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Effects of human bile on bacterial B-glucuronidase activity — An in vitro study
AGAA453
April 2000
2482
2484
FACTORS INFLUENCING THE EXPRESSION OF BACTERIAL B-GLUCURONIDASE ACTIVITIES. Joseph W. Leung, Yan-Iei Liu, Div of Gastroenterology, UC Davis Med Ctr, Sacramento, CA; C W Law Biofilm Research Lab, UCDMC, Sacramento, CA.
EFFECTS OF HUMAN BILE ON BACTERIAL B-GLUCURONIDASE ACTIVITY - AN IN VITRO STUDY. Joseph W. Leung, Van-lei Liu, Div of Gastroenterology, UC Davis Med Ctr, Sacramento, CA; C W Law Biofilm Research Lab, UCDMC, Sacramento, CA. Background: Bacterial f3-glucuronidase deconjugates bilirubin diglucuronide and leads to the precipitation of calcium bilirubinate - an important component of biliary sludge and stones. This process is attributed to aerobic enterobacteriaceae such as E. coli and Klebsiella sp. Using PCR studies, we detected the presence of Clostridium sp. in 48 of 56 intrahepatic stones and cultured Clostridium perfringens from 14 of 18 unblocked biliary stents. Aim:To determine the expression of J3-glucuronidase by different bacteria in human bile. Methods: Between 1994 -1999, 201 bacteria were isolated from human biliary stents and pigment ductal stones. Of these, 55 bacteria that expressed J3-glucuronidase activities in brain heart infusion (BHI) broth were tested. The pH of II human hepatic bile samples were measured. Bacteria were cultured separately in human bile (pH 7.19) under aerobic and anaerobic conditions at 37°C to the early stationary phase. The bacterial cells were collected by centrifugation at 8000 rev min' for 20 min at 4°C, washed twice and re-suspended in 1.0 ml of 0.1 M phosphate buffer (pH 7.5). f3-glucuronidase activities were measured spectrophotometrically using p-Nitrophenyl f3-D glucuronide as substrate and normalized to the optical density at 600 nm (O.D.600 ) to adjust for the number of cells in suspension. The content of each reaction tube was then centrifuged to remove the bacterial cells. The absorbency of p-Nitrophenol in the supernatant was read at 415 nm against a blank. The tests were repeated in triplicates. Results: The mean pH of human hepatic bile was 7.56 (range 7.19-8.51). Of the more commonly isolated aerobic bacteria, 21125(84%) E.coli, 2/14(14%) Klebsiella, 112(50%) Streptococcus but only 4/53(7%) Enterococci were enzyme positive. None of the Pseudomonas produced the enzyme. Of the anaerobic bacteria, 20121 (95%) C. perfringens, 3/3(100%) B. fragilis but none of the C. biferrmentans produced the enzyme. The mean J3-glucuronidase activities of C. perfringens was twice that of B. fragilis, 6.6 times that of E.coli, and 28 times that of Enterococcus. Conclusion: When tested in human bile at physiological pH, the anaerobic bacteria especially C. perfringens produced significantly more f3-glucuronidaseactivities than aerobic bacteria and thus may play an important role in biliary sludge and stones formation.
