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Treatment options for gonorrhoea are scarce. Drug repurposing of bioactive molecules approved for other conditions might therefore be of value. We developed a method for wide-scale, systematic drug ...

To assess the in vitro effect of select antimicrobials on the growth ofTime-kill assays were performed on two referenceCeftriaxone, ertapenem and fosfomycin showed gradual death overtime at higher antimicrobial concentrations with a relatively highBased on time-kill assays, high-dosage ceftriaxone could be used to treat

For Neisseria gonorrhoeae susceptibility testing, Etest, comparable to agar dilution, is frequently used. In recent years, newer MIC gradient strip tests have been commercialized. However, these tests have not been appropriately evaluated for gonococci. We evaluated the sensitivity, specificity, accuracy, quality, availability of antimicrobials and cost of the MIC Test Strip (Liofilchem), M.I.C.Evaluator (Oxoid) and Ezy MIC Strip (HiMedia), compared to the reference Etest (bioMerieux), for gonococcal susceptibility testing. The MICs of eight antimicrobials in 103 gonococcal international reference strains (n = 29) and clinical isolates (n = 74) were examined. Coefficient of determination (R2 ), complete agreement, essential agreement, SIR categorical agreement, sensitivity, specificity and accuracy were calculated. R2 of the MICs for the antimicrobials ranged between 0.674-0.996, 0.617-0.993, and 0.643-0.994 for the MIC Test Strip, M.I.C.Evaluator strips and Ezy MIC Strips respectively. The essential agreement (SIR categorical agreement) was 99.6% (88.6%), 100% (87.1%) and 93.0% (83.1%) respectively. M.I.C.Evaluator strips for gonococcal key antimicrobials were lacking and the Ezy MIC Strips showed an inconsistent accuracy, quality and some strips were contaminated. The Liofilchem MIC Test Strips had limitations, but might be relatively accurate alternatives to Etest for gonococci. Strict quality assurance (at manufacturing and testing laboratory), including quality controls, are required.

Treatment options for gonorrhoea are scarce. Drug repurposing of bioactive molecules approved for other conditions might therefore be of value. We developed a method for wide-scale, systematic drug repurposing screen to identify molecules with activity against Neisseria gonorrhoeae and screened the Prestwick Chemical Library (1200 FDA-approved drugs). As a proof-of-concept, we further examined one promising and interesting screening hit (auranofin; antirheumatic agent). Three WHO gonococcal reference strains (WHO F, O, P) were used for the Library screening. The strains were grown in presence of a fixed concentration of the library drugs in 384-well plates for 12 h, and the remaining bacterial respiration, to reflect growth, was then quantitatively measured using optical density (OD) 450 nm and a resazurin assay. The activity of auranofin was further examined using in vitro susceptibility testing (minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)) against genetically diverse antimicrobial-resistant N. gonorrhoeae strains and time-kill assays. Sixty-eight molecules significantly inhibited bacterial growth of WHO F, O and P. Auranofin showed potent in vitro bactericidal activity (in MIC-, MBC- and time-kill assays) against four WHO reference strains. No cross-resistance between auranofin and any antimicrobial currently or previously used for gonorrhoea treatment was found when examining 51 selected antimicrobial-resistant gonococcal strains. In conclusion, this is the first wide-scale systematic screening effort for repurposing drugs for future treatment of gonorrhoea. Additional studies examining mechanism(s) of action, resistance development, in vivo anti-gonococcal activity and pharmacokinetics/pharmacodynamics for gonococcal infections of auranofin and several other significant screening hits would be valuable.

Background Resistance in Neisseria gonorrhoeae to all therapeutic antimicrobials for gonorrhoea has emerged. Novel antimicrobials for treatment are imperative and the first-in-class spiropyrimidinetrione zoliflodacin appears promising. Zoliflodacin could be introduced in dual antimicrobial therapies to prevent the emergence and/or spread of resistance. We investigated the in vitro activity and induction/selection of resistance to zoliflodacin alone and in combination with six novel, currently or previously used therapeutic antimicrobials against N. gonorrhoeae. Methods The international gonococcal reference strains examined were WHO F (wild-type), and WHO O, WHO V, and WHO X (strains with different AMR profiles). Zoliflodacin was evaluated alone or in combination with ceftriaxone, spectinomycin, gentamicin, tetracycline, cethromycin, and sitafloxacin in checkerboard assays, time-kill curve analysis, and induction/selection of resistance studies. Results Zoliflodacin alone or in combination with all six antimicrobials showed rapid rates of in vitro bacterial killing against all examined strains in time-kill studies. Tetracycline or cethromycin combined with zoliflodacin decreased the rate of zoliflodacin growth inhibition, while ceftriaxone or gentamicin increased the rate of cell killing. The frequency of induced/selected zoliflodacin resistance mutations was low for zoliflodacin and further reduced for all antimicrobial combinations. All resistant mutants contained the GyrB mutations D429N, K450T or K450N, resulting in zoliflodacin MICs of 0.5–4 mg/L consistent with previous results. Conclusion Zoliflodacin, alone or in combination with STI therapeutic antimicrobials has a rapid and high in vitro efficacy against gonococci with low resistance emergence. Zoliflodacin remains a promising novel oral therapeutic for gonorrhoea monotherapy and as part of dual antimicrobial therapy with low resistance emergence potential. A phase III clinical trial evaluating efficacy and safety of zoliflodacin for uncomplicated gonorrhoea treatment is planned in 2019. Disclosure No significant relationships.

