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PubMed2006

Identification and differentiation of Lactobacillus, Streptococcus and Bifidobacterium species in fermented milk products with bifidobacteria.

The aim of this study was to identify and discriminate bacteria contained in commercial fermented milks with bifidobacteria by the use of amplified ribosomal DNA restriction analysis (ARDRA) and randomly amplified polymorphic DNA (RAPD) techniques. ARDRA of the 16S rDNA gene and RAPD were performed on 13 Lactobacillus strains, 13 Streptococcus and 13 Bifidobacterium strains isolated from commercial fermented milk. Lactobacillus delbrueckii, Streptococcus thermophilus and Bifidobacterium animalis isolates were identified by genus- and species-PCR and also, they were differentiated at genus and species level by ARDRA using MwoI restriction enzyme. The ARDRA technique allowed for the discrimination among these three related genus with the use of only one restriction enzyme, since distinctive profiles were obtained for each genus. Therefore it can be a simple, rapid and useful method for routine identification. Also, RAPD technique allowed the discrimination of all bacteria contained in dairy products, at genus- and strain-level by the performance of one PCR reaction.

Bifidobacterium↗
PubMed2004

An improved method of microencapsulation and its evaluation to protect Lactobacillus spp. in simulated gastric conditions.

An improved method of microencapsulation was developed to increase the efficacy of capsules in protecting the encapsulated bacteria under simulated gastric conditions. Lactobacillus acidophilus CSCC 2400 was encapsulated in calcium alginate and tested for its survival in simulated gastric conditions. The effects of different capsule sizes (200, 450, 1000 microm), different sodium alginate concentrations (0.75%, 1%, 1.5%, 1.8% and 2% w/v) and different concentrations of calcium chloride (0.1, 0.2, 1.0 M) on the viability of encapsulated bacteria were investigated. The viability of the cells in the microcapsules increased with an increase in alginate capsule size and gel concentration. There was no significant difference (p>0.05) in the viability of encapsulated cells when the concentration of calcium chloride was increased. Increase in cell load during encapsulation increased the number of bacterial survivors at the end of 3-h incubation in simulated gastric conditions. Hardening the capsule in calcium chloride solution for a longer time (8 h) had no impact on increasing the viability of encapsulated bacteria in a simulated gastric environment. The release of encapsulated cells at different phosphate buffer concentrations was also studied. When encapsulated L. acidophilus CSCC 2400 and L. acidophilus CSCC 2409 were subjected to low pH (pH 2) and high bile concentration (1.0% bile) under optimal encapsulation conditions (1.8% (w/v) alginate, 10(9) CFU/ml, 30 min hardening in 0.1 M CaCl(2) and capsule size 450 microm), there was a significant increase (p<0.05) in viable cell counts, compared to the free cells under similar conditions. Thus the encapsulation method described in this study may be effectively used to protect the lactobacillus from adverse gastric conditions.

Alginates↗
PubMed2003

Sequence and genetic organization of the 19.3-kb erythromycin- and dalfopristin-resistance plasmid pLME300 from Lactobacillus fermentum ROT1.

Lactobacillus fermentum ROT1 was isolated from a raw milk dairy product. It is resistant to novobiocin, tetracycline, erythromycin and dalfopristin. A chromosomal tetracycline-resistance determinant was identified as tetM. A 19,398-bp plasmid (pLME300), present in several erythromycin-resistant strains of Lb. fermentum, was isolated from strain ROT1 and completely sequenced. Based on putative open reading frames, pLME300 contains at least four different functional regions. In region I, ORF1 shows high homologies to replication proteins of different theta-replicating plasmids. In addition, a tandem repeat of a 22-bp sequence appears 4.5 times. In region II, ORF3 may code for a methylase, and ORF4 has homologies to Mrr restriction system proteins of Deinococcus radiodurans and Escherichia coli suggesting a restriction-modification system. Region III harbours antibiotic-resistance genes, coding for a macrolide-lincosamide-streptogramin B (MLS) methylase Erm(LF) and the streptogramin A acetyltransferase Vat(E), which is identical to Vat(E) from Enterococcus faecium. Furthermore, region III shows a 91% nucleotide sequence identity to an erm-vat linkage of E. faecium. Region IV carries ORFs that appear to be involved in plasmid mobilization as characterized by a putative origin of transfer and a mobilization protein. pLME300 is the largest completely sequenced multi-resistance plasmid isolated from any Lactobacillus strain so far.

