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PubMed1986

Lactobacillus endocarditis.

Two cases of endocarditis due to Lactobacillus plantarum and Lactobacillus casei var. rhamnosus are described. Both patients were successfully treated medically. The response of the two isolates to a range of concentrations of penicillin in an apparatus which continuously monitored growth is reported. The strain of L. casei var. rhamnosus was found to be penicillin tolerant. The use of a combination of a penicillin with gentamicin to treat endocarditis due to penicillin tolerant strains of lactobacillus is discussed. The criteria for selecting oral agents to follow intravenous therapy are assessed also.

Adult↗

PubMed1999

Isolation and identification of lactobacilli from novel-type probiotic and mild yoghurts and their stability during refrigerated storage.

A total of 26 Lactobacillus strains were isolated from various mild yoghurts and novel-type probiotic dairy products and from a starter culture preparation and were identified by using DNA-DNA hybridization technique. The species present in those products were found to be Lactobacillus acidophilus, L. johnsonii, L. crispatus, L. casei, L. paracasei and L. rhamnosus. DNA homology analysis revealed that some strains had been misclassified by their investigators. Three strains designated as L. acidophilus (L. acidophilus LA-1, L. acidophilus ATCC 43121 and the Lactobacillus strain from Biogarde culture) were found to belong to L. johnsonii and L. acidophilus L1 to be L. crispatus. Strains designated as L. casei were found to be members of three separate species: L. casei, L. paracasei and L. rhamnosus. Viable numbers of lactobacilli in mild and probiotic yoghurts varied greatly including some products with very low Lactobacillus counts. The majority of the probiotic yoghurts, however, contained viable counts above 10(5) per g even at the end of the best before use period.

Animals↗

PubMed2000

Evaluation of numerical analyses of RAPD and API 50 CH patterns to differentiate Lactobacillus plantarum, Lact. fermentum, Lact. rhamnosus, Lact. sake, Lact. parabuchneri, Lact. gallinarum, Lact. casei, Weissella minor and related taxa isolated from kocho and tef.

AIM: The study was carried out to assess the agreement of API 50 CH fermentation data of food lactobacilli with their RAPD profiles to determine whether the system could be used alone as a reliable taxonomic tool for this genus. METHODS AND RESULTS: API 50 CH, RAPD and DNA:DNA reassociation data for 42 lactobacilli from tef and kocho were compared with 30 type strains. Discrepancies were observed between the three methods in assigning strains of Lactobacillus plantarum, Lact. fermentum, Weissella minor and Lact. gallinarum, and Lact. fermentum, Lact. amylophilus, Lact. casei subsp. pseudoplantarum and Lact. rhamnosus. DNA reassociation data agreed well with RAPD results. CONCLUSIONS: API 50 CH profiles should be complemented with molecular genetic results for effective identification in Lactobacillus. SIGNIFICANCE AND IMPACT OF THE STUDY: The study suggested less dependability of metabolic data alone as an identification tool.

Bacterial Typing Techniques↗

PubMed1985

The threshold growth response of Lactobacillus casei to 5-methyl-tetrahydrofolic acid: implications for folate assays.

