The novel 2024 WHO Neisseria gonorrhoeae reference strains for global quality assurance of laboratory investigations and superseded WHO N. gonorrhoeae reference strains—phenotypic, genetic and reference genome characterization

Abstract Objectives MDR and XDR Neisseria gonorrhoeae strains remain major public health concerns internationally, and quality-assured global gonococcal antimicrobial resistance (AMR) surveillance is imperative. The WHO global Gonococcal Antimicrobial Surveillance Programme (GASP) and WHO Enhanced GASP (EGASP), including metadata and WGS, are expanding internationally. We present the phenotypic, genetic and reference genome characteristics of the 2024 WHO gonococcal reference strains (n = 15) for quality assurance worldwide. All superseded WHO gonococcal reference strains (n = 14) were identically characterized. Material and Methods The 2024 WHO reference strains include 11 of the 2016 WHO reference strains, which were further characterized, and four novel strains. The superseded WHO reference strains include 11 WHO reference strains previously unpublished. All strains were characterized phenotypically and genomically (single-molecule PacBio or Oxford Nanopore and Illumina sequencing). Results The 2024 WHO reference strains represent all available susceptible and resistant phenotypes and genotypes for antimicrobials currently and previously used (n = 22), or considered for future use (n = 3) in gonorrhoea treatment. The novel WHO strains include internationally spreading ceftriaxone resistance, ceftriaxone resistance due to new penA mutations, ceftriaxone plus high-level azithromycin resistance and azithromycin resistance due to mosaic MtrRCDE efflux pump. AMR, serogroup, prolyliminopeptidase, genetic AMR determinants, plasmid types, molecular epidemiological types and reference genome characteristics are presented for all strains. Conclusions The 2024 WHO gonococcal reference strains are recommended for internal and external quality assurance in laboratory examinations, especially in the WHO GASP, EGASP and other GASPs, but also in phenotypic and molecular diagnostics, AMR prediction, pharmacodynamics, epidemiology, research and as complete reference genomes in WGS analysis.


Introduction
4][25][26][27] Most of the currently identified ceftriaxone-resistant strains contain a mosaic penA-60.001[6][7][8] The WHO 3 and ECDC 29,30 have developed global and regional action plans, respectively, to control the transmission and impact of AMR gonococcal strains.One key component is to expand, improve and quality-assure the gonococcal AMR surveillance at local, national and global levels.The WHO global Gonococcal Antimicrobial Surveillance Programme (GASP) was relaunched in 2009 (www.7][8] Furthermore, the WHO Enhanced GASP (EGASP) 26,[31][32][33] is currently being expanded internationally (www.who.int/publications/i/item/9789240021341).WHO EGASP includes isolate AMR data linked to patient metadata and WGS, which is already implemented in some regional GASPs. 9,10To fulfil all the aims of WHO GASP and EGASP, valid, internationally comparable and quality-assured AMR data are imperative.This is enabled through the use of WHO reference strains. 34,35In 2016, the latest WHO gonococcal reference strain panel was published. 35erein, the 2024 WHO gonococcal reference strain panel is presented and characterized in detail.This panel includes 11 of the 2016 WHO reference strains (n = 14), 35 which were further characterized, and four novel WHO reference strains.These novel WHO strains represent highly relevant AMR phenotypes and/or genotypes that were not available for inclusion in the previous WHO reference strain panels. 34,35The novel WHO strains include the internationally spreading ceftriaxone-resistant, mosaic penA-60.001-containingFC428 strain (associated with several ceftriaxone treatment failures), 5,10,[19][20][21][22] one strain expressing ceftriaxone resistance due to a new penA mutation (associated with cefixime treatment failure), 36 the first cultured strain with ceftriaxone resistance plus high-level azithromycin resistance (mosaic penA-60.001-containingand with 23S rRNA gene A2059G mutations, associated with ceftriaxone 1 g plus doxycycline treatment failure) 24 and one internationally spreading azithromycin-resistant strain with a mosaic MtrRCDE efflux pump, i.e. with Neisseria lactamica-like mosaic 2 mtrR promoter and mtrD sequence. 10,37,38The 2024 WHO gonococcal reference strains were characterized in detail phenotypically {e.g.antibiograms [25 antimicrobials] and genetically [e.g.AMR determinants, multi-locus sequence typing (MLST), 39,40 N. gonorrhoeae multiantigen sequence typing (NG-MAST), 40,41 N. gonorrhoeae sequence typing for AMR (NG-STAR) 42 and NG-STAR clonal complexes (CCs) 43 ]}.Complete and characterized reference genomes are also described.These 2024 WHO gonococcal reference strains are recommended for internal and external quality assurance in all types of laboratory investigation, especially in the GASPs, e.g. the WHO global GASP, [6][7][8] WHO EGASP 26,[31][32][33] and other international or national GASPs but also for phenotypic and molecular diagnostics, AMR prediction, pharmacodynamics, epidemiology, research and genomics.All superseded WHO gonococcal reference strains (n = 14), including 11 not previously published WHO reference strains that have been used internationally, were characterized similarly.

