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PubMed2005

Cloning, expression, purification, and analysis of mannitol dehydrogenase gene mtlK from Lactobacillus brevis.

The commercial production of mannitol involves high-pressure hydrogenation of fructose using a nickel catalyst, a costly process. Mannitol can be produced through fermentation by microorganisms. Currently, a few Lactobacillus strains are used to develop an efficient process for mannitol bioproduction; most of the strains produce mannitol from fructose with other products. An approach toward improving this process would be to genetically engineer Lactobacillus strains to increase fructose-to-mannitol conversion with decreased production of other products. We cloned the gene mtlK encoding mannitol-2-dehydrogenase (EC 1.1.1.67) that catalyzes the conversion of fructose into mannitol from Lactobacillus brevis using genomic polymerase chain reaction. The mtlK clone contains 1328 bp of DNA sequence including a 1002-bp open reading frame that consisted of 333 amino acids with a predicted molecular mass of about 36 kDa. The functional mannitol-2-dehydrogenase was produced by overexpressing mtlK via pRSETa vector in Escherichia coli BL21pLysS on isopropyl-beta-D-thiogalactopyranoside induction. The fusion protein is able to catalyze the reduction of fructose to mannitol at pH 5.35. Similar rates of catalytic reduction were observed using either the NADH or NADPH as cofactor under in vitro assay conditions. Genetically engineered Lactobacillus plantarum TF103 carrying the mtlK gene of L. brevis indicated increased mannitol production from glucose. The evaluation of mixed sugar fermentation and mannitol production by this strain is in progress.

Cloning, Molecular↗
PubMedDate not supplied

[Influence of a xylanase feed additive on Lactobacillus species in the jejunum of piglets].

rRNA extracts from the jejunum of rearing piglets fed a wheat/rye based diet with or without a xylanase feed additive were hybridized against eight Lactobacillus spp. specific digoxigenin labelled oligonucleotide probes. Known concentrations of sample- or culture RNA extracts were used to calibrate and quantify hybridization signals from different sample extracts and hybridizations. The xylanase feed additive tended to increase total eubacterial rRNA in the terminal jejunum, probably a result of increased Lactobacillus spp. rRNA in the central and terminal jejunum. However, individual animal variations were high. L. reuteri was the major Lactobacillus species followed by L. acidophilus and L. mucosae. L. amylophilus and L. crispatus were not detected in all RNA extracts. The results of this study show that the xylanase feed additive modifies Lactobacillus spp. populations on the species level in piglets in addition to their well known effects on digesta viscosity and nutrient digestibility. The implications of these effects for the intestinal microbiota require further analysis.

Animal Feed↗
PubMed1990

Evidence for the involvement of thiocyanate in the inhibition of Candida albicans by Lactobacillus acidophilus.

Lactobacillus acidophilus has been found to inhibit Candida albicans when grown on MRS agar plates. Attempts to isolate an active factor responsible for this inhibition from liquid culture and agar plates were not successful. The addition of sodium thiocyanate to the agar was found to increase the inhibition offered by the lactobacillus. The results indicate that hydrogen peroxide produced by the lactobacillus is being used to convert the thiocyanate to hypothiocyanate which is more toxic. The involvement of a lactobacillus peroxidase in this conversion is postulated.

Antibiosis↗
PubMed1990

Microbiologic characteristics of Lactobacillus products used for colonization of the vagina.

Lactobacilli have long been considered to be the protective flora in the vagina. Women with vaginal infections have used various non-prescription products in an attempt to restore their normal vaginal flora. Products that contain lactobacilli include dairy products (yogurt, acidophilus milk) and commercially available Lactobacillus powders and tablets. Recently, Lactobacillus species that produce hydrogen peroxide (H2O2) have been associated with normal vaginal flora. In this study, we compared 16 non-prescription products containing lactobacilli for H2O2 production, purity, and identifiable contaminants. All 16 products contained lactobacilli, of which ten (62%) produced H2O2. At least one contaminant was detected in 11 of 16 (69%) of the products: Enterococcus faecium (ten), Clostridium sporogenes (one), Streptococcus mitis (one), and Pseudomonas species (one). Although all of the products tested contained lactobacilli, only four of the products contained Lactobacillus acidophilus. Most of the lactobacilli-containing products currently available either do not contain the Lactobacillus species advertised and/or contain other bacteria of questionable benefit. We conclude that commercially available products may not be appropriate for recolonization of the vagina.

