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PubMed2001

Diet-dependent shifts in the bacterial population of the rumen revealed with real-time PCR.

A set of PCR primers was designed and validated for specific detection and quantification of Prevotella ruminicola, Prevotella albensis, Prevotella bryantii, Fibrobacter succinogenes, Selenomonas ruminantium-Mitsuokella multiacida, Streptococcus bovis, Ruminococcus flavefaciens, Ruminobacter amylophilus, Eubacterium ruminantium, Treponema bryantii, Succinivibrio dextrinosolvens, and Anaerovibrio lipolytica. By using these primers and the real-time PCR technique, the corresponding species in the rumens of cows for which the diet was switched from hay to grain were quantitatively monitored. The dynamics of two fibrolytic bacteria, F. succinogenes and R. flavefaciens, were in agreement with those of earlier, culture-based experiments. The quantity of F. succinogenes DNA, predominant in animals on the hay diet, fell 20-fold on the third day of the switch to a grain diet and further declined on day 28, with a 57-fold reduction in DNA. The R. flavefaciens DNA concentration on day 3 declined to approximately 10% of its initial value in animals on the hay diet and remained at this level on day 28. During the transition period (day 3), the quantities of two ruminal prevotella DNAs increased considerably: that of P. ruminicola increased 7-fold and that of P. bryantii increased 263-fold. On day 28, the quantity of P. ruminicola DNA decreased 3-fold, while P. bryantii DNA was still elevated 10-fold in comparison with the level found in animals on the initial hay diet. The DNA specific for another xylanolytic bacterium, E. ruminantium, dropped 14-fold during the diet switch and was maintained at this level on day 28. The concentration of a rumen spirochete, T. bryantii, decreased less profoundly and stabilized with a sevenfold decline by day 28. The variations in A. lipolytica DNA were not statistically significant. After an initial slight increase in S. dextrinosolvens DNA on day 3, this DNA was not detected at the end of the experiment. S. bovis DNA displayed a 67-fold increase during the transition period on day 3. However, on day 28, it actually declined in comparison with the level in animals on the hay ration. The amount of S. ruminantium-M. multiacida DNA also increased eightfold following the diet switch, but stabilized with only a twofold increase on day 28. The real-time PCR technique also uncovered differential amplification of rumen bacterial templates with the set of universal bacterial primers. This observation may explain why some predominant rumen bacteria have not been detected in PCR-generated 16S ribosomal DNA libraries.

Animal Feed↗

PubMed2002

Extensive set of 16S rRNA-based probes for detection of bacteria in human feces.

For the detection of six groups of anaerobic bacteria in human feces, we designed seven new 16S rRNA-based oligonucleotide probes. This set of probes extends the current set of probes and gives more data on the composition of the human gut flora. Probes were designed for Phascolarctobacterium and relatives (Phasco741), Veillonella (Veil223), Eubacterium hallii and relatives (Ehal1469), Lachnospira and relatives (Lach571), and Eubacterium cylindroides and relatives (Ecyl387), and two probes were designed for Ruminococcus and relatives (Rbro730 and Rfla729). The hybridization conditions for the new probes were optimized for fluorescent in situ hybridization, and the probes were validated against a set of reference organisms. The probes were applied to fecal samples of 11 volunteers to enumerate their target bacterial groups. The Phasco741 and Veil223 probes both detected average numbers below 1% of the total number of bacteria as determined with the bacterial kingdom-specific Bact338 probe. The Ecyl387 probe detected about 1.4%, the Lach571 and Ehal1469 probes detected 3.8 and 3.6%, respectively, and a combination of the Rbro730 and Rfla729 probes detected 10.3%. A set of 15 probes consisting of probes previously described and those presented here were evaluated in hybridization with the fecal samples of the same volunteers. Together, the group-specific probes detected 90% of the total bacterial cells.

Bacteria↗

PubMed2005

Culture-dependent and -independent methods to investigate the microbial ecology of Italian fermented sausages.

