Intracellular survival of Staphylococcus aureus within cultured enterocytes

BACKGROUND: Candida albicans is a polymorphic fungus that frequently causes systemic infection in postsurgical and trauma patients. Others have reported that Escherichia coli lipopolysaccharide (LPS) acts as a copathogen to enhance the virulence of parenteral C. albicans. Experiments were designed to clarify the effect of parenteral LPS on systemic candidiasis initiated via the oral route. MATERIALS AND METHODS: Antibiotic-treated mice were orally inoculated with C. albicans CAF2 (wild-type) or mutant HLC54 (defective in filament formation), and were given 100 microg parenteral LPS 16 h before sacrifice. Separate groups of mice were additionally exposed to intermittent hypoxia prior to LPS. At sacrifice, cecal flora and microbial translocation to the mesenteric lymph nodes were quantified. C. albicans adherence to cultured HT-29 and Caco-2 enterocytes (pretreated with LPS, or calcium-free medium to expose the enterocyte lateral surface, or both) was quantified by enzyme-linked immunoabsorbent assay. RESULTS: All mice had high numbers of cecal C. albicans, and LPS was associated with an additional increase in cecal concentrations of HLC54 but not CAF2. Translocation of HLC54, but not CAF2, appeared facilitated by hypoxia, but LPS did not facilitate translocation in any treatment group. Exposure of the lateral surface of cultured enterocytes had no effect on C. albicans adherence, although LPS consistently decreased adherence of both C. albicans strains. CONCLUSIONS: In contrast to experiments where systemic candidiasis was initiated by the parenteral route, parenteral LPS did not act as a copathogen in mice with systemic candidiasis initiated by the oral route, and these results might be related to LPS-induced alterations in C. albicans adherence to host enterocytes.     

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 toxin: cytoskeletal changes and lactate dehydrogenase release in hepatocytes

BACKGROUND: We have found that Clostridium difficile toxins can evoke hepatocyte acute-phase protein synthesis, and that this effect is dependent on a functioning interleukin-1 (IL-1) receptor. The present study was undertaken to determine if C. difficile toxicity, as determined by actin rearrangement and lactate dehydrogenase (LDH) release, also requires a functioning IL-1 receptor. METHODS: Primary hepatocyte cultures were prepared from normal mice, knockout mice deficient in the IL-1-converting enzyme (ICE), and knockout mice deficient in the IL-1 p80 receptor. Hepatocytes were treated for 24 h with C. difficile culture extract, purified C. difficile toxin A, or purified C. difficile toxin B. The actin cytoskeleton was examined using confocal microscopy, and LDH release was measured by spectrophotometric analysis. RESULTS: C. difficile culture extract, toxin A, and toxin B induced collapse of the actin cytoskeleton in hepatocytes from normal mice. Hepatocytes from both the ICE-deficient mice and the IL-1 p80 receptor-deficient mice demonstrated similar responses to both toxins. These toxins also induced significant LDH release in a concentration-dependent fashion in the normal hepatocytes and the ICE-deficient hepatocytes. However, no significant increase in LDH release was observed in hepatocytes from IL-1 p80 receptor-deficient mice. CONCLUSIONS: C. difficile toxins induce actin cytoskeletal collapse independent of IL-1 or the IL-1 receptor. In contrast, toxin-stimulated LDH release was dependent on the presence of the IL-1 receptor. Thus, separate pathways appear to mediate toxic effects as manifested by actin rearrangement and LDH release.     

