The novel 2016 WHONeisseria gonorrhoeaereference 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 (n¼14) 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 (n¼ 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 (n¼ 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.

Deciphering the Function of New Gonococcal Vaccine Antigens Using Phenotypic Microarrays

“…The Δngo1205 mutant and the complemented strains were generated for this study as described below or in a previous study (5,6). All mutations were introduced into the FA1090 background and WHO X (H041 [11]). Isolates of N. gonorrhoeae were streaked from frozen stocks stored in glycerol at Ϫ80°C onto gonococcal base (GCB) agar solid medium (Difco) with Kellogg's supplements I and II, diluted 1:100 and 1:1,000, respectively (116).…”

“…Treatment failures with the last viable class of antibiotics, third-generation cephalosporins, have been encountered in several countries (7,8). The development of a protective vaccine(s) is of paramount importance (9)(10)(11)(12)(13)). An exciting development in vaccine research has been the recent success of an experimental vaccine composed of membrane vesicles derived from N. gonorrhoeae FA1090 (14) coadministered with interleukin-12.…”

Peptide Inhibitors Targeting the Neisseria gonorrhoeae Pivotal Anaerobic Respiration Factor AniA

“…Expression of AniA was assessed in a panel of geographically, temporally, and genetically diverse N. gonorrhoeae isolates, including commonly used laboratory strains FA1090 (65), MS11 (75), 1291 (76), and F62 (77); clinical isolates LGB1, LG2, LG14, LG20, and LGB26, which were collected from two public health clinics in Baltimore, MD, from 1991 to 1994 and differ in their porB variable region types and pulsed-field gel electrophoresis patterns (36, 79); 13 isolates from patients attending the Public Health-Seattle & King County, WA, Sexually Transmitted Disease clinic from 2011 to 2013 (the UW strains in Fig. 2) (44); and the 2016 WHO reference strains (46). All strains were cultured concurrently on solid medium collected from GCB plates for 20 h in 5% CO 2 at 37°C, and whole-cell lysates were prepared in SDS sample buffer in the presence of 50 mM dithiothreitol, normalized by the number of OD 600 units, and separated in 4 to 20% mini-Protean TGX precast gels (Bio-Rad).…”

“…deposited into the PubMLST database (http://pubmlst.org/neisseria/, as of 25 January 2017). Further, expression of aniA was tested in whole-cell lysates derived from 36 aerobically grown different N. gonorrhoeae strains isolated from patients at different times and from different geographic locations, including the 2016 WHO reference strains (46), by SDS-PAGE and immunoblotting with polyclonal anti-AniA antiserum (36). Antiserum against AniA cross-reacted with all clinical isolates, albeit with noticeable differences in AniA levels being found between the strains (Fig.…”

Sexually transmitted infections: challenges ahead

“…Effective STI control will require the political will to prioritise and invest in new 150 rapid molecular methods for predicting AMR (for AMR surveillance but ideally also to inform individualized treatment), [190][191][192] rapid point of care tests for diagnosis of gonorrhoea (ideally with combined prediction of AMR); 190,191 ideal phylogenomics of gonococci and their AMR (also in non-cultured samples); 159,457,[459][460][461][462][463][464] and appropriate models for pharmacokinetics/ pharmacodynamics (urogenital and extragenital sites) and prediction of AMR induction/selection, evolution and biological fitness. -Data are for principal diagnosis, recorded using the ICD-9-CM from 1993-94 to 1997-98, and recording using the ICD-10-AM (Australian modification) from 1998-99.…”