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Strains of indigenous Escherichia coli, Bacteroides, and Lactobacillus were isolated from the gastrointestinal tracts of specific pathogen-free (SPF) mice. Nonvaccinated SPF mice exhibited in their spleens low numbers of plaque-forming cells (PFC) and rosette-forming cells reacting with antigens of these andigenous bacteria. PFC reacting with these bacterial antigens were not detected in infant SPF mice until 7 days after birth. Compared with nonvaccinated controls, SPF mice vaccinated parenterally with indigenous E. coli or Bacteroides produced a moderate increase in the numbers of specific PFC. Thus, the SPF mouse is capable of responding immunologically after vaccination with microbes indigenous to its intestinal tract. However, more PFC reacting with homologous vaccine antigens were detected after parenteral vaccination of SPF mice with nonindigenous E. coli O127:B8, E.coli O14, or B. fragilis than after parenteral vaccination with indigenous E. coli or Bacteroides. Gnotobiotic mice orally monoassociated with these nonindigenous bacteria exhibited greater immune responses to antigens of the bacteria used to monoassociation than did gnotobiotes monoassociated with the indigenous microbes. The results are consistent with the hypothesis that mice are more responsive immunologically to antigens of nonindigenous bacteria than they are to antigens of certain microbes indigenous to their gastrointestinal tracts.

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.

Germfree CD-1 mice challenged by the oral route with Salmonella typhimurium had ceca that were abnormal in appearance and reduced in size compared to those of germfree controls. Similarly, germfree mice injected with heat-killed S. typhimurium or gnotobiotes associated with three indigenous microbes (Lactobacillus, Bacteroides, Clostridium), and subsequently challenged with S. typhimurium also had small ceca. By contrast, gnotobiotic mice that had been both injected with the heat-killed S. typhimurium and associated with the three indigenous microbes before challenge with S. typhimurium had ceca similar in size and appearance to germfree mice. Thus, indigenous microorganisms could interfere with the mechanism by which the pathogen induced the decrease in cecal size, but could do so only in mice injected with heat-killed bacteria. This phenomenon suggests synergism between the interference effected by the indigenous bacteria and the resistance mechanisms of the animal.

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 Salmonella typhimurium were examined in a similar manner. Three strains of mice (CD-1, Ha/ICr, and C57BL) were used. Control animals previously inoculated with S. typhimurium had low population levels of Salmonella bacteria in the small and large bowel. Mice previously inoculated with Salmonella and then deprived of food, water, and bedding for 48 h harbored high population levels of these bacteria in their small and large bowels. Coliforms increased in numbers in the large bowel of stressed mice inoculated with Salmonella and in the jejunum-ileum and cecum of stressed mice not previously inoculated with Salmonella. Control mice had high population levels of lactobacilli inhabiting the keratinized squamous epithelium of the stomach. Stressed mice showed dramatic reductions in these populations of lactobacilli. Populations of fusiform-shaped bacteria associated with the mucosal epithelium of the cecum and colon in control mice were reduced in stressed mice as determined by microscope examination of histological sections. Total anaerobic counts were similar, however, in both stressed and control animals. Environmental and dietary stress markedly alter the gastrointestinal microbiota in mice. Therefore, such stressful conditions profoundly affect the factors that regulate the localization and population levels of microorganisms in the stomach and intestines.

Torulopsis pintolopesii is a yeast indigenous to the gastrointestinal tracts of conventional mice and rats from many colonies. In such natively colonized animals, the organism forms layers on the surface of the epithelium in the secreting portion of the stomach and can be cultured from all areas of the gastrointestinal tract. When given in water or food to germfree mice or specific pathogen-free mice possessing an indigenous microbiota free of yeast, T. pintolopesii also can be cultured from all areas of the tract at population levels ranging from 10(5) to 10(8) cells per g (wet weight). Likewise, as in its native hosts, the organism forms layers on gastric surfaces in the associated animals. The layers form on the secreting surface in both the specific pathogen-free and monoassociated ex-germfree mice. In the latter animal, however, a layer of yeast also forms on the nonsecreting gastric surface. In tests of its capacity to adhere to gastrointestinal surfaces in vitro, the organism adheres to epithelia from all areas of the mouse tract. These findings support an hypothesis that the capacity of T. pintolopesii to adhere to epithelial surfaces may be only one determinant influencing it to form layers on the gastric secreting surface in its native hosts.