In this study, the microbial ecology of three naturally fermented sausages produced in northeast Italy was studied by culture-dependent and -independent methods. By plating analysis, the predominance of lactic acid bacteria populations was pointed out, as well as the importance of coagulase-negative cocci. Also in the case of one fermentation, the fecal enterocci reached significant counts, highlighting their contribution to the particular transformation process. Yeast counts were higher than the detection limit (> 100 CFU/g) in only one fermented sausage. Analysis of the denaturing gradient gel electrophoresis (DGGE) patterns and sequencing of the bands allowed profiling of the microbial populations present in the sausages during fermentation. The bacterial ecology was mainly characterized by the stable presence of Lactobacillus curvatus and Lactobacillus sakei, but Lactobacillus paracasei was also repeatedly detected. An important piece of evidence was the presence of Lactococcus garvieae, which clearly contributed in two fermentations. Several species of Staphylococcus were also detected. Regarding other bacterial groups, Bacillus sp., Ruminococcus sp., and Macrococcus caseolyticus were also identified at the beginning of the transformations. In addition, yeast species belonging to Debaryomyces hansenii, several Candida species, and Willopsis saturnus were observed in the DGGE gels. Finally, cluster analysis of the bacterial and yeast DGGE profiles highlighted the uniqueness of the fermentation processes studied.

Bacteria↗

PubMed2005

Genetic diversity of viable, injured, and dead fecal bacteria assessed by fluorescence-activated cell sorting and 16S rRNA gene analysis.

A novel approach combining a flow cytometric in situ viability assay with 16S rRNA gene analysis was used to study the relationship between diversity and activity of the fecal microbiota. Simultaneous staining with propidium iodide (PI) and SYTO BC provided clear discrimination between intact cells (49%), injured or damaged cells (19%), and dead cells (32%). The three subpopulations were sorted and characterized by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene amplicons obtained from the total and bifidobacterial communities. This analysis revealed that not only the total community but also the distinct subpopulations are characteristic for each individual. Cloning and sequencing of the dominant bands of the DGGE patterns showed that most of clones retrieved from the live, injured, and dead fractions belonged to Clostridium coccoides, Clostridium leptum, and Bacteroides. We found that some of the butyrate-producing related bacteria, such as Eubacterium rectale and Eubacterium hallii, were obviously viable at the time of sampling. However, amplicons affiliated with Bacteroides and Ruminococcus obeum- and Eubacterium biforme-like bacteria, as well as Butyrivibrio crossotus, were obtained especially from the dead population. Furthermore, some bacterial clones were recovered from all sorted fractions, and this was especially noticeable for the Clostridium leptum cluster. The bifidobacterial phylotypes identified in total samples and sorted fractions were assigned to Bifidobacterium adolescentis, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium pseudocatenulatum, and Bifidobacterium bifidum. Phylogenetic analysis of the live, dead, and injured cells revealed a remarkable physiological heterogeneity within these bacterial populations; B. longum and B. infantis were retrieved from all sorted fractions, while B. adolescentis was recovered mostly from the sorted dead fraction.

Adult↗

PubMed1966

Importance of the isovalerate carboxylation pathway of leucine biosynthesis in the rumen.

Certain anaerobic ruminal bacteria synthesize the leucine carbon skeleton by use of a pathway different from that described in other microorganisms. These organisms carboxylate the intact carbon skeleton of isovalerate, synthesizing leucine-2-C(14) from isovalerate-1-C(14). Strains of Bacteroides ruminicola and Peptostreptococcus elsdenii were like Ruminococcus flavefaciens in that they incorporated appreciable amounts of C(14) from isovalerate-1-C(14) into cellular protein and in that the only labeled amino acid found was leucine. The specific activity of beta-isopropylmalate dehydrogenase in extracts from R. flavefaciens and from the mixed bacterial population from the rumen was very low as compared with the specific activity of this enzyme in extracts from Escherichia coli. This suggests that the pathway of leucine biosynthesis that operates in many aerobic and facultative microorganisms is not the major pathway in rumen bacteria. This was supported by the finding that after fermentation of whole rumen contents with acetate-2-C(14), leucine from the bacterial cells had a specific activity lower than one would expect if acetate was incorporated directly into carbons 1 and 2 of leucine.

