Liver and circulating NK1.1(+)CD3(-) cells are increased in infection with attenuated Salmonella typhimurium and are associated with reduced tumor in murine liver cancer.

An attenuated (DeltacyA, Deltacrp) strain of Salmonella typhimurium (chi4550) containing a gene for human IL-2 (chi4550pIL2) reduces hepatic tumor burden when orally inoculated into mice with liver cancer; however, wild-type S. typhimurium is also associated with cancer regression. Therefore, experiments were designed to clarify the invasiveness and the anti-tumor properties of three strains of S. typhimurium. S. typhimurium chi4550pIL2, chi4550, or wild type (WT) was incubated with mature Caco-2 and HT-29 enterocytes, and S. typhimurium internalization was assessed. For infectivity experiments, mice were orally inoculated with saline or 10(9)S. typhimurium chi4550pIL2, chi4550, or WT; 48 h later mice were sacrificed for analysis of cecal bacteria and S. typhimurium translocation to mesenteric lymph nodes. For experiments involving tumor implantation, four groups were studied: saline control, tumor alone, chi4550pIL2+tumor, and chi4550+tumor. Mice were orally inoculated with saline or S. typhimurium and underwent laparotomy 24 h later with 5 x 10(4) MCA38 murine adenocarcinoma cells injected into the spleen. On day 14, liver tumors were counted and peripheral blood and hepatic lymphocyte populations were analyzed by FACScan. Attenuated S. typhimurium exhibited decreased internalization by cultured enterocytes and decreased infectivity after oral inoculation. Mice treated with chi4550pIL2 or chi4550 had fewer liver tumors and increased populations of hepatic and circulating NK1.1(+)CD3(-) lymphocytes compared to mice treated with saline (P < 0.01). These data suggest that attenuated S. typhimurium may have an application as an anti-tumor agent.

Clostridium difficile toxins and enterococcal translocation in vivo and in vitro.

BACKGROUND: Clostridium difficile toxins alter permeability in cultured enterocytes and may alter intestinal epithelial permeability to bacteria in vivo. Experiments were designed to test the effects of C. difficile toxins on in vitro interactions of Enterococcus gallinarum with cultured enterocytes, as well as on translocation of E. gallinarum in mice. MATERIALS AND METHODS: Mature Caco-2 and HT-29 enterocytes were pretreated with C. difficile toxin A or toxin B followed by incubation with E. gallinarum. E. gallinarum-enterocyte interactions were assessed by quantitative culture. For in vivo experiments, antibiotic-treated mice were orally inoculated with C. difficile or saline, and all mice were orally inoculated 24 h later with E. gallinarum and sacrificed after another 24 h for analysis of cecal bacteria, cecal C. difficile toxin, and enterococcal translocation. Cecal C. difficile toxin was assayed as cytopathic effects on human foreskin fibroblasts. RESULTS: Although neither toxin had a noticeable effect on bacterial internalization by cultured enterocytes, C. difficile toxins were associated with increased E. gallinarum transmigration across confluent enterocyte cultures. Mice orally inoculated with saline rather than C. difficile (n = 29) had no detectable cecal toxin, while mice orally inoculated with C. difficile (n = 30) had detectable cecal toxin. Viable E. gallinarum was recovered from the mesenteric lymph nodes of 97% of mice orally inoculated with saline followed by oral E. gallinarum, but only 37% of mice orally inoculated with C. difficile followed by oral E. gallinarum (P < 0.01). CONCLUSIONS: These results suggested that observations with cultured enterocytes, demonstrating that C. difficile toxins facilitated bacterial migration across the intestinal epithelium, might have little in vivo relevance in a mouse model of antibiotic-induced C. difficile overgrowth.

Clostridium difficile toxins A and B can alter epithelial permeability and promote bacterial paracellular migration through HT-29 enterocytes.

