Colonic Anaerobes Research Articles

Prevalence of methanogens and associated factors in patients with irritable bowel syndrome and healthy controls in a Southeastern Mexican population

IntroductionMethane (CH4) is an inert gas produced by colonic anaerobes and has been associated with different intestinal diseases, including irritable bowel syndrome (IBS). According to geographic region, the prevalence of methanogens varies, being higher in Africa (80%) and lower in the United States (35-40%). In Mexico, the prevalence of methanogens is unknown. AimTo evaluate the prevalence of CH4 producers and associated factors in a group of patients with IBS and controls in a Mexican population. Materials and methodsA baseline fasting measurement of alveolar H2 and CH4 gas was carried out, by gas chromatography (stationary phase), in consecutive patients diagnosed with IBS and a control group. Subjects with baseline levels of H2 of 0 ppm and CH4 ≥ 5 ppm were classified as methanogenic. ResultsA total of 132 controls (53.8% women) and 67 patients with IBS (76% women) were included. The overall prevalence (n = 199) of methanogenic subjects was 38% (n = 76) (95% CI: 0.31-0.45) and they had a greater prevalence of overweight/obesity (56.5 vs 39.8%, P = .028). The prevalence of methanogens in the healthy controls was 41.6% (95% CI: 0.33-0.49), whereas, in the patients with IBS, it was 31.4% (n = 21, 71% IBS-C and 29% IBS-M). ConclusionsThe prevalence of methanogens in our study on a Mexican population was comparable to that reported in other populations and was associated with overweight/obesity. One-third of the patients with IBS presented with methanogens. Said microorganisms were particularlyassociated with the constipation-predominant IBS subtype.

Prevalencia de metanógenos y factores asociados en pacientes con síndrome de intestino irritable y controles sanos en una población del sureste de México

IntroductionMethane (CH4) is an inert gas produced by colonic anaerobes and has been associated with different intestinal diseases, including irritable bowel syndrome (IBS). According to geographic region, the prevalence of methanogens varies, being higher in Africa (80%) and lower in the United States (35-40%). In Mexico, the prevalence of methanogens is unknown. AimTo evaluate the prevalence of CH4 producers and associated factors in a group of patients with IBS and controls in a Mexican population. Materials and methodsA baseline fasting measurement of alveolar H2 and CH4 gas was carried out, by gas chromatography (stationary phase), in consecutive patients diagnosed with IBS and a control group. Subjects with baseline levels of H2 of 0ppm and CH4≥5ppm were classified as methanogenic. ResultsA total of 132 controls (53.8% women) and 67 patients with IBS (76% women) were included. The overall prevalence (n=199) of methanogenic subjects was 38% (n=76) (95%CI: 0.31-0.45) and they had a greater prevalence of overweight/obesity (56.5 vs 39.8%, P=.028). The prevalence of methanogens in the healthy controls was 41.6% (95%CI: 0.33-0.49), whereas, in the patients with IBS, it was 31.4% (n=21, 71% IBS-C and 29% IBS-M). ConclusionsThe prevalence of methanogens in our study on a Mexican population was comparable to that reported in other populations and was associated with overweight/obesity. One-third of the patients with IBS presented with methanogens. Said microorganisms were particularly associated with the constipation-predominant IBS subtype.

β-Glucan is a major growth substrate for human gut bacteria related to Coprococcus eutactus.

A clone encoding carboxymethyl cellulase activity was isolated during functional screening of a human gut metagenomic library using Lactococcus lactis MG1363 as heterologous host. The insert carried a glycoside hydrolase family 9 (GH9) catalytic domain with sequence similarity to a gene from Coprococcus eutactus ART55/1. Genome surveys indicated a limited distribution of GH9 domains among dominant human colonic anaerobes. Genomes of C. eutactus-related strains harboured two GH9-encoding and four GH5-encoding genes, but the strains did not appear to degrade cellulose. Instead, they grew well on β-glucans and one of the strains also grew on galactomannan, galactan, glucomannan and starch. Coprococcus comes and Coprococcus catus strains did not harbour GH9 genes and were not able to grow on β-glucans. Gene expression and proteomic analysis of C. eutactus ART55/1 grown on cellobiose, β-glucan and lichenan revealed similar changes in expression in comparison to glucose. On β-glucan and lichenan only, one of the four GH5 genes was strongly upregulated. Growth on glucomannan led to a transcriptional response of many genes, in particular a strong upregulation of glycoside hydrolases involved in mannan degradation. Thus, β-glucans are a major growth substrate for species related to C. eutactus, with glucomannan and galactans alternative substrates for some strains.

