Antimicrobial-resistant Neisseria gonorrhoeae in Europe in 2020 compared with in 2013 and 2018: a retrospective genomic surveillance study.

BACKGROUND: Regular quality-assured whole-genome sequencing linked to antimicrobial resistance (AMR) and patient metadata is imperative to elucidate the shifting gonorrhoea epidemiology, both nationally and internationally. We aimed to examine the gonococcal population in the European Economic Area (EEA) in 2020, elucidate emerging and disappearing gonococcal lineages associated with AMR and patient metadata, compare with 2013 and 2018 whole-genome sequencing data, and explain changes in gonococcal AMR and gonorrhoea epidemiology. METHODS: In this retrospective genomic surveillance study, we analysed consecutive gonococcal isolates that were collected in EEA countries through the European Gonococcal Antimicrobial Surveillance Programme (Euro-GASP) in 2020, and made comparisons with Euro-GASP data from 2013 and 2018. All isolates had linked AMR data (based on minimum inhibitory concentration determination) and patient metadata. We performed whole-genome sequencing and molecular typing and AMR determinants were derived from quality-checked whole-genome sequencing data. Links between genomic lineages, AMR, and patient metadata were examined. FINDINGS: 1932 gonococcal isolates collected in 2020 in 21 EEA countries were included. The majority (81·2%, 147 of 181 isolates) of azithromycin resistance (present in 9·4%, 181 of 1932) was explained by the continued expansion of the Neisseria gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) clonal complexes (CCs) 63, 168, and 213 (with mtrD/mtrR promoter mosaic 2) and the novel NG-STAR CC1031 (semi-mosaic mtrD variant 13), associated with men who have sex with men and anorectal or oropharyngeal infections. The declining cefixime resistance (0·5%, nine of 1932) and negligible ceftriaxone resistance (0·1%, one of 1932) was largely because of the progressive disappearance of NG-STAR CC90 (with mosaic penA allele), which was predominant in 2013. No known resistance determinants for novel antimicrobials (zoliflodacin, gepotidacin, and lefamulin) were found. INTERPRETATION: Azithromycin-resistant clones, mainly with mtrD mosaic or semi-mosaic variants, appear to be stabilising at a relatively high level in the EEA. This mostly low-level azithromycin resistance might threaten the recommended ceftriaxone-azithromycin therapy, but the negligible ceftriaxone resistance is encouraging. The decreased genomic population diversity and increased clonality could be explained in part by the COVID-19 pandemic resulting in lower importation of novel strains into Europe. FUNDING: European Centre for Disease Prevention and Control and Örebro University Hospital.

In Vitro Bactericidal Activity of Biogenic Copper Oxide Nanoparticles for Neisseria gonorrhoeae with Enhanced Compatibility for Human Cells.

Resistance to antibiotics and antimicrobial compounds is a significant problem for human and animal health globally. The development and introduction of new antimicrobial compounds are urgently needed, and copper oxide nanoparticles (CuO NPs) have found widespread application across various sectors including biomedicine, pharmacy, catalysis, cosmetics, and many others. What makes them particularly attractive is the possibility of their synthesis through biogenic routes. In this study, we synthesized biogenic green tea (GT, Camellia sinensis)-derived CuO NPs (GT CuO NPs) and examined their biophysical properties, in vitro toxicity for mammalian cells in culture, and then tested them against Neisseria gonorrhoeae, an exemplar Gram-negative bacterium from the World Health Organization's Priority Pathogen List. We compared our synthesized GT CuOP NPs with commercial CuO NPs (Com CuO NPs). Com CuO NPs were significantly more cytotoxic to mammalian cells (IC50 of 7.32 µg/mL) than GT CuO NPs (IC50 of 106.1 µg/mL). GT CuO NPs showed no significant increase in bax, bcl2, il6, and il1ß mRNA expression from mammalian cells, whereas there were notable rises after treatment with Com CuO NPs. GT-CuO NPs required concentrations of 0.625 and 3.125 µg/mL to kill 50 and 100% of bacteria, respectively, whereas Com-CuO NPs needed concentrations of 15.625 and 30 µg/mL to kill 50 and 100% of bacteria, and the antibiotic ceftriaxone killed 50 and 100% with 3.125 and 30 µg/mL. Gonococci could be killed within 30 min of exposure to GT CuO NPs and the NPs could kill up to 107 within 1 h. In summary, this is the first report to our knowledge that describes the bioactivity of biogenic CuO NPs against N. gonorrhoeae. Our data suggest that biogenic nanoparticle synthesis has significant advantages over traditional chemical routes of synthesis and highlights the potential of GT-CuO NPs in addressing the challenges posed by multidrug-resistant Neisseria gonorrhoeae infections.

