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PubMed2006

Application of 16S rRNA gene-targetted fluorescence in situ hybridization and restriction fragment length polymorphism to study porcine microbiota along the gastrointestinal tract in response to different sources of dietary fibre.

A total of 32 pigs of 15+/-0.38 kg body weight were fed for 6 weeks one of four diets differing in their source of dietary fibre. FISH was used to quantify the main bacterial groups in the pig gut using the following probes: Eub338, Bac303, Rfla729, Rbro730, Erec482, Fprau645, Prop853, Str493 and Lab158. FISH counts revealed important differences at four sites along the pig gastrointestinal tract, but we were unable to show differences related to diets. Stomach and jejunal samples gave total bacterial counts of 0.1-5.3 x 10(8) g(-1) of contents. In the stomach, streptococci and lactobacilli were predominant, and the clostridial cluster IX group was abundant (14-41% of total bacterial count). Clostridial cluster IX bacteria were present elsewhere in the gastrointestinal tract at 1-8%. The other groups were generally more abundant in the proximal colon and rectum: Bacteroides/Prevotella (5-10%), clostridial cluster XIVa (10-19%), and cluster IV relatives of Faecalibacterium prausnitzii (1-4%) and ruminococcus (4-10%). Restriction fragment length polymorphism profiles showed changes related to diet, with pigs fed wheat bran having the lowest richness of all diets (P=0.008).

Animals↗

PubMed2006

Analysis of aerotactic band formation by Desulfovibrio desulfuricans in a stopped-flow diffusion chamber.

Aerotactic band formation by Desulfovibrio desulfuricans (DSM 9104) was studied in a stopped-flow diffusion chamber. This chamber allowed us to create reproducible, steep oxygen gradients in a flat capillary, time-lapse video recordings and spatio-temporal analysis of band formation. The cells formed two types of bands. Bands of the first type evolved quickly after starting the experiment and were located near the oxic-anoxic interface. Bands of the second type typically appeared several minutes later and a few millimeters inside the initially anoxic volume of the capillary. Band formation depended on metabolism and could be stimulated by lactate addition, and thus appears to be energy taxis. Mathematical modeling of oxygen diffusion and respiration within the chamber revealed that bands formed preferentially at oxygen concentrations close to 4% air saturation. The swimming speed of the cells was determined by digital single-cell tracking and found to be highest (up to 58 mum s(-1)) close to the oxic-anoxic interfaces. Motility patterns were influenced by surfaces, at which cells accumulated. Bioconvection sometimes occurred if very dense bands had formed. The ecological implications of these two phenomena are unknown.

Aerobiosis↗

PubMed1992

The inhibition of fungal cellulolysis by cell-free preparations from ruminococci.

The degradation of filter paper by the anaerobic fungus Neocallimastix frontalis strain RE1 was reduced by the addition of cell-free supernates from cultures of Ruminococcus albus strain J6 and R. flavefaciens strains 17 and 007. Fungal uptake of, and growth on, glucose was not affected. After gel permeation and anion exchange chromatography, inhibitory activity towards fungal cellulolysis was recovered in a fraction from strain 17 that contained at least five negatively charged polypeptide components, molecular mass 45-68 kDa, on SDS-PAGE.

Animals↗

PubMed1997

13C-NMR study of glucose and pyruvate catabolism in four acetogenic species isolated from the human colon.

Glucose fermentation by four acetogenic species (two Clostridium strains, one Streptococcus strain and Ruminococcus hydrogenotrophicus) isolated from the human colon was of a mixed-acid type, whereas pyruvate metabolism was characterised by homoacetogenesis. Acetate formation from [1-13C] and [2-13C]glucose was consistent with the formation of acetyl-SCoA from pyruvate generated by the Embden-Meyerhof-Parnas pathway. Labelling of lactate and ethanol demonstrated that these metabolites were formed by reduction of pyruvate and acetyl-SCoA, respectively. In contrast, the reductive pathway of acetate formation was the preferential means of re-oxidising cofactors formed during [1-13C]pyruvate catabolism.

