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PubMed1988

Detection of a Lactobacillus substance that inhibits Escherichia coli.

Recent studies have shown that certain lactobacilli strains have the ability to interfere with the adherence and growth of uropathogenic bacteria. This interaction is believed to be important in the maintenance of a normal urogenital flora and in the prevention of infection in females. In the present study, Lactobacillus casei ssp. rhamnosus GR-1 and Lactobacillus acidophilus 76 were found to exert an inhibitory effect on pyelonephritogenic mutant Escherichia coli Hu 734 and E. coli ATCC 25922. The bioactivity of the inhibitor produced by strain GR-1 was retained under pH buffered conditions and was bactericidal. The bioactive substance was heat labile, not precipitated by up to 80% ammonium sulphate, and extractable in chloroform. The data indicated that the inhibitor is not lactic acid or hydrogen peroxide and has a molecular weight greater than 12,000-14,000. Human urine supported production of the inhibitor and reduced and delayed outgrowth of the E. coli. The ability of L. casei GR-1 and possibly other lactobacilli strains to produce inhibitors of uropathogenic bacteria may have clinical importance and significance in the microbial ecology of the urogenital tract.

Antibiosis↗
PubMed2002

Utilization of Lactobacillus sp. for steroid glycoalkaloids degradation by lactic acid fermentation.

The degradation fo steroid glycoalkaloids (SGAs) has been studied in model solutions. The number of colony forming units (CFU) was determined using a nondirect (cultivation) method during all stages of fermentation. The changes in SGAs content were observed by HPLC on the Supelcosil LC-NH2 column. The changes in alpha-tomatine concentration added to fermented Lactobacillus MRS broth have been studied. A mathematical model of steroid glycoalkaloids degradation during lactic fermentation was proposed. The mathematical model was based on the experimental data of SGA and glucose concentration and should be used for study and prediction of SGA concentration changes of fermented samples. The ratio of SGA degradation rate by fermentation and by lactic acid hydrolysis was calculated. The experimental data evaluated by proposed mathematical model for the selected strain of Lactobacillus plantarum 976H show real feasibility for SGA degradation by lactic acid fermentation.

Alkaloids↗
PubMed2002

Pulsed-field gel electrophoretic analysis of the genome of Lactobacillus gasseri ATCC33323, and construction of a physical map.

Some species of Lactobacillus are of major industrial and health significance as fermenting agents in the manufacturing of food products, as food preservatives, as "probiotic" bacteria or as vaccine delivery vehicles. In spite of their importance, there is a paucity of published information on their genome organization and structure. In this study, a combination of pulsed field gel electrophoresis (PFGE) and hybridization approaches was used to investigate the genome of L. gasseri neotype strain ATCC33323. PFGE analysis of chromosomal DNA (after digestion with the rare-cutting restriction enzymes I-CeuI, CspI, SmaI, ApaI, and SgrAI) allowed the chromosome size of L. gasseri to be estimated at 1.96 Mbp, and also revealed the presence of a linear plasmid of 48.5 kbp. A physical map of the L. gasseri chromosome, containing 6 sites for the enzymes I-CeuI and 12 for CspI, was constructed. Placed on the map were the genes dnaA and gyrB (usually located close to the origin of replication on the bacterial chromosome) and 18 ribosomal RNA (rrn) genes. Mapping analysis also revealed that the chromosome contained six rrn operons, and that one of them was inverted in orientation with respect to the others. Each rrn operon contained a single copy of each of the three rrn genes, 23S rRNA (rrl), 16S rRNA (rrs) and 55 rRNA (rrf) gene. The constructed physical map should be a useful foundation for genomic and genetic studies of the lactobacilli and provides a platform for applied research, such as the engineering of Lactobacillus strains with improved characteristics for industrial and probiotic applications.

DNA Restriction Enzymes↗
PubMed2002

Feasibility study using a natural compound (reuterin) produced by Lactobacillus reuteri in sterilizing and crosslinking biological tissues.

