Abstract
Prolonged use of broad-spectrum antibiotics disrupts the indigenous gut microbiota, which consequently enables toxigenic Clostridium difficile species to proliferate and cause infection. The burden of C. difficile infections was exacerbated with the outbreak of hypervirulent strains that produce copious amounts of enterotoxins and spores. In recent past, membrane-active agents have generated a surge of interest due to their bactericidal property with a low propensity for resistance. In this study, we capitalized on the antimicrobial property and low oral bioavailability of salicylanilide anthelmintics (closantel, rafoxanide, niclosamide, oxyclozanide) to target the gut pathogen. By broth microdilution techniques, we determined the MIC values of the anthelmintics against 16 C. difficile isolates of defined PCR-ribotype. The anthelmintics broadly inhibited C. difficile growth in vitro via a membrane depolarization mechanism. Interestingly, the salicylanilides were bactericidal against logarithmic- and stationary-phase cultures of the BI/NAP1/027 strain 4118. The salicylanilides were poorly active against select gut commensals (Bacteroides, Bifidobacterium and Lactobacillus species), and were non-hemolytic and non-toxic to mammalian cell lines HepG2 and HEK 293T/17 within the range of their in vitro MICs and MBCs. The salicylanilide anthelmintics exhibit desirable properties for repositioning as anti-C. difficile agents.
Highlights
Clostridium difficile infections (CDI) has plagued nearly half a million Americans that resulted in 29,300 deaths in 20111, and the propensity of nosocomial CDI recurrence has been observed in up to 50% of patients[2]
We show that the salicylanilide derivatives efficiently inhibited the growth of C. difficile via membrane depolarization, and more importantly, killed both logarithmic- and stationary-phase cells in a concentration-dependent manner
C. difficile ribotype 027 was the predominant ribotype found in CDI fecal samples across North America and Europe[30], in recent years, other ribotypes are becoming increasingly prevalent[31]
Summary
In vitro susceptibilities of C. difficile isolates. Over the last decade, C. difficile ribotype 027 was the predominant ribotype found in CDI fecal samples across North America and Europe[30], in recent years, other ribotypes are becoming increasingly prevalent[31]. We evaluated sixteen C. difficile clinical isolates of defined PCR ribotype (including the more prevalent ribotypes 027, 002, 014, 020, 078 and 106)[31] for their in vitro susceptibilities to anthelmintics closantel, rafoxanide, niclosamide and oxyclozanide (Supplementary Table S1). Based on the MIC against 16 isolates of C difficile, we determined the MIC90 values to be 0.25, 0.13, 2 and 1 μg/mL for closantel, rafoxanide, niclosamide and oxyclozanide, respectively (Table 1 and Supplementary Table S1). In order to ascertain that the observed activity of the salicylanilides occurs through dissipation of the bacterial membrane potential, we prepared analogues 5 and 6 (Fig. 2) as previously described[32], and evaluated their growth inhibitory activity against C. difficile strains 630 (CD630, ATCC BAA-1382-FZ, ribotype 012) and 4118 (CD4118, ATCC BAA-1870, ribotype 027).
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