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Bacterial Biofilm Within Diseased Pancreatic and Biliary Tracts

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Bacterial Biofilm Within Diseased Pancreatic and Biliary Tracts 388 PANCREAS Gut: first published as 10.1136/gut.2004.043059 on 11 February 2005. Downloaded from Bacterial biofilm within diseased pancreatic and biliary tracts A Swidsinski, P Schlien, A Pernthaler, U Gottschalk, E Ba¨rlehner, G Decker, S Swidsinski, J Strassburg, V Loening-Baucke, U Hoffmann, D Seehofer, L P Hale, H Lochs ............................................................................................................................... Gut 2005;54:388–395. doi: 10.1136/gut.2004.043059 Background: Bacterial community structures in human pancreatic and biliary tracts were evaluated. Methods: Gall bladder stones from 153 patients, 20 gall bladder walls, six common duct stones, 52 biliary stents, 21 duodenal biopsies, nine pancreatic duct biopsies, and five bile ducts were investigated using fluorescence in situ hybridisation (FISH) with ribosomal RNA targeted Cy3/Cy5 (carbocyanine) labelled See end of article for authors’ affiliations oligonucleotide probes. ....................... Result: Duodenal, gall bladder, and bile duct walls were free of bacteria. A dense multispecies bacterial biofilm was present within the pancreatic duct of patients with calcific pancreatitis and within biliary stents, Correspondence to: Dr A Swidsinski, Innere irrespective of diagnosis. The concentration, density, and amenability of the biofilm to FISH and DNA Klinik, Gastroenterologie, staining declined progressively with the grade of stent occlusion. The lowest detectable bacterial Charite´, 10098 Berlin, concentrations were found by FISH in completely occluded stents and brown/mixed gall stones. Bacteria Germany; alexander.swidsinski@ were not detectable with FISH in cholesterol gall stones. charite.de Conclusions: A wide range of different branches and groups of bacteria participate in the development of biofilms on the surfaces of foreign bodies, such as biliary stents, mixed gall stones, or calcific pancreatic Revised version received ducts, but not on the surface of pure cholesterol gall stones. Occlusion of stents leads to progressive 21 April 2004 Accepted for publication extinction of the biofilm and mummification of its components. Deposition of cholesterol or other 26 May 2004 substances within the biofilm matrix may be a novel mechanism of host defence against bacteria present in ....................... these biofilms. acterial biofilms are increasingly identified as a source of endoscopically removed common duct stones were also many recalcitrant bacterial infections such as perio- studied. In addition, we studied 52 endoscopically removed dontal disease, endocarditis, chronic obstructive lung biliary stents, 12 corresponding duodenal biopsies, nine B 12 disease, foreign body related infections, etc. Gall stones are pancreatic duct and corresponding nine duodenal biopsies http://gut.bmj.com/ naturally occurring foreign bodies. The incidence, distribu- from patients with calcific pancreatitis, and five common bile tion, and impact of bacteria in gall stone formation are poorly ducts from patients operated on for cholangiocarcinoma. All understood. Currently accepted concepts of bacterial involve- patients with chronic pancreatitis had strictures of the ment are derived mainly from culture studies from the 1960s, pancreatic duct and had undergone previous manipulations, supported by electron microscopy studies34 and molecular- such as sphincterotomy in all and endoprosthesis placement genetic methods using polymerase chain reaction (PCR), in four. None had pancreatic surgery. with subsequent cloning and sequencing in the 1990s.5 The main disadvantage of both culture and PCR techniques on September 23, 2021 by guest. Protected copyright. Tissue preparation is that they do not provide any information about spatial Surgically removed gall bladders and biopsies of the organisation of the bacteria either to each other or to the pancreas, duodenum, and bile duct tissues were fixed in surface to which the biofilm is attached. They are therefore of Carnoy solution7 and initially sent to the Department of limited use in evaluation of complex biofilms. Pathology for histological investigation. Thereafter paraffin Our aims were to study the composition and spatial blocks of these tissues were subjected to FISH evaluation. organisation of bacterial biofilms on the surface of duodenal, pancreatic, and bile duct epithelium within biliary stents, common duct/gall bladder stones, and on the gall bladder Gall stone processing wall. We used fluorescence in situ hybridisation (FISH) with All gall stones were placed in 10% neutral buffered formalin rRNA targeted fluorescent probes at the domain and group immediately after surgery. A total of 