Two assays were designed with which isogenic laboratory strains of Escherichia coli K-12 with and without ColV plasmids were compared for their ability to adhere in vitro to mouse intestinal epithelium. In both assays, disks of intestinal tissue were exposed to bacteria. In the first, disks were homogenized and the numbers of viable bacteria adherent to them were estimated from colony counts of plates inoculated with dilutions of the homogenates. In the second, bacteria were labeled with [(14)C]aspartic acid; the number of adherent cells per disk was estimated by liquid scintillation spectrometry. Data from each assay were compared by analysis of variance. In both assays, strains bearing the ColV plasmid adhered in two- to threefold-greater numbers than isogenic strains without the plasmid. These differences were highly significant statistically. A non-colicinogenic strain free of the ColV plasmid was selected by treatment of a ColV strain with sodium dodecyl sulfate. In the radioisotopic assay, the ColV strain associated with the epithelium in significantly greater numbers than the cured derivative. A ColV strain was created by conjugation; in the radioisotopic assay this strain bound to epithelium in significantly greater numbers than the recipient strain without the plasmid. The original ColV strain, when negatively stained and examined by electron microscopy, had pili that adsorbed male-specific bacteriophage, whereas its isogenic variant without ColV did not. Some such properties, coded by the plasmid, may increase the virulence of the bacteria.

Indigenous segmented filamentous microbes attach to murine ileal epithelial cells. These microbes can be seen on the epithelial surface with a scanning electron microscope. They colonize preferentially the distal ileum in mice. Penicillin, placed in the animal's drinking water, eliminates the microbes from the mouse ileum, but recolinization of the ileum is observed 4 to 5 weeks after the penicillin treatment is stopped. Within 3 to 5 h after rats are given penicillin, the morphology of the microbes is changed. Their external surfaces are wrinkled or broken. Vacated and partially vacated attachment sites are observed. Almost all of the organisms disappear from murine ilea after the animals are exposed to penicillin for 10 h. These observations are discussed in relation to the microbe itself and in its interaction with ileal epithelial cells.

Scanning electron microscopy has been used to visualize the residents of microbial communities populating habitats on epithelial surfaces in the gastrointestinal tracts of mice. In the stomach, bacteria form a dense layer on the stratified squamous epithelium of the nonsecreting area. Microbes of at least three morphological types can be seen in this layer, including short rods with round ends, rods in chains, and tapering filaments composed of repeating units of rod- or coccal-shaped elements varying in size from large at one end of the filament to small at the other end. These three forms all attach by one end to the epithelium. The latter two forms can be found only so attached; in both cases, the end is inserted into a hole or depression in the keratinized epithelium. In the small intestine, a microbe of morphology similar to that of the tapering filaments found in the stomach can be seen attached end-on to the epithelium. Again each filament has one end inserted into a hole in the epithelium. In this case, however, the repeating elements of each filament are all about the same size. In the cecum and colon, predominantly fusiform- and spiral-shaped microbes can be seen mixed together in layers on the epithelium. At least three types of fusiform-shaped microbes can be distinguished on the basis of surface texture, and one type of spiral-shaped microbe can be found. These microorganisms appear to be attached to each other and to the epithelium by weblike filaments. The numerous microbial types present in the various epithelial habitats associate intimately surface-to-surface with each other and with the epithelium. Such surface-surface association may be an important autogenic factor contributing to the stability of the murine gastrointestinal ecosystem.

