Bacterial Beta-glucuronidase Activity Research Articles

Association of Gut Bacterial Beta Glucuronidase Activity with Mycophenolate Mofetil Induced Cytopenia

<h3>Purpose</h3> Mycophenolate mofetil (MMF) is the anti-metabolite of choice in heart transplantation (HT). MMF tolerability is limited by its toxicity, including bone marrow suppression. MMF metabolism is dependent on gastrointestinal flora, particularly pathogens carrying β-D-glucuronidases (GUS). We investigated association between cytopenia and gut microbial composition focusing on GUS bacteria in HT pts. <h3>Methods</h3> 45 HT pts (53±12.9yo, 78.6% M) with 1-month post HT stool samples were included. Stool samples were analyzed using 16S rRNA sequencing. Gut alpha-diversity was defined using Shannon Index (SI). GUS bacteria abundance was computed as sum of individual known GUS-producing bacteria, matched by genus and species. The DESeq2 package and linear regression models were used to identify differentially abundant taxa and analyze difference in SI and GUS abundance by cytopenia occurrence. <h3>Results</h3> Leukopenia and neutropenia occurred in 30(66.7%) and 26(57.8%) pts. Mean SI±SD was similar in pts with leukopenia vs not (5.78±0.59 vs 5.58±0.58, p=0.29) and neutropenia vs not (5.73±0.57 vs 5.68±0.62, p=0.76). In DESeq2 analyses, 8 bacteria species were found to be significantly more abundant among those who had leukopenia, including two known GUS producing taxa <i>Bacteroides dorei</i> and <i>Bacteroides cellulosilyticus</i> (<b>Fig 1A</b><i>).</i>Seven bacteria species were significantly more abundant among those who had neutropenia, including <i>Bacteroides dorei</i> (<b>Fig 1B</b>)<i>.</i> Patients with leukopeniat had higher abundance of GUS bacteria 0.21±0.16 vs 0.12±0.18 (p=0.09) (<b>Fig 1C</b>). This difference became significant following adjustment for valcyte (p=0.04). Patients who experienced neutropenia vs not had a similar abundance of GUS bacteria 0.17±0.14 and 0.18±0.20 (p=0.88) (<b>Fig 1D</b>). <h3>Conclusion</h3> GUS bacteria were expanded among HT recipients with cytopenia, potentially highlighting mechanisms of MMF toxicity. Robust clinical trials delineating the effects of gut microbiome on MMF metabolism are warranted.

Genetic diversity of &lt;FONT FACE=Symbol&gt;b&lt;/font&gt;-glucuronidase activity among 14 strains of the dominant human gut anaerobe Ruminococcus gnavus

Bacterial beta-glucuronidase activity in the gut increases the enterohepatic circulation of toxic compounds and plays a major role in the etiology of colon cancer. Previously, we had found that the gus gene, which codes for beta-glucuronidase in a dominant anaerobic species of the gut microbiota, Ruminococcus gnavus strain E1, is transcribed as part of an operon that includes three ORFs that code for beta-glucoside permeases of the phosphotransferase systems. This genetic organization had never been described. We have now compared beta-glucuronidase activity and the genetic environment of the gus gene in 14 strains of Ruminococcus gnavus.We found that five out of the seven glucuronidase-positive R. gnavus strains possessed another glucuronidase gene different from the gusA operon of R. gnavus E1. This dominant commensal intestinal species appears to have a high degree of genetic diversity in the genes that control beta-glucuronidase activity.

Influence of sodium chloride on the beta-glucuronidase activity of Clostridium perfringens and Escherichia coli.

While the beta-glucuronidase activity of intact cells of Clostridium perfringens was higher in 0.95% sodium chloride (NaCl) than that in 0, 0.1 or 0.5%, that of Escherichia coli was higher in 0.1% NaCl than that in 0, 0.5 or 0.95% NaCl in 0.1 mol l-1 KH2PO4. However, the enzyme activity of both species of intact cells was higher in buffer containing 16 mEq sodium, 134 mEq potassium and 16 mEq chloride per litre than in that containing 146 mEq sodium, 13 mEq potassium and 146 mEq chloride. These findings suggest that bacterial cells are affected by the presence of NaCl and that the effect of NaCl on the activity of bacterial beta-glucuronidase may differ by location in the large intestine.

