Establishment of specific pathogen-free guinea-pig colonies using limited-flora guinea-pigs associated with conventional guinea-pig flora, and monitoring of their cecal flora.

Six groups of limited flora (LF) Hartley guinea-pigs were produced by inoculation of hysterectomy-derived GF guinea-pigs with various combinations of cecal bacteria of conventional (CV) guinea-pigs to determine the effective bacterial cocktails for the establishment of a specific pathogen free (SPF) colony. Bifidobacterium magnum (Bif) isolated from CV guinea-pigs was used for pretreatment. The mortality of LF guinea-pigs inoculated with only Bif was 75%, and that of those inoculated with Bif plus chloroform-treated cecal suspension (CHF) or Bif plus CHF plus 32 isolates from CV guinea-pigs was 40 to 66.7%. These three groups were in an unhealthy condition with mucoid enteritis-like diarrhea. However, the mortality of LF guinea-pigs inoculated with the anaerobic growth on EG plates injected with 10(-5) dilution of cecal contents (CF) or inoculated with Bif plus CF was 6.3 and 15%, respectively. These latter two groups of LF guinea-pigs were transferred to separate barrier rooms and some of the LF guinea-pigs were maintained in isolators as a source of intestinal flora for SPF guinea-pigs. The composition of cecal flora of LF guinea-pigs was stable for a long time, and bacteroidaceae and peptococcaceae were maintained as predominant components. The basic composition of the cecal flora of SPF guinea-pigs originated from LF guinea-pigs, which consists mainly of the anaerobic bacteria, was not changed over a long period, and the flora composition became similar to that in CV guinea-pigs. Guinea-pig-specific pathogens from the SPF colonies were not detected during experiments.     

Characteristic faecal flora of NC mice

The composition of faecal flora of NC mice was compared with that of CF #1 mice. NC- and CF #1-germfree (GF) mice were cage-mated with NC- or CF #1-conventional (CV) mice in an isolator. The faecal flora of these ex-GF mice was dependent on the recipient mouse strain modifying colonization by the donor mouse bacteria. Although NC- and CF #1-pups removed by hysterectomy were fostered to different strains, almost all these mice at 8 weeks old had a strain characteristic pattern of faecal flora regardless of the foster strains. In GF mice mono-associated with a Lactobacillus strain or a Bifidobacterium strain isolated from faeces of CV mice, the numbers of these bacteria in the stomach and small intestine of NC mice were lower than those of CF #1 mice. In GF mice associated with chloroform-treated faeces of CV mice, and a Lactobacillus strain or a Bifidobacterium strain, the numbers of these bacteria in the stomach and all parts of the intestine of NC mice were considerably lower than those of CF #1 mice. These results suggested that the composition of faecal flora of NC mice were characteristic, i.e. the fact that the numbers of lactobacilli were low compared with CF #1 mice with ordinary faecal flora and the colonization of bifidobacteria, peptococcaceae and eubacteria on ES agar in NC mice intestine differed, was due to genetic factors.     

Efficiency of various bacterial suspensions derived from cecal floras of conventional chickens in reducing the population level of Salmonella typhimurium in gnotobiotic mice and chicken intestines

The antagonistic effect exerted towards Salmonella typhimurium by the flora issued from conventional chickens was studied in gnotobiotic animals. In germfree chickens and mice inoculated with S. typhimurium, the highest bacterial counts were observed in ceca, and were not significantly different in either host. The protection afforded by the inoculation of cecal flora issued from a conventional chicken was more effective when this flora was inoculated first into germfree chickens than when it was given only after inoculation with S. typhimurium. Administration of a cecal flora from a 15-day-old chick to gnotobiotic mice and chicken resulted in the inhibition of a further intestinal colonization by S. typhimurium in both hosts. Sixteen strains were isolated among the predominant populations of the fecal flora from chicken flora recipient mice. Association of 14 strains of strictly anaerobic bacteria with 2 strains of Escherichia coli and Streptococcus faecium only decreased the number of S. typhimurium in the ileum of gnotobiotic mice, but not in their cecum. Anaerobe cultures were obtained from 10(-6) and 10(-8) dilutions prepared from the fecal flora of gnotobiotic recipient mice. Antagonistic bacteria were present only in cultures from the 10(-6) dilution. Cecal concentrations of volatile fatty acids were shown not to be the sole factor implicated in the antagonistic effect against S. typhimurium.     

