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Isolation and characterisation of a Lactobacillus helveticus ITG LH1 peptidase-rich sub-proteome.

Lactobacillus helveticus strains, one of the most nutritionally fastidious lactic acid bacteria, have a potent proteolytic system that makes them very interesting for different uses in the dairy industry. Its applications concern from cheese ripening to the preparation of fermented milk products with biologically active peptides. The cell-free extract (CFE) of Lactobacillus helveticus strain ITG LH1 was analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), using IPG immobiline dry strips (pH 4-7). With the aim to study the proteolytic enzymes expressed by Lactobacillus helveticus ITG LH1 grown in milk medium, a two step-chromatography methodology, based on ion exchange and affinity chromatography, was developed for the preparation of a peptidase-rich sub-proteome from the CFE of stationary growing cells. Several affinity chromatography columns were tested and among them a HiTrap Chelating column was selected as it provided the best performance for the enrichment in peptidases. Peptidase activities were studied using different beta-Naphtylamide (beta-NA) derivatives and specific activities were increased 50- to 100-fold by this chromatographic procedure. Sub-proteome characterisation was performed by 2D-PAGE, pH 4-7, followed by protein digestion with trypsin, analysis by MALDI-TOF mass spectrometry and subsequent database searches using peptide mass fingerprints. Among the most abundant proteins seven peptidases were present, namely the two general aminopeptidases (PepN, PepC), three dipeptidases (PepDA, PepV, PepQ) and two endopeptidases (PepO, PepO3), all of them corresponding to the catalytic classes of metallo- or cysteine-peptidases. Several stress proteins (such as heat shock proteins DnaK and GroEL) and other enzymes implied in bacterial metabolism, namely in the carbohydrate pathways (such as LDH), were also identified in the peptidase-rich sub-proteome.

Aminopeptidases↗

Lactobacillus endocarditis: case report and review of cases reported since 1992.

Lactobacillus is a rare cause of endocarditis with only 78 reported cases in the literature. We report a case of Lactobacillus endocarditis and review reported adult cases since the introduction of the Duke's criteria for endocarditis. An analysis is presented with a focus on outcomes. Mortality from reported cases of Lactobacillus endocarditis was found to have substantially improved since 1992. Surgical rates for persistent disease remain the same. Possible reasons for this improvement include better antibiotics, earlier recognition of cases, and more aggressive treatment regimens.

Bacteremia↗

Effects of the probiotic strain Lactobacillus johnsonii strain La1 on autonomic nerves and blood glucose in rats.

Oral administration of Lactobacillus casei reportedly reduces blood glucose concentrations in a non-insulin-dependent diabetic KK-Ay mouse model. In order to determine if other lactobacillus strains affect glucose metabolism, we evaluated the effect of the probiotic strain Lactobacillus johnsonii La1 (LJLa1) strain on glucose metabolism in rats. Oral administration of LJLa1 via drinking water for 2 weeks inhibited the hyperglycemia induced by intracranial injection of 2-deoxy-D-glucose (2DG). We found that the hyperglucagonemic response induced by 2DG was also suppressed by LJLa1. Oral administration of LJLa1 for 2 weeks also reduced the elevation of blood glucose and glucagon levels after an oral glucose load in streptozotocin-diabetic rats. In addition, we recently observed that intraduodenal injection of LJLa1 reduced renal sympathetic nerve activity and enhanced gastric vagal nerve activity, suggesting that LJLa1 might affect glucose metabolism by changing autonomic nerve activity. Therefore, we evaluated the effect of intraduodenal administration of LJLa1 on adrenal sympathetic nerve activity (ASNA) in urethane-anesthetized rats, since the autonomic nervous system, including the adrenal sympathetic nerve, may be implicated in the control of the blood glucose levels. Indeed, we found that ASNA was suppressed by intraduodenal administration of LJLa1, suggesting that LJLa1 might improve glucose tolerance by reducing glucagon secretion via alteration of autonomic nerve activities.

Administration, Oral↗

Clinical study comparing probiotic Lactobacillus GR-1 and RC-14 with metronidazole vaginal gel to treat symptomatic bacterial vaginosis.