Bioprostheses derived from biological tissues have to be fixed and subsequently sterilized before they can be implanted in humans. Currently available crosslinking agents and sterilants used in the fixation or sterilization of biological tissues such as glutaraldehyde and formaldehyde are all highly cytotoxic, which may impair the biocompatibility of bioprostheses. Therefore, it is desirable to provide an agent suitable for use in biomedical applications that is of low cytotoxicity and may form sterile and biocompatible crosslinked products. To achieve this goal, a natural compound (reuterin), produced by Lactobacillus reuteri in the presence of glycerol, was used by our group. It is known that reuterin has antibacterial, antimycotic, and antiprotozoal activities. Additionally, as in the case with formaldehyde, reuterin may react with the free amino groups in biological tissues by using its aldehyde functional group. Therefore, it was speculated that reuterin could be used as a crosslinking agent and a sterilant for biological tissues in the same way as glutaraldehyde and formaldehyde. In the study, the production of reuterin, produced by Lactobacillus reuteri under control conditions, was reported. Preparative chromatography was used to purify reuterin. Also, the minimal inhibitory concentration and minimal bactericidal concentration of reuterin and its antimicrobial activity on a contaminated tissue were investigated. In addition, the cytotoxicity of reuterin was evaluated. Glutaraldehyde, the most commonly used sterilant in the sterilization of biological tissues, was employed as a control. Furthermore, the feasibility of using reuterin as a crosslinking agent in fixing biological tissues was studied. Fresh and the glutaraldehyde-fixed tissues were used as controls. The results obtained in the minimal inhibitory concentration and minimal bactericidal concentration studies and in the sterilization study of a contaminated tissue indicated that the antimicrobial activity of reuterin is significantly superior to its glutaraldehyde counterpart. In addition, the results obtained in the 3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide assay showed that reuterin is significantly less cytotoxic than glutaraldehyde. Additionally, it was found that reuterin is an effective crosslinking agent for biological tissue fixation. The reuterin-fixed tissue had comparable free amino group content, denaturation temperature, and resistance against enzymatic degradation as the glutaraldehyde-fixed tissue. In conclusion, the results obtained in this study indicate that reuterin is an effective agent in the sterilization and fixation of biological tissues.

3T3 Cells↗
PubMed1996

Production of hydroxy fatty acid (10-hydroxy-12(Z)-octadecenoic acid) by Lactobacillus plantarum from linoleic acid and its cardiac effects to guinea pig papillary muscles.

Lactobacillus plantarum produced 10-hydroxy-12(Z)-octadecenoic acid (10-OHODA) from linoleic acid. It was suspected that 10-OHODA might be closely related to leukotoxin (9,10-epoxy-12-octa-decenoic acid (LTx)) which was regarded as a toxic and/or defensive substance in living beings. The cardiac effect of 10-OHODA, which was biosynthesized by Lactobacillus plantarum, on an isolated guinea-pig papillary muscle was determined. Its effect on the contractile force of the tissue preparations was examined both in terms of its concentration and the time of exposure. A decrease in muscular tension was observed immediately after administration of 10-OHODA at concentrations of 30, 100, and 300 microM. We also compared the effect of 10-OHODA on muscle tension with that of LTx and its isomer 12,13-epoxy-9-octadecenoic acid (LTx'). Each inhibitory effect on the contractile force of the papillary muscle was statistically significant in the concentration of 300 microM 5 min after administration compared to the control in the absence of each fatty acid, suggesting that 10-OHODA is likely to exert some influence on the circulatory system together with LTx and LTx'.

Animals↗
PubMed1994

Molecular characterization of a plasmid-borne (pGT633) erythromycin resistance determinant (ermGT) from Lactobacillus reuteri 100-63.

