Wide-Inhibitory Spectra Bacteriocins Produced by Lactobacillus gasseri K7

The aim of our study was to determine the genetic characterization and classification of Lb. gasseri K7 bacteriocins, comparison with bacteriocins of the Lb. gasseri LF221 strain and other related strains. Bacteriocin-encoding genes were amplified by PCR, subjected to DNA sequencing, and BLAST sequence analysis was performed to search the database for homologous peptides. Lb. gasseri K7 produces two two-peptide bacteriocins, named gassericin K7 A and gassericin K7 B. Their nucleotide sequences were deposited at GenBank, under accession numbers EF392861 for the gassericin K7 A and AY307382 for the gassericin K7 B. Analysis of gene clusters of bacteriocins in Lb. gasseri K7 strain revealed a 100 percent sequence identity with bacteriocins in LF221 strain. An active peptide of gassericin K7 B is homologous to the complementary peptide of gassericin T, and a complementary peptide of gassericin K7 B is homologous to the active peptide of gassericin T. Another surprising finding was that the sakacin T-beta peptide is partly homologous to the active peptide of gassericin K7 A, while the other sakacin T peptide (alfa) is partly homologous to the complementary peptide of gassericin K7 B. Gassericins of Lb. gasseri K7 strain were both classified as two-peptide bacteriocins. Human probiotic strains Lb. gasseri K7 and LF221 are different isolates but with identical bacteriocin genes. They produce wide-inhibitory spectra bacteriocins that are new members of two-peptide bacteriocins with some homologies to other bacteriocins in this group. Described bacteriocins offer a great potential in applications in food industry, pharmacy and biomedicine.     

Isolation and characterization of a new bacteriocin from Lactobacillus gasseri KT7

A bacteriocin-producing Lactobacillus gasseri strain, KT7, was isolated from infant faeces. The supernatant fluid showed inhibitory activity not only against some lactic acid bacteria but also, against some pathogenic and food-spoilage species, including Clostridium, Listeria and Enterococcus. An antimicrobial peptide designated gassericin KT7 was isolated from Lactobacillus gasseri KT7. It was purified to homogeneity by a single four-step procedure: a crude supernatant fluid obtained from early stationary-phase culture in MRS medium was subjected to ammonium sulphate fractionation, CM-Sephadex cation-exchange chromatography, Phenyl-Sepharose hydrophobic chromatography and reverse-phase HPLC chromatography. Gassericin KT7 was sensitive to proteolytic enzymes, resistant to heat, active over a wide range of pH, and migrated as a 4.5-5.0 kDa peptide on SDS-PAGE. The bacteriocin was produced constitutively during exponential growth. It was bactericidal to sensitive cells and the bactericidal effect was not produced by cell lysis. The amino acid composition of the bacteriocin was determined and no modified amino acid was found among the residues identified.     

Detection of antimicrobial activities and bacteriocin structural genes in faecal enterococci of wild animals

The production of antimicrobial activities as well as the presence of bacteriocin structural genes (entA, entB, entP, entQ, cylL, entAS-48, bac31, and entL50A/B) were studied in 140 non-selected faecal enterococcal isolates recovered from wild animals. Eight different indicator strains (including Listeria monocytogenes, Pediococcus pentosaceus, and different enterococcal species) were used for antimicrobial activity detection. Twenty-five of the 140 enterococci (18%) showed antimicrobial activity against L. monocytogenes and 33 additional isolates (24%) showed antimicrobial activity against other indicator strains, but Listeria. At least one bacteriocin structural gene was detected in 17 of the 25 enterococci with antimicrobial activity against L. monocytogenes and different combinations of entA, entB, entP, entQ, entL50A/B, and cylL genes were detected; entA and entB were the most prevalent detected genes, and they were generally associated. Bacteriocin structural genes were detected in 10 of 33 isolates with antimicrobial activity against indicator strains other than Listeria, and the cylL gene was the most prevalent one, especially in E. faecalis isolates.     