Prevention of colonic preneoplastic lesions by the probiotic Lactobacillus acidophilus NCFMTM in F344 rats.

The experiments described here were aimed at developing novel probiotic strains that may aid in the reduction of colon cancer risk. We assessed the potential anticancer properties of Lactobacillus acidophilus NCFMTM in male F344 rats using inhibition of the formation of azoxymethane (AOM)-induced aberrant crypt foci (ACF) in the colon as the measure of preventive efficacy. At 6 weeks of age, groups of rats were fed the experimental diets containing 0, 2% or 4% lyophilized cultures of L. acidophilus NCFMTM. At 7 weeks of age, all animals in each dietary group, except the vehicle-treated rats, were s.c. injected with AOM (15 mg/kg body weight) once weekly for two weeks. The vehicle-treated groups were given s.c. injections of normal saline. All rats were necropsied 10 weeks after the last AOM injection and ACF in formalin-fixed, methylene blue-stained colonic tissues were counted under the light microscope. The contents of the cecum were analyzed for bacterial beta-glucuronidase activity. Diet supplementation with the probiotic strain NCFMTM significantly suppressed AOM-induction of colonic ACF, in terms of total number, as well as crypt multiplicity and number of ACF/cm2 colon (P<0.01 - 0.001). NCFMTM inhibited AOM-induced colonic ACF formation in a dose-dependent manner (P<0.01). A significant dose-dependent reduction of cecal beta-glucuronidase activities was observed in the rats fed 2% (P<0.04) and 4% (P<0.0001) NCFMTM. These results suggest that Lactobacillus acidophilus NCFMTM may potentially prevent colon cancer development. Further studies are warranted to determine the full potential of this probiotic strain in preclinical efficacy studies.

Biliary sludge and pigment stone formation in bile duct-ligated guinea pigs.

We studied the effects of bile stasis in a guinea pig model of pigment gallstone. The common bile ducts of guinea pigs were partially ligated, and the guinea pigs killed one or two weeks later. Biliary sludge or stones were examined with the Fourier transform infrared spectroscopy and the scanning electromicroscopy. The bile was analyzed for pH, free calcium, bile acids and bilirubin fractions, and the activities of both bacterial and endogenous beta-glucuronidase. After bile duct ligation, calcium bilirubinate precipitates or stones formed in all except one of the animals studied. The bile pH and the proportion of unconjugated bilirubin rose after bile duct ligation, with a concomitant fall of bilirubin monoglucuronide. The activity of bacterial beta-glucuronidase decreased after ligation, while the activity of endogenous beta-glucuronidase rose at week 2. Our results imply that precipitation of calcium bilirubinate in this animal model was induced by an increased bile pH and the nonenzymatic hydrolysis of conjugated bilirubin.

Prevention of colonic aberrant crypt foci and modulation of large bowel microbial activity by dietary coffee fiber, inulin and pectin.

The present experiments were aimed at developing novel dietary fibers to aid in reduction of colon cancer risk. We assessed the effects of coffee (non-fiber fraction), coffee fiber (arabino-galactose polymer) and inulin (oligo-fructose) in male F344 rats using formation of azoxymethane (AOM)-induced aberrant crypt foci (ACF) in the colon as the measure of preventive efficacy (or lack of such). At 5 weeks of age, groups of rats were fed the AIN-76A (control) and experimental diets that contained 1% coffee, 10% coffee fiber, 10% inulin, 10% pectin (positive control for fiber) or 200 p.p.m. piroxicam (a known ACF inhibitor). At 7 weeks of age, all animals were s.c injected with AOM (15 mg/kg body wt) once weekly for 2 weeks. All rats were killed 8 weeks after the last AOM injection and ACF were counted. The contents of the cecum were analyzed for bacterial beta-glucuronidase activity and short-chain fatty acids (SCFAs). Dietary administration of coffee fiber significantly suppressed AOM-induction of colonic ACF, in terms of total number, as well as crypt multiplicity and number of ACF/cm2 colon (P < 0.01-0.001). Inulin diet had no significant effect on total ACF, but had reduced the number of ACF/cm2 (P < 0.05). Whereas coffee had no effect on ACF formation, 10% pectin diet and 200 p.p.m. piroxicam significantly suppressed colonic ACF (P < 0.001) as had been expected. A significant reduction of cecal beta-glucuronidase activity was observed in the rats fed coffee, coffee fiber and pectin diets. Further, coffee fiber, inulin and pectin increased cecal SCFA levels 3- to 5-fold. These results suggest that coffee fiber can prevent colon cancer risk. Further studies are warranted to determine the full potential of this fiber in pre-clinical efficacy studies.

