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Isolation of Methanobrevibacter smithii from human feces.

Fecal specimens from nine adults were examined for the presence of methanogenic bacteria. Enrichment cultures of five specimens produced methane in 5 days. Of these five specimens, three were tested and produced methane during a short-term incubation. Four specimens did not produce methane in either short-term incubation or in enrichment culture. Each methanogenic culture contained methanogens similar in morphology to organisms of the genus Methanobrevibacter and showed factor-420 fluorescence by fluorescence microscopy. Pure cultures were obtained from four of the five methanogenic enrichment cultures. Each isolate grew and formed methane from either H2-CO2 or formate, but growth obtained with formate was poor. None of the isolates used acetate, methanol, or trimethylamine. All isolates grew in the presence of bile salts. In immunological studies, each isolate was closely related to the type strain of Methanobrevibacter smithii, a finding consistent with the physiological and morphological similarities between the isolates and the type strain.

Adult↗

Rutin-induced beta-glucosidase activity in Streptococcus faecium VGH-1 and Streptococcus sp. strain FRP-17 isolated from human feces: formation of the mutagen, quercetin, from rutin.

A fecal isolate, Streptococcus sp. strain FRP-17, and strain VGH-1 of Streptococcus faecium were shown to contain beta-glucosidases which converted rutin (quercetin-3-O-beta-D-glucose-alpha-L-rhamnose) to quercetin and were active against o-nitrophenyl-beta-D-glucose. The activity against rutin could be measured by increased mutagenicity in the Ames assay or visualized on thin-layer chromatography plates. In both organisms, the beta-glucosidase activities were inducible by the addition of rutin to the growth media. Several closely related strains of Streptococcus spp. lacked any beta-glucosidase activity. In cell preparations of the active organisms, activities with rutin and o-nitrophenyl-beta-D-glucose were optimal at pH 6.8 and could be enhanced by increasing the ionic strength of the assay system. At low ionic strengths, both quercetin and a new product (intermediate between the polarities of rutin and quercetin) were formed by the incubation of rutin with cell preparations of either active organism. This product disappeared with increased ionic strength, suggesting that it may be a reaction intermediate, quercetin-3-O-beta-D-glucose. These results suggest that the beta-glucosidase active against rutin and that active against o-nitrophenyl-beta-D-glucose are the same.

Bacterial Proteins↗

Bacterial strains from human feces that reduce CO2 to acetic acid.

We used dilutions of fecal suspensions from a human volunteer to enrich cultures for bacteria that reduce CO2 to acetate in the colon. The soluble enrichment substrates used were glucose, methanol, formate, and vanillate, which were used with a gas phase that contained 80% N2 and 20% CO2. The gaseous enrichment substrates used were 80% H2-20% CO2 and 50% CO-50% CO2. We isolated three different strains that produced acetate from CO2. One strain produced acetate from methanol, vanillate, H2-CO2, glucose, and other sugars. The other two strains did not form acetate from methanol or vanillate. Both of the latter strains formed acetate from glucose and other sugars, but only one of these strains formed acetate from H2-CO2. Both of these strains cometabolized formate. However, none of the enrichment cultures or pure cultures used CO or formate as a substrate for growth. The two strains that produced acetate from H2 and CO2 grew slowly when the gases alone were used as substrates, but they rapidly cometabolized H2 and CO2 when they were grown with organic substrates. The ability of all of the strains to produce acetate from CO2 and/or other one-carbon precursors was verified by determining the radioactivity of the methyl and carboxyl groups of the acetate formed after growth with 14CO2 or other radioactively labeled one-carbon precursors.

Acetates↗

Amounts of viable anaerobes, methanogens, and bacterial fermentation products in feces of rats fed high-fiber or fiber-free diets.