Clostridium difficile toxins influence hepatocyte protein synthesis through the interleukin 1 receptor

HYPOTHESIS: Clostridium difficile toxins require interleukin 1 (IL-1) production or a functioning IL-1 receptor to elicit acute-phase protein production by murine hepatocytes. DESIGN: Experimental study. SETTING: Research laboratory at the DVA Medical Center, St Louis, Mo. CELLS STUDIED: Hepatocytes prepared from normal mice, from knockout mice deficient in IL-1 production due to loss of IL-1 converting enzyme, or from knockout mice deficient in the IL-1 p80 receptor. INTERVENTIONS: Cells were treated with lipopolysaccharide, a crude C difficile toxin extract, or purified C difficile toxins A or B for 24 hours in vitro, then radiolabeled with (35)S methionine. Newly synthesized acute-phase proteins were identified by electrophoresis and autoradiography. MAIN OUTCOME MEASURES: Synthesis of a 23-kd acute-phase protein in response to the various stimuli. RESULTS: Lipopolysaccharide, C difficile culture extract, and purified toxins A and B stimulated the synthesis of the 23-kd acute-phase protein by hepatocytes from normal mice and by hepatocytes from knockout mice deficient in the IL-1 converting enzyme. However, hepatocytes from knockout mice deficient in the IL-1 p80 receptor failed to produce this acute-phase protein when treated with the C difficile toxins, although they responded fully to lipopolysaccharide. CONCLUSIONS: Stimulation of acute-phase protein synthesis by C difficile toxins does not require IL-1 production, but does require a functioning IL-1 p80 receptor. This suggests that some of the actions of these toxins are mediated by this receptor.     

Detection of Clostridium difficile in stool samples from patients in the early period after liver transplantation

OBJECTIVE: We examined the frequency of detection of Clostridium difficile (CD) toxins compared with the recovery of C. difficile in stool specimen cultures among orthotopic liver transplant (OLT) patients with nosocomial diarrhea in the early period. MATERIALS AND METHODS: The study included stool samples obtained during the first 30 days after OLT in adults who were suspected of CD-associated diseases. The identification of cultured CD strains was performed by standard microbiological methods. The presence of CD toxins was assayed using a commercial immunoassay. RESULTS: All patients were followed prospectively for CD infections from the date of OLT for the first 4 weeks after surgery. Among 54 samples, 16.7% were culture-positive for CD. CD toxins were tested on 54 samples, yielding 63% toxin-positive samples and 30% toxin- and culture-negative results. In the first week after OLT, samples from 19 patients were subjected to CD investigation. Among 19 samples positive for toxin, 52.6% of all samples were culture-negative. We analyzed 35 samples from the second to the fourth week after OLT in 31 recipients. Among 35 samples, 68.6% and 25.7% were positive for CD toxin and for culture, while 20% of samples were negative for toxin and culture. CONCLUSION: In our study, 63% of samples were toxin-positive with 16.7% yielding growth of CD and 30% being negative for toxins and cultures.     

Prevalence of clostridium difficile in excluded colons

Clostridium difficile infection is associated with substantial morbidity and mortality, increased duration of hospitalization, and a marked economic impact. Several case reports and case series have described C. difficile infection in excluded bowels or immediately after reversal of defunctioning ileostomy. The aim of this prospective study is to detect whether the excluded colon is associated with a higher rate of C. difficile colonization than the normal population, which may increase the risk of C. difficile infection. Patients with defunctioning loop ileostomy, undergoing closure of ileostomy to restore bowel continuity, were prospectively recruited. Two stool samples were collected from the ileostomy effluent before closure of ileostomy and two after the procedure including the first bowel movement. All samples were cultured for C. difficile and analyzed for toxins A and B by a Premier EIA test. Demographic data and possible confounding factors were observed and recorded. Twenty-fine adult patients were recruited to this study; five patients were subsequently excluded. Two patients had positive stool cultures for C. difficile in the postoperative samples and another patient developed clinical pseudomembranous colitis with positive toxin. This indicates a possible colonization rate of 3 to 38 per cent (95% confidence interval). Four observed cases out of the 20 subjects taking part in this study would confidently conclude that C. difficile colonization in the excluded colon is 6 to 44 per cent, i.e., higher than the incidence in the healthy adult population, which is 3 per cent. However, the findings of this study prompt larger and well-powered studies to confirm these findings.     

