Studies on Streptococci

The population levels of bacteria in the contents and the walls of the gastrointestinal tract of gnotobiotic rats inoculated with lactic acid bacteria (streptococci, lactobacilli, and bifidobacteria) from humans and rats were determined. Lactobacilli as well as streptococci isolated from rats colonized in the digestive tracts of the gnotobiotic rats at a high population level, characteristically highest in the stomach. On the other hand, in the rats inoculated with human lactic acid bacteria, streptococci were dominant in the lower tract. The human lactobacilli or bifidobacteria did not colonize when the organisms in each genus were inoculated together with streptococci. However, when all three genera were inoculated together, lactobacilli and bifidobacteria colonized. Observations on the species of streptococci showed that the intestinal type of streptococci was found to colonize at a high population level, but the oral type was not. Strains of the same genus of lactic acid bacteria from humans and from rats showed different colonization patterns.     

Diversity, vitality and activities of intestinal lactic acid bacteria and bifidobacteria assessed by molecular approaches

While lactic acid bacteria and bifidobacteria have been scientifically important for over a century, many of these are marketed today as probiotics and have become a valuable and rapidly expanding sector of the food market that is leading functional foods in many countries. The human gastro-intestinal tract with its various compartments and complex microbiota is the primary target of most of these functional foods containing lactic acid bacteria and bifidobacteria (LAB&B). In addition, their use as vectors for delivery of molecules with therapeutic value to the host via the intestinal tract is being studied. This review focuses on molecular approaches for the investigation of the diversity of lactic acid bacteria and bifidobacteria in the human intestine, as well as tracking of probiotic bacteria within this complex ecosystem. Moreover, methodologies to determine the viability of the lactic acid bacteria and bifidobacteria and molecular approaches to study the mechanisms by which they adapt, establish and interact with the human host via the digestive tract, are described.     

In vitro fermentation of rice bran combined with Lactobacillus acidophilus 14 150B or Bifidobacterium longum 05 by the canine faecal microbiota

The fermentability of rice bran (RB), alone or in combination with one of two probiotics, by canine faecal microbiota was evaluated in stirred, pH-controlled, anaerobic batch cultures. RB enhanced the levels of bacteria detected by probes Bif164 (bifidobacteria) and Lab158 (lactic acid bacteria); however, addition of the probiotics did not have a significant effect on the predominant microbial counts compared with RB alone. RB sustained levels of Bifidobacterium longum 05 throughout the fermentation; in contrast, Lactobacillus acidophilus 14 150B levels decreased significantly after 5-h fermentation. RB fermentation induced changes in the short-chain fatty acid (SCFA) profile. However, RB combined with probiotics did not alter the SCFA levels compared with RB alone. Denaturing gradient gel electrophoresis analysis of samples obtained at 24 h showed a treatment effect with RB, which was not observed in the RB plus probiotic systems. Overall, the negative controls displayed lower species richness than the treatment systems and their banding profiles were distinct. This study illustrates the ability of a common ingredient found in pet food to modulate the canine faecal microbiota and highlights that RB may be an economical alternative to prebiotics for use in dog food.     

The viability of bifidobacteria introduced into kimchi

The viability of bifidobacteria in mul-kimchi, a type of kimchi with added water, was investigated under various conditions. When a mul-kimchi preparation was inoculated with five strains of Bifidobacterium at a concentration of 10(7) cfu ml-1, Bif. longum JK-2 showed the highest viability, maintaining a population of 10(6) cfu ml-1 after 1 week at 4 degrees C. The influence of NaCl concentration and initial pH on viability was further investigated in mul-kimchi inoculated with Bif. longum JK-2; NaCl concentrations greater than 3% (w/w) reduced viability considerably. In kimchi started with an initial pH of 6.5, the cells showed the highest survival. When mul-kimchi containing 2% NaCl (w/w) was inoculated with 10(8) cfu ml-1 Bif. longum JK-2, there was a 10-fold reduction in viability during 10 d of incubation at 4 degrees C. These results demonstrate acceptable levels of the organism in the product, suggesting the possible use of selected strains of bifidobacteria in commercial kimchi production.     