Anti-Bacterial Agents↗
PubMed2004

Characterization of pC7 from Lactobacillus paraplantarum C7 derived from Kimchi and development of lactic acid bacteria--Escherichia coli shuttle vector.

A cryptic plasmid pC7 was characterized, which was isolated from Lactobacillus paraplantarum C7 derived from Kimchi, a traditional korean vegetable food. The plasmid pC7 is a circular molecule of 2,134 base-pairs in length with a G + C content of 38.5%. The nucleotide sequence analysis revealed the presence of an open reading frame encoding a putative 317 amino acids protein homologous to replication proteins RepA. Furthermore, a putative double and a single-strand origin were identified. Together with features of nucleotide sequences, the detection of single-stranded intermediate DNA in Lb. paraplantarum demonstrated that pC7 replicates via a rolling circle mechanism. A cloning vector for lactic acid bacteria was developed on the basis of the pC7 replicon, into which were inserted an erythromycin resistance gene as a marker, multiple cloning sites, and Escherichia coli ColE1 replication origin. E. coli and several species of Lactobacillus and Leuconostoc can be transformed with the resultant vector. Therefore, pC7 derivatives may be useful LAB-E. coli shuttle vectors, which are essential in engineering important strains in food fermentation.

Amino Acid Sequence↗
PubMed2005

Characterisation of a novel plasmid p9785S from Lactobacillus johnsonii FI9785.

Lactobacillus johnsonii FI9785, a strain originally isolated from poultry gastrointestinal tract for its probiotic function as a competitive excluder of pathogens, was found to contain two cryptic plasmids of 3.5 and 25.6 kb. Nucleotide sequence analysis of the entire small plasmid, designated p9785S (3471 bp), indicated a G+C content of 35.8%, and revealed two open reading frames (orfs). The product of orf1 exhibited similarity to the relaxases of mobilizable plasmids, whereas the product of orf2 displayed significant homology to replication proteins of plasmids which use the rolling circle mode of replication. A conserved double-strand origin of replication was also present in p9785S. A definite minus origin was not identified although a region with extensive intrastrand base pairing potential was revealed. A 1.4 kb fragment encoding the chloramphenicol resistance gene was cloned into p9785S and the resulting vector, pFI2431, was stably maintained when introduced into the parent Lactobacillus cells.

Amino Acid Sequence↗
PubMed2005

Sequence analysis of the plasmid genome of the probiotic strain Lactobacillus paracasei NFBC338 which includes the plasmids pCD01 and pCD02.

Lactobacillus paracasei NFBC338 is a probiotic strain that was isolated from the human gastrointestinal tract (GIT) and contains a plasmid genome of 80kb. Using a shotgun sequencing approach, two of the plasmids, pCD01 (19,882bp) and pCD02 (8554bp) have been completely sequenced, and four contiguous sequences (Contigs) have been assembled. Bioinformatic analysis of pCD01 revealed that it contains 23 putative open reading frames (ORFs) and that it contains regions characterised by potential replication functions and multidrug resistance (MDR). In contrast, the content of pCD02 is mainly cryptic, although, it does contain two insertion sequence (IS) elements. Indeed, up to 17% of the entire plasmid genome encodes putative transposable elements. In addition, there are a number of interesting ORFs distributed over the four Contigs that show significant homology to genes such as those involved in adherence and biotin metabolism, which may prove beneficial to Lb. paracasei NFBC338 under certain environmental conditions. This study provides a novel insight into the rich plasmid complement of this probiotic Lactobacillus strain, which may potentially be exploited as the basis for development of improved genetic tools for probiotic lactobacilli.

