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A new technique is described for evaluating bacterial cell distribution in fecal samples. Spatial relationships of cells within an area rather than number of cells per unit volume or weight are measured by this technique. Measurements of cell distribution by this method indicated that bacteria occurred in freshly voided swine feces as pure, discrete colonies rather than as single cells distributed randomly or uniformly throughout the sample.
Medium 10 (M10), developed for rumen bacteria and containing small amounts of sugars, starch, volatile fatty acids, hemin, Trypticase, yeast extract, cysteine, and sulfide, plus agar, minerals and CO(2)-HCO(3)-buffer, was used with the Hungate anaerobic method as a basal medium to evaluate the efficacy of various ingredients. Three-day-old colony counts from adults on normal diets (17 samples) were 0.55 x 10(11) to 1.7 x 10(11) per g (mean, 1.15 x 10(11)) for M10. Single deletion of volatile fatty acids, Trypticase, yeast extract, or sulfide did not reduce counts. Deletion of hemin or both Trypticase and yeast extract significantly lowered counts. Addition of fecal extract, rumen fluid, 1% dehydrated Brain Heart Infusion (BHI) or 2 to 6% liver infusion did not increase counts; 1% dehydrated bile or 3.7% BHI markedly depressed them. Decreasing the gas-phase CO(2) concentration from 100 to 5% with N(2) and correspondingly lowering the HCO(3) had little effect. Counts in supplemented Brewer Thioglycollate (Difco), BHI, and Trypticase soy agar were similar or lower than in M10; ease in counting was best in M10. Comparison of features of 88 predominant strains of fecal bacteria randomly isolated indicated that M10 supported growth of as many or more species of bacteria as compared to supplemented BHI. The results suggest that predominant bacteria of human feces, in general, are not as nutritionally fastidious as rumen bacteria and indicate that media for counts or isolation containing large amounts of rich organic materials are neither necessary nor desirable when adequate anaerobic techniques are used.
The incidence of Salmonella and Edwardsiella tarda in gull feces on the Oregon coast was studied to determine the role these birds may have in the contamination of fish products with these organisms. Approximately 2.1 and 0.4% of 521 fecal specimens examined were found to contain Salmonella or E. tarda, respectively.
Enterocytozoon bieneusi is clinically the most significant among the microsporidia causing chronic diarrhea, wasting, and cholangitis in individuals with human immunodeficiency virus/AIDS. Microscopy with either calcofluor or modified trichrome stains is the standard diagnostic test for microsporidiosis and does not allow species identification. Detection of E. bieneusi infection based on PCR is limited to a few reference laboratories, and thus it is not the standard diagnostic assay. We have recently reported the development and characterization of a panel of monoclonal antibodies against E. bieneusi, and in this publication we evaluated the specificity and sensitivity of an immunofluorescence assay (IFA), compared with PCR, in simian immunodeficiency virus-infected macaques. The IFA, which correlated with the primary PCR method, with a detection limit of 1.5 x 10(5) spores per gram of feces, will simplify considerably the detection of E. bieneusi spores in clinical and environmental specimens and in laboratory and epidemiological investigations.
Hepatitis A antigen (HA Ag) was purified from feces collected during acute illness from patients with naturally occurring viral hepatitis, type A. Positive fecal specimens were identified by immune electron microscopy, but for detection of HA Agduring purification immune adherence hemagglutination (IAHA) and microtiter solid-phase radioimmunoassay were used. Isopycnic banding in cesium chloride, rate-zonal separation in sucrose, and preparative zonal electrophoresis were used in various combinations for successive purification, and the purified antigen was successfully used in a test for antibody by IAHA. Seronconversions to HA Ag were demonstrated by IAHA in 20 instances of hepatitis A virus infection, but in none of six cases of type B hepatitis or three cases of post-transfusion hepatitis unrelated to heaptitis A or B viruses, nor in two individuals without hepatitis. In addition, the temporal pattern of antibody development during type A hepatitis was studied in serial sera from an experimentally infected chimpanzee. Antibody titers by IAHA correlated well with antibody ratings determined by immune electron microscopy.
An enzyme-linked immunosorbent assay was developed to allow direct detection of class-specific antirotavirus antibodies. In colostrum and in milk, antirotavirus antibodies were found in the three immunoglobulin classes. Antirotavirus immunoglobulins G and M were predominant in colostrum, whereas antirotavirus immunoglobulin A was predominant in milk and feces.
The relative absence of Streptococcus sanguis in human feces, in contrast to its high concentration on teeth, supports the concept of the mouth as the likely bacterial source in cases of subacute bacterial endocarditis involving this organism.