Molecular profiling of Lactobacillus, Streptococcus, and Bifidobacterium species in feces of active racehorses

Diversity and compositions of the Lactobacillus, Streptococcus, and Bifidobacterium group in the feces of six healthy, actively racing horses (Thoroughbreds) were analyzed by using PCR-denaturing gradient gel electrophoresis (DGGE) and real-time PCR with primer sets specific for each group. PCR-DGGE analysis of the feces showed that Lactobacillus equi, Lactobacillus johnsonii, a phylogenetic relative of Lactobacillus salivarius, a phylogenetic relative of Lactobacillus gastricus, and Weissella confusa were predominant in almost all of the feces tested, and Streptococcus bovis/Streptococcus equinus was predominant in the Streptococcus group. The Bifidobacterium group was not detected by single-PCR but atypical species of the group were found in three of the six Thoroughbreds tested by nested-PCR. Calculation and estimation of lactic acid bacteria and bifidobacteria revealed that lactic acid bacteria were predominant in the feces and bifidobacteria were minor. These results indicate that the community of lactic acid bacteria and bifidobacteria in horse feces are unique because of the presence of specific species for horse feces and a minority of the Bifidobacterium group. Repeated tests of the feces from the same horse over 3 months showed that the diversity and composition of lactic acid bacteria and bifidobacteria in the feces was basically stable throughout the test period.     

The Effect of Lactic Acid Sprays on Campylobacter jejuni Inoculated onto Poultry Carcasses

Spraying poultry carcasses with 1 % lactic acid 10 min after inoculation with Campylobacter jejuni, resulted in a significant reduction in the number of the bacteria after 4 h at 4°C. Some of the inoculated cells, however, survived for at least 144 h. Spraying 10 min after inoculation with 2% lactic acid, totally eliminated all inoculated C. jejuni within 24 h. On the other hand, spraying 24 h after inoculation, with either 1 % or 2 % lactic acid did not eliminate all the bacteria. Inoculated C. jejuni on poultry carcasses not sprayed with lactic acid, survived at 4°C throughout the sampling period (up to 144 h) and showed little tendency to decrease in number even when the carcasses started to deteriorate. Resident Campylobacters on poultry carcasses were significantly reduced by the lactic acid treatment. Frozen and thawed chickens appeared to show a graying of the skins immediately after spraying with lactic acid, slightly stronger with 2 % lactic acid, but the colour reverted to normal after 24 h. We were not able to observe any colour change on the fresh broiler chickens after lactic acid treatment. Our results indicated that lactic acid had a significant bactericidal effect on C. jejuni on both naturally and artificially contaminated poultry carcasses. This effect, however, became manifest only several hours after acid treatment.     

Immunoregulatory mechanisms underlying prevention of colitis-associated colorectal cancer by probiotic bacteria

Background

Inflammatory bowel disease (IBD) increases the risk of colorectal cancer. Probiotic bacteria produce immunoregulatory metabolites in vitro such as conjugated linoleic acid (CLA), a polyunsaturated fatty acid with potent anti-carcinogenic effects. This study aimed to investigate the cellular and molecular mechanisms underlying the efficacy of probiotic bacteria in mouse models of cancer.

Methodology/Principal Findings

The immune modulatory mechanisms of VSL#3 probiotic bacteria and CLA were investigated in mouse models of inflammation-driven colorectal cancer. Colonic specimens were collected for histopathology, gene expression and flow cytometry analyses. Immune cell subsets in the mesenteric lymph nodes (MLN), spleen and colonic lamina propria lymphocytes (LPL) were phenotypically and functionally characterized. Mice treated with CLA or VSL#3 recovered faster from the acute inflammatory phase of disease and had lower disease severity in the chronic, tumor-bearing phase of disease. Adenoma and adenocarcinoma formation was also diminished by both treatments. VSL#3 increased the mRNA expression of TNF-α, angiostatin and PPAR γ whereas CLA decreased COX-2 levels. Moreover, VSL#3-treated mice had increased IL-17 expression in MLN CD4+ T cells and accumulation of Treg LPL and memory CD4+ T cells.

