Neisseria gonorrhoeae FC428 Subclone, Vietnam, 2019–2020

Among 114 clinical Neisseria gonorrhoeae isolates collected in Vietnam during 2019–2020, we detected 15 of subclone sequence type 13871 of the FC428 clonal complex. Fourteen sequence type 13871 isolates with mosaic penA allele 60.001 were ceftriaxone or cefixime nonsusceptible, and 3/14 were azithromycin nonsusceptible. Emergence of this subclone threatens treatment effectiveness.

detect antimicrobial resistance genes. We identified multilocus sequence typing (MLST) records from the Neisseria typing scheme PubMLST (https://pubmlst. org). We performed de novo assembly on the processed reads by using Shovill version 1.1.0 with SPades version 3.14 (GitHub) as the assembler. We used MOB-suite version 3.0.0 (GitHub) to reconstruct chromosome and plasmids from the assemblies. We identified N. gonorrhoeae multi-antigen sequence type (NG-MAST) by using NGMaster version 0.5.5, and we used N. gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR) with pyngSTar (GitHub). We used the closest complete genome of N. gonorrhoeae searched by ReferenceSeeker (GitHub) as reference in Snippy 4.6.0 (GitHub) for variant calling. We created the core-genome alignment by using snippy-core with a provided mask of repeated regions and mobile elements. We used Gubbin version 2.3.4 (GitHub) to filter out the recombination in the alignment and fed it into IQTREE2 (GitHub) to reconstruct a maximumlikelihood phylogenetic tree. We used BEAST version 10.4 and TreeAnnotator (https://beast.community) to estimate the time to the most recent common ancestor (tMRCA), and ggtree version 3.0.2 (GitHub) in R (https://www.r-project.org) for visualization. Sequencing data are available from the European Nucleotide Archive (https://www.ebi.ac.uk/ena; project no. PRJEB45627).
Of 114 N. gonorrhoeae isolates, 15 were typed by MLST as ST13871 (Table). All patients recovered clinically after receiving 1 dose of intramuscular ceftriaxone (1 g) and oral azithromycin (1 g), although microbiological clearance of N. gonorrhoeae was unknown. However, because neither test-of-cure nor pharyngeal testing was performed, persistent asymptomatic infection may have been missed.
In all 15 isolates, we found mutations in gyrA and parC genes, conferring resistance to ciprofloxacin, including gyrA: S91F/G95A and parC: S87R, and 2 mutations (parC: V596I, L479F) not previously reported. We found no plasmidborne tetM causing resistance to tetracycline and no other gene except for mtrR promoter −35Adel conferring resistance to azithromycin.
According to time-scaled Bayesian phylogeny of 17 ST13871 sequences from Vietnam (n = 15), France (n = 1), and Singapore (n = 1) (Figure), samples clustered into 3 distinct clades. Clade 1a contained the 2 previously reported ST13871 isolates from Singapore and France/Cambodia, as well as 3 isolates from this study, including the one sharing the same typing as the international isolates. Median tMRCA of this clade was calculated as March 2017 (95% highest  . Estimated median tMRCA for the 17 ST13871 isolates was September 2014. One clade 1b isolate came from a patient who reported having had sexual contact in Laos 1 week before diagnosis. Two of the XDR isolates belonged to clade 2 but were not closely related.

Conclusions
We detected the globally disseminated FC428-related resistant N. gonorrhoeae clone in Vietnam in 2019-2020. Among 114 N. gonorrhoeae isolates collected, 15 were ST13871 and 14 were related to the FC428 clone by harboring the mosaic penA-60.001 gene conferring resistance to ESCs. The ceftriaxone MICs for these 14 isolates were similar to those for globally reported FC428 isolates, but cefixime MICs were lower (6,12). We found 3 XDR ST13871 isolates, nonsusceptible to azithromycin and ESCs but susceptible to spectinomycin. Resistance determinants to other antimicrobial drugs in all isolates from Vietnam were similar to those of the FC428 clone (5,7,8,13,14).
Our phylogenetic analysis showed that all 17 ST13871 isolates arose from the rooted FC428 strain and were distributed into 3 clades with a common ancestor estimated in 2014, consistent with estimates of other FC428-like isolates (13). These results suggest that ST13871 has been circulating in Southeast Asia for several years. Emergence of multidrug-resistant FC428 subclone (ST13871) in Vietnam possibly threatens effectiveness of the current presumptive treatment. Therefore, regular monitoring of antimicrobial drug susceptibility of N. gonorrhoeae is necessary. Controlling the spread of resistant N. gonorrhoeae may be enhanced by follow-up visits, postrecovery culturing, and partner counseling.
This work was conducted with funding from Wellcome (106680), the Fleming Fund pilot grant Vietnam (Department of Health and Social Care, UK), and the Vietnam Fleming Fund Country Grant (Department of Health and Social Care, UK), managed by Mott MacDonald and awarded to FHI360 (FHI360 FF15/184) with a subcontract to OUCRU/University of Oxford (PO20001070), although the views expressed in this publication do not necessarily reflect those of FHI360 or Mott MacDonald. The funders had no role in study design, data collection and analysis, or the decision to publish.

About the Author
Dr. Trinh is a medical doctor at National Hospital of Dermatology and Venereology, Hanoi, Vietnam, and a member of the project of technical assistance and capacity building for identification and antimicrobial susceptibility testing for N. gonorrhoeae in the Dermatology and Venereology system in Vietnam. Her research interests are surveillance and molecular mechanisms of antimicrobial resistance of N. gonorrhoeae in Vietnam.