Probing fosfomycin's potential: a study on susceptibility testing and resistance in Staphylococcus epidermidis from prosthetic joint infections.

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.

Pharmacodynamic evaluation of ceftriaxone single-dose therapy (0.125-1 g) to eradicate ceftriaxone-susceptible and ceftriaxone-resistant Neisseria gonorrhoeae strains in a hollow fibre infection model for gonorrhoea.

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.

In silico gepotidacin target mining among 33 213 global Neisseria gonorrhoeae genomes from 1928 to 2023 combined with gepotidacin MIC testing of 22 gonococcal isolates with different GyrA and ParC substitutions.

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.

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.

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.

Diverse proinflammatory response in pharyngeal epithelial cells upon interaction with Neisseria meningitidis carriage and invasive isolates.

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-1ß 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.

High-level in vitro resistance to gentamicin acquired in a stepwise manner in Neisseria gonorrhoeae.

OBJECTIVES: Gentamicin is used in several alternative treatments for gonorrhoea. Verified clinical Neisseria gonorrhoeae isolates with gentamicin resistance are mainly lacking and understanding the mechanisms for gonococcal gentamicin resistance is imperative. We selected gentamicin resistance in gonococci in vitro, identified the novel gentamicin-resistance mutations, and examined the biofitness of a high-level gentamicin-resistant mutant. METHODS: Low- and high-level gentamicin resistance was selected in WHO X (gentamicin MIC = 4 mg/L) on gentamicin-gradient agar plates. Selected mutants were whole-genome sequenced. Potential gentamicin-resistance fusA mutations were transformed into WT strains to verify their impact on gentamicin MICs. The biofitness of high-level gentamicin-resistant mutants was examined using a competitive assay in a hollow-fibre infection model. RESULTS: WHO X mutants with gentamicin MICs of up to 128 mg/L were selected. Primarily selected fusA mutations were further investigated, and fusAR635L and fusAM520I + R635L were particularly interesting. Different mutations in fusA and ubiM were found in low-level gentamicin-resistant mutants, while fusAM520I was associated with high-level gentamicin resistance. Protein structure predictions showed that fusAM520I is located in domain IV of the elongation factor-G (EF-G). The high-level gentamicin-resistant WHO X mutant was outcompeted by the gentamicin-susceptible WHO X parental strain, suggesting lower biofitness. CONCLUSIONS: We describe the first high-level gentamicin-resistant gonococcal isolate (MIC = 128 mg/L), which was selected in vitro through experimental evolution. The most substantial increases of the gentamicin MICs were caused by mutations in fusA (G1560A and G1904T encoding EF-G M520I and R635L, respectively) and ubiM (D186N). The high-level gentamicin-resistant N. gonorrhoeae mutant showed impaired biofitness.

Genomic surveillance and antimicrobial resistance determinants in Neisseria gonorrhoeae isolates from Uganda, Malawi and South Africa, 2015-20.

OBJECTIVES: Global antimicrobial resistance (AMR) surveillance in Neisseria gonorrhoeae is essential. In 2017-18, only five (10.6%) countries in the WHO African Region reported to the WHO Global Gonococcal Antimicrobial Surveillance Programme (WHO GASP). Genomics enhances our understanding of gonococcal populations nationally and internationally, including AMR strain transmission; however, genomic studies from Africa are extremely scarce. We describe the gonococcal genomic lineages/sublineages, including AMR determinants, and baseline genomic diversity among strains in Uganda, Malawi and South Africa, 2015-20, and compare with sequences from Kenya and Burkina Faso. METHODS: Gonococcal isolates cultured in Uganda (n = 433), Malawi (n = 154) and South Africa (n = 99) in 2015-20 were genome-sequenced. MICs were determined using ETEST. Sequences of isolates from Kenya (n = 159), Burkina Faso (n = 52) and the 2016 WHO reference strains (n = 14) were included in the analysis. RESULTS: Resistance to ciprofloxacin was high in all countries (57.1%-100%). All isolates were susceptible to ceftriaxone, cefixime and spectinomycin, and 99.9% were susceptible to azithromycin. AMR determinants for ciprofloxacin, benzylpenicillin and tetracycline were common, but rare for cephalosporins and azithromycin. Most isolates belonged to the more antimicrobial-susceptible lineage B (n = 780) compared with the AMR lineage A (n = 141), and limited geographical phylogenomic signal was observed. CONCLUSIONS: We report the first multi-country gonococcal genomic comparison from Africa, which will support the WHO GASP and WHO enhanced GASP (EGASP). The high prevalence of resistance to ciprofloxacin (and empirical use continues), tetracycline and benzylpenicillin, and the emerging resistance determinants for azithromycin show it is imperative to strengthen the gonococcal AMR surveillance, ideally including genomics, in African countries.