Conclusions/Significance

Both CLA and VSL#3 suppressed colon carcinogenesis, although VSL#3 showed greater anti-carcinogenic and anti-inflammatory activities than CLA. Mechanistically, CLA modulated expression of COX-2 levels in the colonic mucosa, whereas VSL#3 targeted regulatory mucosal CD4+ T cell responses.     

Probiotic bacteria produce conjugated linoleic acid locally in the gut that targets macrophage PPAR γ to suppress colitis

Background

Inflammatory bowel disease (IBD) therapies are modestly successful and associated with significant side effects. Thus, the investigation of novel approaches to prevent colitis is important. Probiotic bacteria can produce immunoregulatory metabolites in vitro such as conjugated linoleic acid (CLA), a polyunsaturated fatty acid with potent anti-inflammatory effects. This study aimed to investigate the cellular and molecular mechanisms underlying the anti-inflammatory efficacy of probiotic bacteria using a mouse model of colitis.

Methodology/Principal Findings

The immune modulatory mechanisms of VSL#3 probiotic bacteria and CLA were investigated in a mouse model of DSS colitis. Colonic specimens were collected for histopathology, gene expression and flow cytometry analyses. Immune cell subsets in the mesenteric lymph nodes (MLN), spleen, blood and colonic lamina propria cells were phenotypically and functionally characterized. Fecal samples and colonic contents were collected to determine the effect of VSL#3 and CLA on gut microbial diversity and CLA production. CLA and VSL#3 treatment ameliorated colitis and decreased colonic bacterial diversity, a finding that correlated with decreased gut pathology. Colonic CLA concentrations were increased in response to probiotic bacterial treatment, but without systemic distribution in blood. VSL#3 and CLA decreased macrophage accumulation in the MLN of mice with DSS colitis. The loss of PPAR γ in myeloid cells abrogated the protective effect of probiotic bacteria and CLA in mice with DSS colitis.

Conclusions/Significance

Probiotic bacteria modulate gut microbial diversity and favor local production of CLA in the colon that targets myeloid cell PPAR γ to suppress colitis.     

Production of hydrolases by lactic acid bacteria and bifidobacteria and their antibiotic resistance

It was demonstrated that bifidobacteria and lactic acid bacteria B. adolescentis and Lactobacillus sp. synthesized extracellular enzymes cleaving glycoside bonds in the molecules of dextran, pectic acid, and soluble starch. The maximal production of extracellular beta-galactosidase by B. adolescentis 91-BIM and 94-BIM at a rate of 0.08 and 0.03 U/mg h was observed during the exponential growth phase at 5 and 12 h of cultivation, respectively. The cultures of bifidobacteria retained 60-70% of beta-galactosidase and alpha-amylase activities after six months of storage. The bifidobacterium strains studied were resistant to amphotericin and aminoglycosides (gentamicin, kanamycin, and netromycin). The lactam antibiotics (ampicillin, benzylpenicillin, bicillin 3, bicillin 5, and carbenicillin), the preparations inhibiting protein synthesis at the level of ribosomes (lincomycin), RNA polymerase inhibitors (rifampin), cephalosporin, and Maxipime inhibited the growth of bifidobacteria. Rifampin, erythromycin, amphotericin, Maxipime, Fortum, doxycycline, levomycetin, streptomycin, and the aminoglycosides netromycin, gentamicin, and kanamycin did not have an effect on the growth of Lactobacillus sp., whereas semisynthetic derivatives of penicillin, carbenicillin and ampicillin, inhibited its growth as well as Oxamp and lincomycin. The lactam antibiotics benzylpenicillin, bicillin 3, and bicillin 5 inhibited the growth of lactic acid bacilli by 30-90%.     

