title: Transmucosal passage of bacteria across rat intestinal epithelium in the Ussing chamber: effect of nutritional factors and bacterial virulence.
abstract: Transmucosal passage of bacteria across the intestine, the essential and prerequisite step for bacterial translocation, cannot be effectively studied using in vivo models of translocation. We have adapted the Ussing chamber into a fresh, sterile organ culture system that can facilitate the study of bacterial-epithelial interactions. Intestinal membranes were mounted in the Ussing chamber and perfused with a solution rich in putative mucosal micronutrients. The transmembrane potential difference was constantly monitored as a marker of intestinal integrity. Transmucosal passage of various bacteria across the normal intestinal epithelium was quantitated, and the mucosal membrane was examined by light and transmission electron microscopy. The addition of potassium cyanide to the mucosal perfusate resulted in an irreversible loss of potential difference. Oxygen deprivation also led to a precipitous drop in potential difference, but it was reversible with prompt reoxygenation. In contrast, intestinal membranes perfused with a solution consisting of Dulbecco's modified Eagle's medium + 20 mM glutamine maintained their potential difference for a sustained period (>180 min). Both the viability and structural integrity of the ileal intestinal membrane were maintained in culture ex vivo using this perfusate. Qualitative differences were observed in the mechanism of transmucosal passage of mild to moderately virulent bacteria such as Escherichia coli C-25 and Proteus mirabilis M-13, which pass through the intestinal epithelium without causing overt damage to the mucosa, and more virulent organisms such as Salmonella typhimurium, which cause extensive mucosal damage by light and transmission electron microscopy. The Ussing system should provide a useful model of intact organ culture for the study of the mechanisms of bacterial translocation and the pathogenesis of enteric infections.
authors.affiliation: Department of Surgery, University of Pittsburgh School of Medicine and The Children's Hospital of Pittsburgh, Pennsylvania 15213, USA.
authors.forename: A G
authors.initials: AG
authors.lastname: Kurkchubasche
authors.forename: M
authors.initials: M
authors.lastname: Cardona
authors.forename: S C
authors.initials: SC
authors.lastname: Watkins
authors.forename: S D
authors.initials: SD
authors.lastname: Smith
authors.forename: C T
authors.initials: CT
authors.lastname: Albanese
authors.forename: R L
authors.initials: RL
authors.lastname: Simmons
authors.forename: M I
authors.initials: MI
authors.lastname: Rowe
authors.forename: H R
authors.initials: HR
authors.lastname: Ford
mesh_terms: D000818:Animals; D001120:Arginine / Q000494:pharmacology; D018988:Bacterial Translocation / Q000502:physiology*; D002462:Cell Membrane / Q000187:drug effects / Q000473:pathology; D019069:Cell Respiration; D015194:Diffusion Chambers, Culture / Q000379:methods; D004563:Electrochemistry; D004848:Epithelium / Q000382:microbiology; D004926:Escherichia coli; D005947:Glucose / Q000494:pharmacology; D005973:Glutamine / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000166:cytology / Q000382:microbiology*; D008297:Male; D009584:Nitrogen / Q000494:pharmacology; D011190:Potassium Cyanide / Q000494:pharmacology; D011513:Proteus mirabilis; D051381:Rats; D017207:Rats, Sprague-Dawley; D012486:Salmonella typhimurium
chemical_list: D005973:Glutamine; D001120:Arginine; D005947:Glucose; D011190:Potassium Cyanide; D009584:Nitrogen