The novel 2016 WHO Neisseria gonorrhoeae reference strains for global quality assurance of laboratory investigations: phenotypic, genetic and reference genome characterization

Abstract

Objectives: Gonorrhoea and MDR Neisseria gonorrhoeae remain public health concerns globally. Enhanced, quality-assured, gonococcal antimicrobial resistance (AMR) surveillance is essential worldwide. The WHO global Gonococcal Antimicrobial Surveillance Programme (GASP) was relaunched in 2009. We describe the phenotypic, genetic and reference genome characteristics of the 2016 WHO gonococcal reference strains intended for quality assurance in the WHO global GASP, other GASPs, diagnostics and research worldwide.

Methods: The 2016 WHO reference strains (n = 14) constitute the eight 2008 WHO reference strains and six novel strains. The novel strains represent low-level to high-level cephalosporin resistance, high-level azithromycin resistance and a porA mutant. All strains were comprehensively characterized for antibiogram (n = 23), serovar, prolyliminopeptidase, plasmid types, molecular AMR determinants, N. gonorrhoeae multiantigen sequence typing STs and MLST STs. Complete reference genomes were produced using single-molecule PacBio sequencing.

Results: The reference strains represented all available phenotypes, susceptible and resistant, to antimicrobials previously and currently used or considered for future use in gonorrhoea treatment. All corresponding resistance genotypes and molecular epidemiological types were described. Fully characterized, annotated and finished references genomes (n = 14) were presented.

Conclusions: The 2016 WHO gonococcal reference strains are intended for internal and external quality assurance and quality control in laboratory investigations, particularly in the WHO global GASP and other GASPs, but also in phenotypic (e.g. culture, species determination) and molecular diagnostics, molecular AMR detection, molecular epidemiology and as fully characterized, annotated and finished reference genomes in WGS analysis, transcriptomics, proteomics and other molecular technologies and data analysis.

Figure 1.

BLAST atlas of the 2016 WHO N. gonorrhoeae reference genomes (n = 14). (a) A genome comparison of the 2016 WHO reference strains presented in this study using WHO F as reference and (b) a comparison of the up to three plasmids (named pCryptic, pBlaTEM and pConjugative^,,) identified in the same strains. WHO G pCryptic (cryptic plasmid), WHO M pBlaTEM (β-lactamase-producing plasmid) and WHO G pConjugative [conjugative plasmid including tet(M) in WHO G and N] were used as references, respectively. For each, GC skew is shown in the inner rings. The position in the genomes is shown for genetic resistance determinants and loci used for molecular diagnostics and epidemiological characterization, i.e. NG-MAST STs and MLST STs. The presence of the GGI is indicated in red. An approximately 500 bp region with lower nucleotide conservation (∼75% identity) is shown with lighter colours in WHO M and WHO O pBlaTEM plasmids corresponding to a hypothetical protein.

Figure 2.

Phylogenetic tree of the 2016 WHO N. gonorrhoeae reference core genomes (n = 14). The tree is rooted using an N. meningitidis genome as outgroup (not shown). The number of SNPs is shown on each branch. Highlighted nodes show bootstrap supports higher than 80%. Typing, genomic features and antibiotic resistance patterns of the 2016 WHO reference strains are shown alongside the tree. Only antimicrobials with a SIR categorization assigned are displayed.