Intestinal and immunological effects of daily oral administration of Lactobacillus salivarius CECT5713 to healthy adults

There is an increasing interest in the intestinal and immunological effects of probiotics. The aim of the present study is to evaluate the tolerance and beneficial effects in healthy adults of the strain, Lactobacillus salivarius CECT5713 isolated from breast milk. A phase II, randomized, double-blinded, placebo-controlled human clinical trial was carried out in 40 healthy adults. The Probiotic group received a daily dose of 2 × 10⁸ CFU of L. salivarius CECT5713 in capsules during 4 weeks while volunteers of the control received only a placebo. Gastrointestinal and immunological parameters were analyzed. Results showed that L. salivarius CECT5713 was well tolerated and no adverse effects were detected. Consumption of the probiotic strain increased fecal lactobacilli counts (7.9 ± 0.1 vs. 7.05 ± 0.2 CFU/g feces, P = 0.001). Also, an improvement in the frequency of defecation (P = 0.04) was observed. Probiotic treatment induced significantly the percentage of NK cells and monocytes, as well as the plasmatic levels of immunoglobulins M, A and G, and the regulatory cytokine IL-10 (72.3 ± 11.7 in probiotic group vs. 27.3 ± 6.4 pg/mL in control group, P < 0.01). Thus, it can be concluded that daily administration of L. salivarius CECT5713 to healthy adults is safe and improve gut microbiota and different parameters related to immune response.     

Complete Genome Sequence of Lactobacillus salivarius CECT 5713, a Probiotic Strain Isolated from Human Milk and Infant Feces

Lactobacillus salivarius is a homofermentative lactic acid bacterium and is frequently isolated from mucosal surfaces of healthy humans. L. salivarius CECT 5713, a strain isolated simultaneously from breast milk and infant feces of a healthy mother-infant pair, has immunomodulatory, anti-inflammatory, and anti-infectious properties, as revealed by several in vitro and in vivo assays. Here, we report its complete and annotated genome sequence.In the last years, culture-dependent and -independent analyses of the bacterial diversity of human milk and colostrum have revealed that these biological fluids are a source of live staphylococci, streptococci, lactic acid bacteria, and bifidobacteria in the infant gut (5, 6, 8, 9, 11, 13), where they play a key role in the initiation and development of the gut microbiota (12). In a previous study, we isolated L. salivarius CECT 5713 from human milk and infant feces of a mother-child pair (10). Subsequent studies revealed that this strain was a good probiotic candidate since it achieved high survival rates when exposed to the gastrointestinal tract conditions, showed a strong adherence to intestinal cells, stimulated the expression of mucin-encoding genes, produced antimicrobial compounds (lactate, acetate, and hydrogen peroxide), and displayed in vivo and in vitro immunomodulatory, anti-inflammatory, and antibacterial properties against pathogenic bacteria (2, 10, 15). Moreover, oral administration of L. salivarius CECT 5713 appears to be an efficient alternative for the treatment of infectious mastitis in lactating women (7). Similarly, studies with other L. salivarius strains in animal models and clinical trials have demonstrated their probiotic function and, particularly, their anti-inflammatory effects (3, 14, 16).In order to interrogate the genome sequence of L. salivarius CECT 5713 with regard to its probiotic properties, the complete genome sequence was determined by a whole-genome shotgun strategy using pyrosequencing technology (454 Life Sciences, Banford, CT). The initial draft assembly provided by 454 Life Sciences was based on 444,604 high-quality pyrosequencing reads, which assembled into 59 contigs. The genome sequence of L. salivarius UCC118 (1), a well-characterized probiotic strain, was used to order these contigs into large scaffolds.The genome of L. salivarius CECT 5713 consists of a circular chromosome of 1,828,169 bp, two plasmids (pHN1, 44,581 bp; pHN2, 20,426 bp), and a megaplasmid (pHN3, 242,962 bp). The overall GC content of the chromosome is 32.93%, similar to that of the megaplasmid but lower than those of the plasmids (>38%). The entire genome of CECT 5713 contains 1,558 protein-, 87 tRNA-, and 51 rRNA-encoding genes. A comparison between the genomes of L. salivarius CECT 5713 and UCC118 revealed the presence of 52 protein-encoding genes that are exclusive for CECT 5713, including genes encoding a 6-phospho-β-glucosidase and three collagen-binding proteins, which may explain the high potential for competitive exclusion of pathogens displayed by this strain. The genes responsible for the bacteriocin activity of L. salivarius CECT 5713 are located in pHN3. This megaplasmid contains six open reading frames (ORFs) closely related, but not identical, to the genes responsible for the biosynthesis of salivaricin ABP-118, a two-component class II bacteriocin (4), in L. salivarius UCC118. Globally, several features of the L. salivarius CECT 5713 genome suggest a strong probiotic potential in humans.     