The survival of Listeria monocytogenes in cottage cheese

Because of the difficulty of ensuring that cottage cheese is produced in conditions that prevent contamination with Listeria monocytogenes, the ability of this bacterium to survive in cottage cheese from three sources was investigated (a) during shelf-life at chill temperature and (b) in conditions of temperature abuse. Three batches of creamed cottage cheese, from three sources, received within 24 h of production, were inoculated with L. monocytogenes strain F6861 and stored at 4, 8 or 12 degrees C for 14 d. The three batches differed in their initial pH, titratable acidity and content of lactic acid and of lactic acid bacteria. No increase in numbers of L. monocytogenes occurred in the cottage cheeses during storage in these conditions. The numbers of listeria decreased; the rate of decrease differed in products from the three sources and was least in the product with the highest pH and lowest content of lactic acid. Acid formation by lactic acid bacteria during storage of the products probably contributed to the inhibition of listeria.     

The survival of Listeria monocytogenes in cottage cheese

Because of the difficulty of ensuring that cottage cheese is produced in conditions that prevent contamination with Listeria monocytogenes , the ability of this bacterium to survive in cottage cheese from three sources was investigated (a) during shelf-life at chill temperature and (b) in conditions of temperature abuse. Three batches of creamed cottage cheese, from three sources, received within 24 h of production, were inoculated with L. monocytogenes strain F6861 and stored at 4, 8 or 12°C for 14 d. The three batches differed in their initial pH, titratable acidity and content of lactic acid and of lactic acid bacteria. No increase in numbers of L. monocytogenes occurred in the cottage cheeses during storage in these conditions. The numbers of listeria decreased; the rate of decrease differed in products from the three sources and was least in the product with the highest pH and lowest content of lactic acid. Acid formation by lactic acid bacteria during storage of the products probably contributed to the inhibition of listeria.     

Genetic and structural comparison of linoleate isomerases from selected food‐grade bacteria

Aims: To advance the understanding of the molecular mechanisms underlying the capacity of bifidobacteria and lactic bacteria to convert linoleic acid (LA) into conjugated linoleic acid (CLA) by the linoleate isomerase (LI). Methods and Results: The potential LI enzymes of selected Lactobacillus, Bifidobacterium and Leuconostoc strains were compared at the genetic, amino acid sequence and functional levels. Genetic analysis was achieved by insertional mutagenesis and hybridization studies using a Lact. reuteri LI probe. Biotransformation studies monitored by gas chromatography showed that Bif. breve is a major CLA producer after the reference Lact. reuteri strain. The putative Bif. breve LI gene was PCR isolated and sequenced. Conclusions: The putative LI gene identified in this study seems essential for bacterial growth. Comparative studies indicate that the deduced protein is membrane bound and reveal the presence of several highly conserved domains among a wide range of Gram‐positive bacteria. Significance and Impact of the Study: Given the multiple health benefits of CLA, the capability of some bacteria to convert LA into CLA is of great relevance. Nevertheless, the yields of CLA remain low, and the regulation of the process is far from being understood. A deeper knowledge of this capacity by the genetic studies is revealing the identity of the LI and will eventually contribute to its control.     

Microbiological evaluation of jejunal aspirates and faecal samples after oral administration of bifidobacteria and lactic acid bacteria

A double-blind placebo controlled investigation was carried out to study the effect of peroral colonization. Human volunteers were given mixtures of bifidobacteria and lactic acid bacteria. Measurements were made over a 1 week treatment period and for another week after the end of the treatment. Two different bacteriological preparations were used, one consisted of Enterococcus faecium and Bifidobacterium longum (a total of 6.4 times 10⁸ cfu d^-1); the other consisted of Lactobacillus acidophilus, Bif. bifidum, Lact. delbrueckii ssp. bulgaricus , and Streptococcus thermophilus (a total of 9 times 10⁹ cfu d^-1). Together with a placebo preparation, they were given to 24 healthy controls (eight in each group). Microbiological examinations of jejunal aspirates showed that viable counts of most species were below the detection limit. However, the test preparation containing Ent. faecium and Bif. longum significantly reduced the anaerobe: aerobe ratio in faeces by a factor of three during treatment ( P = 0.03), and increased it by a factor of 30 during the following week ( P <0.02>). This study shows that peroral administration of certain bacterial cultures may affect the distal intestinal microflora.     