Comparative studies of some functional and morphological parameters in the livers of germfree, conventional and ex-germfree mice.

Some parameters of hepatic function and morphology were studied to compare germfree (GF) and conventional (CV) BALB/c mice. The levels of lipid peroxide (LPO) and aniline-hydroxylase (AH) activity in the livers and the serum total cholesterol (TC), triglyceride (TG) and phospholipid (PL) were significantly lower in GF than in CV 8-week-old mice. There were no significant differences in the histology and lectin-histochemistry of the livers in the GF and CV mice. On the other hand, in ex-GF mice which were induced by housing 4-week-old GF mice together with age-matched CV mice, the levels of LPO and AH activity in the liver and the serum TC, TG and PL contents increased rapidly within the first week and then approached values almost identical to those in CV mice 4 weeks later (i.e. at 8 weeks of age). The histologic picture of the liver was similar among the GF, CV and ex-GF mice.     

Atropine inhibits the degranulation of Paneth cells in ex-germ-free mice

Summary Previous studies have shown that the secretory products of Paneth cells contain antibacterial agents (lysozyme, IgA) that are affected by the bacterial milieu in the intestine. To investigate whether Paneth-cell secretion is controlled via cholinergic mechanisms, the ultrastructure of Paneth cells was studied in four animal groups: (1) germfree (GF) control mice (Jcl: ICR [GN], male, 13 weeks old), (2) GF mice injected subcutaneously with atropine sulfate (200 mg/kg body weight, dissolved in physiological saline 20 mg/ml), (3) ex-GF mice inoculated with feces from specific-pathogen-free (SPF) mice, and (4) ex-GF mice injected with atropine and inoculated with feces from SPF mice. In ex-GF mice inoculated with feces, 70–90% of the Paneth cells showed fewer secretory granules than those from GF mice (p<0.01). Approximately 30% of the Paneth cells had a large vacuole (3–10 m diameter) in the apical cytoplasm. Exocytosed electron-dense material from secretory granules was observed in a few crypt lumens. In ex-GF mice inoculated with feces and given atropine, about 90% of the Paneth cells contained numerous secretory granules, like those in GF control mice, but vacuolated Paneth cells and exocytotic figures were rare; thus the secretion of Paneth cells was blocked by atropine. It is therefore possible that the bacterial milieu in the intestine affects the secretory activity of Paneth cells via cholinergic mechanisms.     

Effect of intestinal flora on megaenteron of mice.

CF#1 germfree (GF) and conventional (CV) mice as well as offspring of conventionalized GF (GF-CV) mice were orally inoculated with Escherichia coli 0115a, c: K(B), a causative agent of megaenteron in mice. Although CV and GF mice showed no clinical signs and survived, all of the GF-CV mice died with diarrhea by day 14 after inoculation. Thickened wall of the large intestine, microscopically showing proliferation of crypt type cells, was seen in GF and GF-CV mice but not in CV mice. In addition, in GF-CV mice, hemorrhage and severe erosion with marked inflammatory reactions were observed. While the inoculated E. coli could not colonize in CV mice, a level of 10⁸ cells/g feces was maintained in GF mice from day 1 after inoculation to the end of examination (on day 28) and in GF-CV mice from day 5 to the time of death. Newly prepared germfree (GF-CV-GF) mice obtained hysterectomy from GF-CV mice showed a low sensitivity as comparable to that in GF mice. On the other hand, ex-germfree mice produced by oral administration of feces of GF-CV mice showed severe infection as comparable to that seen in GF-CV mice. These results suggest that the intestinal flora may play roles both on protecting from the infection of pathogenic E. coli and on enhancing the infection.     

Absence of cecal secondary bile acids in gnotobiotic mice associated with two human intestinal bacteria with the ability to dehydroxylate bile acids in vitro.

Germ-free mice were orally inoculated with human intestinal 7alpha-dehydroxylating bacterial strains to evaluate their ability to transform bile acids in vivo. Three weeks after inoculation of the bacteria, cecal bile acids were examined. Among free-form bile acids, only beta-muricholic acid was detected in the cecal contents of gnotobiotic mice associated with Bacteroides distasonis strain K-5. No secondary bile acid was observed in the cecal contents of any of the gnotobiotic mice associated with 7alpha-dehydroxylating bacteria, Clostridium species strain TO-931 or Eubacterium species strain 36S.     

