TEMPO EC for the enumeration of Escherichia coli in foods: collaborative study.

The automated method for enumeration of Escherichia coli, TEMPO EC, in foods uses a dehydrated culture medium and enumeration card containing 48 wells across three different dilutions for the automatic determination of the most probable number (MPN). The alternative method was compared in a multilaboratory collaborative study to AOAC Official Method 966.24. Six food types were artificially contaminated with E. coli: raw ground beef, bagged lettuce, cooked chicken, pasteurized crabmeat, frozen green beans, and pasteurized whole milk. All foods were analyzed for E. coli counts by 11 collaborating laboratories throughout the United States. Test portions from the six food types each contaminated at four different contamination levels were evaluated. The study demonstrated that the TEMPO EC method is a reliable, automated assay for the enumeration of E. coli in foods.

Evaluation of the VIDAS Listeria (LIS) immunoassay for the detection of Listeria in foods using demi-Fraser and Fraser enrichment broths, as modification of AOAC Official Method 999.06 (AOAC Official Method 2004.06).

A multilaboratory study was conducted to compare the VIDAS LIS immunoassay with the standard cultural methods for the detection of Listeria in foods using an enrichment modification of AOAC Official Method 999.06. The modified enrichment protocol was implemented to harmonize the VIDAS LIS assay with the VIDAS LMO2 assay. Five food types--brie cheese, vanilla ice cream, frozen green beans, frozen raw tilapia fish, and cooked roast beef--at 3 inoculation levels, were analyzed by each method. A total of 15 laboratories representing government and industry participated. In this study, 1206 test portions were tested, of which 1170 were used in the statistical analysis. There were 433 positive by the VIDAS LIS assay and 396 positive by the standard culture methods. A Chi-square analysis of each of the 5 food types, at the 3 inoculation levels tested, was performed. The resulting average Chi square analysis, 0.42, indicated that, overall, there are no statistical differences between the VIDAS LIS assay and the standard methods at the 5% level of significance.

Evaluation of the BAX system for detection of Listeria monocytogenes in foods: collaborative study.

A multilaboratory study was conducted to compare the automated BAX system and the standard cultural methods for detection of Listeria monocytogenes in foods. Six food types (frankfurters, soft cheese, smoked salmon, raw, ground beef, fresh radishes, and frozen peas) were analyzed by each method. For each food type, 3 inoculation levels were tested: high (average of 2 CFU/g), low (average of 0.2 CFU/g) and uninoculated controls. A total of 25 laboratories representing government and industry participated. Of the 2335 samples analyzed, 1109 were positive by the BAX system and 1115 were positive by the standard method. A Chi square analysis of each of the 6 food types, at the 3 inoculation levels tested, was performed. For all foods, except radishes, the BAX system performed as well as or better than the standard reference methods based on the Chi square results.

Gastrointestinal colonization by Candida albicans mutant strains in antibiotic-treated mice.

Antibiotic-treated mice orally inoculated with one of three Candida albicans strains (including two mutant strains) or indigenous Candida pelliculosa showed levels of candidal gastrointestinal colonization that were strain specific. However, regardless of strain, the numbers of viable candida were intermediate to high in the stomach, were consistently lowest in the upper small intestine, and increased progressively down the intestinal tract.

The Candida albicans INT1 gene facilitates cecal colonization in endotoxin-treated mice.