Adhesins, Bacterial↗
PubMed2005

Intranasal immunisation using recombinant Lactobacillus johnsonii as a new strategy to prevent allergic disease.

We have previously demonstrated the induction of a specific anti-IgE response in vivo by parenteral immunisation of rhesus monkeys using short IgE mimotopes or an anti-idiotypic antibody mimicking an IgE epitope. Such specific anti-IgE responses may be of clinical benefit for atopic patients. In this study, we examined the potential for a more convenient therapy via mucosal immunisation using live recombinant Lactobacillus johnsonii (Lj) as a vaccine delivery vehicle. Either an anti-idiotypic scFv or an IgE mimotope were expressed on the surface of Lj as fusion proteins using the cell wall anchored proteinase PrtB from Lactobacillus delbrueckii subsp. bulgaricus. The recombinant Lj were shown to express the heterologous fusion proteins and were specifically recognised by the corresponding anti-human IgE monoclonal antibody. Subcutaneous and intranasal immunisation of mice with recombinant Lj, expressing these fusion proteins induced a systemic IgG response against human IgE. Our data suggest that recombinant Lactobacilli expressing IgE epitopes may represent a novel means of vaccination to induce a beneficial anti-IgE response.

Amino Acid Sequence↗
PubMed2002

[Submental abscess by Lactobacillus acidophilus/jensenii].

The different species of Lactobacillus genus are bacteria of a low pathogenicity. They are found in the gastrointestinal tract, the female genitourinary tract, and also as part of the anaerobic flora in the mouth. Due to this low pathogenicity, they are generally related with infections together with other with more virulent microorganisms, or in immunocompromised patients. In our case, Lactobacillus appears alone as an etiological agent in an immunocompetent patient with a submental abscess.

Abscess↗
PubMed1988

Preservation of intestinal integrity during radiotherapy using live Lactobacillus acidophilus cultures.

Twenty-four female patients suffering from gynaecological malignancies and scheduled for internal and external irradiation of the pelvic area (pelvic dose 5000 cGy) were selected for a study on prevention of intestinal side-effects by live Lactobacillus acidophilus cultures. The patients were randomised into two groups. Both groups received dietary counselling recommending a low-fat and low-residue diet during radiotherapy. The control group received dietary counselling only. The test group received 150 ml of a fermented milk test product supplying them with at least 2 X 10(9) live Lactobacillus acidophilus bacteria daily and 6.5% lactulose as substrate for the bacteria. The results indicated that the test product appeared to prevent radiotherapy-associated diarrhoea. However, flatulence was increased probably due to lactulose ingestion in the test group.

Adult↗
PubMed2003

Cervical Lactobacillus and leukocyte infiltration in preterm premature rupture of membranes.

OBJECTIVES: To evaluate the possible association between microorganisms present in the cervical secretions and amniotic fluid of pregnant women with preterm premature rupture of membranes (PPROM), and histologic chorioamnionitis. METHODS: Thirty-seven pregnant women with PPROM and 21 healthy pregnant women were studied. Secretions from the cervical canal and amniotic fluid were collected to isolate microorganisms present in the genital tract. Cervical smears were Gram stained and evaluated microscopically. At delivery, chorioamniotic membranes were collected for histopathologic analysis. RESULTS: Microscopic examination of the cervical secretion smears obtained from the PPROM group showed a low rate of Lactobacillus species, large numbers of leukocytes, and a wide diversity of microorganisms compared with the control group. The PPROM group presented an 80% rate of chorioamnionitis. Staphylococcus aureus isolation in cervical secretion was associated with intense inflammatory infiltrate in the membranes and might play a role in the pathogenesis of PPROM. CONCLUSIONS: The low colonization of cervical flora by Lactobacillus species associated with an intense leukocyte infiltrate detected in Gram-stained cervical smears can be considered a rapid method of detecting chorioamnionitis in pregnant women with PPROM.

Amniotic Fluid↗
PubMed2002

Domain closure, substrate specificity and catalysis of D-lactate dehydrogenase from Lactobacillus bulgaricus.