Evaluation of Potential Probiotics Isolated from Human Milk and Colostrum

Several studies have demonstrated a diversity of bacterial species in human milk, even in aseptically collected samples. The present study evaluated potential probiotic bacteria isolated from human milk and associated maternal variables. Milk samples were collected from 47 healthy women and cultured on selective and universal agar media under aerobic and anaerobic conditions. Bacterial isolates were counted and identified by Biotyper Matrix-Assisted Laser Desorption Ionization–Time of Flight mass spectrometry and then tested for probiotic properties. Total bacteria in human milk ranged from 1.5 to 4.0 log₁₀ CFU/mL. The higher bacterial counts were found in colostrum (mean = 3.9 log₁₀ CFU/mL, 95% CI 3.14–4.22, p = 0.00001). The most abundant species was Staphylococcus epidermidis (n = 76). The potential probiotic candidates were Lactobacillus gasseri (n = 4), Bifidobacterium breve (n = 1), and Streptococcus salivarius (n = 4). Despite the small sample size, L. gasseri was isolated only in breast milk from mothers classified into a normal weight range and after a vaginally delivered partum. No potential probiotics showed antagonism against pathogens, but all of them agglutinated different pathogens. Nine bacterial isolates belonging to the species L. gasseri, B. breve, and S. salivarius were selected as potential probiotics. The present study confirms the presence in breast milk of a bacterial microbiota that could be the source of potential probiotic candidates to be used in the formula of simulated maternal milk.     

Antimicrobial potential of four Lactobacillus strains isolated from breast milk

AIMS: The antimicrobial potential of four lactobacilli (Lactobacillus salivarius CECT5713, Lactobacillus gasseri CECT5714, L. gasseri CECT5715 and Lactobacillus fermentum CECT5716), isolated from fresh human breast milk, was evaluated in this study and compared with Lactobacillus coryniformis CECT5711, a reuterin-producing strain isolated from an artisan goat's cheese. METHODS AND RESULTS: Agar diffusion tests, competitive adhesion assays and mucin expression assays were carried out in order to value the antibacterial properties of the lactobacilli strains. The antibacterial capability of the strains was tested in vivo by using a murine infection model with Salmonella choleraesuis. The results revealed that all the strains studied, displayed antibacterial properties against pathogenic bacteria. However, the antibacterial potential varied among the lactobacilli tested and, in fact, L. salivarius CECT5713 showed not only the best in vitro antibacterial activity, but also the highest protective effect against a Salmonella strain in the murine infection model. CONCLUSION: The four breast-milk lactobacilli, and particularly L. salivarius CECT5713, possess potent antibacterial activities that result in a higher protection against S. choleraesuis CECT4155 in a mouse infection model. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that lactobacilli from breast milk could contribute to an anti-infective protection in neonates and would be excellent candidates for the development of infant probiotic products.     

The consumption of two new probiotic strains, Lactobacillus gasseri CECT 5714 and Lactobacillus coryniformis CECT 5711, boosts the immune system of healthy humans.