Influence of bile salts on beta-glucuronidase activity of intestinal bacteria.

The beta-glucuronidase activity of intact cells of Escherichia coli and Clostridium perfringens was increased in the presence of bile salts. In contrast, bile salts had inhibitory effects on the activity of beta-glucuronidase extracted from the lysed cells. These results suggest that the permeability of the bacterial cells is increased by the presence of bile salts, and that bile salts may significantly enhance bacterial beta-glucuronidase activity in the intestinal tract.

Cholelithiasis in Taiwan

The nature and occurrence of gallstones in Taiwan and their etiologic factors might not be the same as in Western countries and warranted a systematic investigation. Gallbladder biles and gallstones were obtained at surgery from 100 and 74 patients, respectively. Common duct bile and stones were either drained through an indwelling common duct T-tube or aspirated through a nasobiliary catheter in 108 patients. Gallstones were analyzed for bilirubin, cholesterol, bile acid, calcium, and residue, and biles for bile acid, cholesterol, phospholipid, bilirubin, and beta-glucuronidase. There were four major kinds of gallstones in Taiwan: cholesterol/mixed stones, high-residue black formed pigment stones, low-residue brown formed pigment stones, and muddy pigment stones. The surgical incidence of all types of stones increased steadily during the past four decades. During the past 15 years the relative frequencies for mixed, formed pigment, and muddy pigment stones had been roughly 40, 40, and 20%, respectively, with a further increase in the mixed stones and a decrease in the muddy pigment stones in recent years. Improvement of nutritional status and living standards might contribute to such changes. Cholesterol content in the common duct and gallbladder biles was higher in the mixed stone group than in other groups. Bacterial beta-glucuronidase activity was detected in 53% of patients with muddy pigment stones. Endogenous beta-glucuronidase activity and concentration were also highest in this group, intermediate in the formed pigment and mixed stone group, and lowest in the control. We concluded that hypercholesterobilia was responsible for increasing incidence of mixed stones during the past two decades, while both bacterial and human beta-glucuronidase might contribute to pigment cholelithiasis.

Inhibitory effect of Bifidobacterium longum cultures on the azoxymethane-induced aberrant crypt foci formation and fecal bacterial beta-glucuronidase.

Epidemiologic and experimental studies suggest that consumption of fermented milk products and lactic bacterial cultures that are used to ferment the dairy products, decrease the incidence of certain types of cancer. The present study was designed to determine the effect of lyophilized cultures of Bifidobacterium longum (B. longum), a lactic bacteria, on the azoxymethane (AOM)-induced preneoplastic lesions such as aberrant crypt foci (ACF) formation in the colon and on fecal bacterial beta-glucuronidase activity in male F344 rats. At 5 weeks of age, groups of animals were fed the AIN-76A (control) and the experimental diets containing 1.5% and 3% lyophilized cultures of B. longum. At 10 weeks of age, all animals received sc injection of AOM dissolved in normal saline at a dose rate of 20 mg/kg body wt, once weekly for 2 weeks. The animals were necropsied 6 weeks after the last AOM injection, and the ACF were visualized under light microscopy in the formalin-fixed, unsectioned methylene blue-stained colons where they were distinguished by their increased size, more prominent epithelial cells, and pericryptal space. The cecal contents were analyzed for bacterial beta-glucuronidase activity. The feeding of lyophilized cultures of B. longum significantly inhibited the ACF formation (53%) and the crypt multiplicity in the colon. A significant decrease in the fecal bacterial beta-glucuronidase was also observed in the animals fed the diets containing Bifidobacterium supplements as compared with control diet. These results demonstrate that B. longum in diet influences the metabolic activity of certain types of intestinal microflora that are involved in the production of beta-glucuronidase. Furthermore, the findings also suggest that B. longum supplements inhibit ACF formation, an early preneoplastic marker of malignant potential in the process of colon carcinogenesis.

