Healthy gut ecosystems

Scope of the method

Alternative method relates to
  • Animal health
  • Human health
Alternative method is situated in
  • Basic Research
  • Translational - Applied Research
Type of alternative method
  • In vitro - Ex vivo
This method makes use of
  • Other (e.g. bacteria)
ex vivo simulation of part of the human or animal gut microbial ecosystem

Description

Method keywords
  • gut
  • bioreactor
  • ex vivo
  • gut microbiota
  • cecum
Scientific area keywords
  • microbiology
  • antimicrobial resistance
  • probiotics
  • prebiotics
  • digestion
Method description

With a one vessel fermentor (BioFlo 110, New Brunswick), an in vitro simulation of a certain part of the gastrointestinal tract (e.g. cecum, colon) is performed. In this fermentation system, physiological gut parameters (pH, temperature) are kept constant, while chemico-physical characteristics of the gut chime are simulated by adding pork mucin and fibre to the fermentation medium. For a first screening, the fermentation system can be performed in short time (24-48 h) batch mode. The system is inoculated with cecal or fecal material from animals or humans, and can be inoculated with a pathogen like Salmonella. Batch fermentations are monitored by periodic sampling on a quantitative level by dilution plating on selective media for certain bacterial groups (e.g. lactic acid bacteria, bifidobacteria, Enterobacteriaceae, clostridia) as well as for Salmonella, and on a qualitative level for the total microbiota by cultivation independent 16S rDNA based metagenomics; production of short chain fatty acids is measured by GC. From these batch fermentations, for more detailed investigation long-term (up to 2 weeks) a continuous fermentation system is used which simulates even better the gastrointestinal tract of monogastrics like the pig because it takes into account the retention time (or conversely flow of intestinal content) in a certain part of the gut. A continuous fermentation is started in batch mode as described above after which fresh medium is added with a peristaltic pump to the fermentor vessel at a fixed dilution speed and fermented medium is wasted with the same speed to maintain the medium volume in the vessel. After a few days, a steady state is reached and bacteriological and fatty acid parameters can be monitored over several days (or weeks) as described above. All fermentations (batch and continuous) are performed at least twice and for every condition or component, a blank fermentation is included.

Lab equipment
  • 1 liter bioreactors with peristaltic pumps for in and outflow of medium;
  • L2 lab;
  • Incubators;
  • -80°C freezer;
  • UPLC-MS/MS.
Method status
  • History of use
  • Internally validated
  • Published in peer reviewed journal

Pros, cons & Future potential

Advantages
  • No animal experiments are needed to investigate the gut microbiota;
  • Ease of sampling and reproducible conditions can be executed;
  • Two modes of operation are possible depending on the time and resources available:
    • 1/ batch fermentation for screening is possible in a few days;
    • 2/ continuous fermentation is possible for more in depth studies, taking a few weeks
Challenges

The ex vivo system only simulates the gut luminal microbial ecosystem without an animal or human host component (except for addition of porcine mucin to the medium) like gut epithelial cells and host immune system.

Modifications

Addition of mucin beads is possible for simulation of attached bacteria to the mucin layer.

Future & Other applications

Applications in biofilm research are possible, both in the gut ecosystem as on inert surfaces.

References, associated documents and other information

References

W. Messens, J. Goris, N. Dierick, L. Herman and M. Heyndrickx. 2010. Inhibition of Salmonella typhimurium by medium-chain fatty acids in an in vitro simulation of the porcine cecum. Veterinary Microbiology 141: 73-80.

A. Smet, G. Rasschaert, A. Martel, D. Persoons, J. Dewulf, P. Butaye, B. Catry, F. Haesebrouck, L. Herman and M. Heyndrickx. 2010. In situ ESBL conjugation from avian to human Escherichia coli during cefotaxime administration. J. Applied Microbiol. 110: 541-549.

J. Robyn, G. Rasschaert, W. Messens, F. Pasmans and M. Heyndrickx. 2012. Screening for lactic acid bacteria capable of inhibiting Campylobacter jejuni in in vitro simulations of the broiler chicken caecal environment. Beneficial Microbes 3: 299-308.

J. Robyn, G. Rasschaert, D. Hermans, F. Pasmans and M. Heyndrickx. 2013. Is allicin able to reduce Campylobacter jejuni colonization in broilers when added to drinking water? Poultry Science 92: 1408-1418.

L. Peeters, T. De Mulder, E. Van Coillie, J. Huygens, A. Smet, E. Daeseleire, J. Dewulf, H. Imberechts, P. Butaye, F. Haesebrouck, S. Croubels, M. Heyndrickx, and G. Rasschaert. 2017. Selection and transfer of an IncI1-tet(A) plasmid of Escherichia coli in an ex vivo model of the porcine cecum at doxycycline concentrations caused by cross-contaminated feed. Journal of Applied Microbiology 123: 1312-1320.

De Mulder T., Rasschaert G., Van Coillie E., Van den Meersche T., Haegeman A., Ruttink T., de Wiele T.V., Heyndrickx M. 2018. Impact of cross-contamination concentrations of doxycycline hyclate on the microbial ecosystem in an ex vivo model of the pig’s cecum. Microbial Drug Resistance doi/10.1089/mdr.2018.0034

Associated documents
Campy-allicine.pdf Campy-Enterococcus screening.pdf ESBL conjugatie.PDF Salmonella-in vitro.pdf Selection and transfer of an IncI1-tet(A) plasmid.pdf cross contamination doxycycline ex vivo model.pdf
Links
In Vitro Screening and Gastro-Intestinal Simulations

Contact person

Marc Heyndrickx

Organisations

Flanders Research Institute for Agriculture
Technology and Food Science
Belgium