The comparative and absolute growth response of Lactobacillus casei was measured nephelometrically for time periods of 17-23 h in the microbiological assay of folic acid and 5-methyl-tetrahydrofolic acid at concentrations of 0-8 ng/10 ml basal medium at pH 6.8. At concentrations of 0-1 ng/10 ml the comparative growth response to 5-methyl-tetrahydrofolic acid was markedly depressed whereas growth was the same at 2 ng/10 ml and above. Comparative growth was unaffected by the length of assay incubation, depressed growth being due to differences in log-phase growth rates with the rate-plot for 5-methyl-tetrahydrofolic acid being sigmoidal and for folic acid being a rectangular hyperbola with linearity only in the 0-1 ng/10 ml range. The reciprocal rate-plot for folic acid was linear whereas that for 5-methyl-tetrahydrofolic acid was coincidental only in part, giving rise to the same estimate of maximum velocity and substrate concentration for half-maximum velocity, with the exhibition of a strong threshold at low concentration. A previous observation (Phillips & Wright, 1982) that the L. casei growth response to 5-methyl-tetrahydrofolic acid may be significantly less than that to folic acid is confirmed as is the long-established view that the response to both folates may be equal. In the light of current knowledge regarding folate-binding, transport and metabolism by L. casei, it is argued that the intracellular oxidation of 5-methyl-tetrahydrofolic acid to 5, 10-methylene-tetrahydrofolic acid is a rate-limiting step at low substrate concentrations, subsequently giving rise to a threshold growth response peculiar to 5-methyl-tetrahydrofolic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Assay↗

PubMed1991

Pteroylpolyglutamates in human milk.

Considerable variation exists in reported values for total folate content and the pteroylpolyglutamate (PteGlun) content of human milk. We investigated possible methodological sources of this variation. In two laboratories, milk folate content (with and without folate conjugase) was determined microbiologically. No differences in total milk folate or PteGlun (n greater than 3) content were found between laboratories. PteGlun was found to comprise a significant fraction of total milk folate (28%). Use of rennin did not alter total folate content nor the percent of PteGlun in human milk. Heating (121 degrees C for 5 min) increased folate concentrations (190%, P less than 0.0001), indicating that release of folate from binding protein is necessary for folate utilization by Lactobacillus casei. Although human milk folate conjugase, (FC) activity was approximately one-twentieth that of plasma FC activity, it was not sufficient to autolyze endogenous PteGlun. Thus, microbiological protocols that do not use folate conjugase and do not release folate from binding proteins will seriously underestimate milk folate values.

False Negative Reactions↗

PubMed1993

Cloning and sequence determination of the valS gene, encoding valyl-tRNA synthetase in Lactobacillus casei.

The DNA sequence of the valS gene from Lactobacillus casei and the predicted amino acid sequence of its valyl-tRNA synthetase product have been determined. An open reading frame coding for a protein of 901 amino acids was found. A clone containing the intact L. casei valS gene functionally complemented the temperature-sensitive growth of the valS mutant strain 236c of Escherichia coli. The valS gene and the downstream folylpolyglutamate synthetase gene are transcribed in the same direction but are separated by a putative transcription terminator.

Amino Acid Sequence↗

PubMed2006

Modulation of apoptosis in intestinal lymphocytes by a probiotic bacteria in Crohn's disease.

Apoptosis of active T lymphocytes constitutes a major control mechanism of immune homeostasis and tolerance. In Crohn's disease, abnormal activation of mucosal T lymphocytes against enteric bacteria is the key event triggering intestinal inflammation. Resistance of lymphocytes to apoptosis has been proposed as the pathogenetic defect. We examined the influence of bacteria-mucosa interactions on apoptosis of mucosal T lymphocytes. Ileal specimens were obtained at surgery from 12 patients with Crohn's disease. Mucosal explants from each specimen were cultured with nonpathogenic Escherichia coli ATCC 35345, Lactobacillus casei DN-114 001, or no bacteria. Cytokine release was measured in supernatant, and mononuclear cells were isolated for phenotypic characterization and Bcl-2 family protein expression. Coculture of inflamed tissue with L. casei significantly reduced the release of interleukin (IL)-6 and tumor necrosis factor alpha (P < 0.05). In addition, coculture with L. casei significantly reduced the number of T cells displaying the IL-2 receptor in the lamina propria. Expression of the antiapoptotic protein Bcl-2 in lamina propria lymphocytes was also reduced after coculture with L. casei, and the percentage of deoxyuridine triphosphate nick-end labeling positive lymphocytes increased. The nonpathogenic E. coli strain had no significant effect. In conclusion, L. casei reduces the number of activated T lymphocytes in the lamina propria of Crohn's disease mucosa. A balanced, local microecology may restore immune homeostasis.

