Although the intestinal mucosa forms a crucial barrier between the host and the environment, bacterial translocation (BT) occurs frequently in neonates and may be a source of sepsis. The intestinal mucous gel layer is thought to be a vital component of the gut barrier and is composed, in part, of a family of glycoproteins known as mucins. Our aim was to study the effects of mucin on BT in an enterocyte cell-culture model using a fetal (I-407) and an adult (Caco-2) intestinal cell line. I-407 and Caco-2 cells were grown to confluence on porous filters in a two-chamber Transwell system. The integrity of the monolayers was confirmed by transepithelial electrical resistance (TEER) and permeability using the macromolecule dextran blue. Cells were treated with mucin (40 mg/ml) prior to inoculation of 1 x 10(6) Escherichia coli C25. The magnitude of BT was determined quantitatively by culturing the samples from the basal chamber of the wells and was expressed as log 10 [Colony Forming Units (CFU)/ml]. Statistical analysis was performed by the Mann-Whitney U test with statistical significance at P < 0.05. Mucin inhibited BT across both fetal and adult cultured enterocyte monolayers; however, the inhibitory effect was less on the fetal cells compared to the adult cells. Dextran-blue studies showed that monolayers were intact throughout the experiments. Despite 98% inhibition of BT, mucin had a statistically significant effect on post-bacterial inoculation TEER in Caco-2 cells and no effect in I-407 cells. The ability of mucin, a mucous-barrier glycoprotein, to inhibit BT across immature intestinal enterocytes, as in the neonate, may be diminished compared to mature adult enterocytes.

The activity of phospholipase (PL)A2 is elevated in the intestinal epithelia of patients with inflammatory bowel disease (IBD). Recently, we reported that lysophosphatidylcholine (L-PC), the PLA2 hydrolysis product of phosphatidylcholine (PC), stimulates bacterial translocation (BT) in an enterocyte cell-culture model. These two observations stimulated us to examine the effects of extracellular PLA2 on intestinal epithelial permeability. Human Caco-2 enterocytes were grown to confluence on porous filters in the apical chamber of a two-chamber cell-culture system. Monolayer integrity and tight-junction permeability were measured by dextran blue (DB) permeability and transepithelial electric resistance (TEER). Monolayers were treated with PC, L-PC, or PLA2 with and without PC. The magnitude of BT was determined 2 h after treatment by adding Escherichia coli to the apical chamber followed by quantitatively culturing basal chamber samples. Thin-layer chromatography (TLC) was utilized to verify PLA2 hydrolysis of PC to L-PC. Statistical analysis was performed by one-way analysis of variance. The magnitude of BT across monolayers pretreated with PLA2 + PC significantly increased compared to either PC or PLA2 (6.83 +/- 0.069, 2.41 +/- 0.46, and 3.06 +/- 1.14 log10 colony forming units/ml, respectively, P < 0.05). Absence of DB-permeability in any group confirmed monolayer integrity. TLC of PL samples harvested from the apical monolayer surface confirmed PC hydrolysis. PLA2 mediates hydrolysis of PC to L-PC when both are applied to the apical surface of cultured enterocyte monolayers, resulting in increased BT and increased TEER with no damage to monolayer integrity. These observations may have implications in the pathogenesis and treatment strategies for IBD.

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Effects of protease-resistant antimicrobial substances (PRA) produced by Lactobacillus plantarum and Leuconostoc citreum on rumen methanogenesis were examined using the in vitro continuous methane quantification system. Four different strains of lactic acid bacteria, i) Lactococcus lactis ATCC19435 (Control, non-antibacterial substances), ii) Lactococcus lactis NCIMB702054 (Nisin-Z), iii) Lactobacillus plantarum TUA1490L (PRA-1), and iv) Leuconostoc citreum JCM9698 (PRA-2) were individually cultured in GYEKP medium. An 80 ml aliquot of each supernatant was inoculated into phosphate-buffered rumen fluid. PRA-1 remarkably decreased cumulative methane production, though propionate, butyrate and ammonia N decreased. For PRA-2, there were no effects on CH 4 and CO 2 production and fermentation characteristics in mixed rumen cultures. The results suggested that PRA-1 reduced the number of methanogens or inhibited utilization of hydrogen in rumen fermentation.