To Örebro University

OBJECTIVES: Doxycycline post-exposure prophylaxis (doxycycline-PEP) can reduce incident cases of syphilis, chlamydia and possibly gonorrhoea especially among men who have sex with men with recent bacterial sexually transmitted infections (STIs). Owing to potential implementation of doxycycline-PEP internationally, global tetracycline/doxycycline resistance data for contemporary Neisseria gonorrhoeae isolates has become imperative. We report tetracycline resistance data for gonococcal isolates (n = 2993) from eight WHO Enhanced Gonococcal Antimicrobial Surveillance Programme (EGASP) countries in three WHO regions in 2021-2024, i.e. to estimate potential impact of doxycycline-PEP on the incident gonorrhoea cases in these WHO EGASP countries.

METHODS: WHO EGASP isolates cultured from men with urethral discharge in Cambodia (n = 482), Indonesia (n = 101), Malawi (n = 121), The Philippines (n = 843), South Africa (n = 597), Thailand (n = 250), Uganda (n = 350) and Vietnam (n = 249) in 2021-2024 were examined. MICs (mg/L) of tetracycline were determined using Etest.

RESULTS: The tetracycline resistance (range) using the current EUCAST (MIC > 0.5 mg/L) and CLSI (MIC > 1 mg/L) clinical resistance breakpoints in the eight WHO EGASP countries was 92.2% (83.5%-99.6%) and 80.6% (66.3%-98.6%), respectively. Using a previous minocycline-PEP resistance breakpoint (MIC > 2 mg/L) and breakpoint for high-level plasmid (tetM)-mediated tetracycline resistance (MIC > 8 mg/L), the tetracycline resistance (range) was 77.3% (47.4%-98.6%) and 74.3% (31.3%-98.6%), respectively.

CONCLUSIONS: The exceedingly high levels of gonococcal tetracycline resistance (independent of resistance breakpoint used) in the eight WHO EGASP countries elucidate that doxycycline-PEP will unlikely significantly reduce the gonorrhoea cases in these countries. Furthermore, doxycycline-PEP might rapidly select for additional gonococcal strains with tetracycline resistance (low- and high-level) and MDR/XDR strains, i.e. because these strains are mostly resistant to tetracycline.

OBJECTIVES: Zoliflodacin, a novel spiropyrimidinetrione, showed non-inferiority compared with recommended ceftriaxone plus azithromycin treatment in a recent global phase III randomized controlled trial for gonorrhea treatment. We evaluated the susceptibility of zoliflodacin among 2993 contemporary gonococcal isolates collected in 2021-2024 in eight World Health Organization (WHO) Enhanced Gonococcal Antimicrobial Surveillance Programme countries in the WHO Southeast Asian Region (Indonesia, Thailand), WHO Western Pacific Region (Cambodia, the Philippines, Viet Nam), and WHO African Region (Malawi, South Africa, Uganda).

METHODS: Minimum inhibitory concentrations (MICs) of zoliflodacin were determined using the agar dilution technique, and the zoliflodacin target gene (gyrB) was examined with Illumina sequencing.

RESULTS: Zoliflodacin exhibited high activity: MICs ranging from 0.001 to 1 mg/l and a modal MIC of 0.032 mg/l. The zoliflodacin MIC distribution showed mostly a wild-type profile; however, two isolates from Cambodia had MICs of 0.5 mg/l and 1 mg/l. These isolates also harbored the GyrB D429N mutation, associated with increased zoliflodacin MICs.

CONCLUSIONS: We show a high susceptibility to zoliflodacin internationally, including against ceftriaxone- and azithromycin-resistant gonococcal strains. Our findings support the continued clinical development of zoliflodacin as a treatment for gonorrhea, although cautious and monitored introduction and continuous international resistance surveillance are imperative.

Microbiology reference laboratories perform a crucial role within public health systems. This role was especially evident during the COVID-19 pandemic. In this Viewpoint, we emphasise the importance of microbiology reference laboratories and highlight the types of digital data and expertise they provide, which benefit national and international public health. We also highlight the value of surveillance initiatives among collaborative international partners, who work together to share, analyse, and interpret data, and then disseminate their findings in a timely manner. Microbiology reference laboratories have substantial impact at regional, national, and international levels, and sustained support for these laboratories is essential for public health in both pandemic and non-pandemic times.