We investigated the impact of dietary fiber on the fecal output of microorganisms and microbial fermentation products of rats. Two groups of five male Wistar rats were fed high-fiber (HF) and fiber-free (FF) diets in the following order: (group 1) lab chow-->HF-->FF-->HF and (group 2) lab chow-->FF-->HF-->FF. Daily fecal output of total viable anaerobes was 71 times higher with the HF diet. Daily output of methanogens was 1.4 times higher for the HF diet than for the FF diet. Daily excretion of total fermentation acid products (acetate, propionate, butyrate, lactate, succinate, and formate) was 2.4 and 0.1 mmol for HF and FF diets, respectively. The ratios of acetate/propionate/butyrate were 69:21:10 for the HF diet and 92:7:1 for the FF diet. The results show that an HF diet significantly increases microbial growth in the colon and influences the proportions of organic acid products. The HF diet did not increase the ratio of methanogens to total anaerobes. We suggest that the contribution of host-derived substrates to colonic microbial growth and fermentation is insignificant.

Animals↗

PCR amplification and species determination of microsporidia in formalin-fixed feces after immunomagnetic separation.

The term microsporidia is used to describe several species of opportunistic protozoan parasites. Encephalitozoon intestinalis and Enterocytozoon bieneusi have been found in stools of more than 40% of AIDS patients with diarrhea. Diagnosis of infection with these small protozoans has been difficult, and until recently their occurrence has not been well documented. Formalin is widely used to preserve clinical specimens, but due to the nature of the fixation process, subsequent analysis, especially analysis by the PCR, is difficult. This study evaluated methods used to prepare formalin-fixed fecal specimens for PCR amplification of microsporidial DNA. Two methods were devised to allow PCR detection and subsequent identification of microsporidia in formalin-fixed fecal specimens to the species level. One method involved immunomagnetic separation to concentrate microsporidial spores from fecal specimens. In the second method Chelex resin (Bio-Rad, Hercules, Calif.) was used to remove inhibitory substances, followed by a DNA concentration step. Both methods resulted in reproducible, confirmed detection of microsporidia in formalinized fecal specimens and subsequent species determination by PCR sequencing. The detection sensitivity was two in vitro culture-derived spores (Encephalitozoon intestinalis) for the direct PCR. The reproducible detection sensitivity for DNA amplification from formalin-fixed fecal samples was 200 spores for either the Chelex method or the immunomagnetic bead separation method. Thus, we developed two methods for rapid, inexpensive detection of microsporidial spores in formalin-fixed fecal specimens.

AIDS-Related Opportunistic Infections↗

Occurrence of male-specific bacteriophage in feral and domestic animal wastes, human feces, and human-associated wastewaters.

Male-specific bacteriophage (MSB) densities were determined in animal and human fecal wastes to assess their potential impact on aquatic environments. Fecal samples (1,031) from cattle, chickens, dairy cows, dogs, ducks, geese, goats, hogs, horses, seagulls, sheep, and humans as well as 64 sewerage samples were examined for MSB. All animal species were found to harbor MSB, although the great majority excreted these viruses at very low levels. The results from this study demonstrate that in areas affected by both human and animal wastes, wastewater treatment plants are the principal contributors of MSB to fresh, estuarine, and marine waters.

Animals↗

Comparison of sensitivity of immunofluorescent microscopy to that of a combination of immunofluorescent microscopy and immunomagnetic separation for detection of Cryptosporidium parvum oocysts in adult bovine feces.

A direct immunofluorescence assay (DFA) (Merifluor; Meridian Diagnostics, Inc., Cincinnati, Ohio) was compared to an immunomagnetic separation (IMS) assay (Dynabeads; Dynal, Inc., Lake Success, N.Y.) coupled with immunofluorescent microscopy (Waterborne, Inc., New Orleans, La.) for their ability to detect low concentrations of Cryptosporidium parvum oocysts in adult bovine fecal material. IMS-DFA resulted in a 2-log-unit increase in sensitivity (10 oocysts/g) compared to DFA alone (1,000 oocysts/g). The higher sensitivity obtained with IMS-DFA resulted from testing 2 g of fecal material instead of the 13 to 19 mg of fecal material tested in the DFA; the increased sensitivity was not attributable to a higher percent recovery.