Bacterial vaginosis (BV) is particularly common in black women, and in Nigeria it is often caused by Mycoplasma, as well as Atopobium, Prevotella and Gardnerella sp. Antimicrobial metronidazole oral therapy is poorly effective in eradicating the condition and restoring the Lactobacillus microbiota in the vagina. In this study, 40 women diagnosed with BV by discharge, fishy odor, sialidase positive test and Nugent Gram stain scoring, were randomized to receive either two dried capsules containing Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 each night for 5 days, or 0.75% metronidazole gel, applied vaginally twice a day (in the morning and evening). Follow-up at day 6, 15 and 30 showed cure of BV in significantly more probiotic treated subjects (16, 17 and 18/20, respectively) compared to metronidazole treatment (9, 9 and 11/20: P=0.016 at day 6, P=0.002 at day 15 and P=0.056 at day 30). This is the first report of an effective (90%) cure of BV using probiotic lactobacilli. Given the correlation between BV and HIV, and the high risk of the latter in Nigeria, intravaginal use of lactobacilli could provide women with a self-use therapy, similar to over-the-counter anti-yeast medication, for treatment of urogenital infections.

Administration, Intravaginal↗

Safety evaluation of Lactobacillus delbrueckii subsp. lactis UO 004, a probiotic bacterium.

Lactobacillus delbrueckii subsp. lactis UO 004 was evaluated for its use as a potential probiotic from a safety point of view. The strain did not exhibit mucinolytic or other enzymatic activities that might be detrimental, such as those involving glycosidases (beta-D-glucosaminidase or alpha-D-galactosidase) or arylamidases (factor Xa and quimotrypsin-like activities), frequently present in Lactobacillus strains isolated from patients with endocarditis, although it was able to express protein Ca and kallikrein-like activities. On the other hand, the presence of the strain did not interfere with the growth of certain species of normal intestinal microbiota, such as Enterococcus fecalis, Escherichia coli, Bifidobacterium bifidum or Bacteroides fragilis. Moreover, the potential probiotic strain UO 004 is sensitive to antibiotics with transmissible resistance mechanisms in Lactobacillus such as chloramphenicol, erythromycin, tetracycline and vancomycin. In addition, strain L. delbrueckii UO 004 was not able to translocate towards the intestinal barrier of mice or produce changes in their activity or general health status.

Animals↗

DNA-based classification and sequence heterogeneities in the 16S rRNA genes of Lactobacillus casei/paracasei and related species.

The sequence differences within the 16S rRNA genes of Lactobacillus casei/paracasei and related species, Lactobacillus zeae and Lactobacillus rhamnosus, were investigated. Thirty-seven strains of mostly human or cheese origin were grouped by restriction endonuclease analysis (REA) of the total chromosomal DNA and by temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rRNA gene fragments. REA verified that all strains were genomically unique and singled out three major clusters, one L. rhamnosus-cluster and two clusters containing L. paracasei strains. The groups obtained by TTGE corresponded with one exception to the REA-clusters. In the TTGE clustering all L. paracasei strains formed one general group with one TTGE-band in common, and this group was sub-divided into five subgroups due to the presence of more than one TTGE-band in four of the subgroups. The occurrence of multiple TTGE-bands was investigated by amplifying and cloning of the 16S rRNA genes from the strains showing this phenomenon, thereby 12 clones from each strain were sequenced, demonstrating polymorphisms in almost all the cases. Subjecting the clones displaying sequence variations to TTGE as well as sequencing of 16S rDNA revealed by ribotyping of the strains, verified the presence of polymorphisms within the 16S rRNA genes. The migration characteristic of amplified DNA from a single clone corresponded to a specific band in the TTGE-pattern of the strain from which the clone originated. Southern blot hybridisation with a 16S rDNA probe demonstrated the presence of at least five 16S rRNA genes in L. casei/paracasei. A higher degree of variable positions than previously reported was observed in the 16S rRNA gene fragments of the members in the complex. Sequence comparison between the 16S rRNA gene copies of L. casei (CCUG 21451T) and L. zeae (CCUG 35515T) demonstrated that the two species shared almost the same sequence in some copies while the others were more different. Our results provide one explanation for the difficulties in reaching clear-cut taxa within the L. casei/paracasei complex.

