Genetic Manipulation of Neisseria gonorrhoeae and Commensal Neisseria Species.

The sexually transmitted pathogen, Neisseria gonorrhoeae, undergoes natural transformation at high frequency. This property has led to the rapid dissemination of antibiotic resistance markers and the panmictic structure of the gonococcal population. However, high-frequency transformation also makes N. gonorrhoeae one of the easiest bacterial species to manipulate genetically in the laboratory. Techniques have been developed that result in transformation frequencies >50%, allowing the identification of mutants by screening and without selection. Constructs have been created to take advantage of this high-frequency transformation, facilitating genetic mutation, complementation, and heterologous gene expression. Similar methods have been developed for N. meningitidis and nonpathogenic Neisseria including N. mucosa and N. musculi. Techniques are described for genetic manipulation of N. gonorrhoeae and commensal Neisseria species, as well as for growth of these fastidious organisms. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Spot transformation of Neisseria gonorrhoeae on agar plates Basic Protocol 2: Spot transformation of commensal Neisseria on agar plates Basic Protocol 3: Transformation of Neisseria gonorrhoeae in liquid culture Basic Protocol 4: Electroporation of Neisseria gonorrhoeae Basic Protocol 5: Creation of unmarked mutations using a positive and negative selection cassette Basic Protocol 6: In vitro mutagenesis of Neisseria gonorrhoeae chromosomal DNA using EZ-Tn5 Basic Protocol 7: Chemical mutagenesis Basic Protocol 8: Complementation on the Neisseria gonorrhoeae chromosome Alternate Protocol 1: Complementation with replicating plasmids Alternate Protocol 2: Complementation on the Neisseria musculi or Neisseria mucosa chromosome Basic Protocol 9: Preparation of chromosomal DNA from Neisseria gonorrhoeae grown on solid medium Alternate Protocol 3: Preparation of chromosomal DNA from Neisseria gonorrhoeae grown in broth Support Protocol: Preparing PCR templates from Neisseria gonorrhoeae colonies.

Sulfonamide inhibitors of bacterial carbonic anhydrases.

The increasing prevalence of antibiotic-resistant bacteria necessitates the exploration of novel therapeutic targets. Bacterial carbonic anhydrases (CAs) have been known for decades, but only in the past ten years they have garnered significant interest as drug targets to develop antibiotics having a diverse mechanism of action compared to the clinically used drugs. Significant progress has been made in the field in the past three years, with the validation in vivo of CAs from Neisseria gonorrhoeae, and vancomycin-resistant enterococci as antibiotic targets. This chapter compiles the state-of-the-art research on sulfonamide derivatives described as inhibitors of all known bacterial CAs. A section delves into the mechanisms of action of sulfonamide compounds with the CA classes identified in pathogenic bacteria, specifically α, ß, and γ classes. Therefore, the inhibitory profiling of the bacterial CAs with classical and clinically used sulfonamide compounds is reported and analyzed. Another section covers various other series of sulfonamide CA inhibitors studied for the development of new antibiotics. By synthesizing current research findings, this chapter highlights the potential of sulfonamide inhibitors as a novel class of antibacterial agents and paves the way for future drug design strategies.

Neisseria gonorrhoeae carbonic anhydrase inhibition.

Carbonic anhydrases (CAs) are ubiquitous enzymes that are found in all kingdoms of life. Though different classes of CAs vary in their roles and structures, their primary function is to catalyze the reaction between carbon dioxide and water to produce bicarbonate and a proton. Neisseria gonorrhoeae encodes for three distinct CAs (NgCAs) from three different families: an α-, a ß-, and a γ-isoform. This chapter details the differences between the three NgCAs, summarizing their subcellular locations, roles, essentiality, structures, and enzyme kinetics. These bacterial enzymes have the potential to be drug targets; thus, previous studies have investigated the inhibition of NgCAs-primarily the α-isoform. Therefore, the classes of inhibitors that have been shown to bind to the NgCAs will be discussed as well. These classes include traditional CA inhibitors, such as sulfonamides, phenols, and coumarins, as well as non-traditional inhibitors including anions and thiocarbamates.

Antimicrobial activity of compounds identified by artificial intelligence discovery engine targeting enzymes involved in Neisseria gonorrhoeae peptidoglycan metabolism.