Background: Bacterial f3-glucuronidase plays an important role in the formation of biliary sludge and stones through deconjugation of bilirubin diglucuronide. E.coli and Clostridium perfringens are common bacteria isolated from sludge and stones, and both produce significant J3-glucuronidase activities. Aim: To determine the effect of pH, culture media and phenotypic changes of the bacteria on the expression of J3-glucronidase activities by E. coli and C. perfringens. Methods:Six clinical strains of C.peifringens(3) and E.coli(3), isolated from human biliary stents or stones were studied. The E.coli were grown individually in Brain Heart Infusion (BHI) broth (pH 7.3), Tryptic Soya Agar plates and human bile under aerobic conditions at 37°C to the early stationary phase. The C. perfringens were cultured individually in BHI broth (pH 7.4), Brucella anaerobic plates and human bile under anaerobic conditions at 3rC. The cells were collected by centrifugation at 8000 rev min" for 20 min at 4°C, washed twice and re-suspended in 1.0 ml of 0.1 M phosphate buffer. J3-glucuronidase activities of the different bacteria were measured using p-Nitrophenyl J3-D glucuronide as subtrate and normalized to optical density at 600 nm (O.D.6QO) to adjust for number of suspended cells. The content of each reaction tube was centrifuged to remove the bacterial cells. The absorbency of p-Nitrophenol in the supernatant was read at 415 nm against a blank. The effects of changing pH, different culture media, cell lysis following treatment with Triton and phenotypic change from planktonic to sessile bacteria on f3-glucuronidase expression were studied. Results: !.The optimal pH for f3-glucuronidase produced by E.coli is 6.8. The enzyme produced by Cperfringens has a wider pH range (7-10). 2. Phenotypic change from planktonic to sessile form increased overall enzyme activities from 0.171 to 0.299 for E. coli and from 0.574 to 0.995 for C. perfringens respectively. 3. Cell lysis increased the enzyme activities from 0.604 to 2.402 for E.coli and from 0.446 to 1.634 for C. perfringens. 4. Enzyme activities for E.coli increased from 0.745 in BHI broth to 1.601 in human bile; and for C. perfringens from 0.556 in BHI to 2.804 in human bile. Conclusions: I. f3-glucuronidase activities of C. perfringens has a broader optimal pH than E.coli. 2. Bacteria expressed more enzyme activities in sessile form than planktonic cells. 3. Cell lysis enhanced enzyme activities and 4. Human bile induces the expression of f3-glucuronidase activities by E.coli and C. perfringens.
2483 THE ROLE OF GLYCOCALYX IN BACTERIAL ADHERENCE. Joseph W. Leung, Yan-lei Liu, Div of Gastroenterology, UC Davis Med Ctr, Sacramento, CA; C W Law Biofilm Research Lab, UCDMC, Sacramento, CA. Background: Bacterial biofilm plays an important role in biliary sludge and stones formation. Bacteria attach with their surface pili and/or through production of glycocalyx - an exopolysaccharide or slime that glue the bacteria to a surface and to each other. The glycocalyx appears as electron dense particles surrounding the bacteria on Transmission Electron Microscopy (TEM). Aim: To determine the influence of glycocalyx production on bacterial attachment to polyethylene stents (PTFEs). Methods: Three clinical strains of E.coli viz. piliated and glycocalyx producing [P+G+], non-piliated and glycocalyx producing [p-G+] and non-piliated and non glycocalyx producing [P-G-] were used in the in vitro studies. Bacterial adherence on PTFEs were measured using the Modified Robbins Device (MRD) as previously described. Exp.!. Different E.coli were grown separately in Brain Heart Infusion (BHI) broth and perfused individually through the MRD, using a peristaltic pump at a rate of 60 ml/h. The PTFEs were removed at regular intervals (2,4,6,8,24 and 30 hrs) and processed to determine the number of attached bacteria using the viable count method. Exp.2. The (P+G+) E.coli was cultured in BHI broth, Modified Vogel and Bonner s Medium (MVBM) and human bile (HB). Adherence studies were repeated using the MRD and the number of attached bacteria were measured. The experiments were repeated in triplicates. Production of glycocalyx by the E.coli in different media was assessed using TEM and scored. Results: 1. (P+G+) E.coli adhered significantly better than (P-G+) or (P-G-) E.coli, with a 10-100 fold difference. 2. With the (P+G+) E.coli, glycocalyx production was increased in MVBM and HB. MVBM which stimulates glycocalyx production increased the adherence of (P+G+) E.coli by 10 fold. Although HB stimulates f3-glucuronidaseand glycocalyx production, there was a 2 log reduction in bacterial adherence. TEM showed that the bacteria appeared healthy in BHI and MVBM but were sick looking in human bile with ballooning of the cells. Conclusions: bacteria adhere best if they possess pili and produce glycocalyx. Stimulation of glycocalyx production increases bacterial adherence. Despite increased glycocalyx production, bacterial adherence is reduced in human bile probably because of the toxic effects of bile salts on the bacteria.