Isolation of bacterial DNA
Genomic DNA for short-read and long-read sequencing was isolated using the QIAsymphony instrument (Qiagen, Hilden, Germany) and Nanobind CBB kit (PacBio, Menlo Park, CA, USA), respectively.Purified DNA was stored at 4°C before WGS.

Unemo et al.
Pacbio SMRT Tools v.7.0.1 indexed the long-read raw sequencing data in bam format using pbindex and convert it to fastq with bam2fastq.Genome assembly of these long reads were performed using both HGAP v.4.0 60 and Canu v.1.9. 61Complete chromosomes were circularized starting on the dnaA using Circlator v1.5.5. 62Illumina short reads were mapped against the circularized chromosome with BWA-MEM v.0.7.17 63 and the output filtered with samtools v.1.11 64to only keep proper-paired reads that map with a mapping quality of ≥25.These mappings were used to detect and fix base errors, small insertions/deletions (indels), local misassemblies and fill gaps in the initial long-read assembly using Pilon v.1.23. 65A minimum base and mapping qualities of 20 were required, and ≥25% of the reads mapping had to support a single nucleotide polymorphism (SNP) or indel.HGAP and Canu assemblies were compared using ACT v.18.1. 66To resolve discrepancies, we ran Trycycler v.0.4.1 67 using the raw long-read data and both chromosome sequences from each strain.No changes were needed by Pilon on the Trycycler consensus assemblies.When required, a hybrid assembly approach with Unicycler v.0.4.9b 68 was performed using the long-and short-read data.Depth of coverage was obtained by mapping to the final chromosome assemblies using pbmm2 (https://github.com/PacificBiosciences/pbmm2, based on minimap2 69 ), and BWA-MEM, respectively, followed by the samtools depth command.
Finalized circular chromosomes and plasmids were annotated using the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline v.6.6, 72 which also re-annotated the 2016 WHO gonococcal reference strains. 35Mapping of Illumina reads over the final assemblies was visually inspected using Artemis and sequencing depth across the genomes was obtained with samtools v.1.11.The core genome among the 29 strains was inferred using Panaroo v.1.2.6 73 with default parameters and strict mode, polymorphic sites were obtained using SNP-sites 74 and a maximum-likelihood tree was reconstructed from them using IQ-TREE v.2.0.3 75 with automatic detection of the best substitution model 76 (best-fit model TVM + F + ASC + R7) and 1000 ultrafast bootstrap replicates. 77Long-read sequencing data for WHO S2 was generated on a MinION Mk1C device (Oxford Nanopore Technologies) using a v.R10 flow cell (FLO-MIN114).The sequencing library was prepared without DNA fragmentation, and selection of long fragments (>3 kb) using duplex Nanopore chemistry (SQK-LSK114).Sequence data were deposited at the NCBI under BioProject PRJNA1067895.
Molecular sequence types (NG-MAST, NG-STAR and MLST) [39][40][41][42] and AMR determinants were obtained from the N. gonorrhoeae scheme at Pathogenwatch. 10,78 NG-STAR CCs were assigned using eBURST clustering on the NG-STAR ST database downloaded on 29 February 2024 (https:// ngstar.canada.ca/). 43The number of copies of the 23S rRNA gene mutations, pip gene mutants and the presence of the cppB gene in the pCryptic plasmid were inspected manually in Artemis using the finalized assemblies.Individual genome characteristics were also obtained using Artemis.DNA uptake sequences (DUSs) were located in each chromosome using the EMBOSS application fuzznuc. 79
The phenotypic characteristics of the superseded WHO reference strains (n = 14) are described in Table S1 (available as Supplementary data at JAC Online).
The genetic characteristics of the superseded WHO reference strains (n = 14) are described in Table S2.