Lactobacillus reuteri 100-63 contains a 9.8 kb plasmid (pGT633) encoding resistance to the macrolide-lincosamide-streptogramin (MLS) type B antibiotics. The restriction endonucleases AccI, BamHI, BclI, EcoRI, EcoRV, HhaI, HindII, HpaI, KpnI, SalI, and SspI were employed to establish a physical map of pGT633. Next, genetic transfer experiments were conducted to establish the host range of pGT633. The results revealed that pGT633 replicated autonomously and encoded constitutive resistance to erythromycin in a variety of bacterial hosts, including Bacillus subtilis BD170, Streptococcus sanguis DL1, Staphylococcus aureus RN4220, and Enterococcus faecalis 19433, as well as several Lactobacillus spp. A 2.2 kb BamHI fragment of pGT633 containing the genetic determinant (ermGT) encoding resistance to erythromycin was cloned into Escherichia coli. Determination of the nucleotide sequence of ermGT revealed a methylase gene 731 bp in length that was highly related (ca. 81% nucleotide and ca. 78% amino acid identity) to the ermC gene from S. aureus plasmid pE194. The leader sequences of ermGT and ermC were identical except for a single base change at nt 51.

Amino Acid Sequence↗
PubMed1996

Molecular characterization of a plasmid-borne (pTC82) chloramphenicol resistance determinant (cat-TC) from Lactobacillus reuteri G4.

Lactobacillus reuteri G4 contains a 7.0-kb plasmid (pTC82) encoding resistance to chloramphenicol (Cm). Determination of the nucleotide sequence of the genetic determinant (cat-TC) encoding resistance to Cm on pTC82 revealed an open reading frame for a 238-amino-acid Cm acetyltransferase (CAT) monomer. This structural cat gene, 714 bp in length, was highly related (ca. 95% nucleotide and ca. 81% amino acid identity) to the 648-bp cat gene from Staphylococcus aureus plasmid pC194. A total of 6 bp transversions and 4 bp deletions was observed along the whole DNA sequence of cat-TC compared to that of cat-pC194. To determine the activity of the putative cat-TC gene, recombinant plasmid pUC8217 containing the cat determinant from pTC82 was subjected to a maxicell analysis. The observed molecular mass of the synthesized protein, based on electrophoretic mobility, was in reasonable agreement with the 27.3 kDa predicted from the DNA sequence. This is the first reported nucleotide sequence of a Cm-resistance determinant from L. reuteri and also the first evidence of adding Lactobacillus to the list of versatile bacterial genera which naturally acquire the cat-pC194 gene in the microbial ecological system.

Amino Acid Sequence↗
PubMed1994

Expression of a chitinase gene from Serratia marcescens in Lactococcus lactis and Lactobacillus plantarum.

A chitinase gene from the Gram-negative bacterium Serratia marcescens BJL200 was cloned in Lactococcus lactis subsp. lactis MG1363 and in the silage inoculum strain Lactobacillus plantarum E19b. The chitinase gene was expressed as an active enzyme at a low level in Lactococcus lactis, when cloned in the same transcriptional orientation as the gene specifying the replication protein of the vector pIL253. Using the expression vectors pMG36e and pGKV259 with lactococcal promoter fragments p32 and p59, the expression in L. lactis was increased nine- and 27-fold, respectively. An additional twofold increase was obtained after cloning the gene under the control of p59 in the high-copy number replicon pIL253. In Lactobacillus plantarum, chitinase activity was expressed from p32, and the activity was at the same level as under p32 control in L. lactis.

Amino Acid Sequence↗
PubMed1993

Cloning and sequence analysis of the X-prolyl-dipeptidyl-aminopeptidase gene (pepX) from Lactobacillus delbrückii ssp. lactis DSM7290.