Increased intestinal colonization with Candida albicans is believed to be a major predisposing factor to systemic candidiasis. Previous evidence has implicated the C. albicans INT1 gene in hyphal development, epithelial adherence, and mouse virulence. The effect of INT1 on mouse cecal colonization was measured using a parent strain (CAF2, INT1/INT1), an int1 deletion homozygote (CAG3, int1/int1), and a heterozygous reintegrant (CAG5, int1/int1 + INT1). Forty-eight hours after oral inoculation of 10(7) C. albicans into normal mice, only low numbers of each strain were recovered from the cecal flora. In mice pretreated with oral bacitracin/streptomycin, cecal colonization of each C. albicans strain was increased compared to the corresponding strain inoculated into untreated mice, with the CAF2 parent strain greater (P < 0.01) than the two mutant strains, and with the heterozygous and homozygous mutants not different from each other. In mice pretreated with parenteral lipopolysaccharide (LPS), in addition to oral antibiotics, numbers of cecal CAF2, CAG5, and CAG3 were increased (P < 0.01) compared to the corresponding strain inoculated into mice treated with antibiotics alone. In LPS-treated mice, numbers of cecal C. albicans CAF2 (INT1/INT1) were greater (P < 0.05) than C. albicans CAG3 (int1/int1). Thus, parenteral LPS had an additive effect on C. albicans cecal colonization in antibiotic-treated mice, and the presence of two functional copies of the INT1 gene appeared to facilitate colonization in both antibiotic-treated mice and in mice treated with antibiotics plus parenteral endotoxin.

Effect of oral genistein and isoflavone-free diet on cecal flora and bacterial translocation in antibiotic-treated mice.

BACKGROUND: There are several reports indicating that the isoflavone genistein may augment the integrity of the intestinal epithelial barrier as well inhibit bacterial internalization by cultured enterocytes. We speculated that oral genistein might enhance the integrity of the intestinal epithelial barrier as monitored by the extraintestinal dissemination of intestinal bacteria. METHODS: Mice were treated with oral antibiotics to induce cecal bacterial overgrowth accompanied by bacterial translocation of antibiotic-resistant enterobacteria, especially Escherichia coli. These mice were divided into separate groups that included chow-fed mice orally inoculated either with saline, vehicle, or genistein, and mice fed isoflavone-free diet and orally inoculated with either saline, vehicle, or genistein. Intestinal bacterial overgrowth was monitored by quantitative culture of excised ceca and bacterial translocation was monitored by quantitative culture of draining mesenteric lymph nodes. RESULTS: Mice fed the isoflavone-free diet had decreased populations of cecal bacteria compared with chow-fed mice, and bacterial translocation was reduced in chow-fed mice compared with mice fed isoflavone-free diet. However, bacterial translocation was similar in mice given oral genistein compared with appropriate control mice. CONCLUSIONS: Oral genistein had no noticeable effect on bacterial translocation in this model. However, the isoflavone-free diet had an antibacterial effect on cecal flora, and the isoflavone-free diet was associated with decreased numbers of cecal bacteria and decreased incidence of bacterial translocation.

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.

Systemic infection following intravenous inoculation of mice with Candida albicans int1 mutant strains.

The Candida albicans gene INT1 is associated with epithelial adhesion, hyphal formation, and virulence. C. albicans strains carrying two, one, or no functional INT1 alleles were used to assess the association between mortality and C. albicans persistence in the liver and kidney of intravenously inoculated mice. Mice were injected with 10(5) C. albicans CAF2 (parent strain, INT1/INT1), C. albicans CAG3 (homozygous disruptant, Int1/int1), or C. albicans CAG5 (heterozygous reintegrant, int1/int1 + INT1). Mortality was monitored and mice were sacrificed on Days 1, 7, 14, and 21 for quantitative analysis of kidney and liver microbes, with histologic analysis of these tissues as well. Mortality was highest for mice injected with the wild-type strain CAF2 (INT1/INT1) and lowest for mice injected with the homozygous disruptant CAG3 (int/int1). Yeast were readily cleared from the liver of all mice injected with any of the three C. albicans strains. Although the mutant strains CAG3 and CAG5 are defective for hyphal formation in vitro, there was histological evidence of abundant hyphal formation in the renal pelvis of mice injected with these strains. Compared to the wild-type strain, mutant strains were associated with reduced mortality but increased C. albicans persistence in the kidney. Thus, the absolute ability to form hyphae in the kidney did not appear to modulate either C. albicans-induced mortality or the course of progressive infection in the kidney. In addition, reduced virulence was paradoxically associated with increased, not decreased, persistence of C. albicans in the kidney.

The isoflavone genistein inhibits internalization of enteric bacteria by cultured Caco-2 and HT-29 enterocytes.