ObjectivesResistance in Neisseria gonorrhoeae to all gonorrhoea therapeutic antimicrobials has emerged. Novel therapeutic antimicrobials are imperative and the first-in-class spiropyrimidinetrione zoliflodacin appears promising. Zoliflodacin could be introduced in dual antimicrobial therapies to prevent the emergence and/or spread of resistance. We investigated the in vitro activity of and selection of resistance to zoliflodacin alone and in combination with six gonorrhoea therapeutic antimicrobials against N. gonorrhoeae.MethodsThe international gonococcal reference strains WHO F (WT) and WHO O, WHO V and WHO X (strains with different AMR profiles) were examined. Zoliflodacin was evaluated alone or combined with ceftriaxone, cefixime, spectinomycin, gentamicin, tetracycline, cethromycin or sitafloxacin in chequerboard assays, time–kill curve analysis and selection-of-resistance studies.ResultsZoliflodacin alone or in combination with all six antimicrobials showed rapid growth inhibition against all examined strains. The time–kill curve analysis indicated that tetracycline or cethromycin combined with zoliflodacin can significantly decrease the zoliflodacin kill rate in vitro. The frequency of selected zoliflodacin-resistance mutations was low when evaluated as a single agent and further reduced for all antimicrobial combinations. All resistant mutants contained the GyrB mutations D429N, K450T or K450N, resulting in zoliflodacin MICs of 0.5–4 mg/L.ConclusionsZoliflodacin, alone or in combination with sexually transmitted infection therapeutic antimicrobials, rapidly kills gonococci with infrequent resistance emergence. Zoliflodacin remains promising for gonorrhoea oral monotherapy and as part of dual antimicrobial therapy with low resistance emergence potential. A Phase III trial evaluating efficacy and safety of zoliflodacin for uncomplicated gonorrhoea treatment is planned in 2019.

The spread of antimicrobial resistance has become a serious public health concern, making once-treatable diseases deadly again and undermining the achievements of modern medicine. Drug combinations can help to fight multi-drug-resistant bacterial infections, yet they are largely unexplored and rarely used in clinics. Here we profile almost 3,000 dose-resolved combinations of antibiotics, human-targeted drugs and food additives in six strains from three Gram-negative pathogens-Escherichia coli, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa-to identify general principles for antibacterial drug combinations and understand their potential. Despite the phylogenetic relatedness of the three species, more than 70% of the drug-drug interactions that we detected are species-specific and 20% display strain specificity, revealing a large potential for narrow-spectrum therapies. Overall, antagonisms are more common than synergies and occur almost exclusively between drugs that target different cellular processes, whereas synergies are more conserved and are enriched in drugs that target the same process. We provide mechanistic insights into this dichotomy and further dissect the interactions of the food additive vanillin. Finally, we demonstrate that several synergies are effective against multi-drug-resistant clinical isolates in vitro and during infections of the larvae of the greater wax moth Galleria mellonella, with one reverting resistance to the last-resort antibiotic colistin.

Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is common, compromising gonorrhoea treatment internationally. Rapid characterisation of AMR strains could ensure appropriate and personalised treatment, and support identification and investigation of gonorrhoea outbreaks in nearly real-time. Whole-genome sequencing is ideal for investigation of emergence and dissemination of AMR determinants, predicting AMR, in the gonococcal population and spread of AMR strains in the human population. The novel, rapid and revolutionary long-read sequencer MinION is a small hand-held device that generates bacterial genomes within one day. However, accuracy of MinION reads has been suboptimal for many objectives and the MinION has not been evaluated for gonococci. In this first MinION study for gonococci, we show that MinION-derived sequences analysed with existing open-access, web-based sequence analysis tools are not sufficiently accurate to identify key gonococcal AMR determinants. Nevertheless, using an in house-developed CLC Genomics Workbench including de novo assembly and optimised BLAST algorithms, we show that 2D ONT-derived sequences can be used for accurate prediction of decreased susceptibility or resistance to recommended antimicrobials in gonococcal isolates. We also show that the 2D ONT-derived sequences are useful for rapid phylogenomic-based molecular epidemiological investigations, and, in hybrid assemblies with Illumina sequences, for producing contiguous assemblies and finished reference genomes.