Lactobacillus delbrückii ssp. lactis DSM7290 possesses an X-prolyl-dipeptidyl-aminopeptidase, designated PepX, which catalyses the hydrolytic removal of N-terminal dipeptidyl residues from peptides containing proline in the penultimate position. Using the specific substrate L-Ala-L-Pro-p-nitroanilide, PepX was purified by a four-step procedure including ammonium sulphate fractionation, hydrophobic interaction chromatography, ion exchange chromatography, and affinity chromatography. The N-terminus of the purified protein was sequenced. Screening of a gene library of chromosomal Lactobacillus delbrückii ssp. lactis DSM7290 DNA in the low-copy-number vector pLG339 resulted in the identification of the pepX gene in Escherichia coli using a specific plate assay with Gly-L-Pro-beta-naphthylamide as substrate. Nucleotide sequence analysis revealed an open reading frame of 2376 bp, coding for a protein of 792 amino acids with a molecular mass of 88449 Da.

Amino Acid Sequence↗
PubMed1996

DNA sequence analysis, expression, distribution, and physiological role of the Xaa-prolyldipeptidyl aminopeptidase gene from Lactobacillus helveticus CNRZ32.

Lactobacillus helveticus CNRZ32 possesses an Xaa-prolyldipeptidyl aminopeptidase (PepX), which releases amino-terminal dipeptides from peptides containing proline residues in the penultimate position. The PepX gene, designated pepX, from Lb. helveticus CNRZ32 was sequenced. Analysis of the sequence identified a putative 2379-bp pepX open-reading frame, which encodes a polypeptide of 793 amino acid residues with a deduced molecular mass of 88,111 Da. The gene shows significant sequence identity with sequenced pepX genes from lactic acid bacteria. The product of the gene contains a motif that is almost identical with the active-site motif of the serine-dependent PepX from lactococci. The introduction of pepX into Lactococcus lactis LM0230 on either pGK12 (a low-copy-number plasmid vector) or pIL253 (a high-copy-number plasmid vector) did not result in a significant increase in PepX activity, while the introduction of pepX into CNRZ32 on pGK12 resulted in a four-fold increase in PepX activity. Southern hybridization experiments revealed that the pepX gene from CNRZ32 is well conserved in lactobacilli, pediococci and streptococci. The physiological role of PepX during growth in lactobacillus MRS (a rich medium containing protein hydrolysates along with other ingredients) and milk was examined by comparing growth of CNRZ32 and a CNRZ32 PepX-negative derivative. No difference in growth rate or acid production was observed between CNRZ32 and its PepX-negative derivative in MRS. However, the CNRZ32 PepX-negative derivative grew in milk at a reduced specific growth rate when compared to wild-type CNRZ32. Introduction of the cloned PepX determinant into the CNRZ32 PepX-negative derivative resulted in a construct with a specific growth rate similar to that of wild-type CNRZ32.

Amino Acid Sequence↗
PubMed1993

Gene disruption in Lactobacillus plantarum strain 80 by site-specific recombination: isolation of a mutant strain deficient in conjugated bile salt hydrolase activity.

A chloramphenicol-resistance gene (cml) was introduced into the Lactobacillus plantarum gene encoding conjugated bile acid hydrolase (cbh) on a ColE1 replicon. This plasmid which is nonreplicative in Lactobacillus was used to transform L. plantarum strain 80. A homologous double cross-over recombination event resulted in replacement of the chromosomal cbh gene by the cml-containing cbh gene. The transformants obtained were unable to synthesize active conjugated bile acid hydrolase (Cbh). The Cbh- CmlR phenotype was stably maintained for more than 100 generations under nonselective conditions.

Amidohydrolases↗
PubMed1991

Spontaneous mutations changing the raffinose metabolism of Lactobacillus plantarum.