Animals↗

PubMed1967

Rate of isolated hemicellulose degradation and utilization by pure cultures of rumen bacteria.

Rate studies on the utilization or degradation (or both) of isolated hemicelluloses were conducted with six strains of rumen cellulolytic bacteria. Utilization was estimated by total pentose loss, and degradation values were based on solubilization of the hemicellulose in acidified 80% ethyl alcohol. With the various strains of ruminococci, degradation of flax and fescue grass hemicellulose was near the maximum within the first 12 hr of incubation. However, where applicable, the rates of utilization were considerably slower. Both degradation and utilization of corn hull hemicellulose occurred at much slower rates than observed with the other two substrates. With flax and fescue grass hemicellulose, the rates of degradation did not appear to be influenced by the organism's ability, or inability, to utilize the substrate as an energy source. The rates and extent of isolated hemicellulose degradation and utilization were compared between the cellulolytic ruminococci and three strains of bacteria isolated from the rumen with a xylan medium. Similar values were obtained with both types of bacteria. These observations would suggest that the cellulolytic ruminococci may be important in the overall fermentation of forage hemicelluloses in the rumen. The acidified 80% ethyl alcohol supernatant fluids, obtained from fermentations of isolated fescue grass hemicellulose by two strains of Ruminococcus flavefaciens, of which only one was able eventually to utilize the substrate, were investigated by thin-layer chromatography. Results indicated that soluble oligosaccharides were produced, which were observed to disappear gradually with time in fermentations with the utilizing strain and to accumulate in fermentations with the nonutilizing strain. Examination of the acidified 80% ethyl alcohol-insoluble residue hydrolysates, obtained from fermentations with the utilizing strain, revealed that the concentration of all the constituent sugars decreased uniformly.

Journal Article↗

PubMed1972

Fermentation of isolated pectin and pectin from intact forages by pure cultures of rumen bacteria.

Studies on the rate and extent of galacturonic acid and isolated pectin digestion were carried out with nine strains of rumen bacteria (Butyrivibrio fibrisolvens H10b and D16f, Bacteroides ruminicola 23 and D31d, Lachnospira multiparus D15d, Peptostreptococcus sp. D43e, B. succinogenes A3c, Ruminococcus flavefaciens B34b, and R. albus 7). Only three strains, 23, D16f, and D31d, utilized galacturonic acid as a sole energy source, whereas all strains except A3c and H10b degraded (solubilized) and utilized purified pectin. Nutrient composition of the basal medium and separate sterilization of the substrate affected the rate and extent of fermentation for both substrates. Pectin degradation and utilization were measured with two maturity stages each of intact bromegrass and alfalfa. For bromegrass I, all strains tested (B34b, 23, D16f, D31d, D15d, and D43e) degraded a considerable amount of pectin and, with the exception of B34b, utilized most of what was degraded. Similar, but lower, results were obtained with bromegrass II, except for the two strains of B. ruminicola, 23 and D31d, which were unable to degrade and utilize pectin from this forage. All strains were able to degrade and utilize pectin from both maturity stages of alfalfa; however, values were considerably lower for strains 23 and D31d. Synergism studies, in which a limited utilizing strain, B34b, was combined with the limited degrading strain, D31d, resulted in a slight increase in degradation and a very marked increase in utilization of the pectin in all four forages. Similar results were obtained on both alfalfa substrates with a combination of strains B34b and D16f; however, no increases were observed with this combination on bromegrass.

Anaerobiosis↗

PubMed1973

An anaerobic chemostat that permits the collection and measurement of fermentation gases.