Antimicrobial resistant (AMR) Neisseria gonorrhoeae strains are common and compromise gonorrhoea treatment internationally. Rapid identification and characterisation of AMR gonococcal strains could ensure appropriate and even personalised treatment, and support identification and investigation of gonorrhoea outbreaks in nearly real-time. Whole-genome sequencing is ideal for investigation of the emergence and dissemination of AMR determinants that predict AMR in the gonococcal population and spread of AMR strains in the human population. The novel, rapid and revolutionary long-read sequencer MinION is a small hand-held device that can generate bacterial genomes within one day. However, the accuracy of MinION reads has been suboptimal for many objectives and the MinION has not been evaluated for gonococci. In this first MinION study for gonococci, we show that MinION-derived sequences analysed with existing open-access, web-based sequence analysis tools are not sufficiently accurate to identify key gonococcal AMR determinants. Nevertheless, using an in house-developed CLC Genomics Workbench, we show that ONT-derived sequences can be used for accurate prediction of decreased susceptibility or resistance to recommended therapeutic antimicrobials. We also show that the ONT-derived sequences can be useful for rapid phylogenomic-based molecular epidemiological investigations, and, in hybrid assemblies with Illumina sequences, for producing contiguous assemblies and finished reference genomes.

Objectives Rapid, cost-effective and objective methods for antimicrobial susceptibility testing of Neisseria gonorrhoeae would greatly enhance surveillance of antimicrobial resistance. Etest, disc diffusion and agar dilution methods are subjective, mostly laborious for large-scale testing and take ∼24 h. We aimed to develop a rapid broth microdilution assay using resazurin (blue), which is converted into resorufin (pink fluorescence) in the presence of viable bacteria. Methods The resazurin-based broth microdilution assay was established using 132 N. gonorrhoeae strains and the antimicrobials ceftriaxone, cefixime, azithromycin, spectinomycin, ciprofloxacin, tetracycline and penicillin. A regression model was used to estimate the MICs. Assay results were obtained in ∼7.5 h. Results The EC 50 of the dose-response curves correlated well with Etest MIC values (Pearson's r = 0.93). Minor errors resulting from misclassifications of intermediate strains were found for 9% of the samples. Major errors (susceptible strains misclassified as resistant) occurred for ceftriaxone (4.6%), cefixime (3.3%), azithromycin (0.6%) and tetracycline (0.2%). Only one very major error was found (a ceftriaxone-resistant strain misclassified as susceptible). Overall the sensitivity of the assay was 97.1% (95% CI 95.2-98.4) and the specificity 78.5% (95% CI 74.5-82.9). Conclusions A rapid, objective, high-throughput, quantitative and cost-effective broth microdilution assay was established for gonococci. For use in routine diagnostics without confirmatory testing, the specificity might remain suboptimal for ceftriaxone and cefixime. However, the assay is an effective low-cost method to evaluate novel antimicrobials and for high-throughput screening, and expands the currently available methodologies for surveillance of antimicrobial resistance in gonococci.

Background Gonorrhoea is a sexually transmitted infection caused by the Gram-negative bacterium Neisseria gonorrhoeae. Resistance to first-line empirical monotherapy has emerged, so robust methods are needed to evaluate the activity of existing and novel antimicrobials against the bacterium. Pharmacodynamic models describing the relationship between the concentration of antimicrobials and the minimum growth rate of the bacteria provide more detailed information than the MIC only. Results In this study, a novel standardised in vitro time-kill curve assay was developed. The assay was validated using five World Health Organization N. gonorrhoeae reference strains and a range of ciprofloxacin concentrations below and above the MIC. Then the activity of nine antimicrobials with different target mechanisms was examined against a highly antimicrobial susceptible clinical strain isolated in 1964. The experimental time-kill curves were analysed and quantified with a previously established pharmacodynamic model. First, the bacterial growth rates at each antimicrobial concentration were estimated with linear regression. Second, we fitted the model to the growth rates, resulting in four parameters that describe the pharmacodynamic properties of each antimicrobial. A gradual decrease of bactericidal effects from ciprofloxacin to spectinomycin and gentamicin was found. The beta-lactams ceftriaxone, cefixime and benzylpenicillin showed bactericidal and time-dependent properties. Chloramphenicol and tetracycline were purely bacteriostatic as they fully inhibited the growth but did not kill the bacteria. We also tested ciprofloxacin resistant strains and found higher pharmacodynamic MICs (zMIC) in the resistant strains and attenuated bactericidal effects at concentrations above the zMIC. Conclusions N. gonorrhoeae time-kill curve experiments analysed with a pharmacodynamic model have potential for in vitro evaluation of new and existing antimicrobials. The pharmacodynamic parameters based on a wide range of concentrations below and above the MIC provide information that could support improving future dosing strategies to treat gonorrhoea. Electronic supplementary material The online version of this article (doi:10.1186/s12866-016-0838-9) contains supplementary material, which is available to authorized users.