Cells of strains of Escherichia coli K-12 291 bearing one of three colicin V plasmids, pF54, pH247, or pF70, were tested in comparison with cells of strain 291 for their ability to adhere to murine intestinal tissue in vitro. The plasmids were either repressed or derepressed in conjugal genetic transfer functions. The strains bearing pF54 and pH247 repressed in transfer functions (pColVF54 luminal diameter r and pColVH247 luminal diameter r) adhered in higher numbers to the murine tissue than did the host strain lacking the plasmid or the strains containing the plasmids with active transfer functions (pColVF54drd and pColVH247drd). The number of cells (CFU) of strain 291(pColVF54 luminal diameter r) adherent to the tissue was related directly to the time of incubation (up to 30 min) and to the number of cells (CFU) to which the tissues were exposed. As indicated by tests for sensitivity to F factor (F)-pilus-specific bacteriophages, the cells of strains bearing the plasmids derepressed for conjugal functions had F pili on their surfaces, while such structures were missing from cells of the parental strain (291) and the strains containing the plasmids in repressed form. This finding was supported by transmission electron microscopy of cells of strain 291, 291(pColVF54 luminal diameter r), and 291(pColVF54drd). F pili could be seen on cells of the latter strain but not on those of the parental strain or the strain bearing pColVF54 luminal diameter r. Pili other than F pili were not seen on cells of the strains bearing pF54 in either form. Strains 291(pColVF70drd) and 291(pColVF70 luminal diameter r) adhered to the tissues in numbers comparable to those of strain 291. Nevertheless, these findings are further evidence that for certain colicin V plasmids (A. M. Nilius and D. C. Savage, Infect. Immun. 43:947-953, 1984), conjugal genetic transfer functions influence properties that may be important in the pathogenesis of invasive E. coli strains.

Germfree mice housed in isolators under controlled environmental and nutritional conditions were associated with an intestinal microflora. These associated animals and germfree mice drawn from the same population were used in experiments in which saline extracts of cells from the small intestine were assayed for alkaline phosphatase activity and for protein and DNA content. Epithelial cells were harvested from the intestines sequentially from the villous tips to the crypts of Lieberkühn. In all preparations, germfree animals yielded from one and one-third to one and one-half times the mass (wet weight) of cells yielded by associated mice. Likewise, for all preparations, extracts of the mass of cells from germfree mice contained more protein and DNA per milliliter than did extracts from associated animals. The ratio of the amount of extractable protein or DNA per milliliter of extract to the total wet weight of the cells in milligrams was about the same, however, for preparations from germfree and associated animals. All preparations from germfree animals yielded higher total alkaline phosphatase activities than those from associated mice. When related to the amount of DNA in the cells, the enzymatic activities were slightly but not significantly higher in preparations from germfree animals, except for preparations of cells removed from the tips of the villi. When related to the amount of protein in the extracts, the enzymatic activity (i.e., specific activity) was about the same in preparations from germfree and from associated mice, except (again) for preparations of cells removed from the tips of the villi. In the latter preparations, the specific alkaline phosphatase activities and enzymatic activities calculated relative to the amount of DNA were substantially higher for germfree animals than for mice with a microflora. Individual intestinal epithelial cells from germfree and associated animals, except those close to the villous tips, contain about the same alkaline phosphatase activity. Therefore, germfree mice must yield more activity of such microvillous enzymes than do mice with a microflora, partly because enterocytes at the tips of the villi in germfree mice contain more protein with enzymatic activity than do cells in a comparable location in mice with a microflora. In addition, the small intestines of germfree mice contain more activity of enzymes such as alkaline phosphatase than do those of associated animals because the small intestines of the former animals contain more enterocytes than do those of the latter.

Mg-dependent and (Na+ + K+)-stimulated adenosine triphosphatase (ATP-ase) activities were assayed in butanol extracts of duodenal tissue from germ-free, specific-pathogen-free, and ex-germfree mice associated with an indigenous microflora from specific-pathogen-free mice. In the germfree mice the levels of both ATPase activities were significantly higher than the levels in specific-pathogen-free mice. By contrast, the ex-germfree animals colonized by an entire indigenous microflora, the values fell to levels close to those for specific-pathogen-free animals. (Na+ + K+)-stimulated ATPase was not inhibited by ouabain in extracts from any of the three kinds of mice. These findings show that the indigenous microbial flora influences the intestinal ATPase activity of mice and, because of the connection between (Na+ + K+-stimulate ATPase and active transpoort, undoubtedly affects the process of absorption in the intestinal tract.