Evidence for Rho protein regulation of renal tubular epithelial cell function

BACKGROUND: Rho proteins are small guanine 5'-triphosphate (GTP)-binding proteins felt to be important regulators of several aspects of cell function, including the organization of the actin cytoskeleton. The effects of Rho proteins on the regulation of renal tubular epithelial cell function are not known. METHODS: Selected bacterial toxins that inhibit Rho protein function were used to examine the effect of Rho in cultured renal tubular epithelial cells. RESULTS: Clostridium difficile toxin A significantly and dose dependently inhibited LLC-PK(1) cell (3)H-thymidine uptake and healing of small wounds made in confluent monolayers, and it induced apoptosis. A second Clostridium difficile toxin (toxin B) that acted via a different receptor also impaired LLC-PK(1) thymidine uptake and wound healing, and it induced apoptosis. A third bacterial toxin, C3 toxin from Clostridium botulinum, also impaired LLC-PK(1) thymidine uptake and stimulated apoptosis in LLC-PK(1) cells. Since Rho inhibition disrupted organization of the actin cytoskeleton, we examined the effects of another agent that disrupted the actin cytoskeleton (cytochalasin D) and found significant dose-dependent effects that impaired LLC-PK1 thymidine uptake and wound healing and that induced apoptosis. The effects of toxin A and cytochalasin D to induce apoptosis were not associated with significant changes in expression of Bcl-2, BAD, or BAK proteins and were significantly attenuated by a pancaspase inhibitor. CONCLUSIONS: Our results suggest that Rho proteins are important endogenous regulators of several aspects of renal tubular epithelial cell function, including proliferation, migration, and apoptosis. Further studies are needed to clarify the cellular mechanisms of Rho regulation of renal epithelial cell function.     

Endotoxin induces bacterial translocation and increases xanthine oxidase activity

Previously, we documented that endotoxin induces bacterial translocation from the gut and that inhibition or inactivation of xanthine oxidase activity reduces endotoxin-induced bacterial translocation. Consequently, experiments were performed to correlate endotoxin-induced bacterial translocation with changes in intestinal mucosal structure and xanthine dehydrogenase and oxidase activity. Segments of the jejunum, ileum, cecum, proximal colon, distal colon, and liver were harvested from ICR mice 24 hr after IP administration of E. coli 0111:B4 endotoxin (0.1 mg). Xanthine dehydrogenase and oxidase activities were measured in these samples and correlated with intestinal morphology. Bacteria translocated from the intestines to extraintestinal organs in 70% of the mice receiving endotoxin, while the organs of control mice were sterile (p less than 0.01). Endotoxin injured primarily the ileal and cecal mucosa and increased ileal and hepatic xanthine dehydrogenase and cecal oxidase activities (p less than 0.05). These results suggest that xanthine oxidase-induced mucosal damage plays a role in endotoxin-induced bacterial translocation.     

Antibiotic prophylaxis diminishes bacterial translocation but not mortality in experimental burn wound sepsis

Pseudomonas (PSA) burn wound sepsis results in prolonged bacterial translocation (BT) of enteric organisms such as E. coli to the mesenteric lymph nodes (MLN) and organs in rats. Intestinal decontamination with oral antibiotics may improve mortality after burn injury, perhaps due to decreased BT. To determine the effect of oral antibiotic prophylaxis effective against E. coli but not PSA on BT and subsequent mortality in a model of PSA burn wound sepsis, rats were given a 30% scald burn and wound inoculation with 10(8) PSA followed by randomization to either ampicillin (50 mg/kg/d) or saline gavage. Cultures of MLN, organs, blood, and cecal contents were obtained on days 1, 4, and 7 after injury, with additional animals observed for 14-day mortality. Although oral antibiotic prophylaxis resulted in increased cecal colony counts, the incidence of BT was unchanged. The number of organisms present in both the MLN and organs, however, was significantly reduced with prophylaxis, indicating cecal overgrowth by non-translocating bacteria. Reduction of the number of translocating organisms did not result in improved mean survival time after injury, suggesting that mortality from PSA burn wound sepsis occurs independently of bacterial translocation.     