The dietary isoflavone genistein is the focus of much research involving its role as a potential therapeutic agent in a variety of diseases, including cancer and heart disease. However, there is recent evidence that dietary genistein may also have an inhibitory effect on extraintestinal invasion of enteric bacteria. To study the effects of genistein on bacterial adherence and internalization by confluent enterocytes, Caco-2 and HT-29 enterocytes (cultivated for 15-18 d and 21-24 d, respectively) were pretreated for 1 h with 0, 30, 100, or 300 micromol/L genistein, followed by 1-h incubation with pure cultures of Listeria monocytogenes, Salmonella typhimurium, Proteus mirabilis, or Escherichia coli. Pretreatment of Caco-2 and HT-29 enterocytes with genistein inhibited bacterial internalization in a dose-dependent manner (r = 0.60-0.79). Compared to untreated enterocytes, 1-h pretreatment with 300 micromol/L genistein was generally associated with decreased bacterial internalization (P < 0. 05) without a corresponding decrease in bacterial adherence. Using Caco-2 cell cultures, decreased bacterial internalization was associated with increased integrity of enterocyte tight junctions [measured by increased transepithelial electrical resistance (TEER)], with alterations in the distribution of enterocyte perijunctional actin filaments (visualized by fluorescein-labeled phalloidin), and with abrogation of the decreased TEER associated with S. typhimurium and E. coli incubation with the enterocytes (P < 0.01). Thus, genistein was associated with inhibition of enterocyte internalization of enteric bacteria by a mechanism that might be related to the integrity of the enterocyte tight junctions, suggesting that genistein might function as a barrier-sustaining agent, inhibiting extraintestinal invasion of enteric bacteria.

Cytochalasin-induced actin disruption of polarized enterocytes can augment internalization of bacteria.

Cytochalasin-induced actin disruption has often been associated with decreased bacterial internalization by cultured epithelial cells, although polarized enterocytes have not been systematically studied. In assays using confluent polarized HT-29 enterocytes, cytochalasin D appeared to increase internalization of wild-type Salmonella typhimurium, Proteus mirabilis, and Escherichia coli. HeLa and HEp-2 epithelial cells, as well as HT-29 and Caco-2 enterocytes, were used to clarify this unexpected observation. Resulting data showed that cytochalasin D was associated with increased internalization of S. typhimurium and P. mirabilis by both HT-29 and Caco-2 enterocytes and with increased internalization of E. coli by HT-29 enterocytes; with either HeLa or HEp-2 cells, cytochalasin was associated with no change or a decrease in internalization of these same bacterial strains. Cytochalasin caused decreased internalization of Listeria monocytogenes by HT-29, Caco-2, HeLa, and HEp-2 cells, indicating that cytochalasin did not consistently augment bacterial internalization by polarized enterocytes. Fluorescein-labeled phalloidin confirmed marked disruption of filamentous actin in cytochalasin-treated HT-29, Caco-2, HeLa, and HEp-2 cells. Cytochalasin had no noticeable effect on epithelial viability but caused distorted apical microvilli, cell rounding, and separation of adjacent enterocytes in confluent cultures (with a corresponding decrease in transepithelial electrical resistance). Scanning electron microscopy showed that cytochalasin-induced enhanced bacterial internalization was associated with preferential bacterial adherence on the exposed enterocyte lateral surface. Colchicine, used to disrupt microtubules, had no noticeable effect on bacterial internalization by HT-29 or Caco-2 enterocytes. These data indicated that for HT-29 and Caco-2 enterocytes, cytochalasin-induced disruption of filamentous actin might augment internalization of some bacterial species by a mechanism that appeared to involve exposure of the enterocyte lateral surface.

Intracellular survival of enteric bacteria in cultured human enterocytes.

