A gut feeling

The human gastrointestinal (GI) tract contains a diverse array of bacteria with up to 1 x 1012 bacteria per gram of gut contents in the colon. Bacteria may live in the gut content or on the surface of the gut lining. There is currently a great deal of scientific interest in this huge bacterial population and its effect on the human health.

Gut microbiota stimulate the immune system, produce vitamins and short chain fatty acids, help the digestion process and are involved in prevention of the colonisation of potential pathogenic bacteria by competitive exclusion.

If these pathogenic bacteria are allowed to reach sufficient numbers they have the potential to cause disease.

The whole study of gut microbiology is very complex as the gut may contain as many as 400 different bacterial species. One of the main barriers for research on gut microbiota has been the number of unculturable bacteria present in the GI tract.

It is claimed that up to 70% of the bacteria present are unculturable by traditional techniques of selective and non-selective culturing methods.

There are several reasons why many of the gut microbiota cannot be cultured, these include being strict anaerobes (requiring the absence of oxygen), others are thought to be living in symbiosis with other bacteria. For some microbes scientists have, as yet, not designed culture media that allow these organisms to grow.

Now, with the advent of molecular methods, a whole new set of avenues have opened up for researchers to study the GI tract microbiota.Much of the advancement in this area of science is due to the discovery of DNA and the ability to manipulate and gain information from it. Scientists can make multiple copies of any specifically desired DNA sequences through a process known as PCR (Polymerase Chain Reaction).

This is often based around a stretch of DNA present in all bacteria known as the 16S ribosomal DNA (rDNA). The 16S rDNA sequences are useful in bacterial identification as they are conserved among members of the same species, and differ from more distantly related genera. The basic principles of PCR and 16S rDNA can then be applied and modified to gain increased insight into the GI tract bacterial community.

Scientists in the microbial genetics group led by Professor Mike Gasson at the Institute of Food Research (IFR) are developing and using molecular techniques for profiling the GI tract. Approaches employed at IFR include Denaturing Gradient Gel Electrophoresis (DGGE), percent Guanidine and Cytosine (GC) ratio analysis combined with DGGE, and DNA microarray technology.

These methods can be used to study bacteria in both human and animal GI tracts, and in many ways complement each other with the information they provide.

Using DNA microarray technology to profile GI tract microbiota is a new adaptation of the microarray technology. Microarrays have traditionally been used to study the entire genome from one organism, for example observing which genes are induced or repressed under stress conditions. This is achieved by labelling DNA or RNA with a fluorescent marker and hybridising these products to a glass slide, which has the complete set of the organism' genes represented as single separate gene spots.

The labelled DNA or RNA bind specifically to its corresponding gene spots, fluoresces at different wavelengths and intensities, indicating to the researcher the expression level of a particular gene.

However at IFR we are modifying this technique to study a single gene common to all bacteria, and applying the data to identify bacterial presence, or absence, in the GI tract samples (Figure 1).

 
Figure 1: Microarray hybridised with DNA from
two diferent faecal samples. One is labelled red
and the other is green. A yellow colour spot is
obtained by red and green labelled DNA binding to
the same spot of the slide. Each spot represents
a different gut bacterium

DGGE gels again take advantage of PCR. A special PCR reaction is performed whereby a clamp can be attached to the amplified rDNA fragments DNA.

The DNA is then electrophoresed through a gel containing increasing concentrations of denaturants such as urea. The resulting product separation (visualised as bands on the gel) is caused by differences in bacterial rDNA sequences, based on the varying melting temperatures of the DNA. Patterns of banding alone can give indications of either a subset or more general information on the bacterial population.

Specific information on bacterial identity may be obtained if the DNA bands are excised from the gel, sequenced, and then compared to known DNA sequences in computer databases. This method has alerted researchers to many previously unknown bacterial strains, revealing just how little scientists understand about the GI tract populations, but also giving them a powerful tool to study the population further.

Dr Tracy Eaton and colleagues at IFR are now combining the power of percent GC analysis and DGGE methods to further increase the sensitivity and resolution of the bacterial community in the GI tract (Figure 2).

 
Figure 2 : Faecal bacteria community profiles generated
by gradient gel electrophoresis. Each lane represets a
sample from different human subject

The molecular finger printing techniques described above are proving invaluable in understanding the function and properties of probiotic bacteria for use in both human and animals. A probiotic is defined as a live microbial feed supplement, which beneficially affects the host animal by improving its intestinal microbial balance1. There are many benefits of probiotics as discussed previously.

The most frequently used probiotic organisms are bifidobacteria and the lactic acid bacteriumlactobacillus. IFR has over 20 years expertise in studying lactic acid bacteria (LAB). In collaboration with Veterinary Laboratory agency, Dr Arjan Narbad published interesting results describing the discovery of a new probiotic strain of Lactobacillus Johnsonii.

This lactobacillus strain is capable of removing the pathogenic bacterium Clostridium perfringens from the gut of chicks2 that often cause necrotic lesions in poultry and some strains also cause food poisoning in humans (Figure 3).

Figure 3: Adhesion of Lactobacillus
Johnsonii to human tissue cells
(X 400 magnification)

IFR also has an established expertise in the use of LAB as GI tract delivery vehicles for a range of bioactive agents, with the objectives of developing LAB-based products for vaccination, prevention and treatment of allergy, treatment of metabolic disorders and delivery of therapeutic antioxidants.

In collaboration with the Gut Immunology group led by Dr Claudio Nicoletti we are now looking at the interaction of human gut bacteria with their host. As molecular methods advance, we are increasingly gaining an insight into the world of gut microbiota and as research develops further, scientists increasingly understand how bacteria interact with each other and with the host. These new techniques in conjunction with traditional culturing methods, are allowing us to build up a picture of complex microflora of the human and animal GI tract.

References

1 Fuller,R. (1989) Probiotics in Man and Animals Journal of Applied Bacteriology 66: 365-378.
2 La Ragione,R.M.; Narbad,A.; Gasson,M.J.; Woodward,M.J. (2004) In vivo characterization of Lactobacillus johnsonii FI9785 for use as a defined competitive exclusion agent against bacterial pathogens in poultry. Letters in Applied Microbiology. 38: 197-205

By Carl Harrington, post-graduate research scientist at the Institute of Food Research
enquiry number 02465