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Significant differences between Lactobacillus casei subsp. casei ATCC 393T and a commonly used plasmid-cured derivative revealed by a polyphasic study.

Many studies on Lactobacillus casei subsp. casei (L. casei) have been carried out using strain ATCC 393 (pLZ15-). Four strains of L. casei ATCC 393T and three of ATCC 393 (pLZ15-) were compared using phenotypic methods and many of the available genotyping techniques. These tests showed that strains of ATCC 393T obtained from independent public type-culture collections were significantly different from the plasmid-free (pLZ15-) strains of ATCC 393T. These findings were confirmed by sequencing the first 580 nt (domain I) of the 16S and 23S rDNAs of the strains. Complete sequencing of the 16S rDNA of one representative strain from each group revealed that strain ATCC 393T from culture collections was 99% similar to Lactobacillus zeae ATCC 15820T and that the strain so far considered as L. casei ATCC 393 (pLZ15-) was, in turn, 100% similar to L. casei ATCC 334 and Lactobacillus paracasei subsp. paracasei ATCC 4022. All data obtained in this work indicate that the ancestral strain of ATCC 393 (pLZ15-) might never have been the strain that is now available from culture collections.

Base Sequence↗

Case of aortic endocarditis caused by Lactobacillus casei.

A case of Lactobacillus aortic valve endocarditis in a 53-year-old immunocompetent patient with past history of rheumatic fever is reported. Clinical symptoms began after a dental extraction and the patient's diet included several yogurts per day. Blood, bone marrow cultures and the replaced aortic valve were positive for Lactobacillus: The clinical isolate was identified as Lactobacillus casei by 16S rDNA sequencing.

Aortic Valve↗

A synthetic promoter library for constitutive gene expression in Lactobacillus plantarum.

A synthetic promoter library (SPL) for Lactobacillus plantarum has been developed, which generalizes the approach for obtaining synthetic promoters. The consensus sequence, derived from rRNA promoters extracted from the L. plantarum WCFS1 genome, was kept constant, and the non-consensus sequences were randomized. Construction of the SPL was performed in a vector (pSIP409) previously developed for high-level, inducible gene expression in L. plantarum and Lactobacillus sakei. A wide range of promoter strengths was obtained with the approach, covering 3-4 logs of expression levels in small increments of activity. The SPL was evaluated for the ability to drive beta-glucuronidase (GusA) and aminopeptidase N (PepN) expression. Protein production from the synthetic promoters was constitutive, and the most potent promoters gave high protein production with levels comparable to those of native rRNA promoters, and production of PepN protein corresponding to approximately 10-15 % of the total cellular protein. High correlation was obtained between the activities of promoters when tested in L. sakei and L. plantarum, which indicates the potential of the SPL for other Lactobacillus species. The SPL enables fine-tuning of stable gene expression for various applications in L. plantarum.

Aminopeptidases↗

Natural killer cell activities of synbiotic Lactobacillus casei ssp. casei in conjunction with dextran.

We have reported previously that Lactobacillus casei ssp. casei, together with specific substrate dextran, exhibited an adjuvant effect of stimulating humoral immune responses against bovine serum albumin (BSA) as a model antigen in BALB/c mice. In the present study, among the Lactobacillus species tested, L. casei ssp. casei with dextran significantly elevated the natural killer (NK) cell activities in spleen mononuclear cells from BALB/c mice in comparison to L. casei ssp. casei alone or other Lactobacillus species with or without dextran. Oral administration of L. casei ssp. casei together with dextran also resulted in a significant increase of NK cell activities in healthy human volunteers. Further, L. casei ssp. casei induced significant production of interleukin (IL)-12 in human peripheral blood mononuclear cells and IL-15 mRNA expression in the human intestinal epithelial cell line Caco-2. L. casei ssp. casei with dextran in food also significantly elevated the survival rate of BALB/c mice bearing Meth-A cells. Taken together, these results demonstrate that dietary synbiotic supplementation which is a combination of the L. casei ssp. casei used as a probiotic together with the dextran, a specific substrate as a prebiotic, efficiently elicits murine and human NK cell activities.