Animals↗

Identification and characterization of a bile acid 7alpha-dehydroxylation operon in Clostridium sp. strain TO-931, a highly active 7alpha-dehydroxylating strain isolated from human feces.

Clostridium sp. strain TO-931 can rapidly convert the primary bile acid cholic acid to a potentially toxic compound, deoxycholic acid. Mixed oligonucleotide probes were used to isolate a gene fragment encoding a putative bile acid transporter from Clostridium sp. strain TO-931. This DNA fragment had 60% nucleotide sequence identity to a known bile acid transporter gene from Eubacterium sp. strain VPI 12708, another bile acid-7alpha-dehydroxylating intestinal bacterium. The DNA (9.15 kb) surrounding the transporter gene was cloned from Clostridium sp. strain TO-931 and sequenced. Within this larger DNA fragment was a 7.9-kb region, containing six successive open reading frames (ORFs), that was encoded by a single 8.1-kb transcript, as determined by Northern blot analysis. The gene arrangement and DNA sequence of the Clostridium sp. strain TO-931 operon are similar to those of a Eubacterium sp. strain VPI 12708 bile acid-inducible operon containing nine ORFs. Several genes in the Eubacterium sp. strain VPI 12708 operon have been shown to encode products required for bile acid 7alpha-dehydroxylation. In Clostridium sp. strain TO-931, genes potentially encoding bile acid-coenzyme A (CoA) ligase, 3alpha-hydroxysteroid dehydrogenase, bile acid 7alpha-dehydratase, bile acid-CoA hydrolase, and a bile acid transporter were similar in size and exhibited amino acid homology to similar gene products from Eubacterium sp. strain VPI 12708 (encoded by baiB, baiA, baiE, baiF, and baiG, respectively). However, no genes similar to Eubacterium sp. strain VPI 12708 biaH or baiI were found in the Clostridium sp. strain TO-931 bai operon, and the two putative Eubacterium sp. strain VPI 12708 genes, baiC and baiD, were arranged in one continuous ORF in Clostridium sp. strain TO-931. Intergene regions showed no significant DNA sequence similarity, but primer extension analysis identified a region 115 bp upstream from the first ORF that exhibited 58% identity to a bai operator/promoter region identified in Eubacterium sp. strain VPI 12708. These results indicate that the gene organization, gene product amino acid sequences, and promoters of the bile acid-inducible operons of Clostridium sp. strain TO-931 and Eubacterium sp. strain VPI 12708 are highly conserved.

3-Hydroxysteroid Dehydrogenases↗

Ruminococcin A, a new lantibiotic produced by a Ruminococcus gnavus strain isolated from human feces.

When cultivated in the presence of trypsin, the Ruminococcus gnavus E1 strain, isolated from a human fecal sample, was able to produce an antibacterial substance that accumulated in the supernatant. This substance, called ruminococcin A, was purified to homogeneity by reverse-phase chromatography. It was shown to be a 2,675-Da bacteriocin harboring a lanthionine structure. The utilization of Edman degradation and tandem mass spectrometry techniques, followed by DNA sequencing of part of the structural gene, allowed the identification of 21 amino acid residues. Similarity to other bacteriocins present in sequence libraries strongly suggested that ruminococcin A belonged to class IIA of the lantibiotics. The purified ruminococcin A was active against various pathogenic clostridia and bacteria phylogenetically related to R. gnavus. This is the first report on the characterization of a bacteriocin produced by a strictly anaerobic bacterium from human fecal microbiota.

Amino Acid Sequence↗

Application of new primer-enzyme combinations to terminal restriction fragment length polymorphism profiling of bacterial populations in human feces.