Promotion by burn stress of the translocation of bacteria from the gastrointestinal tracts of mice

A mouse burn model was established to test the effect of nonlethal thermal injury on the translocation of indigenous bacteria from the gastrointestinal (GI) tract to other organs. Specific pathogen-free (SPF) mice were given 15% or 30% total body surface area burns, and the mesenteric lymph nodes (MLNs), spleens, livers, blood, and peritoneal cavities were cultured for translocated bacteria at various time intervals. No viable aerobic, facultatively anaerobic, or strictly anaerobic bacteria of the indigenous flora grew in cultures from the MLNs of these mice. Consequently, SPF mice were antibiotic decontaminated and then colonized with Escherichia coli to develop a model in which E coli maintains abnormally high cecal population levels and translocates continuously to the MLN. These mice received 15% or 30% thermal burns four days after colonization with E coli. The incidence of bacterial translocation and the numbers of E coli translocating to the MLN, spleen, liver, blood, and peritoneal cavity increased with increasing burn area compared with controls. Mice receiving 15% burns could not clear intravenously challenged E coli from their bloodstream, MLN, or liver. Thus, burn stress promotes the translocation of bacteria from the GI tract to other organs to cause bacteremia.     

Surgical stress shifts the intestinal Escherichia coli population to that of a more adherent phenotype: role in barrier regulation.

BACKGROUND: We have shown that the combination of surgical stress and starvation in mice is associated with a defect in epithelial permeability and increased numbers of mucosa-associated Escherichia coli in the cecum. The aim of this study was to determine the specific role of mucosa-associated E coli on epithelial barrier dysfunction in this model. METHODS: Cecal E coli were harvested from mice 48 hours after a sham operation (control mice) or after a 30% surgical hepatectomy with only water provided ad libitum (short-term starvation) after the surgical procedure. Strains were tested for their ability to adhere to and alter the transepithelial electrical resistance (TEER) of cultured young adult mouse colon epithelial cells. TEER changes were further characterized by mannitol fluxes to confirm a defect in paracellular permeability. RESULTS: Strains of cecal E coli harvested from hepatectomy-starved mice adhered to and altered the permeability of young adult mouse colon cells, whereas E coli from the cecum of control mice were less adherent and had no effect on epithelial permeability. The effect of the strains harvested from mice after hepatectomy on the TEER of young adult mouse colon cells was inhibited by mannose and reversed by ciprofloxacin. CONCLUSION: The combination of surgical stress and short-term starvation is associated with a greater abundance of adherent and barrier-altering strains of E coli in the mouse cecum.     

Studies on the tissue culture of murine sarcoma (Dunn sarcoma)--a fix cell line established in vitro (DSK) and its cytokinetic findings (author's transl)

Dunn osteosarcoma, transplanted successively from the original spontaneous murine osteosarcoma, was cultured for generations and serial changes of cultured cells were observed morphologically and also from the view points of chromosomes and cytokinetics. 1) In the early generations of subculture, the cultured cells were predominantly of spindle shape, proliferating in a mesh-like pattern. However, with generations the epitheloid cells became dominant with a slight increase in their proliferating rate, and from 16th generation thereafter, the cellular pattern became almost uniform. The nuclei, being in the center of the cytoplasm, varied in their size and number, and several mitoses were also noted. In the 8th and 9th generations, the multinucleated giant cells were greatest in their size, and their nuclei were most numerous. 2) The cells of each generation, inoculated into the mice, developed tumors in the mice with the same histological appearance as that of original tumors. From the 17th generation thereafter, these inoculations (in vivo back inoculation) were constantly successful in producing tumors. Meanwhile, the cells of the tumor induced by in vivo back inoculation could be cultured, and showed the same morphological patterns as that of the serially cultured cells. 3) The chromosomes of the cultured cells were prepared by treating the tissue with colchicine followed by 0.2% hypotonic saline. From the primary culture to the first ten generations, the mode of the chromosome number varied from diploidy to high diploidy, while it became stable in high diploidy from 16th generation thereafter. The shapes of the chromosomes were various, and there were a few bar-shaped chromosomes. No constant marker chromosome was detectable. 4) The mouse spleen cells, cultured with the inactivated cultured tumor cells were activated by the target, recognizing the tissue specific transplantation antigen of the target. Considering these findings, this series of tissue culture is regarded as a fixed cell line. Therefore, it is concluded that an experimental system of Dunn osteosarcoma has been established in vitro, and it could be named as DSK (Dunn sarcoma in vitro, KITASATO) by the author. The duration of cell cycle of DSK, using the cumulative labeling method, is 11 hours, which is about two hours shorter than that of in vivo.     