Epoxy allergy, investigation of a modern industry.

BACKGROUND: Five workers from an industry manufacturing various articles from carbon fibre reinforced epoxy plastics were referred to our department because of suspected occupational allergic contact dermatitis (OACD). When patch tested, four of them had positive reactions to components of epoxy resin systems (ERSs) that could explain their current skin problems. All of them had been working at the same workstation at a specially designed pressing machine, with operations including manually mixing epoxy resin with hardener. Multiple cases of OACD in the plant prompted an investigation including all workers with possible risk exposures at the plant. OBJECTIVES: To investigate the prevalence of occupational dermatoses and contact allergies among the workers at the plant. MATERIALS AND METHODS: Totally 25 workers underwent investigation that included a brief consultation with a standardized anamnesis and clinical examination followed by patch testing. RESULTS: ERSs-related reactions were found in 7 of the 25 investigated workers. None of the seven had a history of previous exposure to ERSs and they are regarded as sensitized through work. CONCLUSIONS: Twenty-eight percent of investigated workers showed reactions to ERSs. Of these the majority would have been missed if supplementary testing would not have been added to testing with the Swedish base line series.

Multidrug-resistant Neisseria gonorrhoeae isolate SE690: mosaic penA-60.001 gene causing ceftriaxone resistance internationally has spread to the more antimicrobial-susceptible genomic lineage, Sweden, September 2022.

We report a ceftriaxone-resistant, multidrug-resistant urogenital Neisseria gonorrhoeae in a female sex worker in Sweden, September 2022, who was treated with ceftriaxone 1 g, but did not return for test-of-cure. Whole genome sequencing of isolate SE690 identified MLST ST8130, NG-STAR CC1885 (new NG-STAR ST4859) and mosaic penA-60.001. The latter, causing ceftriaxone resistance in the internationally spreading FC428 clone, has now also spread to the more antimicrobial-susceptible genomic lineage B, showing that strains across the gonococcal phylogeny can develop ceftriaxone resistance.

GyrB in silico mining in 27 151 global gonococcal genomes from 1928-2021 combined with zoliflodacin in vitro testing of 71 international gonococcal isolates with different GyrB, ParC and ParE substitutions confirms high susceptibility.

OBJECTIVES: Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a global threat and novel treatment alternatives are imperative. Herein, susceptibility to the novel antimicrobial zoliflodacin, currently in a global Phase 3 randomized controlled clinical trial for gonorrhoea treatment, was investigated by screening for zoliflodacin GyrB target mutations in publicly available gonococcal genomes and, where feasible, determination of the associated zoliflodacin MIC. METHODS: The European Nucleotide Archive was queried using the search term 'Taxon: 485'. DNA sequences from 27 151 gonococcal isolates were analysed and gyrB, gyrA, parC and parE alleles characterized. RESULTS: GyrB amino acid alterations were rare (97.0% of isolates had a wild-type GyrB sequence). GyrB V470L (2.7% of isolates) was the most prevalent alteration, followed by S467N (0.12%), N. meningitidis GyrB (0.092%), V470I (0.059%), Q468R/P (0.015%), A466T (0.0074%), L425I + L465I (0.0037%), L465I (0.0037%), G482S (0.0037%) and D429V (0.0037%). Only one isolate (0.0037%) carried a substitution in a resistance-associated GyrB codon (D429V), resulting in a zoliflodacin MIC of 8 mg/L. None of the other detected gyrB, gyrA, parC or parE mutations caused a zoliflodacin MIC outside the wild-type MIC distribution. CONCLUSIONS: The zoliflodacin target GyrB was highly conserved among 27 151 global gonococcal isolates cultured in 1928-2021. The single zoliflodacin-resistant clinical isolate (0.0037%) was cultured from a male patient in Japan in 2000. Evidently, this strain has not clonally expanded nor has the gyrB zoliflodacin-resistance mutation disseminated through horizontal gene transfer to other strains. Phenotypic and genomic surveillance, including gyrB mutations, of zoliflodacin susceptibility are imperative.