Lactobacillus plantarum ATCC 8014 grew poorly on raffinose agar plates, but large mutant colonies appeared in high frequency from a thin film of background growth. The alpha-galactosidase and beta-galactosidase activities of L. plantarum ATCC 8014 and a mutant strain were studied in static cultures and pH-controlled fermenter cultures. Both alpha-galactosidase and beta-galactosidase production were inducible in the parental strain; the induction was not needed in the mutant. The alpha-galactosidase activity of both strains was repressed by glucose but not by alpha-methyl-D-glucoside. The mutant phenomenon might be an obstacle in connection to traditional Lactobacillus identification by means of carbohydrate fermentation.

Colony Count, Microbial↗
PubMed1994

Cloning, characterization and insertional inactivation of the Lactobacillus helveticus D(-) lactate dehydrogenase gene.

A plasmid, designated pSUW100, encoding the D(-)lactate dehydrogenase [D(-)-LDH; NAD+ oxidoreductase, EC 1.1.1.28] from Lactobacillus helveticus CNRZ32 was identified from a genomic library by complementation of Escherichia coli FMJ39. The D(-)LDH gene was localized by Tn5 mutagenesis and subcloning to a 1.4-kb region of pSUW100. A 2-kb DraI fragment of pSUW100 encoding D(-)LDH activity was subcloned and its nucleotide sequence determined. Analysis of this sequence identified a putative 1,014-bp D(-)LDH open reading frame that encodes a polypeptide of 337 amino acid residues with a deduced molecular mass of 38 kDa. The distribution of homology to the CNRZ32 D(-)LDH gene in several lactic acid bacteria was determined by Southern hybridization using an internal fragment of the D(-)LDH gene as a probe. Hybridization was detected in leuconostocs and pediococci but not in lactococci or Lactobacillus casei. An integration plasmid was constructed from pSA3 and a 0.60-kb internal fragment of the D(-)LDH gene. This plasmid was used to construct a D(-)LDH-negative derivative of L. helveticus CNRZ 32 by gene disruption; this derivative was determined as producing only L(+)lactic acid. No significant difference in growth or total lactic acid production was observed between CNRZ32 and its D(-)LDH mutant.

Amino Acid Sequence↗
PubMed1997

Lactobacillus species as emerging pathogens in neutropenic patients.

The intensive use of broad-spectrum antibiotics in the context of prolonged and severe neutropenia has contributed to the emergence of unusual pathogens. Four new cases of severe Lactobacillus infections-three of septicemia and one of pneumonia-are reported. They occurred in patients with acute leukemia who had chemotherapy-induced neutropenia. All patients were treated in the same intensive care unit and received the same antimicrobial prophylaxis which included a total bowel decontamination containing vancomycin. The four patients were treated with a combination of intravenous ceftazidime and vancomycin prior to the development of Lactobacillus infection. Improvement in the condition of all patients was obtained with a treatment including penicillin and concurrent recovery of granulopoiesis.

Adult↗
PubMed1997

Assessment of a bacteriocin-producing Lactobacillus strain in the control of spoilage of a cereal-based African fermented food.

As a part of a program to develop starter cultures aiding in the spoilage control and sanitation of African fermented foods, a cereal-based food ('ogi' and its solid form 'agidi' or 'eko') was prepared using a bacteriocin-producing Lactobacillus strain as the starter culture. The survival of an enterotoxigenic Escherichia coli strain was investigated in the naturally fermented food and in food fermented with the starter bacteriocin-producing Lactobacillus strain. An inhibition of E. coli was observed within 2 h of incubation in 'ogi' fermented with the bacteriocin producing strain. After 6 h, the viable count of E. coli in locally fermented 'ogi' was log 6.41 (2.54 X 10(6) CFU/mL), whereas in 'ogi' fermented with the bacteriocin producer it was reduced to log 1.70 (0.5 x 10(2) CFU/mL). Comparison of the shelf life of 'agidi' prepared from the naturally fermented food with that bacteriocin-producing starter culture showed that the latter had a better shelf life (kept for 11 d before spoilage occurred as compared with 7 d for the natural one). The results are discussed in terms of the potential of bacteriocin-producing cultures in the control and retardation of spoilage and food-forne infections in some African fermented foods.