Survival ofLactobacillus species (strain GG) in human gastrointestinal tract

A newly isolated strain of a species of Lactobacillus of human origin, designated GG (Lactobacillus GG), has been studied to determine its ability to survive in the human gastrointestinal tract. When fed to 76 volunteers as a frozen concentrate or as a fermented preparation in milk or whey, Lactobacillus GG was recovered in the feces of all subjects receiving the fermented milk or whey and in 86% receiving the frozen concentrate when a single fecal specimen was cultured. The organism was also present in the feces of subjects concurrently receiving ampicillin. After terminating feeding of the organism, Lactobacillus GG persisted in the feces of 87% of volunteers four days later and in 33% of subjects seven days later. Lactobacillus GG lowered fecal bacterial beta-glucuronidase activity by approximately 80% in volunteers given the organism for four weeks. These studies demonstrate that Lactobacillus GG can survive and temporarily colonize the human gastrointestinal tract and can affect the metabolic activity of the resident microflora.

Beta-Glucuronidase Activity in the Bile of Gallstone Patients Both with and without Duodenal Diverticula

Patients with juxtapapillary duodenal diverticula have an increased occurrence of calcium bilirubinate gallstones. One possible hypothesis to explain this observation is enzymatic deconjugation of bilirubin conjugates in the bile. Beta-glucuronidase of human or bacterial origin may lead to deconjugation of the bilirubin glucuronides in bile. This, in turn, may increase the amounts of unconjugated, water-insoluble bilirubin which can precipitate as calcium bilirubinate, the main component of brown pigment stones. In this study we compared gallstone patients with and without duodenal diverticula treated with endoscopic papillotomy. Increased occurrence of bacteria producing beta-glucuronidase (p less than 0.01) and increased activity of bacterial beta-glucuronidase (pH 7.0) in the bile itself (p less than 0.01) were found in patients with duodenal diverticula. When the activity of the enzyme at pH 4.5, the optimum of the human enzyme, was measured, no such difference was found. The results support the hypothesis of bacterial glucuronidase as an etiologic factor in pigment gallstone disease in patients with duodenal diverticula. The high activity of bacterial enzyme found in the bile in some patients without diverticula suggests bacteria as an etiologic factor, independent of the presence of diverticula.

Beta-Glucuronidase Activity Related to Bacterial Growth in Common Bile Duct Bile in Gallstone Patients

Beta-glucuronidase activity in the bile may be of importance in the etiology of pigment gallstones. This enzyme is of hepatic or bacterial origin. We have described a method to measure the activity of bacterial beta-glucuronidase in human bile, using 4-nitrophenyl-beta-D-glucopyranosiduronic acid as substrate. The method was used to measure the beta-glucuronidase activity in the bile from 51 patients with gallstone disease. This activity was related to the presence of beta-glucuronidase-producing bacteria in the bile. Escherichia coli, Bacteroides species, and Clostridium perfringens were the only species found to produce beta-glucuronidase. Patients with beta-glucuronidase-producing bacteria had on an average significantly higher enzyme activity in the bile than patients without such bacteria (p less than 0.01). The limitations of using artificial substrates in this type of studies are discussed.

Effects of pH on the deconjugation of conjugated bilirubin in human bile.

The enzymatic activity of bacterial beta-glucuronidase plays an essential role in the formation of calcium bilirubinate in bile. There are, however, many unsettled problems such as methodology of the assay for its enzymatic activity. In the present study (1) the azopigments from monoconjugated bilirubin (MCB) and unconjugated bilirubin (UCB) in native bile were semiquantitatively determined, (2) the deconjugation of conjugated bilirubin (CB) in bile was estimated with azopigment analysis and (3) factors affecting the deconjugation of CB in bile, especially for pH value, were investigated. CB in bile was stable at physiologic pH during 6-hr incubation at 37 degrees C, but was hydrolyzed at alkaline pH. At physiologic pH, addition of beta-glucuronidase from E. coli hydrolyzed CB in bile and increased MCB and UCB in bile. Based upon the results mentioned above, it is suggested that alkaline pH and enzymatic activity of beta-glucuronidase should cause the increase of UCB in bile. It can be said that beta-glucuronidase is essential for the formation of calcium bilirubinate gallstone at physiologic pH.

