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Colonization of the gastrointestinal tract by bacteria of the normal flora was followed by bacteriological and special histological techniques in mice from several colonies. These histological techniques were designed to preserve the intimate associations that become established between particular strains of microorganisms and the epithelium of the mucosa of certain areas of the gut. The findings were as follows: 1. The various strains of bacteria of the normal flora became established in the different areas of the guts of infant mice according to a definite time sequence. 2. The first types of bacteria that could be cultured from the gut were lactobacilli and Group N streptococci. Within the first day after birth, these bacteria colonized the entire digestive tract and formed layers on the stratified squamous epithelium of the nonsecreting portion of the stomach and of the distal esophagus. 3. The bacterial types that appeared next were coliforms and enterococci. From about the 9th to the 18th day after birth, these bacteria could be cultured in extremely high numbers from the cecum and the colon. Histological sections of those organs taken during the first 2 or 3 days of that interval revealed microcolonies of Gram-positive cocci in pairs and tiny Gram-negative rods embedded in the mucous layer of the epithelium. The microcolonies were well separated from the mixture of digesta and bacteria that occupied the center of the lumen; they may have consisted of the coliforms and enterococci mentioned above; but this possibility remains to be proved. 4. Histological sections also revealed that, at about the 12th day after birth, long, thin Gram-variable rods with tapering ends were present, side by side, with the small Gram-negative rods and Gram-positive cocci in the mucous layer. By the 15th day after birth, the fusiform bacteria formed thick layers in the mucus, and seemed to be the only bacteria remaining in that location. It has not yet been possible to enumerate these tapered rods by culture methods, but as judged by visual appearances in the histological sections, they seemed to outnumber all other bacteria in the cecum and the colon by a factor of as much as 1000. It must be stressed that these bacterial layers are readily disrupted and even washed away by conventional histological techniques; their discovery was largely due to the use of the special histological techniques described in the text. The bacteriological and histological findings described here constitute further evidence for the hypothesis that symbiotic associations exist between microorganisms and animals, and that a very large percentage of the bacteria in the gastrointestinal tract constitutes a true autochthonous flora. The constant occurrence of several distinct associations of bacteria with the special histological structures of the animal host renders obsolete the notion that the intestine constitutes a chemostat in which the bacterial populations are randomly mixed. For a full understanding of the ecology of the normal microflora, it is necessary to think of body surfaces as distinct microenvironments in which virtually pure cultures of a few species of microorganisms interact with their host and the adjacent microbial populations. Experiments based on this hypothesis are admittedly difficult to design, but on the other hand studies based on the assumption that microorganisms exist as mixtures in the gastrointestinal tract will be only of limited value and may often be misleading.

Aerobic and anaerobic cultural techniques and histological methods were used in a study of the effects of environmental and dietary stress on the indigenous microbiota of the gastrointestinal tract of mice. Mice previously inoculated with on July 15, 2020 by guest http://iai.asm.org/ Downloaded from on July 15, 2020 by guest http://iai.asm.org/ Downloaded from