2485 MAGNETIC COMPRESSION ANASTOMOSIS FOR BENIGN OBSTRUCTION OF THE COMMON BILE DUCT:A CASE REPORT. Yoichiro Matsuo, Sonshin Takao, Hiroyuki Shinchi, Takashi Aiko, Takeshi Iseji, Eigoro Yamanouchi, Kagoshima Univ Sch of Medicine, Kagoshima, Japan; Tokyo Women's Med Univ, Tokyo, Japan. Introduction: It has been impossible for interventional radiology(IVR) to anastomosis of internal organs. Recently, the magnets have made possible a novel method of the anastomosis between the bile duct and the small intestine with the advance of IVR. We demonstrated the case that magnetic compression anastomosis was carried out for the benign obstruction of the common bile duct repeated cholangititis after subgastrectomy. Case report: The patient was 70-yeas-old man. In August 1997, He had subgastrectomy and Billroth II reconstruction for gastric cancer. After the discharge from hospital, he had repeated cholangititis with high fever. In July 1998, jaundice was appeared, so he had a cholecystectomy in nearby hospital. However, to investigate the continuous jaundice, the cholangioscopy showed the narrowing of common bile duct(CBD). After admission to our hospital, endoscopic retrograde cholangiopancreatography(ERCP) and magnetic resonance cholangiopancreatography(MRCP) revealed the complete obstruction of CBD caused by the repeated cholangititis. Therefor, we performed the anastomosis between the hilar bile duct and 2nd portion of the duodenum using the magnets. Parent magnet moved forward the afferent loop of the duodenum, and daughter magnet was put into the obstructed CBD. Consequently, two magnets have mated transmurally. These magnets ware Samarium-Cobalt rare-earth magnets. No complications ensured and the completely patent anastomosis was established at 32 postoperative days. Discussion:Magnetic compression anastomosis has made possible non-invasive bloodless treatment. This novel treatment has the advantages of non-invasion, simple and easy suggesting a good indication for biliary obstruction. The magnetic compression might be spread throughout various branches of medicine in the future.
April 2000
2482
2484
FACTORS INFLUENCING THE EXPRESSION OF BACTERIAL B-GLUCURONIDASE ACTIVITIES. Joseph W. Leung, Yan-Iei Liu, Div of Gastroenterology, UC Davis Med Ctr, Sacramento, CA; C W Law Biofilm Research Lab, UCDMC, Sacramento, CA.
EFFECTS OF HUMAN BILE ON BACTERIAL B-GLUCURONIDASE ACTIVITY - AN IN VITRO STUDY. Joseph W. Leung, Van-lei Liu, Div of Gastroenterology, UC Davis Med Ctr, Sacramento, CA; C W Law Biofilm Research Lab, UCDMC, Sacramento, CA. Background: Bacterial f3-glucuronidase deconjugates bilirubin diglucuronide and leads to the precipitation of calcium bilirubinate - an important component of biliary sludge and stones. This process is attributed to aerobic enterobacteriaceae such as E. coli and Klebsiella sp. Using PCR studies, we detected the presence of Clostridium sp. in 48 of 56 intrahepatic stones and cultured Clostridium perfringens from 14 of 18 unblocked biliary stents. Aim:To determine the expression of J3-glucuronidase by different bacteria in human bile. Methods: Between 1994 -1999, 201 bacteria were isolated from human biliary stents and pigment ductal stones. Of these, 55 bacteria that expressed J3-glucuronidase activities in brain heart infusion (BHI) broth were tested. The pH of II human hepatic bile samples were measured. Bacteria were cultured separately in human bile (pH 7.19) under aerobic and anaerobic conditions at 37°C to the early stationary phase. The bacterial cells were collected by centrifugation at 8000 rev min' for 20 min at 4°C, washed twice and re-suspended in 1.0 ml of 0.1 M phosphate buffer (pH 7.5). f3-glucuronidase activities were measured spectrophotometrically using p-Nitrophenyl f3-D glucuronide as substrate and normalized to the optical density at 600 nm (O.D.600 ) to adjust for the number of cells in suspension. The content of each reaction tube was then centrifuged to remove the bacterial cells. The absorbency of p-Nitrophenol in the supernatant was read at 415 nm against a blank. The tests were repeated in triplicates. Results: The mean pH of human hepatic bile was 7.56 (range 7.19-8.51). Of the more commonly isolated aerobic bacteria, 21125(84%) E.coli, 2/14(14%) Klebsiella, 112(50%) Streptococcus but only 4/53(7%) Enterococci were enzyme positive. None of the Pseudomonas produced the enzyme. Of the anaerobic bacteria, 20121 (95%) C. perfringens, 3/3(100%) B. fragilis but none of the C. biferrmentans produced the enzyme. The mean J3-glucuronidase activities of C. perfringens was twice that of B. fragilis, 6.6 times that of E.coli, and 28 times that of Enterococcus. Conclusion: When tested in human bile at physiological pH, the anaerobic bacteria especially C. perfringens produced significantly more f3-glucuronidaseactivities than aerobic bacteria and thus may play an important role in biliary sludge and stones formation.