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.

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.

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.

Phenotypic and genotypic characterization of Neisseria gonorrhoeae isolates among individuals at high risk for sexually transmitted diseases in Zurich, Switzerland.

BACKGROUND: While ceftriaxone resistance remains scarce in Switzerland, global Neisseria gonorrhoeae (NG) antimicrobial resistance poses an urgent threat. This study describes clinical characteristics in MSM (men who have sex with men) diagnosed with NG infection and analyses NG resistance by phenotypic and genotypic means. METHODS: Data of MSM enrolled in three clinical cohorts with a positive polymerase chain reaction test (PCR) for NG were analysed between January 2019 and December 2021 and linked with antibiotic susceptibility testing. Bacterial isolates were subjected to whole genome sequencing (WGS). RESULTS: Of 142 participants, 141 (99%) were MSM and 118 (84%) living with HIV. Participants were treated with ceftriaxone (N = 79), azithromycin (N = 2), or a combination of both (N = 61). No clinical or microbiological failures were observed. From 182 positive PCR samples taken, 23 were available for detailed analysis. Based on minimal inhibitory concentrations (MICs), all isolates were susceptible to ceftriaxone, gentamicin, cefixime, cefpodoxime, ertapenem, zoliflodacin, and spectinomycin. Resistance to azithromycin, tetracyclines and ciprofloxacin was observed in 10 (43%), 23 (100%) and 11 (48%) of the cases, respectively. Analysis of WGS data revealed combinations of resistance determinants that matched with the corresponding phenotypic resistance pattern of each isolate. CONCLUSION: Among the MSM diagnosed with NG mainly acquired in Switzerland, ceftriaxone MICs were low for a subset of bacterial isolates studied and no treatment failures were observed. For azithromycin, high occurrences of in vitro resistance were found. Gentamicin, cefixime, cefpodoxime, ertapenem, spectinomycin, and zoliflodacin displayed excellent in vitro activity against the 23 isolates underscoring their potential as alternative agents to ceftriaxone.

Prevalence of mutations associated with macrolide and fluoroquinolone resistance in Neisseria gonorrhoeae with Allplex™ NG&DR Assay (Seegene®) in a tertiary hospital from Madrid, Spaina / Prevalencia de mutaciones asociadas a resistencia a macrólidos y fluoroquinolonas en Neisseria gonorrhoeae con el kit Allplex™ NG&DR Assay (Seegene®) en un hospital terciario de la Comunidad de Madrid, España

Background. The prevalence of drug-resistant Neisseria gonorrhoeae (NG) infections is increasing. Studies report the prevalence of NG strains presenting A2059G/C2611T (rRNA 23S) and S91F (parC) mutations conferring resistance to azith romycin and ciprofloxacin. Material and methods. We conducted a prospective cohort study evaluating first void-urine urines, rectal, and oropharyngeal swabs collected from a cohort of patients in a tertiary hospital in Madrid between October 2022 and January 2023. Samples were screened by Allplex™ 7-STI Essential As say (Seegene®). Drug resistances were performed by Allplex™ NG&DR Assay (Seegene®). Results. A total of 1,415 patients were included, of which 112 had a positive sample for NG infection. One patient had a C2611T mutation (0.9%) and neither patient showed A2059G mutation. We found 67 (59.8%) S91F-positive patients. For ty-four patients (39.3%) not had any mutations. Conclusions. We report a low-prevalence of mutations A2059G/C2611T to macrolides and a high-prevalence to S91F in NG infections. Molecular methods for the detection of NG resistance could be useful in direct non-culturable samples (AU)