Africa↗
PubMed2003

Production of plantaricin NC8 by Lactobacillus plantarum NC8 is induced in the presence of different types of gram-positive bacteria.

Lactobacillus plantarum NC8 was shown to produce plantaricin NC8 (PLNC8), a recently purified and genetically characterized inducible class IIb bacteriocin, only when it was co-cultured with other gram-positive bacteria. Among 82 strains belonging to the genera Bacillus, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Staphylococcus, and Streptococcus, 41 were shown to induce PLNC8 production in L. plantarum NC8. There was apparently no relationship between the sensitivity of the strains and their ability to induce the bacteriocin, indicating that the inducer and sensitive phenotypes may not be linked. In some instances, induction was promoted by both living and heat-killed cells of the inducing bacteria. However, no PLNC8-inducing activity was found in the respective cell-free, pure culture supernatants. Inducer strains also promoted the production of a PLNC8-autoinducing activity by L. plantarum NC8, which was found only in the cell-free co-culture supernatants showing inhibitory activity. This PLNC8-autoinducing activity was diffusible, heat resistant, and of a proteinaceous nature, and was different from the bacteriocin itself. Taken together, the results suggest that the presence of specific gram-positive bacteria acts as an environmental stimulus activating both PLNC8 production by L. plantarum NC8 and a PLNC8-autoinducing activity, which in turn triggers or maintains bacteriocin production in the absence of inducing cells.

Antibiosis↗
PubMed2006

Quantification of Lactobacillus in fermented milk by multivariate image analysis with least-squares support-vector machines.

This paper reports an approach for quantification of Lactobacillus in fermented milk, grown in a selective medium (MRS agar), by use of digital colour images of Petri plates easily obtained by use of a flatbed scanner. A one-dimensional data vector was formed to characterize each digital image on the basis of the frequency-distribution curves of the red (R), green (G), and blue (B) colour values, and quantities derived from them, for example lightness (L), relative red (RR), relative green (RG), and relative blue (RB). The frequency distributions of hue, saturation, and intensity (HSI) were also calculated and included in the data vector used to describe each image. Multivariate non-linear modelling using the least-squares support vector machine (LS-SVM) and a linear model based on PLS regression were developed to relate the microbiological count and the frequency vector. Feasibly models were developed using the LS-SVM and errors were below than 10% for Lactobacillus quantification, indicating the proposed approach can be used for automatic counting of colonies.

Animals↗
PubMed2003

In vitro evaluation of the fermentation properties of galactooligosaccharides synthesised by alpha-galactosidase from Lactobacillus reuteri.

Stirred, pH-controlled anaerobic batch cultures were used to evaluate the in vitro utilisation by canine gut microflora of novel alpha-galactooligosaccharides synthesised with an enzyme extract from a canine Lactobacillus reuteri strain. Fructooligosaccharides (FOS), melibiose and raffinose were used as reference carbohydrates for the prebiotic properties of the synthesised oligosaccharide (galactosyl melibiose mixture-GMM). Addition of Lactobacillus acidophilus was used as control for the evaluation of the synbiotic properties of the oligosaccharide with L. reuteri. Populations of predominant gut bacterial groups were monitored over 48 h of batch culture by fluorescent in situ hybridisation, and short-chain fatty acid (SCFA) production was measured. GMM showed a higher increase in bifidobacteria and lactobacilli population number and size as well as a higher decrease in clostridia population number and size compared to the commercial prebiotics (FOS, melibiose, raffinose). This prebiotic effect was further increased by the addition of L. reuteri followed by a change in the SCFA production pattern compared to GMM alone or GMM with L. acidophilus. The observed change in SCFA production was in accordance with the fermentation properties of L. reuteri, suggesting that the novel synbiotic had a significant effect on the canine gut microflora fermentation.

Acetic Acid↗