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.

World Health Organization Enhanced Gonococcal Antimicrobial Surveillance Programme, Cambodia, 2023.

To determine antimicrobial susceptibility of Neisseria gonorrhoeae, we analyzed phenotypes and genomes of 72 isolates collected in Cambodia in 2023. Of those, 9/72 (12.5%) were extensively drug resistant, a 3-fold increase from 2022. Genomic analysis confirmed expansion of newly emerging resistant clones and ongoing resistance emergence across new phylogenetic backbones.

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.

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.

Zap the clap with DNA: a novel microbicide for preventing Neisseria gonorrhoeae infection.

Each year, Neisseria gonorrhoeae (Ngo) causes over 1.5 million new infections in the United States, and >87 million worldwide. The absence of a vaccine for preventing gonorrhea, the rapid emergence of multidrug-resistant and extremely drug-resistant Ngo strains, and the limited number of antibiotics available for treating gonorrhea underscore the importance of developing new modalities for addressing Ngo infection. Here, we describe DNA-based microbicides that kill Ngo but not commensals. Previously, we showed that Ngo is killed when it takes up differentially methylated DNA with homology to its genome. We exploited this Achilles heel to develop a new class of microbicides for preventing Ngo infection. These microbicides consist of DNA molecules with specific sequences and a methylation pattern different from Ngo DNA. These DNAs kill low-passage and antibiotic-resistant clinical isolates with high efficiency but leave commensals unharmed. Equally important, the DNAs are equally effective against Ngo whether they are in buffered media or personal lubricants. These findings illustrate the potential of this new class of practical, low-cost, self-administered DNA-based microbicides for preventing Ngo transmission during sexual intercourse.

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.

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.

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.

Investigating the cause of increased tetracycline-resistant Neisseria gonorrhoeae in England, 2016-20.

BACKGROUND: Antimicrobial resistance in Neisseria gonorrhoeae is a global public health concern. Tetracycline resistance (TetR) increased from 39.4% to 75.2% between 2016 and 2021 in N. gonorrhoeae isolates collected through national surveillance in England, despite the absence of use of tetracyclines for the treatment of gonorrhoea. OBJECTIVES: We investigated whether there was correlation between bacterial sexually transmitted infection (STI) tests performed and treatment with antimicrobials, with increased TetR in N. gonorrhoeae. METHODS: We examined correlations between bacterial STI tests, antimicrobial treatment and TetR in N. gonorrhoeae, using national surveillance data from three large sexual health services (SHS) in London during 2016-20. Doxycycline prescribing data and antibiograms of a non-STI pathogen from distinct patient groups (sexual health, obstetric and paediatric), at a large London hospital, were analysed to identify if doxycycline use in SHS was associated with resistance in a non-STI organism. RESULTS: A substantial increase in TetR was observed, particularly in isolates from gay, bisexual and other MSM (GBMSM). Strong positive correlations were observed exclusively in GBMSM between N. gonorrhoeae TetR and both bacterial STI tests (r = 0.97, P = 0.01) and antimicrobial treatment (r = 0.87, P = 0.05). Doxycycline prescribing increased dramatically during the study period in SHS. Prevalence of TetR in Staphylococcus aureus was higher in isolates sourced from SHS attendees than those from other settings. CONCLUSIONS: Frequent screening of GBMSM at higher risk of STIs, such as those on pre-exposure prophylaxis (PrEP) leading to/and increased use of doxycycline for the treatment of diagnosed infections, may account for the increase in TetR in N. gonorrhoeae.

Recent dynamics in Neisseria gonorrhoeae genomic epidemiology in Brazil: antimicrobial resistance and genomic lineages in 2017-20 compared to 2015-16.

