Human colonic microbiota associated with diet, obesity and weight loss.

BACKGROUND: It has been proposed that the development of obesity in humans is influenced by the relative proportions of the two major phyla of bacteria (Bacteroidetes and Firmicutes) present in the large intestine. OBJECTIVE: To examine the relationships between body mass index, weight loss and the major bacterial groups detected in fecal samples. DESIGN: Major groups of fecal bacteria were monitored using fluorescent in situ hybridization (FISH) in obese and non-obese subjects under conditions of weight maintenance, and in obese male volunteers undergoing weight loss on two different reduced carbohydrate weight-loss diets given successively for 4 weeks each. RESULTS: We detected no difference between obese and non-obese individuals in the proportion of Bacteroidetes measured in fecal samples, and no significant change in the percentage of Bacteroidetes in feces from obese subjects on weight loss diets. Significant diet-dependent reductions in a group of butyrate-producing Firmicutes were, however, detected in fecal samples from obese subjects on weight loss diets. CONCLUSIONS: Diets designed to achieve weight loss in obese subjects can significantly alter the species composition of the gut microbiota, but we find no evidence that the proportions of Bacteroidetes and Firmicutes among fecal bacteria have a function in human obesity.

Understanding the effects of diet on bacterial metabolism in the large intestine.

Recent analyses of ribosomal RNA sequence diversity have demonstrated the extent of bacterial diversity in the human colon, and have provided new tools for monitoring changes in the composition of the gut microbial community. There is now an excellent opportunity to correlate ecological niches and metabolic activities with particular phylogenetic groups among the microbiota of the human gut. Bacteria that associate closely with particulate material and surfaces in the gut include specialized primary degraders of insoluble substrates, including resistant starch, plant structural polysaccharides and mucin. Butyrate-producing bacteria found in human faeces belong mainly to the clostridial clusters IV and XIVa. In vitro and in vivo evidence indicates that a group related to Roseburia and Eubacterium rectale plays a major role in mediating the butyrogenic effect of fermentable dietary carbohydrates. Additional cluster XIVa species can convert lactate to butyrate, while some members of the clostridial cluster IX convert lactate to propionate. The metabolic outputs of the gut microbial community depend not only on available substrate, but also on the gut environment, with pH playing a major role. Better understanding of the colonic microbial ecosystem will help to explain and predict the effects of dietary additives, including nondigestible carbohydrates, probiotics and prebiotics.

Cultivable bacterial diversity from the human colon.

Knowledge of the composition of the colonic microbiota is important for our understanding of how the balance of these microbes is influenced by diet and the environment, and which bacterial groups are important in maintaining gut health or promoting disease. Molecular methodologies have advanced our understanding of the composition and diversity of the colonic microbiota. Importantly, however, it is the continued isolation of bacterial representatives of key groups that offers the best opportunity to conduct detailed metabolic and functional studies. This also permits bacterial genome sequencing which will accelerate the linkage to functionality. Obtaining new human colonic bacterial isolates can be challenging, because most of these are strict anaerobes and many have rather exact nutritional and physical requirements. Despite this many new species are being isolated and described that occupy distinct niches in the colonic microbial community. This review focuses on these under-studied yet important gut anaerobes.

Dietary effects on the microbiological safety of food.

The high mortality rate associated with human infections caused by Escherichia coli strains of the serotype O157:H7 has brought to public attention the importance of ruminants as reservoirs of food-borne pathogens. In addition to established examples such as salmonella, campylobacter and listeria, recent evidence is emerging of the role of food in the transmission of Helicobacter pylori and Mycobacterium paratuberculosis. Food-borne pathogens harboured by ruminants are spread through shedding in the faeces and subsequent faecal contamination of raw food. Ruminant shedding appears to be affected by diet and, of particular concern, may be increased during fasting regimens imposed during transport to the slaughterhouse. The survival of food-borne pathogens in the ruminant gut is affected by many factors including microbe-microbe interactions, interactions involving plant metabolites and the presence of inhibitory end-product metabolites such as short-chain fatty acids. The potential importance of digesta flow and bacterial detachment in shedding of food-borne pathogens is discussed. Experimental procedures with dangerous pathogens have constraints, particularly in animal experimentation. This situation may be overcome by the use of rumen-simulating fermentors. One such system which, like the natural rumen, has a different turnover rate for solid and liquid digesta, was found to maintain rumen-like variables over an 11 d period. This system may prove useful for the study of dietary effects on food-borne pathogens.