NAD-dependent Lactobacillus bulgaricus D-Lactate dehydrogenase (D-LDHb) catalyses the reversible conversion of pyruvate into D-lactate. Crystals of D-LDHb complexed with NADH were grown and X-ray data collected to 2.2 A. The structure of D-LDHb was solved by molecular replacement using the dimeric Lactobacillus helveticus D-LDH as a model and was refined to an R-factor of 20.7%. The two subunits of the enzyme display strong asymmetry due to different crystal environments. The opening angles of the two catalytic domains with respect to the core coenzyme binding domains differ by 16 degrees. Subunit A is in an "open" conformation typical for a dehydrogenase apo enzyme and subunit B is "closed". The NADH-binding site in subunit A is only 30% occupied, while in subunit B it is fully occupied and there is a sulphate ion in the substrate-binding pocket. A pyruvate molecule has been modelled in the active site and its orientation is in agreement with existing kinetic and structural data. On domain closure, a cluster of hydrophobic residues packs tightly around the methyl group of the modelled pyruvate molecule. At least three residues from this cluster govern the substrate specificity. Substrate binding itself contributes to the stabilisation of domain closure and activation of the enzyme. In pyruvate reduction, D-LDH can adapt another protonated residue, a lysine residue, to accomplish the role of the acid catalyst His296. Required lowering of the lysine pK(a) value is explained on the basis of the H296K mutant structure.

Binding Sites↗
PubMed1994

Crystallization of D-lactate dehydrogenase from Lactobacillus bulgaricus.

The D-lactate dehydrogenase (D-LDH) from Lactobacillus bulgaricus has been purified and co-crystallized with its cofactor NAD+. Crystals suitable for X-ray diffraction experiments have been obtained from an ammonium sulfate solution by the hanging-drop method. The crystals belong to the orthorhombic space group C222 (or C222(1)) with cell dimensions a = 76.5 A, b = 93.3 A, c = 118.4 A and one monomer of 37,000 daltons per asymmetric unit. They diffract beyond 3.0 A resolution. Sequence comparison suggests that D-LDHs have no evolutionary relationship to L-LDHs and belong instead to the family of the D-2-hydroxyacid dehydrogenases. The X-ray crystallographic structure of the D-LDH from Lactobacillus bulgaricus will be a decisive test of this hypothesis.

Crystallization↗
PubMed1997

Inhibition of initial adhesion of uropathogenic Enterococcus faecalis to solid substrata by an adsorbed biosurfactant layer from Lactobacillus acidophilus.

OBJECTIVES: The potency of the Lactobacillus acidophilus RC14 biosurfactant "surlactin" to reduce the initial adhesion of Enterococcus faecalis 1131 was investigated on a hydrophilic and a hydrophobic substratum in a parallel-plate flow chamber, using phosphate-buffered saline and pooled human urine as a suspending fluid. METHODS: A parallel-plate flow chamber with a glass or silicone rubber bottom plate was filled with different biosurfactant solutions of 0, 0.01, 0.1, or 1.0 mg/mL for overnight adsorption (18 hours). Subsequently, the adhesion of E. faecalis on thus prepared biosurfactant layers was observed in situ in phosphate-buffered saline or in urine by automated image analysis. RESULTS: Adsorbed biosurfactant layers caused an important, dose-related inhibition of the initial deposition rate of E. faecalis and the number of adherent bacteria after 4 hours on both hydrophilic glass and hydrophobic silicone rubber, although this effect was stronger in buffer than in urine. For the experiments carried out in urine, the inhibitory effect of the biosurfactant layer was largest when silicone rubber was used rather than glass, whereas no influence of the substratum hydrophobicity on the inhibition of E. faecalis adhesion was noticed for experiments performed in buffer. CONCLUSIONS: The biosurfactant surlactin, as released by several Lactobacillus isolates, might open the way to the development of antiadhesive biologic coatings for catheter materials. It should be considered, however, that these results are preliminary and that the efficiency of the biosurfactant is probably affected not only by the hydrophobicity of the substratum and the suspending fluid, but also by the type of uropathogen involved.

