The Application of Lactobacillus reuteri CCM 8617 and Flaxseed Positively Improved the Health of Mice Challenged with Enterotoxigenic E. coli O149:F4.

The aim of our study was to monitor the effects of dietary synbiotics on experimentally infected mice. Sixty mice were divided into the following three groups: negative control group C1, positive control group C2 (mice infected with enterotoxigenic Escherichia coli O149:F4NAL), and experimental group LF (Lactobacillus reuteri CCM 8617RIF + 10% flaxseed + E. coli O149:F4NAL). Supplements were administered for 42 days. Microbiological, hematological, and biochemical analyses, electrophoretic analysis of lactate dehydrogenase (LDH) isoenzymes, and analysis of fatty acids using gas chromatography and isotachophoresis were performed. We recorded higher numbers of jejunal and ileal lactic acid bacteria, lower Enterobacteriaceae counts in the feces of the animals, and an increased production of organic acids in the synbiotic-fed group. The supplements applied favored n-3 polyunsaturated fatty acid (PUFA) metabolism and inhibited n-6 PUFA metabolism; thus, they influenced the n-6 to n-3 and eicosapentaenoic to arachidonic acid ratios. Additionally, the incorporation of n-3 PUFAs to the cell membrane decreased the activity of LDH, transaminases, and alkaline phosphatase. Results obtained in our study indicate the positive effect of continuous supplementation of combination of probiotic cheese enriched with L. reuteri CCM 8617RIF and crushed flaxseed on composition of intestinal microflora and alleviation of the course of infection induced by pathogenic bacterium E. coli O149:F4NAL.

Systemic immune response of gnotobiotic mice infected with porcine circovirus type 2 after administration of Lactobacillus reuteri L26 Biocenol™.

In our previous study we confirmed an antiviral activity of probiotic Lactobacillus reuteri L26 which was mediated by stimulation of local intestinal immunity. The aim of this paper was to evaluate the influence of L. reuteri L26 on the systemic immune response in gnotobiotic mice infected with porcine circovirus type 2 (PCV2). A total of 30 germ-free mice were divided into 3 groups and animals in noninfected and infected control groups (NC and IC; n=10) received sterile de Man-Rogosa-Sharpe broth for 7 days and animals in experimental group L+PCV (n=10) were inoculated with L. reuteri L26. Subsequently, mice in L+PCV and IC groups were infected with PCV2; however, mice in the control group received virus cultivation medium (mock). The results showed an increase of percentage of cytotoxic cells (CD8+ and CD49b+CD8-) and oxidative burst of phagocytes, up-regulation of the gene expression of RANTES, granulocyte-macrophage colony-stimulating factor, interferon-γ and immunoglobulin A in blood above all in the later phase of infection (14 dpi) in L+PCV group accompanied by higher load of PCV2 in the serum. These findings indicate that L. reuteri L26 has a potential to induce systemic immune reaction, but in gnotobiotic mice immune stimulation can increase virus replication.

Influence of Lactobacillus reuteri L26 Biocenol™ on immune response against porcine circovirus type 2 infection in germ-free mice.

Probiotic bacteria are frequently used for prevention of bacterial infections of the gastrointestinal tract, but there are only limited studies on their efficacy against viral gut infections in animals. The aim of this study was to investigate the effect of probiotic Lactobacillus reuteri L26 BiocenolTM on the innate and adaptive immune responses in germ-free Balb/c mice, experimentally infected by porcine circovirus type 2 (PCV2), which confers immunosuppressive effect. A total of 30 six-week-old female mice were divided into 3 groups and animals in experimental group LPCV (n=10) were inoculated with L. reuteri L26, animals in the control group (C; n=10) and experimental group PCV (n=10) received sterile De Man-Rogosa-Sharpe broth for 7 days. Subsequently, mice from both experimental groups were infected with PCV2; however, mice in the control group received virus cultivation medium (mock). Virus load in faeces, ileum and mesenteric lymph nodes (MLN); as well as gene expression of selected cytokines, immunoglobulin A (IgA) and polymeric Ig receptor (PIgR) in the ileum, and percentage of CD8+, CD19+ and CD49b+CD8- cells in the MLN were evaluated. Our results showed that L. reuteri significantly decreased the amount of PCV2 in faeces and in the ileum, and up-regulated the gene expression of chemokines, interferon (IFN)-γ, IgA and PIgR in the ileum. Increased IFN-γ mRNA level was accompanied by higher proportion of natural killer cells and up-regulated IgA and PIgR gene expressions were in accordance with significantly higher percentage of CD19+ lymphocytes in the MLN. These findings indicate that probiotic L. reuteri has an antiviral effect on PCV2 in the intestine which is mediated by stimulation of local gut immune response.