A chemostat was designed to allow anaerobic growth in the culture vessel in the absence of a continuous stream of O(2)-free gas. Produced gases were collected within the culture and collection vessels, and pressure build-up was prevented by allowing gases to expand into a collapsed football bladder. The culture overflow was collected in a flask, held at 0 C, that was emptied by applying a positive CO(2) pressure to the system. Ruminococcus albus, a H(2) and CO(2)-producing anaerobe, was used to test the operation of the apparatus. H(2) production was measured by sampling the various gas spaces of known volume and measuring H(2) concentration by gas chromatography. Measurement of accumulated fermentation gases and the effects of the accumulation on fermentations can be studied with the apparatus.

Anaerobiosis↗

PubMed1974

Predominant gram-positive bacteria in human feces: numbers, variety, and persistence.

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.

Adult↗

PubMed1974

Tryptophan biosynthesis from indole-3-acetic acid by anaerobic bacteria from the rumen.

Microbes in ruminal contents incorporated (14)C into cells when they were incubated in vitro in the presence of [(14)C]carboxyl-labeled indole-3-acetic acid (IAA). Most of the cellular (14)C was found to be in tryptophan from the protein fractions of the cells. Pure cultures of several important ruminal species did not incorporate labeled IAA, but all four strains of Ruminococcus albus tested utilized IAA for tryptophan synthesis. R. albus did not incorporate (14)C into tryptophan during growth in medium containing either labeled serine or labeled shikimic acid. The mechanism of tryptophan biosynthesis from IAA is not known but appears to be different from any described biosynthetic pathway. We propose that a reductive carboxylation, perhaps involving a low-potential electron donor such as ferredoxin, is involved.

Anaerobiosis↗

PubMed1990

Cloning and sequencing of a Bacteroides ruminicola B(1)4 endoglucanase gene.

Bacteroides ruminicola B(1)4, a noncellulolytic rumen bacterium, produces an endoglucanase (carboxymethylcellulase [CMCase]) that is excreted into the culture supernatant. Cultures grown on glucose, fructose, maltose, mannose, and cellobiose had high specific activities of CMCase (greater than 3 mmol of reducing sugar per mg of protein per min), but its synthesis was repressed by sucrose. B. rumincola did not grow on either ball-milled or acid-swollen cellulose even though the CMCase could hydrolyze swollen cellulose. The CMCase gene was cloned into Escherichia coli, and its nucleotide sequence contained a single open reading frame coding for a protein of 40,481 daltons. The enzyme was overproduced in E. coli under the control of the tac promoter and purified to homogeneity. The N-terminal sequence, amino acid composition, and molecular weight of the purified enzyme were similar to the values predicted from the open reading frame of the DNA sequence. However, the CMCase present in B. ruminicola was found to have a monomer molecular weight of 88,000 by Western immunoblotting. This discrepancy appeared to have resulted from our having cloned only part of the CMCase gene into E. coli. The amino acid sequence of the CMCase showed homology to sequences of beta-glucanases from Ruminococcus albus and Clostridium thermocellum.

Amino Acid Sequence↗

PubMed1993

The xynC gene from Fibrobacter succinogenes S85 codes for a xylanase with two similar catalytic domains.

The xynC gene of Fibrobacter succinogenes S85 codes for a 66.4-kDa xylanase which consists of three distinct domains separated by two flexible regions rich in serine residues. Domains A and B of XynC code for catalytic domains with 56.5% identity and 9.6% similarity with each other, and both domains share homology with xylanases of Ruminococcus flavefaciens, Neocallimastix patriciarum, Clostridium acetobutylicum, Bacillus pumilus, Bacillus subtilis, and Bacillus circulans. More than 88% of the xylanase activity of Escherichia coli cells carrying the original 13-kb recombinant plasmid was released from intact cells by cold water washes. The major products of hydrolysis of xylan by both domains were xylose and xylobiose, indicating that the xynC gene product exhibits catalytic properties similar to those of the XynA xylanases from R. flavefaciens and N. patriciarum. So far, these features are not shared broadly with bacteria from other environments and may indicate specific selection for this domain structure in the highly competitive environment of the rumen.

Amino Acid Sequence↗

PubMed1966

Enzymatic studies of pure cultures of rumen microorganisms.