Clostridium difficile toxins A and B are the widely recognized etiologic agents of antibiotic-associated diseases ranging from diarrhea to pseudomembranous colitis. We hypothesized that C. difficile toxins may alter intestinal epithelial permeability and facilitate bacterial penetration of the intestinal epithelial barrier. Experiments were designed to clarify the effects of C. difficile toxins A and B on the flux of inert particles across HT-29 enterocyte monolayers, and to correlate these results with bacteria-enterocyte interactions. In all experiments, mature, confluent HT-29 cultures were preincubated 16 h with toxin A or B (1-100 ng/mL). To study alterations in epithelial permeability, toxin-treated enterocytes were incubated with 5 pM solutions of 10- and 40-kD inert dextran particles. Toxin A, but not toxin B, was associated with increased dextran flux through enterocyte monolayers. To study bacteria-enterocyte interactions, toxin-treated enterocytes were incubated with 10(8) Salmonella typhimurium, Proteus mirabilis, or Escherichia coli. Although numbers of internalized bacteria were generally unaffected, both toxins were associated with increased bacterial adherence, as well as increased bacterial transmigration through enterocyte monolayers. Bacterial transmigration was significantly greater using toxin A- compared to toxin B-treated enterocytes, consistent with the observation that dextran flux was significantly greater using toxin A- compared to toxin B-treated enterocytes. Thus intestinal colonization with toxigenic C. difficile may facilitate bacterial penetration of the intestinal epithelium by a mechanism involving increased permeability of the intestinal epithelial barrier.

Clostridium difficile toxins may augment bacterial penetration of intestinal epithelium.

BACKGROUND: Clostridium difficile can be recovered from many high-risk hospitalized patients receiving broad-spectrum antibiotic therapy. Clostridium difficile toxins A and B have been associated with increased intestinal permeability in vitro and there is growing evidence that increased intestinal permeability may be a common mechanism whereby enteric bacteria penetrate the intestinal epithelium. HYPOTHESIS: Clostridium difficile-induced alterations in the intestinal barrier facilitate microbial penetration of the intestinal epithelium, which in turn facilitates the translocation of intestinal bacteria. DESIGN: Mature Caco-2 enterocytes were pretreated with varying concentrations of toxin A or toxin B followed by 1 hour of incubation with pure cultures of either Salmonella typhimurium, Escherichia coli, or Proteus mirabilis. The effects of toxins A and B on enterocyte viability, cytoskeletal actin, and ultrastructural topography were assessed using vital dyes, fluorescein-labeled phalloidin, and scanning electron microscopy, respectively. The toxins' effects on bacterial adherence and bacterial internalization by cultured enterocytes were assessed using enzyme-linked immunosorbent assay and quantitative culture, respectively. Epithelial permeability was assessed by changes in transepithelial electrical resistance and by quantifying paracellular bacterial movement through Caco-2 enterocytes cultivated on permeable supports. RESULTS: Neither toxin A nor toxin B had a measurable effect on the numbers of enteric bacteria internalized by Caco-2 enterocytes; however, both toxins were associated with alterations in enterocyte actin, decreased transepithelial electrical resistance, and increased bacterial adherence and paracellular transmigration. CONCLUSION: Clostridium difficile toxins A or B may facilitate bacterial adherence and penetration of the intestinal epithelial barrier.

Acute acalculous cholecystitis (AAC) resulting in gallbladder perforation in a solid organ transplant recipient: a case report.

Gallbladder perforation is a frequent complication of acute acalculous cholecystitis (AAC), resulting in substantially increased morbidity and mortality. Two groups of patients are at increased risk for perforation: those with systemic diseases (especially peripheral vascular disease, intrinsic heart disease, or diabetes) and those who are chronically immunosuppressed. The current population of solid organ transplant recipients meets both criteria. We describe an unusual case of gallbladder perforation as a complication of AAC in an otherwise healthy kidney transplant recipient. Because transplant recipients are at increased risk for gallbladder perforation, maintaining a high index of suspicion for this complication will help avoid the increased morbidity and mortality associated with this diagnosis.

Bacteroides fragilis enterotoxin modulates epithelial permeability and bacterial internalization by HT-29 enterocytes.