Background: Bacterial f3-glucuronidase plays an important role in the formation of biliary sludge and stones through deconjugation of bilirubin diglucuronide. E.coli and Clostridium perfringens are common bacteria isolated from sludge and stones, and both produce significant J3-glucuronidase activities. Aim: To determine the effect of pH, culture media and phenotypic changes of the bacteria on the expression of J3-glucronidase activities by E. coli and C. perfringens. Methods:Six clinical strains of C.peifringens(3) and E.coli(3), isolated from human biliary stents or stones were studied. The E.coli were grown individually in Brain Heart Infusion (BHI) broth (pH 7.3), Tryptic Soya Agar plates and human bile under aerobic conditions at 37°C to the early stationary phase. The C. perfringens were cultured individually in BHI broth (pH 7.4), Brucella anaerobic plates and human bile under anaerobic conditions at 3rC. The cells were collected by centrifugation at 8000 rev min" for 20 min at 4°C, washed twice and re-suspended in 1.0 ml of 0.1 M phosphate buffer. J3-glucuronidase activities of the different bacteria were measured using p-Nitrophenyl J3-D glucuronide as subtrate and normalized to optical density at 600 nm (O.D.6QO) to adjust for number of suspended cells. The content of each reaction tube was centrifuged to remove the bacterial cells. The absorbency of p-Nitrophenol in the supernatant was read at 415 nm against a blank. The effects of changing pH, different culture media, cell lysis following treatment with Triton and phenotypic change from planktonic to sessile bacteria on f3-glucuronidase expression were studied. Results: !.The optimal pH for f3-glucuronidase produced by E.coli is 6.8. The enzyme produced by Cperfringens has a wider pH range (7-10). 2. Phenotypic change from planktonic to sessile form increased overall enzyme activities from 0.171 to 0.299 for E. coli and from 0.574 to 0.995 for C. perfringens respectively. 3. Cell lysis increased the enzyme activities from 0.604 to 2.402 for E.coli and from 0.446 to 1.634 for C. perfringens. 4. Enzyme activities for E.coli increased from 0.745 in BHI broth to 1.601 in human bile; and for C. perfringens from 0.556 in BHI to 2.804 in human bile. Conclusions: I. f3-glucuronidase activities of C. perfringens has a broader optimal pH than E.coli. 2. Bacteria expressed more enzyme activities in sessile form than planktonic cells. 3. Cell lysis enhanced enzyme activities and 4. Human bile induces the expression of f3-glucuronidase activities by E.coli and C. perfringens.
