Inhibitory effect of a copper-dipeptide complex on the establishment of a Clostridium perenne strain in the intestinal tract of gnotobiotic mice.

A semisynthetic diet fed to axenic mice was found to prevent the establishment of a Clostridium perenne strain in their intestinal tract. This inhibitory effect did not occur when axenic mice were preinoculated with a strain of Clostridium difficile. The inhibitory effect was related to the presence in the intestinal contents of axenic mice of both dietary copper and a dipeptide, aspartic-epsilon-lysine. When C. difficile was inoculated into axenic mice, the dipeptide disappeared from the digesta, and C. perenne became established even in the presence of high concentrations of copper.     

Inhibitory effect of a copper-dipeptide complex on the establishment of a Clostridium perenne strain in the intestinal tract of gnotobiotic mice

A semisynthetic diet fed to axenic mice was found to prevent the establishment of a Clostridium perenne strain in their intestinal tract. This inhibitory effect did not occur when axenic mice were preinoculated with a strain of Clostridium difficile. The inhibitory effect was related to the presence in the intestinal contents of axenic mice of both dietary copper and a dipeptide, aspartic-epsilon-lysine. When C. difficile was inoculated into axenic mice, the dipeptide disappeared from the digesta, and C. perenne became established even in the presence of high concentrations of copper.     

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.     

Differentiation of Clostridium difficile toxin from Clostridium botulinum toxin by the mouse lethality test

The mouse lethality test is the most sensitive method for confirming the diagnosis of infant botulism. Both Clostridium difficile and Clostridium botulinum produce heat-labile toxins which are lethal for mice and can be found in the feces of infants. These two toxins can be distinguished from one another in this assay when both are present in the same fecal specimen because they appear to be immunologically distinct toxins.     

高脂饮食中添加短链菊粉对小鼠肠道菌群的影响

目的:探究高脂饮食中添加短链菊粉对小鼠肠道菌群的影响。方法:选择8周龄雄性小鼠,5只喂食高脂饲料,5只喂食10%菊粉复合型高脂饲料,喂食8周后收集小鼠粪便,检测小鼠粪便中三种主要的短链脂肪酸。同时,提取小鼠粪便中的细菌基因组,对菌群基因组16S rRNA基因V4高变区进行测序,对数据进行PCoA分析、Alpha多样性分析、LEfSe分析和16S功能预测。结果:菊粉添加后,小鼠粪便中含有的细菌DNA量增多,短链脂肪酸增加。菊粉组和对照组PCoA图可以看到明显聚类。菊粉组物种多样性低于对照组。菊粉组小鼠粪便中S24_7菌科丰度上升;Lachnospiraceae(毛螺菌科),Ruminococcaceae(瘤胃菌科)和Deferribacteraceae(脱铁杆菌科)丰度下降。16S基因功能预测发现22个第二层级的KEGG通路发生变化。结论:高脂饮食情况下短链菊粉的添加会改变小鼠肠道菌群,继而影响肠道菌群的功能。     

Differentiation of Clostridium difficile Toxin from Clostridium botulinum Toxin by the Mouse Lethality Test

The mouse lethality test is the most sensitive method for confirming the diagnosis of infant botulism. Both Clostridium difficile and Clostridium botulinum produce heat-labile toxins which are lethal for mice and can be found in the feces of infants. These two toxins can be distinguished from one another in this assay when both are present in the same fecal specimen because they appear to be immunologically distinct toxins.     