Effect of Neomycin on Azoxymethane-Induced Colon Carcinogenesis in F344 Rats&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;&lt;xref ref-type="fn" rid="FN3"&gt;3&lt;/xref&gt;

The effect of oral administration of neomycin (100 and 200 micrograms/ml in drinking water) on colon tumors induced by azoxymethane [(AOM); CAS: 25843-45-2] was studied in female F344 rats. Five-week-old rats were fed NIH-07 diet and given daily in drinking water 0, 100, and 200 micrograms neomycin/ml (0, 100, and 200 ppm). At 7 weeks of age, all animals except vehicle-treated groups received weekly sc injections of 8 mg AOM/kg body weight for 8 weeks. The AOM- or vehicle-treated groups were necropsied 30 weeks after the last injection of AOM. The combined incidence of adenomas and adenocarcinomas of the colon did not differ significantly among the 3 groups. The animals in the groups given 100 and 200 micrograms neomycin had a higher incidence of colon adenocarcinomas than did those in the control group. Colonic and cecal bacterial beta-glucuronidase activity was significantly lower in the group given 200 micrograms neomycin than it was in the control group. The excretion of fecal cholesterol, total bile acids, and deoxycholic acid was increased significantly in animals given 100 and 200 micrograms neomycin as compared to animals given no neomycin. These results suggest that long-term oral administration of neomycin increases the incidence of colon adenocarcinomas.

Effect of High-Fat, High-Beef Diet and of Mode of Cooking of Beef in the Diet on Fecal Bacterial Enzymes and Fecal Bile Acids and Neutral Sterols

The effect of a high-fat, high-beef diet and of method of preparation of beef in the diet on the fecal bile acids and neutral sterols and on the activities of fecal bacterial beta-glucuronidase, cholesterol dehydrogenase and 7 alpha-dehydroxylase was studied in healthy men and women, 24-41 years old, who were consuming a customary mixed-western diet. The experimental diets were high in fat and beef, which was cooked rare, medium or well-done. The sequence of dietary regimen was selected at random and each diet phase lasted for 4 weeks. Individual 24-hour fecal specimens were collected from each volunteer for the final 3 days during each dietary period, including pre- and post-experimental periods while they were consuming a customary mixed-western diet. The mode of cooking beef in the high-fat, high-beef experimental diets had no influence on the fat and protein content of the diets but the fat content of experimental diets was high compared to customary mixed-western diet. Fecal bacterial beta-glucuronidase activity and fecal secondary bile acid and cholesterol metabolite levels were significantly higher during the experimental diet periods but the fecal bacterial activities of 7 alpha-dehydroxylase and cholesterol dehydrogenase were unaffected. The mode of cooking beef in experimental diets had no influence on the fecal bacterial enzymes and on the excretion of fecal bile acids and cholesterol metabolites.

Fecal constituents of a high-risk North American and a low-risk finnish population for the development of large bowel cancer

Feces collected in Kuopio, Finland, a low-risk population for colon cancer, and in the New York metropolitan area, a high-risk population for colon cancer, were compared. The dietary intake of fat and protein were the same in the two populations, but the sources of fat were different, a greater portion coming from meat in New York, and from dairy products in Kuopio. The daily stool output was higher in Kuopio due to the high intake of cereal products rich in fiber. The concentration of fecal secondary bile acids and the bacterial beta-glucuronidase activity were lower in Kuopio, but the daily output of these constituents was the same in the two groups. The daily fecal excretion of neutral sterols was higher in Kuopio than in New York. Our data suggest that the greater fecal bulk in Kuopio may dilute tumorigenic compounds which come in direct contact with the colon mucosa.

Fecal Bacterial β-Glucuronidase: Control by Diet

The effect of a mixed Western, high meat diet or a nonmeat diet on the intestinal bacterial beta-glucuronidase activity was studied in human volunteers. This enzyme was significantly higher in stools of subjects on a high meat diet as compared to the nonmeat regimen. Thus, intestinal flora of subjects on a high meat diet was more able to hydrolyze glucuronide conjugates than that of individuals on a nonmeat diet. This, in turn, may raise the amount of substances, such as carcinogens, within the colonic lumen.