Genomic surveillance and antimicrobial resistance in Neisseria gonorrhoeae isolates in Bangkok, Thailand in 2018.

OBJECTIVES: Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a substantial global public health problem. Gonococcal infections acquired in or from Asia represent most verified ceftriaxone treatment failures, and several ceftriaxone-resistant strains have emerged in Asia and subsequently spread globally. Additionally, in Thailand the gonorrhoea incidence remains high. Herein, we investigate the genomic diversity, AMR and AMR determinants in gonococcal isolates cultured in 2018 in Bangkok, Thailand. METHODS: Gonococcal isolates from males (n = 37) and females (n = 62) were examined by Etest and WGS. AMR determinants and molecular epidemiological STs were characterized. For phylogenomic comparison, raw sequence data were included from China (432 isolates), Japan (n = 270), Vietnam (n = 229), Thailand (n = 3), a global dataset (n = 12 440) and the 2016 WHO reference strains plus WHO Q (n = 15). RESULTS: In total, 88, 66 and 41 different NG-MAST, NG-STAR and MLST STs, respectively, and 31 different NG-STAR clonal complexes were found. A remarkably high frequency (88%) of ß-lactamase TEM genes was detected and two novel TEM alleles were found. The phylogenomic analysis divided the isolates into the previously described lineages A and B, with a large proportion of Thai isolates belonging to the novel sublineage A3. CONCLUSIONS: We describe the first molecular epidemiological study using WGS on gonococcal isolates from Thailand. The high prevalence of AMR and AMR determinants for ciprofloxacin, tetracycline and benzylpenicillin, and some strains belonging to clones/clades especially in sublineage A2 that are prone to develop resistance to extended-spectrum cephalosporins (ESCs) and azithromycin, should prompt continued and strengthened AMR surveillance, including WGS, of N. gonorrhoeae in Thailand.

Significant increase in azithromycin "resistance" and susceptibility to ceftriaxone and cefixime in Neisseria gonorrhoeae isolates in 26 European countries, 2019.

BACKGROUND: The European Gonococcal Antimicrobial Surveillance Programme (Euro-GASP) performs annual sentinel surveillance of Neisseria gonorrhoeae susceptibility to therapeutically relevant antimicrobials across the European Union/European Economic Area (EU/EEA). We present the Euro-GASP results from 2019 (26 countries), linked to patient epidemiological data, and compared with data from previous years. METHODS: Agar dilution and minimum inhibitory concentration (MIC) gradient strip methodologies were used to determine the antimicrobial susceptibility (using EUCAST clinical breakpoints, where available) of 3239 N. gonorrhoeae isolates from 26 countries across the EU/EEA. Significance of differences compared with Euro-GASP results in previous years was analysed using Z-test and the Pearson's χ2 test was used to assess significance of odds ratios for associations between patient epidemiological data and antimicrobial resistance. RESULTS: European N. gonorrhoeae isolates collected between 2016 and 2019 displayed shifting MIC distributions for; ceftriaxone, with highly susceptible isolates increasing over time and occasional resistant isolates each year; cefixime, with highly-susceptible isolates becoming increasingly common; azithromycin, with a shift away from lower MICs towards higher MICs above the EUCAST epidemiological cut-off (ECOFF); and ciprofloxacin which is displaying a similar shift in MICs as observed for azithromycin. In 2019, two isolates displayed ceftriaxone resistance, but both isolates had MICs below the azithromycin ECOFF. Cefixime resistance (0.8%) was associated with patient sex, with resistance higher in females compared with male heterosexuals and men-who-have-sex-with-men (MSM). The number of countries reporting isolates with azithromycin MICs above the ECOFF increased from 76.9% (20/26) in 2016 to 92.3% (24/26) in 2019. Isolates with azithromycin MICs above the ECOFF (9.0%) were associated with pharyngeal infection sites. Following multivariable analysis, ciprofloxacin resistance remained associated with isolates from MSM and heterosexual males compared with females, the absence of a concurrent chlamydial infection, pharyngeal infection sites and patients ≥ 25 years of age. CONCLUSIONS: Resistance to ceftriaxone and cefixime remained uncommon in EU/EEA countries in 2019 with a significant decrease in cefixime resistance observed between 2016 and 2019. The significant increase in azithromycin "resistance" (azithromycin MICs above the ECOFF) threatens the effectiveness of the dual therapy (ceftriaxone + azithromycin), i.e., for ceftriaxone-resistant cases, currently recommended in many countries internationally and requires close monitoring.