Adult↗

PubMed2000

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.

Animals↗

PubMed2004

Monocyte function in healthy middle-aged people receiving fermented milk containing Lactobacillus casei.

BACKGROUND: Lactic acid bacteria have been suggested as a dietary strategy to enhance immune system activity. OBJECTIVE: The aim of the current work was to test the effects of a Lactobacillus casei fermented milk consumption on monocyte activity of middle-aged volunteers. DESIGN: Forty-five healthy volunteers, 24 women and 21 men (aged: 51 - 58 years), were randomized in two groups to receive three cups per day of a fermented milk containing L. casei DN114001 (108 - 1010/g) (n = 23), or placebo (n = 22), during 8 weeks. White blood cell count and the oxidative burst capacity of monocytes and granulocytes were examined with a FACScalibur. Measurements were performed at baseline and after the nutritional intervention, at day fifty-six. RESULTS: After the trial, no changes in immune cell proportions were detected in both groups, as well as in monocyte activity after the placebo consumption (p = 0.625). However, volunteers included in the probiotic-treated group increased (p = 0.029) their oxidative burst capacity of monocytes, and this increment inversely and significantly correlated with the intensity registered at baseline (r = -0.653, p = 0.004). CONCLUSIONS: Results showed that daily intake of fermented milk containing Lactobacillus casei was able to module the oxidative burst capacity of monocyte subset in healthy middle-aged people, particularly in subjects with lower initial levels. Thus, this nutritional strategy could be considered to maintain immune competence in ageing.

Aging↗

PubMed1990

Pentose synthesis in glucose-grown cells of Lactobacillus casei.

The pathway of pentose synthesis in glucose-grown cells of Lactobacillus casei was ascertained. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were present in glucose-grown cells, while transaldolase and transketolase were present only in traces. This suggested that only the oxidative arm of this pathway was operative in glucose-grown cells. On the other hand, in ribose-grown cells, transaldolase was induced with a concomitant suppression of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. These results were confirmed by the detection of labelled CO2 produced by L. casei grown on [1-14C]glucose. The activities of the enzymes of the oxidative pentose phosphate pathway as also the rate of CO2 formation were higher in the exponential phase of growth as compared to the stationary phase, when the requirement of the cells for pentoses for the formation of DNA and RNA was higher.

Glucose↗

PubMed1988

Animal models of vasculitis. Lessons we can learn to improve our understanding of Kawasaki disease.

Kawasaki disease is an inflammatory illness of unknown etiology which is often complicated by coronary arteritis. Systematic study of the pathologically similar coronary arteritis induced in mice by a single intraperitoneal injection of Lactobacillus casei cell wall fragments may provide useful clues to the pathogenesis and appropriate treatment of Kawasaki disease. Studies of mouse strains with inbred immune deficiencies suggest that the coronary artery lesions may result from macrophage-mediated injury rather than immune complexes or a disturbance of T cell or B cell function.

Animals↗

PubMed1976

The clinical value of the radioassay of serum folate.

Measurement of serum folate levels in humans using a radioassay has now been frequenly reported. However, very little data is available assessing the diagnostic value of this test in comparison with other laboratory and clinical information pertaining to folate status. A radioassay, using the folate binder in dried milk was used in parallel with the well established Lactobacillus casei microbiological assay. Three slightly different modifications of the radioassay were tested. At first, a partially purified milk binder was used, in the second the unpurified milk and, third, the unpurified milk with variation of the incubation periods during the assay, were utilized. A total of 159 sera were tested by one of the modifications of the radioassay as well as by the L. casei method. Duplicate estimations were done in all cases. Using the first method, the correlation between the radio- and microbiological assays was 0.89, with the second 0.52, and with the third 0.85. The final assay system gave the most reliable results when compared to the L. casei assay which was taken as the standard of reference. However, in a number of cases, normal levels by L. casei gave low levels by radioassay and vice versa. In 13 such instances, clinical and other laboratory data were assessed and it was concluded that the microbiological assay gave the more accurate indication of folate stores in humans. Some reasons for the discrepancies between the assay systems were discussed.

