Molecular assessment of intestinal microflora

Molecular assessment of intestinal microflora1–3 Gerald W Tannock KEY WORDS Probiotics, intestinal microflora, molecular typing, lactobacillus, bifidobacteria, enterobacteria INTESTINAL MICROFLORA OF HUMANS The intestinal microflora comprises a diverse collection of microbial species that are mostly bacterial and are commonly detected in human feces. Such bacterial genera are as follows (1): Acidaminococcus, Bacteroides, Bifidobacterium, Clostridium, Coprococcus, Enterobacter, Enterococcus Escherichia, Eubacterium, Fusobacterium, Klebsiella, Lactobacillus, Megamonas, Megasphaera, Peptostreptococcus, Proteus, Ruminococcus, and Veillonella. Some of these microbial groups attain high population levels (1  1010 colony-forming units per gram wet weight of intestinal contents or feces). Besides the use of electron microscopy in studies of the gastrointestinal microflora of experimental animals, the study of intestinal microflora composition has relied almost exclusively on the quantitative culture of microbes from fecal samples. Culture results obtained in these studies compose between 50% and 80% of the total microscopic count. Enumeration of particular microbial genera or species relies on the use of selective media. The inability to culture all of the microbes present in samples and the use of a limited range of reliable selective media doubtless introduces bias into analyses of the composi410S tion of the normal microflora (2). Even when cultivable, the identification of isolates to species level can be difficult because of the considerable variation in biochemical attributes (fermentation profiles) that seem to occur among strains currently considered to represent the same species. Differentiation of isolates into species, especially in the case of the obligate anaerobes, is always logistically difficult and is subject to intuitive interpretations. The microflora has marked influences on the animal host, which has been observed in experiments in which the characteristics of germfree (absence of a microflora) and conventional (presence of a microflora) animals were made. These comparisons showed that many biochemical, physiologic, and immunologic characteristics of the animal host are strongly influenced by the presence of the normal microflora (Table 1, Table 2, and Table 3). Because of this, although sometimes overlooked, biochemical assays of microflora-associated activities provide a suitable method of analyzing the overall functioning of the intestinal microflora (Table 2). MOLECULAR METHODS AND THE ANALYSIS OF ECOSYSTEMS Analysis of terrestrial and aquatic ecosystems in more recent years has benefited from the use of molecular methods with which community profiles have been established (17). The molecular methods involve the amplification by polymerase chain reactions (PCR) of 16S ribosomal RNA genes (16S rDNA) from microbial DNA extracted from samples collected from particular habitats. The amplified 16S rDNA sequences are cloned and should contain copies of the gene from all of the species represented in the sample. The 16S rDNA clones are screened (some sequences are cloned more than once) and representative clones are sequenced. Because 16S rDNA sequences are one of the cornerstones of microbial taxonomy, alignment of the sequences with those stored in databanks permits the recognition of which species are represented in the habitat, including those that cannot be cultivated by conventional techniques. Interestingly, 1 From the Department of Microbiology, University of Otago, Dunedin, New Zealand. 2 Presented at the symposium Probiotics and Prebiotics, held in Kiel, Germany, June 11–12, 1998. 3 Address correspondence to GW Tannock, Department of Microbiology, University of Otago, Dunedin, New Zealand. E-mail: gerald.tannock@stonebow. otago.ac.nz. Am J Clin Nutr 2001;73(suppl):410S–4S. Printed in USA. © 2001 American Society for Clinical Nutrition ABSTRACT The application of molecular methodologies to intestinal microflora analysis should enable the development of a detailed knowledge of the microbial ecology of the human colon. This knowledge is essential to derive scientifically valid probiotics. Molecular typing (genetic fingerprinting) methods, eg, ribotyping and pulsed field gel electrophoresis of DNA digests, provide a means of distinguishing bacterial strains inhabiting the intestinal tract. Analysis of lactobacillus, bifidobacterial, and enterobacterial populations with the use of these methods has shown that human and porcine subjects harbor a characteristic collection of bacterial strains. Additionally, perturbations and transitions that occur in these populations and are caused by antibiotic administration or by autogenic or allogenic factors can be detected by molecular analysis of the intestinal microflora. In future studies, molecular typing methods could be used to analyze the composition of bacterial populations before, during, and after the administration of the probiotic product. This experimental approach would provide information on the effect of the probiotic on indigenous strains inhabiting the intestinal tract of humans and other animals. Am J Clin Nutr 2001;73(suppl):410S–4S. MOLECULAR ANALYSIS OF MICROFLORA 411S TABLE 1 Comparison of selected properties of germfree (absence of microflora) and conventional (presence of microflora) animals1 Biochemical, physiologic, and immunologic host characteristics Bile acid metabolism Conventional Short-chain fatty acids Tryptic activity Urease -Glucuronidase (pH 6.5) Organ weights (heart, lung, and liver) Cardiac output and oxygen utilization Mucin content of intestinal mucus Extent of degradation of mucins Cecal size (rodents) Enzyme activities associated with duodenal enterocytes Intestinal wall Intestinal mucosal surface area Rate of enterocyte replacement Peristaltic movement of contents through small bowel Body temperature Serum cholesterol concentration Lymph nodes -Globulin fraction in blood Large amounts of several acids Little activity Present Present High High High More Small Low Thick Great Fast Fast High Low Large More 1 Absence of deconjugation, dehydrogenation, and dehydroxylation Little deconjugation; absence of reduction Absence of coprostanol Present Absence of hydrogen and methane; less carbon dioxide Small amounts of a few acids High activity Absent Absent Low Low Low Less Large High Thin Small Slow Slow Low High Small Less Data from references 3–5. Wilson and Blitchington (18) compared culture and 16S rDNA sequence analysis as methods of analysis of human fecal samples. They found that, overall, there was good agreement in the biodiversity of the samples analyzed by the 2 methods. Temperature-gradient gel electrophoresis and denaturing-gradient gel electrophoresis are being developed as an additional means of intestinal microflora analysis. In this technique, 16S rDNA is amplified by PCR from the DNA of the bacterial cells in a sample. The various molecul (...truncated)