Patterns of early gut colonization shape future immune responses of the host

The most important trigger for immune system development is the exposure to microbial components immediately after birth. Moreover, targeted manipulation of the microbiota can be used to change host susceptibility to immune-mediated diseases. Our aim was to analyze how differences in early gut colonization patterns change the composition of the resident microbiota and future immune system reactivity. Germ-free (GF) mice were either inoculated by single oral gavage of caecal content or let colonized by co-housing with specific pathogen-free (SPF) mice at different time points in the postnatal period. The microbiota composition was analyzed by denaturing gradient gel electrophoresis for 16S rRNA gene followed by principal component analysis. Furthermore, immune functions and cytokine concentrations were analyzed using flow cytometry, ELISA or multiplex bead assay. We found that a single oral inoculation of GF mice at three weeks of age permanently changed the gut microbiota composition, which was not possible to achieve at one week of age. Interestingly, the ex-GF mice inoculated at three weeks of age were also the only mice with an increased pro-inflammatory immune response. In contrast, the composition of the gut microbiota of ex-GF mice that were co-housed with SPF mice at different time points was similar to the gut microbiota in the barrier maintained SPF mice. The existence of a short GF postnatal period permanently changed levels of systemic regulatory T cells, NK and NKT cells, and cytokine production. In conclusion, a time window exists that enables the artificial colonization of GF mice by a single oral dose of caecal content, which may modify the future immune phenotype of the host. Moreover, delayed microbial colonization of the gut causes permanent changes in the immune system.     

The role of encapsulated anaerobic bacteria in synergistic infections

Abstract: The effect of encapsulation on the virulence, survival, and protection of anaerobic bacteria from phagocytosis is reviewed. Support for the importance of encapsulated Gram-negative anaerobic rods ( Bacteroides sp., Prevotella sp. and Porphyromonas sp.), anaerobic and facultative Gram-positive cocci (AFGPC) was provided by their higher recovery rate in oropharyngeal infections, abscesses and blood, compared to their number in the normal flora. The pathogenicity of Bacteroides, Fusobacterium, Clostridium , and AFGPC was studied by inoculating them into mice and observing their ability to induce subcutaneous abscesses. Encapsulated Bacteroides, Fusobacteria , and AFGPC generally induced abscesses, whereas non-encapsulated organisms did not. However, many of the strains that had only a minimal number of encapsulated organisms (< 1%) survived in the abscesses, and they became heavily encapsulated when inoculated with other viable or non-viable encapsulated bacteria. Thereafter, these strains were able to induce abscesses when injected alone. Encapsulated Gram-negative anaerobic rods and AFGPC-induced bacteraemia and translocation, and increased the mortality of the infected animals more often than did the non-encapsulated form of the same strains. As determined by using selective antimicrobial therapy and quantitative cultures of abscesses induced in mice, possession of a capsule generally made Gram-negative anaerobic rods more important than their aerobic counterparts. Synergistic potentials were seen between encapsulated Gram-negative anaerobic rods and all tested aerobic bacteria and most AFGPC, and also between most AFGPC and Pseudomonas aeruginosa or Staphylococcus aureus . These studies demonstrated the importance of encapsulated anaerobes in mixed infections.     

Susceptibility of Germ-free Mice to Infectious Megaenteron

Germ-free (GF)-ICR mice were shown to be less susceptible to oral inoculation with a pathogenic strain of Escherichia coli (E. coli 0115a, c: K(B)) than GF-CF#1 mice. In GF-CF#1 mice a large number of organisms were recovered from the intestinal wall from the cecum to the rectum 3 to 7 days after inoculation. Unlike those in GF-CF#1 mice, lesions in GF-ICR mice were localized in a part of the cecum and organisms were recovered only from the cecal wall and rarely from organs other than those of the alimentary tract. In both strains of mice, however, organisms were recovered in large number from the intestinal contents. Histopathology and immunofluorescence revealed organisms closely attached to the surface of the cecum, colon and rectal epithelia in GF-CF#1 mice but only in a part of the cecal epithelium in GF-ICR mice. After being in contact with conventional CF#1 mice for 21 days and then inoculated orally with the pathogenic E. coli, ex-GF-CF#1 mice died within 14 days with severe intestinal lesions, but ex-GF-ICR mice survived without lesions.     

