Antagonism among the normal anaerobic bacteria of the mouse gastrointestinal tract determined by immunofluorescence.

Strictly anaerobic Bacteroides sp., Eubacterium sp., and Fusobacterium sp. were isolated from the cecum of a conventional mouse. An immunofluorescent method utilizing rabbit antisera specific for each of these three strains was developed to determine their population levels in the gastrointestinal tracts of gnotobiotic mice. Population levels of these anaerobes in groups of gnotobiotic mice colonized with either Bacteroides, Eubacterium, or Fusobacterium were compared with those of gnotobiotes colonized with all three strains. Bacteroides population levels in gnotobiotes colonized with all three strains were 100-fold less than the Bacteroides population level in gnotobiotes colonized with only the Bacteroides strain. Eubacterium or Fusobacterium population levels were not reduced by the presence of the other anaerobic strains. Thus, strictly anaerobic Eubacterium sp. and Fusobacterium sp. that colonized gnotobiotic mice caused a reduction in the in vivo population levels of a strictly anaerobic Bacteroides sp.     

Antagonistic effect exerted by three strictly anaerobic strains against various strains of Clostridium perfringens in gnotobiotic rodent intestines

Our purpose was to study bacterial antagonism between a limited number of strictly anaerobic strains and Clostridium perfringens in the intestinal tract of gnotobiotic rodents. Gnotobiotic mice harboring a Bacteroides thetaiotaomicron, a Fusobacterium necrogenes, and a Clostridium sp. strain were protected against pathogenic B, C, and D C. perfringens serotypes. A drastic antagonistic effect of this three-strain association was also observed against a nonpathogenic C. perfringens serotype A (CpA). It was less efficient in gnotobiotic rats than in mice and less efficient in gnotobiotic mice fed an autoclaved diet than in mice fed the same diet sterilized by irradiation. No diffusible inhibitory substances against CpA were detected in feces of gnotobiotic mice harboring the three antagonistic strains, and no nutrient depletion was demonstrated in filtrates prepared from 10-fold diluted feces of these mice. In vitro mixed cultures of the three antagonistic strains failed to inhibit growth of CpA, whereas CpA did not multiply in a 10-fold diluted feces from gnotobiotic mice. A reverse correlation between the initial number of antagonistic strains and the division number of CpA was determined using serially diluted fecal suspensions. Thus, large numbers of viable cells of both antagonistic strains were required to inhibit the target strain in fecal suspensions as was also found in gnotobiotic mice intestines. However, no diffusible inhibitory substance was detectable nor could depletion of growth factors be identified as causing antagonism. Whatever factors that may be responsible for antagonism were found to be influenced by the host and its diet.     

Influence of certain indigenous gastrointestinal microorganisms on duodenal alkaline phosphatase in mice.

Alkaline phosphatase activity was assayed in duodenal homogenates or extracts from adult specific pathogen-free (SPF) and germfree mice and gnotobiotic mice monoassociated with a Lactobacillus sp., a Bacteroides sp., or a coliform strain indigenous to SPF mice. Activity levels of the enzyme were much higher in the preparations from germfree mice than in those from the SPF controls. In the gnotobiotes monoassociated either with a freshly isolated Lactobacillus sp. or a Bacteroides sp., the levels of alkaline phosphatase activity were intermediate between the values for germfree and SPF mice. By contrast, in the gnotobiotes monoassociated with a coliform strain, alkaline phosphatase activity remained at high germfree levels. Butanol extracts of duodenal tissue from SPF mice, germfree mice, and exgermfree mice associated with an indigenous microflora from SPF mice (conventionalized) were subjected to acrylamide gel electrophoresis. A stain for alkaline phosphatase activity revealed three major bands in the gels prepared with extracts from SPF and conventionalized mice, but only two in the gels prepared with extracts from germfree mice. All three bands may have been present in the latter gels. One of the bands (the middle one) may have been obscured, however, by high activity in the slowest moving band. As determined by densitometric scanning, the slowest moving band had much higher activity in the preparations from germfree animals than in those from SPF or conventionalized mice. These findings suggest that the indigenous microbial flora affects not only quantitatively, but also qualitatively, the activity of alkaline phosphatases in the mouse intestinal mucosa.     

