The Efficacy of Probiotics in Modulating Clostridium difficile Spore Germination, Growth and Toxin Production in an in vitro Human Gut Model

Abstract

Background and aims Clostridium difficile infection (CDI) remains a healthcare burden and recurrent CDI (rCDI) still affects 20-30% of patients. Probiotics are live microorganisms that confer a host health benefit, but evidence of their efficacy in CDI prevention/treatment is controversial. Non-toxigenic C. difficile (NTCD) have been used successfully used in animals/humans to reduce CDI. The present study aimed to assess efficacy of two probiotics, Lactobacillus casei Shirota (LcS, Yakult) and NTCD, in preventing simulated CDI in an in vitro human gut model. Methods C. difficile-negative pooled faeces from healthy volunteers (>65 yrs) was used to inoculate the gut model. Two probiotics, LcS (6.2 x 109 cfu) and NTCD (1 x 108 spores), were dosed into separate gut models prior to C. difficile (CD) ribotype 027 spores (RT027, 1 x 108). LcS was chosen given its commercial availability, in addition to studies that report the prevention of antibiotic-associated diarrhoea by LcS, and its role in preventing CDI. Similarly, NTCD was chosen given reports of its antagonism against toxigenic C. difficile. Probiotic dosing was for 28d; LcS once-daily and NTCD spores once-weekly. The rationale for dosing is consistent with literature and the ease of obtaining the probiotic. For example LcS is commercially available whereas NTCD had to be prepared using a rigorous process over a duration of 7 – 10 days. Various antimicrobial groups such as lincosamides, aminopenicillins, fluoroquinolones, and third generation cephalosporins was used to disrupt the gut microflora in separate experiments. Gut model contents were assayed for microflora composition using viable counting techniques and CD cytotoxin production using a Vero cell cytotoxicity assay. Probiotic dosing ceased 14 days before the end of the experiment. Results LcS dosing resulted in marked increases in lactobacilli and bifidobacterial viable counts. However, during clindamycin (DA) dosing these viable counts declined by 4-log10cfu/mL. RT027 spore germination and cell proliferation was observed during/after antimicrobial instillation. Interestingly, another cycle of growth/cytotoxin was observed after LcS dosing ceased. NTCD did not colonise the gut model prior to DA instillation; spores were quiescent and washed out. NTCD spores germinated and vegetative cells multiplied, whereas, RT027 spores did not germinate and no cytotoxin was produced. NTCD remained vegetative until the end of the experiment and isolated cells retained their non-toxigenic phenotype. Conclusions Instillation of NTCD prevented primary CDI in a human gut model, whereas dosing with LcS did not. LcS, if beneficial in the antagonism of CD, is unlikely to be due to nutrient/adhesion competition or production of antimicrobial substances. NTCD may be beneficial not only in treating rCDI but also in the prevention of primary infection. Further work is needed to better understand the protection that NTCD might confer.