Establishment of specific pathogen-free (SPF) rat colonies using gnotobiotic techniques.

Gnotobiotic Wistar rats were produced using gnotobiotic techniques, which were established in the production of a SPF mouse colony, in order to establish a barrier-sustained colony. One strain of Escherichia coli, 28 strains of Bacteriodaceae (B-strains), three strains of Lactobacillus (L-strains) and a chloroform-treated fecal suspension (CHF, Clostridium mixture) were prepared from conventional Wistar rats as the microflora source. Two groups of limited-flora rats, E. coli plus B-strains and E. coli plus CHF, were produced. After confirmation that Clostridium difficile was not detected in the CHF-inoculated rats, two groups of limited-flora rats were transferred to an isolator and housed together in a cage. These rats were then orally inoculated with L-strains. The gnotobiotic rats showed colonization resistance to Pseudomonas aeruginosa, and the number of E. coli in the feces was 10(5) to 10(6)/g. The gnotobiotic rats were transferred to a barrier room as a source of intestinal flora for SPF colonies. In the SPF rats, basic cecal flora was mainly composed of Bacteroidaceae, clostridia, fusiform-shaped bacteria and lactobacilli, and did not change over a long period. Their flora became similar to that of conventional rats.     

Isolation, Culture Characteristics, and Identification of Anaerobic Bacteria from the Chicken Cecum

Studies on the anaerobic cecal microflora of the 5-week-old chicken were made to determine a suitable roll-tube medium for enumeration and isolation of the bacterial population, to determine effects of medium components on recovery of total anaerobes, and to identify the predominant bacterial groups. The total number of microorganisms in cecal contents determined by direct microscope cell counts varied (among six samples) from 3.83 x 10(10) to 7.64 x 10(10) per g. Comparison of different nonselective media indicated that 60% of the direct microscope count could be recovered with a rumen fluid medium (M98-5) and 45% with medium 10. Deletion of rumen fluid from M98-5 reduced the total anaerobic count by half. Colony counts were lower if chicken cecal extract was substituted for rumen fluid in M98-5. Supplementing medium 10 with liver, chicken fecal, or cecal extracts improved recovery of anaerobes slightly. Prereduced blood agar media were inferior to M98-5. At least 11 groups of bacteria were isolated from high dilutions (10(-9)) of cecal material. Data on morphology and physiological and fermentation characteristics of 90% of the 298 isolated strains indicated that these bacteria represented species of anaerobic gram-negative cocci, facultatively anaerobic cocci and streptococci, Peptostreptococcus, Propionibacterium, Eubacterium, Bacteroides, and Clostridium. The growth of many of these strains was enhanced by rumen fluid, yeast extract, and cecal extract additions to basal media. These studies indicate that some of the more numerous anaerobic bacteria present in chicken cecal digesta can be isolated and cultured when media and methods that have been developed for ruminal bacteria are employed.     

Bacteremia After Tooth Extractions Studied with the Aid of Prereduced Anaerobically Sterilized Culture Media

Both prereduced molten agar and broth and aerobic molten agar and broth were inoculated with blood samples collected from patients with periodontitis, but in otherwise good health, both before and after extraction of two or more teeth. Postoperative blood samples from 23 of 25 patients sampled yielded anaerobic and facultative species. Colony counts from nine samples yielded from less than 1 to over 100 colonies per ml of blood. Organisms detected were species belonging to the genera Bacteroides, Fusobacterium, Peptostreptococcus, Leptotrichia, Propionibacterium, Peptococcus, Veillonella, plus Streptococcus mitis, S. salivarius, vibrio forms, and strains resembling S. mutans. The data indicate that prereduced anaerobically sterilized culture medium with polyanethol sulfonate is effective for detecting anaerobic species in bacteremia and that anaerobic species can be prevalent in bacteremias immediately after tooth extraction in patients with periodontitis.     