Introducción. La infección por Neisseria gonorrhoeae (NG) resistente está aumentando. Se ha descrito la prevalencia de cepas de NG con mutaciones A2059G/C2611T (rRNA 23S) y S91F (parC) que confieren resistencia a azitromicina y cipro floxacino. Material y métodos. Realizamos un estudio prospecti vo evaluando orinas de primera micción, hisopos anales y fa ríngeos recogidos de una cohorte de pacientes en un hospital terciario de Madrid entre octubre de 2022 y enero de 2023. El cribado de las muestras se realizó mediante Allplex™ 7-STI Es sential Assay (Seegene®). Las resistencias a macrólidos y fluo roquinolonas se realizaron mediante Allplex™ NG&DR Assay (Seegene®). Resultados. Se incluyeron 1.415 pacientes, de los cua les 112 fueron positivos para NG. Un paciente presentaba una mutación C2611T (0,9%) y en ningún paciente se detec tó A2059G. Encontramos 67 pacientes (59,8%) positivos pa ra S91F. Cuarenta y cuatro pacientes (39,3%) no presentaban mutaciones. Conclusiones. Reportamos una baja prevalencia de mu taciones A2059G/C2611T a macrólidos y una alta prevalencia de S91F en NG. Los métodos moleculares para la detección de resistencias en NG podrían ser útiles en muestras directas no cultivables (AU)

Australian Gonococcal Surveillance Programme Annual Report, 2022.

The Australian Gonococcal Surveillance Programme (AGSP) has continuously monitored antimicrobial resistance in Neisseria gonorrhoeae for more than 40 years. In 2022, a total of 8,199 isolates from patients in the public and private sectors, in all jurisdictions, were tested for in vitro antimicrobial susceptibility by standardised methods. The current treatment recommendation for gonorrhoea, for the majority of Australia, continues to be dual therapy with ceftriaxone and azithromycin. In 2022, of N. gonorrhoeae isolates tested, 0.51% (42/8,199) met the WHO criterion for ceftriaxone decreased susceptibility (DS), defined as a minimum inhibitory concentration value ≥ 0.125 mg/L. Resistance to azithromycin was reported in 3.9% of N. gonorrhoeae isolates, proportionally stable since 2019. There were nine isolates with high-level resistance to azithromycin (MIC value ≥ 256 mg/L) reported in Australia: Queensland (4), New South Wales (3), Victoria (1) and non-remote Western Australia (1). This is the highest number detected annually by the AGSP. In 2022, penicillin resistance was found in 38.8% of gonococcal isolates, and ciprofloxacin resistance in 63.3%, however, there was considerable variation by jurisdiction. In some remote settings, penicillin resistance remains low; in these settings, penicillin continues to be recommended as part of an empiric therapy strategy. In 2022, in remote Northern Territory, one penicillin-resistant isolate was reported; in remote Western Australia, 11.8% of gonococcal isolates (9/76) were penicillin resistant. There were three ciprofloxacin-resistant isolates reported from remote Northern Territory; ciprofloxacin resistance rates remain comparatively low in remote Western Australia (6/76; 7.9%).

Rapid expansion of Neisseria gonorrhoeae ST7827 clone in Australia, with variable ceftriaxone phenotype unexplained by genotype.

BACKGROUND: Neisseria gonorrhoeae is identified as a priority pathogen due to its capacity to rapidly develop antimicrobial resistance (AMR). Following the easing of SARS-CoV-2 pandemic travel restrictions across international borders in the state of New South Wales (NSW), Australia, a surge of gonococcal isolates with raised ceftriaxone MIC values were detected. METHODS: All N. gonorrhoeae isolates (n = 150) with increased ceftriaxone MIC values in NSW between 1 January 2021 and July 2022 from males and females from all sites were sequenced. RESULTS: A new emergence and rapid expansion of an N. gonorrhoeae ST7827 clone was documented within NSW, Australia and provides further evidence of the ability of N. gonorrhoeae to undergo sufficient genomic changes and re-emerge as a geographically restricted subclone. Mapping AMR determinants to MIC results did not reveal any genomic pattern that correlated with MIC values. CONCLUSIONS: The rapid dissemination and establishment of this clone at the population level is a new and concerning demonstration of the agility of this pathogen, and underscores concerns about similar incursions and establishment of MDR clones. Moreover, it is notable that in this context the AMR genotype-phenotype correlates remain unclear, which requires further investigation to enable better understanding of genomic aspects of AMR in N. gonorrhoeae.

Interactions between Loci Contributing to Antimicrobial Resistance and Virulence in Neisseria gonorrhoeae.