Adult↗

Identification and nucleotide sequence of genes involved in the synthesis of lactocin 705, a two-peptide bacteriocin from Lactobacillus casei CRL 705.

The structural gene determinants of lactocin 705, a bacteriocin produced by Lactobacillus casei CRL 705, have been amplified from a plasmid of approximately 35 kb and sequenced. Lactocin 705 is a class IIb bacteriocin, whose activity depends upon the complementation of two peptides (705alpha and 705beta) of 33 amino acid residues each. These peptides are synthesized as precursors with signal sequences of the double-glycine type, which exhibited high identities with the leader peptides of plantaricin S and J from Lactobacillus plantarum, brochocin C from Brochotrix campestris, sakacin P from Lactobacillus sake, and the competence stimulating peptides from Streptococcus gordonii and Streptococcus mitis. However, the two mature bacteriocins 705alpha and 705beta do not show significant similarity to other sequences in the databases.

Amino Acid Sequence↗

Efficacy of Lactobacillus GG as a Diarrheal Preventive in Travelers.

Traveler's diarrhea can be a debilitating problem for individuals on international trips. Retrospective and prospective studies have shown the incidence of traveler's diarrhea to range from 15-56%. 1,2A placebo-controlled, double-blinded study in Finnish travelers found that the probiotic Lactobacillus GG decreases the incidence of traveler's diarrhea.3 Lactobacillus GG, initially isolated from healthy humans, is remarkable in its ability to resist acid and bile degradation and to adhere to the intestinal mucosa.4 To assess the efficacy of Lactobacillus GG in preventing diarrhea in American tourists, a study was conducted at the Travel and Immunization Center of the Long Island Jewish Medical Center (LIJMC).

Journal Article↗

Expression of the immunity protein of plantaricin 423, produced by Lactobacillus plantarum 423, and analysis of the plasmid encoding the bacteriocin.

Plantaricin 423 is a class IIa bacteriocin produced by Lactobacillus plantarum isolated from sorghum beer. It has been previously determined that plantaricin 423 is encoded by a plasmid designated pPLA4, which is now completely sequenced. The plantaricin 423 operon shares high sequence similarity with the operons of coagulin, pediocin PA-1, and pediocin AcH, with small differences in the DNA sequence encoding the mature bacteriocin peptide and the immunity protein. Apart from the bacteriocin operon, no significant sequence similarity could be detected between the DNA or translated sequence of pPLA4 and the available DNA or translated sequences of the plasmids encoding pediocin AcH, pediocin PA-1, and coagulin, possibly indicating a different origin. In addition to the bacteriocin operon, sequence analysis of pPLA4 revealed the presence of two open reading frames (ORFs). ORF1 encodes a putative mobilization (Mob) protein that is homologous to the pMV158 superfamily of mobilization proteins. Highest sequence similarity occurred between this protein and the Mob protein of L. plantarum NCDO 1088. ORF2 encodes a putative replication protein that revealed low sequence similarity to replication proteins of plasmids pLME300 from Lactobacillus fermentum and pYIT356 from Lactobacillus casei. The immunity protein of plantaricin 423 contains 109 amino acids. Although plantaricin 423 shares high sequence similarity with the pediocin PA-1 operon, no cross-reactivity was recorded between the immunity proteins of plantaricin 423 and pediocin PA-1.

Amino Acid Sequence↗

Production of succinic Acid from citric Acid and related acids by lactobacillus strains.