Parasitic nematode worms (Aspicularis tetraptera) were observed microscopically in histological sections of the colons of C57BL/6StCrl female mice. These nematodes have gram-positive bacteria intimately associated with the mucosal epithelium in their alimentary tract.

A Lactobacillus strain isolated from a mouse (indigenous) and another strain isolated from swine feces (nonindigenous) were compared in two model systems for their ability to adhere in vitro and in vivo to keratinizing squamous and columnar epithelia of mouse stomachs. In one model, stomachs dissected from specific-pathogen-free or germfree mice were injected with suspensions of lactobacilli labeled with [(3)H]thymidine and incubated at 37 degrees C. Thereafter, the non-secreting and secreting tissues were separated and washed vigorously. The radioactivity remaining with each tissue was counted by liquid scintillation spectrometry. When the radioactivity remaining with these tissues ranged between 500 and 100,000 cpm, the calculated radioactivity (disintegrations per minute) was related linearly to the number of lactobacilli adhering to the tissue. The estimate of the number of bacteria adherent to the tissue was not influenced significantly by artifacts in the techniques used. In this model, both Lactobacillus strains adhered in equally high numbers to both types of epithelial surfaces from stomachs from germfree mice. In contrast, in the second model, in which germfree mice were monoassociated with one or the other of the Lactobacillus strains, only the strain indigenous to the mouse formed dense layers on the epithelia of the nonsecreting portions of the stomachs, although both strains maintained high population levels throughout the gastrointestinal tracts of the animals. The capacity to adhere to the mucosal surface is undoubtedly necessary for lactobacilli to colonize gastric epithelia in mice. Our findings suggest, however, that nutritional or environmental conditions dictate whether particular Lactobacillus strains can colonize particular surfaces in the stomachs of living animals.

Eight colicin V plasmids were conjugated into a plasmidless Escherichia coli (K-12) strain that was susceptible to the bactericidal effects of normal rabbit serum. The resulting colicin V-positive strains were examined for their capacity to resist the lethal effects of serum. Serum resistance was assessed as growth of the bacterial strain in medium containing 5% normal rabbit serum inoculated from a culture in the early exponential phase. Only three of the eight colicin V plasmids were found to confer the serum resistance phenotype on the host strain. Derepression of the transfer system was associated with serum resistance in two of the plasmids. Two other derepressed plasmids did not confer serum resistance on the host bacterium. Therefore, such derepression alone was insufficient to produce serum resistance. The factor(s) encoded by colicin V plasmids and responsible for the serum resistance of the bacterial strains bearing the plasmids was shown to be a property associated with the cell and not an extracellular factor excreted into the growth medium.

The concentration of protein and the activities of alkaline phosphatase, maltase, and sucrase were measured in saline extracts of the proximal small intestine of germfree and ex-germfree mice colonized with an indigenous microflora. The two populations of animals were maintained in plastic film isolators under tightly controlled environmental and nutritional conditions. Samples were taken at 0, 4, 8, 12, and 17 days and at 3, 4, 5, 6, 7, and 8 weeks after association. The activities were expressed as specific activities and as total units per segment of small intestine. Enzymatic activities expressed in both ways fluctuated considerably in the samples taken from one time to the next in animals of both types. The activities expressed as total units per segment of bowel of all three enzymes had decreased from levels in germfree animals by as early as 4 days after association. The total units of activity per segment of bowel tested continued to decrease for approximately 3 weeks in the associated animals to levels two- to fivefold lower than those of germfree animals. However, the specific activities of the three enzymes in the animals of the two types became less disparate at later sample times. This latter result is predictable because the concentration of protein extractable from the small intestines of the mice of the two types was the same at the beginning of the experiment, but by the later sampling times, the concentration of protein extractable from small bowels of ex-germfree mice was significantly lower than that from germfree mice. The fluctuations in levels of the enzymatic activities, even under controlled environmental and nutritional conditions, point to the necessity of using such conditions and a kinetic approach in studies of the effects of the microbiota on the activities of enzymes in the microvillous membranes of small bowel enterocytes. The changes in protein concentrations suggest that such activities and the amounts of protein extractable from the mucosa are influenced by different properties of the microflora. Thus, studies in which the enzymes are extracted from the entire mucosa and the activities are expressed as units per weight of extractable protein may give misleading results concerning the influence of the microbiota on the enterocyte membranes.