Joyner, A. E., Jr. (University of California, Davis), and R. L. Baldwin. Enzymatic studies of pure cultures of rumen microorganisms. J. Bacteriol. 92:1321-1330. 1966.-The activities of enzymes representing the major pathways of carbohydrate metabolism and anaerobic electron transport in cell-free extracts of whole rumen contents have been reported. The effects of diet upon the activities of several enzymes suggested that enzymatic measurements might prove useful for the study of rumen metabolism. In the present study, the distribution and characteristics of aldolase, succinate dehydrogenase, glutamate dehydrogenase, lactyl-coenzyme A dehydrase, lactate dehydrogenase, and other enzymes were measured in cell-free extracts of pure cultures of Ruminococcus flavefaciens, R. albus, Bacteroides succinogenes, B. ruminicola, B. amylophilus, Butyrivibrio fibrisolvens, Peptostreptococcus elsdenii, Streptococcus bovis, and Selenomonas ruminantium. Some enzymes were widely distributed (aldolase, glutamate dehydrogenase), whereas others were observed in one or two species (lactyl-coenzyme A dehydrase). The cofactor requirements and kinetic characteristics of enzymes varied considerably with species. Enzymes that vary with species might be employed as indices for estimating the activities of various groups of microorganisms in whole rumen contents.

Animals↗

PubMed1967

Volatile fatty acid requirements of cellulolytic rumen bacteria.

A gas chromatographic method was developed which could separate the isomers isovaleric and 2-methylbutyric acid. Subsequent analyses revealed that most commercially available samples of these acids were cross-contaminated; however, one sample of each acid was found to be pure by this criterion. The growth response of seven strains of cellulolytic rumen bacteria (three strains of Bacteroides succinogenes, three strains of Ruminococcus flavefaciens, and one strain of R. albus) to additions of isobutyric, isovaleric, 2-methylbutyric, valeric, and combinations of valeric and a branched-chain acid was determined. Strains of B. succinogenes required a combination of valeric plus either isobutyric or 2-methylbutyric acid. Isovaleric acid was completely inactive. Either isobutyric or 2-methylbutyric acid was required for the growth of R. albus 7. Strain C-94 of R. flavefaciens grew slowly in the presence of any one of the three branched-chain acids, but a combination of isobutyric and 2-methylbutyric acids appeared to satisfy this organism's growth requirements. None of the individual acids or mixtures of straight- and branched-chain acids allowed growth of R. flavefaciens strain C1a which would approach the response obtained from the total mixture of acids. Further work indicated that all three branched-chain acids were required for optimal growth by this strain, although isovaleric acid only influenced the rate of maximal growth. Either 2-methylbutyric or isovaleric acid allowed growth of nearly the same magnitude as that of the positive control for R. flavefaciens B34b. The presence of acetic acid had little influence on the rate or extent of growth of any of the strains except R. albus 7, for which the extent of growth was markedly increased. Determination of the quantitative fatty acid requirements for the three B. succinogenes strains indicated that 0.1 mumole of valeric per ml and 0.05 mumole of 2-methylbutyric per ml permitted maximal growth. However, with isobutyric acid as the branched-chain component, strains A3c and B21a required 0.1 mumole/ml in contrast to S-85 which exhibited optimal growth at the 0.05 mumole/ml level. By use of mixtures of isobutyric and 2-methylbutyric acids, good growth of C-94 was obtained at concentrations of 0.1 and 0.01 mumole/ml, respectively. About 0.3 mumole/ml of each acid was required for satisfactory growth of C1a.

Animals↗

PubMed2026

Resistant starch types 2 and 4 induce distinct and reversible changes in the human gut microbiome.