We developed a rapid in vitro test for determining the association of Campylobacter jejuni and C. coli with HeLa cells. Association was expressed as a weighted mean of the number of bacteria associated with one cell in an association index (AI). The reproducibility of the AI was checked by repeating the test six times, using four strains chosen at random. Means and standard deviations of the means were 7.3 +/- 1.2, 6.8 +/- 0.9, 1.8 +/- 1.2, and 0.1 +/- 0.2. The experimental conditions for which the results are reliable have been standardized. Among 42 strains from human feces, two groups appeared: for 22 nonassociative strains (52%), AI values ranged from 0.0 to 2.1 (mean +/- SD, 0.5 +/- 0.6); for 20 associative strains (48%), AI values ranged from 3.5 to 8.3 (mean +/- SD, 6.2 +/- 1.4). Of these 42 strains, 17 were clinically documented. Diarrhea occurred more frequently in patients infected with associative strains than in those infected with noninvasive strains (7/7 versus 3/10, P = 0.01). Fever also occurred more frequently in patients infected with associative strains (6/7 versus 2/10, P = 0.03). Transmission electron microscopy and viable counts made after killing of extracellular bacteria by gentamicin support the fact that associated Campylobacter spp. are adherent to the cell membrane and are internalized into cytoplasmic vacuoles. The described test seems to be a convenient and rapid method for estimating the pathogenicity of a given strain.
Escherichia coli strains isolated in Sao Paulo, Brazil, from feces of patients with diarrhea and from food samples produced toxin(s) that was shown to be related both immunologically and genetically to the recently characterized type II heat-labile enterotoxin of E. coli. The new isolates of type II heat-labile enterotoxin-producing E. coli belonged to five different serotypes and did not represent a single clone.
To test the canine reservoir hypothesis of extraintestinal pathogenic Escherichia coli (ExPEC), 63 environmental canine fecal deposits were evaluated for the presence of ExPEC by a combination of selective culturing, extended virulence genotyping, hemagglutination testing, O serotyping, and PCR-based phylotyping. Overall, 30% of canine fecal samples (56% of those that yielded viable E. coli) contained papG-positive E. coli, usually as the predominant E. coli strain and always possessing papG allele III (which encodes variant III of the P-fimbrial adhesin molecule PapG). Multiple other virulence-associated genes typical of human ExPEC were prevalent among the canine fecal isolates. According to serotyping, virulence genotyping, and random amplified polymorphic DNA analysis, over 50% of papG-positive fecal E. coli could be directly correlated with specific human clinical isolates from patients with cystitis, pyelonephritis, bacteremia, or meningitis, including archetypal human ExPEC strains 536, CP9, and RS218. Five canine fecal isolates and (clonally related) archetypal human pyelonephritis isolate 536 were found to share a novel allele of papA (which encodes the P-fimbrial structural subunit PapA). These data confirm that ExPEC representing known virulent clones are highly prevalent in canine feces, which consequently may provide a reservoir of ExPEC for acquisition by humans.
The predominant gram-positive bacteria in 47 fecal specimens from 10 healthy men were studied by microscopic and cultural counts, by the characterization and tentative identification of isolates, and by the use of fluorescein isothiocyanate (FITC)-conjugated globulins prepared using some of the isolates. Gram-positive bacteria averaged 10(10.5+/-0.4(sd)/g (wet weight) of feces with significant variation from host to host. Characterization of 865 isolates, all strict anaerobes and carbohydrate fermenters, showed 12 to 39 distinguishable strains from each host and indicated that some strains were present the full period of about 18 months. Sixty percent of the isolates belonged to one of five types, tentatively identified with five species-Bifidobacterium adolescentis, Eubacterium aerofaciens, E. rectale, Peptostreptococcus productus, and Ruminococcus bromii. There was distinct host idiosyncrasy in the pattern of estimated counts of these five types. Certain strains resembling B. adolescentis, E. aerofaciens, and P. productus, distinguished with FITC conjugates, were resident in their hosts for many months. In direct smears each strain constituted about 1% of the total bacteria.
A methanogenic coccus isolated from human feces requires H2 and CH3OH for growth and uses H2 to reduce CH3OH to CH4. Growth does not occur with CH3OH alone. The organism does not grow or produce CH4 from acetate or methylamines without or with H2 or from H2 and CO2 or formate. In a complex medium. CO2 is required for formation of approximately 50% of cell carbon, whereas the methyl carbon from methanol is not incorporated into cell carbon.
Here we present the first metagenomic analyses of an uncultured viral community from human feces, using partial shotgun sequencing. Most of the sequences were unrelated to anything previously reported. The recognizable viruses were mostly siphophages, and the community contained an estimated 1,200 viral genotypes.