Local Anesthetics Reduce Mortality and Protect against Renal and Hepatic Dysfunction in Murine Septic Peritonitis

Background: Mortality from sepsis frequently results from multiple organ injury and dysfunction. Cecal ligation and puncture is an established murine model of septic peritonitis that produces septic shock characterized by an initial hyperinflammatory response. In addition to their anesthetic properties, local anesthetics have been shown to attenuate inflammatory responses both in vivo and in vitro. In the current study, the ability of local anesthetic infusions to protect against sepsis-induced mortality, as well as renal and hepatic dysfunction after cecal ligation and puncture, was investigated.

Methods: C57BL/6 mice received mini-osmotic pumps containing saline (vehicle), 10% lidocaine, or 1% bupivacaine and were subjected to cecal ligation and puncture. Twenty-four hours after cecal ligation and puncture, renal and hepatic functions were assessed as well as markers of inflammation (proinflammatory cytokine protein and mRNA concentrations and myeloperoxidase activity). Renal apoptosis and 7-day survival was also assessed.

Results: Mice treated with lidocaine or bupivacaine infusion showed improved survival and had significantly lower plasma creatinine, aspartate aminotransferase, and alanine aminotransferase concentrations compared with mice receiving vehicle alone. Significant reduction in plasma tumor necrosis factor-[alpha] and keratinocyte-derived chemokine, as well as reductions in myeloperoxidase activity, intracellular adhesion molecule-1 protein expression, mRNA concentrations of proinflammatory markers, and apoptosis were observed in renal cortices from both local anesthetic groups.     

Macrophage antigen presentation and interleukin 1 production after cecal ligation and puncture in C3H/HeN and C3H/HeJ mice

Following cecal ligation and puncture with a 25-gauge needle, endotoxin-sensitive C3H/HeN mice have a 45% mortality compared with no mortality in endotoxin-resistant C2H/HeJ mice. Macrophage production of interleukin 1 and antigen presentation were studied in these two strains of mice following cecal ligation and puncture at 2, 4, 8, 16, and 24 hours and at 2, 4, 6, and 8 days. Splenic macrophages were cultured with a T-helper cell clone (D10.G4.1), and antigen presentation and interleukin 1 production were measured by D10.G4.1 proliferation. Macrophage antigen presentation by C2H/HeJ mice was markedly increased compared with that in C3H/HeN mice at all times after cecal ligation and puncture, most strikingly at 2 days (185m740 cpm for C3H/HeJ mice vs 30,300 for C2H/HeN mice). Macrophage interleukin 1 production was significantly increased in C3H/HeJ mice vs C3H/HeN mice at all times after cecal ligation and puncture (except at 2 days) and was maximal at 8 days (25,000 cpm for C3H/HeJ mice vs 5190 for C3H/HeN mice). These data suggest that the differences in mortality after cecal ligation and puncture between these two strains of mice may relate to a supranormal response of macrophages of C3H/HeJ mice or to an inadequate response of macrophages of C3H/HeN mice.     

