Fidaxomicin, recently approved for the treatment of C. difficile associated diarrhea, demonstrates bactericidal activity against C. difficile and was superior in clinical trials to vancomycin (VAN) in its sustained clinical response. In this study, we showed its mechanism of antimicrobial activity to be via inhibition of RNA polymerase (RNAP). Since this mechanism may also lead to inhibition of virulence factors, we examined toxin levels and toxin gene expressions following exposure to FDX.
Transcription inhibition studies were performed with purified C. difficile RNAP in presence of FDX. Impact of FDX on toxin production was determined by adding drugs to C.difficile ATCC 43255 (a high toxin producing strain) and UK14 (NAP1/BI/027 epidemic strain) at early stationary phase of growth followed by incubation ~7 days. Culture supernatants were tested by commercial ELISAtgcBiomics for the presence of toxins A and B. Toxin gene expression was assessed in presence of drugs (qRTPCR) in C. difficile UK1 (NAP1/BI/027 type epidemic strain) and in its close nonepidemic relative, CD196. Toxin gene transcript levels were normalized to levels of 16s rRNA and rpoC mRNA.
Toxin production in the absence of drugs peaked between 3 and 5 days in C. difficile ATCC 43255, however, in the presence of FDX and its major metabolite OP1118 (both at 1/4xMIC), production of TcdB and TcdA levels were strongly suppressed. In contrast, VAN did not inhibit toxin production. Similar inhibitory effects on TcdA and TcdB expression by FDX and OP1118 but not VAN, or metronidazole (MTZ) were seen in the UK14 strain. Addition of FDX (2xMIC) or OP1118 (2.5xMIC) at the end of exponential growth phase led to nearly complete inhibition of subsequent accumulation of transcripts from the pathogenicity locus, i.e. tcdR, tcdA, tcdB and tcdC. By contrast, addition of vancomycin (2.5xMIC) at the end of exponential growth phase had little or no significant effect on subsequent toxin production. Transcription inhibition studies with C. difficile RNA polymerase demonstrated inhibition at a low IC50 (~1mM); thus the reduction in toxin production may be mediated via the inhibition of RNAP.
Results demonstrate that both FDX and OP1118, but not VAN or MTZ, block C. difficile toxin synthesis even when added to stationary phase cells.
To cite this abstract:Gomez A, Sonenshein A, Sims C, Babakhani F, Seddon J, Bouillaut L, Sears P. Fidaxomicin Inhibits Production of Toxin a and Toxin B in Clostridium Difficile. Abstract published at Hospital Medicine 2012, April 1-4, San Diego, Calif. Abstract 97620. Journal of Hospital Medicine. 2012; 7 (suppl 2). https://www.shmabstracts.com/abstract/fidaxomicin-inhibits-production-of-toxin-a-and-toxin-b-in-clostridium-difficile/. Accessed May 26, 2019.