Cultured Representatives of Two Major Phylogroups of Human Colonic Faecalibacterium prausnitzii Can Utilize Pectin, Uronic Acids, and Host-Derived Substrates for Growth

Faecalibacterium prausnitzii is one of the most abundant commensal bacteria in the healthy human large intestine, but information on genetic diversity and substrate utilization is limited. Here, we examine the phylogeny, phenotypic characteristics, and influence of gut environmental factors on growth of F. prausnitzii strains isolated from healthy subjects. Phylogenetic analysis based on the 16S rRNA sequences indicated that the cultured strains were representative of F. prausnitzii sequences detected by direct analysis of fecal DNA and separated the available isolates into two phylogroups. Most F. prausnitzii strains tested grew well under anaerobic conditions on apple pectin. Furthermore, F. prausnitzii strains competed successfully in coculture with two other abundant pectin-utilizing species, Bacteroides thetaiotaomicron and Eubacterium eligens, with apple pectin as substrate, suggesting that this species makes a contribution to pectin fermentation in the colon. Many F. prausnitzii isolates were able to utilize uronic acids for growth, an ability previously thought to be confined to Bacteroides spp. among human colonic anaerobes. Most strains grew on N-acetylglucosamine, demonstrating an ability to utilize host-derived substrates. All strains tested were bile sensitive, showing at least 80% growth inhibition in the presence of 0.5 μg/ml bile salts, while inhibition at mildly acidic pH was strain dependent. These attributes help to explain the abundance of F. prausnitzii in the colonic community but also suggest factors in the gut environment that may limit its distribution.

An extreme and life-threatening case of recurrent D-lactate encephalopathy

D-lactic acidosis has been reported in patients after a variety of gastrointestinal surgeries, particularly jejunoileal bypass. An insufficient length of small intestine to metabolize ingested carbohydrates leads to an abnormal carbohydrate load in the colon. These carbohydrates are metabolized by colonic anaerobes (especially Lactobacillus species) into the dextrorotary isomer of lactate. Unlike its levorotary counterpart, D-lactate has neurotoxic effects and patients suffering from a significant D-lactate burden may suffer encephalopathic symptoms. These symptoms are usually mild and self-limiting in patients with normal renal function. We present here a case of D-lactic acidosis in a patient with end-stage renal disease who developed recurrent and life-threatening respiratory failure due to severe D-lactic acid encephalopathy. To our knowledge, no previously reported case has been sufficiently severe to necessitate endotracheal intubation and mechanical ventilation. An array of treatments including hemodialysis effected a prompt reversal of sensorium to baseline. We describe the potential treatments for D-lactic acidosis, which can be viewed as a paradigm of substrate, catalyst and pathologic product and review reports of their relative efficacy.

Novel tetracycline resistance gene, tet(32), in the Clostridium-related human colonic anaerobe K10 and its transmission in vitro to the rumen anaerobe Butyrivibrio fibrisolvens.

A novel tetracycline resistance gene, designated tet(32), which confers a high level of tetracycline resistance, was identified in the Clostridium-related human colonic anaerobe K10, which also carries tet(W). tet(32) was transmissible in vitro to the rumen anaerobe Butyrivibrio fibrisolvens 2221(R). The predicted gene product of tet(32) has 76% amino acid identity with Tet(O). PCR amplification indicated that tet(32) is widely distributed in the ovine rumen and in porcine feces.

The consequences of suppression of anaerobic bacteria.