Two strains of Listeria monocytogenes, one that formed smooth colonies on agar surfaces and a varient of it that formed rough colonies, colonized the gastrointestinal tracts of germfree mice. Within 24 h after mice were inoculated orally with about 100 bacteria, the population levels per gram (wet weight) of tissue of both strains were 10(5) to 10(7) in the stomach and ileum and 10(8) to 10(9) in the cecum and colon, respectively. As detected in Gram-stained histological sections, in such gnotobiotes, the bacteria colonized the lumen in all areas of the tract and much of the mucus layer on the epithelial surface in the proximal colon. The strain that formed smooth colonies did not colonize the tracts of specific-pathogen-free mice, but did colonize, to the same levels as in germfree mice, the stomachs and bowels of ex-germfree mice previously associated with two members of the indigenous flora (Bacteroides and Clostridium). In the latter animals, however, the listeria did not form layers on the colonic epithelium as efficiently as they did in monoassociated gnotobiotes.

A wild-type strain of Salmonella typhimurium and three mutant rough colonial variants of the wild type were compared for their ability to become associated with and invade the ileal mucosa of germfree and specific-pathogen-free mice. The rough-mutant strains differed from the wild type in having incomplete lipopolysaccharides lacking one or more sugars in the polysaccharide moiety. The wild-type and mutant strains also differed one from the other in the types of appendages (flagella, pili) on their surfaces. Depending upon the dosage of bacteria given, all mutant strains as well as the wild type could associate with and invade the intestinal mucosa of infected gnotobiotic mice. If the infecting dosage was high enough, at least two of the mutant strains and the wild type invade the intestinal mucosa of the specific-pathogen-free animals. O antigen, flagella, or pili do not appear to be essential for the association of S. typhimurium with the mucosal surface of the mouse ileum. O antigen on the bacterial cell surface may be important, but not essential, for invasion of the ileal mucosa.

Enlargement and microbial colonization of the cecum were examined in neonatal mice suckling mothers drinking either water or an aqueous solution of penicillin. The full ceca increased in weight at the same rate in both drug-treated and control mice during the first 15 to 17 days after birth. Thereafter, cecal weight increased at a greater rate in the drug-treated animals than in the untreated controls. At weaning, the ceca in treated mice were two to three times the size of control organs and remained enlarged as long as penicillin was given. The enlarged ceca did not differ histologically from those in controls. From birth, the cecal microflora in the drug-treated mice differed qualitatively and quantitatively and in colonization pattern from the flora of control mice. The ceca of untreated animals were colonized primarily by large populations of lactobacilli during the first week after birth, small populations of coliforms and enterococci during the second week, and enormous populations of bacteroides and certain gram-negative fusiform-shaped anaerobic bacteria during the third week. In contrast, the organs of the treated mice were populated by large populations of coliforms and enterococci during the first week and enormous populations of clostridia and unusual gram-negative nonsporeforming bacteria during the third week. These large abnormal populations were present in the ceca as they enlarged during the third week after birth in the drug-treated animals. These findings confirm that only certain populations of anaerobic bacteria can act to maintain cecal size in normal animals.