A number of Lactobacillus strains produced succinic acid in de Man-Rogosa-Sharpe broth to various extents. Among 86 fresh isolates from fermented cane molasses in Thailand, 30 strains (35%) produced succinic acid; namely, 23 of 39 Lactobacillus reuteri strains, 6 of 18 L. cellobiosus strains, and 1 of 6 unidentified strains. All of 10 L. casei subsp. casei strains, 5 L. casei subsp. rhamnosus strains, 6 L. mali strains, and 2 L. buchneri strains did not produce succinic acid. Among 58 known strains including 48 type strains of different Lactobacillus species, the strains of L. acidophilus, L. crispatus, L. jensenii, and L. parvus produced succinic acid to the same extent as the most active fresh isolates, and those of L. alimentarius, L. collinoides, L. farciminis, L. fructivorans (1 of 2 strains tested), L. malefermentans, and L. reuteri were also positive, to lesser extents. Diammonium citrate in de Man-Rogosa-Sharpe broth was determined as a precursor of the succinic acid produced. Production rates were about 70% on a molar basis with two fresh strains tested. Succinic acid was also produced from fumaric and malic acids but not from dl-isocitric, alpha-ketoglutaric, and pyruvic acids. The present study is considered to provide the first evidence on the production of succinic acid, an important flavoring substance in dairy products and fermented beverages, from citrate by lactobacilli.

Journal Article↗

Effect of Indoleacetic Acid and Related Indoles on Lactobacillus sp. Strain 11201 Growth, Indoleacetic Acid Catabolism, and 3-Methylindole Formation.

A study was conducted to determine the activity of the 3-methylindole (3MI)-forming enzyme in Lactobacillus sp. strain 11201. Cells were incubated anaerobically with 17 different indolic and aromatic compounds. Indoleacetic acid (IAA), 5-hydroxyindoleacetic acid, 5-methoxy-3-indoleacetic acid, indole-3-pyruvate, or indole-3-propionic acid induced 3MI-forming activity. The highest total enzyme activity induced by IAA was observed in cells incubated with an initial concentration of 1.14 mM IAA. Peak activity of the 3MI-forming enzyme occurred 4 h after bacteria were incubated with either 0.114 or 1.14 mM IAA. Enzyme activity peaked earlier (2 h) and disappeared more rapidly at 5.7 mM IAA than at other concentrations of IAA. The effects of IAA and 3MI on the growth of Lactobacillus sp. strain 11201 and formation of 3MI from IAA also were determined. Bacterial growth and 3MI formation from IAA were reduced in medium containing exogenous 3MI. The growth depression observed in medium containing 5.7 mM IAA appears to be due to the toxicity of 3MI rather than IAA. The formation of 3MI in this ruminal Lactobacillus sp. is mediated by an inducible enzyme, and as 3MI accumulates, bacterial growth and rates of 3MI formation from IAA are reduced.

Journal Article↗

Differentiation of Lactobacillus Species by Molecular Typing.

A total of 64 type, reference, clinical, health food, and stock isolates of microaerophilic Lactobacillus species were examined by restriction fragment length polymorphisms. Of particular interest were members of six of the eight species most commonly recovered from the vaginas of healthy premenopausal women, namely, Lactobacillus jensenii, L. casei, L. rhamnosus, L. acidophilus, L. plantarum, and L. fermentum. Six main groupings were identified on the basis of ribotyping. This technique was able to classify fresh isolates to the species level. In the case of the ribotype A grouping for L. rhamnosus, differences between strains were evident by chromosome typing (chromotyping). Many isolates did not possess plasmids. Six L. rhamnosus strains isolated from four different health food products appeared to be identical to L. rhamnosus ATCC 21052. The molecular typing system is useful for identifying and differentiating Lactobacillus isolates. Studies of strains of potential importance to the urogenital flora should include molecular characterization as a means of comparing genetic traits with those of strains whose characteristics associated with colonization and antagonism against pathogens have been defined.

Journal Article↗

In vitro and in vivo inhibition of Helicobacter pylori by Lactobacillus casei strain Shirota.