Role of the probiotic strain Lactobacillus paracasei LMGP22043 carried by artichokes in influencing faecal bacteria and biochemical parameters in human subjects

Aims: To evaluate the positive influence of the probiotic strain Lactobacillus paracasei LMGP22043 carried by artichokes into the human gut with special reference to faecal bacterial balance, short‐chain fatty acid concentrations and enzyme activities in a randomized, double‐blind human trial in comparison with probiotic‐free artichokes (control). Methods: Twenty subjects were randomized into two groups, which consumed daily 180 g of the artichoke product (probiotic or control) during two 15‐day study periods (periods 1 and 2) separated by a 15‐day washout in a crossover manner. Faecal samples were subjected to microbiological and biochemical analyses, and a strain‐specific PCR was performed to monitor the probiotic strain. Results: The probiotic strain, transported by the vegetable matrix, transiently colonized the gut of 17/20 subjects (median 6·87 log CFU g^?1 faeces), antagonized Escherichia coli and Clostridium spp. and increased the genetic diversity of lactic population based on REP‐PCR profiles, mainly after period 1. Conclusions: The probiotic L. paracasei LMGP22043 successfully colonized the human gut and positively influenced faecal bacteria and biochemical parameters. Significance and Impact of the Study: The association of the probiotic L. paracasei with a food carrier rich in fibre can represent a new strategy for favouring a daily supply of probiotics and attracting more consumers to vegetable food fortified with probiotic strains.     

植物乳杆菌R260产细菌素发酵条件的研究

目的 获取植物乳杆菌R260产细菌素的最佳发酵条件.方法用琼脂扩散法测定发酵液对苏云金芽胞杆菌的抑菌效价.结果 产细菌素的最佳培养基是MRS培养基,最适起始Ph为6.5,最适接种量和接种种龄分别为3%和12 h,产细菌素最适发酵温度和时间分别为30℃和20 h：细菌素在对数期开始产生,稳定期产量达到最大值.结论 通过优化发酵条件提高了细菌素的产量,达1656 IU/ml.     

Detection of Lactobacillus gasseri OLL2716 strain administered with yogurt drink in gastric mucus layer in humans

In animal models and human trials, Lactobacillus gasseri OLL2716 (LG21) strain suppressed Helicobacter pylori colonization in the stomach. The aim of the present study was to clarify whether orally administered LG21 strain can enter the gastric mucus layer. Biopsy samples were taken from the gastric antrum and corpus of two healthy volunteers (H. pylori infected and non-infected) who drank yogurt supplemented with LG21 strains. DNA of LG21 and H. pylori in the mucus layer was detected using the laser-assisted microdissection and non-contact pressure catapulting (LMPC) method and the semi-nested PCR method with primer sets of RNA helicases of superfamily II gene-Insertion sequence for LG21 strain and those of ureA gene for H. pylori. In the volunteer with H. pylori infection, DNA fragments of LG21- and H. pylori-specific regions from both antrum and corpus were amplified, whereas in a non-infected volunteer, only the LG21 DNA from the antrum was amplified. The present study demonstrated that LG21 strains administered through a yogurt drink can enter into the gastric mucus layer. Our novel method may be useful in studying gastric probiotics for H. pylori infection.     

Primary amino acid and DNA sequences of gassericin T, a lactacin F-family bacteriocin produced by Lactobacillus gasseri SBT2055

A broad-spectral bacteriocin, named gassericin T, produced by Lactobacillus gasseri SBT 2055 (from human feces) was isolated to homogeneity from the culture supernatant by hydrophobic chromatography. By SDS-PAGE and in situ activity assay, the purified gassericin T migrated as a single band with bacteriocin activity and molecular size of 5,400. A 2.9-kbp HindIII-HindIII fragment of chromosome DNA was hybridized with the oligonucleotide probe designed from the partial N-terminal amino acid sequence of gassericin T and was cloned. Six ORFs including the structural gene of gassericin T were deduced by computer analysis and the data bases. The structural gene of gassericin T (gatA) was identified as the fourth ORF, which encoded a protein composed of 75 amino acids that included the GG motif of the cleavage site. Chemical sequencing analysis of the complete amino acid sequence showed that gassericin T (57 amino acids) had a disulfide bond in the molecule and no modified amino acid residues, making it a class II bacteriocin. The gassericin T had 60% sequence similarity to mature LafA (57 amino acids, lactacin F, bacteriocins produced by L. johnsonii VPI11088), and the sequences around the processing site and C-terminal area were well conserved. The fifth ORF was designated as gatX, encoded as a peptide composed of 65 amino acids containing the GG motif of the putative cleavage site, however mature GatX and its antibacterial activity were not detected in the culture supernatant. GatX has higher similarity with LafX than with lactobin A (50 amino acids) belonging to the first lactacin F-family. These results indicated that gassericin T belongs to the hydrophobic class II bacteriocins and the most vicinal lactacin F-family.     