Base Sequence↗

Immune responses generated by Lactobacillus as a carrier in DNA immunization against foot-and-mouth disease virus.

To exploit Lactobacillus acidophilus as a carrier in DNA immunization against foot-and-mouth disease virus (FMDV), a recombinant eukaryotic expression plasmid (pRc/CMV2-VP1-Rep. 8014) harboring pRc/CMV2 vector, the FMDV VP1 gene, and a replication origin from Lactobacillus plantarum ATCC 8014 strain was constructed. To detect the VP1 protein, pRc/CMV2-VP1-Rep. 8014 was expressed in PK 15 cells and transfected into a L. acidophilus SW1 strain (L. acidophilus SFMD-1). To evaluate the immunization effect of L. acidophilus SFMD-1, anti-FMDV VP1 antibody, T-cell proliferation, antigen-specific delayed-type hypersensitivity (DTH), and tissue distribution were investigated using intramuscular, intraperitoneal, intranasal, and oral administration delivery routes. The results showed that L. acidophilus SFMD-1 was able to elicit a detectable antibody level on day 21. The VP1 antibody levels induced by L. acidophilus SFMD-1 and commercial inactivated FMDV vaccine rose rapidly to 0.84 and 0.70, respectively, by day 42, then sustained a high level by day 49. The route of administration had an impact on the magnitude of the systemic antigen-specific IgG responses, with intramuscularly applied L. acidophilus SFMD-1 generating the greatest FMDV VP1 antibody response, followed by intraperitoneal, intranasal, and oral administration delivery routes. Using the T-cell proliferation assay, the stimulation index of a group immunized with L. acidophilus SFMD-1 reached 2.78 versus 5.08 in a group immunized with pRc/CMV2-VP1-Rep. 8014 plasmid. Mice immunized with L. acidophilus SFMD-1 were able to induce T-cell-mediated antigen-specific DTH. In addition, the VP1 gene was detected in the muscle, kidney, spleen, and heart, but not in the liver. The results demonstrate clearly that Lactobacillus as a carrier is a promising approach of DNA vaccination, and is a potentially guard against FMDV.

Animals↗

Life on the salvage path: the deoxynucleoside kinase of Lactobacillus acidophilus R-26.

In Lactobacillus acidophilus R-26, the synthesis of DNA precursor deoxynucleotides occurs exclusively by salvage of deoxynucleosides, beginning with phosphorylation by four deoxynucleoside kinases. Subunits bearing three of these activities are uniquely organized into two heterodimers, deoxyadenosine/deoxycytidine kinase (dAK/dCK) and deoxyadenosine/deoxyguanosine kinase (dAK/dGK), which, along with a distinct deoxythymidine kinase (TK), catalyze the parallel first committed steps of dNTP biosynthesis. Whereas TK is common to most prokaryotes (and eukaryotes), the other three activities that are the emphasis of this review are quite unusual in bacteria. Each activity is regulated in cis by its homologous end-product (dNTP) which is understood to act as a multisubstrate inhibitor capable of binding to both nucleoside and phosphate subsites. Conversely, the inactive dAK subunit is progressively activated by 1) association with a dGK or dCK subunit and 2) the conformationally driven heterotropic affect of dGuo or dCyd bound to the opposing subunit. Limited proteolysis has proven to be a powerful probe of conformational states. Further indication of conformational or structural differences between dAK and dGK (or dCK) is that the former follows an ordered kinetic path, while dGK or dCK exhibits rapid-equilibrium random kinetics. The multi-substrate behavior of end-product binding provides a convenient new diagnostic tool for distinguishing kinetic mechanisms. Tandem dak-dgk genes have been cloned from Lactobacillus DNA and expressed in Escherichia coli as dAK/dGK, utilizing the associated promoter. Sequence alignments reveal 65% identity in their DNA and 61% in their derived amino acid sequences. Encoded N-terminal sequences are identical for the first 18 residues, and both subunits share conserved sequences in common with adenylate kinase and viral TK. A more unusual conserved element, which appears to play a role in the activation of dAK, resembles the G2 loop of p21 ras. Remarkably, no homologous gene(s) for the dAK/dCK pair could be found. Comparisons of amino acid sequences, isoelectric pHs and subunit masses strongly indicated that native dCK and dGK are identical in sequence, except at their extreme N-termini (M-IVL for dCK and -TVIVL for dGK), suggesting that processing of a common precursor occurs in Lactobacillus. Accordingly, deletion of codons 2 and 3 from dgk resulted in the expression of dAK/dCK in the E. coli host; its kinetic properties are indistinguishable from those of native dAK/dCK. Subcloning the dgk or engineered dck gene resulted in expression of active dGK or dCK homodimers, each with a virtually unchanged Km toward its primary deoxynucleoside. However, in common with human dCK, dCK (or dGK) homodimer exhibits secondary activities with much larger Kms towards dAdo and dGuo (or dCyd). dCTP (or dGTP) is the best inhibitor of all three activities of the respective homodimer. Fully active heterodimers can be reconstituted simply by mixing a homodimer with independently expressed (inactive) dAK.