BACKGROUND: Neisseria gonorrhoeae (Ng) causes the sexually transmitted disease gonorrhoea. There are no vaccines and infections are treated principally with antibiotics. However, gonococci rapidly develop resistance to every antibiotic class used and there is a need for developing new antimicrobial treatments. In this study we focused on two gonococcal enzymes as potential antimicrobial targets, namely the serine protease L,D-carboxypeptidase LdcA (NgO1274/NEIS1546) and the lytic transglycosylase LtgD (NgO0626/NEIS1212). To identify compounds that could interact with these enzymes as potential antimicrobials, we used the AtomNet virtual high-throughput screening technology. We then did a computational modelling study to examine the interactions of the most bioactive compounds with their target enzymes. The identified compounds were tested against gonococci to determine minimum inhibitory and bactericidal concentrations (MIC/MBC), specificity, and compound toxicity in vitro. RESULTS: AtomNet identified 74 compounds that could potentially interact with Ng-LdcA and 84 compounds that could potentially interact with Ng-LtgD. Through MIC and MBC assays, we selected the three best performing compounds for both enzymes. Compound 16 was the most active against Ng-LdcA, with a MIC50 value < 1.56 µM and MBC50/90 values between 0.195 and 0.39 µM. In general, the Ng-LdcA compounds showed higher activity than the compounds directed against Ng-LtgD, of which compound 45 had MIC50 values of 1.56-3.125 µM and MBC50/90 values between 3.125 and 6.25 µM. The compounds were specific for gonococci and did not kill other bacteria. They were also non-toxic for human conjunctival epithelial cells as judged by a resazurin assay. To support our biological data, in-depth computational modelling study detailed the interactions of the compounds with their target enzymes. Protein models were generated in silico and validated, the active binding sites and amino acids involved elucidated, and the interactions of the compounds interacting with the enzymes visualised through molecular docking and Molecular Dynamics Simulations for 50 ns and Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA). CONCLUSIONS: We have identified bioactive compounds that appear to target the N. gonorrhoeae LdcA and LtgD enzymes. By using a reductionist approach involving biological and computational data, we propose that compound Ng-LdcA-16 and Ng-LtgD-45 are promising anti-gonococcal compounds for further development.

Molecular Basis of Influence of A501X Mutations in Penicillin-Binding Protein 2 of Neisseria gonorrhoeae Strain 35/02 on Ceftriaxone Resistance.

The increase in the resistance of mutant strains of Neisseria gonorrhoeae to the antibiotic ceftriaxone is pronounced in the decrease in the second-order acylation rate constant, k2/KS, by penicillin-binding protein 2 (PBP2). These changes can be caused by both the decrease in the acylation rate constant, k2, and the weakening of the binding affinity, i.e., an increase in the substrate constant, KS. A501X mutations in PBP2 affect second-order acylation rate constants. The PBP2A501V variant exhibits a higher k2/KS value, whereas for PBP2A501R and PBP2A501P variants, these values are lower. We performed molecular dynamic simulations with both classical and QM/MM potentials to model both acylation energy profiles and conformational dynamics of four PBP2 variants to explain the origin of k2/KS changes. The acylation reaction occurs in two elementary steps, specifically, a nucleophilic attack by the oxygen atom of the Ser310 residue and C-N bond cleavage in the ß-lactam ring accompanied by the elimination of the leaving group of ceftriaxone. The energy barrier of the first step increases for PBP2 variants with a decrease in the observed k2/KS value. Submicrosecond classic molecular dynamic trajectories with subsequent cluster analysis reveal that the conformation of the ß3-ß4 loop switches from open to closed and its flexibility decreases for PBP2 variants with a lower k2/KS value. Thus, the experimentally observed decrease in the k2/KS in A501X variants of PBP2 occurs due to both the decrease in the acylation rate constant, k2, and the increase in KS.

Cyclization increases bactericidal activity of arginine-rich cationic cell-penetrating peptide for Neisseria gonorrhoeae.