Some indigenous microorganisms localize on epithelial surfaces in various areas of the digestive tracts of animals. One of these, a segmented, filamentous microbe, localizes on the epithelium in the small bowels of mice and rats. These filamentous microbes colonize mice at weaning time and persist in adult animals for at least 2 months. Results of the study of light and electron micrographs suggest that the microorganisms are procaryotic, and that they interact with small bowel epithelial cells to form an attachment site. This site consists of modified epithelial cell membrane and apical cytoplasm adjacent to the attached bacterium. The microbe fills the site with part of its first segment. This segment has a nipple-like appendage on the end inserted into the epithelial cell. The other segments, which compose the rest of the filament, are usually separated by septa. Many of the individual segments contain intrasegmental bodies that appear to be procaryotic cells. Some of these intrasegmental bodies are similar in morphology to the first segment of each filament inserted into an epithelial cell. These intracellular bodies may be components in the life cycle of the microorganism. The organism has not yet been cultured in recognizable form. Therefore, such a hypothesis cannot be proved as yet, nor can the microbe be classified with certainty. Because it localizes in an epithelial habitat in the small bowel, however, it may be a particularly important microbial type in the gastrointestinal ecosystem of laboratory rodents. Adult murine gastrointestinal tracts contain many types of indigenous microorganisms living in relatively stable communities localized in specific regions of the tract (5, 7, 10, 15, 16, 18). The various microbial types in these communities colonize the tracts of neonatal mice in a characteristic and reproducible succession. The succession in suckling mice has been reported for lactic acid bacteria, coliforms, enterococci, bacteroides, fusiform, and spiral-shaped microbes. The habitats of these microorganisms in suckling and adult mice have been described (4, 7, 17, 19). Another microbial type can be found on the epithelial surfaces of villi in the small intestines of adult rats (13, 14), mice (11; C. P. Davis, S. Erlandsen, and D. C. Savage, Abstr. Annu. Meet. Amer. Soc. Microbiol. 1973, p. 57), and chickens (8). This organism has never been identified with certainty. Moreover, little is known about its morphology, habitat, and ecology. In this report, we detail the ultrastructure, habitat, succession, and attachment to epithe-lial cells of these segmented, filamentous microbes in the small bowels of laboratory rodents. MATERIALS AND METHODS Animals. Specific pathogen-free male young adult rats were purchased from four different suppliers

Bacteria of numerous species isolated from the human gastrointestinal tract express bile salt hydrolase (BSH) activity. How this activity contributes to functions of the microorganisms in the gastrointestinal tract is not known. We tested the hypothesis that a BSH protects the cells that produce it from the toxicity of conjugated bile salts. Forty-nine strains of numerous Lactobacillus spp. were assayed to determine their capacities to express BSH activities (taurodeoxycholic acid [TDCA] hydrolase and taurocholic acid [TCA] hydrolase activities) and their capacities to resist the toxicity of a conjugated bile acid (TDCA). Thirty of these strains had been isolated from the human intestine, 15 had been recovered from dairy products, and 4 had originated from other sources. Twenty-six of the strains expressed both TDCA hydrolase and TCA hydrolase activities. One strain that expressed TDCA hydrolase activity did not express TCA hydrolase activity. Conversely, in one strain for which the assay for TDCA hydrolase activity gave a negative result there was evidence of TCA hydrolase activity. Twenty-five of the strains were found to resist the toxicity of TDCA. Fourteen of these strains were of human origin, nine were from dairy products, and two were from other sources. Of the 26 strains expressing both TDCA hydrolase and TCA hydrolase activities, 15 were resistant to TDCA toxicity, 6 were susceptible, and 5 gave inconclusive results. Of the 17 strains that gave negative results for either of the enzymes, 7 were resistant to the toxicity, 9 were susceptible, and 1 gave inconclusive results. These findings do not support the hypothesis tested. They suggest, however, that BSH activity is important at some level for lactobacillus colonization of the human intestine.

Lactobacillus johnsonii strain 100-100 expresses two antigenically distinct conjugated bile salt hydrolases (BSH), α and β, that combine to form native homo-and heterotrimers. This paper reports characterization of loci within the genome that encode this capacity. A locus that encodes BSHβ (cbsHβ), a partial (cbsT1) and a complete conjugated bile salt transporter (cbsT2) was identified previously. DNA sequence analysis at this locus was extended and revealed a complete ORF for cbsT1 and no other ORFs in tandem. The three genes, cbsT1, cbsT2 and cbsHβ, probably constitute an operon ; a putative promoter was identified upstream of cbsT1. A second locus that expresses BSH activity in strain 100-100 was identified. Sequence analysis of the clone predicted a 978 nt ORF that did not share tandem organization with other ORFs, was similar in sequence to other BSH genes, and matched, in predicted protein sequence, the first 25 amino acids of BSHα. A phenotypic screen for BSH activity and a genetic screen for the cbsHβ locus were performed on 50 Lactobacillus isolates from humans or dairy products. Nearly all of the isolates that were positive for cbsHβ were from human sources. Variability in the BSH phenotype and cbsHβ genotype was identified in isolates of the same species. DNA sequence was obtained and analysed from the cbsHβ locus of one human isolate, L. acidophilus strain KS-13. This organism has cbsT1, cbsT2 and cbsβ genes that are 84, 87 and 85 % identical in DNA sequence to those of strain 100-100. DNA sequence identity to strain 100-100 ends in regions flanking this locus. The findings of this study suggest that BSH genes have been acquired horizontally and that BSH activity is important at some level for lactobacilli to colonize the lower gastrointestinal tract.