In a recent mBio article, Ayala et al. (mBio 13:e00276-22, 2022, https://doi.org/10.1128/mbio.00276-22) identified a single nucleotide variant in the repressor gdhR in Neisseria gonorrhoeae that reduces binding to the promoter of the virulence factor lctP and thereby increases its expression. The allele (gdhR6) frequently co-occurs with mutations in the mtr operon promoter that reduce expression of another repressor, mtrR, resulting in overexpression of the efflux pump-encoding mtrCDE and increased antimicrobial resistance. Because mtrR also represses gdhR, a decline in mtrR would decrease expression of lctP. Hypothesizing that gdhR6 arose to circumvent the impact of mtrR promoter mutations on lctP expression, the authors analyzed these loci in genomes of N. gonorrhoeae isolates from the preantibiotic era. Surprisingly, they found isolates with gdhR6 prior to selection for mtrR resistance-associated alleles. These results suggest that independent and perhaps interacting pressures have influenced the co-occurrence of these alleles.

Ribonucleotide reductase, a novel drug target for gonorrhea.

Antibiotic-resistant Neisseria gonorrhoeae (Ng) are an emerging public health threat due to increasing numbers of multidrug resistant (MDR) organisms. We identified two novel orally active inhibitors, PTC-847 and PTC-672, that exhibit a narrow spectrum of activity against Ng including MDR isolates. By selecting organisms resistant to the novel inhibitors and sequencing their genomes, we identified a new therapeutic target, the class Ia ribonucleotide reductase (RNR). Resistance mutations in Ng map to the N-terminal cone domain of the α subunit, which we show here is involved in forming an inhibited α4ß4 state in the presence of the ß subunit and allosteric effector dATP. Enzyme assays confirm that PTC-847 and PTC-672 inhibit Ng RNR and reveal that allosteric effector dATP potentiates the inhibitory effect. Oral administration of PTC-672 reduces Ng infection in a mouse model and may have therapeutic potential for treatment of Ng that is resistant to current drugs.

Single gene targeted nanopore sequencing enables simultaneous identification and antimicrobial resistance detection of sexually transmitted infections.

OBJECTIVES: To develop a simple DNA sequencing test for simultaneous identification and antimicrobial resistance (AMR) detection of multiple sexually transmitted infections (STIs). METHODS: Real-time PCR (qPCR) was initially performed to identify Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), Mycoplasma genitalium (MG) and Trichomonas vaginalis (TV) infections among a total of 200 vulvo-vaginal swab samples from female sex workers in Ecuador. qPCR positive samples plus qPCR negative controls for these STIs were subjected to single gene targeted PCR MinION-nanopore sequencing using the smartphone operated MinIT. RESULTS: Among 200 vulvo-vaginal swab samples 43 were qPCR positive for at least one of the STIs. Single gene targeted nanopore sequencing generally yielded higher pathogen specific read counts in qPCR positive samples than qPCR negative controls. Of the 26 CT, NG or MG infections identified by qPCR, 25 were clearly distinguishable from qPCR negative controls by read count. Discrimination of TV qPCR positives from qPCR negative controls was poorer as many had low pathogen loads (qPCR cycle threshold >35) which produced few specific reads. Real-time AMR profiling revealed that 3/3 NG samples identified had gyrA mutations associated with fluoroquinolone resistance, 2/10 of TV had mutations related to metronidazole resistance, while none of the MG samples possessed 23S rRNA gene mutations contributing to macrolide resistance. CONCLUSIONS: Single gene targeted nanopore sequencing for diagnosing and simultaneously identifying key antimicrobial resistance markers for four common genital STIs shows promise. Further work to optimise accuracy, reduce costs and improve speed may allow sustainable approaches for managing STIs and emerging AMR in resource poor and laboratory limited settings.

Dissemination and genome analysis of high-level ceftriaxone-resistant penA 60.001 Neisseria gonorrhoeae strains from the Guangdong Gonococcal antibiotics susceptibility Programme (GD-GASP), 2016-2019.