We previously reported that a linear cationic 12-amino acid cell-penetrating peptide (CPP) was bactericidal for Neisseria gonorrhoeae. In this study, our objectives were to determine the effect of cyclization of the linear CPP on its antibacterial activity for N. gonorrhoeae and cytotoxicity for human cells. We compared the bactericidal effect of 4-hour treatment with the linear CPP to that of CPPs cyclized by a thioether or a disulfide bond on human challenge and multi-drug resistant (MDR) strains of N. gonorrhoeae grown in cell culture media with 10% fetal bovine serum (FBS). The effect of lipooligosaccharide (LOS) sialylation on bactericidal activity was analyzed. We determined the ability of the CPPs to treat human cells infected in vitro with N. gonorrhoeae, to reduce the inflammatory response of human monocytic cells to gonococci, to kill strains of three commensal Neisseria species, and to inhibit gonococcal biofilms. The cyclized CPPs killed 100% of gonococci from all strains at 100 µM and >90% at 20 µM and were more potent than the linear form. The thioether-linked but not the disulfide-linked CPP was less cytotoxic for human cervical cells compared to the linear CPP. LOS sialylation had minimal effect on bactericidal activity. In treating infected human cells, the thioether-linked CPP at 20 µM killed >60% of extra- and intracellular bacteria and reduced TNF-α expression by THP-1 cells. The potency of the CPPs for the pathogenic and the commensal Neisseria was similar. The thioether-linked CPP partially eradicated gonococcal biofilms. Future studies will focus on determining efficacy in the female mouse model of gonorrhea.IMPORTANCENeisseria gonorrhoeae remains a major cause of sexually transmitted infections with 82 million cases worldwide in 2020, and 710,151 confirmed cases in the US in 2021, up 25% from 2017. N. gonorrhoeae can infect multiple tissues including the urethra, cervix, rectum, pharynx, and conjunctiva. The most serious sequelae are suffered by infected women as gonococci ascend to the upper reproductive tract and cause pelvic inflammatory disease, chronic pelvic pain, and infertility in 10%-20% of women. Control of gonococcal infection is widely recognized as increasingly challenging due to the lack of any vaccine. N. gonorrhoeae has quickly developed resistance to all but one class of antibiotics and the emergence of multidrug-resistant strains could result in untreatable infections. As such, gonorrhea is classified by the Center for Disease Control (CDC) as an urgent public health threat. The research presented herein on new therapeutics for gonorrhea has identified a cyclic cell-penetrating peptide (CPP) as a potent molecule targeting N. gonorrhoeae.

Assessing thresholds of resistance prevalence at which empiric treatment of gonorrhea should change among men who have sex with men in the US: A cost-effectiveness analysis.

BACKGROUND: Since common diagnostic tests for gonorrhea do not provide information about susceptibility to antibiotics, treatment of gonorrhea remains empiric. Antibiotics used for empiric therapy are usually changed once resistance prevalence exceeds a certain threshold (e.g., 5%). A low switch threshold is intended to increase the probability that an infection is successfully treated with the first-line antibiotic, but it could also increase the pace at which recommendations are switched to newer antibiotics. Little is known about the impact of changing the switch threshold on the incidence of gonorrhea, the rate of treatment failure, and the overall cost and quality-adjusted life-years (QALYs) associated with gonorrhea. METHODS AND FINDINGS: We developed a transmission model of gonococcal infection with multiple resistant strains to project gonorrhea-associated costs and loss in QALYs under different switch thresholds among men who have sex with men (MSM) in the United States. We accounted for the costs and disutilities associated with symptoms, diagnosis, treatment, and sequelae, and combined costs and QALYs in a measure of net health benefit (NHB). Our results suggest that under a scenario where 3 antibiotics are available over the next 50 years (2 suitable for the first-line therapy of gonorrhea and 1 suitable only for the retreatment of resistant infections), changing the switch threshold between 1% and 10% does not meaningfully impact the annual number of gonorrhea cases, total costs, or total QALY losses associated with gonorrhea. However, if a new antibiotic is to become available in the future, choosing a lower switch threshold could improve the population NHB. If in addition, drug-susceptibility testing (DST) is available to inform retreatment regimens after unsuccessful first-line therapy, setting the switch threshold at 1% to 2% is expected to maximize the population NHB. A limitation of our study is that our analysis only focuses on the MSM population and does not consider the influence of interventions such as vaccine and common use of rapid drugs susceptibility tests to inform first-line therapy. CONCLUSIONS: Changing the switch threshold for first-line antibiotics may not substantially change the health and financial outcomes associated with gonorrhea. However, the switch threshold could be reduced when newer antibiotics are expected to become available soon or when in addition to future novel antibiotics, DST is also available to inform retreatment regimens.

