Efficacy of alternative fidaxomicin dosing regimens for treatment of simulated Clostridium difficile infection in an in vitro human gut model.

BACKGROUND: Fidaxomicin treatment reduces the risk of recurrent Clostridium difficile infection (CDI) compared with vancomycin. Extending duration of fidaxomicin therapy may further reduce recurrence. We compared the efficacy of four extended fidaxomicin regimens in an in vitro model of CDI. METHODS: Four gut models were primed with human faeces, spiked with C. difficile spores (PCR ribotype 027) and clindamycin instilled (33.9 mg/L, four-times daily, 7 days) to induce simulated CDI. Four extended fidaxomicin treatment regimens were evaluated: model 1, 20 days, 200 mg/L twice daily; model 2, 5 days 200 mg/L twice daily, 5 days rest, 5 days 200 mg/L twice daily; model 3, 5 days 200 mg/L twice daily, 5 days rest, 10 days 200 mg/L once daily; and model 4, 5 days 200 mg/L twice daily, 20 days 200 mg/L once every other day. C. difficile populations, toxin, gut microbiota and antimicrobial levels were monitored daily. RESULTS: All fidaxomicin regimens successfully resolved simulated CDI without recurrence. Five days of fidaxomicin instillation was barely sufficient to resolve CDI (models 2-4). A second pulse or tapered dosing further reduced C. difficile and toxin detection. All regimens were sparing of microbiota, affecting only enterococci and bifidobacteria. Pulsed or tapered regimens allowed greater bifidobacteria recovery than the extended (20 day) regimen. Bioactive fidaxomicin persisted throughout the experiment in all models at concentrations inhibitory to C. difficile. CONCLUSIONS: Pulsed or tapered fidaxomicin regimens may enhance suppression of C. difficile whilst allowing microbiota recovery; clinical studies are required to ascertain the potential of this approach in further reducing recurrent CDI.

Successful treatment of simulated Clostridium difficile infection in a human gut model by fidaxomicin first line and after vancomycin or metronidazole failure.

OBJECTIVES: Fidaxomicin reduces the risk of recurrent Clostridium difficile infection (CDI) compared with vancomycin. We investigated fidaxomicin primary or secondary treatment efficacy using a gut model. METHODS: Four triple-stage chemostat gut models were inoculated with faeces. After clindamycin induction of CDI, fidaxomicin (200 mg/L twice daily), vancomycin (125 mg/L four times daily) or metronidazole (9.3 mg/L three times daily) was administered for 7 days. Following failure/CDI recurrence, fidaxomicin (200 mg/L twice daily, 7 days) was instilled. C. difficile (CD) total viable counts (TVC), spore counts (SP), toxin titres (CYT), gut bacteria counts and antimicrobial concentrations were measured throughout. RESULTS: Fidaxomicin instillation reduced CD TVC/SP and CYT below the limit of detection (LOD) after 2 and 4 days, respectively, with no CDI recurrence. Metronidazole instillation failed to decrease CD TVC or CYT. Vancomycin instillation reduced CD TVC and CYT to LOD by day 4, but SP persisted. Recurrence occurred 13 days after vancomycin instillation; subsequent fidaxomicin instillation reduced CD TVC/SP/CYT below the LOD from day 2. CD was isolated sporadically, with no evidence of spore recrudescence or toxin production. Fidaxomicin had a minimal effect on the microflora, except for bifidobacteria. Fidaxomicin was detected for at least 21 days post-instillation, whereas other antimicrobials were undetectable beyond ∼4 days. CONCLUSIONS: Fidaxomicin successfully treated simulated primary and recurrent CDI. Fidaxomicin was superior to metronidazole in reducing CD TVC and SP, and superior to vancomycin in reducing SP without recurrence of vegetative cell growth. Fidaxomicin, but not vancomycin or metronidazole, persisted in the gut model for >20 days after instillation.

Kytococcus sedentarius, the organism associated with pitted keratolysis, produces two keratin-degrading enzymes.

AIMS: To determine characteristics of the extracellular enzyme activity of Kytococcus sedentarius on human callus. METHODS AND RESULTS: A concentrate of a continuous culture supernatant fluid of K. sedentarius, which had callus-degrading activity, was subjected to a series of chromatographic purification procedures. The enzyme activity was found to be attributable to two proteases. These were capable of degrading both native callus and extracted keratin polypeptides and were purified to homogeneity, as shown by SDS-PAGE with silver staining. The enzymes P1 and P2 were 30 kDa and 50 kDa in size with isoelectric points of 4.6 and 2.7, respectively. The optimum conditions for callus-degrading activity were 40 degrees C, pH 7.1 for P1 and 50 degrees C, pH 7.5 for P2. P2 displayed increased activity in the presence of 800 mmol l(-1) NaCl and both enzymes were inhibited by PMSF (1 mmol(-1) Phenylmethylsulphoryl fluoride) and 1 mmol l(-1) EDTA. The main enzyme cleavage sites were Lys-Trp, Val-Lys, Gly-Asp and Asp-Arg, as determined after incubation of P1 and P2 with the beta-chain of insulin. CONCLUSIONS: K. sedentarius produces two extracellular enzymes that independently degrade natural, insoluble human callus. Both enzymes are serine proteases and have cleavage preference sites that are present in a range of human keratins. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification, in K. sedentarius cultures, of two enzymes which can degrade human callus strengthens the hypothesis that this organism is responsible for the pitting in human epidermis observed in pitted keratolysis. These enzymes may be of commercial use in the biodegradation of a range of keratin polymers, biological washing powders and in the treatment of unwanted callus on human skin.