Acetates↗

PubMed2001

Interspecies H2 transfer in cellulose degradation between fibrolytic bacteria and H2-utilizing microorganisms from the human colon.

Interspecies H2 transfer between two newly isolated fibrolytic strains (18P13 and 18P16) and H2-utilizing methanogen or acetogen from the human colon was investigated during in vitro cellulose degradation. Both H2-consuming microorganisms utilized efficiently H2 produced from cellulose fermentation by the fibrolytic species. H2 utilization by Methanobrevibacter smithii did not change the metabolism and the cellulolytic activity of strain 18P16 whereas it induced a metabolic shift in strain 18P13. However, this metabolic shift was not associated with enhancement of cellulose degradation. In contrast, an increase in cellulose breakdown was observed when strain 18P13 was cultivated with Ruminococcus hydrogenotrophicus. This stimulating effect could be attributed to both the autotrophic and the heterotrophic metabolism of the acetogen in the coculture.

Anaerobiosis↗

PubMed1996

Identification and characterization of cellulose-binding domains in xylanase A of Clostridium stercorarium.

The xynA gene encoding a major xylanase of Clostridium stercorarium F-9 was sequenced. The structural gene consists of an open reading frame of 1533 bp encoding a protein of 511 amino acids with an M(r) of 56,519. XynA consists of a catalytic domain belonging to family G at the NH2-terminus and two direct repeats of about 90 amino acids with a short spacing at the COOH-terminus. The repeated sequences, CBDI and CBDII, were not homologous with amino acid sequences of the CBDs classified into families I to V. Nevertheless, XynA showed an affinity for insoluble cellulose such as Avicel. Binding of XynA to Avicel was strongly dependent on the concentration of the incubation buffer and was inhibited by Triton X-100. XynA bound to Avicel (2.4 nmol/g-cellulose) and acid-swollen cellulose (180 nmol/g-cellulose), suggesting that this enzyme has higher affinity for amorphous cellulose than for crystalline cellulose. Functions of CBDI and CBDII were investigated by constructing the mutant enzymes and evaluating the cellulose-binding ability of each of them. XynA4 lacking CBDI and XynA5 lacking CBDII bound to Avicel to a lesser extent than the parental enzyme XynA; but XynA6, devoid of both CBDs, did not bind at all, indicating that CBDI and CBDII each functioned independently as CBD in XynA and their binding capacity was additive. Although the Ruminococcus albus endoglucanase EgIV that was joined to CBDs of XynA acquired cellulose-binding ability, the substrate specificity of EgIV was not altered in the presence or absence of CBDs.

Amino Acid Sequence↗

PubMed2005

Comparison of three PCR primer sets for identification of vanB gene carriage in feces and correlation with carriage of vancomycin-resistant enterococci: interference by vanB-containing anaerobic bacilli.