Susceptibility of Neisseria gonorrhoeae against a dual treatment antibiotics regimen in primary health centres in Surabaya, Indonesia.

BACKGROUND AND OBJECTIVES: There were 82.4 million new gonorrhoea cases worldwide in 2020. Dual treatment with ceftriaxone or cefixime and azithromycin or doxycycline is currently recommended for gonorrhoea in Indonesia. However, reduced susceptibility and resistance to cephalosporins and azithromycin are increasing. We evaluated the susceptibility pattern of Neisseria gonorrhoeae to cefixime, ceftriaxone, azithromycin and doxycycline. METHOD: N. gonorrhoeae isolates were obtained from 19 male participants with clinically and laboratory-confirmed gonorrhoea. Antibiotic susceptibility testing was conducted by disc diffusion and interpreted according to Clinical and Laboratory Standards Institute and Centers for Disease Control and Prevention criteria. RESULTS: Reduced susceptibility or resistance was observed against doxycycline in 19 isolates (100%), cefixime in six (31.6%), ceftriaxone in three (15.8%) and azithromycin in zero (0%) isolates. DISCUSSION: A dual treatment regimen with ceftriaxone and azithromycin can still be recommended as first-line therapy for gonorrhoea in Indonesia. Antibiotic susceptibility surveillance of N. gonorrhoeae should be routinely conducted.

Data quality assessment of the Enhanced Gonococcal Antimicrobial Surveillance Programme (EGASP), Thailand, 2015-2021.

BACKGROUND: Quality assessments of gonococcal surveillance data are critical to improve data validity and to enhance the value of surveillance findings. Detecting data errors by systematic audits identifies areas for quality improvement. We designed and implemented an internal audit process to evaluate the accuracy and completeness of surveillance data for the Thailand Enhanced Gonococcal Antimicrobial Surveillance Programme (EGASP). METHODS: We conducted a data quality audit of source records by comparison with the data stored in the EGASP database for five audit cycles from 2015-2021. Ten percent of culture-confirmed cases of Neisseria gonorrhoeae were randomly sampled along with any cases identified with elevated antimicrobial susceptibility testing results and cases with repeat infections. Incorrect and incomplete data were investigated, and corrective action and preventive actions (CAPA) were implemented. Accuracy was defined as the percentage of identical data in both the source records and the database. Completeness was defined as the percentage of non-missing data from either the source document or the database. Statistical analyses were performed using the t-test and the Fisher's exact test. RESULTS: We sampled and reviewed 70, 162, 85, 68, and 46 EGASP records during the five audit cycles. Overall accuracy and completeness in the five audit cycles ranged from 93.6% to 99.4% and 95.0% to 99.9%, respectively. Overall, completeness was significantly higher than accuracy (p = 0.017). For each laboratory and clinical data element, concordance was >85% in all audit cycles except for two laboratory data elements in two audit cycles. These elements significantly improved following identification and CAPA implementation. DISCUSSION: We found a high level of data accuracy and completeness in the five audit cycles. The implementation of the audit process identified areas for improvement. Systematic quality assessments of laboratory and clinical data ensure high quality EGASP surveillance data to monitor for antimicrobial resistant Neisseria gonorrhoeae in Thailand.

Impact of mutations in the mtrR, rpdlVD and rrl genes on azithromycin resistance in Neisseria gonorrhoeae.