Bacteria↗
PubMed1989

[Reciprocal effect of Klebsiella pneumoniae and Lactobacillus on their cytadhesion].

Reciprocal effect of 4 strains of Klebsiella pneumoniae and 6 Lactobacillus strains on their cytadhesion in mixed populations was studied on a model of formalinized human erythrocytes. The Lactobacillus strains included 2 strains of Lactobacillus casei subsp. casei, 2 strains of L. plantarum and 2 strains of L. fermentum. It was shown that adhesion of both the Klebsiella and the Lactobacillus strains changed under their reciprocal effect. The changes were characterized by the strain differences and depended on the quantitative ratio of the microorganisms.

Bacterial Adhesion↗
PubMed1983

Pyruvoyl-dependent histidine decarboxylases from Clostridium perfringens and Lactobacillus buchneri. Comparative structures and properties.

Histidine decarboxylase (EC 4.1.1.22) was purified to homogeneity from Clostridium perfringens and also from Lactobacillus buchneri. Both enzymes are composed of alpha and beta subunits, with an essential pyruvoyl group bound to the alpha subunit. In this respect and also in molecular weight of both the alpha and beta subunits and the native enzyme, they closely resemble the previously described (Riley, W.D., and Snell, E. E. (1970) Biochemistry 9, 1485-1491) histidine decarboxylase from Lactobacillus 30a. Rabbit antibodies to the latter enzyme cross-react incompletely with the decarboxylase from L. buchneri but not with that from C. perfringens in double diffusion tests. The clostridial decarboxylase differs substantially from the Lactobacillus 30a enzyme in amino acid composition and, unlike the latter enzyme, requires high ionic strength (I approximately 1.4 M) for maximum activity. The enzymes also differ in rates of electrophoretic migration. A proenzyme for the decarboxylase similar to that previously found in Lactobacillus 30a was detected in immunoprecipitates of extracts of L. buchneri. We conclude that these proteins arise from pyruvate-free precursor proteins by similar mechanisms and probably have diverged from a common ancestral protein.

Amino Acids↗
PubMed1993

[Characteristics of lactobacillus species strains isolated from reproductive organs in various clinical cases].

The study was aimed at detection of presence of Lactobacillus species bacteria in 167 samples of material derived from vagina of patients with cervix uteri (22 samples), chronic vaginosis (100 samples) and from women after treatment of inflammatory diseases (45 samples). Out of 100 isolated strains of Lactobacillus only 13 belonged to L. acidophilus. They were mainly present (10 strains) in patients after effective treatment of inflammatory diseases of reproductive organs. Remaining strains belonged to L. jensenii (18 strains) and Lactobacillus sp. (69 strains) and did not cleave glycogen. This was detected by quantitative determinations of lactic acid in cultures of these bacteria in the fluid media containing 0.25% of glycogen. It seems that treatment of bacterial infections of reproductive organs should be concentrated on not only elimination of pathogenic bacteria, but also on noneffective strains from Lactobacillus species (strains unable to cleave glycogen with production of lactic acid).

Chronic Disease↗
PubMed1996

[Liver abscess caused by Lactobacillus acidophilus].

Lactobacillus acidophilus is usually considered to be non pathogenic. We report a case of liver abscess due to Lactobacillus acidophilus in a 39 year-old man with chronic pancreatitis complicated by both endocrine and exocrine insufficiency, and with a choledoco-duodenostomy. Lactobacillus acidophilus was isolated in blood and liver samples. Complete recovery of the liver abscess occurred after antibiotherapy. We suggest that the abnormally low duodenal pH secondary to pancreatic insufficiency may have promoted both Lactobacillus acidophilus adhesion and multiplication in this patient. The choledoco-duodenostomy may then have promoted biliary tract colonisation.

Adult↗
PubMed2005

Ribotyping of Lactobacillus casei group strains isolated from dairy products.

