Development and validation of a chemostat gut model to study both planktonic and biofilm modes of growth of Clostridium difficile and human microbiota

Copyright: 2014 Crowther et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.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.Peer reviewe

Evaluation of antimicrobial activity of ceftaroline against Clostridium difficile and propensity to induce C. difficile infection in an in vitro human gut model

OBJECTIVES: To examine the effects of exposure to ceftaroline or ceftriaxone on the epidemic Clostridium difficile strain PCR ribotype 027 and the indigenous gut microflora in an in vitro human gut model. Additionally, the MICs of ceftriaxone and ceftaroline for 60 C. difficile isolates were determined. METHODS: Two triple-stage chemostat gut models were primed with human faeces and exposed to ceftaroline (10 mg/L, twice daily, 7 days) or ceftriaxone (150 mg/L, once daily, 7 days). Populations of indigenous gut microorganisms, C. difficile total viable counts, spore counts, cytotoxin titres and antimicrobial concentrations were monitored throughout. MICs were determined by a standard agar incorporation method. RESULTS: In the gut model, both ceftaroline and ceftriaxone induced C. difficile spore germination, proliferation and toxin production, although germination occurred 5 days later in the ceftaroline-exposed model. Toxin detection was sustained until the end of the experimental period in both models. No active antimicrobial was detected in vessel 3 of either model, although inhibitory effects on microflora populations were observed. Ceftaroline was ∼8-fold more active against C. difficile than ceftriaxone (geometric mean MICs, 3.38 versus 28.18 mg/L; MIC90s, 4 versus 64 mg/L; and MIC ranges, 0.125-16 versus 8-128 mg/L). CONCLUSIONS: Ceftaroline, like ceftriaxone, can induce simulated C. difficile infection in a human gut model. However, low in vivo gut concentrations of ceftaroline and increased activity against C. difficile in comparison with ceftriaxone mean that the true propensity of this novel cephalosporin to induce C. difficile infection remains unclear.Peer reviewe

Efficacy of novel antimicrobial agent SMT-19969 (SMT) against simulated Clostridium difficile infection in an in vitro human gut model

Background: C. difficile infection (CDI) is the leading cause of nosocomial diarrhoea. Current CDI therapy is limited to metronidazole (M) or vancomycin. Recent evidence suggests reduced M efficacy and emergence of reduced susceptibility in epidemic CD strains. Evaluation of new therapies for CDI is essential. Methods: A 3-stage chemostat human gut model was inoculated with pooled faeces (5 healthy elderly volunteers). Clindamycin (CLIN, 33.9 mg/L qid,7d) was dosed to simulate CDI by CD ribotype 027 (NAP1/BI) (SMT MIC = 0.125mg/L). Two 7d SMT dosing regimens were evaluated: 31.25 & 62.5 mg/L QID. Gut bacteria, CD vegetative cells (V) and spores (SP), cytotoxin titres (CYT) & antimicrobial levels (HPLC & bioassay) were determined. Emergence of CD SMT resistance was monitored on breakpoint agar (4xMIC). Results: CLIN elicited CD CYT (4 relative units, RU) in both experiments at which point SMT dosing started. Both SMT regimens caused little adverse antimicrobial activity against gut microflora groups except total clostridia (1.4-2 log cfu/mL decrease). During SMT dosing, CD V and SP declined by ~1.5 log10cfu/mL over 7 d in all vessels of both models. CD V were detectable thereafter, increasing slightly by the end of both experiments. CYT declined to undetectable (ND) or 1 RU during SMT dosing, but recurred at low levels after stopping (<2 RU). CYT was ND for the last 5-6 days of both models. No SMT resistant CD were isolated. SMT persisted in both models to experiment end, with higher concentrations in the SMT 62.5 mg/L model. HPLC & bioassay indicated incomplete drug solubility. Conclusions: SMT treatment was sparing of gut microflora, and by the end of SMT dosing, all groups except total clostridia had recovered to normal levels; this could be expected to have benefit in prevention of recurrent disease. Inhibition of CD growth and CYT, without cell death, indicate SMT is bacteriostatic for CD. There was no evidence of SMT resistance. Evaluation of further dosing regimens are warranted to optimise SMT efficacyNon peer reviewe

Recurrence of dual-strain Clostridium difficile infection in an in vitro human gut model