INTRODUCTION: Bacterial sexually transmitted infections (STIs) pose a major public health problem. The emergence of antibiotic-resistant strains of Neisseria gonorrhoeae represents a serious threat to successful treatment and epidemiological control. The first extensively drug-resistant (XDR) strains (ceftriaxone-resistant and high-level azithromycin-resistant [HLR AZY]) have been reported. AIMS: To identify molecular mechanisms implicated in azithromycin resistance in strains isolated from patients over a three-year period in a university hospital in Switzerland. MATERIAL AND METHODS: From January 2020 to December 2022, 34 isolates (one per patient) were recovered from samples analyzed at the University Hospital of Lausanne. Eight genes involved in azithromycin resistance were sequenced: mtrR repressor (mtrCDE operon repressor) and his promotor mtrR-pr, rplD gene (L4 ribosomal protein), rplV gene (L22 ribosomal protein) and the four alleles of the rrl gene (23S rRNA). RESULTS: With a cutoff value of 1 mg/L, 15 isolates were considered as being resistant to azithromycin, whereas the remaining 19 were susceptible. The C2597T mutation in 3 or 4 of the rrl allele confer a medium-level resistance to azithromycin (MIC = 16 mg/L, N = 2). The following mutations were significantly associated with MIC values ≥1 mg/L: the three mutations V125A, A147G, R157Q in the rplD gene (N = 10) and a substitution A->C in the mtrR promotor (N = 9). Specific mutations in the mtrR repressor and its promotor were observed in both susceptible and resistant isolates. CONCLUSIONS: Resistance to azithromycin was explained by the presence of mutations in many different copies of 23S RNA ribosomal genes and their regulatory genes. Other mutations, previously reported to be associated with azithromycin resistance, were documented in both susceptible and resistant isolates, suggesting they play little role, if any, in azithromycin resistance.

Surge in Ceftriaxone-Resistant Neisseria gonorrhoeae FC428-Like Strains, Asia-Pacific Region, 2015-2022.

Ceftriaxone-resistant Neisseria gonorrhoeae FC428-like strains have disseminated across the Asia-Pacific region, with a continuous rise in prevalence during 2015-2022. To mitigate the effect of these strains, we advocate for enhanced molecular diagnostics, expanded surveillance networks, and a regionally coordinated effort to combat the global spread of FC428-like strains.

Combination of compound screening with an animal model identifies pentamidine to prevent Chlamydia trachomatis infection.

Chlamydia trachomatis (Ct) is the most common cause for bacterial sexually transmitted infections (STIs) worldwide with a tremendous impact on public health. With the aim to unravel novel targets of the chlamydia life cycle, we screen a compound library and identify 28 agents to significantly reduce Ct growth. The known anti-infective agent pentamidine-one of the top candidates of the screen-shows anti-chlamydia activity in low concentrations by changing the metabolism of host cells impairing chlamydia growth. Furthermore, it effectively decreases the Ct burden upon local or systemic application in mice. Pentamidine also inhibits the growth of Neisseria gonorrhea (Ng), which is a common co-infection of Ct. The conducted compound screen is powerful in exploring antimicrobial compounds against Ct in a medium-throughput format. Following thorough in vitro and in vivo assessments, pentamidine emerges as a promising agent for topical prophylaxis or treatment against Ct and possibly other bacterial STIs.

Prevalence and Molecular Characterization of Pharyngeal Gonorrhea in Korean Men With Urethritis.

Background: Pharyngeal infection is more difficult to diagnose and treat than genital or rectal infection and can act as a reservoir for gonococcal infection. We determined the prevalence of pharyngeal gonorrhea in Korean men with urethritis and analyzed the molecular characteristics and antimicrobial susceptibility of the isolates. Methods: Seventy-two male patients with symptoms of urethritis who visited a urology clinic in Wonju, Korea, between September 2016 and March 2018 were included. Urethral and pharyngeal gonococcal cultures, antimicrobial susceptibility testing, Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST), and multiplex real-time PCR (mRT-PCR) were performed. Results: Among the 72 patients, 59 tested positive for gonococcus by mRT-PCR. Of these 59 patients, 18 (30.5%) tested positive in both the pharynx and urethra, whereas 41 tested positive only in the urethra. NG-MAST was feasible in 16 out of 18 patients and revealed that 14 patients had the same sequence types in both urethral and pharyngeal specimens, whereas two patients exhibited different sequence types between the urethra and pharynx. Of the 72 patients, 33 tested culture-positive. All patients tested positive only in urethral specimens, except for one patient who tested positive in both. All culture-positive specimens also tested positive by mRT-PCR. All isolates were susceptible to azithromycin and spectinomycin, but resistance rates to ceftriaxone and cefixime were 2.9% and 14.7%, respectively. Conclusions: The prevalence of pharyngeal gonorrhea in Korean men with gonococcal urethritis is as high as 30.5%, highlighting the need for pharyngeal screening in high-risk groups. Ceftriaxone is the recommended treatment for pharyngeal gonorrhea.

Interactions between Zoliflodacin and Neisseria gonorrhoeae Gyrase and Topoisomerase IV: Enzymological Basis for Cellular Targeting.