Extensively drug-resistant (XDR) Neisseria gonorrhoeae causing possible gonorrhoea treatment failure with ceftriaxone plus azithromycin in Austria, April 2022.

We describe a gonorrhoea case with ceftriaxone plus high-level azithromycin resistance. In April 2022, an Austrian heterosexual male was diagnosed with gonorrhoea after sexual intercourse with a female sex worker in Cambodia. Recommended treatment with ceftriaxone (1 g) plus azithromycin (1.5 g) possibly failed. Worryingly, this is the second strain in an Asian Neisseria gonorrhoeae genomic sublineage including high-level azithromycin-resistant strains that developed ceftriaxone resistance by acquisition of mosaic penA-60.001. Enhanced resistance surveillance and actions are imperative to prevent spread.

The European response to control and manage multi- and extensively drug-resistant Neisseria gonorrhoeae.

Because cefixime and ceftriaxone resistance in Neisseria gonorrhoeae and gonorrhoea treatment failures were increasing, a response plan to control and manage multidrug-resistant N. gonorrhoeae (MDR-NG) in Europe was published in 2012. The three main areas of the plan were to: (i) strengthen surveillance of antimicrobial resistance (AMR), (ii) implement monitoring of treatment failures and (iii) establish a communication strategy to increase awareness and disseminate AMR results. Since 2012, several additional extensively drug-resistant N. gonorrhoeae (XDR-NG) strains have emerged, and strains with high-level ceftriaxone resistance spread internationally. This prompted an evaluation and review of the 2012 European Centre for Disease Prevention and Control (ECDC) response plan, revealing an overall improvement in many aspects of monitoring AMR in N. gonorrhoeae; however, treatment failure monitoring was a weakness. Accordingly, the plan was updated in 2019 to further support European Union/European Economic Area (EU/EEA) countries in controlling and managing the threat of MDR/XDR-NG in Europe through further strengthening of AMR surveillance and clinical management including treatment failure monitoring. The plan will be assessed biennially to ensure its effectiveness and its value. Along with prevention, diagnostic, treatment and epidemiological surveillance strategies, AMR surveillance is essential for effective control of gonorrhoea.

Ceftriaxone-resistant, multidrug-resistant Neisseria gonorrhoeae with a novel mosaic penA-237.001 gene, France, June 2022.

We report a ceftriaxone-resistant, multidrug-resistant urogenital gonorrhoea case in a heterosexual woman in France, June 2022. The woman was successfully treated with azithromycin 2 g. She had unprotected sex with her regular partner, who developed urethritis following travel to Vietnam and Switzerland. Whole genome sequencing of the gonococcal isolate (F92) identified MLST ST1901, NG-STAR CC-199, and the novel mosaic penA-237.001, which caused ceftriaxone resistance. penA-237.001 is 98.7% identical to penA-60.001, reported in various ceftriaxone-resistant strains, including the internationally spreading FC428 clone.