Spatial distribution and stability of the eight microbial species of the altered schaedler flora in the mouse gastrointestinal tract

The overall complexity of the microbial communities in the gastrointestinal (GI) tracts of mammals has hindered observations of dynamics and interactions of individual bacterial populations. However, such information is crucial for understanding the diverse disease-causing and protective roles that gut microbiota play in their hosts. Here, we determine the spatial distribution, interanimal variation, and persistence of bacteria in the most complex defined-flora (gnotobiotic) model system to date, viz., mice colonized with the eight strains of the altered Schaedler flora (ASF). Quantitative PCR protocols based on the 16S rRNA sequence of each ASF strain were developed and optimized to specifically detect as few as 10 copies of each target. Total numbers of the ASF strains were determined in the different regions of the GI tracts of three C.B-17 SCID mice. Individual strain abundance was dependent on oxygen sensitivity, with microaerotolerant Lactobacillus murinus ASF361 present at 10(5) to 10(7) cells/g of tissue in the upper GI tract and obligate anaerobic ASF strains being predominant in the cecal and colonic flora at 10(8) to 10(10) cells/g of tissue. The variation between the three mice was small for most ASF strains, except for Clostridium sp. strain ASF502 and Bacteroides sp. strain ASF519 in the cecum. A comparison of the relative distribution of the ASF strains in feces and the colon indicated large differences, suggesting that fecal bacterial levels may provide a poor approximation of colonic bacterial levels. All ASF strains were detected by PCR in the feces of C57BL/6 restricted flora mice, which had been maintained in an isolator without sterile food, water, or bedding for several generations, providing evidence for the stability of these strains in the face of potential competition by bacteria introduced into the gut.     

The Occurence and Properties of Uric Acid Decomposing Anaerobic Bacteria in the Avian Caecum

S ummary . Anaerobic bacteria which decompose uric acid have been found in the caeca of chickens, turkeys, ducks, pheasants and guinea-fowl at levels between 5.4 × 10⁸ and 1.8 × 10¹⁰/g of caecal material (wet weight). A study has been made of the properties and identity of 35 strains which were present in high numbers in the chicken caecum. The isolates included strains of Bacteroides clostridiiformis var. clostridiiformis, Fusobacterium plauti, Clostridium malenominatum, Peptostreptococcus productus and a number of types which could not be identified with known species. Of these 3 were Bacteroides spp., 3 Eubacterium spp., 2 Peptostreptococcus spp. and 1 was an anaerobic Streptococcus . None of the strains had an absolute requirement for uric acid.     

Azoreductase activity of anaerobic bacteria isolated from human intestinal microflora.

A plate assay was developed for the detection of anaerobic bacteria that produce azoreductases. With this plate assay, 10 strains of anaerobic bacteria capable of reducing azo dyes were isolated from human feces and identified as Eubacterium hadrum (2 strains), Eubacterium spp. (2 species), Clostridium clostridiiforme, a Butyrivibrio sp., a Bacteroides sp., Clostridium paraputrificum, Clostridium nexile, and a Clostridium sp. The average rate of reduction of Direct Blue 15 dye (a dimethoxybenzidine-based dye) in these strains ranged from 16 to 135 nmol of dye per min per mg of protein. The enzymes were inactivated by oxygen. In seven isolates, a flavin compound (riboflavin, flavin adenine dinucleotide, or flavin mononucleotide) was required for azoreductase activity. In the other three isolates and in Clostridium perfringens, no added flavin was required for activity. Nondenaturing polyacrylamide gel electrophoresis showed that each bacterium expressed only one azoreductase isozyme. At least three types of azoreductase enzyme were produced by the different isolates. All of the azoreductases were produced constitutively and released extracellularly.     

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.     

Azoreductase activity of anaerobic bacteria isolated from human intestinal microflora

A plate assay was developed for the detection of anaerobic bacteria that produce azoreductases. With this plate assay, 10 strains of anaerobic bacteria capable of reducing azo dyes were isolated from human feces and identified as Eubacterium hadrum (2 strains), Eubacterium spp. (2 species), Clostridium clostridiiforme, a Butyrivibrio sp., a Bacteroides sp., Clostridium paraputrificum, Clostridium nexile, and a Clostridium sp. The average rate of reduction of Direct Blue 15 dye (a dimethoxybenzidine-based dye) in these strains ranged from 16 to 135 nmol of dye per min per mg of protein. The enzymes were inactivated by oxygen. In seven isolates, a flavin compound (riboflavin, flavin adenine dinucleotide, or flavin mononucleotide) was required for azoreductase activity. In the other three isolates and in Clostridium perfringens, no added flavin was required for activity. Nondenaturing polyacrylamide gel electrophoresis showed that each bacterium expressed only one azoreductase isozyme. At least three types of azoreductase enzyme were produced by the different isolates. All of the azoreductases were produced constitutively and released extracellularly.     

