Grace S. Crowther

Evaluation of NVB302 versus vancomycin activity in an in vitro human gut model of Clostridium difficile infection

OBJECTIVES First-line treatment options for Clostridium difficile infection (CDI) are limited. NVB302 is a novel type B lantibiotic under evaluation for the treatment of CDI. We compared the responses to NVB302 and vancomycin when used to treat simulated CDI in an in vitro gut model. METHODS We used ceftriaxone to elicit simulated CDI in an in vitro gut model primed with human faeces. Vancomycin and NVB302 were instilled into separate gut models and the indigenous gut microbiota and C. difficile total viable counts, spores and toxin levels were monitored throughout. RESULTS Ceftriaxone instillation promoted C. difficile germination and high-level toxin production. Commencement of NVB302 and vancomycin instillation reduced C. difficile total viable counts rapidly with only C. difficile spores remaining within 3 and 4 days, respectively. Cytotoxin was reduced to undetectable levels 5 and 7 days after vancomycin and NVB302 instillation commenced in vessel 2 and 3, respectively, and remained undetectable for the remainder of the experiments. C. difficile spores were unaffected by the presence of vancomycin or NVB302. NVB302 treatment was associated with faster resolution of Bacteroides fragilis group. CONCLUSIONS Both NVB302 and vancomycin were effective in treating simulated CDI in an in vitro gut model. C. difficile spore recrudescence was not observed following successful treatment with either NVB302 or vancomycin. NVB302 displayed non-inferiority to vancomycin in the treatment of simulated CDI, and had less deleterious effects against B. fragilis group. NVB302 warrants further clinical investigation as a potentially novel antimicrobial agent for the treatment of CDI.

In vitro activity of cadazolid against clinically relevant Clostridium difficile isolates and in an in vitro gut model of C. difficile infection

OBJECTIVES We investigated the in vitro activity of cadazolid against 100 Clostridium difficile isolates and its efficacy in a simulated human gut model of C. difficile infection (CDI). METHODS MICs of cadazolid, metronidazole, vancomycin, moxifloxacin and linezolid were determined using agar incorporation for 100 C. difficile isolates, including 30 epidemic strains (ribotypes 027, 106 and 001) with reduced metronidazole susceptibility, 2 linezolid-resistant isolates and 2 moxifloxacin-resistant isolates. We evaluated the efficacy of two cadazolid dosing regimens (250 versus 750 mg/L twice daily for 7 days) to treat simulated CDI. Microflora populations, C. difficile total viable counts and spores, cytotoxin titres, possible emergence of cadazolid, linezolid or quinolone resistance, and antimicrobial concentrations were monitored throughout. RESULTS Cadazolid was active against all (including linezolid- and moxifloxacin-resistant) C. difficile strains (MIC90 0.125, range 0.03-0.25 mg/L). The cadazolid geometric mean MIC was 152-fold, 16-fold, 9-fold and 7-fold lower than those of moxifloxacin, linezolid, metronidazole and vancomycin, respectively. Both cadazolid dosing regimens rapidly reduced C. difficile viable counts and cytotoxin with no evidence of recurrence. Cadazolid levels persisted at 50-100-fold supra-MIC for 14 days post-dosing. Cadazolid inhibition of enumerated gut microflora was limited, with the exception of bifidobacteria; Bacteroides fragilis group and Lactobacillus spp. counts were unaffected. There was no evidence for selection of strains resistant to cadazolid, quinolones or linezolid. CONCLUSIONS Cadazolid activity was greater than other tested antimicrobials against 100 C. difficile strains. Cadazolid effectively treated simulated CDI in a gut model, with limited impact on the enumerated gut microflora and no signs of recurrence or emergence of resistance within the experimental timeframe.

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.

Efficacy of surotomycin in an in vitro gut model of Clostridium difficile infection

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.

Evaluation of the effect of oritavancin on Clostridium difficile spore germination, outgrowth and recovery

OBJECTIVES 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. METHODS 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 Braziers modified CCEYL agar. Persistence of oritavancin activity on spores after washing was determined by measuring activity against a Staphylococcus aureus lawn. RESULTS 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 surface CONCLUSIONS Oritavancin 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.

Development and Validation of a Chemostat Gut Model To Study Both Planktonic and Biofilm Modes of Growth of Clostridium difficile and Human Microbiota

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.