New primer-enzyme combinations for terminal restriction fragment length polymorphism (T-RFLP) targeting of the 16S rRNA gene were constructed by using the T-RFLP analysis program (designated TAP T-RFLP) located at the Ribosomal Database Project website, and their performance was examined empirically. By using the fluorescently labeled 516f primer (Escherichia coli positions 516 to 532) and 1510r primer (positions 1510 to 1492), the 16S rRNA gene was amplified from human fecal DNA. The resulting amplified product was digested with RsaI plus BfaI or with BslI. When the T-RFLP was carried out with fecal DNAs from eight individuals, eight predominant operational taxonomic units (OTUs) were detected with RsaI and BfaI digestion and 14 predominant OTUs were detected with BslI digestion. The distribution of the OTUs was consistent with the results of the computer simulations with TAP T-RFLP. The T-RFLP analyses of the fecal DNAs from individuals gave characteristic profiles, while the variability of the T-RFLP profiles between duplicate DNA preparations from the same samples were minimal. This new T-RFLP method made it easy to predict what kind of intestinal bacterial group corresponded to each OTU on the basis of the terminal restriction fragment length compared with the conventional T-RFLP and, moreover, made it possible to identify the bacterial species that an OTU represents by cloning and sequencing.

Bacteria↗

Selective plating underestimates abundance and shows differential recovery of bifidobacterial species from human feces.

The aim of the present work was to compare the efficacies and levels of selectivity of different culture-dependent and -independent methods for analyzing bifidobacteria in human stool samples. The three different culture media used here significantly differed from each other, particularly with regard to the recovery of Bifidobacterium adolescentis. Bifidobacterium medium failed to recover B. adolescentis; Beerens medium recovered some B. adolescentis organisms (17% of total bifidobacteria), whereas tomato-Eugon medium recovered mainly B. adolescentis organisms (58% of total bifidobacteria). A culture-independent method that combines GC fractionation of bacterial community DNA and 16S rRNA sequencing indicated that B. adolescentis organisms accounted for 85% of all bifidobacteria. Methodological biases, such as those described in this paper, should be taken into account in interpreting earlier studies and designing future experiments.

Bifidobacterium↗

Phylogenetic analysis of the hypervariable region of the 18S rRNA gene of Cryptosporidium oocysts in feces of Canada geese (Branta canadensis): evidence for five novel genotypes.

To assess genetic diversity in Cryptosporidium oocysts from Canada geese, 161 fecal samples from Canada geese in the United States were analyzed. Eleven (6.8%) were positive for Cryptosporidium spp. following nested PCR amplification of the hypervariable region of the 18S rRNA gene. Nine PCR products from geese were cloned and sequenced, and all nine diverged from previously reported Cryptosporidium 18S rRNA gene sequences. Five sequences were very similar or identical to each other but genetically distinct from that of Cryptosporidium baileyi; two were most closely related to, but genetically distinct from, the first five; and two were distinct from any other sequence analyzed. One additional sequence in the hypervariable region of the 18S rRNA gene isolated from a cormorant was identical to that of C. baileyi. Phylogenetic analysis provided evidence for new genotypes of Cryptosporidium species in Canada geese. Results of this study suggest that the taxonomy of Cryptosporidium species in geese is complex and that a more complete understanding of genetic diversity among these parasites will facilitate our understanding of oocyst sources and species in the environment.

Animals↗

Real-time PCR quantitation of clostridia in feces of autistic children.