INTRODUCTION: Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a global public health concern and enhanced global gonococcal AMR surveillance is imperative. As in many African countries, regular, representative and quality-assured gonococcal AMR is lacking in Ethiopia. We describe the AMR in gonococcal isolates from five cities across Ethiopia, 2021-22, and patient epidemiological data.

METHODS: Urethral discharge from males and cervical discharge from females were collected from October 2021 to September 2022. Epidemiological data were collected using a questionnaire. MIC determination (ETEST; eight antimicrobials) was performed on gonococcal isolates and EUCAST breakpoints (v13.1) were used.

RESULTS: From 1142 urogenital swab samples, 299 species-identified gonococcal isolates were identified; 78.3% were from males and 21.7% from females. The median age for males and females was 25 and 23 years, respectively. Most isolates (61.2%) were identified in Addis Ababa, followed by Gondar (11.4%), Adama (10.4%), Bahir Dar (10.0%) and Jimma (7.0%). The resistance level to ciprofloxacin, tetracycline and benzylpenicillin was 97.0%, 97.0% and 87.6%, respectively, and 87.6% of isolates were producing β-lactamase. All isolates were susceptible to ceftriaxone, cefixime, azithromycin and spectinomycin. Recommended therapy [ceftriaxone (250 mg) plus azithromycin (1 g)] was used for 84.2% of patients.

CONCLUSIONS: We present the first national quality-assured gonococcal AMR data from Ethiopia. Resistance levels to ciprofloxacin, tetracycline and benzylpenicillin were exceedingly high. However, all isolates were susceptible to ceftriaxone, cefixime, azithromycin and spectinomycin. In Ethiopia, it is essential to strengthen the gonococcal AMR surveillance by including further epidemiological data, more isolates from different cities, and WGS.

BACKGROUND: Regular quality-assured whole-genome sequencing linked to antimicrobial resistance (AMR) and patient metadata is imperative to elucidate the shifting gonorrhoea epidemiology, both nationally and internationally. We aimed to examine the gonococcal population in the European Economic Area (EEA) in 2020, elucidate emerging and disappearing gonococcal lineages associated with AMR and patient metadata, compare with 2013 and 2018 whole-genome sequencing data, and explain changes in gonococcal AMR and gonorrhoea epidemiology.

METHODS: In this retrospective genomic surveillance study, we analysed consecutive gonococcal isolates that were collected in EEA countries through the European Gonococcal Antimicrobial Surveillance Programme (Euro-GASP) in 2020, and made comparisons with Euro-GASP data from 2013 and 2018. All isolates had linked AMR data (based on minimum inhibitory concentration determination) and patient metadata. We performed whole-genome sequencing and molecular typing and AMR determinants were derived from quality-checked whole-genome sequencing data. Links between genomic lineages, AMR, and patient metadata were examined.

FINDINGS: 1932 gonococcal isolates collected in 2020 in 21 EEA countries were included. The majority (81·2%, 147 of 181 isolates) of azithromycin resistance (present in 9·4%, 181 of 1932) was explained by the continued expansion of the Neisseria gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) clonal complexes (CCs) 63, 168, and 213 (with mtrD/mtrR promoter mosaic 2) and the novel NG-STAR CC1031 (semi-mosaic mtrD variant 13), associated with men who have sex with men and anorectal or oropharyngeal infections. The declining cefixime resistance (0·5%, nine of 1932) and negligible ceftriaxone resistance (0·1%, one of 1932) was largely because of the progressive disappearance of NG-STAR CC90 (with mosaic penA allele), which was predominant in 2013. No known resistance determinants for novel antimicrobials (zoliflodacin, gepotidacin, and lefamulin) were found.