Discussion
Herein, the 2024 WHO N. gonorrhoeae reference strains (and superseded WHO gonococcal reference strains) and their detailed phenotypic, genetic and reference genome characteristics are described.The utility of these strains includes internal and external quality assurance in all types of laboratory investigation, especially in the AMR testing (phenotypic and genetic) in GASPs, such as the WHO global GASP [6][7][8] and WHO EGASP, 26,[31][32][33] but also for phenotypic (e.g.culture, species verification) and molecular (e.g.NAATs) diagnostics, AMR prediction, pharmacodynamics, epidemiology, research and genomics.The strains include all important global susceptible; susceptible, increased exposure; and resistant phenotypes and the ranges of resistances seen for most antimicrobials currently or previously recommended in national and international gonorrhoea treatment guidelines or antimicrobials in advanced clinical development for future treatment of gonorrhoea.However, the consensus MIC values (Table 1 and Table S1) were determined using one MIC-based method only (Etest).Accordingly, these MIC values may vary slightly using other MIC-based methods, however, the resistance phenotypes should be consistent.The 2024 WHO gonococcal reference strains are available through WHO sources and from the National Collection of Type Cultures (https://www.culturecollections.org.uk).
4][125] The genetic AMR determinants that result in the different AMR phenotypes in the 2024 WHO gonococcal reference strains were characterized in detail and included most known gonococcal AMR determinants.Accordingly, the 2024 WHO reference strains can be used for internal and external quality assurance and quality controls of both conventional phenotypic AMR surveillance and surveillance using molecular AMR prediction.Molecular AMR methods can never entirely replace phenotypic culture-based AMR testing because they only detect known AMR determinants and new ones will continue to evolve.

Figure 1 .
Figure 1.Phylogenomic tree of the 2024 WHO Neisseria gonorrhoeae reference core genomes (n = 15).Typing, key genetic determinants of AMR and phenotypic AMR patterns of the 2024 WHO gonococcal reference strains are shown alongside the tree.Only antimicrobials with EUCAST breakpoints (v.14.0, https:// www.eucast.org/clinical_breakpoints)are displayed.This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Table 1 .
Serogroup, PIP production and antimicrobial susceptibility/resistance phenotypes displayed by the 2024 WHO Neisseria gonorrhoeae reference strains (n = 15), which are relevant for susceptibility testing of current, previous and novel therapeutic antimicrobials

Table 2 .
clinical susceptibility/resistance breakpoints stated by the EUCAST (v.14.0; https://www.eucast.org/clinical_breakpoints),where available.The reported MIC values are mean MICs (rounded to whole MIC doubling dilution) and the acceptable range of the MICs for each antimicrobial and the different strains is ±1 MIC doubling dilution.Note: the consensus MICs shown should be used and interpreted with caution because these were derived using one Etest method only and, consequently, may slightly differ using other methods.Genetic characteristics of relevance for epidemiology, diagnostics and AMR in the 2024 WHO Neisseria gonorrhoeae reference strains (n = 15), which are relevant for susceptibility testing of current, previous and novel therapeutic antimicrobials Characteristics b Do not produce the enzyme prolyliminopeptidase (PIP), which can result in doubtful and/or false-negative species identification of N. gonorrhoeae using biochemical or enzyme-substrate test.Global transmission of PIP-negative N. gonorrhoeae strains has been documented.45 c PPNG, penicillinase-producing N. gonorrhoeae (always considered resistant to all penicillins independent on identified MIC value, which might slightly vary).d Resistance phenotypes based on MIC (mg/L) using Etest and agar dilution (zoliflodacin, gepotidacin, lefamulin), and e No susceptibility/resistance breakpoints stated by the EUCAST (v.14.0; https://www.eucast.org/clinical_breakpoints).2024 WHO N. gonorrhoeae reference strains

Table 3 .
35clude some previously published results,35however, many additional genes and mutations, and reference genomes have been characterized in the present paper., not applicable due to frame-shift mutation that causes a premature stop codon and truncated peptide.Escherichia coli numbering (A2045 and C2597, respectively, in N. gonorrhoeae).Number of the four alleles of the 23S rRNA gene with mutations is shown in parenthesis.2024WHON. gonorrhoeae reference strains General characteristics of the reference genomes of the 2024 WHO Neisseria gonorrhoeae reference strains (n = 15) a b N/Ac N/A, not applicable because these strains were of serogroup WI (PorB1a).d