (5,9).
Isolate NM7, which had elevated MICs to both ceftriaxone and cefixime, harbored the penA-2840 allele type. That allele is closely related (93.6% sequence similarity) to the mosaic penA-60 allele, which can cause elevated MICs to ceftriaxone and cefixime in N. gonorrhoeae (14,15). Ceftriaxone is the recommended first-line treatment for N. gonorrhoeae and N. meningitidis infections. NM7 was also part of the NmUC-B subclade.
We also analyzed genomic features associated with N. meningitidis urethral colonization. All 16 isolates were nongroupable, and in silico prediction showed they had lost the N. meningitidis capsule. Eleven of the 16 isolates carried the insertion sequence element IS1301 in the capsule polysaccharide (cps) locus, disrupting capsule biosynthesis (Appendix Table 2). We determined that 14 of the 15 CC11 isolates carried the aniA/norB denitrification cassette associated with microaerobic and anaerobic growth, as well as urethral colonization (Appendix Table 5). Those 14 isolates were all part of the NmUC-B subclade and also harbored the ≈3-kb gonococcal partial operon NEIS1446–NEIS1442 and the gonococcal argB (NEIS1038) alleles. The single CC35 isolate did not contain the hallmark genomic features associated with urethral colonization commonly found in the NmUC. However, we found that isolate had lost its capsule and therefore was nongroupable via in silico prediction.
Our study indicates that the ongoing, global NmUC clade expanded into Thailand as early as 2017. That clade, which originated from a 2015 outbreak in the United States, has not only continued to spread to globally but also continued to evolve to create the NmUC-B subclade of isolates that have increased urethral adaptability because of more homologous recombination events with gonococcal DNA (6). That increased urethral adaptability could have contributed to the observed increase in the local prevalence of N. meningitidis–associated urethritis in Thailand.
We also identified concerning AMR genomic markers with corresponding elevated MICs to both ciprofloxacin and extended-spectrum cephalosporins in the NmUC-B isolates from Thailand. That finding is a public health concern because ciprofloxacin is used as prophylaxis for invasive N. meningitidis infections, and extended-spectrum cephalosporins are used to treat N. gonorrhoeae–associated urethritis. One isolate had a mosaic penA allele that has been associated with decreased susceptibility to ceftriaxone in N. meningitidis and cefixime in N. gonorrhoeae, demonstrating that N. meningitidis could be a reservoir for AMR variants and contribute to the spread of AMR among Neisseria spp. bacteria. Those findings raise concerns for both gonococcal and meningococcal disease control.
In summary, we found increased urethral adaptability and AMR markers among N. meningitidis isolates from Thailand. Continued global surveillance is needed to monitor the spread of urethral N. meningitidis and the possibility of further AMR in both N. meningitidis and N. gonorrhoeae.
Mr. Cartee is as a researcher in the STD Laboratory Reference and Research Branch, Division of STD Prevention, National Center for HIV, Viral Hepatitis, STD, and Tuberculosis Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, USA. His research interests include microbial population genomics specifically in Neisseria gonorrhoeae, and antimicrobial resistance and the global spread of antimicrobial resistance genes. Dr. Cherdtrakulkiat was the SCC EGASP coordinator, in Thailand under the Epidemiological Unit, Behavioral & Clinical Research Section, Division of HIV Prevention, Thailand MOPH–US CDC Collaboration, Thailand under the Division of HIV Prevention, NCHHSTP, CDC, Atlanta, Georgia, USA. She is currently the Research Project Manager at the Department of Research and Innovation, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. Her research interests include molecular epidemiology, AMR surveillance of N. gonorrhoeae, and clinical research study.
We thank the Silom Community Clinic (SCC) and the Bangrak STI Center (BSC) patients for contributing their samples and information and the SCC and BSC clinical teams for their efforts in sample and data collection. We also thank Pattaraporn Nimsamer and Worarat Kruasuwan for performing DNA QC management, library preparation for sequencing, nanopore long-read sequencing at Siriraj Long-read Lab (Si-LoL), Division of Medical Bioinformatics, Research and Innovation Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, and sending to Novogene, China, for Novaseq short-read sequencing. We also thank Eileen Dunne for the initiative and encouragement of the N. meningitidis isolate collection.
This activity was reviewed by CDC, deemed research not involving human subjects, and was conducted consistent with applicable federal law and CDC policy. The Thailand Ministry of Public Health reviewed and approved the EGASP protocol as a routine disease surveillance activity. The activity was considered a public health practice and routine surveillance. No personal identifier information was collected in the analysis database.
All fastq files can be found in the National Center for Biotechnology Information (BioProject no. PRJNA1237777). Accession numbers are provided (Appendix Table 1).
This work was supported by the National Biobank of Thailand, National Science and Technology Development Agency (NSTDA), Thailand and Siriraj Research Development Fund (project no. (OI) R016436003), Faculty of Medicine Siriraj Hospital, Mahidol University, and Siriraj Foundation fund (no. D003474). Additional support was provided by the US Centers for Disease Control and Prevention’s Antimicrobial Resistance Solutions Initiative through the Global Antimicrobial Resistance Laboratory and Response Network (cooperative agreement no. 1 NU3HCK000017-01-00).
At the time of this work, T.W. and I.M. were staff members of the World Health Organization. The authors alone are responsible for the views expressed in this article, and they do not necessarily represent the decisions, policy, or views of the World Health Organization. Use of trade names are for identification only and do not imply endorsement by any of the groups named above. This manuscript and the work described within were completed on December 5, 2024.