Amino Acid Sequence↗

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↗

Rapid PCR-based procedure to identify lactic acid bacteria: application to six common Lactobacillus species.

The goal of this study was to develop a method allowing rapid identification of the lactic acid bacteria strains in use in the laboratory (Lactobacillus plantarum NCIMB8826; L. fermentum KLD; L. reuteri 100-23; L. salivarius UCC43321; L. paracasei LbTGS1.4; L. casei ATCC393), based on PCR amplification of 16S RNA coding sequences. First, specific forward oligonucleotides were designed in the variable regions of 16S RNA coding sequences of six Lactobacillus strains. The reverse oligonucleotide was designed in the region where the sequences were homologous for the six strains. The expected size of the amplification product was +/-1000 bp. The specificity of the method was tested on total chromosomal DNA. For five out of the six strains, the amplification of the fragment was strain-specific and the method was directly applicable to colonies. For the strain L. casei ATCC393, an additional argument to the classification of this bacteria in the paracasei group could be proposed. Validation of the developed method was performed by applying it to six Lactobacillus reference strains and to various species of bacteria.

Animals↗

Effects of Lactobacillus strains on the ripening and organoleptic characteristics of Arzúa-Ulloa cheese.

Seven batches of Arzúa-Ulloa, a short-ripened soft cow's milk cheese produced in Galicia (NW Spain), were prepared from pasteurized milk. Two control batches of cheese (CB) were made with an acid-aromatic starter containing Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. lactis var. diacetylactis, isolated from raw-milk Arzúa-Ulloa cheeses. Five batches of cheese (LB) were made with the acid-aromatic starter plus one of five strains of mesophilic homofermentative Lactobacillus spp.: four of them isolated from raw-milk Arzúa-Ulloa cheese (characterized in previous works) and the remaining was a commercial Lactobacillus strain. Higher counts of mesophilic viable bacteria, lactic acid bacteria and citrate-fermenting bacteria were found on days 1 or 15 of ripening, while higher counts of lactobacilli were found on day 30 of ripening. On day 1 of ripening the highest diacetyl-acetoin content was noted in the CB, but after day 15 the diacetyl-acetoin content was similar or higher in three of the five LB. The mean degradation of beta-casein in CB was higher than in LB, while the degradation of alpha(s1)-casein was higher in LB. The mean contents of nitrogen-soluble fractions were slightly higher in the LB than in the CB. Volatile free fatty acid (VFFA) contents were, in general, greater in LB than in CB and maximum amounts were determined on day 15 of maturation. Sensorial analysis indicated a more acid taste was in LB, while bitter and astringent tastes were more intense in CB. A positive correlation was found between beta-casein degradation and bitter taste. Yogurt and butter aromas were more intense in CB and in two of the five LB. Firmness was lower in LB and a negative correlation was found between this parameter and alpha(s1)-casein degradation. Crumbliness showed a positive correlation with beta-casein degradation. The use of the Lactobacillus strains assessed in this study is recommended for Arzúa-Ulloa cheese manufacture, in order to enhance the desirable characteristics of this cheese variety, i.e., a soft texture due to alpha(s1)-casein proteolysis but without the bitter taste due to beta-casein degradation and a spicy and slightly rancid aroma and taste.