We assessed the sensitivities and specificities of three previously described PCR primers on enrichment broth cultures of feces for the accurate detection of fecal carriage of vancomycin-resistant enterococci (VRE). In addition, we investigated specimens that were vanB PCR positive but VRE culture negative for the presence of other vanB-containing pathogens. Feces from 59 patients (12 patients carrying vanB Enterococcus faecium strains and 47 patients negative for VRE carriage) were cultured for 36 h in aerobic brain heart infusion (BHI) broth, anaerobic BHI (AnO(2)BHI) broth, or aerobic Enterococcosel (EC) broth. DNA was extracted from the cultures and tested for the presence of vanB by using the PCR primers of Dutka-Malen et al. (S. Dutka-Malen, S. Evers, and P. Courvalin, J. Clin. Microbiol. 33:24-27, 1995), Bell et al. (J. M. Bell, J. C. Paton, and J. Turnidge, J. Clin. Microbiol. 36:2187-2190, 1998), and Stinear et al. (T. P. Stinear, D. C. Olden, P. D. R. Johnson, J. K. Davies, and M. L. Grayson, Lancet 357:855-856, 2001). The sensitivity (specificity) of PCR compared with the results of culture on BHI, AnO(2)BHI, and EC broths were 67% (96%), 50% (94%), and 17% (100%), respectively, with the primers of Dutka-Malen et al.; 92% (60%), 92% (45%), and 92% (83%), respectively, with the primers of Bell et al.; and 92% (49%), 92% (43%), and 100% (51%) respectively, with the primers of Stinear et al. The primers of both Bell et al. and Stinear et al. were significantly more sensitive than those of Dutka-Malen et al. in EC broth (P = 0.001 and P < 0.001, respectively). The poor specificities for all primer pairs were due in part to the isolation and identification of six anaerobic gram-positive bacilli, Clostridium hathewayi (n = 3), a Clostridium innocuum-like organism (n = 1), Clostridium bolteae (n = 1), and Ruminococcus lactaris-like (n = 1), from five fecal specimens that were vanB positive but VRE culture negative. All six organisms were demonstrated to contain a vanB gene identical to that of VRE. VanB-containing bowel anaerobes may result in false-positive interpretation of PCR-positive fecal enrichment cultures as VRE, regardless of the primers and protocols used.

Bacteria, Anaerobic↗

PubMed1981

Isolation and presumptive identification of adherent epithelial bacteria ("epimural" bacteria) from the ovine rumen wall.

One hundred sixty-one strains of adherent bacteria were isolated under anaerobic conditions from four sites on the rumen epithelial surface of sheep fed hay or a hay-grain ration. Before isolation of bacteria, rumen tissue was washed six times in an anaerobic dilution solution, and viable bacteria suspended in the washings were counted. Calculation indicated that unattached bacteria would have been removed from the tissue by this procedure, but a slow and progressive release of attached bacteria also occurred. Nevertheless, a wide range of characteristic morphological types remained associated with the epithelium as demonstrated by scanning electron microscopy. Most of these types were represented among the isolates. Characterization and presumptive identification of the isolates showed that 95.0% belonged to previously described genera of functionally significant rumen bacteria, including Butyrivibrio sp. (31.1%), Bacteroides sp. (22.4%), Selenomonas ruminantium (9.9%), Succinivibrio dextrinosolvens (8.7%), Streptococcus bovis (8.1%), Propionibacterium sp. (4.3%), Treponema sp. (3.1%), and Eubacterium sp., Lachnospira multiparus, and Ruminococcus flavefaciens (2.5% each). Eight isolates (5.0%) were not identified. L. multiparus was recovered only from hay-fed animals; all other genera were obtained from animals fed either ration. All S. bovis strains and two strains each of Bacteroides sp. and Butyrivibrio sp. were aerotolerant; all other strains were strictly anaerobic. Bacteria representing the gram-positive, facultatively anaerobic flora associated with rumen wall tissue (R. J. Wallace, K.-J. Cheng, D. Dinsdale, and E. R. Ørskov, Nature (London) 279:424-426, 1979) were therefore not recovered by the techniques used; instead a different fraction of the adherent population was isolated. The term "epimural" is proposed to describe the flora associated with the rumen epithelium.

Anaerobiosis↗

PubMed1982

Cellulolytic and non-cellulolytic bacteria in rat gastrointestinal tracts.

Lactobacillus and Bifidobacterium species were the predominant organisms isolated from small intestinal (jejunal) contents of rats, and lactic acid was the only organic acid detected. The numbers of cellulolytic bacteria in small intestines were low (approximately 10(3)/g). The fermentation in ceca was different from that in intestines, as, in addition to small amounts of lactic acid, high concentrations of volatile fatty acids were detected. The mixed cecal microflora was able to digest cellulose (pebble-milled Whatman no. 1) and cabbage. High numbers of cellulolytic bacteria were found (0.5 X 10(8) to 12.2 X 10(8)/g; 6% of total viable bacteria). The predominant celluloytic organism isolated was Bacteroides succinogenes. Ruminococcus flavifaciens was isolated from a few animals. The kinds and numbers of the predominant non-cellulolytic organisms isolated from rat ceca were similar to those described by previous workers.

