Action of nitroheterocyclic drugs against Clostridium difficile

Abstract

The nitroheterocyclic classes of drugs have a long history of use in treating anaerobic infections, as exemplified by metronidazole as a first-line treatment for mild-to-moderate Clostridium difficile infection (CDI). Since direct comparisons of the three major classes of nitroheterocyclic drugs (i.e. nitroimidazole, nitazoxanide and nitrofurans) and nitrosating agents against C. difficile are under-examined, in this study their actions against C. difficile were compared. Results show that whilst transient resistance occurs to metronidazole and nitazoxanide, stable resistance arises to nitrofurans upon serial passage. All compounds killed C. difficile at high concentrations in addition to the host defence nitrosating agent S-nitrosoglutathione (GSNO). This suggests that GSNO killing of C. difficile contributes to its efficacy in murine CDI. Although nitric oxide production could not be detected for the nitroheterocyclic drugs, the cellular response to metronidazole and nitrofurans has some overlap with the response to GSNO, causing significant upregulation of the hybrid-cluster protein Hcp that responds to nitrosative stress. These findings provide new insights into the action of nitroheterocyclic drugs against C. difficile.

Keywords: Anti-Clostridium difficile action; Nitazoxanide; Nitrofurans; Nitroimidazole; Nitrosative stress.

Fig. 1

Time–kill kinetics against log-phase Clostridium difficile CD196. Data are shown for metronidazole (MTZ) and nitazoxanide (NTZ) at their minimum bactericidal concentration (MBC), nitrofurantoin (NFT) at 8× and 16× its minimum inhibitory concentration (MIC) and S-nitrosoglutathione (GSNO) at its MIC. Data are the mean of at least two independent experiments, with error bars showing the standard error of the mean.

Fig. 2

Changes in the susceptibility of Clostridium difficile strains to nitroheterocyclic drugs during serial passage for 20 days. Values represent the mean of three independent experiments, with error bars showing the standard error of the mean. MTZ, metronidazole; OND, ornidazole; NTZ, nitazoxanide; NFT, nitrofurantoin; NFZ, nitrofurazone; NIF, nifuroxazide; FZD, furazolidone; RFX, rifaximin. Mean starting minimum inhibitory concentrations (MICs) against CD196, BAA-1875 and R20291 were 0.01, 0.005 and 0.03 µg/mL, respectively.

Fig. 3

Gene expression analysis in response to metronidazole (MTZ), nitrofurantoin (NFT), nitazoxanide (NTZ) and S-nitrosoglutathione (GSNO). The bar depicts the relative quantities of mRNA for respective genes, measured by quantitative reverse transcription PCR and normalised against the 16S rRNA gene. The mean of three independent experiments is shown, with error bars showing the standard error of the mean.