The potential for the control of Escherichia coli O157 in farm animals.

The presence of Escherichia coli O157 in the faeces of farm animals appears to provide a primary route for human infection, either through physical contact or by contamination of the food chain. Controlling the survival and proliferation of this pathogen in the ruminant gut could offer a measure of protection in the short term, and ultimately complement alternative biotechnological based solutions. Normally, E. coli is greatly outnumbered in the ruminant gut by anaerobic bacteria, producers of weak acids inhibitory to the growth of this species. Withdrawal of feed prior to animal slaughter reduces the concentration of these acids in the gut and may be accompanied by the proliferation of E. coli. There are conflicting reports concerning the effects of changes in the ruminant diet upon faecal shedding of E. coli O157. It is contended that it is important to identify animal husbandry methods or feed additives that may be accompanied by an increased risk of proliferation of this pathogen. Greater understanding of the mechanisms involved in bacterial survival in the presence of weak acids, in the interactions between E. coli and other gut bacteria, and of the effects of some antibacterial plant secondary plant compounds on E. coli, could lead to the development of novel control methods.

Phylogenetic relationships of butyrate-producing bacteria from the human gut.

Butyrate is a preferred energy source for colonic epithelial cells and is thought to play an important role in maintaining colonic health in humans. In order to investigate the diversity and stability of butyrate-producing organisms of the colonic flora, anaerobic butyrate-producing bacteria were isolated from freshly voided human fecal samples from three healthy individuals: an infant, an adult omnivore, and an adult vegetarian. A second isolation was performed on the same three individuals 1 year later. Of a total of 313 bacterial isolates, 74 produced more than 2 mM butyrate in vitro. Butyrate-producing isolates were grouped by 16S ribosomal DNA (rDNA) PCR-restriction fragment length polymorphism analysis. The results indicate very little overlap between the predominant ribotypes of the three subjects; furthermore, the flora of each individual changed significantly between the two isolations. Complete sequences of 16S rDNAs were determined for 24 representative strains and subjected to phylogenetic analysis. Eighty percent of the butyrate-producing isolates fell within the XIVa cluster of gram-positive bacteria as defined by M. D. Collins et al. (Int. J. Syst. Bacteriol. 44:812-826, 1994) and A. Willems et al. (Int. J. Syst. Bacteriol. 46:195-199, 1996), with the most abundant group (10 of 24 or 42%) clustering with Eubacterium rectale, Eubacterium ramulus, and Roseburia cecicola. Fifty percent of the butyrate-producing isolates were net acetate consumers during growth, suggesting that they employ the butyryl coenzyme A-acetyl coenzyme A transferase pathway for butyrate production. In contrast, only 1% of the 239 non-butyrate-producing isolates consumed acetate.

Characteristics of sheep-rumen isolates of Pseudomonas aeruginosa inhibitory to the growth of Escherichia coli O157.

Screening facultative sheep-rumen bacteria which inhibit growth of Escherichia coli produced 11 strains of Pseudomonas aeruginosa. The isolates showed three different pulsed-field gel electrophoresis patterns and strains from different sheep produced pyocins that varied in strain specificity. Representative strains were resistant to ampicillin, methicillin, erythromycin, fusidic acid and augmentin, but not to tetracycline or nalidixic acid. Tested strains attached in large numbers to cultured rumen epithelial cells, potentially providing a means of survival in this ecosystem.

Inhibitory activity of gut bacteria against Escherichia coli O157 mediated by dietary plant metabolites.

Under both aerobic and anaerobic conditions, the growth of Escherichia coli O157 strain NCTC 12,900 was inhibited by the coumarins esculetin, umbelliferone and scopoletin, but not by the coumarin glycoside esculin. Esculin-hydrolysing bacteria from the rumen, the pig gut and the human gut inhibited growth of E. coli in an overlay-plate assay in the presence of esculin. The combined effect of esculetin and volatile fatty acids was greater than the effect of either factor alone suggesting that coumarin glycosides in the diet might reduce the growth or survival of E. coli O157 in the gut. Adding esculin to incubations of mixed rumen contents significantly reduced the survival of E. coli O157.