High susceptibility to zoliflodacin and conserved target (GyrB) for zoliflodacin among 1209 consecutive clinical Neisseria gonorrhoeae isolates from 25 European countries, 2018.

OBJECTIVES: Novel antimicrobials for treatment of gonorrhoea are imperative. The first-in-class spiropyrimidinetrione zoliflodacin is promising and currently in an international Phase 3 randomized controlled clinical trial (RCT) for treatment of uncomplicated gonorrhoea. We evaluated the in vitro activity of and the genetic conservation of the target (GyrB) and other potential zoliflodacin resistance determinants among 1209 consecutive clinical Neisseria gonorrhoeae isolates obtained from 25 EU/European Economic Area (EEA) countries in 2018 and compared the activity of zoliflodacin with that of therapeutic antimicrobials currently used. METHODS: MICs of zoliflodacin, ceftriaxone, cefixime, azithromycin and ciprofloxacin were determined using an agar dilution technique for zoliflodacin or using MIC gradient strip tests or an agar dilution technique for the other antimicrobials. Genome sequences were available for 96.1% of isolates. RESULTS: Zoliflodacin modal MIC, MIC50, MIC90 and MIC range were 0.125, 0.125, 0.125 and ≤0.004-0.5 mg/L, respectively. The resistance was 49.9%, 6.7%, 1.6% and 0.2% to ciprofloxacin, azithromycin, cefixime and ceftriaxone, respectively. Zoliflodacin did not show any cross-resistance to other tested antimicrobials. GyrB was highly conserved and no zoliflodacin gyrB resistance mutations were found. No fluoroquinolone target GyrA or ParC resistance mutations or mutations causing overexpression of the MtrCDE efflux pump substantially affected the MICs of zoliflodacin. CONCLUSIONS: The in vitro susceptibility to zoliflodacin was high and the zoliflodacin target GyrB was conserved among EU/EEA gonococcal isolates in 2018. This study supports further clinical development of zoliflodacin. However, additional zoliflodacin data regarding particularly the treatment of pharyngeal gonorrhoea, pharmacokinetics/pharmacodynamics and resistance selection, including suppression, would be valuable.

Evaluation of the SpeeDx ResistancePlus® GC and SpeeDx GC 23S 2611 (beta) molecular assays for prediction of antimicrobial resistance/susceptibility to ciprofloxacin and azithromycin in Neisseria gonorrhoeae.

BACKGROUND: Accurate molecular assays for prediction of antimicrobial resistance (AMR)/susceptibility in Neisseria gonorrhoeae (Ng) can offer individualized treatment of gonorrhoea and enhanced AMR surveillance. OBJECTIVES: We evaluated the new ResistancePlus® GC assay and the GC 23S 2611 (beta) assay (SpeeDx), for prediction of resistance/susceptibility to ciprofloxacin and azithromycin, respectively. METHODS: Nine hundred and sixty-seven whole-genome-sequenced Ng isolates from 20 European countries, 143 Ng-positive (37 with paired Ng isolates) and 167 Ng-negative clinical Aptima Combo 2 (AC2) samples, and 143 non-gonococcal Neisseria isolates and closely related species were examined with both SpeeDx assays. RESULTS: The sensitivity and specificity of the ResistancePlus® GC assay to detect Ng in AC2 samples were 98.6% and 100%, respectively. ResistancePlus® GC showed 100% sensitivity and specificity for GyrA S91 WT/S91F detection and 99.8% sensitivity and specificity in predicting phenotypic ciprofloxacin resistance. The sensitivity and specificity of the GC 23S 2611 (beta) assay for Ng detection in AC2 samples were 95.8% and 100%, respectively. GC 23S 2611 (beta) showed 100% sensitivity and 99.9% specificity for 23S rRNA C2611 WT/C2611T detection and 64.3% sensitivity and 99.9% specificity for predicting phenotypic azithromycin resistance. Cross-reactions with non-gonococcal Neisseria species were observed with both assays, but the analysis software solved most cross-reactions. CONCLUSIONS: The new SpeeDx ResistancePlus® GC assay performed well in the detection of Ng and AMR determinants, especially in urogenital samples. The GC 23S 2611 (beta) assay performed relatively well, but its sensitivity, especially for predicting phenotypic azithromycin resistance, was suboptimal and further optimizations are required, including detection of additional macrolide resistance determinant(s).