INTERPRETATION: Azithromycin-resistant clones, mainly with mtrD mosaic or semi-mosaic variants, appear to be stabilising at a relatively high level in the EEA. This mostly low-level azithromycin resistance might threaten the recommended ceftriaxone-azithromycin therapy, but the negligible ceftriaxone resistance is encouraging. The decreased genomic population diversity and increased clonality could be explained in part by the COVID-19 pandemic resulting in lower importation of novel strains into Europe.

BACKGROUND: Invasive meningococcal disease (IMD), including sepsis and meningitis, can develop when Neisseria meningitidis bacteria breach the barrier and gain access to the circulation. While IMD is a rare outcome of bacterial exposure, colonization of the oropharynx is present in approximately 10% of the human population. This asymptomatic carriage can be long or short term, and it is unknown which determining factors regulate bacterial colonization. Despite descriptions of many bacterial virulence factors and recent advances in detailed genetic identification and characterization of bacteria, the factors mediating invasion and disease vs. asymptomatic carriage following bacterial colonization remain unknown. The pharyngeal epithelia play a role in the innate immune defense against pathogens, and the aim of this study was to investigate the proinflammatory response of pharyngeal epithelial cells following meningococcal exposure to describe the potential inflammatory mediation performed during the initial host‒pathogen interaction. Clinically relevant isolates of serogroups B, C, W and Y, derived from patients with meningococcal disease as well as asymptomatic carriers, were included in the study.

RESULTS: The most potent cellular response with proinflammatory secretion of TNF, IL-6, CXCL8, CCL2, IL-1β and IL-18 was found in response to invasive serogroup B isolates. This potent response pattern was also mirrored by increased bacterial adhesion to cells as well as induced cell death. It was, however, only with serogroup B isolates where the most potent cellular response was toward the IMD isolates. In contrast, the most potent cellular response using serogroup Y isolates was directed toward the carriage isolates rather than the IMD isolates. In addition, by comparing isolates from outbreaks in Sweden (epidemiologically linked and highly genetically similar), we found the most potent proinflammatory response in cells exposed to carriage isolates rather than the IMD isolates.

CONCLUSION: Although certain expected correlations between host‒pathogen interactions and cellular proinflammatory responses were found using IMD serogroup B isolates, our data indicate that carriage isolates invoke stronger proinflammatory activation of the epithelial lining than IMD isolates.

Objectives: The novel dual-target triazaacenaphthylene, gepotidacin, recently showed promising results in its Phase III randomized controlled trial for the treatment of gonorrhoea. We investigated alterations in the gepotidacin GyrA and ParC targets in gonococci by in silico mining of publicly available global genomes (n = 33 213) and determined gepotidacin MICs in isolates with GyrA A92 alterations combined with other GyrA and/or ParC alterations.

Methods: We examined gonococcal gyrA and parC alleles available at the European Nucleotide Archive. MICs were determined using the agar dilution method (gepotidacin) or Etest (four antimicrobials). Models of DNA gyrase and topoisomerase IV were obtained from AlphaFold and used to model gepotidacin in the binding site.

Results: GyrA A92 alterations were identified in 0.24% of genomes: GyrA A92P/S/V + S91F + D95Y/A/N (0.208%), A92P + S91F (0.024%) and A92P (0.003%), but no A92T (previously associated with gepotidacin resistance) was found. ParC D86 alterations were found in 10.6% of genomes: ParC D86N/G (10.5%), D86N + S87I (0.051%), D86N + S88P (0.012%) and D86G + E91G (0.003%). One isolate had GyrA A92P + ParC D86N alterations, but remained susceptible to gepotidacin (MIC = 0.125 mg/L). No GyrA plus ParC alterations resulted in a gepotidacin MIC > 4 mg/L. Modelling of gepotidacin binding to GyrA A92/A92T/A92P suggested that gepotidacin resistance due to GyrA A92T might be linked to the formation of a new polar contact with DNA.

Conclusions: In silico mining of 33 213 global gonococcal genomes (isolates from 1928 to 2023) showed that A92 is highly conserved in GyrA, while alterations in D86 of ParC are common. No GyrA plus ParC alterations caused gepotidacin resistance. MIC determination and genomic surveillance of potential antimicrobial resistance determinants are imperative.