Effects of Coumarin and Sparteine on Attachment to Cellulose and Cellulolysis by Neocallimastix frontalis RE1.

The plant secondary metabolites coumarin and sparteine reduced attachment to cellulose, cellulose solubilization, and the proportion of lactate in the fermentation products of the anaerobic fungus Neocallimastix frontalis RE1. Neither compound directly inhibited the endoglucanase or lactate dehydrogenase activities of cell extracts of the fungus.

Isolation and characteristics of a wheatbran-degrading Butyrivibrio from human faeces.

Screening over 100 isolates from human faeces for cellulolytic activity led to the isolation of a weakly cellulolytic anaerobic, curved, motile bacterium which produced H2, lactate and butyrate from wheatbran. The mol% of G + C in the DNA was 39-42. These properties, together with the Gram-positive cell wall ultrastructure and SDS-PAGE profile, are consistent with the genus Butyrivibrio. The isolate is believed to be the most active wheatbran-degrading bacterium so far described.

Interactions between rumen bacterial strains during the degradation and utilization of the monosaccharides of barley straw cell-walls.

Pure cultures and pair-combinations of strains representative of the rumen cellulolytic species Ruminococcus flavefaciens, Fibrobacter succinogenes and Butyrivibrio fibrisolvens were grown on cell-wall materials from barley straw. Of the pure cultures, R. flavefaciens solubilized straw most rapidly. The presence of B. fibrisolvens, which was unable to degrade straw extensively in pure culture, increased the solubilization of dry matter by R. flavefaciens and the solubilization of cell-wall carbohydrates by both R. flavefaciens and F. succinogenes. During fermentation, both R. flavefaciens and F. succinogenes released bound glucose and free and bound arabinose and xylose into solution. The accumulation of these sugars, especially arabinose and xylose, was greatly reduced in co-cultures containing B. fibrisolvens, suggesting that significant interspecies cross feeding of the products of hemicellulose hydrolysis (particularly soluble bound xylose released by F. succinogenes) occurs during straw degradation by mixed cultures containing this species.

Binding and degradation of lectins by components of rumen liquor.

The binding of 15 different plant lectins to feed particles and microbes in rumen liquor, and their degradation were studied in vitro. The rate of degradation assessed from the label released when radioactive iodine-labelled lectins were incubated with rumen liquor conflicted with the rates calculated from measurements of the survival of the antigenic structure (immuno-rocket electrophoresis) or the biological function (haemagglutination) of the lectins. Thus solubilization of the radioactive label indicated that Concanavalin A (Con A), but not the soyabean agglutinin, SBA, or kidney bean phytohaemagglutinin, PHA-E3L, was stable to rumen proteolysis. In contrast, both SBA and PHA-E3L were shown by immuno-rocket electrophoresis or haemagglutination tests to be highly resistant to breakdown, while the degradation of Con A proceeded at a constant slow rate under the same conditions. This was in accord with the previously established general stability of lectins in the gut of single-stomach animals. Of the 15 lectins, SBA, favin (Vicia faba lectin) and Con A were bound by hay and the particle fraction of rumen liquor. This was, in part, specific and reversible in the presence of appropriate sugars. Most pure bacterial strains preferentially bound lectins with specificity for glucose/mannose (favin and Con A), while rumen fungi reacted with SBA. The level of binding was low with other lectins. However, inter-strain differences of lectin-binding were found in Selenomonas ruminantium and Ruminococcus flavefaciens. Clearly, as some lectins were not fully degraded in the rumen, they could be expected to depress the utilization of the diet not only in single-stomach animals but, possibly, also in ruminants.

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.

Prevention of fungal colonization and digestion of cellulose by the addition of methylcellulose.