Variations of Bacterial Populations in Human Feces Measured by Fluorescent In Situ Hybridization with Group-Specific 16S rRNA-Targeted Oligonucleotide Probes

Six 16S rRNA-targeted oligonucleotide probes were designed, validated, and used to quantify predominant groups of anaerobic bacteria in human fecal samples. A set of two probes was specific for species of the Bacteroides fragilis group and the species Bacteroides distasonis. Two others were designed to detect species of the Clostridium histolyticum and the Clostridium lituseburense groups. Another probe was designed for the genera Streptococcus and Lactococcus, and the final probe was designed for the species of the Clostridium coccoides-Eubacterium rectale group. The temperature of dissociation of each of the probes was determined. The specificities of the probes for a collection of target and reference organisms were tested by dot blot hybridization and fluorescent in situ hybridization (FISH). The new probes were used in initial FISH experiments to enumerate human fecal bacteria. The combination of the two Bacteroides-specific probes detected a mean of 5.4 × 10¹⁰ cells per g (dry weight) of feces; the Clostridium coccoides-Eubacterium rectale group-specific probe detected a mean of 7.2 × 10¹⁰ cells per g (dry weight) of feces. The Clostridium histolyticum, Clostridium lituseburense, and Streptococcus-Lactococcus group-specific probes detected only numbers of cells ranging from 1 × 10⁷ to 7 × 10⁸ per g (dry weight) of feces. Three of the newly designed probes and three additional probes were used in further FISH experiments to study the fecal flora composition of nine volunteers over a period of 8 months. The combination of probes was able to detect at least two-thirds of the fecal flora. The normal biological variations within the fecal populations of the volunteers were determined and indicated that these variations should be considered when evaluating the effects of agents modulating the flora.     

Cockroaches as vectors of Sarcocystis muris and of other coccidia in the laboratory.

To assess the roles of the German cockroach (Blatella germanica) and the American cockroach (Periplaneta americana) in the transmission of Sarcocystis muris and of 3 other coccidia of cats-Toxoplasma gondii, Isospora felis, and Isospora rivolta, cockroaches exposed to feces containing these coccidia were periodically fed to mice, as was a portion of the fecal matter. Sarcocystis muris sporocysts, which in feces remained infectious for at least 20 days, were also transmitted to mice by P. americana for at least 20 days and by B. germanica for 5 days after exposure to infectious feces. Toxoplasma gondii oocysts were transmitted by P. americana intermittently up to 10 days, but by B. germanica only immediately after exposure to feces. Oocysts of 2 species of Isospora, when associated with fecal matter, remained infectious for 20 days. Those of I. rivolta were transmitted by both cockroach species for 10 days, but I. felis was transmitted only by by B. germanica, and for only 2 days.     

Concentrations of pathogens and indicators in animal feces in the Sydney watershed

A fecal analysis survey was undertaken to quantify animal inputs of pathogenic and indicator microorganisms in the temperate watersheds of Sydney, Australia. The feces from a range of domestic animals and wildlife were analyzed for the indicator bacteria fecal coliforms and Clostridium perfringens spores, the pathogenic protozoa Cryptosporidium and Giardia, and the enteric viruses adenovirus, enterovirus, and reovirus. Pathogen and fecal indicator concentrations were generally higher in domestic animal feces than in wildlife feces. Future studies to quantify potential pathogen risks in drinking-water watersheds should thus focus on quantifying pathogen loads from domestic animals and livestock rather than wildlife.     

Concentrations of Pathogens and Indicators in Animal Feces in the Sydney Watershed

A fecal analysis survey was undertaken to quantify animal inputs of pathogenic and indicator microorganisms in the temperate watersheds of Sydney, Australia. The feces from a range of domestic animals and wildlife were analyzed for the indicator bacteria fecal coliforms and Clostridium perfringens spores, the pathogenic protozoa Cryptosporidium and Giardia, and the enteric viruses adenovirus, enterovirus, and reovirus. Pathogen and fecal indicator concentrations were generally higher in domestic animal feces than in wildlife feces. Future studies to quantify potential pathogen risks in drinking-water watersheds should thus focus on quantifying pathogen loads from domestic animals and livestock rather than wildlife.     