Background: After Neisseria gonorrhoeae FC428 was first found in Japan, ceftriaxone-resistant strains disseminated globally, and the gonococcal resistance rate increased remarkably. Epidemiological investigations are greatly significant for the analysis of antimicrobial resistance (AMR) trends, molecular features and evolution. Objectives: To clarify the AMR trend from 2016-2019 and reveal the molecular characteristics and evolution of ceftriaxone-resistant penA 60.001 isolates. Methods: The minimum inhibitory concentrations (MICs) of antibiotics against 4113 isolates were detected by the agar dilution method. N. gonorrhoeae multiantigen sequence typing (NG-MAST), multilocus sequence typing (MLST) and N.gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) were used to identify the sequence types. Genome analysis was conducted to analyze resistance genes, virulence factors, and evolutionary sources. Results: Isolates with decreased ceftriaxone susceptibility have increased from 2.05% (2016) to 16.18% (2019). Six ceftriaxone-resistant isolates possessing penA 60.001 appeared in Guangdong Province, and were resistant to ceftriaxone, penicillin, tetracycline, ciprofloxacin and cefixime, but susceptible to azithromycin and spectinomycin. Single-nucleotide polymorphisms (SNPs) in the porB gene were the major cause of different NG-MAST types. ST1903 was the main NG-STAR genotype and only strain-ZH545 was ST7365, with molecular features consistent with the MICs. Furthermore, different MLSTs suggested diverse evolutionary sources. Genome analysis revealed a set of virulence factors along with the resistance genes "penA" and "blaTEM-1B". Half of penA 60.001 strains were fully mixed with global FC428-related strains. Conclusions: Global FC428-related clones have disseminated across Guangdong, possibly causing decreased ceftriaxone susceptibility. Enhanced gonococcal surveillance will help elucidate the trajectory of transmission and curb further dissemination.

Coumarins effectively inhibit bacterial α-carbonic anhydrases.

Coumarins are known to act as prodrug inhibitors of mammalian α-carbonic anhydrases (CAs, EC 4.2.1.1) but they were not yet investigated for the inhibition of bacterial α-CAs. Here we demonstrate that such enzymes from the bacterial pathogens Neisseria gonorrhoeae (NgCAα) and Vibrio cholerae (VchCAα) are inhibited by a panel of simple coumarins incorporating hydroxyl, amino, ketone or carboxylic acid ester moieties in various positions of the ring system. The nature and the position of the substituents in the coumarin ring were the factors which strongly influenced inhibitory efficacy. NgCAα was inhibited with KIs in the range of 28.6-469.5 µM, whereas VchCAα with KIs in the range of 39.8-438.7 µM. The two human (h)CA isoforms included for comparison reason in the study, hCA I and II, were less prone to inhibition by these compounds, with KIs of 137-948.9 µM for hCA I and of 296.5-961.2 µM for hCA II, respectively. These findings are relevant for discovering coumarin bacterial CA inhibitors with selectivity for the bacterial over human isoform, with potential applications as novel antibacterial agents.

Large Increase in Azithromycin-Resistant Neisseria gonorrhoeae in Northern Spain.

The aim of this study was to characterize the evolution of gonorrhea in the general population by correlating epidemiological, genotypic, and antimicrobial resistance data of Neisseria gonorrhoeae isolates collected in northern Spain from 2014 to 2018. One hundred ninety-four strains underwent antimicrobial susceptibility testing and were genetically analyzed by N. gonorrhoeae multiantigen sequence typing. Increasing cases of gonococcal infections have been observed after 2015. Most occurred in male with urethritis. Sequence type (ST)-9972 and ST-1576, the predominant genotypes identified, have not been previously described as epidemic clones. Of great concern was the significant increase in azithromycin-resistant N. gonorrhoeae. More than 30% of these isolates were obtained from men who have sex with men (MSM). ST-12302 was the most prevalent clone among the azithromycin-resistant strains, and was also resistant to penicillin, ciprofloxacin, and tetracycline. This multidrug-resistant clone was exclusively isolated from MSM during 2018. The incidence rates of gonorrhea and azithromycin-resistant N. gonorrhoeae have significantly increased due to the emergence of new clones. ST-12302 has recently been recognized as an epidemic clone; therefore, its surveillance could be the key in controlling further dissemination of azithromycin resistance. These data highlight the need to perform local studies to update treatment guidelines and reinforce preventive measures against gonorrhea.

Promising Antifungal and Antibacterial Agents Based on 5-Aryl-2,2'-bipyridines and Their Heteroligand Salicylate Metal Complexes: Synthesis, Bioevaluation, Molecular Docking.