Gyrase and topoisomerase IV are the cellular targets for fluoroquinolones, a critically important class of antibacterial agents used to treat a broad spectrum of human infections. Unfortunately, the clinical efficacy of the fluoroquinolones has been curtailed by the emergence of target-mediated resistance. This is especially true for Neisseria gonorrhoeae, the causative pathogen of the sexually transmitted infection gonorrhea. Spiropyrimidinetriones (SPTs), a new class of antibacterials, were developed to combat the growing antibacterial resistance crisis. Zoliflodacin is the most clinically advanced SPT and displays efficacy against uncomplicated urogenital gonorrhea in human trials. Like fluoroquinolones, the primary target of zoliflodacin in N. gonorrhoeae is gyrase, and topoisomerase IV is a secondary target. Because unbalanced gyrase/topoisomerase IV targeting has facilitated the evolution of fluoroquinolone-resistant bacteria, it is important to understand the underlying basis for the differential targeting of zoliflodacin in N. gonorrhoeae. Therefore, we assessed the effects of this SPT on the catalytic and DNA cleavage activities of N. gonorrhoeae gyrase and topoisomerase IV. In all reactions examined, zoliflodacin displayed higher potency against gyrase than topoisomerase IV. Moreover, zoliflodacin generated more DNA cleavage and formed more stable enzyme-cleaved DNA-SPT complexes with gyrase. The SPT also maintained higher activity against fluoroquinolone-resistant gyrase than topoisomerase IV. Finally, when compared to zoliflodacin, the novel SPT H3D-005722 induced more balanced double-stranded DNA cleavage with gyrase and topoisomerase IV from N. gonorrhoeae, Escherichia coli, and Bacillus anthracis. This finding suggests that further development of the SPT class could yield compounds with a more balanced targeting against clinically important bacterial infections.

Transcriptional responses of Neisseria gonorrhoeae to glucose and lactate: implications for resistance to oxidative damage and biofilm formation.

Understanding how bacteria adapt to different environmental conditions is crucial for advancing knowledge regarding pathogenic mechanisms that operate during infection as well as efforts to develop new therapeutic strategies to cure or prevent infections. Here, we investigated the transcriptional response of Neisseria gonorrhoeae, the causative agent of gonorrhea, to L-lactate and glucose, two important carbon sources found in the host environment. Our study revealed extensive transcriptional changes that gonococci make in response to L-lactate, with 37% of the gonococcal transcriptome being regulated, compared to only 9% by glucose. We found that L-lactate induces a transcriptional program that would negatively impact iron transport, potentially limiting the availability of labile iron, which would be important in the face of the multiple hydrogen peroxide attacks encountered by gonococci during its lifecycle. Furthermore, we found that L-lactate-mediated transcriptional response promoted aerobic respiration and dispersal of biofilms, contrasting with an anaerobic condition previously reported to favor biofilm formation. Our findings suggest an intricate interplay between carbon metabolism, iron homeostasis, biofilm formation, and stress response in N. gonorrhoeae, providing insights into its pathogenesis and identifying potential therapeutic targets.IMPORTANCEGonorrhea is a prevalent sexually transmitted infection caused by the human pathogen Neisseria gonorrhoeae, with ca. 82 million cases reported worldwide annually. The rise of antibiotic resistance in N. gonorrhoeae poses a significant public health threat, highlighting the urgent need for alternative treatment strategies. By elucidating how N. gonorrhoeae responds to host-derived carbon sources such as L-lactate and glucose, this study offers insights into the metabolic adaptations crucial for bacterial survival and virulence during infection. Understanding these adaptations provides a foundation for developing novel therapeutic approaches targeting bacterial metabolism, iron homeostasis, and virulence gene expression. Moreover, the findings reported herein regarding biofilm formation and L-lactate transport and metabolism contribute to our understanding of N. gonorrhoeae pathogenesis, offering potential avenues for preventing and treating gonorrhea infections.

Emergence of Extensively Drug-Resistant Neisseria gonorrhoeae, France, 2023.

Since 2022, Europe has had 4 cases of extensively drug-resistant Neisseria gonorrhoeae, sequence type 16406, that is resistant to ceftriaxone and highly resistant to azithromycin. We report 2 new cases from France in 2023 involving strains genetically related to the 4 cases from Europe as well as isolates from Cambodia.

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.