Actinomycetaceae↗

PubMed1984

Cellulolytic bacteria from pig large intestine.

An anaerobic, cellulose-degrading, gram-negative rod and a gram-positive coccus, identified as Bacteroides succinogenes and Ruminococcus flavefaciens, respectively, were isolated from pig fecal samples. These organisms were isolated from cellulolytic most-probable-number dilutions which represented 4 or 6% of the viable bacterial count when pigs were fed a low- or high-fiber diet, respectively.

Animals↗

PubMed1985

Degradation of bermuda and orchard grass by species of ruminal bacteria.

Fiber degradation in Bermuda grass and orchard grass was evaluated gravimetrically and by scanning and transmission electron microscopy after incubation with pure cultures of rumen bacteria. Lachnospira multiparus D-32 was unable to degrade plant cell wall components. Butyrivibrio fibrisolvens 49 degraded 6 and 14.9% of the fiber components in Bermuda grass and orchard grass, respectively, and Ruminococcus albus 7 degraded 11.4% orchard grass fiber but none in Bermuda grass. Both B. fibrisolvens and R. albus lacked capsules, did not adhere to fiber, and degraded only portions of the more easily available plant cell walls. R. flavefaciens FD-1 was the most active fiber digester, degrading 8.2 and 55.3% of Bermuda and orchard grass fiber, respectively. The microbe had a distinct capsule and adhered to fiber, especially that which is slowly degraded, but was able to cause erosion and disorganization of the more easily digested cell walls, apparently by extracellular enzymes. Results indicated that more digestible cell walls could be partially degraded by enzymes disassociated from cellulolytic and noncellulolytic bacteria, and data were consistent with the hypothesis that the more slowly degraded plant walls required attachment. Microbial species as well as the cell wall architecture influenced the physical association with and digestion of plant fiber.

Journal Article↗

PubMed1986

Effect of 3-phenylpropanoic Acid on growth of and cellulose utilization by cellulolytic ruminal bacteria.

The growth of several cellulolytic species of ruminal bacteria was measured in media containing either cellobiose or cellulose as the energy source and with or without added 3-phenylpropanoic acid (PPA). With Ruminoccoccus albus 7 and 8, the addition of PPA greatly enhanced the rate of cellulose utilization but had little effect on the rate of growth when cellobiose was the energy source. Comparative rates of growth obtained on either cellobiose or cellulose for Ruminococcus flavefaciens FD1 or C94 and Butyrivibrio fibrisolvens 12, 49, or A38 were similar regardless of the PPA content of the growth medium.

Journal Article↗

PubMed1986

Palladium-Mediated Hydrogenation of Unsaturated Hydrocarbons with Hydrogen Gas Released during Anaerobic Cellulose Degradation.

Among five hydrogenation catalysts, palladium on charcoal was the most reactive one when suspended in anaerobic culture medium, and Lindlar catalyst (Pd on CaCO(3)) was the most reactive one when suspended in the gas phase of culture tubes. Palladium on charcoal in the culture medium (40 to 200 mg 10 ml) completely inhibited growth of Neocallimastix frontalis and partly inhibited Ruminococcus albus. Lindlar catalyst (40 to 200 mg per tube) suspended in a glass pouch above the culture medium did not affect the rate of cellulose degradation or the ratio of fermentation products by these organisms. Acetylene added to tubes containing Lindlar catalyst in pouches, and either of the two organisms in monoculture or coculture with Methanospirillum hungatei, was reduced to ethylene and then ethane, followed by hydrogen production. Similar results were obtained with 1-pentene. Neither acetylene nor 1-pentene affected cellulose degradation but both inhibited methanogenesis. In the presence of Lindlar catalyst and propylene or 1-butene, fermenter-methanogen cocultures continued to produce methane at the same rate as controls and no olefin reduction occurred. Upon addition of bromoethanesulfonic acid, methanogenesis stopped and olefin reduction took place followed by hydrogen evolution. In a gas mixture consisting of propylene, 1-butene, and 1-pentene, the olefins were reduced at rates which decreased with increasing molecular size. These results demonstrate the technical feasibility of combining in one reactor the volatile fatty acid production by anaerobic digestion with chemical catalyst-mediated reductions, using the valuable by-product hydrogen.