Local anesthetics reduce mortality and protect against renal and hepatic dysfunction in murine septic peritonitis

BACKGROUND: Mortality from sepsis frequently results from multiple organ injury and dysfunction. Cecal ligation and puncture is an established murine model of septic peritonitis that produces septic shock characterized by an initial hyperinflammatory response. In addition to their anesthetic properties, local anesthetics have been shown to attenuate inflammatory responses both in vivo and in vitro. In the current study, the ability of local anesthetic infusions to protect against sepsis-induced mortality, as well as renal and hepatic dysfunction after cecal ligation and puncture, was investigated. METHODS: C57BL/6 mice received mini-osmotic pumps containing saline (vehicle), 10% lidocaine, or 1% bupivacaine and were subjected to cecal ligation and puncture. Twenty-four hours after cecal ligation and puncture, renal and hepatic functions were assessed as well as markers of inflammation (proinflammatory cytokine protein and mRNA concentrations and myeloperoxidase activity). Renal apoptosis and 7-day survival was also assessed. RESULTS: Mice treated with lidocaine or bupivacaine infusion showed improved survival and had significantly lower plasma creatinine, aspartate aminotransferase, and alanine aminotransferase concentrations compared with mice receiving vehicle alone. Significant reduction in plasma tumor necrosis factor-alpha and keratinocyte-derived chemokine, as well as reductions in myeloperoxidase activity, intracellular adhesion molecule-1 protein expression, mRNA concentrations of proinflammatory markers, and apoptosis were observed in renal cortices from both local anesthetic groups. CONCLUSIONS: The current data demonstrate that local anesthetic infusions confer a protective effect in mice from septic peritonitis by attenuating the hyperacute inflammatory response. This suppression resulted in improved mortality and less progression to acute kidney and liver injury and dysfunction.     

Clostridium difficile infections: what is new?

C. difficile is the most common infectious cause of healthcare-associated diarrhea but now is increasingly recognized as a cause of diarrhea in outpatients and persons without apparent health care contacts. Emergence and spread of new epidemic clones of C. difficile 027 (PCR-ribotype) and 078/126 (toxinotype) with increase toxin production, an aditional binary toxin and high level resistance to fluoroquinolones and increasing incidence of more rapidly progressive severe disease, require prompt clinical recognition and new tools to predict severity and to prevent recurrences. Although antibiotics are effective at inhibiting C. difficile and treating symptoms, these drugs could not reestablish normal bowel flora and the rate of recurrences is 25%. During the past years we assisted to an impressive search for new and more effective therapy that shoud be save, with low potential for the development of resistance, with low levels of systemic absorbtion and high levels of active drug in the colon and should be associated with a low rate of recurrence after treatment. By consequence, different approaches to the management of recurrent infections have been studied such as new antibiotics (fidaxomicin), human monoclonal antibodies against C. difficile toxins A and B, intravenous human immunoglobulin, active immunization, and probiotic therapy.     

Effect of intraluminal antibiotics on translocation of Candida albicans in burned guinea-pigs

Guinea-pigs were pretreated orally with various antibiotics and given 3 x 10(10) Candida albicans by gastric lavage followed by a 40 per cent TBSA full skin thickness burn. The mesenteric lymph nodes, liver and spleen were cultured for the presence of viable organisms. Caecal contents were quantitatively cultured for aerobic bacteria and C. albicans. Clindamycin and penicillin G were the greatest promoters of translocation followed by the combination streptomycin/bacitracin. The mechanism for antibiotic-induced translocation is multifactorial centering on intestinal flora, anaerobic spectrum of the antibiotic and host defense as well as microbe virulence. The systemic use of broad-spectrum antibiotics, particularly those with strong anaerobic activity, should not be taken too lightly. A severely immunocompromised patient on this type of therapy may be prone to a severe fungal infection. This study reaffirms the concept that translocation from the gastrointestinal barrier is a potential source of life-threatening nosocomial infection.     