A series of lactobacilli isolated from dairy products were characterized using biotyping and ribotyping with EcoRI and HindIII restriction enzymes. Biotyping assigned 14 strains as Lactobacillus casei, 6 strains as Lactobacillus paracasei subsp. paracasei and 12 as Lactobacillus rhamnosus. The obtained ribotype patterns separated all analyzed strains into two clearly distinguished groups corresponding to L. rhamnosus and L. casei/L. paracasei subsp. paracasei. The HindIII ribotypes of individual strains representing these two groups were visually very similar. In contrast, EcoRI ribotyping revealed high intraspecies variability. All ribotypes of L. casei and L. paracasei subsp. paracasei dairy strains were very close and some strains even shared identical ribotype profiles. The type strains L. casei CCM 7088T (= ATCC 393T) and Lactobacillus zeae CCM 7069T revealing similar ribopatterns formed a separate subcluster using both restriction enzymes. In contrast, the ribotype profile of L. casei CCM 7089 (= ATCC 334) was very close to ribopatterns obtained from the dairy strains. These results support synonymy of L. casei and L. paracasei species revealed by other studies as well as reclassification of the type strain L. casei CCM 7088T as L. zeae and designation of L. casei CCM 7089 as the neotype strain.

Dairy Products↗
PubMed2006

Effect of Lactobacillus fermentum on serum lipids in subjects with elevated serum cholesterol.

BACKGROUND AND AIMS: There is increasing interest in the use of natural therapies to reduce elevated low density lipoprotein (LDL) cholesterol. This study assessed the effects of PCC Lactobacillus fermentum on LDL cholesterol and other lipid fractions. METHODS AND RESULTS: This was a single centre, double blind, placebo-controlled, parallel design trial in volunteers having total cholesterol > or =4 mmol/L. Subjects (n=46) were randomised to receive either Lactobacillus fermentum 2 capsules twice daily (each capsule containing 2 x 10(9) colony forming units) or matching placebo for a period of 10 weeks. Main outcome measures were percentage changes in LDL cholesterol and other lipids, changes in liver enzymes and other safety tests. Two subjects withdrew early in the study, 1 for personal reasons and 1 because of bowel discomfort. Three other subjects experienced some bowel discomfort but still completed the study. LDL cholesterol showed a modest downward trend on both Lactobacillus fermentum and placebo of 7.0% and 5.2% respectively. This trend did not reach statistical significance over time, nor was there a significant difference between the treatment arms. There were no significant changes over time or between treatments noted in total cholesterol, high density lipoprotein cholesterol or triglycerides. There were no significant changes in liver enzymes or other safety parameters with time or between treatments. CONCLUSION: Lactobacillus fermentum did not appear to produce a major change in serum lipid fractions, but a small effect cannot be excluded.

Adult↗
PubMed1999

Lactobacillus GG in the prevention of antibiotic-associated diarrhea in children.

OBJECTIVE: The objective of this study was to determine the efficacy of Lactobacillus casei sps. rhamnosus (Lactobacillus GG) (LGG) in reducing the incidence of antibiotic-associated diarrhea when coadministered with an oral antibiotic in children with acute infectious disorders. STUDY DESIGN: Two hundred two children between 6 months and 10 years of age were enrolled; 188 completed all phases of the protocol. LGG, 1 x 10(10) - 2 x 10(10) colony forming units per day, or comparable placebo was administered in a double-blind randomized trial to children receiving oral antibiotic therapy in an outpatient setting. The primary caregiver was questioned every 3 days regarding the incidence of gastrointestinal symptoms, predominantly stool frequency and consistency, through telephone contact by blinded investigators. RESULTS: Twenty-five placebo-treated but only 7 LGG-treated patients had diarrhea as defined by liquid stools numbering 2 or greater per day. Lactobacillus GG overall significantly reduced stool frequency and increased stool consistency during antibiotic therapy by the tenth day compared with the placebo group. CONCLUSION: Lactobacillus GG reduces the incidence of antibiotic-associated diarrhea in children treated with oral antibiotics for common childhood infections.

Anti-Bacterial Agents↗
PubMed2002

Lactobacillus species is more cytotoxic to human bladder cancer cells than Mycobacterium Bovis (bacillus Calmette-Guerin).