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.     

Cooperative formation of omega-muricholic acid by intestinal microorganisms.

Three anaerobic bacteria, isolated from the ceca of rats and mice, converted, through a concerted mechanism, beta-muricholic acid, the predominant bile acid in germfree rats, into omega-muricholic acid. One isolate was a Eubacterium lentum strain; the second and third isolates were tentatively identified as atypical Fusobacterium sp. strains. The conversion of beta-muricholic acid into omega-muricholic acid proceeded in two steps: E. lentum oxidized the 6 beta-hydroxyl group of beta-muricholic acid to a 6-oxo group, which was reduced by either of the two other species to a 6 alpha-hydroxyl group, yielding omega-muricholic acid. This transformation occurred both in vitro and in gnotobiotic rats. Monoassociation of germfree rats with the E. lentum strain gave rise to an unidentified fecal bile acid, probably a derivative of beta-muricholic acid having a double bond in the side chain.     

Anaerobic bacteria from the large intestine of mice.

Anaerobic bacteria from the colon of laboratory mice were enumerated and isolated using strict anaerobic techniques. Direct microscopic counts revealed 4.4 X 10(10) organisms in each gram (wet weight) of colon contents. Actual cultural counts averaged 3.2 X 10(10) organisms, which was 73% of the direct microscopic count. The tentatively identified genera were Bacteroides, Eubacterium, Fusobacterium, Lactobacillus, Peptostreptococcus, and Propionibacterium. Strains of Fusobacterium, Lactobacillus, Peptostreptococcus, and Propionibacterium were biochemically homogeneous. Strains of Bacteroides and Eubacterium, on the other hand, were biochemically heterogeneous and were subdivided into several distinct groups. The data indicate that many of the isolates are different from previously described species of the respective genera and may belong to new species.     

小鼠盲肠内容物中正常菌群的分离和定量

本文报道了采用厌氧培养技术,从小鼠盲肠内容物中分离到6株严格厌氧和兼性厌氧菌。鉴定为拟杆菌、乳酸杆菌、双歧杆菌、难辨梭菌、粪链球菌和大肠扦菌。它们在小鼠盲肠内容物的正常菌群中占优势,含菌量与国外文献中报告的相似。     

Colonization resistance against potentially pathogenic bacteria in hexaflora-associated gnotobiotic mice.

A study of colonization resistance against potentially pathogenic bacteria (Escherichia coli and Pseudomonas aeruginosa) was conducted in hexaflora-associated gnotobiotic mice. Groups of germfree AKR mice were swabbed with five bacterial and a single gastrointestinal yeast species: Streptococcus faecalis. Lactobacillus brevis. Staphylococcus epidermidis, Enterobacter aerogenes, Bacteroides fragilis var. vulgatus, and Torulopsis sp. All species became established in the gut in 8 weeks. Later these associated mice were divided and challenged by four graded doses of E. coli or P. aeruginosa. The presence of challenge organism was monitored specifically in the freshly voided fecal specimens of the challenged mice. Escherichia coli colonized the gut of each mouse at each level up to 60 days post challenge. Pseudomonas aeruginosa was completely eliminated from each mouse at each dose level after 30 days post challenge. Evidence suggests that all six species were sufficient to prevent the colonization of P. aeruginosa and not of E. coli in the gut of the gnotobiotic mice.     

Antibiotic susceptibility of clinically relevant anaerobes in Estonia from 1999 to 2001