Culture-independent analysis of fecal microbiota in cattle

The phylogenetic diversity of the fecal bacterial community in Holstein cattle was determined by 16S ribosomal RNA gene sequence analysis. The sequences were affiliated with the following phyla: Firmicutes (81.3%), Bacteroidetes (14.4%), Actinobacteria (2.5%), and Proteobacteria (1.4%). The Clostridium leptum subgroup was the most phylogenetically diverse group in cattle feces. In addition, a number of previously uncharacterized and unidentified bacteria were recognized in clone libraries.     

Comparative analysis of fecal microbiota and intestinal microbial metabolic activity in captive polar bears

The composition of the intestinal microbiota depends on gut physiology and diet. Ursidae possess a simple gastrointestinal system composed of a stomach, small intestine, and indistinct hindgut. This study determined the composition and stability of fecal microbiota of 3 captive polar bears by group-specific quantitative PCR and PCR-DGGE (denaturing gradient gel electrophoresis) using the 16S rRNA gene as target. Intestinal metabolic activity was determined by analysis of short-chain fatty acids in feces. For comparison, other Carnivora and mammals were included in this study. Total bacterial abundance was approximately log 8.5 DNA gene copies·(g feces)-1 in all 3 polar bears. Fecal polar bear microbiota was dominated by the facultative anaerobes Enterobacteriaceae and enterococci, and the Clostridium cluster I. The detection of the Clostridium perfringens α-toxin gene verified the presence of C.?perfringens. Composition of the fecal bacterial population was stable on a genus level; according to results obtained by PCR-DGGE, dominant bacterial species fluctuated. The total short-chain fatty acid content of Carnivora and other mammals analysed was comparable; lactate was detected in feces of all carnivora but present only in trace amounts in other mammals. In comparison, the fecal microbiota and metabolic activity of captive polar bears mostly resembled the closely related grizzly and black bears.     

Detection and quantification of four species of the genus Clostridium in infant feces

To determine the composition of Clostridium in the feces of infants approximately 30 days old, we have developed a detection and quantification method of Clostridium paraputrificum, Clostridium perfringens, Clostridium tertium, and Clostridium difficile by species-specific primers. C. perfringens and C. difficile were detected in four fecal samples from 22 infants (18.2%), whereas C. paraputrificum was detected in three samples (16.7%). C. tertium was detected in two samples (9.1%). Moreover, the occurrences of the four species in bottle-and mix-fed infants were relatively higher than in breast-fed infants (P< 0.05). Subsequently, positive samples detected by nested PCR (polymerase chain reaction) were subjected to realtime PCR. The results showed that the numbers of C. paraputrificum, C. perfringens, C. tertium, and C. difficile ranged from about 1x10(5) to 3x10(7) cells/g wet feces.     

First isolation of Neospora caninum from the feces of a naturally infected dog.

Neospora caninum is a major cause of abortion in cattle worldwide. Cattle become infected with N. caninum by ingesting oocysts from the environment or transplacentally from dam to fetus. Experimentally, dogs can act as definitive hosts, but dogs excrete few oocysts after ingesting tissue cysts. A natural definitive host was unknown until now. In the present study, N. caninum was isolated from the feces of a dog. Gerbils (Meriones unguiculatus) fed feces from the dog developed antibodies to N. caninum in the Neospora caninum agglutination test, and tissue cysts were found in their brains. Neospora caninum was isolated in cell culture and in gamma-interferon gene knockout mice inoculated with brain homogenates of infected gerbils. The DNA obtained from fecal oocysts of the dog, from the brains of gerbils fed dog feces, and from organisms isolated in cell cultures inoculated with gerbil brains was confirmed as N. caninum. The identification of N. caninum oocyst by bioassay and polymerase chain reaction demonstrates that the dog is a natural definitive host for N. caninum.     

Microbiota Composition of the Intestinal Mucosa: Association with Fecal Microbiota?