Objectives: Clostridium 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. Methods: An 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 and antimicrobial activity was monitored throughout the experiment. Results: Both C. difficile strains germinated and proliferated in response to ceftriaxone instillation, with cytotoxin detection during peak vegetative growth. Vancomycin instillation resulted in a rapid decline in vegetative forms of both strains, with only spores remaining 2 days after dosing commencement. Recrudescence of both strains occurred following cessation of vancomycin, although this was observed in one strain sooner, and to a greater extent, than the other strain. Conclusions: Within a human gut model multiple C. difficile strains are able to germinate and proliferate concurrently in response to antibiotic challenge (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

Evaluation of linezolid for the treatment of Clostridium difficile infection caused by epidemic strains using an in vitro human gut model

OBJECTIVES: Therapeutic options in Clostridium difficile infection (CDI) are limited. We examined linezolid activity in vitro and potential therapeutic efficacy using a gut model of CDI.METHODS: MICs were determined by agar incorporation for 118 diverse C. difficile faecal isolates, including epidemic strains and strains with reduced susceptibility to metronidazole. CDI was established in two gut model experiments using C. difficile epidemic strains (ribotypes 027 and 106) and linezolid was dosed to achieve human gut concentrations.RESULTS: Linezolid demonstrated good in vitro activity against 98% of the isolates. Two isolates (PCR ribotypes 023 and 067) demonstrated resistance to linezolid, although supplementary susceptibility testing of ribotype 023 isolates did not detect further resistance. In a gut model that simulates CDI, linezolid reduced the duration of cytotoxin production by C. difficile PCR ribotype 027 without influencing viable counts of vegetative forms of the organism. C. difficile PCR ribotype 106 viable counts declined at a faster rate than those of PCR ribotype 027 following dosing with linezolid, but cytotoxin titres declined at a similar rate to an untreated control. Gut flora perturbation occurring on linezolid exposure reversed after drug cessation. Recrudescence of spore germination with subsequent cytotoxin was seen with the C. difficile ribotype 106 strain. Resistance to linezolid was not detected either during linezolid instillation or post-dosing.CONCLUSIONS: Linezolid may reduce toxin levels, as reported in staphylococci and streptococci. Further evaluation is warranted of the effect of linezolid on expression of C. difficile toxin, and to investigate potential recurrence of CDI following cessation of linezolid.</p

Bacterial populations in model B1.

<p>Mean (± SE) rod-associated populations (log₁₀ cfu/g) of (a) facultative (b) obligate anaerobes within the triple stage biofilm gut model (model B1) at time points Z- X. Planktonic populations of each bacterial group (log₁₀ cfu/g) at each time point are represented by a line. LFE – lactose-fermenting <i>Enterobacteriaceae</i>.</p

Bacterial populations in model B2.

<p>Mean (± SE) rod-associated populations (log₁₀ cfu/g) of (a) facultative (b) obligate anaerobes within the triple stage biofilm gut model (model B2) at time points Z- X. Planktonic populations of each bacterial group (log₁₀ cfu/g) at each time point are represented by a line. LFE – lactose-fermenting <i>Enterobacteriaceae</i>.</p

Experimental designs used in this study.

<p>Experimental design of (a) single stage (model A1 and A2) and (b) triple stage biofilm gut model validation experiments (models B1 and B2). Vertical line represents the last day of each time period. CD, <i>C. difficile</i> PCR ribotype 027 spores.</p

Culture medium used for the isolation and enumeration of indigenous gut microbiota and <i>C. difficile</i> (all agar bases are supplied by Oxoid, with the exception of CCEYL and FAA supplied by LabM, and made according to the manufacturer's instructions).

<p>Culture medium used for the isolation and enumeration of indigenous gut microbiota and <i>C. difficile</i> (all agar bases are supplied by Oxoid, with the exception of CCEYL and FAA supplied by LabM, and made according to the manufacturer's instructions).</p

Mean (log₁₀ cfu/g) and standard error (SE) of rod-associated populations of indigenous gut microbiota at sample points Z, Y and X in the triple stage biofilm gut model B1 and B2. NR – no result.

<p>Mean (log₁₀ cfu/g) and standard error (SE) of rod-associated populations of indigenous gut microbiota at sample points Z, Y and X in the triple stage biofilm gut model B1 and B2. NR – no result.</p