The Probiotic Mixture VSL#3 Accelerates Gastric Ulcer Healing by Stimulating Vascular Endothelial Growth Factor

Studies assessing the effect and mechanism of probiotics on diseases of the upper gastrointestinal tract (GI) including gastric ulcers are limited despite extensive work and promising results of this therapeutic option for other GI diseases. In this study, we investigated the mechanisms by which the probiotic mixture VSL#3 (a mixture of eight probiotic bacteria including Lactobacilli, Bifidobacteria and Streptococcus species) heals acetic acid induced gastric ulcer in rats. VSL#3 was administered orally at low (6×10⁹ bacteria) or high (1.2×10¹⁰ bacteria) dosages from day 3 after ulcer induction for 14 consecutive days. VSL#3 treatments significantly enhanced gastric ulcer healing in a dose-dependent manner. To assess the mechanism(s) whereby VSL#3 exerted its protective effects, we quantified the gene expression of several pro-inflammatory cytokines, protein and expression of stomach mucin-Muc5ac, regulatory cytokine-IL-10, COX-2 and various growth factors. Of all the components examined, only expression and protein production of VEGF was increased 332-fold on day 7 in the ulcerated tissues of animals treated with VSL#3. Predictably, animals treated with VEGF neutralizing antibody significantly delayed gastric ulcer healing in VSL#3 treated animals. This is the first report to demonstrate high efficacy of the probiotic mixture VSL#3 in enhancing gastric ulcer healing. Probiotic efficacy was effective at higher concentrations of VSL#3 by specifically increasing the expression and production of angiogenesis promoting growth factors, primarily VEGF.     

Effects of lactose on colon microbial community structure and function in a four-stage semi-continuous culture system

A semi-continuous four-channel colon simulator was used to study the effects of lactose for the first time on the growth and fermentation dynamics of colonic microbiota. In six separate simulations, lactose supplementation increased the total SCFA concentration by 3-5 fold as compared with the baseline in the respective vessels. The total bacterial density was inversely correlated with lactic acid production (P = 0.003), while production of butyrate (P = 0.007) and propionate (P = 0.02) correlated with higher numbers of bacteria. A major shift in the microbial community structure in the lactose supplemented vessels was demonstrated by bacterial genomic %G+C-profiling of the total population, where lactose supplementation induced a clearly dominant peak in the bifidobacteria prominent area, %G+C 60-65. The transient shift to increased numbers of bifidobacteria (23-27%) of all bacteria in the first two vessels was also confirmed by the bifidobacteria-specific QPCR-method. In conclusion, lactose produced dramatic changes in microbiota composition and activity as compared with the baseline fermentation.     

Experiences with the use of a starter culture in the fermentation of maize for ‘kenkey’ production in Ghana

Controlled fermentation of maize was carried out using six strains of Lactobacillus fermentum and one strain of yeast, Saccharomyces cerevisiae, isolated from traditionally fermented maize dough as starter cultures for inoculum enrichement. The fermentations were monitored by pH, acidity, microbiological analysis and taste panel evaluation of two products, kenkey and koko, prepared from the fermented doughs. The strains of L. fermentum used as starter culture dominated the microflora during fermentation and in most inoculated doughs the required pH was attained by 24 h instead of 48 h of dough fermentation. Higher contents of lactic acid bacteria and yeasts were observed in inoculated doughs at the initial stages of fermentation but the spontaneously fermented doughs attained similar lactic acid bacteria and yeasts counts by 24 h of dough fermentation. The organoleptic quality of kenkey and koko prepared from doughs fermented with starter culture for 48 h was not significantly different from the traditional products. Kenkey prepared from doughs fermented for 24 h with starter culture were found to be unacceptable by the taste panel although similarly produced koko was acceptable.The authors are with the Food Research Institute, Council for Scientific and Industrial Research, P.O Box M 20. Accra, Ghana.     