Journal Article↗

PubMed1987

Seasonal changes in the cecal microflora of the high-arctic Svalbard reindeer (Rangifer tarandus platyrhynchus).

The dominant cecal bacteria in the high-arctic Svalbard reindeer were characterized, their population densities were estimated, and cecal pH was determined in summer, when food quality and availability is good, and in winter, when it is very poor. In summer the total culturable viable bacterial population was (8.9 +/- 5.3) X 10(8) cells ml-1, whereas in winter it was (1.5 +/- 0.7) X 10(8) cells ml-1, representing a decrease to 17% of the summer population density. Of the dominant species of cultured bacteria, Butyrivibrio fibrisolvens represented 23% in summer and 18% in winter. Streptococcus bovis represented 17% in summer and 5% in winter. Bacteroides ruminicola represented 10% in summer and 26% in winter. In summer and winter, respectively, the proportion of the viable population showing the following activities was as follows: fiber digestion, 36 and 48%; cellulolysis, 10 and 6%; xylanolysis, 33 and 48%; and starch utilization, 77 and 71%. The most abundant cellulolytic species in summer was Butyrivibrio fibrisolvens, representing 62% of the total cellulolytic population, and in winter it was Ruminococcus albus, representing 80% of the total cellulolytic population. The most abundant xylanolytic species in summer was Butyrivibrio fibrisolvens, and in winter it was Bacteroides ruminicola, representing 59 and 54% of the xylanolytic isolates in summer and winter, respectively. The cecal bacterial of the Svalbard reindeer have the ability to digest starch and the major structural carbohydrates of the diet that are not digested in the rumen. The cecum in these animals has the potential to contribute very substantially to the digestion of the available plant material in both summer and winter.

Animals↗

PubMed1987

Influence of dietary fiber on xylanolytic and cellulolytic bacteria of adult pigs.

Xylanolytic and cellulolytic bacteria were enumerated over an 86-day period from fecal samples of 10 8-month-old gilts that were fed either a control or a 40% alfalfa meal (high-fiber) diet. Fecal samples were collected from all pigs on days 0, 3, 5, 12, 25, 37, 58, and 86. Overall, the numbers of xylanolytic bacteria producing greater than 5-mm-diameter zones of clearing on 0.24% xylan roll tube medium after 24 to 36 h of incubation were 1.6 X 10(8) and 4.2 X 10(8)/g (dry weight) of feces for the control pigs and those fed the high-fiber diet, respectively. After 1 week of incubation, a large number of smaller zones of clearing (1 to 2 mm) appeared. Besides Bacteroides succinogenes and Ruminococcus flavefaciens, which produced faint zones of clearing in xylan roll tubes, three strains which closely resembled B. ruminicola hydrolyzed and used xylan for growth. The overall numbers of cellulolytic bacteria producing zones of clearing in 0.5% agar roll tube medium were 0.36 X 10(8) and 4.1 X 10(8)/g for the control pigs and those fed the high-fiber diet, respectively. B. succinogenes was the predominant cellulolytic isolate from both groups of pigs, and R. flavefaciens was found in a ratio of approximately 1 to 15 with B. succinogenes. Degradation of xylan and cellulose, measured by in vitro dry matter disappearance after inoculation with fecal samples, was significantly greater for pigs fed the high-fiber diet than that for the controls. These data suggest that the number of fibrolytic microorganisms and their activity in the large intestine of the adult pig can be increased by feeding pigs high-alfalfa-fiber diets and that these organisms are similar to those found in the rumen.