A series of new 5-aryl-2,2'-bipyridines and their (polyfluoro)salicylate complexes of Cu(II), Co(II) and Mn(II) were synthesized. Their antimicrobial activity was evaluated in vitro against six strains of Trichophytons, E. floccosum, M. canis, C. ablicans and Gram-negative bacteria N. gonorrhoeae. Among azo-ligands, Ph-bipy and Tol-bipy showed promising antifungal activity (minimum inhibitory concentration (MIC)<0.8-27 µM). Their antifungal action was found can be realized via binding Fe(III) ions. Tol-bipy suppressed growth of Gram-positive bacteria S. aureus, S. aureus MRSA and their monospecies biofilms (MIC 6-16 µM). Using molecular docking, the anti-staphylococcal action mechanism based on the inhibition of S. aureus DNA gyrase GyrB was proposed for the lead compounds. Among metal complexes, Cu(II) and Mn(II) complexes based on tetrafluorosalicylic acid and Tol-bipy or Ph-bipy had the high antifungal activity (MIC<0.24-32 µM). Mn(SalF4 -2H)2 (Tol-bipy)2 ] suppressed the growth of seven Candida strains at MIC 12-24 µM. [Cu(Sal-2H)(Ph-bipy)] and [Cu(SalF3 -2H)(Ph-bipy)2 ] showed the promising anti-gonorrhoeae activity (MIC 4.2-5.2 µM). (Cu(SalFn -2H)(Tol-bipy)2 ], [Cu(SalF4 -2H)(Ph-bipy)2 ] and [Cu(SalF3 -2H)(Ph-bipy)2 ]) were found active against the bacteria of S. aureus, S. aureus MRSA and their biofilms (MIC 2.4-41.4 µM). The most active compounds were tested for toxicity in vitro against human embryonic kidney (HEK-293) cells and in vivo experiments with CD-1 mice.

The emergence of the ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone by transfer of resistance from an oral Neisseria subflava reservoir of resistance.

BACKGROUND: The ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone was first discovered in Japan in 2015. OBJECTIVES: We investigated the possibility of horizontal gene transfer from Neisseria subflava harbouring the mosaic-like PBP-2 in the emergence of the FC428 clone. We also analysed whether there were fitness costs associated with the sustained international dissemination of the clone. METHODS: Sequencing of the penA gene in ceftriaxone-resistant N. subflava strains was performed. For transformation experiments between donor N. subflava and ciprofloxacin-resistant wild-type penA N. gonorrhoeae recipient, the full-length PCR amplification product of the penA gene, including DUS regions, was used as the donor DNA. Biological fitness of the transformants was measured by growth competition assays. The impact of QRDR and mtrR mutations, which have been reported as compensatory mutations, on fitness was also assessed. RESULTS: The penA mosaic allele of the FC428 clone showed 100%, 91.8%, and 89.8% homology, respectively, with penA genes of three ceftriaxone-resistant N. subflava strains, No. 30, No. 9 and No. 14. Results were consistent with homologous recombination with the donated penA mosaic allele. In co-cultures with the parent strain, transformants showed comparable growth indicating that a gyrA mutation compensates for the fitness cost of mosaic penA alleles. CONCLUSIONS: Our findings support the hypothesis that the FC428 clone was generated by transformation of the mosaic penA allele from oropharyngeal N. subflava to N. gonorrhoeae. Furthermore, it suggests that mutations in the gyrA QRDR region compensate for fitness costs and contribute to the continued transmission of the FC428 clone.

Lipophilic quinolone derivatives: Synthesis and in vitro antibacterial evaluation.

This paper reports on the design of a series of 10 novel lipophilic piperazinyl derivatives of the 1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, their synthesis, their characterisation by 1H, 13C and 19F NMR, IR spectroscopy and HRMS, as well as their biological activity against bacteria of medical interest. Among these derivatives, 2 were as potent as the parent quinolone against Neisseriagonorrhoeae whereas all the compounds displayed lower activity than the parent quinolone against other bacteria of medical interest. Our results showing that the increased lipophilicity was deleterious for antibacterial activity may help to design new quinolone derivatives in the future, especially lipophilic quinolones which have been poorly investigated previously.