We studied the potential inhibitory effect of Lactobacillus casei strain Shirota (from the fermented milk product Yakult [Yakult Ltd., Tokyo, Japan]) on Helicobacter pylori by using (i) in vitro inhibition assays with H. pylori SS1 (Sydney strain 1) and nine H. pylori clinical isolates and (ii) the in vivo H. pylori SS1 mouse model of infection over a period of 9 months. In vitro activity against H. pylori SS1 and all of the clinical isolates was observed in the presence of viable L. casei strain Shirota cells but not in the cell-free culture supernatant, although there was profound inhibition of urease activity. In vivo experiments were performed by oral administration of L. casei strain Shirota in the water supply over a period of 9 months to 6-week-old C57BL/6 mice previously infected with H. pylori SS1 (study group; n = 25). Appropriate control groups of H. pylori-infected but untreated animals (n = 25) and uninfected animals given L. casei strain Shirota (n = 25) also were included in the study. H. pylori colonization and development of gastritis were assessed at 1, 2, 3, 6, and 9 months postinfection. A significant reduction in the levels of H. pylori colonization was observed in the antrum and body mucosa in vivo in the lactobacillus-treated study group, as assessed by viable cultures, compared to the levels in the H. pylori-infected control group. This reduction was accompanied by a significant decline in the associated chronic and active gastric mucosal inflammation observed at each time point throughout the observation period. A trend toward a decrease in the anti-H. pylori immunoglobulin G response was measured in the serum of the animals treated with lactobacillus, although this decrease was not significant.

Animals↗

Isolation and characterization of promoters from the Lactobacillus casei temperate bacteriophage A2.

Random Sau3A1 DNA fragments from the temperate Lactobacillus bacteriophage A2 were cloned into the promoter-probe plasmid pGKV210. Seven DNA fragments with promoter activity were selected, after transformation of Escherichia coli and Lactococcus lactis, subsp, lactis, through the chloramphenicol resistance they conferred to the corresponding clones. The seven promoters were functional in Lactobacillus casei. Their strength was analysed by measuring the levels of chloramphenicol resistance and chloramphenicol acetyltransferase activity induced in each host. The nucleotide sequences of these fragments were determined and primer extension analysis was used to locate the initiation site of transcription from each promoter in E coli. The promoters contained -10 and -35 regions similar to the consensus sequences of E. coli and Lactobacillus promoters.

Bacteriophages↗

Lactobacillus casei subspecies casei endocarditis--a case report.

Lactobacillus sp., generally considered to be a harmless indigenous bacteria of the mucous membrane, occasionally causes serious infections. Lactobacillus endocarditis is a very rare disease, and no case has been reported in Korea. Gram-positive bacilli were isolated from blood cultures of a 41-year-old man with clinically suspected subacute bacterial endocarditis. The patient had a dental procedure 3 months prior to the infection. The isolate was identified as L. casei subsp. casei based on the cultural characteristics and gas liquid chromatography of metabolic products. The patient was treated with ampicillin and improved. When Lactobacillus is isolated from the blood of an endocarditis patient, the significance should be seriously considered.

Adult↗

Clones of Lactobacillus casei and Torulopsis glabrata associated with recurrent abdominal wall abscess.

Infectious disease caused by Lactobacillus sp has not been previously reported in Taiwan. We present a case of recurrent abdominal wall abscess in a chronically ill 36-year-old woman, and review the literature on Lactobacillus infection. Five isolates of L. casei were recovered from blood and pus samples, and two isolates of Torulopsis glabrata were isolated from two blood specimens 3 months apart. Two clones of L. casei and T. glabrata were identified by means of antibiotyping with the E test and molecular methods. The abscess was surgically removed because of poor response to 7 months of antimicrobial therapy for the second infectious episode. Recurrent Lactobacillus infection can occur in chronically ill or immunosuppressed patients. Treatment of these infections may require a longer duration of antibiotic therapy, or surgical intervention.

Abdominal Abscess↗

The role of humoral immunity in Lactobacillus casei cell wall induced arthritis.