Acetoin↗

Susceptibility of Lactobacillus spp. to antimicrobial agents.

Bacteria used as probiotics or in starter cultures may serve as hosts of antibiotic resistance genes, which can be transferred to pathogenic bacteria. Before launching a starter culture or a probiotic product into the market, it is therefore important to verify that the single bacterial isolates (strains) do not contain transferable resistance genes. A study has been undertaken to establish the levels of susceptibility of Lactobacillus spp. to various antimicrobial agents. This is a prerequisite for differentiating putative transferable resistance from natural resistance. A selection of 62 strains has been screened with the use of the Etest (ABBiodisk, Stockholm, Sweden) for their susceptibility to 25 antimicrobial agents. The strains belonged to the following species: Lactobacillus plantarum/pentosus, L. rhamnosus, L. paracasei, L. sakei, L. curvatus and species of the L. acidophilus group: L. johnsonii, L. crispatus, L. gasseri, and L. acidophilus. The results from the Etests have shown that the level of susceptibility to the antimicrobial agents is species-dependent. For the following antimicrobial agents, susceptibility varied several folds between species: vancomycin, teicoplanin, tetracycline, norfloxacin, ciprofloxacin, fusidic acid, and clindamycin. The differences between the species were more subtle for the rest of the tested antimicrobial agents. On the basis of the result, it was possible to suggest minimal inhibition concentrations (MICs) for the individual Lactobacillus species to be used as a microbiological breakpoint when screening strains for transferable resistance genes.

Anti-Bacterial Agents↗

The lactobin A and amylovorin L471 encoding genes are identical, and their distribution seems to be restricted to the species Lactobacillus amylovorus that is of interest for cereal fermentations.

Lactobin A and amylovorin L471 are two bacteriocins produced by the phenotypically different strains Lactobacillus amylovorus LMG P-13139 and L. amylovorus DCE 471, respectively. A 110-bp PCR fragment of the structural gene of lactobin A was obtained from total genomic DNA of L. amylovorus LMG P-13139, which was used as a probe to isolate a 3.6-kb HindIII chromosomal fragment for sequencing. PCR amplification revealed that both the structural genes of both the bacteriocins lactobin A and amylovorin L471 were identical. These bacteriocins will be further referred to as amylovorin L. Amylovorin L can be defined as a small, strongly hydrophobic, antibacterial peptide consisting of 50 amino acids. It is synthesized as a precursor peptide of 65 amino acids processed at a characteristic double-glycine proteolytic cleavage site. Amylovorin L hence belongs to the class II bacteriocins. It has a narrow inhibitory spectrum, being most active towards Lactobacillus delbrueckii subsp. bulgaricus LMG 6901(T). Among 38 strains of the Lactobacillus acidophilus DNA homology group, another 6 L. amylovorus strains were also inhibitory towards the L. delbrueckii subsp. bulgaricus LMG 6901(T) strain. The lactobin A or amylovorin L471 structural genes could be detected in the genomes of three of these L. amylovorus strains, but only after extensive PCR amplification, indicating that the inhibitory substances were slightly different. The bacteriocins were characterized as small (approximately 4800 Da), heat-stable peptides that were active in a wide pH range (2.2-8.0). Finally, preliminary experiments indicated that the production of amylovorin L by L. amylovorus DCE 471 took place during a natural rye fermentation, indicating its potential importance in the development of a functional (probiotic) starter culture for cereal fermentations.

Amino Acid Sequence↗

Adhesion of some probiotic and dairy Lactobacillus strains to Caco-2 cell cultures.