Animal Feed↗

PubMed1988

Effects of Cellulolytic Ruminal Bacteria and of Cell Extracts on Germination of Euonymus americanus L. Seeds.

In past attempts, the experimental germination of the seeds of Euonymus americanus L. in vitro has had little success. However, treatment of seeds with ruminal fluid containing viable microflora has been successful in stimulating germination. In the presence of the cellulolytic ruminal bacterium, Clostridium cellobioparum ATCC 15832, seeds of E. americanus were stimulated to germinate. Subsequent studies were designed to determine whether the bacterium synthesized a cellulolytic enzyme responsible for initiating germination. The cell-free endocellulase from C. cellobioparum induced germination of the seeds. To support the hypothesis that the endocellulase from C. cellobioparum was responsible for triggering germination, a 1,4-beta-d-glucan glucanohydrolase (EC 3.2.1.4) from Penicillum funiculosum was used to treat the seeds. In addition, no germination was obtained from seeds treated with a commercial exocellulase enzyme. Also, Ruminococcus flavefaciens FD-1 was found to initiate germination of E. americanus seeds. Thus, cellulase activity is indicated in the degradation of the testa of the seed, allowing imbibition and germination.

Journal Article↗

PubMed1988

Effect of the novel ionophore tetronasin (ICI 139603) on ruminal microorganisms.

The antimicrobial activity of the novel ionophore tetronasin (formerly ICI 139603) was compared with that of monensin for the growth of ruminal bacteria, protozoa, and an anaerobic fungus. The potency of tetronasin toward most bacteria and the fungus was an order of magnitude or more greater than that of monensin. Lactobacillus casei was 55 times more sensitive to tetronasin than to monensin, indicating a potential role for tetronasin in reversing lactic acidosis. Bacteria with a gram-positive ultrastructure were generally sensitive to the ionophores and unable to adapt to grow in their presence. The exception was the cellulolytic Ruminococcus flavefaciens, which adapted during successive cultivation on media with increasing ionophore concentrations to grow at 100-fold higher concentrations of tetronasin than were initially lethal to the organism. Gram-negative bacteria were more resistant and generally able to adapt to grow in the presence of both ionophores. An in vivo trial with cattle and in vitro growth experiments indicated that the effect of tetronasin on ciliate protozoa was minor. In vitro experiments measuring hydrogen production by Neocallimastix frontalis suggested that this fungus would be unable to survive in ruminants receiving tetronasin.

Animals↗

PubMed1988

Enumeration and isolation of cellulolytic and hemicellulolytic bacteria from human feces.

The fibrolytic microbiota of the human large intestine was examined to determine the numbers and types of cellulolytic and hemicellulolytic bacteria present. Fecal samples from each of five individuals contained bacteria capable of degrading the hydrated cellulose in spinach and in wheat straw pretreated with alkaline hydrogen peroxide (AHP-WS), whereas degradation of the relatively crystalline cellulose in Whatman no. 1 filter paper (PMC) was detected for only one of the five samples. The mean concentration of cellulolytic bacteria, estimated with AHP-WS as a substrate, was 1.2 X 10(8)/ml of feces. Pure cultures of bacteria isolated on AHP-WS were able to degrade PMC, indicating that interactions with other microbes were primarily responsible for previous low success rates in detecting fecal cellulolytic bacteria with PMC as a substrate. The cellulolytic bacteria included Ruminococcus spp., Clostridium sp., and two unidentified strains. The mean concentration of hemicellulolytic bacteria, estimated with larchwood xylan as a substrate, was 1.8 X 10(10)/ml of feces. The hemicellulose-degrading bacteria included Butyrivibrio sp., Clostridium sp., Bacteroides sp., and two unidentified strains, as well as four of the five cellulolytic strains. This work demonstrates that many humans harbor intestinal cellulolytic bacteria and that a hydrated cellulose source such as AHP-WS is necessary for their consistent detection and isolation.

Adult↗