Histo-FISH protocol to detect bacterial compositions and biofilms formation in vivo.

The study of biofilm function in vivo in various niches of the gastrointestinal tract (GIT) is rather limited. It is more frequently used in in vitro approaches, as an alternative to the studies focused on formation mechanisms and function of biofilms, which do not represent the actual in vivo complexity of microbial structures. Additionally, in vitro tests can sometimes lead to unreliable results. The goal of this study was to develop a simple approach to detect bacterial populations, particularly Lactobacillus and Bifidobacterium in biofilms, in vivo by the fluorescent in situ hybridisation (FISH) method. We standardised a new Histo-FISH method based on specific fluorochrome labelling probes which are able to detect Lactobacillus spp. and Bifidobacterium spp. within biofilms on the mucosal surface of the GIT embedded in paraffin in histological slices. This method is also suitable for visualisation of bacterial populations in the GIT internal content. Depending on the labelling probes, the Histo-FISH method has the potential to detect other probiotic strains or pathogenic bacteria. This original approach permits us to analyse bacterial colonisation processes as well as biofilm formation in stomach and caecum of BALB/c and germ-free mice.

Anti-inflammatory and immunoregulatory effects of flax-seed oil and Lactobacillus plantarum - Biocenol™ LP96 in gnotobiotic pigs challenged with enterotoxigenic Escherichia coli.

The aim of this study was to determine the immune response after preventive administration of flax-seed oil (rich in n-3 PUFAs) or probiotic strain Lactobacillus plantarum - Biocenol™ LP96 or their combination in the jejunum of ETEC-challenged gnotobiotic pigs. Subsequently, gene expression of selected cytokines, phagocytic activity of leukocytes from peripheral blood and percentage of CD2(+), CD4(+), CD8(+) and CD4(+)CD25(+) lymphocytes in jejunal mucosa were evaluated. Our results showed that combined treatment down-regulates IL-1α and IL-8 gene expression, up-regulates IFN-γ and tends to regulate inflammation induced by ETEC through cytokine IL-10. In general, changes in cytokine gene expression correlated with the proportions of immune cells isolated from the same part of the jejunal mucosa. Results indicate that probiotic L. plantarum in combination with flax-seed oil rich in n-3 PUFAs has anti-inflammatory properties, stimulates Th1-mediated cell immunity and phagocytosis, and tends to regulate the inflammatory response induced by ETEC.

Effectivity of freeze-dried form of Lactobacillus fermentum AD1-CCM7421 in dogs.

The impact of probiotic supplementation of canine-derived strain Lactobacillus fermentum AD1-CCM7421 in freeze-dried form on quantitative composition of microbiota and short-chain fatty acid profile in feces of dogs was demonstrated by two independent studies (straightforward repeated-measures model; study I: a dose of 2 g per dog for 2 weeks, 10(8) CFU/g, n = 12; study II: 1 g per dog for 1 week, 10(7) CFU/g, n = 11. The results revealed a significant increase of lactic acid bacteria population persisting also after the cessation of probiotic application in both studies. A reduction of clostridia (study I, p (sum) < 0.01) and tested Gram-negative bacterial genera (coliforms, Aeromonas sp., Pseudomonas sp., study II, p < 0.05) was also detected. The strain AD1-CCM7421 colonized the canine digestive tract in sufficient numbers (10(5)-10(6) CFU/g) and it persisted in the majority of dogs after cessation of probiotic application. An increase of short-chain fatty acid concentrations (study I: butyric, succinic, valeric, formic acid) especially in the early post-treatment phase (p < 0.05) most likely led to a decrease of fecal pH value (p < 0.05) without negative influence on fecal consistency throughout the studies.

Effects of the administration of lactobacilli, maltodextrins and fructooligosaccharides upon the adhesion of E. coli O8:K88 to the intestinal mucosa and organic acid levels in the gut contents of piglets.

