A novel method for imaging the pharmacological effects of antibiotic treatment on Clostridium difficile.

Clostridium difficile is a significant cause of nosocomial-acquired infection that results in severe diarrhea and can lead to mortality. Treatment options for C. difficile infection (CDI) are limited, however, new antibiotics are being developed. Current methods for determining efficacy of experimental antibiotics on C. difficile involve antibiotic killing rates and do not give insight into the drug's pharmacologic effects. Considering this, we hypothesized that by using scanning electron microscopy (SEM) in tandem to drug killing curves, we would be able to determine efficacy and visualize the phenotypic response to drug treatment. To test this hypothesis, supraMIC kill curves were conducted using vancomycin, metronidazole, fidaxomicin, and ridinilazole. Following collection, cells were either plated or imaged using a scanning electron microscope (SEM). Consistent with previous reports, we found that the tested antibiotics had significant bactericidal activity at supraMIC concentrations. By SEM imaging and using a semi-automatic pipeline for image analysis, we were able to determine that vancomycin and to a lesser extent fidaxomicin and ridinilazole significantly affected the cell wall, whereas metronidazole, fidaxomicin, and ridinilazole had significant effects on cell length suggesting a metabolic effect. While the phenotypic response to drug treatment has not been documented previously in this manner, the results observed are consistent with the drug's mechanism of action. These techniques demonstrate the versatility and reliability of imaging and measurements that could be applied to other experimental compounds. We believe the strategies laid out here are vital for characterizing new antibiotics in development for treating CDI.

Impact on toxin production and cell morphology in Clostridium difficile by ridinilazole (SMT19969), a novel treatment for C. difficile infection.

OBJECTIVES: Ridinilazole (SMT19969) is a narrow-spectrum, non-absorbable antimicrobial with activity against Clostridium difficile undergoing clinical trials. The purpose of this study was to assess the pharmacological activity of ridinilazole and assess the effects on cell morphology. METHODS: Antibiotic killing curves were performed using the epidemic C. difficile ribotype 027 strain, R20291, using supra-MIC (4× and 40×) and sub-MIC (0.125×, 0.25× and 0.5×) concentrations of ridinilazole. Following exposure, C. difficile cells were collected for cfu counts, toxin A and B production, and morphological changes using scanning electron and fluorescence microscopy. Human intestinal cells (Caco-2) were co-incubated with ridinilazole-treated C. difficile growth medium to determine the effects on host inflammatory response (IL-8). RESULTS: Treatment at supra-MIC concentrations (4× and 40× MIC) of ridinilazole resulted in a significant reduction in vegetative cells over 72 h (4 log difference, P < 0.01) compared with controls without inducing spore formation. These results correlated with a 75% decrease in toxin A production (P < 0.05) and a 96% decrease in toxin B production (P < 0.05). At sub-MIC levels (0.5× MIC), toxin A production was reduced by 91% (P < 0.01) and toxin B production was reduced by 100% (P < 0.001), which resulted in a 74% reduction in IL-8 release compared with controls (P < 0.05). Sub-MIC (0.5×)-treated cells formed filamentous structures ∼10-fold longer than control cells. Following fluorescence labelling, the cell septum was not forming in sub-MIC-treated cells, yet the DNA was dividing. CONCLUSIONS: Ridinilazole had robust killing effects on C. difficile that significantly reduced toxin production and attenuated the inflammatory response. Ridinilazole also elicited significant cell division effects suggesting a potential mechanism of action.

In vivo assessment of SMT19969 in a hamster model of clostridium difficile infection.

SMT19969 [2,2'-bis(4-pyridyl)3H,3'-H 5,5-bibenzimidazole] is a novel narrow-spectrum nonabsorbable antibiotic currently in development for the treatment of Clostridium difficile infection. The comparative activities of SMT19969 and vancomycin against nonepidemic and epidemic strains of C. difficile were studied in an established hamster model. Against nonepidemic (VA11) strains, the survival rates of SMT19969-treated animals ranged from 80% to 95%. Vancomycin exhibited 100% protection during treatment, with relapse observed starting on day 9 and 50% survival at day 20. At 50 mg/kg of body weight, SMT19969 administered orally once daily for 5 days provided full protection of treated animals on the dosing days and through day 12 against epidemic strains. Vancomycin also protected during the dosing interval, but apparent relapse occurred earlier, starting on day 11. SMT19969 exhibited excellent in vitro activity, with MICs of 0.25 µg/ml for all isolates. The MICs for vancomycin were 2- to 4-fold higher at ≤0.5 to 1 µg/ml. All plasma sample concentrations of SMT19969 were below the limit of quantification (25 ng/ml) at all time points, consistent with the reported lack of bioavailability of the compound. Cecal concentrations were significantly above the MIC (ranging from 96 µg/ml to 172 µg/ml).

Automated large-scale culture and medium-throughput chemical screen for modulators of proliferation and viability of human induced pluripotent stem cell-derived neuroepithelial-like stem cells.

The aim of this study was to demonstrate proof-of-concept feasibility for the use of human neural stem cells (NSCs) for high-throughput screening (HTS) applications. For this study, an adherent human induced pluripotent stem (iPS) cell-derived long-term, self-renewing, neuroepithelial-like stem (lt-NES) cell line was selected as a representative NSC. Here, we describe the automated large-scale serum-free culture ("scale-up") of human lt-NES cells on the CompacT SelecT cell culture robotic platform, followed by their subsequent automated "scale-out" into a microwell plate format. We also report a medium-throughput screen of 1000 compounds to identify modulators of neural stem cell proliferation and/or survival. The screen was performed on two independent occasions using a cell viability assay with end-point reading resulting in the identification of 24 potential hit compounds, 5 of which were found to increase the proliferation and/or survival of human lt-NES on both occasions. Follow-up studies confirmed a dose-dependent effect of one of the hit compounds, which was a Cdk-2 modulator. This approach could be further developed as part of a strategy to screen compounds to either improve the procedures for the in vitro expansion of neural stem cells or to potentially modulate endogenous neural stem cell behavior in the diseased nervous system.