gastrointestinal microflora with new molecular techniques

The diverse microbial flora of the gastro intestinal tract contributes to the nutrition, immunology, protection and therefore also health of the host animal. The vast limitations opposed to the investigation of these very important living populations in the gut can be overcome by the use of molecular techniques, which are based on the sequence comparison of nucleic acids (DNA or RNA). These techniques include the denaturing gel electrophoresis (DGGE), a method to display the genetic diversity of a complex microbial population. In contrast to traditional methods, the DGGE approach allows for profiling gut samples from a large number of individual animals. Thus, it is a suitable tool for detecting shifts in the bacterial population as well as nutritional influences on the composition of bacterial communities in the gut.


Farm animals live in symbiosis with an extensive number of microorganisms inhabiting their gut. Thus, a healthy intestinal microflora is a prerequisite for high performance in terms of live weight gain, feed conversion, milk yield or egg production. Beneficial bacteria contribute to overall gut health by counteracting pathogens through competitive exclusion, production of acidic or bactericidal agents, modulation of the immune system or vitamin synthesis (Ewing and Cole, 1994). Thus, modern animal nutrition focuses on the manipulation of the gut microflora through nutritional means. Researchers are investigating the gut microflora in order to understand the interactions of microorganisms with the host and consequently to enable development of strategies to protect animals from enteric diseases.

The development and colonization of the gastrointestinal tract in young animals is a critical time. In pigs, for example, in the period from birth to weaning, the gut is colonized rapidly by populations such as E. coli, clostridia, streptococci, lactobacilli, bacterioides and bifidobacteria (Stewart, 1997). Weaning is a very stressfulprocess in which piglets are separated from the mother sow and are gradually introduced to solid feed. Hence, piglets in the first few weeks of life are highly susceptible to develop intestinal diseases, which may lead to diarrhea and increased mortality. Therefore, a great concern and interest lies in the protection of piglets by providing them with high quality feed, including health promoting additives such as probiotics and prebiotics. Probiotic and prebiotic feed additives are potential alternatives for antibiotic growth promoters (AGP), which are already prohibited in the European Union and will most likely be banned in animal production world wide in the future. There exist several definitions of a probiotic, but generally it is stated as a living microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance. Prebiotics are defined as non-digestible feed ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improve host health (Gibson, 1995). The beneficial impact of probiotic supplementation on weight gain has been shown in a 56-day trial with pigs (Figure 1). 



Figure 1.Effect of probiotics (Enterococcus faecium Biomin® IMB52) on growth performance of pigs (HÖLZL, 2006)



Investigating the intestinal microflora

Although the significance of the gut microflora for health and performance has become more and more evident during the last few years, knowledge about microbial gut communities in different farm animals, as well as factors affecting their composition, is still insufficient. Beyond doubt, traditional methods are inappropriate to draw a precise picture of the intestinal microflora. The development of nutritional strategies first of all requires establishment of rapid and reliable methods for the identification of microorganisms inhabiting the gut.

To study the effects of feed additives, such as probiotics and prebiotics, on the microbial populations in the gut, several methods can be applied. Among microbiologists it is well known that with traditional culture dependent methods only a small fraction of bacteria can be isolated and characterized due to limitations in selective enrichment. Microbial detection is improved by the use of nucleic acid based methodologies that mostly target the 16S ribosomal RNA gene sequence, a sequence marking the signature for bacterial groups (Woese, 1987). Denaturing gradient gel electrophoresis (DGGE) of ribosomal DNA fragments is a promising fingerprinting technique, applied to provide a pattern of genetic diversity of intestinal microorganisms (Simpson et al., 1999). By using DGGE, many samples can be processed simultaneously, making DGGE a powerful tool to monitor the development of bacterial community composition of the host animal over time and to measure possible changes in populations based upon dietary factors, intestinal compartments or age (Muyzer et al., 1993).


DGGE fingerprinting

The 16S RNA gene sequence contains regions which are conserved among all bacteria, but also regions which are variable and can be highly species-specific. Comparing the 16S rDNA sequence similarities therefore serves as an identification and consequently is used to analyze bacterial communities (Raskin et al., 1997).

In a polymerase chain reaction (PCR) the 16S rDNA sequences from the bacterial community of  a sample of gut content are amplified and subsequently can be separated by DGGE. The main steps in DGGE methodology are shown in Figure 2.