Anaerobic bacteria such as Bacteroides fragilis, Peptostreptococcus species, and Fusobacterium species, when accompanied by aerobic bacteria or in the presence of dead tissue, can cause severe infections. This article discusses the most common type of anaerobic infection, i.e., infection after colonic contamination of the abdominal cavity and soft tissues. Colonic anaerobes rarely cause infections as solitary pathogens. Mixed infections of aerobes and anaerobes are treated by source control, surgical drainage and debridement, and combination antibiotic therapy. Antimicrobial treatment should cover both anaerobes and aerobes; treatment of mixed infections with anti-anaerobic agents alone is likely to result in abscess formation. Recent trends toward cost cutting and the advent of antibiotics with good coverage of both aerobes and relevant pathogenic anaerobes have led to increased single-agent therapy with cefoxitin, cefotetan, ampicillin/sulbactam, imipenem/cilastatin, ticarcillin/clavulanate, trovafloxacin/alatrofloxacin, and piperacillin/tazobactam. In the past 15 years, research has begun to focus on the gut barrier, particularly on the beneficial effects of anaerobic microflora. Directing antibiotic therapy against the anaerobe when it is involved in clinical infection is important; however, the negative consequences of anti-anaerobic antibiotic therapy on the beneficial effects of normal distal gut colonization must also be considered.

Changes in intestinal flora after administration of panipenem/betamipron or sulbactam/cefoperazone for treatment of postoperative infections in gastrectomy patients

To determine the effect of antibiotics used for the treatment of postoperative infections on the normal intestinal flora in patients who have undergone gastroenterological surgery, we performed a prospective randomized study in patients who had undergone distal gastrectomy. Cefazoline was administered up to postoperative day 3 for prophylaxis. For the treatment of postoperative infections, either panipenem/betamipron (PAPM/BP; n=5) or sulbactam/ cefoperazone (SBT/CPZ; n=5) was adminstered to ten patients with signs of postoperative infections. Feces were examined for bacteria before and 4 and 7 days after the commencement of administration of these therapeutic antibiotics. The anaerobic bacterial count decreased significantly in four of five patients treated with SBT/CPZ, from 1010.2±0.36/g before treatment to 105.22±3.51/g. In the PAPM/BPtreated group, the anaerobic bacterial count did not change after antibiotic therapy. We also investigated the major genera of colonic anaerobes, and found that the numbers of bifidobacteria were maintained during PAPM/BP administration. However, in the SBT/CPZ group, the number of bifidobacteria decreased to below the detection limit in four of five patients. Little change was observed in the aerobic bacterial count in either group. However, Enterobacteriaceae were replaced by enterococcus or candida in all patients from the SBT/CPZ group and in one of five patients from the PAPM/BP group. Physicians should be alert

Utilization of starch and synthesis of a combined amylase/alpha-glucosidase by the human colonic anaerobe Bacteroides ovatus.

Bacteroides ovatus preferentially utilized starch and pectin when grown on a mixture of polysaccharides in batch culture, indicating that these carbohydrates are important substrates for the bacterium in the human large intestine. Further studies on starch breakdown showed that continuous cultures grew on the polysaccharide when it provided the sole carbohydrate source, to yield a single hydrolytic product at low dilution rates (D = 0.04 h-1), with an estimated molecular mass of 13 kDa. In contrast, two major types of oligomeric products were formed at higher dilution rates (D = 0.44 h-1), with approximate molecular weights of 11 and 140 kDa. Analysis of cell-associated starch-degrading enzymes produced by Bact. ovatus using ion exchange chromatography and HPLC gel-filtration showed that amylase and alpha-glucosidase activities eluted in the same fractions. The single peak containing amylase and alpha-glucosidase activities obtained by HPLC gel-filtration chromatography corresponded to a molecular mass of approximately 140 kDa, and activity staining of gels for alpha-glucosidase activity after polyacrylamide gel electrophoresis, in the presence of sodium dodecyl sulphate, gave an estimated molecular mass of 70 kDa, indicating this enzyme to be a dimer. After renaturation, the 70 kDa band was cut from the gels and solubilized. The extract hydrolysed gelatinized starch and p-nitrophenyl-alpha-D-glucopyranoside.

