Masaaki Watanuki

Protective Effect of Lactobacillus casei Strain Shirota on Shiga Toxin-Producing Escherichia coli O157:H7 Infection in Infant Rabbits

ABSTRACT We examined colonization patterns of Shiga toxin-producingEscherichia coli (STEC), concentrations of Shiga toxins (Stxs) and specific immunoglobulin A (lgA) against Stxs and STEC bacterial cell surface antigen in various portions of the gastrointestinal tract in an infant rabbit infection model. After inoculation of 3-day-old infant rabbits with STEC strain 89020087 at low doses (∼103 CFU/body), numbers of colonizing STEC bacteria and concentrations of Stxs in the intestine increased dramatically and the animals developed diarrhea within a couple of days after infection. Daily administration ofLactobacillus casei from the day of birth dramatically decreased the severity of diarrhea and lowered STEC colonization levels in the gastrointestinal tract 100-fold day 7 after infection. Both Stx1 and Stx2 concentrations in the intestines and histological damage to the intestinal mucus induced by STEC infection were decreased by the administration of L. casei. Examination of the concentrations of volatile fatty acids and pH of the intestinal contents revealed that the protective effect of L. caseiadministration against STEC infection was not due to fermented products such as lactic acid in the gastrointestinal tract. Administration ofL. casei increased levels of lgAs against Stx1, Stx2, and formalin-killed STEC cells in the colon approximately two-, four-, and threefold, respectively, compared with those of the untreated controls by day 7 after infection. These results suggest that administration ofL. casei strain Shirota enhances the local immune responses to STEC cells and Stxs and leads to elimination of STEC and thus decreases Stx concentrations in the intestines.

Antimicrobial Activity of Intraurethrally Administered Probiotic Lactobacillus casei in a Murine Model of Escherichia coli Urinary Tract Infection

ABSTRACT The antimicrobial activity of the intraurethrally administered probiotic Lactobacillus casei strain Shirota againstEscherichia coli in a murine urinary tract infection (UTI) model was examined. UTI was induced by intraurethral administration ofEscherichia coli strain HU-1 (a clinical isolate from a UTI patient, positive for type 1 and P fimbriae), at a dose of 1 × 106 to 2 × 106 CFU in 20 μl of saline, into a C3H/HeN mouse bladder which had been traumatized with 0.1 N HCl followed immediately by neutralization with 0.1 N NaOH 24 h before the challenge infection. Chronic infection with the pathogen at 106 CFU in the urinary tract (bladder and kidneys) was maintained for more than 3 weeks after the challenge, and the number of polymorphonuclear leukocytes and myeloperoxidase activity in the urine were markedly elevated during the infection period. A single administration of L. casei Shirota at a dose of 108 CFU 24 h before the challenge infection dramatically inhibited E. coli growth and inflammatory responses in the urinary tract. Multiple daily treatments with L. casei Shirota during the postinfection period also showed antimicrobial activity in this UTI model. A heat-killed preparation ofL. casei Shirota exerted significant antimicrobial effects not only with a single pretreatment (100 μg/mouse) but also with multiple daily treatments during the postinfection period. The otherLactobacillus strains tested, i.e., L. fermentum ATCC 14931T, L. jensenii ATCC 25258T, L. plantarum ATCC 14917T, and L. reuteri JCM 1112T, had no significant antimicrobial activity. Taken together, these results suggest that the probiotic L. casei strain Shirota is a potent therapeutic agent for UTI.

Mechanism of fibrin-specific fibrinolysis by staphylokinase: Participation of α2-plasmin inhibitor

When the extent of plasminogen activation by staphylokinase (SAK) or streptokinase (SK) was measured in human plasma, SAK barely induced plasminogen activation, whereas SK activated plasminogen significantly. When the plasma was clotted with thrombin, the plasminogen activation by SAK was markedly enhanced, but that of SK was little enhanced. Similarly, in a purified system composed of plasminogen, fibrinogen and alpha 2-plasmin inhibitor (alpha 2-PI, alpha 2-antiplasmin), such a fibrin clot increased the activity of SAK significantly. However, when alpha 2-PI was removed from the reaction system, enhancement of the SAK reaction was not observed. In addition, SAK as distinct from SK, showed very little interference with the action of alpha 2-PI. Plasminogen activation by SAK is thus essentially inhibited by alpha 2-PI, but this reaction is not inhibited in fibrin clots. These results suggest that SAK forms a complex with plasminogen, which binds to fibrin and induces fibrinolysis.

Lower Plasma Triglyceride Level in Syrian Hamsters Fed on Skim Milk Fermented with Lactobacillus casei strain Shirota

The effect of fermented skim milk (FSM) by Lactobacillus casei strain Shirota on plasma lipids in hamsters was examined. Hamsters fed on cholesterol-free and -enriched diets containing 30% FSM had lower levels of plasma triglyceride than those fed on the control diet. In the experiment with the cholesterol-enriched diet-fed hamsters, the plasma triglyceride level was suppressed by FSM at concentrations of 10% to 30%. Unfermented milk tended to lower the level of triglyceride, but not significantly. The plasma cholesterol concentration was not affected by an FSM and unfermented skim milk supplement to the diet. L. casei strain Shirota grew well in the presence of mixed lipid micelles containing bile acid, but did not have the ability to remove cholesterol from the culture broth. These results indicate that FSM lowered the plasma triglyceride level in hamsters.

Inhibitory effect of gliotoxin on platelet aggregation ―importance of disulfide structure―

In the course of screening for anti-platelet principles produced by micro-organisms, strong anti-platelet activity was detected in the culture broth of Aspergillus fumigatus Fres. The purified active compound was identified as gliotoxin. Gliotoxin inhibited ADP-induced aggregation as well as collagen- or arachidonate-induced aggregation of rabbit platelets (IC50 = about 27 μm) and also accelerated the dissociation of aggregates. Gliotoxin also inhibited the heat hemolysis of rabbit erythrocytes, suggesting that this agent is a membrane-stabilizing anti-aggregant. The disulfide structure in the gliotoxin molecule was responsible for the inhibitory activity, because des- thiogliotoxin had effects on neither platelet aggregation nor heat hemolysis of erythrocytes.