嗜酸乳杆菌SR-1产类细菌素发酵条件及生物学特性的研究

本研究通过正交试验获得嗜酸乳杆菌sR-1分泌类细菌素的最适培养条件,即葡萄搪2％,大豆蛋白胨2．5％,酵母粉0．4％,血浆蛋白2．5％。并首次利用流加培养的方式,改进了类细菌素产生菌的发酵条件,抑菌直径从19mm提高到25mm,同时对类细菌素生物学特性进行了初步的探索。结果表明：嗜酸乳杆菌产生的类细菌素对多种蛋白酶不敏感,在低pH下能抑制革兰阴性、阳性的致病菌,在pH6．5下吸附菌体作用最强。     

Detection and quantification of Bifidobacterium lactis LAFTI B94 in human faecal samples from a consumption trial

A method was developed to allow detection of the probiotic Bifidobacterium lactis LAFTI B94 in human clinical samples. A new probe, Laf94p, was developed to accomplish colony hybridization of B. lactis B94. PCR detection of B94 was also achieved using the species-specific (B. lactis) primer pair. These tests and probes allowed detection and quantification of B94 in the human intestinal flora. The sensitivity of the probe was assessed by monitoring faecal levels of B94 in humans who were fed the culture. In this trial, five volunteers were fed with the probiotic. The presence of B94 was assessed daily. Viable B94 could be detected at high levels (as high as 1.8 x 10(9) cfu g(-1) wet weight) during the feeding period. Four weeks after the feeding stopped, B94 could still be detected in one subject. These results indicate that B94 survives in the human gastrointestinal tract.     

Detection and activity of lactacin B, a bacteriocin produced by Lactobacillus acidophilus

A total of 52 strains of Lactobacillus acidophilus were examined for production of bacteriocins. A majority (63%) demonstrated inhibitory activity against all members of a four-species grouping of Lactobacillus leichmannii, Lactobacillus bulgaricus, Lactobacillus helveticus, and Lactobacillus lactis. Four L. acidophilus strains with this activity also inhibited Streptococcus faecalis and Lactobacillus fermentum, suggesting a second system of antagonism. Under conditions eliminating the effects of organic acids and hydrogen peroxide, no inhibition of other gram-positive or -negative genera was demonstrated by L. acidophilus. The agent produced by L. acidophilus N2 and responsible for inhibition of L. leichmannii, L. bulgaricus, L. helveticus, and L. lactis was investigated. Ultrafiltration studies indicated a molecular weight of approximately 100,000 for the crude inhibitor. The agent was sensitive to proteolytic enzymes and retained full activity after 60 min at 100 degrees C (pH 5). Activity against sensitive cells was bactericidal but not bacteriolytic. These characteristics identified the inhibitory agent as a bacteriocin, designated lactacin B. Examination of strains of L. acidophilus within the six homology groupings of Johnson et al. (Int. J. Syst. Bacteriol. 30:53-68, 1980) demonstrated that production of the bacteriocin lactacin B could not be used in classification of neotype L. acidophilus strains. However, the usefulness of employing sensitivity to lactacin B in classification of dairy lactobacilli is suggested.     

16S ribosomal DNA analysis of the faecal lactobacilli composition of human subjects consuming a probiotic strain Lactobacillus acidophilus NCFM