PURPOSE: We determined if Lactobacillus species has growth inhibitory effects in human bladder cancer cell lines and how this effect compares with the known effects of Mycobacterium bovis, that is bacillus Calmette-Guerin (BCG). MATERIALS AND METHODS: The growth of MGH and RT112 cells were determined by cell counts after 24, 48 and 72 hours of exposure to L. casei strain Shirota (Yakult, Singapore) or L. rhamnosus strain GG (National Collection of Industrial and Marine Bacteria, Ltd., Aberdeen, Scotland) (1 x 10 and 1 x 10 cfu) or BCG (1 x 10 cfu) in the presence and absence of streptomycin. Annexin-V was used to monitor the presence of pre-apoptotic cells. RESULTS: L. rhamnosus GG inhibited MGH proliferation and it was cytotoxic to RT112 cells (p <0.05). L. casei Shirota was cytotoxic to the 2 cell lines (p <0.05). BCG had a similar cytotoxic effect in MGH cells as Lactobacillus species but was not as effective in RT112 cells. Streptomycin abrogated the cytotoxic effect of Lactobacillus species but not that of BCG. Cytotoxic activity was not found in Lactobacilli culture supernates but it was induced in the presence of mammalian cells. L. rhamnosus GG induced apoptosis in RT112 but not in MGH cells. No apoptotic cells were detected after treatment with L. casei Shirota. CONCLUSIONS: Lactobacillus species induced cytotoxic effects in bladder cancer cells. Unlike BCG, it requires bacterial protein synthesis. Like BCG, L. casei Shirota induces cell death primarily via necrosis. The cytoxicity of these lactobacilli in bladder cancer cells raises the possibility of using this species of bacteria as intravesical agents for treating bladder cancer.

BCG Vaccine↗
PubMed1997

Anti-aflatoxigenic activity of Lactobacillus casei pseudoplantarum.

Lactobacillus casei pseudoplantarum 371 isolated from a silage inoculant was found to inhibit aflatoxins B1 and G1 biosynthesis by Aspergillus flavus subsp. parasiticus NRRI. 2999, in liquid medium. The inhibitory activity in the Lactobacillus cell-free supernatant was found to be sensitive to proteolytic enzymes such as trypsin and alpha-chymotrypsin, but resistant to pepsin. Lab-Lemco tryptone broth (LTB), supplemented with 20% of dialyzed protein concentrate of the supernatant, totally inhibited the production of aflatoxins B1 and G1. When the protein concentrate was digested with trypsin, the production of aflatoxins B1 and G1 was restored. The inhibitory activity of the supernatant was inactivated within 10 min at 100 degrees C. A. flavus grown in the Lactobacillus cell-free supernatant did not produce a mutagenic response in the Salmonella mutagenicity test. However, Lactobacillus casei pseudo plantarum 371 did not have an effect on aflatoxin production and mold growth as measured by ergosterol and plate count, when the organisms were inoculated together on sterile steamed rice.

Aflatoxin B1↗
PubMed2001

Identification of salivary Lactobacillus rhamnosus species by DNA profiling and a specific probe.

The Lactobacillus genus has been shown to be associated with the dental carious process, but little is known about the species related to the decay, although Lactobacillus rhamnosus is suspected to be the most implicated species. Conventional identification methods based on biochemical criteria lead to ambiguous results, since the Lactobacillus species found in saliva are phenotypically close. To clarify the role of this genus in the evolution of carious disease, this work aimed to find a rapid and reliable method for identifying the L. rhamnosus species. Methods based on hybridization with DNA probes and DNA amplification by PCR were used. The dominant salivary Lactobacillus species (reference strains from the ATCC) were selected for this purpose as well as some wild strains isolated from children's saliva. DNA profiling using semirandom polymorphic DNA amplification (semi-RAPD) generated specific patterns for L. rhamnosus ATCC 7469. The profiles of all L. rhamnosus strains tested were similar and could be grouped; these strains shared four common fragments. Wild strains first identified with classic methods shared common patterns with the L. rhamnosus species and could be reclassified. One fragment of the profile was purified, cloned, used as a probe and found to be specific to the L. rhamnosus species. These results may help to localize this species within its ecological niche and to elucidate the progression of the carious process.

Bacterial Typing Techniques↗
PubMed2003

A comparative study and phage typing of silage-making Lactobacillus bacteriophages.

