Species-specific activity of antibacterial drug combinations

International audienceThe spread of antimicrobial resistance has become a serious public health concern, making once treatable diseases deadly again and undermining breakthrough achievements of modern medicine 1,2. Drug combinations can aid in fighting multi-drug resistant (MDR) bacterial infections, yet, are largely unexplored and rarely used in clinics. To identify general principles for antibacterial drug combinations and understand their potential, we profiled ~3,000 dose-resolved combinations of antibiotics, human-targeted drugs and food additives in 6 strains from three Gram-negative pathogens, Escherichia coli, Salmonella Typhimurium and Pseudomonas aeruginosa. Despite their phylogenetic relatedness, more than 70% of the detected drug-drug interactions 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 targeting different cellular processes, whereas synergies are more conserved and enriched in drugs targeting the same process. We elucidate mechanisms underlying this dichotomy and further use our resource to dissect the interactions of the food additive, vanillin. Finally, we demonstrate that several synergies are effective against MDR clinical isolates in vitro and during Galleria mellonella infections with one reverting resistance to the last-resort antibiotic, colistin

A new rapid resazurin-based microdilution assay for antimicrobial susceptibility testing of Neisseria gonorrhoeae.

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

A new rapid resazurin-based microdilution assay for antimicrobial susceptibility testing of Neisseria gonorrhoeae

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 EC50 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

Pharmacodynamics of Ceftriaxone, Ertapenem, Fosfomycin and Gentamicin in <i>Neisseria gonorrhoeae</i>

Objectives: To assess the in vitro effect of select antimicrobials on the growth of N. gonorrhoeae and its pharmacodynamic parameters. Methods: Time–kill assays were performed on two reference N. gonorrhoeae strains (ceftriaxone-resistant WHO X and ceftriaxone-susceptible WHO F) and one clinical N. gonorrhoeae strain (ceftriaxone-susceptible CS03307). Time–kill curves were constructed for each strain by measuring bacterial growth rates at doubling antimicrobial concentrations of ceftriaxone, ertapenem, fosfomycin and gentamicin. Inputs from these curves were used to estimate minimal bacterial growth rates at high antimicrobial concentrations (ψmin), maximum bacterial growth rates in the absence of antimicrobials (ψmax), pharmacodynamic minimum inhibitory concentrations (zMIC), and Hill’s coefficients (κ). Results: Ceftriaxone, ertapenem and fosfomycin showed gradual death overtime at higher antimicrobial concentrations with a relatively high ψmin, demonstrating time-dependent activity. Compared to WHO F, the ψmin for WHO X was significantly increased, reflecting decreased killing activity for ceftriaxone, ertapenem and fosfomycin. At high ceftriaxone concentrations, WHO X was still efficiently killed. CS03307 also showed a high ψmin for ceftriaxone in spite of a low MIC and no difference in ψmin for fosfomycin in spite of significant MIC and zMIC differences. Gentamicin showed rapid killing for all three strains at high concentrations, demonstrating concentration-dependent activity. Conclusions: Based on time–kill assays, high-dosage ceftriaxone could be used to treat N. gonorrhoeae strains with MIC above breakpoint, with gentamicin as a potential alternative. Whether ertapenem or fosfomycin would be effective to treat strains with a high MIC to ceftriaxone is questionable

Additional file 1: of Time-kill curve analysis and pharmacodynamic modelling for in vitro evaluation of antimicrobials against Neisseria gonorrhoeae

Figure S1. Growth curves for five WHO reference strains. WHO G (A), WHO K (B), WHO L (C), WHO M (D), WHO N (E). Data from three independent experiments are shown. CFU/ml for each time-point are shown in circles (experiment 1), triangles (experiment 2) and diamonds (experiment 3). A Gompertz growth model was fit to the data from three independent experiments (solid lane, pooled data). Individual fits from each of the experiments are shown as well in dashed lines. Growth rates were estimated in log phase between 2 and 20 h (WHO G = 0.75 [h-1], WHO K = 0.72 [h-1], WHO L = 0.57 [h-1], WHO M = 0.75 [h-1], WHO N = 0.70 [h-1]). The maximal bacterial density was estimated as upper asymptote of the Gompertz model (WHO G = 9.74*109 [CFU/ml], WHO K = 1.32*109 [CFU/ml], WHO L = 6.57*107 [CFU/ml], WHO M = 1.32*109 [CFU/ml], WHO N = 5.32*1011 [CFU/ml]). Table S1. Parameter estimates from nine different antimicrobials in DG666 and model based standard errors. Table S2. Parameter estimates from ciprofloxacin in five WHO reference strains and model based standard errors. (PDF 489 kb

In vitro activity and time-kill curve analysis of sitafloxacin against a global panel of antimicrobial-resistant and multidrug-resistant Neisseria gonorrhoeae isolates.