Based on the hypothesis that intestinal clostridia play a role in late-onset autism, we have been characterizing clostridia from stools of autistic and control children. We applied the TaqMan real-time PCR procedure to detect and quantitate three Clostridium clusters and one Clostridium species, C. bolteae, in stool specimens. Group- and species-specific primers targeting the 16S rRNA genes were designed, and specificity of the primers was confirmed with DNA from related bacterial strains. In this procedure, a linear relationship exists between the threshold cycle (CT) fluorescence value and the number of bacterial cells (CFU). The assay showed high sensitivity: as few as 2 cells of members of cluster I, 6 cells of cluster XI, 4 cells of cluster XIVab, and 0.6 cell of C. bolteae could be detected per PCR. Analysis of the real-time PCR data indicated that the cell count differences between autistic and control children for C. bolteae and the following Clostridium groups were statistically significant: mean counts of C. bolteae and clusters I and XI in autistic children were 46-fold (P = 0.01), 9.0-fold (P = 0.014), and 3.5-fold (P = 0.004) greater than those in control children, respectively, but not for cluster XIVab (2.6 x 10(8) CFU/g in autistic children and 4.8 x 10(8) CFU/g in controls; respectively). More subjects need to be studied. The assay is a rapid and reliable method, and it should have great potential for quantitation of other bacteria in the intestinal tract.

Autistic Disorder↗

Detection of Salmonella strains and Escherichia coli O157:H7 in feces of small ruminants and their isolation with various media.

Salmonella strains and Escherichia coli O157:H7 were detected in 17 and 5 small ruminants in Virginia, respectively, of 287 tested. Background microflora interfered with the fecal analysis. The combination of Salmonella enzyme immunoassay (EIA) detection and xylose-lysine-deoxycholate agar isolation was satisfactory. Modifying enrichment to a 1:100 dilution enabled effective E. coli O157:H7 detection by EIA and isolation by sorbitol-MacConkey agar with cefixime-tellurite.

Animals↗

Movement and fixation of intestinal microbiota after administration of human feces to germfree mice.

Human flora-associated (HFA) mice have been considered a tool for studying the ecology and metabolism of intestinal bacteria in humans, although they have some limitations as a model. Shifts in dominant species of microbiota in HFA mice after the administration of human intestinal microbiota was revealed by 16S rRNA gene sequence and terminal restriction fragment length polymorphism (T-RFLP) analyses. Characteristic terminal restriction fragments (T-RFs) were quantified as the proportion of total peak area of all T-RFs. Only the proportion of the T-RF peak at bp 366, identified as the Gammmaproteobacteria group and the family Coriobacteriaceae, was reduced in this study. Increased T-RFs over time at bp 56, 184, and 196 were affiliated with the Clostridium group. However, most of the isolated bacteria with unique population shifts were phylotypes. The vertical transmission of the intestinal microbiota of the mouse offspring was also investigated by dendrogram analysis derived from the similarity of T-RFLP patterns among samples. As a result, the intestinal microbiota of HFA mice and their offspring reflected the composition of individual human intestinal bacteria with some modifications. Moreover, we revealed that human-derived lactobacilli (HDL), which have been considered difficult to colonize in the HFA mouse intestine in previous studies based on culture methods, could be detected in the HFA mouse intestine by using a lactic acid bacterium-specific primer and HDL-specific primers. Our results indicate that the intestinal microbiota of HFA mice represents a limited sample of bacteria from the human source and are selected by unknown interactions between the host and bacteria.

Animals↗

Specificity of a Bacteroides thetaiotaomicron marker for human feces.

A bacterial primer set, known to produce a 542-bp amplicon specific for Bacteroides thetaiotaomicron, generated this product in PCR with 1 ng of extracted DNA from 92% of 25 human fecal samples, 100% of 20 sewage samples, and 16% of 31 dog fecal samples. The marker was not detected in 1 ng of fecal DNA from 61 cows, 35 horses, 44 pigs, 24 chickens, 29 turkeys, and 17 geese.

Animals↗

Comparison of cultures from rectoanal-junction mucosal swabs and feces for detection of Escherichia coli O157 in dairy heifers.

Fecal culture for Escherichia coli O157:H7 was compared to rectoanal mucosal swab (RAMS) culture in dairy heifers over a 1-year period. RAMS enrichment culture was as sensitive as fecal culture using immunomagnetic separation (IMS) (P = 0.98, as determined by a chi-square test). RAMS culture is less costly than fecal IMS culture and can yield quantitative data.

Anal Canal↗