OBJECTIVES: Regular quality-assured WGS with antimicrobial resistance (AMR) and epidemiological data of patients is imperative to elucidate the shifting gonorrhoea epidemiology, nationally and internationally. We describe the dynamics of the gonococcal population in 11 cities in Brazil between 2017 and 2020 and elucidate emerging and disappearing gonococcal lineages associated with AMR, compare to Brazilian WGS and AMR data from 2015 to 2016, and explain recent changes in gonococcal AMR and gonorrhoea epidemiology. METHODS: WGS was performed using Illumina NextSeq 550 and genomes of 623 gonococcal isolates were used for downstream analysis. Molecular typing and AMR determinants were obtained and links between genomic lineages and AMR (determined by agar dilution/Etest) examined. RESULTS: Azithromycin resistance (15.6%, 97/623) had substantially increased and was mainly explained by clonal expansions of strains with 23S rRNA C2611T (mostly NG-STAR CC124) and mtr mosaics (mostly NG-STAR CC63, MLST ST9363). Resistance to ceftriaxone and cefixime remained at the same levels as in 2015-16, i.e. at 0% and 0.2% (1/623), respectively. Regarding novel gonorrhoea treatments, no known zoliflodacin-resistance gyrB mutations or gepotidacin-resistance gyrA mutations were found. Genomic lineages and sublineages showed a phylogenomic shift from sublineage A5 to sublineages A1-A4, while isolates within lineage B remained diverse in Brazil. CONCLUSIONS: Azithromycin resistance, mainly caused by 23S rRNA C2611T and mtrD mosaics/semi-mosaics, had substantially increased in Brazil. This mostly low-level azithromycin resistance may threaten the recommended ceftriaxone-azithromycin therapy, but the lack of ceftriaxone resistance is encouraging. Enhanced gonococcal AMR surveillance, including WGS, is imperative in Brazil and other Latin American and Caribbean countries.

Impact of molecular ciprofloxacin resistance testing in management of gonorrhoea in a large urban clinic.

OBJECTIVES: Antibiotic resistance in gonorrhoea is of significant public health concern with the emergence of resistance to last-line therapies such as ceftriaxone. Despite around half of Neisseria gonorrhoeae isolates tested in the UK being susceptible to ciprofloxacin, very little ciprofloxacin is used in clinical practice. Testing for the S91F mutation associated with ciprofloxacin resistance is now available in CE-marked assays and may reduce the requirement for ceftriaxone, but many patients are treated empirically, or as sexual contacts, which may limit any benefit. We describe the real-world impact of such testing on antimicrobial use and clinical outcomes in people found to have gonorrhoea in a large urban UK sexual health clinic. METHODS: Molecular ciprofloxacin resistance testing (ResistancePlus GC assay (SpeeDx)) was undertaken as an additional test after initial diagnosis (m2000 Realtime CT/NG assay (Abbott Molecular)) in those not already known to have had antimicrobial treatment. Data from a 6-month period (from March to September 2022) were analysed to determine treatment choice and treatment outcome. RESULTS: A total of 998 clinical samples tested positive for N. gonorrhoeae in 682 episodes of infection. Of the 560 (56%) samples eligible for resistance testing, 269 (48.0%) were reported as wild-type, 180 (32.1%) were predicted to be resistant, 63 (11.3%) had an indeterminate resistance profile, and in 48 (8.6%) samples, N. gonorrhoeae was not detected. Ciprofloxacin was prescribed in 172 (75%) of 228 episodes in which the wild-type strain was detected. Four (2%) of those treated with ciprofloxacin had a positive test-of-cure sample by NAAT, with no reinfection risk. All four had ciprofloxacin-susceptible infection by phenotypic antimicrobial susceptibility testing. CONCLUSIONS: In routine practice in a large UK clinic, molecular ciprofloxacin resistance testing led to a significant shift in antibiotic use, reducing use of ceftriaxone. Testing can be targeted to reduce unnecessary additional testing. Longer term impact on antimicrobial resistance requires ongoing surveillance.

Trends of Antimicrobial Susceptibility of Neisseria gonorrhoeae Isolates Between 2012 and 2023: Results From an Open Italian Cohort.