Associations between antimicrobial susceptibility/resistance of Neisseria gonorrhoeae isolates in European Union/European Economic Area and patients' gender, sexual orientation and anatomical site of infection, 2009-2016.

BACKGROUND: The emergence and spread of antimicrobial resistance (AMR) in Neisseria gonorrhoeae, nationally and internationally, is a serious threat to the management and control of gonorrhoea. Limited and conflicting data regarding the epidemiological drivers of gonococcal AMR internationally have been published. We examined the antimicrobial susceptibility/resistance of gonococcal isolates (n = 15,803) collected across 27 European Union/European Economic Area (EU/EEA) countries in 2009-2016, in conjunction to epidemiological and clinical data of the corresponding patients, to elucidate associations between antimicrobial susceptibility/resistance and patients' gender, sexual orientation and anatomical site of infection. METHODS: In total, 15,803 N. gonorrhoeae isolates from the European Gonococcal Antimicrobial Surveillance Programme (Euro-GASP), 2009-2016, were examined. Associations between gonococcal susceptibility/resistance and patients' gender, sexual orientation and anatomical site of infection were investigated using univariate and multivariate logistic regression analysis. Statistical significance was determined by Pearson χ2-test or Fisher's exact test with two-tailed p-values of < 0.05 indicating significance. RESULTS: The overall gonococcal resistance from 2009 to 2016 was 51.7% (range during the years: 46.5-63.5%), 7.1% (4.5-13.2%), 4.3% (1.8-8.7%), and 0.2% (0.0-0.5%) to ciprofloxacin, azithromycin, cefixime, and ceftriaxone, respectively. The level of resistance combined with decreased susceptibility to ceftriaxone was 10.2% (5.7-15.5%). Resistance to cefixime and ciprofloxacin, and resistance combined with decreased susceptibility to ceftriaxone were positively associated with urogenital infections and heterosexual males, males with sexual orientation not reported and females (except for ciprofloxacin), i.e. when compared to men-who-have-sex-with-men (MSM). Azithromycin resistance was positively associated with heterosexual males, but no association was significant regarding anatomical site of infection. CONCLUSIONS: Overall, sexual orientation was the main variable associated with gonococcal AMR. Strongest positive associations were identified with heterosexual patients, particularly males, and not MSM. To provide evidence-based understanding and mitigate gonococcal AMR emergence and spread, associations between antimicrobial susceptibility/resistance and patients' gender, sexual orientation and anatomical site of infection need to be further investigated in different geographic settings. In general, these insights will support identification of groups at increased risk and targeted public health actions such as intensified screening, 3-site testing using molecular diagnostics, sexual contact tracing, and surveillance of treatment failures.

Antimicrobial resistance and molecular epidemiological typing of Neisseria gonorrhoeae isolates from Kyrgyzstan in Central Asia, 2012 and 2017.