Translocating enteric bacteria have been visualized within intact intestinal epithelial cells in animal models of bacterial translocation. Although the ability of the enterocyte to engulf bacteria has been well documented in both in vivo and in vitro experimental models, relatively little is known about the enterocyte's ability to degrade internalized bacteria. Intracellular survival of eight strains of enteric bacteria (two strains of Listeria monocytogenes, Salmonella typhimurium, Proteus mirabilis, two strains of Escherichia coli, and two strains of Enterococcus faecalis) was quantified over a 20 h period using two different types of terminally differentiated polarized enterocytes considered relevant in vitro models of human small intestinal epithelium, namely Caco-2 and HT-29 cells. Caco-2 enterocytes were generally more permissive for bacterial uptake when compared with HT-29 enterocytes. However, bacterial survival was similar within each type of enterocyte, and most strains of enteric bacteria remained viable within enterocytes for the 20 h duration of the assay. In addition, with the exception of one strain of L. monocytogenes in Caco-2 cells, intracellular enteric bacteria had no noticeable effect on host enterocyte viability for this 20 h duration. Transmission electron microscopy was used to visualize both intact and degraded bacteria within individual enterocytes, suggesting that prolonged bacterial survival might have resulted from simultaneous bacterial degradation and replication. Thus, although enterocytes internalize enteric bacteria, enterocytes might not kill internalized bacteria as efficiently as leukocytes. Observations of bacterial intracellular survival supported the hypothesis that the enterocyte might be a portal of entry for translocating microbes, and observations of intracellular bacterial degradation might have implications for the role of the enterocyte as an antigen-presenting cell.

Effect of hypoxia on enterocyte endocytosis of enteric bacteria.

OBJECTIVE: To clarify the effect of hypoxia on bacteria-enterocyte interactions. DESIGN: Randomized. SETTING: Research laboratory. SUBJECTS: Enteric bacterial and cultured human intestinal epithelial cells, HT-29 cells. INTERVENTIONS: The effect of hypoxia on bacterial internalization and intracellular survival was studied, using enterocytes cultured for 21 days in either 20%, 10%, or 5% oxygen. The effect of bacterial growth conditions on bacterial internalization by enterocytes was studied, using bacterial cells in either the log phase or stationary phase of aerobic growth, and using bacterial cells in stationary phase, grown either under low oxygen conditions or under anaerobic conditions. MEASUREMENTS AND MAIN RESULTS: Individual strains of enteric bacteria were incubated with HT-29 cells for 1 hr. Numbers of internalized bacteria were subsequently quantified after enterocyte lysis. Bacterial growth conditions (anaerobic vs. aerobic and log-phase vs. stationary-phase bacterial cells) had no noticeable effect on the numbers of Salmonella typhimurium, Proteus mirabilis, and Escherichia coli internalized by enterocytes. Enterocytes cultivated in 20%, 10%, or 5% oxygen were >95% viable. Enterocytes cultivated in 20% oxygen were confluent, but those enterocytes cultivated in hypoxia were not confluent and were fewer in number compared with enterocytes cultivated in normoxia. Compared with enterocytes grown in normoxia, enterocytes cultivated in 5% and 10% oxygen internalized greater numbers of each of seven strains of enteric bacteria, including Listeria monocytogenes (two strains), Enterococcus faecalis (two strains), and P. mirabilis, E. coli (two strains), with statistically significant increases noted for five of these seven bacterial strains. Intracellular survival of L. monocytogenes and P. mirabilis was assayed. Both species survived intracellularly for 22 hrs, with no noticeable differences in the numbers of intracellular bacteria recovered from enterocytes cultivated in 20%, 10%, and 5% oxygen. CONCLUSION: These in vitro results suggest that augmented bacterial endocytosis by enterocytes might at least partially explain the increased frequency of bacterial translocation associated with tissue ischemia.

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 in a large-animal model of small-bowel transplantation. Portal vs systemic venous drainage and the effect of tacrolimus immunosuppression.