Production of hydrolases by lactic acid bacteria and bifidobacteria and their antibiotic resistance

It was demonstrated that bifidobacteria and lactic acid bacteria B. adolescentis and Lactobacillus sp. synthesized extracellular enzymes cleaving glycoside bonds in the molecules of dextran, pectic acid, and soluble starch. The maximal production of extracellular β-galactosidase by B. adolescentis 91-BIM and 94-BIM at a rate of 0.08 and 0.03 U/mg per h was observed during the exponential growth phase at 5 and 12 h of cultivation, respectively. The cultures of bifidobacteria retained 60–70% of β-galactosidase and α-amylase activities after six months of storage. The bifidobacterium strains studied were resistant to amphotericin and aminoglycosides (gentamicin, kanamycin, and netromycin). The lactam antibiotics (ampicillin, benzylpenicillin, bicillin 3, bicillin 5, and carbenicillin), the preparations inhibiting protein synthesis at the level of ribosomes (lincomycin), RNA polymerase inhibitors (rifampin), cephalosporin, and Maxipime inhibited the growth of bifidobacteria. Rifampin, erythromycin, amphotericin, Maxipime, Fortum, doxycycline, levomycetin, streptomycin, and the aminoglycosides netromycin, gentamicin, and kanamycin did not have an effect on the growth of Lactobacillus sp., whereas semisynthetic derivatives of penicillin, carbenicillin and ampicillin, inhibited its growth as well as Oxamp and lincomycin. The lactam antibiotics benzylpenicillin, bicillin 3, and bicillin 5 inhibited the growth of lactic acid bacilli by 30–90%.     

Evaluation of bifidobacteria for the production of antimicrobial compounds and assessment of performance in cottage cheese at refrigeration temperature

Twelve strains of bifidobacteria were identified which exhibited a broad spectrum of antagonistic activity against both Gram-positive and Gram-negative indicators, especially Pseudomonas species, using deferred antagonism spot plate assays. Inhibitory action was shown to be unrelated to hydrogen peroxide production and not solely dependent on acidity. However, attempts to detect inhibitory activity in cell-free supernatant fluids from these strains were unsuccessful. The production of inhibitory compound(s) by Bifidobacterium infantis NCFB 2255 was shown to be an unstable trait resulting in phenotypic alternation between production and non-production. Results from food trials using commercial cottage cheese which was inoculated with the inhibitor-producing strains of Bif. infantis NCFB 2255 and Bif. breve NCFB 2258 indicated that levels of Pseudomonas were reduced, but this observation was species-dependent. The viability of bifidobacteria themselves during storage in cottage cheese at refrigeration temperature was found to be a strain variable trait.     

Effects of Lactose on Colon Microbial Community Structure and Function in a Four-Stage Semi-Continuous Culture System

A semi-continuous four-channel colon simulator was used to study the effects of lactose for the first time on the growth and fermentation dynamics of colonic microbiota. In six separate simulations, lactose supplementation increased the total SCFA concentration by 3–5 fold as compared with the baseline in the respective vessels. The total bacterial density was inversely correlated with lactic acid production (P=0.003), while production of butyrate (P=0.007) and propionate (P=0.02) correlated with higher numbers of bacteria. A major shift in the microbial community structure in the lactose supplemented vessels was demonstrated by bacterial genomic %G+C-profiling of the total population, where lactose supplementation induced a clearly dominant peak in the bifidobacteria prominent area, %G+C 60–65. The transient shift to increased numbers of bifidobacteria (23–27%) of all bacteria in the first two vessels was also confirmed by the bifidobacteria-specific QPCR-method. In conclusion, lactose produced dramatic changes in microbiota composition and activity as compared with the baseline fermentation.     

Study of the intestinal microflora in residents of the city of Vladikavkaz

Microbiological study revealed high occurrence of dysbacteriosis with the decreased amount of Escherichia coli (80.9%) and the presence of E. coli hemolytic forms (76.6%). Rather often the elevated content of E. coli with decreased fermentative properties was noted (51.1%). In all cases E. coli with decreased biochemical activity had hemolytic activity, which was accompanied by the deficiency of bifidobacteria (79.2% of cases) and the combined deficiency of lactic acid bacteria and bifidobacteria (75% of cases).