Developmental toxicity in vitro assays have hitherto been established as stand-alone systems, based on a limited number of toxicants. Within the embryonic stem cell-based novel alternative tests project, we developed a test battery framework that allows inclusion of any developmental toxicity assay and that explores the responses of such test systems to a wide range of drug-like compounds. We selected 28 compounds, including several biologics (e.g., erythropoietin), classical pharmaceuticals (e.g., roflumilast) and also six environmental toxicants. The chemical, toxicological and clinical data of this screen library were compiled. In order to determine a non-cytotoxic concentration range, cytotoxicity data were obtained for all compounds from HEK293 cells and from murine embryonic stem cells. Moreover, an estimate of relevant exposures was provided by literature data mining. To evaluate feasibility of the suggested test framework, we selected a well-characterized assay that evaluates 'migration inhibition of neural crest cells.' Screening at the highest non-cytotoxic concentration resulted in 11 hits (e.g., geldanamycin, abiraterone, gefitinib, chlorpromazine, cyproconazole, arsenite). These were confirmed in concentration-response studies. Subsequent pharmacokinetic modeling indicated that triadimefon exerted its effects at concentrations relevant to the in vivo situation, and also interferon-β and polybrominated diphenyl ether showed effects within the same order of magnitude of concentrations that may be reached in humans. In conclusion, the test battery framework can identify compounds that disturb processes relevant for human development and therefore may represent developmental toxicants. The open structure of the strategy allows rich information to be generated on both the underlying library, and on any contributing assay. © 2014 The Author(s). Chemicals/CAS: abiraterone, 154229-19-3; arsenic trioxide, 1303-24-8, 1327-53-3, 13464-58-9, 15502-74-6; chlorpromazine, 50-53-3, 69-09-0; gefitinib, 184475-35-2, 184475-55-6, 184475-56-7; geldanamycin, 30562-34-6; triadimefon, 43121-43-3

Introduction Neisseria gonorrhoeae shows increasing resistance to first line empirical treatment, which demonstrates the need for robust methods to evaluate antibiotic treatment regimens. Antibiotic efficacy is traditionally determined in vitro by measuring minimum inhibitory concentrations (MICs). Time-kill curve assays for N. gonorrhoeae have been difficult to standardise. We developed a new time-kill curve assay and used pharmacodynamics functions to analyse the relationship between antibiotic concentration and bacterial net growth rate. Methods We used a defined medium (Graver-Wade medium) and grew bacteria in 96-well microtiter plates. To measure colony forming units over a time course of six hours, we used a previously described drop plate method and spotted 10 µl droplets on chocolate agar with a multichannel pipette. The assay was validated using 16 reference strains, representing diffent clades of the N. gonorrhoeae phylogenetic tree. We then studied a highly sensitive strain isolated in 1954 in Denmark in detail. Penicillin G, spectinomycin, gentamicin, tetracyline, chloramphenicol, ciprofloxacin, azithromycin, cefixime and ceftriaxone were examined in concentrations from 0.016× to 16×MIC. A pharmacodynamic function was fitted to the net bacterial growth rates at each concentration, resulting in four parameters that describe the pharmacodynamic properties of each antibiotic. Results Our time-kill curve assay was reproducible for all the N. gonorrhoeae strains tested. Ciprofloxacin and spectinomycin induced the strongest bactericidal effect during the first six hours. Tetracycline and chloramphenicol were the only antibiotics that showed a purely bacteriostatic effect. Differences in the shape of the pharmacodynamic functions illustrate the time and concentration dependent properties of the antibiotics. Conclusion We developed a standardised, robust and quality-assured time-kill curve assay for N. gonorrhoeae . Pharmacodynamic functions allowed classification of nine antibiotics according to their antimicrobial properties. These methods can be used to investigate new antibiotic regimens and help to improve dosing strategies. Disclosure of interest statement This work was supported by SystemsX, Switzerland, Orebro County Council Research Committee and the Foundation for Medical Research at Orebro University Hospital, Sweden.