BACKGROUND: Gonorrhoea and antimicrobial resistance (AMR) in Neisseria gonorrhoeae are significant public health concerns globally. Nearly no gonococcal AMR data are available from Central Asia, and no data from Kyrgyzstan has been published. We examined, for the first time, AMR and molecular epidemiology of N. gonorrhoeae isolates cultured in Kyrgyzstan in 2012 and 2017, in order to inform refinements of the Kyrgyz national gonorrhoea management guidelines. METHODS: N. gonorrhoeae isolates cultured in 2012 (n = 84) and 2017 (n = 72) in Kyrgyzstan were examined. MICs of nine antimicrobials were determined using Etest and, where available, clinical breakpoints from the EUCAST were applied. N. gonorrhoeae multiantigen sequence typing (NG-MAST) was also performed. RESULTS: The overall resistance levels were high to ciprofloxacin (88.5%), tetracycline (56.9%), benzylpenicillin (39.1%), and kanamycin (4.7%). Resistance to cefixime (0.6%, n = 1 isolate), azithromycin (0.6%, n = 1), and gentamicin (0.6%, n = 1) was rare. No resistance to ceftriaxone or spectinomycin was found. However, the proportion of isolates with decreased susceptibility (MIC = 0.125 mg/L) to ceftriaxone and cefixime was 12.8 and 11.5%, respectively. Gonococcal isolates were assigned 69 sequence types, of which 52 (75.4%) were new. CONCLUSIONS: The gonococcal population in Kyrgyzstan in 2012 and 2017 showed a high genetic diversity. Ceftriaxone, 500-1000 mg, in combination with azithromycin 2 g or doxycycline, particularly when chlamydial infection has not been excluded, should be recommended as empiric first-line treatment. Spectinomycin 2 g could be an alternative treatment, and given with azithromycin 2 g if pharyngeal gonorrhoea has not been excluded. Fluoroquinolones, aminoglycosides, benzylpenicillin, or tetracyclines should not be used for empiric treatment of gonorrhoea in Kyrgyzstan. Timely updating and high compliance to national gonorrhoea treatment guidelines based on quality-assured AMR data is imperative. Expanded and improved gonococcal AMR surveillance in Kyrgyzstan is crucial.

Antimicrobial resistance in Neisseria gonorrhoeae isolates and gonorrhoea treatment in the Republic of Belarus, Eastern Europe, 2009-2019.

BACKGROUND: Limited antimicrobial resistance (AMR) data for Neisseria gonorrhoeae are available in Eastern Europe. We investigated AMR in N. gonorrhoeae isolates in the Republic of Belarus from 2009 to 2019, antimicrobial treatment recommended nationally, and treatment given to patients with gonorrhoea. METHODS: N. gonorrhoeae isolates (n = 522) cultured in three regions of Belarus in 2009-2019 were examined. Determination of minimum inhibitory concentrations (MICs) of eight antimicrobials was performed using Etest. Resistance breakpoints from the European Committee on Antimicrobial Susceptibility Testing were applied where available. A Nitrocefin test identified ß-lactamase production. Gonorrhoea treatment for 1652 patients was also analysed. Statistical significance was determined by the Z-test, Fisher's exact test, or Mann-Whitney U test with p-values of < 0.05 indicating significance. RESULTS: In total, 27.8% of the N. gonorrhoeae isolates were resistant to tetracycline, 24.7% to ciprofloxacin, 7.0% to benzylpenicillin, 2.7% to cefixime, and 0.8% to azithromycin. No isolates were resistant to ceftriaxone, spectinomycin, or gentamicin. However, 14 (2.7%) isolates had a ceftriaxone MIC of 0.125 mg/L, exactly at the resistance breakpoint (MIC > 0.125 mg/L). Only one (0.2%) isolate, from 2013, produced ß-lactamase. From 2009 to 2019, the levels of resistance to ciprofloxacin and tetracycline were relatively high and stable. Resistance to cefixime was not identified before 2013 but peaked at 22.2% in 2017. Only sporadic isolates with resistance to azithromycin were found in 2009 (n = 1), 2012 (n = 1), and 2018-2019 (n = 2). Overall, 862 (52.2%) patients received first-line treatment according to national guidelines (ceftriaxone 1 g). However, 154 (9.3%) patients received a nationally recommended alternative treatment (cefixime 400 mg or ofloxacin 400 mg), and 636 (38.5%) were given non-recommended treatment. CONCLUSIONS: The gonococcal resistance to ciprofloxacin and tetracycline was high, however, the resistance to azithromycin was low and no resistance to ceftriaxone was identified. Ceftriaxone 1 g can continuously be recommended as empiric first-line gonorrhoea therapy in Belarus. Fluoroquinolones should not be prescribed for treatment if susceptibility has not been confirmed by testing. Timely updating and high compliance with national evidence-based gonorrhoea treatment guidelines based on quality-assured AMR data are imperative. The need for continued, improved and enhanced surveillance of gonococcal AMR in Belarus is evident.