The fecal and mucosal microbiota of infants with rectal bleeding and the fecal microbiota of healthy age-matched controls were investigated by fluorescent in situ hybridization. Bifidobacteria were the main genus in both the feces and mucosa. The other genera tested, Bacteroides, Clostridium, Escherichia coli and lactobacilli/enterococci, represented only minor constituents. No differences in fecal microbiota were observed between patients and controls. In the patients, however, four times greater numbers of bifidobacteria were observed in the feces when compared to the mucosa. Notwithstanding this difference, a strong positive correlation prevailed for bifidobacteria in feces and mucosal samples. The genera assessed accounted for 16% of total bacterial counts on mucosal samples and for 47% of total bacterial counts in feces. This indicates that the unidentified part of the microbiota, especially on the mucosa, deserves more attention.     

Intestinal microbiota and secretory immunoglobulin A in feces of exclusively breast-fed infants with blood-streaked stools

Episodes of blood‐streaked stools are not uncommon in exclusively breast‐fed infants under 6 months of age. Such bleeding is thought to be associated with food protein‐induced proctocolitis, however the pathomechanism remains unclear. The aim of this study was to investigate intestinal microbiota and secretory immunoglobulin A in the feces of exclusively breast‐fed infants with blood‐streaked stools. Fecal specimens from 15 full‐term infants with blood‐streaked stools and 15 breast‐fed healthy infants were studied and the results compared. All infants had been delivered vaginally and exclusively breast‐fed. The fecal microbiota were investigated by phylogenetic analysis combined with culture methods for some bacterial species, and feces were assessed for the presence of fecal secretory immunoglobulin A by enzyme‐linked immunosorbent assay. Phylogenetic cluster analysis revealed four major clusters of fecal bacteria, cluster A being found only in healthy infants. The Bacteroides fragilis group was observed more frequently in controls than in patients (P < 0.05). In the controls, the predominant species belonging to the Enterobacteriaceae group was Escherichia coli, whereas in the patients it was Klebsiella (P < 0.05). Concentrations of secretory immunoglobulin A were high in one third of the healthy controls. In conclusion, the pathomechanism of rectal bleeding in exclusively breast‐fed infants may be related to differences in the composition of their intestinal flora.     

Dynamic pathways of selenium metabolism and excretion in mice under different selenium nutritional statuses

The selenoprotein, cellular glutathione peroxidase (cGPx), has an important role in protecting organisms from oxidative damage through reducing levels of harmful peroxides. The liver and kidney in particular, have important roles in selenium (Se) metabolism and Se is excreted predominantly in urine and feces. In order to characterize the dynamics of these pathways we have measured the time-dependent changes in the quantities of hepatic, renal, urinary, and fecal Se species in mice fed Se-adequate and Se-deficient diets after injection of (82)Se-enriched selenite. Exogenous (82)Se was transformed to cGPx in both the liver and kidney within 1 h after injection and the synthesis of cGPx decreased 1 to 6 h and continued at a constant level from 6 to 72 h after injection. The total amount of Se associated with cGPx in mice fed Se-deficient diets was found to be less than in mice fed Se-adequate diets. This finding indicated that cGPx synthesis was suppressed under Se-deficient conditions and did not recover with selenite injection. Excess Se was associated with selenosugar in liver and transported to the kidney within 1 h after injection, and then excreted in urine and feces within 6 h after injection. Any excess amount of Se was excreted mainly as a selenosugar in urine.     

Microbial community analysis and identification of alternative host-specific fecal indicators in fecal and river water samples using pyrosequencing