A selective medium, Clostridium botulinum isolation (CBI) agar, was developed for the isolation of C. botulinum from human feces. This medium contains cycloserine (250 microgram/ml), sulfamethoxazole (76 microgram/ml), and trimethoprim (4 microgram/ml) as selective inhibitory agents. Qualitative tests indicated complete recovery of C. botulinum types A, B, F, and G on CBI medium. It was more difficult to recognize type G colonies on the medium because of their lack of lipase activity. Except for a few species of Clostridium, the growth of other obligate anaerobes and of the facultative anaerobes tested on CBI medium was suppressed. Quantitative studies of C. botulinum on the selective medium yielded counts comparable to those obtained on egg yolk agar control plates. Isolation of C. botulinum types A, B, and F from seeded fecal specimens was easily achieved with CBI medium. The use of CBI agar should aid the rapid isolation of C. botulinum from fecal specimens associated with foodborne and infant botulism.
Fecal specimens from 223 subjects were evaluated for the presence of Clostridium difficile by use of a selective medium developed in our laboratory and for the presence of C. difficile cytotoxin. C. difficile and cytotoxin were detected in 89 and 83%, respectively, of patients with antimicrobial agent-associated pseudomembranous colitis (PMC). In patients in whom PMC was not documented, C. difficile and cytotoxin were present in only 37 and 21%, respectively. C. difficile and cytotoxin were also recovered from the feces of 6 and 3, respectively, of 13 antimicrobial recipients who did not have diarrhea. Although C. difficile appears to be a major cause of PMC, it is not responsible for at least some two-thirds of cases of antimicrobial agent-associated diarrhea in which PMC is not documented. Neither the recovery of C. difficile nor the detection of its cytotoxin should be considered diagnostic for C. difficile-induced disease.
To determine optimal temperature and atmospheric conditions for isolating Campylobacter jejuni from fecal specimens of humans, we studied six laboratory isolates and 19 fecal specimens that were known to contain C. jejuni. We compared incubations in 5% oxygen, the CampyPak II (BBL Microbiology Systems, Cockeysville, Md.) with 6 plates per jar (CP-6) and 12 plates per jar (CP-12), and candle jars at 37 and 42 degrees C. At both temperatures, the colony sizes for the laboratory strains were larger in the 5% O2 and the CP-6 than under the other two conditions. For the primary isolations, CP-12 failed to detect one and two campylobacters at 42 and 37 degrees C, respectively, whereas the candle jar failed to detect one at 42 degrees C and four at 37 degrees C. Colony size was again larger in the 5% O2 and the CP-6. For all four atmospheric conditions tested, colonies were significantly larger at 42 degrees C than at 37 degrees C. These studies showed that incubation at 42 degrees C in either 5% O2 or the CampyPak II with six plates per jar was optimal for primary isolation of C. jejuni from fecal specimens of humans. The candle jars incubated at 42 degrees C appeared to be satisfactory for primary isolation of C. jejuni from human feces, but incubation at 37 degrees C was not acceptable.
Hepatitis A virus in feces remained viable after being dried and then stored at 25 degrees C and 42% relative humidity for 30 days, as evidenced by infection of two marmosets inoculated with 1 ml of the treated fecal material. The virus was excreted in stool specimens from these animals, and seroconversion to antibody to hepatitis A virus occurred 28 and 35 days post-inoculation.
A total of 258 formalinized stool specimens received in our clinical laboratory were examined for parasites by direct smears and by the standard Formalin-ethyl acetate (FEAc) concentration method. Microconcentration (MC), a miniaturization of the FEAc method, was compared with the standard method for efficiency of parasite recovery. MC employed 0.25 to 0.50 ml of formalinized stools, 0.5 ml of Formalin, and 0.25 ml of ethyl acetate; the washing steps were omitted, whereas the rest of the procedure remained the same as the FEAc method. A total of 36 (13.9%) specimens were positive for parasites; of these, 23 (63.9%) were negative on direct examination. In 14 of these 23 specimens, the FEAc and MC methods were equivalent in detecting parasites. MC failed to detect parasites in eight specimens that were positive by FEAc and detected a parasite in one specimen that was negative by FEAc. Of 14 specimens positive by both concentration methods, FEAc detected additional parasite species in 2 specimens and MC did so in 1 specimen. The reduced sensitivity of parasite concentration evident in the MC we believe to be exclusively due to the drastically reduced sample size. We propose MC as an alternative to the FEAc concentration method when only small amounts of feces can be obtained.