The influence of the administration of Lactobacillus plantarum, maltodextrin Maldex 150 and Raftifeed IPX fructooligosaccharides on the inhibition of adhesion of E. coli O8:K88 to the mucosa of the jejunum, ileum and colon as well as on the organic acid levels was investigated in 33 conventional piglets. The counts of E. coli K88 adhering to the jejunal mucosa were significantly decreased (p < 0.05) in Lact. plantarum + Maldex 150 and Lact. plantarum + Maldex 150 + Raftifeed IPX groups. The counts of E. coli K88 adhering to the colonic mucosa of Lact. plantarum + Maldex 150 + Raftifeed IPX and Lact. plantarum + Raftifeed IPX groups were significantly lower (p < 0.05) than in Lact. plantarum and Lact. plantarum + Maldex 150 animals. The acetic acid levels in the ileum and colon of the Lact. plantarum + Maldex 150 + Raftifeed IPX group and Lact. plantarum + Raftifeed IPX group were significantly higher (p < 0.05) than in the Lact. plantarum and Lact. plantarum + Maldex 150 group. The combination of Lact. plantarum, maltodextrin Maldex 150 and Raftifeed IPX proved to be the most effective one to inhibit the counts of E. coli O8:K88 adhering to the intestinal mucosa of the jejunum and colon of conventional piglets.

Improvement of the probiotic effect of micro-organisms by their combination with maltodextrins, fructo-oligosaccharides and polyunsaturated fatty acids.

Probiotics could represent an effective alternative to the use of synthetic substances in nutrition and medicine. The data concerning the efficacy of probiotics are often contradictory. This paper focuses on the enhancement of the efficacy of probiotics by their combination with synergistically acting components of natural origin. Maltodextrins can be obtained by enzymatic hydrolysis of starch and are suitable for consumption. Administration of Lactobacillus paracasei together with maltodextrin decreased the number of Escherichia coli colonising the jejunal mucosa of gnotobiotic piglets by 1 logarithm compared to the control group. Fructo-oligosaccharides (FOS) are naturally occurring oligosaccharides, mainly of plant origin. L. paracasei administered in combination with FOS significantly increased counts of Lactobacillus spp., Bifidobacterium spp., total anaerobes and total aerobes compared to the control group as well as the L. paracasei group. It also significantly decreased Clostridium and Enterobacterium counts in the faeces of the weanling piglets compared with the control group. Dietary lipids influence the gastrointestinal microbiota and specifically the population of lactic acid bacteria. In gnotobiotic piglets the oral administration of an oil containing polyunsaturated fatty acids (PUFA) significantly increased the number of L. paracasei adhering to jejunal mucosa compared to the control group. Our results showed that maltodextrin KMS X-70 and PUFA can be used to enhance the effect of probiotic micro-organisms in the small intestine, and similarly FOS enhance the effect of probiotic micro-organisms in the large intestine.

Study of the effect of Lactobacillus paracasei and fructooligosaccharides on the faecal microflora in weanling piglets.

The influence of administration of Lactobacillus paracasei alone and mixture of Lactobacillus paracasei and fructooligosaccharide on faecal bacteria counts in the weanling pigs was investigated. The administration of Lactobacillus paracasei alone significantly decreased Clostridium (p < 0.05) and Enterobacteriaceae (p < 0.05) counts as compared to the control. Lactobacillus paracasei administered in combination with fructooligosaccharide significantly increased Lactobacillus (p < 0.01-p < 0.05), Bifidobacterium (p < 0.05), total anaerobes (p < 0.05), and total aerobes (p < 0.05) counts compared to control group as well as Lactobacillus paracasei group and significantly decreased Clostridium (p < 0.05) and Enterobacteriaceae (p < 0.01) counts compared to control group. The results obtained point out to a synergic effect of the combination of Lactobacillus paracasei and fructooligosaccharide on numbers of bacterial populations observed in the faeces of the weanling pigs.

Potentiation of the effectiveness of Lactobacillus casei in the prevention of E. coli induced diarrhea in conventional and gnotobiotic pigs.

The influence of preventive administration of Lactobacillus casei subsp. casei and maltodextrin KMS X-70 on Escherichia coli 08: K88 adhesion in the gastrointestinal tract of 11 conventional and 6 gnotobiotic piglets was investigated. The preventive administration of L. casei alone had almost no inhibitory effect on the adherence of E. coli to the jejunal mucosa of gnotobiotic and conventional piglets while the lactobacilli administered together with maltodextrin decreased the number of E. coli colonising jejunal mucosa of gnotobiotic piglets by 1 logarithm (4.95 log 10/cm2) in comparison with the control group (5.96 log 10/cm2). L. casei administered in combination with maltodextrin decreased the number of E. coli colonising the jejunum of conventional piglets by more than two and half logarithm (4.75 log 10/cm2, p < 0.05) in comparison with the control (7.42 log 10/cm2). The inhibitory effect of Lactobacillus casei and maltodextrin KMS X-70 on the adhesion of E. coli to the intestinal mucosa of conventional and gnotobiotic pigs was probably mediated by Lactobacillus--produced antibacterial substances and stimulation of immunity.