2483 THE ROLE OF GLYCOCALYX IN BACTERIAL ADHERENCE. Joseph W. Leung, Yan-lei Liu, Div of Gastroenterology, UC Davis Med Ctr, Sacramento, CA; C W Law Biofilm Research Lab, UCDMC, Sacramento, CA. Background: Bacterial biofilm plays an important role in biliary sludge and stones formation. Bacteria attach with their surface pili and/or through production of glycocalyx - an exopolysaccharide or slime that glue the bacteria to a surface and to each other. The glycocalyx appears as electron dense particles surrounding the bacteria on Transmission Electron Microscopy (TEM). Aim: To determine the influence of glycocalyx production on bacterial attachment to polyethylene stents (PTFEs). Methods: Three clinical strains of E.coli viz. piliated and glycocalyx producing [P+G+], non-piliated and glycocalyx producing [p-G+] and non-piliated and non glycocalyx producing [P-G-] were used in the in vitro studies. Bacterial adherence on PTFEs were measured using the Modified Robbins Device (MRD) as previously described. Exp.!. Different E.coli were grown separately in Brain Heart Infusion (BHI) broth and perfused individually through the MRD, using a peristaltic pump at a rate of 60 ml/h. The PTFEs were removed at regular intervals (2,4,6,8,24 and 30 hrs) and processed to determine the number of attached bacteria using the viable count method. Exp.2. The (P+G+) E.coli was cultured in BHI broth, Modified Vogel and Bonner s Medium (MVBM) and human bile (HB). Adherence studies were repeated using the MRD and the number of attached bacteria were measured. The experiments were repeated in triplicates. Production of glycocalyx by the E.coli in different media was assessed using TEM and scored. Results: 1. (P+G+) E.coli adhered significantly better than (P-G+) or (P-G-) E.coli, with a 10-100 fold difference. 2. With the (P+G+) E.coli, glycocalyx production was increased in MVBM and HB. MVBM which stimulates glycocalyx production increased the adherence of (P+G+) E.coli by 10 fold. Although HB stimulates f3-glucuronidaseand glycocalyx production, there was a 2 log reduction in bacterial adherence. TEM showed that the bacteria appeared healthy in BHI and MVBM but were sick looking in human bile with ballooning of the cells. Conclusions: bacteria adhere best if they possess pili and produce glycocalyx. Stimulation of glycocalyx production increases bacterial adherence. Despite increased glycocalyx production, bacterial adherence is reduced in human bile probably because of the toxic effects of bile salts on the bacteria.
2485 MAGNETIC COMPRESSION ANASTOMOSIS FOR BENIGN OBSTRUCTION OF THE COMMON BILE DUCT:A CASE REPORT. Yoichiro Matsuo, Sonshin Takao, Hiroyuki Shinchi, Takashi Aiko, Takeshi Iseji, Eigoro Yamanouchi, Kagoshima Univ Sch of Medicine, Kagoshima, Japan; Tokyo Women's Med Univ, Tokyo, Japan. Introduction: It has been impossible for interventional radiology(IVR) to anastomosis of internal organs. Recently, the magnets have made possible a novel method of the anastomosis between the bile duct and the small intestine with the advance of IVR. We demonstrated the case that magnetic compression anastomosis was carried out for the benign obstruction of the common bile duct repeated cholangititis after subgastrectomy. Case report: The patient was 70-yeas-old man. In August 1997, He had subgastrectomy and Billroth II reconstruction for gastric cancer. After the discharge from hospital, he had repeated cholangititis with high fever. In July 1998, jaundice was appeared, so he had a cholecystectomy in nearby hospital. However, to investigate the continuous jaundice, the cholangioscopy showed the narrowing of common bile duct(CBD). After admission to our hospital, endoscopic retrograde cholangiopancreatography(ERCP) and magnetic resonance cholangiopancreatography(MRCP) revealed the complete obstruction of CBD caused by the repeated cholangititis. Therefor, we performed the anastomosis between the hilar bile duct and 2nd portion of the duodenum using the magnets. Parent magnet moved forward the afferent loop of the duodenum, and daughter magnet was put into the obstructed CBD. Consequently, two magnets have mated transmurally. These magnets ware Samarium-Cobalt rare-earth magnets. No complications ensured and the completely patent anastomosis was established at 32 postoperative days. Discussion:Magnetic compression anastomosis has made possible non-invasive bloodless treatment. This novel treatment has the advantages of non-invasion, simple and easy suggesting a good indication for biliary obstruction. The magnetic compression might be spread throughout various branches of medicine in the future.