AIMS: The aims of this study were to evaluate the ability of exogenous Lactobacillus acidophilus strain NCFM to survive through the human gastro-intestinal (GI) tract, and to evaluate the selectivity of Rogosa SL medium for faecal lactobacilli. METHODS AND RESULTS: The composition of the faecal lactobacilli of 10 healthy subjects was monitored for two weeks prior to, two weeks during and two weeks after the administration of the Lact. acidophilus strain NCFM consumed with skim milk (daily dose 10(10) viable cells). Fresh faecal samples were collected, processed and cultured on Rogosa SL selective medium for lactobacilli enumeration. Colonies demonstrating various morphologies were identified and purified for 16S ribosomal DNA sequence analysis for speciation of colonial genotype. The species composition of cultivable faecal lactobacilli changed considerably during consumption of the strain NCFM. CONCLUSIONS: The probiotic Lact. acidophilus strain NCFM can survive through the human GI tract, but cannot colonize itself during the two-week consumption. Rogosa SL medium is selective for faecal lactobacilli. However, genetic analysis is required for colony speciation. SIGNIFICANCE AND IMPACT OF THE STUDY: It is demonstrated that continuous consumption is necessary to maintain a high population of the probiotic strain, and that the Rogosa SL medium is reliable.     

Quantitative strain-specific detection of Lactobacillus rhamnosus GG in human faecal samples by real-time PCR

Aims:  To develop a strain-specific rapid assay for identification and quantification of Lactobacillus rhamnosus GG in human faecal samples.
Methods and Results:  A unique random amplified polymorphic DNA (RAPD) band of the L. rhamnosus GG strain was isolated and sequenced. Strain-specific polymerase chain reaction (PCR) primers and probes were designed based on the sequence. Quantification was performed by the real-time PCR using a fluorescent resonance energy transfer (FRET) system. The specificity of the assay was tested with DNA isolated from a set of known strains and human faecal samples. The analytical sensitivity of the method for L. rhamnosus GG was about 10 CFU per assay, which corresponds to 10⁵ CFU g⁻¹ of wet faeces.
Conclusions:  Quantitative real-time PCR is a suitable method for strain-specific identification of L. rhamnosus GG in human faecal samples.
Significance and Impact of the Study:  Lactobacillus rhamnosus GG is one of the most studied probiotic strains in clinical trials but still lacks a DNA-based identification method. This study describes a real-time PCR method for strain-specific identification and quantification of L. rhamnosus GG in human faecal samples.     

Detection and quantification of Escherichia coli O157:H7 in environmental samples by real-time PCR

AIMS: To apply the real-time Polymerase chain reaction (PCR) method to detect and quantify Escherichia coli O157:H7 in soil, manure, faeces and dairy waste washwater. METHODS AND RESULTS: Soil samples were spiked with E. coli O157:H7 and subjected to a single enrichment step prior to multiplex PCR. Other environmental samples suspected of harbouring E.coli O157:H7 were also analysed. The sensitivity of the primers was confirmed with DNA from E.coli O157:H7 strain 3081 spiked into soil by multiplex PCR assay. A linear relationship was measured between the fluorescence threshold cycle (C T ) value and colony counts (CFU ml(-1)) in spiked soil and other environmental samples. The detection limit for E.coli O157:H7 in the real-time PCR assay was 3.5 x 10(3) CFU ml(-1) in pure culture and 2.6 x 10(4) CFU g(-1) in the environmental samples. Use of a 16-h enrichment step for spiked samples enabled detection of <10 CFU g(-1) soil. E. coli colony counts as determined by the real-time PCR assay, were in the range of 2.0 x 10(2) to 6.0 x 10(5) CFU PCR (-1) in manure, faeces and waste washwater. CONCLUSIONS: The real-time PCR-based assay enabled sensitive and rapid quantification of E. coli O157:H7 in soil and other environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to quantitatively determine cell counts of E.coli O157:H7 in large numbers of environmental samples, represents considerable advancement in the area of pathogen quantification for risk assessment and transport studies.     

Selection of a potential probiotic Lactobacillus strain and subsequent in vivo studies

The probiotic potential of a Lactobacillus strain, isolated from pig faeces, was assessed as a probiotic in piglets. The strain was examined for resistance to pH 2.0, 0.5% oxgall and antibiotics, and antimicrobial activities against enteric pathogenic bacteria. The probiotic strain, L. reuteri BSA131, was administered through the feed to 25 1-month-old Landrace piglets. The piglets were divided into five groups of five piglets each and fed with different diets for 28 days. The daily consumption of L. reuteriBSA131 was assigned into two groups by the concentration of 10⁶ or 10⁸ freeze-dried bacteria. Fecal samples were collected before, during, and after consumption. Lactobacilli and enterobacteria cell counts were determined in the fecal samples. The liveweight gains and feed consumption of the piglets were recorded daily. This study showed that strain BSA 131 enhanced liveweight gains and feed conversion rates in piglets. It also showed a significant increase in lactobacilli cell counts and decreases in enterobacterial numbers in the fecal samples. Strain BSA 131 was considered to be a potential probiotic for piglets, especially after weaning.     

