2 new assays to assess the effects of test compounds on Clostridium difficile infection

Clostridium difficile, the causative agent of C. difficile infection (CDI), is a Gram-positive, anaerobic, spore forming bacterium. CDI results from the oral intake of acid-resistant spores in patients with a dysbiosed gut community. The organism developed a sporulation pathway, enabling it to withstand aerobic conditions and survive the acidic conditions of the stomach. After passage through the stomach, small intestine and proximal colon region, the pathogen finally reaches the distal colon region of the gastrointestinal tract, where it causes infection through the production of cytotoxins. CDI manifests in different ways, varying from mild diarrhea to life-threatening conditions such as pseudomembranous colitis and toxic megacolon. During the last few years, CDI has become a leading cause of antibiotic-associated diarrhea in Europe and North-America.

  1. difficile typically causes infection when the healthy balance of the normal gut microbiota is disrupted, for instance due to the intake of antimicrobial agents.

While treatments are available to cure CDI, typical first-line treatments (i.e. vancomycin and metronidazole) have the disadvantage of having broad-spectrum activity and thus typically further disrupting the gut microbiota. Moreover, with the emergence of antibiotic resistant enterococci, the use of vancomycin has been restricted. Disruption of the microbiota is one of the leading causes of recurrence of infection, the latter being the main bottleneck for effective treatment of CDI.

Extensive research has been done to develop new treatments, ranging from antibiotic therapy to immunotherapy, vaccines, pre- and probiotics and faecal transplants. Key aspects in the development of new CDI treatments include resistance to microbial degradation and limiting the collateral damage to indigenous microbes involved in prevention of disease recurrence. In order to predict the efficiency of an active in disarming C. difficile and to estimate the effect on the indigenous gut microbiota, ProDigest has developed an innovative screening assay, highly representative of what is observed in vivo using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®).

The ProDigest’s approach

The large intestinal environment is very complex, both in terms of medium and microbial community composition. ProDigest specializes in mimicking the intestinal environment, thus allowing to perform predictive studies on the behavior of actives in the gastrointestinal tract. For this particular field of research, ProDigest aimed not only at predicting the behavior (stability, release, …) of an active in the gastrointestinal tract, but also at assessing the effect of test compounds on C. difficile and the gut microbiota in a representative environment. In order to do so, ProDigest developed two different assays:

  1. A short-term screening assay, consisting of the administration of a single dose of a test product and measuring specific endpoints as outlined below.
  2. A long-term SHIME experiment to assess the effect of repeated administration of a test product and measuring specific endpoints as outlined below.
  1. The short-term screening assay

Since CDI develops in the distal region of the colon, the experiment is designed to simulate that particular environment in a small-scale batch reactor. In the short-term screening assay, C. difficile is allowed to grow under representative conditions in the presence of a microbial community collected from the distal colon reactor of the SHIME®.

The outcome of the assay is an assessment of the effects of a test compound (potentially tested at different concentrations to establish dose-response relationships) on three main endpoints:

  •  Growth of C. difficile over a 48h period (as shown in the figure below).
  •  The composition of the indigenous microbiota. This is performed by molecular techniques (qPCR or 16S-based Illumina sequencing). If desired dose-response curves for different taxonomic groups of bacteria in the gut can be constructed.
  •  Functioning of the indigenous microbiota. This is performed by measuring specific metabolites such as short chain fatty acids, lactate and markers for proteolysis.

These tests provide a fast and cost-effective way to get insight in the efficiency of a variety of test compounds (i.e. APIs, pre- and probiotics, etc…) in eliminating C. difficile in a representative gut environment at an early stage of product development. At the same time the test allows to verify the selectivity and the effect of the test compounds on metabolite production. Efficient elimination of C. difficile, combined with high selectivity and low impact on the composition and activity of the indigenous gut microbiome may highlight potentially interesting products.

  1. Long term SHIME experiment (PathoGut™)

ProDigest developed an in vitro platform that allows to obtain stable C. difficile infection in the SHIME®. This is referred to as the PathoGut™ model. The outcome of these experiments is an assessment of the effects of the test compounds on the following endpoints:

  • The effect of repeated administration of a test compound on growth and germination of C. difficile, and thus CDI.
  • The effect of repeated administration of a test compound on community composition of the indigenous microbiota via molecular techniques (qPCR or 16S-based Illumina sequencing).
  • The effect of repeated administration of a test compound on community functioning of the indigenous microbiota by measuring specific metabolites such as short chain fatty acids, lactate and ammonium.
  •  The efficacy of a test compound on the prevention of CDI recurrence.
  • The stability and availability of an API in a representative colonic environment.

Different experimental designs allow to evaluate both the preventive and the curative nature of a specific test compound.

The PathoGut™ model provides a complete and cost-effective means to get insight in the efficiency of a limited number of test compounds in treating CDI in a representative gut environment at an early stage of product development. Besides that, the model provides first evidence whether disease recurrence is likely to occur. Efficient elimination of C. difficile, in combination with high selectivity and low impact on the composition and activity of the indigenous gut microbiome, may highlight potentially interesting products, which can then be tested in animal and clinical trials.

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