Resistant starch (RS) can confer benefits for the gut microbiome and host cardiometabolic health. However, different types of resistant starch can differentially affect gut microbiome composition and functional capacity, especially given interindividual variability in responses, thus limiting the application of resistant starch in dietary strategies. We used shotgun metagenomics to perform a secondary analysis of samples collected during a previously reported randomized clinical trial to determine the effects of dietary supplementation with two types of resistant starch (RS2 and RS4) and a digestible starch (control) on the gut microbiome. Both resistant starch types induced distinct but transient alterations in the gut microbial community. RS2 enriched the keystone degrader, Ruminococcus bromii, and Blautia glucerasea, whereas RS4 favored Parabacteroides distasonis and known but uncharacterized microbial species such as a Lachnospiraceae bacterium. Moreover, we detected strain-level differences in the response of Bifidobacterium adolescentis to resistant starch. Microbial functional profiling revealed an enhanced capacity for complex carbohydrate utilization following resistant starch intake, including increased abundance of specific α-amylases, glycoside hydrolases, starch utilization systems, and other currently uncharacterized genes. Identifying the bacterial strains and genes that respond to different RS types will help to more accurately predict who will benefit from a given RS type. Our findings demonstrate that RS2 and RS4 differentially shape microbial ecology and metabolic capacity and provide a foundation for microbiome-informed personalization of resistant starch-based dietary interventions.IMPORTANCEDietary intake influences human health by modulating metabolism, partly by shaping the microbiota inhabiting the gut. Resistant starch (RS), a dietary fiber, is associated with metabolic improvements. While previous research has explored how RS alters the gut microbiome, RS comprises five types with differing physical and chemical characteristics, and the distinct impacts of each type on the microbiome and host health have not been fully characterized, particularly using high-resolution approaches such as shotgun metagenomics. In this secondary analysis of samples from a longitudinal crossover intervention study, we link dietary supplementation with RS2 and RS4 with distinct and transient changes in the composition and functional potential of the human gut microbiome. Specifically, we identify species that increase in abundance with each RS type, accompanied by increases in genes and pathways involved in complex carbohydrate utilization. The findings support the development of precision nutrition strategies utilizing RS supplementation to improve metabolic health.This study is registered with ClinicalTrials.gov as NCT05743790.

Humans↗

PubMed2026

Multi-level aggregation analysis of microbiome composition and host gene expression reveals associations with systemic and local immunity.

The human gut microbiome plays a critical role in immune regulation, yet the molecular links between microbiome composition and host gene expression remain incompletely understood. We analyzed associations between host gene expression and microbiome composition in a cohort of 315 healthy individuals, integrating microarray-based gene expression data from three intestinal sites (ileum, transverse colon, and rectum) and six immune cell types with microbiome sequencing data. Using a hierarchical feature aggregation strategy combining principal component analysis, clustering, and covariate correction, we discovered significant associations primarily related to immunity. While microbial profiles were similar across the three intestinal sites, the transverse colon yielded the most "microbiome-host gene expression" associations. Among the immune cell types, CD8+ cells showed the highest number of associations. The first principal component of microbiome composition, reflecting a gradient from commensals (e.g., Ruminococcaceae and Christensenellaceae) to proinflammatory taxa ([Ruminococcus] gnavus and Lachnoclostridium), correlated with the expression of TNF-α-linked genes (HMOX1, CPI17, HSD3B2, and SLC5A1). Among individual genera, Catenibacterium abundance was associated with gene expression in both intestinal and immune cells, including negative associations with MRPS21 (related to mitochondrial function) in the transverse colon and with CD8+ gene programs related to T cell differentiation. These findings align with emerging evidence implicating mitochondrial dysfunction in intestinal inflammation. Our results identify multi-level associations between the gut microbiome and host gene expression, suggesting potential mechanisms by which microbiota shape local and systemic immunity and vice versa. The implicated genes and taxa represent candidates for experimental validation to improve understanding of host-microbiome homeostasis and its disruption in disease.IMPORTANCEThe gut microbiome and immune system are engaged in a complex interplay throughout human life. While most associative studies focus on case-control comparisons-typically examining patients with conditions such as inflammatory bowel disease or metabolic diseases-less is known about the molecular links between the microbiome and immune system in healthy individuals. In this study of a large cohort of healthy individuals, we addressed this gap by applying multiscale modeling to tackle the high dimensionality of host-microbiome data. We identified multi-level associations between microbiome composition and host gene expression in both intestinal tissues and immune cells. These findings offer a valuable reference for understanding baseline host-microbiome communication and highlight molecular candidates-such as TNF-α-related genes and mitochondrial pathways-for future experimental validation.