Formation of Phenolic and Indolic Compounds by Anaerobic Bacteria in the Human Large Intestine

Batch culture incubations were used to investigate the effects of pH (6.8 or 5.5) and carbohydrate (starch) availability on dissimilatory aromatic amino acid metabolism in human fecal bacteria. During growth on peptide mixtures, tyrosine and phenylalanine fermentations occurred optimally at pH 6.8, while individual metabolic reactions were inhibited by up to 80% in the presence of 10 g l−1 starch. Tryptophan metabolites were not detected in these experiments. When free amino acids replaced peptides, phenol production was increased during carbohydrate fermentation, although formation of p-cresol, another tyrosine metabolite was strongly inhibited. Phenylpropionate, which is produced from phenylalanine, was unaffected by starch. Tryptophan was fermented in these studies, although indole production was reduced in the starch fermentors. The importance of different fermentation substrates (casein, peptide mixtures, free amino acids) on aromatic amino acid metabolism was investigated in incubations of material taken from the proximal bowel. The phenylalanine metabolites, phenylacetate and phenylpropionate, were the principal phenolic compounds formed from all three substrates. Phenol was the major tyrosine metabolite produced in casein and peptide fermentations, while hydroxyphenylpropionate was a more important tyrosine product from free amino acids. Indole was the sole product of tryptophan metabolism, but was formed only from the free amino acid. Bacterial metabolism of individual phenolic and indolic compounds was also investigated. Phenol, p-cresol, phenylacetate, phenylpropionate, 4-ethylphenol, indole, indoleacetate, and indolepropionate were not metabolized by colonic bacteria. However, hydroxyphenylacetate was hydrolyzed to p-cresol, while hydroxyphenylpropionate was transformed into phenylpropionate. Indolepyruvate was either converted to indoleacetate or metabolized into indole. Indolepropionate, and to a lesser degree indoleacetate were produced from indolelactate. These data show that human colonic anaerobes are able to extensively degrade either free or peptide-bound aromatic amino acids, with the concomitant formation of toxic metabolic products. These processes are controlled to a significant degree by environmental factors such as pH and carbohydrate availability, and this ultimately influences the types and amounts of fermentation products that can be formed in different regions of the large bowel.

Incrimination of anaerobic bacteria in the induction of experimental colitis.

Commensal bacteria may participate in the pathogenesis of bowel inflammation. We studied the role of bacteria from the rat colonic flora on transmural inflammation induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS). First, bacterial translocation to the colonic wall after induction of colitis was assessed by microbiological and histological methods. Second, rats with a colonic segment excluded from fecal transit were prepared for recolonization with preselected bacteria and used to test the effects of different species on inflammation (eicosanoid release, tissue myeloperoxidase) and damage (histology). Six strains (three aerobes and three anaerobes) were identified in colonic tissue 24 h after induction of colitis. Acridine staining showed bacteria in necrotic areas of the mucosa and invading the submucosa. Rats with excluded colon and sterile culture of luminal washings showed mild inflammation and low mucosal damage in response to TNBS. Rats colonized with anaerobes showed significantly higher eicosanoid release than rats colonized with aerobes only. Moreover, submucosal-lesions were mostly observed in rats with anaerobes. Our findings suggest that colonic anaerobes play a key role in transmural inflammation.

Contribution of a neopullulanase, a pullulanase, and an alpha-glucosidase to growth of Bacteroides thetaiotaomicron on starch.

Bacteroides thetaiotaomicron, a gram-negative colonic anaerobe, can utilize three forms of starch: amylose, amylopectin, and pullulan. Previously, a neopullulanase, a pullulanase, and an alpha-glucosidase from B. thetaiotaomicron had been purified and characterized biochemically. The neopullulanase and alpha-glucosidase appeared to be the main enzymes involved in the breakdown of starch, because they were responsible for most of the starch-degrading activity detected in B. thetaiotaomicron cell extracts. To determine the importance of these enzymes in the starch utilization pathway, we cloned the genes encoding the neopullulanase and alpha-glucosidase. The gene encoding the neopullulanase (susA) was located upstream of the gene encoding the alpha-glucosidase (susB). Both genes were closely linked to another starch utilization gene, susC, which encodes a 115-kDa outer membrane protein that is essential for growth on starch. The gene encoding the pullulanase, pulI, was not located in this region in the chromosome. Disruption of the neopullulanase gene, susA, reduced the rate of growth on starch by about 30%. Elimination of susA in this strain allowed us to detect a low residual level of enzyme activity, which was localized to the membrane fraction. Previously, we had shown that a disruption in the pulI gene did not affect the rate of growth on pullulan. We have now shown that a double mutant, with a disruption in susA and in the pullulanase gene, pulI, was also able to grow on pullulan. Thus, there is at least one other starch-degrading enzyme besides the neopullulanase and the pullulanase. Disruption of the alpha-glucosidase gene, susB, reduced the rate of growth on starch only slightly. No residual alpha-glucosidase activity was detectable in extracts from this strain. Since this strain could still grow on maltose, maltotriose, and starch, there must be at least one other enzyme capable of degrading the small oligomers produced by the starch-degrading enzymes. Our results show that the starch utilization system of B. thetaiotaomicron is quite complex and contains a number of apparently redundant degradative enzymes.