BACKGROUND & AIMS: Enterotoxigenic Bacteroides fragilis has been associated with diarrheal disease, and the enterotoxin has a cytopathic effect on cultured HT-29 enterocytes. Experiments were designed to determine the effect of B. fragilis enterotoxin on bacteria-enterocyte interactions. METHODS: Confluent HT-29 enterocytes were incubated for 1 hour with B. fragilis enterotoxin, followed by 1 hour of incubation with pure cultures of enteric bacteria, namely, Salmonella typhimurium (two strains), Listeria monocytogenes (three strains), Proteus mirabilis, Escherichia coli (three strains), and Enterococcus faecalis. Enterocyte viability was assessed using vital dyes, epithelial permeability was measured using transepithelial electrical resistance, enterocyte morphology and bacteria-enterocyte interactions were visualized using light and electron microscopy, and bacterial internalization was assessed using a quantitative culture of lysed enterocytes. RESULTS: B. fragilis enterotoxin did not affect enterocyte viability but decreased transepithelial electrical resistance, and individual enterocytes pulled apart. Enterotoxin pretreatment decreased internalization of L. monocytogenes (P < 0.01) but increased (P < 0.01) internalization of the other strains of enteric bacteria. Augmented bacterial internalization was associated with preferential bacterial adherence on the exposed lateral surface of enterotoxin-treated enterocytes. CONCLUSIONS: B. fragilis enterotoxin was associated with HT-29 cell rounding and with augmented internalization of selected strains of enteric bacteria that were preferentially adherent on the exposed enterocyte lateral surface.

Bacterial translocation and lipopolysaccharide-induced mortality in genetically macrophage-deficient op/op mice.

Genetically macrophage-deficient op/op mice have a total absence of macrophage colony-stimulating factor (also known as colony-stimulating factor 1 or CSF-1), and therefore an absence of a population of macrophages dependent on CSF-1. op/op mice also have profound secondary deficiencies in certain cytokines secreted by this macrophage population, such as tumor necrosis factor, interleukin-1, and granulocyte colony-stimulating factor. In the present study, op/op mice were used to clarify the role of the macrophage in two clinical processes: (a) bacterial translocation in response to antibiotic-induced intestinal overgrowth, and (b) endotoxin-induced bacterial translocation, morbidity, and mortality. The results were unexpected, in that bacterial translocation and endotoxin-induced morbidity and mortality were similar in op/op mice and their functionally normal littermates. These data indicated either that a specific macrophage population and its cytokines (including tumor necrosis factor and interleukin 1) might not play pivotal roles in the pathogenesis of bacterial translocation and endotoxin-induced septic shock, or alternatively, as yet unknown redundancies in vivo might compensate for the genetic deficiencies associated with the op/op mutation.

Oral infectivity and bacterial interactions with mononuclear phagocytes.

The purpose of this study was to clarify the association between the oral infectivity of a bacterial strain and its susceptibility to ingestion by mononuclear phagocytes or ability to survive within them. Ten bacterial strains tested--all of known oral infectivity--comprised Salmonella typhimurium, Listeria monocytogenes (three strains), Escherichia coli (two strains), Proteus mirabilis, Enterococcus faecalis, Bacteroides fragilis, and a Bacteroides sp. The phagocytic uptake of each strain was measured as the bacteria to phagocyte ratio after mononuclear phagocytes in mouse peritoneal exudate were permitted to ingest bacteria in vivo for 3 min. The three Listeria strains were the most susceptible to phagocytic uptake and the Salmonella strain was relatively resistant. The intracellular survival of each strain was studied during a subsequent 2 h in-vitro incubation of the mononuclear phagocytes that had been permitted to ingest bacteria in vivo. The strains with the best intracellular survival were Ent. faecalis and two of the three Listeria strains. The ability of S. typhimurium to survive intracellularly was intermediate but better than that of the two E. coli strains. Oral infectivity was not consistently correlated with susceptibility to ingestion by mononuclear phagocytes or ability to survive within them.