BACKGROUND: Neisseria gonorrhoeae (Ng) is a public health priority because of the rapid evolution of antimicrobial resistance, the emergence of antibiotic resistance, and the absence of a vaccine against Ng. The aim of this study was to investigate trends in the minimum inhibitory concentration and resistance (R) or reduced susceptibility (DS) of Ng cases to ceftriaxone (CRO), azithromycin (AZM), tetracycline (TET), benzylpenicillin (PenG), and ciprofloxacin (CIP) during a 10-year period. METHODS: We conducted a retrospective analysis on an open cohort of Ng cases diagnosed on rectal, urethral, and pharyngeal samples at San Raffaele Scientific Institute, between September 2012 and February 2023. Minimum inhibitory concentrations of antibiotics were determined by gradient-test strips. Bivariate linear regression models were applied on logarithmic minimum inhibitory concentrations values; Cochran-Armitage test was used to determine a linear trend in the proportions of resistant strains. RESULTS: A total of 436 Ng isolates from 352 individuals were analyzed. Minimum inhibitory concentrations of CRO and PenG reduced over time ( P < 0.001, P = 0.030), AZM increased ( P = 0.001), and CIP and TET did not change ( P = 0.473, P = 0.272). The percentages of resistant strains were as follows: PenG, 89.9%; TET, 90.8%; CIP, 48.2%; AZM, and 4.4%. CRO-DS strains were 8.7%, and only 1 case of CRO-R was identified. The proportion of resistant strains increased over time for AZM ( P = 0.007), TET ( P = 0.001), and CIP ( P < 0.001), whereas it decreased for PenG ( P < 0.001) and CRO-DS/R strains ( P < 0.001). CONCLUSIONS: Ng strains showed high susceptibility to CRO, although we identified cases of DS/R and observed high levels of susceptibility to AZM. Overall, the recommended primary regimen for Ng treatment was confirmed to be effective.

In vitro susceptibility of Neisseria gonorrhoeae to netilmicin and etimicin in comparison to gentamicin and other aminoglycosides.

PURPOSE: Single doses of gentamicin have demonstrated clinical efficacy in the treatment of urogenital gonorrhea, but lower cure rates for oropharyngeal and anorectal gonorrhea. Formulations selectively enriched in specific gentamicin C congeners have been proposed as a less toxic alternative to gentamicin, potentially permitting higher dosing to result in increased plasma exposures at the extragenital sites of infection. The purpose of the present study was to compare the antibacterial activity of individual gentamicin C congeners against Neisseria gonorrhoeae to that of other aminoglycoside antibiotics. METHODS: Antimicrobial susceptibility of three N. gonorrhoeae reference strains and 152 clinical isolates was assessed using standard disk diffusion, agar dilution, and epsilometer tests. RESULTS: Gentamicin C1, C2, C1a, and C2a demonstrated similar activity against N. gonorrhoeae. Interestingly, susceptibility to the 1-N-ethylated aminoglycosides etimicin and netilmicin was significantly higher than the susceptibility to their parent compounds gentamicin C1a and sisomicin, and to any other of the 25 aminoglycosides assessed in this study. Propylamycin, a 4'-propylated paromomycin analogue, was significantly more active against N. gonorrhoeae than its parent compound, too. CONCLUSION: Selectively enriched gentamicin formulations hold promise for a less toxic but equally efficacious alternative to gentamicin. Our study warrants additional consideration of the clinically established netilmicin and etimicin for treatment of genital and perhaps extragenital gonorrhea. Additional studies are required to elucidate the mechanism behind the advantage of alkylated aminoglycosides.

Comparison of gradient diffusion and molecular methods using Allplex™ NG&DR assay (Seegene®) for macrolide and fluoroquinolone screening resistance in Neisseria gonorrhoeae.

Antimicrobial resistance in Neisseria gonorrhoeae (NG) is increasing worldwide. Second-line treatments with macrolides or fluoroquinolones are an option for NG infections in some cases following the STI guideline recommendations. In our study, we compared the gradient diffusion test using EUCAST 2024 breakpoints with a new molecular method using the Allplex™ NG&DR assay (Seegene®) including A2059G/C2611 mutations (23S rRNA) associated with high/moderate-level macrolide resistance and S91F mutation (gyrA) relationship with fluoroquinolone resistance in NG isolates (n = 100). We calculated the sensitivity, specificity, and correlation of the molecular test for fluoroquinolone using the gradient diffusion as the reference method. In twenty-three strains was not detected any mutation associated with macrolides or fluoroquinolone resistance. No A2059G/C2611T mutations were detected, and the S91F mutations were detected in 77 out of the 100 isolates screened. Twenty-three NG isolates were reported to be resistant to azithromycin (ECOFF: >1 mg/L), and 78 NG isolates were resistant to ciprofloxacin (MIC: >0.06 mg/L). The molecular method showed a sensitivity of 96.1% and, a specificity of 90.9% for fluoroquinolone susceptibility, but the statistical analysis between the molecular test and gradient diffusion test was not statistically significant for fluoroquinolone resistance (p = 1). Statistical analysis was not performed for macrolides because of the absence of positive RT-PCR results. According to our data, Allplex™ assay cannot replace the gradient diffusion test for macrolide resistance. However, the assay could be used to test fluoroquinolone resistance in NG isolates as a replacement for phenotypic methods.