BACKGROUND: Antimicrobial resistance in Neisseria gonorrhoeae is threatening the gonorrhoea treatment, and optimizations of the current ceftriaxone-treatment regimens are crucial. We evaluated the pharmacodynamics of ceftriaxone single-dose therapy (0.125-1 g) against ceftriaxone-susceptible and ceftriaxone-resistant gonococcal strains, based on EUCAST ceftriaxone-resistance breakpoint (MIC > 0.125 mg/L), in our hollow fibre infection model (HFIM) for gonorrhoea.

METHODS: Gonococcal strains examined were WHO F (ceftriaxone-susceptible, MIC < 0.002 mg/L), R (ceftriaxone-resistant, MIC = 0.5 mg/L), Z (ceftriaxone-resistant, MIC = 0.5 mg/L) and X (ceftriaxone-resistant, MIC = 2 mg/L). Dose-range HFIM 7 day experiments simulating ceftriaxone 0.125-1 g single-dose intramuscular regimens were conducted.

RESULTS: Ceftriaxone 0.125-1 g single-dose treatments rapidly eradicated WHO F (wild-type ceftriaxone MIC). Ceftriaxone 0.5 and 1 g treatments, based on ceftriaxone human plasma pharmacokinetic parameters, eradicated most ceftriaxone-resistant gonococcal strains (WHO R and Z), but ceftriaxone 0.5 g failed to eradicate WHO X (high-level ceftriaxone resistance). When simulating oropharyngeal gonorrhoea, ceftriaxone 0.5 g failed to eradicate all the ceftriaxone-resistant strains, while ceftriaxone 1 g eradicated WHO R and Z (low-level ceftriaxone resistance) but failed to eradicate WHO X (high-level ceftriaxone resistance). No ceftriaxone-resistant mutants were selected using any ceftriaxone treatments.

CONCLUSIONS: Ceftriaxone 1 g single-dose intramuscularly cure most of the anogenital and oropharyngeal gonorrhoea cases caused by the currently internationally spreading ceftriaxone-resistant gonococcal strains, which should be further confirmed clinically. A ceftriaxone 1 g dose (±azithromycin 2 g) should be recommended for first-line empiric gonorrhoea treatment. This will buy countries some time until novel antimicrobials are licensed. Using ceftriaxone 1 g gonorrhoea treatment, the EUCAST ceftriaxone-resistance breakpoint is too low.

BACKGROUND: There are limited treatment options for prosthetic joint infections (PJI) due to multidrug-resistant Staphylococcus epidermidis (MDRSE). Fosfomycin (FOF) has gained attention as a potential therapy, but there is a paucity of information on the phenotypic and genotypic susceptibility amongst S. epidermidis, including MDRSE.

OBJECTIVES: To investigate phenotypical and genotypical susceptibility to FOF in S. epidermidis isolates prospectively collected from PJIs in Sweden. METHODS: MIC determination was performed using in-house agar dilution (AD) and a commercial AD panel. Genes and gene variants associated with FOF resistance were analysed.

RESULTS: Multidrug resistance was common [74/89 (83%) isolates were MDRSE].FOF inhibited all isolates except one, which had an MIC > 256 mg/L. The commercial AD panel demonstrated good overall performance but tended to overestimate the MIC, resulting in 84% essential agreement with the gold standard. Genomic analysis with publically available tools for whole-genome sequencing (WGS) data suggested genotypic FOF resistance in all isolates, but in-depth analysis revealed that fosB, associated with FOF resistance, was only present in the phenotypically resistant isolate. No other genes or gene variants associated with FOF resistance were detected.

CONCLUSIONS: Phenotypic resistance to FOF and presence of fosB were rare in this collection, indicating FOF's potential as a treatment option for S. epidermidis. The commercial AD panel demonstrated high reproducibility, but EA with the reference method was less than optimal. Findings of genotypic FOF resistance using common tools for WGS data should be critically evaluated and appropriately verified with relevant fosB references for S. epidermidis.

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.