Humans↗

PubMed2004

Lactic acid bacteria secrete metabolites retaining anti-inflammatory properties after intestinal transport.

BACKGROUND: Probiotic bacteria have a beneficial effect on intestinal inflammation. In this study, we have examined the effect of lactic acid and commensal Gram positive (+) bacteria conditioned media (CM) on tumour necrosis factor alpha (TNF-alpha) release and the mechanisms involved. METHODS: Lipopolysaccharide (LPS) induced TNF-alpha secretion by peripheral blood mononuclear cells or the THP-1 cell line was monitored in the presence or absence of bacteria CM obtained from two probiotic strains, Bifidobacterium breve (Bb) and Streptococcus thermophilus (St), and three commensal bacterial strains (Bifidobacterium bifidum, Ruminococcus gnavus, and unidentified Streptococcus). Bb and St bacteria CM were allowed to cross filter grown intestinal epithelial cell monolayers (HT29-19A) to assess intestinal transport of active bacterial products. These products were characterised and their effect on LPS binding to THP-1 cells and nuclear factor kappa B (NF kappa B) activation assessed. RESULTS: Dose dependent inhibition of LPS induced TNF-alpha secretion was noted for both probiotic bacteria CM (64% and 71% inhibition for Bb and St, respectively) and to a lesser extent commensal bacteria CM (21-32% inhibition). Active products from Bb and St were resistant to digestive enzymes and had a molecular mass <3000 Da. Their inhibitory effect was preserved after transepithelial transport across intestinal cell monolayers, mainly in inflammatory conditions. LPS-FITC binding to THP-1 cells and NF kappa B activation were significantly inhibited by Bb and St CM. CONCLUSION: B breve and S thermophilus release metabolites exerting an anti-TNF-alpha effect capable of crossing the intestinal barrier. Commensal bacteria also display a TNF-alpha inhibitory capacity but to a lesser extent. These results underline the beneficial effect of commensal bacteria in intestinal homeostasis and may explain the role of some probiotic bacteria in alleviating digestive inflammation.

Bifidobacterium↗

PubMed1978

Effects of heavy metals and other trace elements on the fermentative activity of the rumen microflora and growth of functionally important rumen bacteria.

The inhibitory effects of high concentrations of essential and non-essential trace elements were tested on the rumen microflora using the rate of fermentation in vitro as the assay. The elements (and the concentration causing 50% inhibition) in decreasing order of toxicity were Hg2+ (20 microgram/ml), Cu2+ (21 microgram/ml), Cr6+ (70 microgram/ml), Se4+ (73 microgram/ml), Ni2+ (160 microgram/ml), Cd2+ (175 microgram/ml), As3+ (304 microgram/ml) and As5+ (1610 microgram/ml). The elements tested that were either weak or noninhibitory at concentrations greater than 400 microgram/ml included Zn2+, Cr2+, Fe2+, Mn2+, Pb2+, and Co2+. Methylmercury was as inhibitory as mercuric chloride to the fermentation. When the inhibitory effect of Cd2+ was tested on separated bacterial and protozoal fractions, it was more inhibitory to the bacteria. The inhibitory effects of trace elements were also determined for a number of axenic cultures of rumen bacteria. The bacteria which most frequently exhibited the greatest sensitivity were Bacteroides succinogenses, Ruminococcus albus, Bacteroides amylophilus, and Eubacterium ruminantium. Those often exhibiting intermediate sensitivities included Butyrivibrio fibrisolvens, Selenomonas ruminantium, and Megasphera elsdenii, while Streptococcus bovis was very refractory to all elements tested. Rumen fluid provided a modest protective effect for the bacteria.

Animals↗