Studies on the Cecal Microflora of Commercial Broiler Chickens

A study was made of the cecal microflora isolated from broilers (5-week-old) reared under typical commercial husbandry conditions. Three hundred and twenty-five bacterial strains (randomly isolated from colonies representing 49 to 81% of the microscopic count) were isolated from cecal digesta of six animals on a rumen fluid roll tube medium (M98-5). Seventy-seven percent of these strains consisted of strict anaerobes: gram-negative, pleomorphic cocci (5.2%), Peptostreptococcus (1.5%), gram-positive rods (36.1% as Propionibacterium acnes and Eubacterium sp.), gram-negative rods (18.6% as Bacteroides clostridiiformis, B. hypermegas and B. fragilis) and sporeforming rods (15.7% as Clostridium sp.). Two types of facultatively anaerobic bacteria (gram-positive cocci and Escherichia coli) were also isolated and constituted 17.5% of the remaining flora. The distribution of the bacterial groups isolated from six cecal samples varied considerably. Data on the growth requirements of anaerobic strains indicated that many could be cultured in a simple medium consisting of an energy source, minerals, reducing agent, Trypticase, and yeast extract (or a vitamin mixture in place of yeast extract). The growth of some of these bacteria was also enhanced by CO(2) and rumen fluid. These preliminary data suggest that some of the more numerous anaerobes isolated from the chicken cecum may not require complex nutrients for growth and, in fact, may be nutritionally similar to rumen anaerobes.     

In vitro anaerobic incubation of Salmonella enterica serotype Typhimurium and laying hen cecal bacteria in poultry feed substrates and a fructooligosaccharide prebiotic

The objective of this study was to investigate the effect of combining a prebiotic with poultry feeds on the growth of Salmonella enterica serotype Typhimurium (ST) in an in vitro cecal fermentation system. Cecal contents from three laying hens were pooled and diluted to a 1:3000 concentration in an anaerobic dilution solution. The cecal dilution was added to sterile test tubes filled with alfalfa and layer ration with and without fructooligosaccharide (FOS). Two controls containing cecal dilutions and anaerobic dilution solution were used. The samples were processed in the anaerobic hood and incubated at 37 degrees C. Samples were inoculated with Salmonella at 0 and 24h after in vitro cecal fermentation and plated at 0 and 24h after inoculation with ST. Plates were incubated for 24h and colony forming units (CFU) enumerated. The samples immediately inoculated with ST without prior cecal fermentation did not significantly lower ST counts 24h later. However, samples pre-incubated for 24h with cecal microflora prior to ST inoculation exhibited reduced ST CFU by approximately 2 logarithms, with the most dramatic decreases seen in alfalfa and layer ration combined with FOS. The addition of FOS to feed substrate diets in combination with cecal contents acted in a synergistic manner to decrease ST growth only after ST was introduced to 24h cecal incubations.     

Intestinal colonization of laboratory rats with Oxalobacter formigenes.

Six strains of Oxalobacter formigenes (anaerobic oxalate-degrading bacteria) were examined for their ability to colonize the gastrointestinal tracts of adult laboratory rats. These rats did not harbor O. formigenes. Strain OxCR6, isolated from the cecal contents of a laboratory rat that was naturally colonized by oxalate-degrading bacteria, colonized the ceca and colons of adult rats fed a diet that contained 4.5% sodium oxalate. Five days after rats were inoculated intragastrically with 10(9) viable cells of strain OxCR6, oxalate degradation rates in cecal and colonic contents increased by 19 and 40 times, respectively. Viable counts of strain OxCR6 from these rats averaged 10(8)/g (dry weight) of cecal contents. Strain OxCR6 was not detected in the cecal contents of inoculated rats fed diets that contained less than 3.0% sodium oxalate. Strains of O. formigenes isolated from the cecal contents of swine, guinea pigs, and wild rats and from human feces also colonized the ceca of laboratory rats; a ruminal strain failed to colonize the rat cecum.     

Identification of Bacteroides fragilisby a modified immuno polymerase chain reaction (mIPCR), using a specific monoclonal antibody

Bacteroides fragilis is the anaerobic species most commonly isolated from human clinical specimens, and is resistant to many antimicrobial agents. A monoclonal antibody, mAb4H8 (IgG3), reacting with a specific epitope in the lipopolysaccharide (LPS) isolated from most of the B. fragilis strains, was produced and employed with modified Immuno Polymerase Chain Reaction (mIPCR) for identification of B. fragilis with a detection limit of 10(4) cfu/mL bacterial suspension. A number of bacterial strains were examined, including B. fragilis, Bacteroides spp. other than B. fragilis and other genera. All the B. fragilis strains with the immunodominant (beta1,6-linked D-galactosyl chain) epitope were positive. None of the other strains showed the positive reaction. The results indicate that mIPCR assay with mAb4H8 has a high specificity and high sensitivity.     

