Sub-lethal doses of surotomycin and vancomycin have similar effects on Clostridium difficile virulence factor production in vitro

Abstract

Purpose: Clostridium difficile is an anaerobic spore-forming bacterial pathogen that causes a spectrum of illness severity ranging from mild diarrhoea to severe life-threatening pseudomembranous colitis. C. difficile infection (CDI) is antibiotic-associated and primarily mediated by two exotoxins, Toxins A and B. We and others have shown that some antibiotics stimulate Toxin A and B production by C. difficile in a strain-specific manner. Still, the effects of newer anti-C. difficile antibiotics on this process and spore formation remain to be investigated.

Methodology: Surotomycin (formally CB-183,315) is a novel, minimally absorbed, narrow-spectrum antibiotic. We determined the effects of surotomycin on C. difficile growth, toxin production and sporulation in historical and BI/NAP1/027 epidemic strains of C. difficile.Results/Key findings. While antibiotic free controls showed toxin production during the stationary phase growth, all strains exposed to sub-inhibitory concentrations of surotomycin and vancomycin demonstrated increased TcdA and TcdB production during early (log phase) growth by all strains. However, this effect was not observed at 24 or 48 h post-treatment by any of the C. difficile strains exposed to either antibiotic. Additionally, all doses of surotomycin and vancomycin suppressed spore formation in all tested strains.

Conclusion: In summary, these findings demonstrate that surotomycin and vancomycin have similar effects on exotoxin production and sporulation by C. difficile in vitro. Furthermore, since spores contribute to recurrent infection, the ability of surotomycin to suppress spore formation may explain its ability to disrupt the reinfection cycle in the clinical setting.

Keywords: Clostridium difficile; exotoxin; sporulation; surotomycin; vancomycin.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Figures

Fig. 1.

Effects of surotomycin on growth of C. difficile historical 9689 and epidemic 9689 and 5325 strains. (a) Antibiotic-free cultures; (b) 1/8× MIC; (c) 1/4× MIC; (d) 1× MIC; and (e) 2× MIC. Surotomycin was added, at the final concentrations indicated, during early log phase growth (designated Time 0). Samples were collected in duplicate over 48 h for quantification of viable C. difficile.

Fig. 2.

Effects of vancomycin ongrowth of C. difficile historical 9689 and epidemic 9689 and 5325 strains. (a) Antibiotic-free cultures; (b) 1/8× MIC; (c) 1/4× MIC; and (d) 1× MIC; and (e) 2× MIC. Vancomycin was added, at the final concentrations indicated, during early log phase growth (designated Time 0). Samples were collected in duplicate over 48 h for quantification of viable C. difficile.

Fig. 3.

Comparison of soluble TcdA/TcdB production following exposure to sub-inhibitory and inhibitory concentrations of surotomycin and vancomycin. Supernatant samples were collected at 6, 12, 24 and 48 h from C. difficile ATCC 9689, BI 5325 and 2989 strain cultures containing either nothing, 1/8×, 1/4× or 1× MIC doses of (a) surotomycin or (b) vancomycin. Collected samples were screened for detection of TcdA/TcdB by commercial ELISA and are given as relative values compared to the drug-free culture. Data shown are the means of 3 replicates from 2 experiments.

Fig. 4.

Comparison of spore production at 24 and 48 h following exposure to sub-inhibitory and inhibitory concentrations of antibiotics. Strains 9689, 5325 (Historical BI) and 2989 (Epidemic BI) strains of C. difficile were exposed to either nothing, surotomycin (a and b) or vancomycin (c and d) at 24 and 48 h, respectively, at the final concentrations indicated. Samples were collected and spores isolated by mixing an equal volume of culture supernatant with 100 % ethanol followed by centrifugation. Harvested spores were enumerated by serially diluting in PBS and plating onto BHI agar plates. Plates were incubated anaerobically at 37 °C for 72 h.