Figure 2.Main steps of DGGE



16S ‘species’ can be distinguished by this electrophoretic method, showing a pattern of bands, which represents the composition of species in the original sample. Therefore, this molecular methodology is also known as genetic fingerprinting.

As with every method also molecular techniques are afflicted with biases and errors. The way of sampling, e.g. the choice of the sample region in the gut (upper, lower ileum or colon) or the handling of the sample under aerobic versus anaerobic conditions, can already have great influence on the results. Furthermore, the extraction of nucleic acids from cells in the sample may be biased due to inefficient cell lysis and removal of contaminants, which may inhibit PCR amplification in subsequent analyzing steps. In general, DGGE will display the fragments from predominant species which constitute at least 1% of the total community (Muyzer et al., 1993). Sensitivity can be improved by the use of group- or species specific primers, which have already been used for the amplification of the 16S ribosomal DNA from Lactobacillus or Bifidobacteria. (Satokariet al.,2001; Heilig et al., 2002). It should be made clear that species identification cannot be achieved with DGGE. Therefore, the DNA fragments from one band of the gel should be excised for subsequent cloning and sequencing of the PCR fragment.

Besides all limitations, DGGE is a very reliable, rapid and reproducible technique to study a complex microflora.


Interpretation of DGGE fingerprints

To obtain an objective interpretation of complex DGGE fingerprints, specialized computer software programs are used. Statistical analysis, cluster analysis or diversity analysis can be achieved, depending on the question of interest. As part of  a current study, a feeding trial was designed to measure changes in intestinal bacterial populations in response to probiotic and prebiotic administration. Additionally, DGGE banding patterns of different gut compartments (ileum and colon) were analyzed in order to see diversity changes independent from feed quality. Therefore, eight pigs from the control feeding group were sacrificed and content of ileum and colon was obtained to be further prepared for DGGE analysis. Figure 3 shows the banding patterns of the described samples, whereat each band represents a different bacterial population and each lane indicates the bacterial fingerprint of the respective gut sample. The much higher bacterial diversity in samples collected from the colon in contrast to the ones collected from the ileum could be revealed by means of the GelcompareII software (Applied Maths).



Figure 3.Dendogram and banding patterns generated from PCR products of the variable region V3 of the 16S rRNA in a 30-60% formamide-urea DGGE. Lanes marked with dots represent fingerprints from ileum, lanes with stars from colon samples, collected from different pigs fed the same diet.

The complexity for a single sample can be expressed by diversity indices (e.g. Shannon’s). With this diversity value, changes in bacterial community composition based on differences in diet or age may be measured. In another study (Konstantinov et. al. 2004) the bacterial communityof ileum and colon of weaning piglets were analyzed in response to addition of four different fermentable carbohydrates (inulin, lactulose, wheat starch, and sugar beet pulp). Using DGGE, based on amplified 16S rRNA genes, a higher number of bands in the colon than in the ileum, as well as a significantly higher diversity in the colonic microflora of pigs fed the fermentable-carbohydrate-enriched diet was observed. However, diversity and community structure may also be influenced by the host genotype, which would mean that the environment has less impact or is just less visible (Zoetendal EG. and Mackie RI.). Nevertheless, this genetic linkage also has to be considered in future investigations.


Summary and conclusion

Due to the significance of a healthy gut microflora for health and performance, modern animal nutrition focuses on the manipulation of gut microbes through nutritional means. DGGE is an effective molecular fingerprinting technique for displaying the composition of the intestinal microbiota and contributes to a better understanding of the very complex bacterial dynamics in the gut. Furthermore, composition of particular populations of interest (e.g. lactic acid bacteria, lactobacilli or bifidobacteria) can be observed by the use of group-specific primers in PCR. In general, DGGE will always give an overview of the composition of dominant species present in the gut. For a more detailed investigation of subdominant populations, species identification and quantification, other nucleic acid based techniques in combination with DGGE need to be implemented in gut microflora research, related to development of functional probiotic products.



Authors: Verity Ann SATTLER¹, Viviana KLOSE¹, Tobias STEINER^2
1 BIOMIN GmbH, BIOMIN Reseach Center, Dep. IFA-Tulln, Konrad-Lorenz-Strasse 20, 3430 Tulln, Austria
² BIOMIN GmbH, Industriestrasse 21, 3130 Herzogenburg, Austria