Bacterial Adhesion↗
PubMed2002

Characterization of the tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 reveals a composite structure.

The 10,877bp tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 was completely sequenced. The sequence revealed a composite structure containing DNA from up to four different sources. The replication region had homology to other plasmids of lactic acid bacteria while the tetracycline resistance region, containing a tet(M) gene, had high homology to sequences from Clostridium perfringens and Staphylococcus aureus. Within the tetracycline resistance region a Lactobacillus IS-element was found. The remaining part of the plasmid contained three open reading frames with unknown functions. The composite structure with several truncated genes suggests a recent assembly of the plasmid. This is the first sequence of an antibiotic resistance plasmid isolated from L. plantarum.

Base Sequence↗
PubMed2000

Characterization of natural isolates of Lactobacillus strains to be used as starter cultures in dairy fermentation.

The technological relevant characteristics of five homofermentative lactobacilli strains, isolated from natural fermented hard cheeses, were studied. Isolates CRL 581 and CRL 654, from Argentinian artesanal hard cheeses, and isolates CRL 1177, CRL 1178, and CRL 1179, from Italian Grana cheeses, were identified as Lactobacillus delbrueckii subsp. lactis and Lactobacillus helveticus, respectively, by physiological and biochemical tests, SDS-PAGE of whole-cell proteins and sequencing of the variable (V1) region of the 16S ribosomal DNA. All strains showed high levels of beta-galactosidase activity. However, proteolytic activity varied widely among isolates. Strains CRL 581, CRL 654, and CRL 1177 hydrolyzed alpha- and beta-caseins and were able to coagulate reconstituted skim milk in less than 16 h at 42 degrees C. According to the substrate specificity, these proteinases have a caseinolytic activity comparable to that of the P(III)-type of lactococcal proteinases. No strains produced inhibitor substances (bacteriocin) and all were insensitive to attack by 14 L. helveticus- and L. delbrueckii subsp. lactis-specific bacteriophages.

Animals↗
PubMed2001

Dry sausage fermented by Lactobacillus rhamnosus strains.

The ability of three probiotic Lactobacillus rhamnosus strains GG, E-97800 and LC-705 and one commercial Pediococcus pentosaceus starter strain (control) to produce dry sausage was studied. During the fermentation process the numbers of inoculated lactic acid bacteria increased from approx. 7 log10 to 8-9 log10 cfu/g and the pH values decreased from 5.6 to 4.9-5.0. The sensory test indicated that the dry sausages fermented by L. rhamnosus LC-705 were inferior to the control sausages. The presence of inoculated experimental strains as predominant organisms in the dry sausages was recognised on the basis of their genetic fingerprints by ribotyping. The concentrations of biogenic amines remained low during the ripening process. These results indicated that the studied Lactobacillus rhamnosus strains, especially strains GG and E-97800, are suitable for use as probiotic starter cultures in fermenting dry sausage.

Animals↗
PubMed2001

Inhibition of in vitro growth of Shiga toxin-producing Escherichia coli O157:H7 by probiotic Lactobacillus strains due to production of lactic acid.

The inhibiting characteristics of lactic acid bacteria on Shiga toxin-producing Escherichia coli (STEC) O157:H7 (three strains, clinically isolated) was investigated by using a batch fermentation system. The species such as Lactobacillus casei strain Shirota or L. acidophilus YIT 0070 exert growth inhibitory and bactericidal activities on STEC. The pH value and undissociated lactic acid (U-LA) concentration of the culture medium of STEC cocultured with L. casei or L. acidophilus dramatically lowered or increased, respectively [corrected], when compared with those of the control culture. The cytotoxic properties of U-LA on STEC strain 89020087 analyzed in vitro was divided into two phases, i.e., the bacteriostatic phase (between 3.2 to 62 mM) and the bactericidal phase (over 62 mM). These data suggest that the bactericidal effect of Lactobacillus on STEC depends on its lactic acid production and pH reductive effect.

Escherichia coli O157↗