The adhesion of 12 different Lactobacillus strains was studied using Caco-2 cell line as an in vitro model for intestinal epithelium. Some of the strains tested have been used as probiotics, and most of them are used in the dairy and food industry. Human and bovine enterotoxigenic Escherichia coli strains were used as positive and negative control, respectively. Bacterial adhesion to Caco-2 cell cultures was quantitated using radiolabelled bacteria. The adherence of bacteria was also observed microscopically after Gram staining. Viability of bacteria prior to adhesion was verified using flow cytometry. Among the tested strains, L. casei (Fyos) was the most adhesive strain and L. casei var. rhamnosus (Lactophilus) was the least adhesive strain, approximately 14 and 3% of the added bacteria adhered to Caco-2 cell cultures, respectively. The corresponding values for positive and negative control E. coli strains were 14 and 4%, respectively. The Lactobacillus strains tested could not be divided into distinctly adhesive or non-adhesive strains, since there was a continuation of adhesion rates. The four most adhesive strains were L. casei (Fyos), L. acidophilus 1 (LC1), L. rhamnosus LC-705 and Lactobacillus GG (ATCC 53103). No significant differences in the percentage adhesion were observed between these strains. Adhesion of all the strains was dependent on the number of bacteria used, since an approximately constant number of Caco-2 cells was used, indicating that the Caco-2 cell binding sites were not saturated. Viability of bacteria was high since approximately 90% of the bacteria were viable with the exception of L. acidophilus 1 which was 74% viable. Microscopic evaluations agreed with the radiolabelled binding as evidenced by observing more bacteria in Gram-stained preparations of good adhering strains compared to poorly adhering strains.

Animals↗

Purification of a novel fructosyltransferase from Lactobacillus reuteri strain 121 and characterization of the levan produced.

Fructosyltransferase (FTF) enzymes have been characterized from various Gram-positive bacteria, but not from Lactobacillus sp. In a screening of 182 lactobacilli for polysaccharide production only one strain, Lactobacillus reuteri strain 121, was found to produce a fructan being a levan. Here we report the first-time identification and biochemical characterization of a Lactobacillus FTF enzyme. When incubated with sucrose the enzyme produced a levan that is identical to that produced by Lb. reuteri strain 121 cells.

Amino Acid Sequence↗

Rapid amplified ribosomal DNA restriction analysis (ARDRA) identification of Lactobacillus spp. isolated from fecal and vaginal samples.

Amplified ribosomal DNA restriction analysis (ARDRA) was used to identify different species of Lactobacillus isolated from human faeces and vagina. PCR-ARDRA was performed using a set of four restriction enzymes, able to differentiate fourteen species of Lactobacillus. The PCR-ARDRA procedure described in this paper was shown to be a reliable and rapid method for identifying Lactobacillus species from intestinal and vaginal microflora at species and subspecies level.

DNA, Bacterial↗

Lactobacillus amylolyticus sp. nov., isolated from beer malt and beer wort.

Some of the strains used for the biological acidification in breweries belong to L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. lactis or L. fermentum. However, more recent studies showed that most strains isolated are physiologically different from the above mentioned species and were tentatively allocated to Lactobacillus amylovorus. Genotypic studies of 25 strains exclusively isolated from beer malts and beer worts, showed, that there were differences to the type strain of L. amylovorus concerning DNA-DNA similarities and the sequences of their 16S and 23S rRNA genes. Therefore, we propose to combine these strains in a new species of the genus Lactobacillus, namely L. amylolyticus. Strain DSM 11664 is proposed as the type strain. An rRNA targeted oligonucleotide probe was designed that allows a fast and reliable identification of Lactobacillus amylolyticus.

Bacterial Typing Techniques↗

Antibacterial activity of Lactobacillus species against Vibrio species.

Forty-one Lactobacillus strains were tested for antagonistic activity against nine strains of Vibrio. L. plantarum and L. casei were the most effective, and L. brevis was the least effective in inhibiting the growth of Vibrio species. L. gasseri and L. helveticus strains showed higher activity, while L. reuteri and L. fermentum showed lower inhibitory activity against Vibrio species. L. acidophilus strains exhibited various degrees of antagonistic activities against Vibrio species. However, none of the Lactobacillus species were able to inhibit the growth of Salmonella enteritidis, S. typhimurium, Escherichia coli, and Staphylococcus aureus. Inhibition of the Vibrio species was probably due to the production of organic acids by the Lactobacillus species.

Bacteriocins↗