Ruminal microbial digestion in free-living, in captive lichen-fed, and in starved reindeer (Rangifer tarandus tarandus) in winter.

In free-living (FL) reindeer eating a natural mixed winter diet dominated by lichens, captive (CF) reindeer fed pure lichens ad libitum, and CF reindeer subsequently starved for 1 day (CS1 reindeer) or 4 days (CS4 reindeer), the dominant rumen anaerobic bacteria were characterized, their population densities were estimated, and ruminal pH and volatile fatty acid concentrations were determined. In the FL reindeer, the total median viable anaerobic bacterial population ranged from 18 x 10(8) to 35 x 10(8) cells per ml of rumen fluid (n = 4), compared with 26 x 10(8) to 34 x 10(8) and 0.09 x 10(8) to 0.1 x 10(8) cells per ml of rumen fluid in CF reindeer (n = 2) and CS4 reindeer (n = 2), respectively. The median bacterial population adhering to the rumen solids ranged from 260 x 10(8) to 450 x 10(8), 21 x 10(8) to 38 x 10(8), and 0.5 x 10(8) cells per g (wet weight) of rumen solids in FL, CF, and CS4 reindeer, respectively. Although there were variations in the rumen bacterial composition among the FL reindeer (n = 4), strains of Bacteroides, Fibrobacter, Streptococcus, and Clostridium dominated in the rumen fluid. Streptococcus spp. and Clostridium spp. were the dominant bacteria in the CF reindeer (n = 2), while in the CS4 reindeer (n = 2) the dominant bacteria were Fusobacterium spp., members of the family Enterobacteriaceae, and Eubacterium spp. Transmission electron micrographs of lichen particles from the rumen of one FL reindeer, one CF reindeer, and one CS4 reindeer show bacteria resembling Bacteroides spp. adhering to the lichen particles, evidently digesting the lichen hyphae from the inside.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of fructooligosaccharide utilization by Lactobacillus and Bacteroides species

The utilization of 1-kestose (GF(2)) and nystose (GF(3)), the main components of fructooligosaccharides (FOS), by Lactobacillus and Bacteroides species was examined. Of seven Lactobacillus and five Bacteroides strains that utilized FOS, L. salivarius, L. rhamnosus, L. casei, and L. gasseri utilized only GF(2), whereas L. acidophilus and all the Bacteroides strains utilized both GF(2) and GF(3). Only the strains able to utilize both GF(2) and GF(3) had β-fructosidase activity in the culture supernatants. The culture supernatants of the Lactobacillus strains had higher β-fructosidase activity for GF(2) than for GF(3), whereas those of the Bacteroides strains had higher activity for GF(3) than for GF(2). Furthermore, β-fructosidase activity of the culture supernatants of the Lactobacillus cells grown in the GF(3) medium was much higher than that of the cells grown in the GF(2) medium, whereas the activity of the culture supernatants of the Bacteroides cells grown in the GF(3) medium was almost the same as that of the cells grown in the GF(2) medium. These results indicate that Lactobacillus species metabolize FOS in a different way from that of Bacteroides species.     

Microorganisms Responsible for Controlling the Populations of Escherichia coli and Enterococcus and the Consistency of Cecal Contents in the Chicken

A study was made to clarify what kinds of intestinal organisms might be responsible for controlling the populations of Escherichia coli and Streptococcus faecalis var. liquefaciens in the cecum and the consistency of the cecal contents of chickens. Germ-free chickens were inoculated orally with various mixtures of bacterial cultures alone or in combination, different dilutions of the cecal contents of chickens, different dilutions of the cecal contents treated by heating or with chloroform, the supernatant of diluted cecal contents, and dilutions of human feces. Factors controlling the E. coli population, enterococcal population, and consistency of the cecal contents were shown to be independent of one another. The ecosystem controlling the E. coli or enterococcal population was more complex than that controlling the consistency of the cecal contents. The former was composed of anaerobic and facultatively anaerobic bacteria isolated and heat- or chloroform-resistant organisms, and the latter of heat- or chloroform-resistant organisms alone, which were inferred not to be prevailing in the cecal contents of chickens. Discussion is made on ecological systems controlling the intestinal flora.