Alkaline phosphatase activity was assayed by two procedures in duodenal homogenates from specific pathogen-free (SPF) mice of various ages, adult germfree mice, adult ex-germ-free mice colonized with an indigenous microflora from the SPF mice, and adult ex-germ-free mice monocontaminated with a Lactobacillus sp. indigenous to the SPF mice. In the SPF neonates, the activity remained at low levels until at least 12 days of age, increased to high levels at 20 days of age, and then fell to adult levels between the early neonatal and later high levels. In the germ-free mice, the activity levels were significantly higher than the levels in SPF mice at any age. In contrast, in the ex-germ-free animals, colonized by an entire indigenous microflora, the values fell within the range for adult SPF animals. In the ex-germ-free mice colonized only by the Lactobacillus sp., the activity levels were intermediate between the values for germ-free and SPF mice. These findings show that the indigenous microbial flora influences considerably the intestinal alkaline phosphatase activity in the mouse.

Some indigenous microorganisms have been shown to localize in certain anatomical sites of the digestive tract of mammals. We studied the ceca of normal adult rats by light and electron microscopy to determine whether any specific bacterial population localizes in this area. All rats studied showed that the crypt was packed with organisms whose morphological character differs from those of the cecal lumen. Organisms localized in the crypt were often identified topographically close to the microvilli of the epithelial cells. These organisms could be differentiated into three types according to their characteristic ultrastructure. Type 1 was a thin spiral-shaped microbe that resembled a Borrelia. Type 2 possessed helically coiled fibers and flagella-like appendages. Type 3 was spiral-shaped but lacked axial fibers. Types 1 and 2 were both capable of penetrating through the crypt epithelium into the lamina propria where they were found in either phagocytes or extracellular locations. These observations are discussed in relation to other host-microflora localization patterns.

Anaerobic bacteria can be detected at population levels of 10(11) organisms per g of cecum or colon in adult mice from four different colonies widely spaced in the United States. Most of these microorganisms are oxygen-intolerant fusiform-shaped bacteria. At least one type of these tapered, rod-shaped bacteria can be seen in layers in the epithelial mucin in frozen-section histological preparations of the large bowels of mice. In addition, such microorganisms can be seen within 0.5 mum of the epithelium in ultrathin sections of colon or cecum examined in an electron microscope. These fusiform-shaped bacteria predominate in the mucin layers. However, spiral-shaped microorganisms can be found as well near the mucosal epithelia in ultrathin sections of colon. Also, such organisms can be seen in negatively-stained preparations of washings of the colonic mucosal epithelia examined in an electron microscope. At least three types of spiral-shaped organisms, including both spiral-shaped bacteria and spirochetes, can be found in preparations from mice from three of the four colonies. Such spiral-shaped microorganisms can be detected at population levels as great as 10(9) organisms per g of cecum or colon in anaerobic cultures of the large bowels of mice from all four colonies. One anaerobic spiral bacterium was isolated in pure culture. This particular organism was found by immunofluorescence to be intermingled with the fusiform-shaped bacteria in the mucin on the mucosal epithelium in the mouse large bowel.

Colonization by indigenous microorganisms of the mucosal epithelia of the large bowels of suckling mice was followed by microbial culture techniques and by light, fluorescence, and electron microscopy. Certain microbes colonize in distinctive patterns the cecal and colonic epithelia in these mice. Coliforms and enterococci colonize the large bowel 7 to 9 days after birth and reach high population levels during the second week. During that period, these facultative anaerobes can be detected by immunofluorescence techniques in microcolonies in the mucin on the epithelium. During the third week, however, after their populations decline to the low levels characteristic of adult mice, coliforms and enteroccoci can be observed only infrequently in the mucous layer. Anaerobic fusiform-shaped bacteria appear in the mucous layers along with the microcolonies of enterococci and coliforms during the second week after birth. These anaerobes increase in numbers in the mucin until they form thick layers on the mucosal epithelium by the end of the third week. They remain in the mucous layer throughout the life of the normal mouse. Anaerobic spiral-shaped microbes also colonize the mucous layer on the cecal and colonic epithelium. But these organisms can be detected by immunofluorescence in 1-week-old mice, well in advance of the time the fusiform-shaped bacteria can be found. In the second week, the latter microbes co-inhabit the mucous layer with the spiral-shaped organisms. The fusiform- and spiral-shaped microbes remain associated in the mucin on the cecal and colonic mucosal epithelia into the adult life of mice.