OBJECTIVE: To study whether bacterial translocation is more prevalent after small-bowel transplantation with systemic venous drainage (SVD) vs portal venous drainage (PVD) and whether it is influenced by immunosuppression. DESIGN: We performed 15 small-bowel transplantations in pigs. Group 1 (n = 5) had SVD and no immunosuppression; group 2 (n = 6), PVD and no immunosuppression; and group 3 (n = 4), PVD and immunosuppression with tacrolimus and methylprednisolone sodium succinate. Portal and systemic blood, portal and mesenteric lymph nodes, and liver were cultured in donors and recipients on postoperative day 0 (POD 0) and in recipients on postoperative day 3 (POD 3). Jejunal and ileal contents were also sampled at these times. SUBJECTS: Outbred male Yorkshire-Landrace pigs. MAIN OUTCOME MEASURES: (1) Blood and tissue bacterial cultures, (2) blood endotoxin levels, and (3) histopathologic examination. RESULTS: Cultures were positive for bacteria in 32% (16/50) of samples on POD 0 and 88% (22/25) on POD 3 in group 1, in 18% (11/60) of samples on POD 0 and 97% (29/30) on POD 3 in group 2, and in 8% (3/40) of samples on POD 0 and 95% (19/20) on POD 3 in group 3. Systemic blood cultures were positive for bacteria on POD 3 in 60% (3/5) of pigs in group 1, 83% (5/6) in group 2, and 100% (4/4) in group 3. Significantly more bacteria were present in the ileum than in the jejunum on POD 0 in group 2; this difference approached significance in groups 1 and 3. Bacterial numbers were identical in the ileum and jejunum by POD 3 in all groups. Circulating endotoxin levels were significantly elevated on POD 3 vs POD 0 only in group 1. Endotoxin levels were not significantly different between the SVD group (group 1) and either PVD group (groups 2 and 3). CONCLUSIONS: Bacterial translocation is prevalent after small-bowel transplantation in pigs whether PVD or SVD is used. Immunosuppression with tacrolimus does not prevent bacterial translocation but may reduce systemic endotoxemia.

Exposure of the lateral enterocyte membrane by dissociation of calcium-dependent junctional complex augments endocytosis of enteric bacteria.

Intestinal bacterial translocation is facilitated in a variety of clinical conditions involving increased intestinal permeability, such as shock and trauma. Because there is both in vivo and in vitro evidence that enteric bacteria can be internalized by intestinal epithelial cells, experiments were designed to test the effect of increased intestinal permeability on enterocyte endocytosis of enteric bacteria. Mature, confluent cultures of HT-29 enterocytes were placed in a calcium-free solution for 1 h. Enterocyte viability was not noticeably altered, but transepithelial electrical resistance was significantly decreased (indicating a decrease in epithelial junctional integrity), and the enterocytes were pulled apart. Electron microscopic observations revealed enteric bacteria preferentially adherent on the exposed enterocyte lateral surface, and the numbers of viable enteric bacteria (Listeria monocytogenes, Salmonella typhimurium, Proteus mirabilis, Escherichia coli, and Enterococcus faecalis) internalized by these enterocytes were significantly increased. Restoration of calcium restored confluency to enterocyte cultures, and bacterial internalization reverted to control levels. Thus, calcium-dependent junctional integrity might play a role in augmenting bacterial translocation in clinical conditions associated with increased intestinal permeability.

Inhibitory effect of bile on bacterial invasion of enterocytes: possible mechanism for increased translocation associated with obstructive jaundice.