Biological Assay↗

PubMedDate not supplied

Comparison of ELISA with activity and ligand-binding methods for the determination of thymidylate synthase concentration.

The determination of enzyme levels in cellular extracts by active site titrations or by catalytic activity measurements is relevant in both science and medicine. However, these techniques assume that enzymes exhibit the same response in crude sample matrices as they do in the purified state. We report here an example of how an enzyme-linked immunosorbent assay (ELISA) was used to determine the true enzyme concentration which was compared to the effective enzyme concentration obtained by ligand binding and catalytic assay methods in a crude bacterial cell extract. Rabbit antibodies specific for Lactobacillus casei thymidylate synthase (TS) were used to develop a highly specific and sensitive heterogeneous noncompetitive ELISA assay with a typical detection limit of 1.4 fmol of TS (100 pg) and a dynamic working range of 3 orders of magnitude. The antibodies showed identical responses for TS, its inhibitory binary complex with 5-fluoro-2'-deoxyuridylate, and its inhibitory ternary complex with 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate in the immunoassay. L. casei cell-free extracts were subjected to extraction with CM-Sephadex and the various fractions were analyzed by ELISA, active-site titrations, and catalytic assays which demonstrated that assays which assumed full catalytic or ligand-binding competence underestimated the true enzyme level.

Blotting, Western↗

PubMed1976

Folate analogues altered in the C9-N10 bridge region. 10-Oxafolic acid and 10-oxaaminopterin.

The unambiguous synthesis of two folate analogues, in which the 10-amino group of folic acid was replaced with oxygen, is described. The synthetic sequence employed commercially available methyl p-hydroxybenzoate and n-(2,3-epoxypropyl)phthalimide as starting materials. The use of cesium bicarbonate as a coreactant in the nucleophilic displacement reaction between bromo ketone 3 and the nucleophile 4 was found to be unique in character. The aminoacetonyl oxime 7 obtained by the hydrazinolysis of 6 was used as a common intermediate for the synthesis of both compounds. The generality of the use of the TFA-HCL mixture to deprotect the carbonyl group of both 10 and 12 reductions involving sodium hydrosulfite in aqueous dmf were further substantiated by conversions of 11 and 13 to 14 and 15 quickly and efficiently without employing catalytic hydrogenations. Subsequent cyclizations, oxidations, and hydrolysis of these reduction products to the pteroate analogues 17 and 19 were carried out efficiently as described for the synthesis of the sulfur analogues. Activation of the carboxyl group of 19 by way of the mixed anhydride 22 and subsequent coupling to glutamic acid was carried out using the solid-phase coupling procedure. However, compound 17 required trifluoroacetylation to 20 prior to the coupling reaction due to solubility problems. Both 10-oxafolic acid (1) and 10-oxaaminopterin (2) showed potent antifolate activity when tested against two folate-requiring organisms. Compound 2 was a very powerful inhibitor of DCM-resistant lactobacillus casei dihydrofolate reductase. The activity was comparable to that of methotrexate while the 4-hydroxy analogue did not show inhibition. 7,8-Dihydro-10-oxafolic acid failed to show any substrate activity to this enzyme and did not inhibit the enzymatic reaction when used with an equimolar concentration of the natural substrate.

Aminopterin↗

PubMed2005

Differential activities of four Lactobacillus casei promoters during bacterial transit through the gastrointestinal tracts of human-microbiota-associated mice.