Fulminant Clostridium difficile: an underappreciated and increasing cause of death and complications

OBJECTIVE: To review the epidemiology and characteristics of patients who died or underwent colectomy secondary to fulminant Clostridium difficile colitis. SUMMARY BACKGROUND DATA: In patients with C. difficile colitis, a progressive, systemic inflammatory state may develop that is unresponsive to medical therapy; it may progress to colectomy or death. METHODS: The authors reviewed 2,334 hospitalized patients with C. difficile colitis from January 1989 to December 2000. Sixty-four patients died or underwent colectomy for pathologically proven C. difficile colitis. RESULTS: In 2000, the incidence of C. difficile colitis in hospitalized patients increased from a baseline of 0.68% to 1.2%, and the incidence of patients with C. difficile colitis in whom life-threatening symptoms developed increased from 1.6% to 3.2%. Forty-four patients required a colectomy and 20 others died directly from C. difficile colitis. Twenty-two percent had a prior history of C. difficile colitis. A recent surgical procedure and immunosuppression were common predisposing conditions. Lung transplant patients were 46 times more likely to have C. difficile colitis and eight times more likely to have severe disease. Abdominal computed tomography scan correctly diagnosed all patients, whereas 12.5% of toxin assays and 10% of endoscopies were falsely negative. Patients undergoing colectomy for C. difficile colitis had an overall death rate of 57%. Significant predictors of death after colectomy were preoperative vasopressor requirements and age. CONCLUSIONS: C. difficile colitis is a significant and increasing cause of death. Surgical treatment of C. difficile colitis has a high death rate once the fulminant expression of the disease is present.     

Effect of INT1 gene on Candida albicans murine intestinal colonization

BACKGROUND: Increased intestinal colonization with Candida albicans is believed to be a major factor predisposing immunocompromised and postsurgical patients to systemic candidiasis, although the mechanisms facilitating C. albicans colonization remain unclear. Because previous studies have linked the C. albicans INT1 gene to filament formation, epithelial adherence, and mouse virulence, experiments were designed to evaluate the effect of INT1 on intestinal colonization. MATERIALS AND METHODS: Mice were orally inoculated with either the parent strain (CAF2, INT/INT1), an int1 heterozygote (CAG1, INT1/int1), an int1 homozygote (CAG3, int1/int1), or a reintegrant (CAG5, int1/int1 + INT1), and sacrificed 3 and 7 days later for quantitative analysis of cecal C. albicans. RESULTS: Following oral inoculation with 10(3) C. albicans, only small numbers of each strain were recovered from the cecal flora of normal mice. However, in mice pretreated with oral antibiotics, cecal colonization of each strain was increased (P < 0.01). In addition, cecal colonization was reduced for all int1 mutant strains compared with the parent strain (P < 0.05). By light microscopy, all four C. albicans strains were easily observed in the ileal lumen as both budding yeast and filamentous forms, although only occasional yeast forms appeared adherent to the intestinal epithelium. CONCLUSIONS: C. albicans readily colonized and replicated in the ceca of antibiotic-treated mice. The presence of two functional copies of INT1 appeared to facilitate C. albicans cecal colonization, suggesting that intestinal colonization may be another virulence factor associated with INT1 and that the gene product may be an attractive target to control C. albicans intestinal colonization.     

Thermal injury promotes bacterial translocation from the gastrointestinal tract in mice with impaired T-cell-mediated immunity

We have shown previously that after thermal trauma viable bacteria will cross the intact gastrointestinal mucosa (bacterial translocation) to invade the mesenteric lymph nodes and other organs if the normal indigenous microflora is disrupted, allowing bacterial overgrowth. To determine whether T-cell-mediated immunity (T-CMI) was important in preventing translocation after thermal injury in animals with an intact normal flora, conventional (+/+), athymic (nu/nu), and heterozygous (nu/+) mice receiving a 30% third-degree burn were killed at various intervals after burn and their organs cultured. Bacterial translocation did not occur in control or burned specific pathogen-free mice with intact T-CMI but did occur in athymic mice with deficient T-CMI. Both the incidence of positive organs and the numbers of translocated bacteria per gram of organ were increased after thermal injury. Bacterial overgrowth was not responsible for these findings, since the levels of cecal enteric bacteria were not different between the burned and nonburned groups. Since translocation occurred to a greater extent in athymic burned mice than control athymic mice, it appears that a thermal injury promotes translocation by impairing other host defense systems in addition to the T-CMI.