Optimization of the freeze-drying media and survival throughout storage of freeze-dried Lactobacillus gasseri and Lactobacillus delbrueckii subsp. delbrueckii for veterinarian probiotic applications

The use of lactic acid bacteria (LAB) as vaginal probiotic cultures depends on the preservation technologies employed by the related industries.A full two-factor analysis of variance (ANOVA), considering medium and strain, of the decrease in bacterial viability during freeze-drying was applied. Lactobacillus gasseri CRL1421 was significantly more resistant than L. gasseri CRL1412 to the process. L. gasseri CRL1412 suspended in skim milk showed a significantly higher resistance than when it was suspended in water, but lactose or sucrose did not significantly increase its viability after lyophilization. Lactobacillus delbrueckii subsp. delbrueckii CRL1461, an autoaggregative strain, was significantly more sensitive to freeze-drying under the assessed conditions.The dried cultures were included in two pharmaceutical forms and viability was monitored during 270 days of storage. Although the microorganisms studied belonged to the same species, the optimal storage conditions were different for each of them.Our results can be applied to the design of a veterinarian probiotic to prevent metritis in diary postpartum cows.     

Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92

AIMS: To investigate aggregation and adhesiveness of Lactobacillus acidophilus M92 to porcine ileal epithelial cells in vitro, and the influence of cell surface proteins on autoaggregation and adhesiveness of this strain. METHODS AND RESULTS: Lactobacillus acidophilus M92 exhibits a strong autoaggregating phenotype and manifests a high degree of hydrophobicity determined by microbial adhesion to xylene. Aggregation and hydrophobicity were abolished upon exposure of the cells to pronase and pepsin. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of cell surface proteins revealed the presence of potential surface layer (S-layer) proteins, approximated at 45 kDa, in L. acidophilus M92. The relationship between autoaggregation and adhesiveness to intestinal tissue was investigated by observing the adhesiveness of L. acidophilus M92 to porcine ileal epithelial cells. Removal of the S-layer proteins by extraction with 5 mol l-1 LiCl reduced autoaggregation and in vitro adhesion of this strain. CONCLUSIONS: These results demonstrate that there is relationship between autoaggregation and adhesiveness ability of L. acidophilus M92, mediated by proteinaceous components on the cell surface. SIGNIFICANCE AND IMPACT OF THE STUDY: This investigation has shown that L. acidophilus M92 has the ability to establish in the human gastrointestinal tract, which is an important determinant in the choice of probiotic strains.     

Evaluation of the passage of Lactobacillus gasseri K7 and bifidobacteria from the stomach to intestines using a single reactor model

Background

The antibacterial activity of bacteriophages has been described rather well. However, knowledge about the direct interactions of bacteriophages with mammalian organisms and their other, i.e. non-antibacterial, activities in mammalian systems is quite scarce. It must be emphasised that bacteriophages are natural parasites of bacteria, which in turn are parasites or symbionts of mammals (including humans). Bacteriophages are constantly present in mammalian bodies and the environment in great amounts. On the other hand, the perspective of the possible use of bacteriophage preparations for antibacterial therapies in cancer patients generates a substantial need to investigate the effects of phages on cancer processes.

Results

In these studies the migration of human and mouse melanoma on fibronectin was inhibited by purified T4 and HAP1 bacteriophage preparations. The migration of human melanoma was also inhibited by the HAP1 phage preparation on matrigel. No response of either melanoma cell line to lipopolysaccharide was observed. Therefore the effect of the phage preparations cannot be attributed to lipopolysaccharide. No differences in the effects of T4 and HAP1 on melanoma migration were observed.

Conclusion

We believe that these observations are of importance for any further attempts to use bacteriophage preparations in antibacterial treatment. The risk of antibiotic-resistant hospital infections strongly affects cancer patients and these results suggest the possibility of beneficial phage treatment. We also believe that they will contribute to the general understanding of bacteriophage biology, as bacteriophages, extremely ubiquitous entities, are in permanent contact with human organisms.