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BackgroundClostridium difficile infection (CDI) is still a major challenge to healthcare facilities. The detection of multiple C. difficile strains has been reported in some patient samples during initial and recurrent CDI episodes. However, the behaviour of individual strains and their contribution to symptomatic disease is unclear.MethodsAn in vitro human gut model was used to investigate the germination and proliferation of two distinct C. difficile strains during initial and recurrent simulated CDI, as well as their response to vancomycin treatment. The gut model was inoculated with a pooled human faecal emulsion and indigenous gut microbiota, C. difficile populations (vegetative and spore forms), cytotoxin levels and antimicrobial activity were monitored throughout the experiment.ResultsBoth C. difficile strains germinated and proliferated in response to ceftriaxone instillation, with cytotoxin detected during the peak vegetative growth. Vancomycin instillation resulted in a rapid decline in the vegetative forms of both strains, with only spores remaining 2 days after the start of dosing. A recrudescence of both strains occurred following the cessation of vancomycin installation, although this was observed more quickly, and to a greater extent, in one strain than the other.ConclusionsWithin a human gut model, multiple C. difficile strains are able to germinate and proliferate concurrently in response to antibiotic challenge (the onset of simulated CDI). Similarly, more than one strain can proliferate during simulated recurrent CDI, although with differences in germination and growth rate and timing. It appears probable that multiple strains can contribute to CDI within an individual patient, with possible implications for management and bacterial transmission.

OBJECTIVES: We investigated the efficacy of the cyclic lipopeptide surotomycin in treating clindamycin-induced Clostridium difficile infection (CDI) using an in vitro gut model. METHODS: Two three-stage chemostat gut models were inoculated with human faeces, spiked with C. difficile spores (∼10(7) cfu/mL, PCR ribotype 027 or 001). Clindamycin (33.9 mg/L, four times daily for 7 days) was dosed to induce CDI. Following high-level toxin production, surotomycin (250 mg/L, twice daily for 7 days) was instilled. Microflora populations, C. difficile vegetative cells and spores, cytotoxin titres and antimicrobial levels (LC-MS/MS and bioassay) were determined. The emergence of C. difficile and enterococci with reduced susceptibility to surotomycin was monitored on breakpoint agar (4 × MIC). RESULTS: Counts of viable C. difficile were reduced to near the limit of detection on Days 1 and 3 of surotomycin instillation, and cytotoxin was undetectable on Days 3 and 4 of surotomycin instillation in the 027 and 001 models, respectively. Recurrence of vegetative growth and toxin production occurred 11 days (001 model) and 15 days (027 model) after surotomycin instillation had ceased, and remained for the duration of the experiment. Surotomycin instillation decreased populations of bifidobacteria, clostridia, enterococci and lactobacilli, but was sparing of Bacteroides fragilis group populations. All enumerated organisms had recovered to steady-state levels by 3 weeks post-surotomycin instillation. No evidence of the emergence of reduced susceptibility to surotomycin was observed. CONCLUSIONS: Surotomycin successfully reduced C. difficile vegetative cell counts and toxin levels in the gut model and was sparing of B. fragilis group populations. There was no evidence of decreased susceptibility to surotomycin during exposure or post-exposure.

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.

Objectives Co-amoxiclav is widely prescribed in hospitals. Although reports have suggested it may be linked to onset of Clostridium difficile infection (CDI), data on the risk of CDI associated with specific antibiotics is difficult to obtain, due to confounding clinical factors. We have examined the propensity of co-amoxiclav to induce CDI using a human gut model. Methods We used a triple-stage chemostat human gut model to study the effects of co-amoxiclav on indigenous gut microorganisms and C. difficile PCR ribotype 027. C. difficile viable counts and spores were evaluated, and cytotoxin titres were assayed. Co-amoxiclav concentrations were measured using a large plate bioassay. Results Co-amoxiclav induced rapid C. difficile germination and high toxin production in the gut model, from 5 days after commencement of instillation. Cell proliferation and toxin production were prolonged and continued throughout the duration of the experiment. Only very low levels of co-amoxiclav antimicrobial activity could be detected within the gut model, despite having a marked effect on gut flora microorganisms. Conclusions Co-amoxiclav induced CDI within the gut model, supporting clinical observations linking co-amoxiclav treatment with CDI onset. This reinforces the value of the gut model as a clinically relevant means of studying CDI. Caution should be exercised in the prescription of co-amoxiclav to patients in high CDI risk settings.