Comparison of planktonic and biofilm-associated communities of Clostridium difficile and indigenous gut microbiota in a triple-stage chemostat gut model

BACKGROUND Biofilms are characteristic of some chronic or recurrent infections and this mode of growth tends to reduce treatment efficacy. Clostridium difficile infection (CDI) is associated with a high rate of recurrent symptomatic disease. The presence and behaviour of C. difficile within intestinal biofilms remains largely unexplored, but may factor in recurrent infection. METHODS A triple-stage chemostat gut model designed to facilitate the formation of intestinal biofilm was inoculated with a pooled human faecal emulsion. Bacterial populations were allowed to equilibrate before simulated CDI was induced by clindamycin (33.9 mg/L, four times daily, 7 days) and subsequently treated with vancomycin (125 mg/L, four times daily, 7 days). Indigenous gut microbiota, C. difficile total viable counts, spores, cytotoxin and antimicrobial activity in planktonic and biofilm communities were monitored during the 10 week experimental period. RESULTS Vancomycin successfully treated the initial episode of simulated CDI, but ∼18 days after therapy cessation, recurrent infection occurred. Germination, proliferation and toxin production were evident within planktonic communities in both initial and recurrent CDI. In contrast, sessile C. difficile remained in dormant spore form for the duration of the experiment. The effects of and recovery from clindamycin and vancomycin exposure for sessile populations was delayed compared with responses for planktonic bacteria. CONCLUSIONS Intestinal biofilms provide a potential reservoir for C. difficile spore persistence, possibly facilitating their dispersal into the gut lumen after therapeutic intervention, leading to recurrent infection. Therapeutic options for CDI could have increased efficacy if they are more effective against sessile C. difficile.

SMT19969 as a treatment for Clostridium difficile infection: an assessment of antimicrobial activity using conventional susceptibility testing and an in vitro gut model

Objectives We investigated the efficacy of the novel antimicrobial agent SMT19969 in treating simulated Clostridium difficile infection using an in vitro human gut model. Methods Concentrations of the predominant cultivable members of the indigenous gut microfloras and C. difficile (total and spore counts) were determined by viable counting. Cytotoxin titres were determined using cell cytotoxicity and expressed as log10 relative units (RU). Clindamycin was used to induce simulated C. difficile PCR ribotype 027 infection. Once high-level cytotoxin titres (≥4 RU) were observed, SMT19969 was instilled for 7 days. Two SMT19969 dosing regimens (31.25 and 62.5 mg/L four times daily) were evaluated simultaneously in separate experiments. MICs of SMT19969 were determined against 30 genotypically distinct C. difficile ribotypes. Results SMT19969 was 7- and 17-fold more active against C. difficile than metronidazole and vancomycin, respectively, against a panel of genotypically distinct isolates (P < 0.05). Both SMT19969 dosing regimens demonstrated little antimicrobial activity against indigenous gut microflora groups except clostridia. SMT19969 inhibited C. difficile growth and repressed C. difficile cytotoxin titres in the gut model. Conclusions These data suggest that SMT19969 is a narrow-spectrum and potent antimicrobial agent against C. difficile. Additional studies evaluating SMT19969 in other models of C. difficile infection are warranted, with human studies to place these gut model observations in context.

Effectiveness of a short (4 day) course of oritavancin in the treatment of simulated Clostridium difficile infection using a human gut model

OBJECTIVES We previously demonstrated that 7 days of oritavancin instillation effectively treats Clostridium difficile infection (CDI) in a human gut model. Oritavancin may be more effective than vancomycin due to apparently increased activity against spores. We compared the efficacy of shortened dosing duration (4 days) of oritavancin and vancomycin for CDI treatment using the gut model. METHODS Clindamycin induced CDI in two triple-stage chemostat gut models primed with pooled human faeces and C. difficile ribotype 027 spores. Oritavancin (64 mg/L twice daily) or vancomycin (125 mg/L four times daily) was instilled for 4 days and the effects on C. difficile proliferation and toxin production, and gut microflora were determined. RESULTS Both oritavancin and vancomycin reduced toxin to undetectable levels. Recurrent C. difficile germination occurred 20 days after vancomycin instillation, with high-level toxin production. Oritavancin reduced C. difficile counts to around the detection limit for the remainder of the experiment, with spores undetectable from day 1 of instillation. Toxin production was reduced to below detectable levels, but was sporadically seen later, despite no evidence of germination. Both oritavancin and vancomycin instillation led to only modest effects on gut microflora. CONCLUSIONS Shortened courses of oritavancin and vancomycin effectively treated CDI in a human gut model, but evidence of recurrence was observed following vancomycin instillation. Oritavancin exposure inhibited the recovery of C. difficile spores, as previously described. Shortened antibiotic exposure minimizes disruption to the gut microflora. These data indicate the possible value of a 4 day oritavancin dosing regimen for CDI treatment.

Co-amoxiclav induces proliferation and cytotoxin production of Clostridium difficile ribotype 027 in a human gut model

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.