Evidence for natural transfer of a tetracycline resistance gene between bacteria from the human colon and bacteria from the bovine rumen.

Previously, we demonstrated conjugal transfer of a specially constructed shuttle vector, pRDB5, from the human colonic anaerobe Bacteroides uniformis to the ruminal anaerobe Prevotella (Bacteroides) ruminicola B(1)4. We have now shown that naturally occurring gene transfer elements in Bacteroides species and Prevotella ruminicola can also be transferred between these two genera. A self-transmissible chromosomal element originally found in a clinical isolate of Bacteroides fragilis (Tcr Emr 12256) was transferred from B. uniformis 0061 to P. ruminicola B(1)4 and from P. ruminicola B(1)4 back to B. uniformis or to another human colonic species, Bacteroides thetaiotaomicron. Similarly, a conjugative plasmid (pRRI4) originally found in P. ruminicola 223 was transferred from P. ruminicola B(1)4 to B. uniformis or B. thetaiotaomicron. pRRI4 could be transferred from the colonic Bacteroides species only if the donor strain contained the Tcr Emr 12256 element in its chromosome. These results show that transfer of naturally occurring elements can be demonstrated under laboratory conditions. Evidence that such transfers may actually have occurred in nature came from our finding that the tetracycline resistance (Tcr) gene on the P. ruminicola plasmid pRRI4 hybridized on high-stringency Southern blots with the Tcr gene found on the Bacteroides Tcr elements. The presence of the same gene in such distantly related genera of bacteria is most likely to have occurred as a result of horizontal transfer.

Colonic fermentation of potato starch after a freeze-thaw cycle

To estimate colonic carbohydrate fermentation following a potato meal, 13 healthy volunteers consumed 375 g potatoes containing 60 g starch on three different occasions in random order: (A) potatoes boiled and consumed fresh at 60 degrees C; (B) potatoes boiled, frozen, thawed and consumed at 20 degrees C; and (C) potatoes boiled, frozen, thawed, reheated to 90 degrees C, and consumed at 60 degrees C. End-expiratory breath hydrogen (H2) was measured every 15 min for 10-14 hr with a selective electrochemical cell. The extent of colonic carbohydrate fermentation (AUC = area under the breath H2 concentration vs time curve) in experiment B was significantly higher (+186%, P less than 0.002) than in experiment A. The breath hydrogen AUC in experiment C was higher than in experiment A (+48%, P less than 0.04) but lower than in experiment B (-94%, P less than 0.003). It is suggested that structural alterations of the starch molecule occur during freezing, thawing, and reheating and alter the availability of carbohydrates for fermentation by colonic anaerobes.

Purification and characterization of a cell-associated, soluble mannanase from Bacteroides ovatus.

Bacteroides ovatus, a human colonic anaerobe, utilizes the galactomannan guar gum as a sole source of carbohydrate. Previously, we found that none of the galactomannan-degrading enzymes were extracellular, and we characterized an outer membrane mannanase which hydrolyzes the backbone of guar gum to produce large fragments. We report here the purification and characterization of a second mannanase from B. ovatus. This enzyme is cell-associated and soluble. Using ion-exchange chromatography, gel filtration, and chromatofocusing steps, we have purified the soluble mannanase to apparent homogeneity. The enzyme has a native molecular weight of 190,000 and a monomeric molecular weight of 61,000. It is distinct from the membrane mannanase not only with respect to cellular location but also with respect to stability and isoelectric point (pI of 6.9 for the membrane mannanase and pI of 4.8 for the soluble mannanase). The soluble mannanase, like the membrane mannanase, hydrolyzed guar gum to produce large fragments rather than monosaccharides. However, if galactosyl side chains were removed from the galactomannan fragments by alpha-galactosidase, both the soluble mannanase and the membrane mannanase could degrade guar gum to monosaccharides. Thus either or both of these two enzymes, working together with alpha-galactosidase, appear to be sufficient for the breakdown of guar gum to the level of monosaccharides.