When the attachment of cellulolytic rumen fungi to cellulose is blocked by the addition of methylcellulose, cellulose digestion is entirely inhibited. Even after these fungi have colonized and penetrated the cellulosic fibers of filter paper, the addition of methylcellulose effectively halts cellulose digestion. This effect of methylcellulose is accompanied by the complete inhibition of fungal attachment to cellulose fibers; the addition of methylcellulose does not affect the growth of these organisms on soluble substrates. We conclude that fungal cellulose digestion, like bacterial cellulose digestion, requires the spatial juxtaposition of the cellulolytic organism and its insoluble substrate. The simultaneous inhibition of both attachment and digestion by the same inhibitor suggests that these two processes are functionally linked in the fungi.

The properties of forms of Ruminococcus flavefaciens which differ in their ability to degrade cotton cellulose.

Two mutant strains of Ruminococcus flavefaciens strain 007 that differ in their ability to hydrolyse cotton fibres have been shown also to differ in their cell-surface topology, in that the cotton degrading form possessed larger and more protuberant cell surface structures. The strains had similar CMCase, cellobiosidase and beta-glucosidase activities. The results indicate the importance of cell-surface properties in cotton degradation by R. flavefaciens.

Safety and efficacy of topical minoxidil in the management of androgenetic alopecia.

Of 149 subjects with androgenetic alopecia, 102 completed 1 year of a double-blind, randomized study comparing 2% minoxidil and 3% minoxidil solutions for safety and efficacy. One third of the subjects used a vehicle placebo for the first 4 months and then switched to 3% minoxidil. At 12 months the 2% minoxidil group switched to a 3% solution. During months 5 to 12 a steady increase in terminal hair counts occurred to an equal degree within the 2% and 3% minoxidil groups and the 3% treatment group switched from placebo. Total hair counts at 12 months increased from a baseline mean of 63.5 to 180.6 in the 2% treatment group, from 61.0 to 179.9 in the 3% group, and from 65.0 to 191.1 in the placebo to 3% crossover group. Although all 102 subjects completing 12 months of the study thought that visible hair growth had resulted, 89 were considered by the investigators to have visible growth. Dense hair growth, defined as hair long enough to cut or comb, was present in 48 subjects by their own evaluation and in 33 subjects by investigator evaluation. There were no serious side effects. Two instances of allergic contact dermatitis and four of pruritus were attributed to use of the drug. Two individuals complained of impotence, which disappeared within a few days of discontinuation of topical minoxidil. This effect has not been reported during the use of minoxidil in its oral form (Loniten) for the treatment of hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of ammonia treatment on the solubilization of straw and the growth of cellulolytic rumen bacteria.

Pre-treatment of straw with anhydrous ammonia increased its susceptibility to solubilization by the predominant cellulolytic bacteria from the rumen, Bacteroides succinogenes, Ruminococcus albus and R. flavefaciens. Ammonia treatment also increased the production of microbial protein and fermentation products by all three species. Scanning electron microscope observations of straw during digestion suggested that the attack of straw by these bacteria was accompanied by the formation of substantial numbers of adherent microcolonies.

Clindamycin phosphate used in combination with tretinoin in the treatment of acne.

Sixty-four patients with acne were studied to evaluate the irritancy, tolerance, and systemic absorption of topical 1% clindamycin phosphate when combined with tretinoin gel 0.025% as compared to either agent alone. All these treatment regimens were effective and well-tolerated. Clindamycin alone was less irritating than tretinoin alone or tretinoin combined with clindamycin. The combination treatment produced fewer complaints from patients than tretinoin alone. No systemic absorption of clindamycin was detected after two weeks and eight weeks of usage.

Benzoyl peroxide reactions in an acne study group.

Benzoyl peroxide patch testing was performed on 60 young adults who had participated in a double-blind acne study. 25% of those who had used a benzoyl peroxide product showed reactivity of +1 or greater (on a 0--3 scale). None of these individuals demonstrated symptoms suggestive of irritancy or allergy greater than those who did not have positive patch tests and who were on the same treatment program. The positive patch tests did not correlate with acne treatment responses. 2 of 44 individuals in the acne study group developed clinical allergy and had dramatically positive patch tests, while the majority of individuals with positive patch tests could use products containing benzoyl peroxide daily without significant adverse effects.