132 patients had levels6 to assess the potential impact of bacteria in stent cholesterol gall stones (cholesterol content .70%), 19 had obliteration and gall stone pathogenesis. mixed pigment gall stones (cholesterol content 30–70%), and two had brown pigment gall stones (,30% cholesterol). Four of the six common duct gall stones were mixed cholesterol METHODS All human samples were removed for clinical indications, and gall stones and two were brown pigment gall stones. A 100 mg section of gall stone was crushed in 1.5 ml of represented in most cases material that would otherwise have been discarded. The use of this histological material for physiological saline and vigorously shaken for five minutes. The mixture was centrifuged at 700 for 30 seconds. Then, research aims and obtaining additional duodenal biopsies g was approved by the Institutional Human Research Committee of the Charite Hospital and all patients gave Abbreviations: Cy3/Cy5, different carbocyanine dyes used in written informed consent. fluorescence microscopy to label the oligonucleotide probes; DAPI, 4,6- Investigations were performed on gall bladder stones from diamidino-2-phenylindole; FISH, fluorescence in situ hybridisation; PCR, 153 patients. Twenty corresponding gall bladder walls and six polymerase chain reaction www.gutjnl.com Biliary and pancreatic biofilms 389 Table 1 Fluorescence in situ hybridisation probes Gut: first published as 10.1136/gut.2004.043059 on 11 February 2005. Downloaded from Name Target Reference No Eub338 Virtually all Bacteria, Kingdom (Eu)Bacteria 9 Alf1b Alpha group of Proteobacteria: Rhodobacter, Acetobacter, Paracoccus, 10 Beta42a Beta subclass of Proteobacteria: Rhodocyclus, Bordetella, Neisseria, Thiobacillus, Alcaligenes, and other 10 Gam42a Gamma subclass of Proteobacteria: Enterobacteriaceae, Proteus, Legionella, Azotobacter 10 Ec1531 Escherichia coli 11 Srb385 Sulphate reducing bacteria 12 Hpy-1 Helicobacter pylori epsilon subclass of Proteobacteria 13 Arc1430 Arcobacter sp epsilon subclass of Proteobacteria 8 HGC Gram positive bacteria with high G+C content: Actinobacteria 14 LGC Gram positive bacteria with low G+C content: Firmicutes 15 Sfb Segmented filamentous bacteria 16 Erec482 Clostridium coccoides-Eubacterium rectale group 17 Chis150 Clostridium histolyticum group 17 Clit135 Clostridium. lituseburense group 17 Lab158 Lactobacillus and Enterococcus group 18 Strc493 Streptococcus 17 Ecyl Eubacterium biforme, Clostridium innocuum, and other 19 Phasco Acidaminococcus fermentans and other 19 Veil Veillonella group 19 Rbvo,Rfla Ruminococcus flavefaciens, Clostridium leptum 19 Bif164 Bifidobacterium 20 Ato291 Atopobium, Coriobacterium, Eggerthell,andCollinsella spp 21 CF319a Cytophaga-Flavobacterium group 22 Bac303 Bacteroides/Prevotella group 22 Bfra602 Bacteroides fragilis group 17 Bdis656 Bacteroides distasonis group 17 Fprau645 Fusobacterium prausnitzii group 23 Non338 Nonsense probe was used to test for non-specific binding 10 750 ml of supernatant were taken, added to 750 ml of fresh volume of the stent piece. The volume of extracted sludge physiological saline, shaken, and centrifuged once more for was divided by the inside volume of the stent to quantify the 30 seconds. Then, 750 ml of supernatant were centrifuged at grade of stent occlusion. Stents with a sludge volume ,30% 6500 g for 10 minutes. The precipitate was resuspended in of stent volume were classified as narrowed. Stents with a 75% ethanol and stored at 220˚C. The remnants of the stones sludge volume of 30–70% were considered partially occluded. and intact stones were stored in absolute alcohol at 220˚C The stent portion was defined as occluded if the sludge and used to prepare smears and slices. volume was .70% of stent volume. Bacteria were extracted from the sludge and stored http://gut.bmj.com/ Biliary stents according to the protocol used for extraction of bacteria from Removed undamaged biliary stents (Wilson-Cook Medical, gall stones. Limerick, Ireland) were placed in 10% neutral buffered A 3 mm section of the adjacent liver ends and duodenal formalin. Two 2 cm pieces were cut off from the liver and ends of the biliary stents were cut and embedded in paraffin duodenal ends of the stent and inspected for signs of for further investigation of bacterial biofilm. occlusion. Physiological saline (2 ml) was passed by syringe through the lumen of the 2 cm stent pieces. The volume of FISH on September 23, 2021 by guest. Protected copyright. the collected sludge was measured. To estimate the inside Fluorescence in situ hybridisation was performed on glass volume of the stent piece, the stent piece was dipped in a dish slides.8 Oligonucleotide probes were synthesised with a Cy3 of water. One of the ends of the stent piece was closed under or Cy5 (carbocyanine) reactive fluorescent dye at the 59 end the water surface. The stent
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