Digestion of proteoglycan by Bacteroides thetaiotaomicron.

It has been shown previously that Bacteroides thetaiotaomicron, a human colonic anaerobe, can utilize the tissue mucopolysaccharide chondroitin sulfate as a source of carbon and energy and that the enzymes involved in this utilization are all cell associated (A. A. Salyers and M. B. O'Brien, J. Bacteriol. 143:772-780, 1980). Since chondroitin sulfate does not generally occur in isolated form in tissue, but rather is bound covalently in proteoglycan, we investigated the extent to which chondroitin sulfate which is bound in such a sterically hindered complex can be utilized by intact bacteria. Intact cells of B. thetaiotaomicron were able to digest chondroitin sulfate in proteoglycan, although at a slightly slower rate than free chondroitin sulfate. Prior digestion of proteoglycan with trypsin to produce small fragments of protein with several chondroitin sulfate chains attached did not increase the rate at which the bound chondroitin sulfate was digested. Accordingly, the slower rate of digestion was probably due to attachment of chondroitin sulfate chains to the protein backbone rather than to steric hindrance by other components of the proteoglycan. When proteoglycan which had been incubated with intact bacteria was treated with sodium borohydride to release the undigested fragments of chondroitin sulfate from the protein backbone, the size and composition of the fragments indicated that intact bacteria were able to digest all but three monosaccharides of the chondroitin sulfate chains. Thus, despite steric hindrance due to attachment of the chondroitin sulfate chains to the protein backbone, digestion of bound chondroitin sulfate by intact bacteria was nearly complete.

Jejunal cAMP-activated sodium secretion via deconjugated bile salts and fatty acids

Jejunal cAMP-activated sodium secretion associated with in vivo intraluminal bile salts and free fatty acid accumulation in the small bowel was studied. Wistar rats (70–90 g) were treated with mecamylamine HCl (20 mg/kg twice daily, × 3 days); this ganglionic blocking agent allows bacterial overgrowth of the small bowel with colonic anaerobes. Controls were given 0.9% NaCl. Half of each group was fed 4% cholestyramine resin in a commercial lab chow; the other half received the same diet without this resin. The contents of the small intestine were assayed for free fatty acids, bile salts, and enterotoxin activity. Jejunal sodium and 3- O-methyl- d-glucose transport rates were assessed by in vivo perfusions. Jejunal mucosa was studied for ATPases and cAMP. Mecamylamine-treated rats had significantly higher jejunal luminal concentrations of free fatty acids and bile salts. Deconjugated bile salts were notably elevated, principally cholate. There was no enterotoxic activity detectable. Mecamylamine treatment was also associated with jejunal sodium secretion and a three-fold increase in mucosal cAMP concentrations, while control rats absorbed sodium and had no alterations in cAMP levels. Dietary cholestyramine treatment ameliorated the intestinal derangements induced by mecamylamine. Resin-treated rats had reduced luminal free fatty acids and deconjugated bile salts, while sodium was absorbed in the jejunum and mucosal cAMP levels were comparable to controls. Mecamylamine treatment did not alter (Na +-K +)-ATPase activity or jejunal transport of 3- O-methyl- d-glucose at a 4 m m concentration. These data indicate that during small bowel overgrowth with colonic bacteria following mecamylamine treatment in rats, there is a cAMP-mediated sodium secretion associated with increased luminal free fatty acids and deconjugated bile salts.

Influence of high luminal PO2 on the anaerobic bacteria of the rat colon

The influence of high luminal PO2 on Colonic anaerobes was investigated by chronically perfusing the colon of unrestrained rats with O2 or N2. A luminal PO2, of approximately 700 mm Hg did not significantly influence the fecal concentration of total anaerobes recovered on rabbit blood agar or of Bifidobacterium sp. and Bacteroides sp. recovered on selective media. However, the O2 infusion reduced the rat's excretion of CH4 (a metabolic product of anaerobic bacteria) to about 5% of the control value, although the feces of O2-treated rats produced CH4 normally. This study suggests that the production of metabolites by the Colonic bacteria may sometimes be more accurately studied in situ rather than in culture. Measurement of volatile bacterial metabolites in breath may provide a simple means of studying the in situ metabolism of a variety of colonic bacteria in man.