In a previous study using fusion of the deregulated lactose promoter lacTp* and reporter genes, we suggested that Lactobacillus casei could initiate de novo protein synthesis during intestinal transit. In order to confirm this finding and extend it to other promoters, we adopted a reverse transcriptase quantitative PCR (RT-QPCR) approach combined with a transcriptional fusion system consisting of luciferase genes under the control of four promoters (ccpA, dlt, ldh, and lacT*) from L. casei DN-114 001. Promoter expression was monitored during cell growth, and variable luciferase activities were detected. In 3-day cultures, all the genetically modified strains survived but without exhibiting luciferase activity. Luciferase mRNA levels determined by RT-QPCR analysis (RNA/CFU) were not significant. The cultures were administered to human-microbiota-associated mice, and the feces were collected 6 h later. L. casei promoters lacTp* and ldhp initiated mRNA synthesis during gastrointestinal transit. The promoters, ccpAp and dltp, exhibited no luciferase activity, nor was de novo-synthesized luciferase mRNA detected in the feces. L. casei seems to adapt its physiology to the gastrointestinal tract environment by modulating promoter activities. The approach (fecal transcriptional analysis) described herein may, moreover, be of value in studying gene expression of transiting bacteria in human fecal specimens.

Animals↗

PubMed2001

Protective effect of Lactobacillus casei strain Shirota on Shiga toxin-producing Escherichia coli O157:H7 infection in infant rabbits.

We examined colonization patterns of Shiga toxin-producing Escherichia 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 (approximately 10(3) 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 of Lactobacillus 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. casei administration against STEC infection was not due to fermented products such as lactic acid in the gastrointestinal tract. Administration of L. 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 of L. 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.

Animals↗

PubMed1976

Evidence for extrachromosomal elements in Lactobacillus.

Three strains of lactobacilli, Lactobacillus casei subsp. casei 64H, L. casei subsp. rhamnosus OC91, and L. coryniformis M34, were examined for the presence of plasmids. Plasmids of molecular weights of 23 x 10(6) and 16 x 10(6) were found in the first two strains respectively. This represents the first evidence for plasmids in lactobacilli; their function is not presently known.

DNA, Bacterial↗

PubMed2006

Effects of Lactobacillus casei Shirota, Bifidobacterium breve, and oligofructose-enriched inulin on colonic nitrogen-protein metabolism in healthy humans.

Pre- and/or probiotics can cause changes in the ecological balance of intestinal microbiota and hence influence microbial metabolic activities. In the present study, the influence of oligofructose-enriched inulin (OF-IN), Lactobacillus casei Shirota, and Bifidobacterium breve Yakult on the colonic fate of NH3 and p-cresol was investigated. A randomized, placebo-controlled, crossover study was performed in 20 healthy volunteers to evaluate the influence of short- and long-term administration of OF-IN, L. casei Shirota, B. breve Yakult, and the synbiotic L. casei Shirota + OF-IN. The lactose[15N,15N]ureide biomarker was used to study the colonic fate of NH3. Urine and fecal samples were analyzed for 15N content by combustion-isotope ratio mass spectrometery and for p-cresol content by gas chromatography-mass spectrometry. RT-PCR was applied to determine the levels of total bifidobacteria. Both short- and long-term administration of OF-IN resulted in significantly decreased urinary p-cresol and 15N content. The reduction of urinary 15N excretion after short-term OF-IN intake was accompanied by a significant increase in the 15N content of the fecal bacterial fraction. However, this effect was not observed after long-term OF-IN intake. In addition, RT-PCR results indicated a significant increase in total fecal bifidobacteria after long-term OF-IN intake. Long-term L. casei Shirota and B. breve Yakult intake showed a tendency to decrease urinary 15N excretion, whereas a significant decrease was noted in p-cresol excretion. In conclusion, dietary addition of OF-IN, L. casei Shirota, and B. breve Yakult results in a favorable effect on colonic NH3 and p-cresol metabolism, which, in the case of OF-IN, was accompanied by an increase in total fecal bifidobacteria.

Adult↗