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Objectives: Until the introduction of fidaxomicin, antimicrobial treatment for Clostridium difficile infection (CDI) was limited to metronidazole and vancomycin. The changing epidemiology of CDI and the emergence of epidemic C. difficile PCR ribotype 027 necessitate continued surveillance to identify shifts in antibiotic susceptibility. ClosER, currently the largest pan-European epidemiological study of C. difficile ribotype distribution and antibiotic susceptibility, aimed to undertake antimicrobial resistance surveillance pre- and post-introduction of fidaxomicin.\ud \ud \ud \ud Methods: Between July 2011 and July 2014, 39 sites across 22 European countries submitted 2830 C. difficile isolates for ribotyping, toxin testing and susceptibility testing to metronidazole, vancomycin, fidaxomicin, rifampicin, moxifloxacin, clindamycin, imipenem, chloramphenicol and tigecycline.\ud \ud \ud \ud Results: Ribotypes 027, 014, 001, 078, 020, 002, 126, 015 and 005 were most frequently isolated, and emergent ribotypes 198 and 356 were identified in Hungary and Italy, respectively. All isolates were susceptible to fidaxomicin, with scarce resistance to metronidazole (0.2%, 6/2694), vancomycin (0.1%, 2/2694) and tigecycline (0%). Rifampicin, moxifloxacin and clindamycin resistance was evident in multiple ribotypes. Lack of ribotype diversity correlated with greater antimicrobial resistance. Epidemic ribotypes (027/001) were associated with multiple antimicrobial resistance, and ribotypes 017, 018 and 356 with high-level resistance. Additional factors may also influence local ribotype prevalence.\ud \ud \ud \ud Conclusions: Fidaxomicin susceptibility was retained post-introduction, and resistance to metronidazole and vancomycin was rare. Continued surveillance is needed, with more accurate classification and clarification of ribotype subtypes to further understand their role in the spread of resistance. Other factors may also influence changes in prevalence of C. difficile ribotypes with reduced antibiotic susceptibility.

Previous work suggests oritavancin may be inhibitory to Clostridium difficile spores. We have evaluated the effects of oritavancin exposure on C. difficile spore germination, outgrowth and recovery.Germination and outgrowth of C. difficile spores exposed to different concentrations of oritavancin, vancomycin, or metronidazole (0.1-10 mg/L) were monitored at 0, 2, 4, 6, 24 and 48 h using phase-contrast microscopy. Recovery of antimicrobial-exposed spores was determined by viable counting on Brazier's modified CCEYL agar. Persistence of oritavancin activity on spores after washing was determined by measuring activity against a Staphylococcus aureus lawn.Oritavancin, vancomycin and metronidazole exposure did not prevent germination of phase-bright spores to phase-dark spores, but did inhibit further outgrowth into vegetative cells. The inhibitory effect of oritavancin persisted after washing, whereas the inhibitory effects of vancomycin and metronidazole did not. Oritavancin exposure affected spore recovery; fewer spores were recovered after washing following oritavancin exposure than vancomycin exposure. The extent of this effect was dependent on PCR ribotype, with recovery of ribotype 078 spores completely prevented, but recovery of ribotype 001 spores only slightly affected. Spores exposed to oritavancin, but not vancomycin, retained antimicrobial activity after washing, indicating adherence of oritavancin, but not vancomycin, to the spore surfaceOritavancin may adhere to spores, potentially causing early inhibition of germinated cells, preventing subsequent vegetative outgrowth and spore recovery. This may prevent some recurrences of symptomatic C. difficile infection that are due to germination of residual spores following antibiotic therapy.

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.

The human gastrointestinal tract harbours a complex microbial community which exist in planktonic and sessile form. The degree to which composition and function of faecal and mucosal microbiota differ remains unclear. We describe the development and characterisation of an in vitro human gut model, which can be used to facilitate the formation and longitudinal analysis of mature mixed species biofilms. This enables the investigation of the role of biofilms in Clostridium difficile infection (CDI). A well established and validated human gut model of simulated CDI was adapted to incorporate glass rods that create a solid-gaseous-liquid interface for biofilm formation. The continuous chemostat model was inoculated with a pooled human faecal emulsion and controlled to mimic colonic conditions in vivo. Planktonic and sessile bacterial populations were enumerated for up to 46 days. Biofilm consistently formed macroscopic structures on all glass rods over extended periods of time, providing a framework to sample and analyse biofilm structures independently. Whilst variation in biofilm biomass is evident between rods, populations of sessile bacterial groups (log10 cfu/g of biofilm) remain relatively consistent between rods at each sampling point. All bacterial groups enumerated within the planktonic communities were also present within biofilm structures. The planktonic mode of growth of C. difficile and gut microbiota closely reflected observations within the original gut model. However, distinct differences were observed in the behaviour of sessile and planktonic C. difficile populations, with C. difficile spores preferentially persisting within biofilm structures. The redesigned biofilm chemostat model has been validated for reproducible and consistent formation of mixed species intestinal biofilms. This model can be utilised for the analysis of sessile mixed species communities longitudinally, potentially providing information of the role of biofilms in CDI.

SUMMARY Ambiguities in Web search tool (more commonly known as “search engine”) terminology are problematic when conducting precise, replicable research or when teaching others to use search tools. Standardized terminology would enable Web searchers to be aware of subtle differences between Web search tools and the implications of these for searching. A categorization and nomenclature for standardized classifications of different aspects of Web search tools is proposed, and advantages and disadvantages of using tools in each category are discussed.

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.