OBJECTIVE: To clarify the effect of bile salts on internalization of enteric bacteria by intestinal epithelial cells. DESIGN: Randomized study. SETTING: Research laboratory. SUBJECTS: Cultured human intestinal epithelial cells, namely HT-29 cells. INTERVENTIONS: The effect of bile was studied by adding bile during the time period in which bacterial cells were permitted to interact with enterocytes. In subsequent experiments, bile was added to the culture medium used to grow bacteria, and bacterial cells were washed before adding bacteria to enterocytes. Three different concentrations of three different bile preparations were tested. MEASUREMENTS AND MAIN RESULTS: Salmonella typhimurium and Proteus mirabilis were each incubated with HT-29 cells for 1 hr; the numbers of internalized bacteria were subsequently quantified following enterocyte lysis. The presence of bile during bacteria-enterocyte incubation had no effect on the numbers of internalized bacteria. However, if S. typhimurium or P. mirabilis were grown in the presence of bile, these washed bacterial cells were generally internalized by HT-29 cells in significantly fewer numbers, compared with bacterial cells grown in medium without bile supplementation. Enterocyte viability and morphologic ultrastructure did not appear to be affected by the presence of bile itself, or by the interaction with bacterial cells that had been cultivated in unsupplemented medium or in bile-supplemented medium. CONCLUSIONS: Exposure to bile during bacterial growth resulted in bacterial cells with decreased invasiveness for cultured intestinal epithelial cells. This observation is consistent with previous in vivo studies of obstructive jaundice, where the absence of bile in the intestinal lumen, not bile duct ligation, appeared to facilitate bacterial translocation in obstructed animals. Thus, the presence of bile in the intestinal lumen may decrease bacterial translocation by a mechanism that involves decreased epithelial internalization of enteric bacteria.

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.

Bacterial translocation in cultured enterocytes: magnitude, specificity, and electron microscopic observations of endocytosis.

Previous in vivo evidence has shown that bacterial phagocytosis by enterocytes may be an initial step in bacterial translocation across the intestinal epithelium. This study analyzed the interactions of cultured enterocytes, namely Caco-2 cells, with nine strains of enteric bacteria, tested in pure culture and in mixed culture. These nine strains had a spectrum of invasive potential and included Salmonella typhimurium, Listeria monocytogenes (three strains), Escherichia coli (three strains), Proteus mirabilis, and Enterococcus faecalis. Numbers of viable intracellular bacteria recovered from Caco-2 cells were: L. monocytogenes > S. typhimurium > P. mirabilis > E. coli > E. faecalis. Uptake of a given microbe by enterocytes was strain-specific and was not influenced by the presence of another strain, regardless of the invasive ability of the coinfecting strain. Electron microscopic visualization of bacterial adherence and uptake by Caco-2 cells indicated that the epithelial interactions of normal enteric bacteria were similar to these observed with invasive strains of salmonella and listeria.

Effect of LPS on epithelial integrity and bacterial uptake in the polarized human enterocyte-like cell line Caco-2.

Bacterial lipopolysaccharide (LPS) has been speculated to facilitate bacterial translocation by a mechanism involving physical disruption of the gut mucosal barrier. Polarized, cultured intestinal epithelial cells (Caco-2 cells) were used to study the effect of LPS on enterocyte structure, viability, and susceptibility to bacterial invasion. Varying concentrations of biologically active LPS were incubated with enterocytes for 1 and 16 hr. LPS had no noticeable effect on enterocyte viability or morphology, as measured by uptake of vital dyes, by distribution of cytoskeletal filamentous actin, and by visualization of subcellular ultrastructure. Transepithelial electrical resistance was similar in enterocyte cultures incubated with LPS for 1 hr, but there was a noticeable decrease after 16 hr, indicating a loss of epithelial integrity after prolonged exposure to LPS. The effect of LPS on bacterial uptake was studied using six strains of enteric bacteria with varying abilities to invade Caco-2 cells: Listeria monocytogenes, Salmonella typhimurium, Proteus mirabilis, Escherichia coli (2 strains), and Enterococcus faecalis. Electron microscopy showed enteric bacteria in intimate association with enterocyte apical microvilli, and internalized bacteria were consistently observed within cytoplasmic, membrane-bound vacuoles. Following a 1-hr incubation of individual strains of enteric bacteria with Caco-2 cells, numbers of viable intracellular bacteria varied significantly between individual bacterial strains, but numbers of intracellular bacteria were similar for each strain incubated with enterocytes exposed to 0, 10, and 100 micrograms LPS for 1 and 16 hr. Thus, although prolonged exposure to LPS might have some effect on enterocyte culture integrity (as measured by decreased electrical resistance), LPS had no discernible effect on enterocyte structure, viability, and susceptibility to bacterial invasion. These results suggested that LPS-induced bacterial translocation might not involve loss of epithelial viability, or facilitated entry of bacteria into intestinal epithelial cells.

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.