At present little or no data is available regarding the resistance profiles of anaerobic bacteria in relation to the general usage of antibiotics. The objective of this study was to assess whether any potential relationship exists between the dynamics of antibiotic resistance of anaerobic bacteria and the consumption of antibiotics during the last 3 years within the Estonian population. In total, 416 anaerobic isolates were investigated from various clinical samples. The anaerobes were isolated on Wilkins-Chalgren Agar, incubated in an anaerobic glove box and identified by standard methods. beta-lactamase negative strains were tested against metronidazole, clindamycin, benzylpenicillin and the positive strains were further tested against metronidazole, clindamycin, and ampicillin/sulbactam by E-tests. The results of the susceptibility tests were interpreted according to the current criteria of NCCLS. Data from the Estonian State Agency of Medicines was used to assess the antibiotic consumption rate in the population (Defined Daily Doses per 1000 inhabitants annually). The following species of anaerobes were isolated: B. fragilis group, Bacteroides sp., Fusobacterium sp., Porphyromonas sp., Prevotella sp., Peptostreptococcus sp., in addition to various unidentified Gram-positive rods. Metronidazole resistance was not found among Gram-negative bacteria despite a relatively high consumption of this antimicrobial agent in Estonia. Only ampicillin/sulbactam demonstrated excellent in vitro activity against all anaerobes. Unexpectedly despite a relatively low rate of consumption of clindamycin a high rate of resistance to this agent occurred; a similar situation was noted for penicillin. In the present study we did not observe a relationship between the changes in antibiotic consumption (DDD/1000) rate and the resistance pattern of anaerobic bacteria to metronidazole, clindamycin, penicillin and ampicillin/sulbactam during a 3-year follow-up period. High resistance to penicillin among some species and also to clindamycin is similar to the global trend and argues for limited use of these antibiotics in empirical treatment. We would suggest that monitoring of local susceptibility pattern is necessary for the selection of initial empirical therapy.     

Urease assay and urease-producing species of anaerobes in the bovine rumen and human feces.

A growth medium and test were developed for rapid detection of urease in fermentative anaerobic bacteria. Using nonselective rumen fluid roll-tube agar medium and the new test, it was confirmed that Peptostreptococcus productus is often the most numerous urease-forming species in human feces. Also, some fecal strains of Ruminococcus albus, Clostridium innocuum, and Clostridium beijerinckii produced urease. Single strains of Fusobacterium prausnitzii, Coprococcus catus, and Streptococcus mitis that were strongly ureolytic on isolation later lost this ability. Urease activity was also detected in many strains of nonselectively isolated rumen species. They include Succinivibrio dextrinosolvens, Treponema sp., Ruminococcus bromii (not previously known to be present in the rumen), Butyrivibrio sp., Bifidobacterium sp., Bacteroides ruminicola, and P. productus. Most P. productus strains contain urease; however, the uniformity of this feature in the other species noted above is not known. The urease in many of these species was not detected if the growth medium contained 0.2% or more (each) yeast extract and Trypticase.     

Distribution and effects of a defined six-member murine-derived microflora in gnotobiotic gerbils.

The gnotobiotic gerbil was selected as a model with which to study the effects of colonization with a defined microflora on organ morphology, histology, and selected blood biochemical parameters. Gerbils were maintained germfree for 13 months but failed to reproduce, presumably because of the enlarged cecum. A colony of gnotobiotic gerbils that was associated with a bacterial flora consisting of Lactobacillus brevis, Streptococcus faecalis, Staphylococcus epidermidis, Bacteroides vulgatus, Enterobacter aerogenes, and a Fusobacterium sp. was established. These gnotobiotic gerbils had smaller ceca than germfree gerbils and proved capable of reproduction. Except for the presence of large numbers of Bacteroides organisms in the stomach and greater numbers of S. epidermidis in gnotobiotic gerbils, the number and location of gastrointestinal bacteria were similar in conventional and gnotobiotic gerbils. Bacteroides sp. was the second most predominant microorganism present in gnotobiotic gerbils, whereas clostridia were reported to be the second most predominant microorganism in conventional gerbils. Microscopic examination of direct-impression smears indicated that fusobacteria were present on mucosal surfaces. Intestines of gnotobiotic gerbils weighed twice as much as the intestines of conventional gerbils. Intestinal tissue water weight values from conventional and gnotobiotic gerbils were similar. Histological examination of gerbil intestinal tissue revealed no cellular hypertrophy and no evidence of inflammation in gnotobiotic gerbil intestines. Spleens of gnotobiotic gerbils showed no germinal center stimulation. Statistical differences in total serum glucose, serum protein, and hematocrit levels were found between conventional and gnotobiotic gerbils.     