To investigate basic characteristics of 10 virulent phages active on silage-making lactobacilli, morphological properties, host ranges, protein composition and genome characterization were separated into five groups based on host ranges and basic properties. The seven phages of groups I, II and V were active on Lactobacillus plantarum and Lactobacillus pentosus. Phage phiPY4 (group III) infected both L. casei and Lactobacillus rhamnosus. Phage phiPY5 (group IV) specifically infected Lactobacillus casei. Morphologically, three phages of groups I belonged to the Myoviridae family, while seven other phages of groups II, III and V belonged to the Siphoviridae family. SDS-PAGE profiles, restriction analysis, G + C contents of DNA and Dot blot hybridization revealed a high degree of homology in each group. Clustering derived from host range analysis was closely related to results of DNA and protein analyses. These phages may be applicable to phage typing for silage-making lactobacilli.

Journal Article↗
PubMed1996

The effect of Lactobacillus GG on the initiation and promotion of DMH-induced intestinal tumors in the rat.

Male Fischer 344 rats were fed a 20% or a 5% corn oil diet and were injected subcutaneously with dimethylhydrazine (DMH) weekly for 16 weeks. In addition, an approximately equal number of animals challenged with DMH were fed daily, until the end of the study, 2 x 10(10) Lactobacillus casei subsp. rhamnosus strain GG starting three weeks before DMH administration or after the ninth weekly injection. The feeding of the Lactobacillus GG before and during carcinogen treatment resulted in a significant decrease in the incidence of colon tumors and the number of small intestinal and colon tumors per tumor-bearing animal for rats fed a 20% corn oil diet. This decrease in tumor incidence or number of tumors was not seen when animals were fed the Lactobacillus after the ninth week of carcinogen treatment. Animals fed a 5% corn oil diet had a lower tumor incidence and number of tumors resulting from the decrease in dietary fat; in addition the feeding of Lactobacillus GG before the carcinogen challenge resulted in a lower incidence of colon tumors. These studies show that a specific strain of L. casei subsp. rhamnosus designated GG can interfere with the initiation or early promotional stages of DMH-induced intestinal tumorigenesis, and this effect is most pronounced for animals fed a high-fat diet.

Animals↗
PubMed1995

Lactobacillus GG promotes recovery from acute nonbloody diarrhea in Pakistan.

A prospective, placebo-controlled, triple blind clinical trial was carried out in Pakistan to determine the effect of Lactobacillus GG on the course of acute diarrhea in hospitalized children. Forty children (mean age, 13 months) were enrolled and after rehydration received either oral Lactobacillus GG (n = 21) or placebo (n = 19) twice daily for 2 days, in addition to the usual diet. The clinical course of diarrhea was followed during the treatment period. Features on admission into the study groups were similar and were characterized by severe diarrhea, malnutrition and inappropriate management before presentation. Response was evident on Day 2 when the frequency of both vomiting and diarrhea was less in the Lactobacillus group. In those who had presented with acute nonbloody diarrhea (n = 32), the percentage of children with persistent watery diarrhea at 48 hours was significantly less in the Lactobacillus group: 31% vs. 75% (P < 0.01). No significant difference was observed by 48 hours in those presenting with bloody diarrhea. The relevance of this finding to the management of diarrhea in the tropics is discussed.

Acute Disease↗
PubMed1991

Characterization of the Cell Wall-Bound Proteinase of Lactobacillus casei HN14.

Lactobacillus casei HN14, which was isolated from homemade cheese, produces an extracellular, cell wall-bound proteinase. The HN14 proteinase can be removed from the cell envelope by washing the cells in a Ca-free buffer. The activity of the crude proteinase extract is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the HN14 proteinase is similar to the lactococcal PI-type enzyme, since it hydrolyzes beta-casein only. Lactobacillus casei HN14 appeared to be plasmid free, which suggests that the proteinase gene is chromosomally located. Chromosomal DNA of this strain hybridizes with DNA probes Q1 (which contains a fragment of the prtM gene) and Q6 and Q92 (which contain fragments of the prtP gene); all three probes originated from the proteinase gene region of Lactococcus lactis subsp. cremoris Wg2. A restriction enzyme map of the proteinase region of Lactobacillus casei HN14 was constructed on the basis of hybridization experiments. Comparison of the restriction enzyme maps of the Lactobacillus casei HN14 proteinase gene region and those of lactococcal proteinase gene regions studied so far indicates that they are highly similar.

Journal Article↗