Policy, practice, and prediction: model-based approaches to evaluating N. gonorrhoeae antibiotic susceptibility test uptake in Australia.

BACKGROUND: Antimicrobial resistance (AMR) represents a significant threat to global health with Neisseria gonorrhoea emerging as a key pathogen of concern. In Australia, the Australian Gonococcal Surveillance Program (AGSP) plays a critical role in monitoring resistance patterns. However, antibiotic susceptibility test (AST) uptake - a crucial component for effective resistance surveillance - remains to be a limiting factor. The study aims to model the processes involved in generating AST tests for N. gonorrhoea isolates within the Australian healthcare system and assess the potential impact of systematic and policy-level changes. METHODS: Two models were developed. The first model was a mathematical stochastic health systems model (SHSM) and a Bayesian Belief Network (BBN) to simulate the clinician-patient dynamics influencing AST initiation. Key variables were identified through systematic literature review to inform the construction of both models. Scenario analyses were conducted with the modification of model parameters. RESULTS: The SHSM and BBN highlighted clinician education and the use of clinical support tools as effective strategies to improve AST. Scenario analysis further identified adherence to guidelines and changes in patient-level factors, such as persistence of symptoms and high-risk behaviours, as significant determinants. Both models supported the notion of mandated testing to achieve higher AST initiation rates but with considerations necessary regarding practicality, laboratory constraints, and culture failure rate. CONCLUSION: The study fundamentally demonstrates a novel approach to conceptualising the patient-clinician dynamic within AMR testing utilising a model-based approach. It suggests targeted interventions to educational, support tools, and legislative framework as feasible strategies to improve AST initiation rates. However, the research fundamentally highlights substantial research gaps in the underlying understanding of AMR.

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.

Isolation and Genomic Characterization of Schaalia turicensis from a Patient with Gonococcal Urethritis, Thailand.

Schaalia turicensis is facultative anaerobic Gram-positive bacillus that commonly inhabits the oropharynx, gastrointestinal, and genitourinary tract of healthy individuals. This organism has been co-isolated with Neisseria gonorrhoeae from 15-year-old Thai male patient with gonococcal urethritis in Bangkok, Thailand. In this study, we characterized the class 1 integron in S. turicensis isolate using whole-genome sequencing and bioinformatics analysis. Sequencing analysis confirmed the presence of an imperfect class 1 integron located on chromosome and a novel 24.5-kb-long composite transposon, named Tn7083. The transposon Tn7083 carried genes encoding chloramphenicol resistance (cmx), sulfonamide resistance (sul1), and aminoglycoside resistance [aph(6)-Id (strB), aph(3'')-Ib (strA), aph(3')-Ia].

New pyridine-based chalcones and pyrazolines with anticancer, antibacterial, and antiplasmodial activities.

New pyridine-based chalcones 4a-h and pyrazolines 5a-h (N-acetyl), 6a-h (N-phenyl), and 7a-h (N-4-chlorophenyl) were synthesized and evaluated by the National Cancer Institute (NCI) against 60 different human cancer cell lines. Pyrazolines 6a, 6c-h, and 7a-h satisfied the pre-determined threshold inhibition criteria, obtaining that compounds 6c and 6f exhibited high antiproliferative activity, reaching submicromolar GI50 values from 0.38 to 0.45 µM, respectively. Moreover, compound 7g (4-CH3) exhibited the highest cytostatic activity of these series against different cancer cell lines from leukemia, nonsmall cell lung, colon, ovarian, renal, and prostate cancer, with LC50 values ranging from 5.41 to 8.35 µM, showing better cytotoxic activity than doxorubicin. Furthermore, the compounds were tested for antibacterial and antiplasmodial activities. Chalcone 4c was the most active with minimal inhibitory concentration (MIC) = 2 µg/mL against methicillin-resistant Staphylococcus aureus (MRSA), while the pyrazoline 6h showed a MIC = 8 µg/mL against Neisseria gonorrhoeae. For anti-Plasmodium falciparum activity, the chalcones display higher activity with EC50 values ranging from 10.26 to 10.94 µg/mL. Docking studies were conducted against relevant proteins from P. falciparum, exhibiting the minimum binding energy with plasmepsin II. In vivo toxicity assay in Galleria mellonella suggests that most compounds are low or nontoxic.

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.