Spatial Distribution and Stability of the Eight Microbial Species of the Altered Schaedler Flora in the Mouse Gastrointestinal Tract

The overall complexity of the microbial communities in the gastrointestinal (GI) tracts of mammals has hindered observations of dynamics and interactions of individual bacterial populations. However, such information is crucial for understanding the diverse disease-causing and protective roles that gut microbiota play in their hosts. Here, we determine the spatial distribution, interanimal variation, and persistence of bacteria in the most complex defined-flora (gnotobiotic) model system to date, viz., mice colonized with the eight strains of the altered Schaedler flora (ASF). Quantitative PCR protocols based on the 16S rRNA sequence of each ASF strain were developed and optimized to specifically detect as few as 10 copies of each target. Total numbers of the ASF strains were determined in the different regions of the GI tracts of three C.B-17 SCID mice. Individual strain abundance was dependent on oxygen sensitivity, with microaerotolerant Lactobacillus murinus ASF361 present at 10⁵ to 10⁷ cells/g of tissue in the upper GI tract and obligate anaerobic ASF strains being predominant in the cecal and colonic flora at 10⁸ to 10¹⁰ cells/g of tissue. The variation between the three mice was small for most ASF strains, except for Clostridium sp. strain ASF502 and Bacteroides sp. strain ASF519 in the cecum. A comparison of the relative distribution of the ASF strains in feces and the colon indicated large differences, suggesting that fecal bacterial levels may provide a poor approximation of colonic bacterial levels. All ASF strains were detected by PCR in the feces of C57BL/6 restricted flora mice, which had been maintained in an isolator without sterile food, water, or bedding for several generations, providing evidence for the stability of these strains in the face of potential competition by bacteria introduced into the gut.     

Urease-producing species of intestinal anaerobes and their activities.

Urease activities of anaerobic bacteria that constituted predominant gut flora were examined. It was demonstrated that some strains of Eubacterium aerofaciens, E. lentum, and Peptostreptococcus products produced urease. They were the most numerous species in human feces. All strains of Bifidobacterium infantis and some strains of Bacteroides multiacidus, B. bifidum, Clostridium symbiosum, Fusobacterium necrophorum, F. varium, Lactobacillus fermentum, Peptococcus asaccharolyticus, and P. prevotii produced urease. The optimum pH of the Lactobacillus urease was found to be 4.0, whereas the pH value of B. multiacidus urease was 8.0.     

Urease-producing species of intestinal anaerobes and their activities.

Urease activities of anaerobic bacteria that constituted predominant gut flora were examined. It was demonstrated that some strains of Eubacterium aerofaciens, E. lentum, and Peptostreptococcus products produced urease. They were the most numerous species in human feces. All strains of Bifidobacterium infantis and some strains of Bacteroides multiacidus, B. bifidum, Clostridium symbiosum, Fusobacterium necrophorum, F. varium, Lactobacillus fermentum, Peptococcus asaccharolyticus, and P. prevotii produced urease. The optimum pH of the Lactobacillus urease was found to be 4.0, whereas the pH value of B. multiacidus urease was 8.0.     

Effects of alternative dietary substrates on competition between human colonic bacteria in an anaerobic fermentor system

Duplicate anaerobic fermentor systems were used to examine changes in a community of human fecal bacteria supplied with different carbohydrate energy sources. A panel of group-specific fluorescent in situ hybridization probes targeting 16S rRNA sequences revealed that the fermentors supported growth of a greater proportion of Bacteroides and a lower proportion of gram-positive anaerobes related to Faecalibacterium prausnitzii, Ruminococcus flavefaciens-Ruminococcus bromii, Eubacterium rectale-Clostridium coccoides, and Eubacterium cylindroides than the proportions in the starting fecal inoculum. Nevertheless, certain substrates, such as dahlia inulin, caused a pronounced increase in the number of bacteria related to R. flavefaciens-R. bromii and E. cylindroides. The ability of three strictly anaerobic, gram-positive bacteria to compete with the complete human fecal flora was tested in the same experiment by using selective plating to enumerate the introduced strains. The Roseburia-related strain A2-183(F) was able to grow on all substrates despite the fact that it was unable to utilize complex carbohydrates in pure culture, and it was assumed that this organism survived by cross-feeding. In contrast, Roseburia intestinalis L1-82(R) and Eubacterium sp. strain A2-194(R) survived less well despite the fact that they were able to utilize polysaccharides in pure culture, except that A2-194(R) was stimulated 100-fold by inulin. These results suggest that many low-G+C-content gram-positive obligate anaerobes may be selected against during in vitro incubation, although several groups were stimulated by inulin. Thus, considerable caution is necessary when workers attempt to predict the in vivo effects of probiotics and prebiotics from their effects in vitro.