Treatment of gonorrhoea is a challenge worldwide because of emergence of resistance in N. gonorrhoeae to all therapeutic antimicrobials available and novel antimicrobials are imperative. The newer-generation fluoroquinolone sitafloxacin, mostly used for respiratory tract infections in Japan, can have a high in vitro activity against gonococci. However, only a limited number of recent antimicrobial-resistant isolates from Japan have been examined. We investigated the sitafloxacin activity against a global gonococcal panel (250 isolates cultured in 1991-2013), including multidrug-resistant geographically, temporally and genetically diverse isolates, and performed time-kill curve analysis for sitafloxacin. The susceptibility to sitafloxacin (agar dilution) and seven additional therapeutic antimicrobials (Etest) was determined. Sitafloxacin was rapidly bactericidal, and the MIC range, MIC50 and MIC90 was ≤0.001-1, 0.125 and 0.25 mg/L, respectively. There was a high correlation between the MICs of sitafloxacin and ciprofloxacin; however, the MIC50 and MIC90 of sitafloxacin were 6-fold and >6-fold lower, respectively. Sitafloxacin might be an option for particularly dual antimicrobial therapy of gonorrhoea and for cases with ceftriaxone resistance or allergy. However, further in vitro and particularly in vivo evaluations of potential resistance, pharmacokinetics/pharmacodynamics and ideal dosing for gonorrhoea, as well as performance of randomized controlled clinical, trials are crucial

Definition of transcriptome-based indices for quantitative characterization of chemically disturbed stem cell development: introduction of the STOP-Tox(ukn) and STOP-Tox(ukk) tests

Stem cell-based in vitro test systems can recapitulate specific phases of human development. In the UKK test system, human pluripotent stem cells (hPSCs) randomly differentiate into cells of the three germ layers and their derivatives. In the UKN1 test system, hPSCs differentiate into early neural precursor cells. During the normal differentiation period (14 days) of the UKK system, 570 genes [849 probe sets (PSs)] were regulated > fivefold; in the UKN1 system (6 days), 879 genes (1238 PSs) were regulated. We refer to these genes as 'developmental genes'. In the present study, we used genome-wide expression data of 12 test substances in the UKK and UKN1 test systems to understand the basic principles of how chemicals interfere with the spontaneous transcriptional development in both test systems. The set of test compounds included six histone deacetylase inhibitors (HDACis), six mercury-containing compounds ('mercurials') and thalidomide. All compounds were tested at the maximum non-cytotoxic concentration, while valproic acid and thalidomide were additionally tested over a wide range of concentrations. In total, 242 genes (252 PSs) in the UKK test system and 793 genes (1092 PSs) in the UKN1 test system were deregulated by the 12 test compounds. We identified sets of 'diagnostic genes' appropriate for the identification of the influence of HDACis or mercurials. Test compounds that interfered with the expression of developmental genes usually antagonized their spontaneous development, meaning that up-regulated developmental genes were suppressed and developmental genes whose expression normally decreases were induced. The fraction of compromised developmental genes varied widely between the test compounds, and it reached up to 60 %. To quantitatively describe disturbed development on a genome-wide basis, we recommend a concept of two indices, 'developmental potency' (D (p)) and 'developmental index' (D (i)), whereby D (p) is the fraction of all developmental genes that are up- or down-regulated by a test compound, and D (i) is the ratio of overrepresentation of developmental genes among all genes deregulated by a test compound. The use of D (i) makes hazard identification more sensitive because some compounds compromise the expression of only a relatively small number of genes but have a high propensity to deregulate developmental genes specifically, resulting in a low D (p) but a high D (i). In conclusion, the concept based on the indices D (p) and D (i) offers the possibility to quantitatively express the propensity of test compounds to interfere with normal development