It is important to know the comprehensive microbial communities of fecal pollution sources and receiving water bodies for microbial source tracking. Pyrosequencing targeting the V1-V3 hypervariable regions of the 16S rRNA gene was used to investigate the characteristics of bacterial and Bacteroidales communities in major fecal sources and river waters. Diversity analysis indicated that cow feces had the highest diversities in the bacterial and Bacteroidales group followed by the pig sample, with human feces having the lowest value. The Bacteroidales, one of the potential fecal indicators, totally dominated in the fecal samples accounting for 31%-52% of bacterial sequences, but much less (0.6%) in the river water. Clustering and Venn diagram analyses showed that the human sample had a greater similarity to the pig sample in the bacterial and Bacteroidales communities than to samples from other hosts. Traditional fecal indicators, i.e., Escherichia coli, were detected in the human and river water samples at very low rates and Clostridium perfringens and enterococci were not detected in any samples. Besides the Bacteroidales group, some microorganisms detected in the specific hosts, i.e., Parasutterella excrementihominis, Veillonella sp., Dialister invisus, Megamonas funiformis, and Ruminococcus lactaris for the human and Lactobacillus amylovorus and Atopostipes sp. for the pig, could be used as potential host-specific fecal indicators. These microorganisms could be used as multiple fecal indicators that are not dependent on the absence or presence of a single indicator. Monitoring for multiple indicators that are highly abundant and host-specific would greatly enhance the effectiveness of fecal pollution source tracking.     

Cholesterol gallstones in alloxan-diabetic mice

Normal and alloxan-diabetic male mice (Crj-ICR) were fed a diet containing 0.5% cholesterol for 5 and 10 weeks, and gallbladder bile was analyzed for cholesterol, phospholipids and bile acids, feces for sterols and bile acids, and plasma and liver for cholesterol, phospholipids, and triglycerides. Normal mice developed no gallstones but the diabetic mice developed cholesterol gallstones with an incidence of 70% by 5 weeks and 80% by 10 weeks after feeding of the cholesterol diet. Diabetic mice fed the ordinary diet also developed stones (23%) by 10 weeks. In the diabetic mice, the gallbladder was enlarged about threefold, and biliary lipid concentration, diet intake, and fecal excretion of sterols and bile acids increased but body weight decreased. Cholic acid and beta-muricholic acid comprised over 40% each of the total biliary bile acids in normal mice, but cholic acid increased to about 80% and beta-muricholic acid decreased to a few percent in the diabetic mice. Fecal excretion of bile acids increased after cholesterol feeding in both normal and diabetic mice, but the increased bile acid in the normal animals was beta-muricholic acid and that in the diabetic mice was deoxycholic acid. The mice that developed gallstones showed a marked increase in biliary cholesterol value and decreases in gallbladder bile and bile acid concentration, but no difference in biliary and fecal bile acid composition, bile acid synthesis, fecal sterols, or plasma and liver lipid levels. Cholesterol absorption was increased in the diabetic mice when examined by plasma 14C/3H ratio and fecal 14C-labeled sterol excretion after a single oral administration of [14C]cholesterol and a simultaneous intravenous injection of [3H]cholesterol. These data led to the conclusion that cholesterol gallstones developed in alloxan-diabetic mice fed excess cholesterol, due to the hyperphagia and the enhancement of cholesterol absorption caused by increases in the synthesis and secretion of cholic acid.     

Oligosaccharides from feces of preterm infants fed on breast milk

Nine neutral and five acidic oligosaccharides were isolated from feces of a preterm (30th postmenstrual week) blood group A nonsecretor infant fed on pooled breast milk. Structural analyses were carried out using sugar and methylation analyses, fast atom bombardment mass spectrometry, and 1H NMR. The acidic oligosaccharides are well-known components of human milk. The neutral oligosaccharides are characteristic of nonsecretor milk. Surprisingly, no secretor gene-dependent oligosaccharides were present in the feces. Another preterm (27th postmenstrual week) blood group A, secretor infant fed on pooled breast milk showed the same fecal oligosaccharide pattern as above during the first week after birth, despite being a secretor individual. Also notable was the absence of blood group A-active oligosaccharides in this sample. Another sample of feces collected 8 weeks later from the latter infant contained the expected blood group A-active oligosaccharides. Furthermore, free sialic acid was present at the cost of the sialyl oligosaccharides seen earlier. Thus, infants born prematurely do not show the same degree of development of oligosaccharide metabolism as their more mature counterparts.