The effect of lactic acid bacteria on intestinal metabolism and metabolic profile in gnotobiotic pigs.

The effect of inoculation of Lactobacillus casei on selected parameters of metabolic profile and intestinal metabolism of gnotobiotic piglets was investigated during the first three weeks of their life. The experiment was carried out on 8 germ-free piglets. The experimental group was inoculated once a day with the Lactobacillus casei subsp. casei strain. The inoculum contained 1 x 10(8) microorganisms in 1 ml. The control group of piglets received no inoculum. Lactobacillus casei colonized jejunum and ileum in the numbers from 5.63 to 6.06 log 10 cm-2 and their numbers in the jejunal and ileal contents were in the range 8.38-9.87 log 10.ml-1. The daily consumption of milk by the inoculated animals was significantly higher (p < 0.001). The average weight of inoculated piglets at the end of the period investigated was higher by 29.7%. Lactobacillus casei affected several parameters investigated. Piglets inoculated with lactobacilli showed significantly lower (p < 0.05-0.01) values of pH of the jejunal content, numbers of erythrocytes, values of haematocrit, urea, glucose, total lipids, cholesterol and calcium in the serum and significantly higher values (p < 0.05-0.01) of lactic acid in the jejunal content. The values of phagocytic activity and the index of phagocytic activity in the piglets of the experimental group were two to three-fold higher in comparison with those detected in the control group. The application of Lactobacillus casei affected positively the growth of gnotobiotic piglets, their intestinal metabolism, the level of cholesterol in the serum and phagocytic activity.

Colonization capability of orally administered Lactobacillus strains in the gut of gnotobiotic piglets.

In the present study, the effect of Lactobacillus casei subsp. casei and Lactobacillus fermentum inoculation on jejunum and ileum colonization in gnotobiotic piglets has been observed. The characteristic features of the strains used were strong adherence to pig epithelial gut cells as well as inhibitory activity against enteropathogenic E. coli under in vitro conditions. Strains were inoculated to 2, 3, and 4 day old gnotobiotic piglets at a dose of 2 ml (1 x 10(8) germs/ml). On the second day after the last inoculation, Lactobacillus casei subsp. casei strain counts adhered to the jejunum mucosa and those adhered to the ileum mucosa were 4.54 log 10.cm-2 and 5.40 log 10.cm-2, respectively. Lactobacillus fermentum counts adhered to the jejunum mucosa and those adhered to the ileum mucosa were 5.73 log 10.cm-2 and 4.01 log 10.cm-2, respectively. On day 5 after the last inoculation, the counts in both strains were by one log higher. The results obtained point out to the fact that Lactobacillus casei subsp. casei and Lactobacillus fermentum colonized the mucosa of both jejunum and ileum and survived in the intestinal tract. The adherence of lactobacilli to gut cells in vitro correlated with their capability to adhere to the mucosa of both jejunum and ileum in vivo.

In vitro studies of porcine lactobacilli for possible probiotic use.

Fourteen strains of lactobacilli isolated from the gut of suckling pigs were identified and studied to determine their susceptibility to antimicrobial feed additives, acid tolerance, adherence to epithelial cells from the porcine intestine and antimicrobial activity. Four strains were identified as Lactobacillus casei subsp. casei, two strains as Lactobacillus rhamnosus as well as Lactobacillus reuteri and three strains as Lactobacillus salivarius. The remaining strains could not be identified. Antimicrobial susceptibility tests showed susceptibility of the lactobacilli to many of the antimicrobial feed additives commonly used in pigs. Strains resistant to cyadox and nourseothricin occurred in high numbers. The tested lactobacilli were acid-resistant at pH 3. Thirteen strains of lactobacilli exhibited strong adherence to epithelial cells from the porcine intestine while one strain was classed as weakly-adherent. All lactobacilli showed inhibitory activity against indicator bacteria in the presence of glucose. Lactate and acetate were the principal final products of glucose fermentation in all strains. Only three strains produced H2O2 in detectable amounts.