We have characterized and purified the bile salt hydrolase from Lactobacillus sp. strain 100-100. Bile salt hydrolase from cells of the strain was purified with column and high-performance liquid chromatography. The activity was assayed in whole cells and cell-free extracts with either a radiochemical assay involving ['4C]taurocholic acid or a nonradioactive assay involving trinitrobenzene sulfonate. The activity was detectable only in stationary-phase cells. Within 20 min after conjugated bile acids were added to stationary-phase cultures of strain 100-100, the activity in whole cells increased to levels three-to fivefold higher than in cells from cultures grown in medium free of bile salts. In cell-free extracts, however, the activity was about equal, 1.41 and 1.53 ,umol/min per mg of protein, respectively, whether or not the cells have been grown with bile salts present. When supernatant solutions from cultures grown in medium containing taurocholic acid were used to suspend cells grown in medium free of the bile salt, the bile salt hydrolase activity detected in whole cells increased two-to threefold. Two forms of the hydrolase were purified from the cells and designated hydrolases A and B. They eluted from anion-exchange high-performance liquid chromatography in two sets of fractions, A at 0.15 M NaCl and B at 0.18 M NaCl. Their apparent molecular weights in nondenaturing polyacrylamide gel electrophoresis were 115,000 and 105,000, respectively. However, discrepancies existed in the apparent molecular weights and number of peptides detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the two forms. Both had similar substrate specificities, highest on taurodeoxycholic and glycocholic acid, and pH optima between 3.8 and 4.5. The kinetic properties were also similar, with Vmaxs of 17 and 53 ,Lmol/min per mg of protein and Kms of 0.76 and 0.95 mM taurocholic acid for A and B, respectively.

A mammal is a complex organism consisting of eukaryotic animal cells and eukaryotic and prokaryotic microbial cells. Most of the microorganisms reside in communities in the gastrointestinal tract. These gastrointestinal microfloras are known to serve nutritional functions in ruminants, pseudoruminants, and monogastric mammals with only modest or no foregut fermentations but with extensive hindgut fermentations in blind cecal pouches. In adult animals, the microflora hydrolyzes exogenous (dietary) and endogenous polymers, and provides the adult with all or at least a significant proportion of its carbon, energy, vitamins, and macromolecular building blocks. The flora also functions as a conservator of nitrogen that would otherwise be excreted as urea. In exchange, the flora competes directly with the host tissues for nutrients ingested in the diet, and also competes indirectly by somewhat repressing the absorptive capacities of the animal tissues. When the synergism is in balance, the animal tissues and the microflora operate in harmony for the health and nutritional welfare of the host as a whole. The system may be unbalanced by antibacterial drugs that destroy the microflora and by diseases of the animal tissues that destroy the controls regulating where indigenous communities localize in the tract, their microbial composition, and their biochemical activities. At such times, the nutrition of the animal tissues can be adversely affected to the extreme. Humans living in developed and developing countries have extensive microfloras in their hindguts. Humans living in developing countries may also have extensive microfloras in their small bowels. Those floras may function in nutrition of the animal tissues of man much the same as do floras in similar locations in the gastrointestinal tracts of mammals other than man. However, animals of some species other than human gain much of the nutritional benefit from their microflora through the practice of coprophagy. Since adult humans do not normally practice coprophagy, any nutritional benefit from the microflora depends upon the capacity of the bowel mucosa, principally that of the large bowel, to absorb bacterial products, e.g. short-chain volatile fatty acids. Such absorption undoubtedly occurs, but is surely not a major source of carbon and energy for the animal tissues of man.