Although both cellular and humoral immunity have been shown to play important roles in the development of collagen induced arthritis, their roles in the pathogenesis of bacterial cell wall induced arthritis remain unclear. Previous studies of humoral immunity in bacterial cell wall induced arthritis were carried out in outbred animals and measured the humoral response to only the eliciting bacteria. We compared the humoral immune responses of arthritis susceptible Lewis and arthritis resistant Fisher rats after the injection of arthritogenic group B Lactobacillus casei cell wall fragments. Both strains developed significant IgG and IgM responses to the Lactobacillus casei cell wall fragments. Neither strain developed a significant immune response to types I, II, IX or 1a2a3a collagens. These data suggest that humoral immune responses to the Lactobacillus casei cell wall fragments and types I, II, IX, or 1a2a3a collagens do not play a significant role in determining the differing arthritis susceptibilities of Lewis and Fisher rats.

Animals↗

Lactobacillus casei pneumonia and sepsis in a patient with AIDS. Case report and review of the literature.

Lactobacilli are ubiquitous gram-positive anaerobic rods present in the normal bacterial flora of the mouth, vagina and gastrointestinal tract. Although they are usually non pathogenic, serious infections have occasionally been described in transplant recipients or severely ill patients. Only 4 cases have been reported involving AIDS: one had predisposing conditions other than AIDS, and none of the others had pure growth of lactobacilli. We report a case of community-acquired Lactobacillus casei pneumonia in a CD4 lymphocyte-depleted AIDS patient. Lactobacillus was isolated in pure growth in repeated blood cultures in an outpatient with no preexisting lung diseases and no known risk factors for Lactobacillus infections (dental procedures, complicated deliveries, gastrointestinal diseases, cardiac prosthetic valves) or consumption of unusual dairy products. Although uncommon, lactobacilli are possible pathogens in HIV-infected patients with very low CD4 counts, and their isolation in clinical specimens must not be neglected. Susceptibility tests are essential because of the variable antibiotic-resistance patterns of these bacteria.

AIDS-Related Opportunistic Infections↗

Construction of an integrative food-grade cloning vector for Lactobacillus acidophilus.

An integrative cloning vector was constructed using a randomly cloned HindIII-digested chromosomal fragment from Lactobacillus acidophilus ADH inserted into an Escherichia coli vector, pBluescript II SK+. Southern hybridization studies demonstrated homology of the inserted fragment with one other L. acidophilus strain and one Bifidobacterium strain. Identification of a SauI site located near the middle of the 1.9-kb ADH chromosomal fragment made it possible to clone the Lactobacillus bulgaricus beta-galactosidase (EC 3.2.1.23) gene into this vector. The vector was unable to replicate in the homologous host, L. acidophilus ADH, following electroporation. The chromosomal fragment allowed the integration of the beta-galactosidase gene (beta gal) into the host chromosome via homologous recombination. The size of the two flanking L. acidophilus ADH chromosomal fragments, approximately 0.95 kb each, was sufficient to allow the double cross-over to take place. Southern hybridization demonstrated that only L. acidophilus and L. bulgaricus DNA had been integrated into the chromosome of the host strain. The beta-galactosidase activity of the transformant was increased approximately 200-fold when compared to the enzyme activity of the wild-type strain. The beta gal gene remained stable in the transformant strain after 30 transfers in growth media without selection pressure. This first-generation integrative cloning vector is constructed solely of DNA from organisms consumed by humans and could be considered a food-grade vector system.

Bacterial Proteins↗

In vitro adherence of Lactobacillus species to vaginal epithelial cells.

Because of the reported use of yogurt douches for the treatment of vaginitis, the adherence of Lactobacillus species to normal human vaginal epithelial cells was tested by in vitro methods. L. acidophilus and L. bulgaricus isolated from three commercial yogurts were compared to L. acidophilus, L. fermentum, L. cellobiosus, and L. casei isolated from clinical specimens and from American Type Culture Collection cultures. L. acidophilus isolated from Lactinex tablets was also tested. No significant difference was found among the 19 clinical isolates, the American Type Culture Collection cultures, and the Lactinex isolate, but all showed significantly higher (p less than 0.01) adherence than any Lactobacillus isolated from yogurt. The presence of Gardnerella vaginalis, which adheres strongly to vaginal epithelial cells, was found to interfere with the adherence of lactobacilli from all sources studied.

Adhesiveness↗