The membrane as a target for controlling hypervirulent Clostridium difficile infections

Abstract

Objectives: The stationary phase of Clostridium difficile, which is primarily responsible for diarrhoeal symptoms, is refractory to antibiotic killing. We investigated whether disrupting the functions of the clostridial membrane is an approach to control C. difficile infections by promptly removing growing and non-growing cells.

Methods: The bactericidal activities of various membrane-active agents were determined against C. difficile logarithmic-phase and stationary-phase cultures and compared with known antibiotics. Their effects on the synthesis of ATP, toxins A/B and sporulation were also determined. The effect of rodent caecal contents on anti-difficile activities was examined using two reutericyclin lead compounds, clofazimine, daptomycin and other comparator antibiotics.

Results: Most membrane-active agents and partially daptomycin showed concentration-dependent killing of both logarithmic-phase and stationary-phase cultures. The exposure of cells to compounds at their MBC resulted in a rapid loss of viability with concomitant reductions in cellular ATP, toxins A/B and spore numbers. With the exception of nisin, these effects were not due to membrane pore formation. Interestingly, the activity of the proton ionophore nigericin significantly increased as the growth of C. difficile decreased, suggesting the importance of the proton gradient to the survival of non-growing cells. The activities of the lipophilic antimicrobials reutericyclins and clofazimine were reduced by caecal contents.

Conclusions: These findings indicate that C. difficile is uniquely susceptible to killing by molecules affecting its membrane function and bioenergetics, indicating that the clostridial membrane is a novel antimicrobial target for agents to alleviate the burden of C. difficile infections.

Figure 1.

Nigericin killing of C. difficile (BAA-1803) increases as cell growth decreases; a 6.8-fold difference in MBC is observed for nigericin against cultures of OD₆₀₀ = 0.2 and 0.8.

Figure 2.

Killing of stationary-phase C. difficile (BAA-1803) by compounds is correlated with reductions in ATP biosynthesis. The percentages of surviving cells and ATP concentrations relative to the starting point are shown. The following MBC_STA concentrations were used for compounds: nigericin, 0.0006 mg/L; DCCD, 16 mg/L; nisin, 0.8 mg/L; valinomycin, 4 mg/L; 867, 0.5 mg/L; clofazimine, 2 mg/L; metronidazole, 8 mg/L; and vancomycin, 10 mg/L (as a control). Due to differences in rates of kill for compounds, samples were recovered at various timepoints, as reflected in the graphs.

Figure 3.

Effects on toxin production by membrane-active agents and comparators. At bactericidal concentrations membrane-active agents decreased the total toxin in cultures of stationary-phase C. difficile BAA-1803 within 24 h, compared with the protein synthesis inhibitors puromycin (150 mg/L) and fusidic acid (10 mg/L). Vancomycin (10 mg/L) did not alter total toxin production. The bactericidal concentrations used for membrane-active compounds are their MBC_STA: nigericin, 0.0006 mg/L; DCCD, 16 mg/L; nisin, 0.8 mg/L; valinomycin, 4 mg/L; 867, 0.5 mg/L; clofazimine, 2 mg/L; and metronidazole, 8 mg/L. Statistical significance (shown by an asterisk) was determined by one-way analysis of variance followed by Tukey's test (P < 0.05) in Graphpad prism 5 with no drug as the control.