Orally ingested probiotic bacteria are able to modulate the immune system. However, differences exist in the immunomodulatory effects of different probiotic strains. Moreover, different regulatory effects, which depend on the health status of the consumer, have been identified. This work describes a randomized, double-blind, placebo-controlled human clinical trial to investigate the immune effects on healthy people of a fermented product containing two new probiotic strains, Lactobacillus gasseri CECT 5714 and Lactobacillus coryniformis CECT 5711, which was compared with another fermented product, a standard yogurt. Consumption of either the new product or yogurt increased the proportion of phagocytic cells, including monocytes and neutrophils, as well as their phagocytic activity. However, combination of the product containing the strains L. gasseri CECT 5714 and L. coryniformis CECT 5711 also induced an increase in the proportion of natural killer (NK) cells and in IgA concentrations. The effects were higher after two weeks of treatment than after 4 weeks, which suggests regulation of the immune system. In addition, the new product enhanced immunity in the participants to a greater extent than did the control standard yogurt.     

Potential probiotic properties of phytase-producing Lactobacillus salivarius FC113

Probiotics have known efficacy as dietary supplements. Here, Lactobacillus strain F113 was characterized for probiotic use. Strain FC113 was selected as having the highest phytase activity (403.6 U) among tested strains showing acid tolerance and nitrite production. FC113 was tentatively identified as Lactobacillus salivarius based on an API 50 CHL assay and 16S rRNA gene analysis. The production of interleukin (IL)-1α and tumor necrosis factor (TNF)-α was measured in in vitro culture experiments. Cytokine production by L. salivarius FC113 at 1?×?10⁷ CFU/ml was approximately 175.5?±?36.40 pg/mL IL-1α and 353.5?±?61.79 pg/mL TNF-α. L. salivarius FC113 was profoundly resistant to artificial gastric juice (pH 2.5, 1 % pepsin), and persisted for 24 h in artificial bile acid. According to results obtained with an API ZYM kit, L. salivarius FC113 did not generate carcinogenic enzymes. L. salivarius F113 had an inhibitory effect on food-borne pathogens, and adhered strongly to HT-29 human intestinal epithelial cell lines. These results show that L. salivarius FC113 has probiotic characteristics, and exhibits high feed bioavailability in the host animal, in addition to an immune-stimulating effect.     

Lactobacillus salivarius CTC2197 Prevents Salmonella enteritidis Colonization in Chickens

A rifampin-resistant Lactobacillus salivarius strain, CTC2197, was assessed as a probiotic in poultry, by studying its ability to prevent Salmonella enteritidis C-114 colonization in chickens. When the probiotic strain was dosed by oral gavage together with S. enteritidis C-114 directly into the proventriculus in 1-day-old Leghorn chickens, the pathogen was completely removed from the birds after 21 days. The same results were obtained when the probiotic strain was also administered through the feed and the drinking water apart from direct inoculation into the proventriculus. The inclusion of L. salivarius CTC2197 in the first day chicken feed revealed that a concentration of 10⁵ CFU g⁻¹ was enough to ensure the colonization of the gastrointestinal tract of the birds after 1 week. However, between 21 and 28 days, L. salivarius CTC2197 was undetectable in the gastrointestinal tract of some birds, showing that more than one dose would be necessary to ensure its presence till the end of the rearing time. Freeze-drying and freezing with glycerol or skim milk as cryoprotective agents, appeared to be suitable methods to preserve the probiotic strain. The inclusion of the L. salivarius CTC2197 in a commercial feed mixture seemed to be a good way to supply it on the farm, although the strain showed sensitivity to the temperatures used during the feed mixture storage and in the chicken incubator rooms. Moreover, survival had been improved after several reinoculations in chicken feed mixture.     

Relative Ability of Orally Administered Lactobacillus murinus To Predominate and Persist in the Porcine Gastrointestinal Tract

Five porcine-derived Lactobacillus or Pediococcus isolates administered to pigs (n = 4), either singly or as a combination at ~10¹⁰ CFU per day varied with respect to intestinal survival and persistence. Two Lactobacillus murinus strains survived best and were excreted at ~10⁷ to 10⁸ CFU/g of feces. In contrast, Pediococcus pentosaceus DPC6006 had the lowest fecal count at ~10⁵ CFU/g and was excreted at a significantly lower level than both L. murinus strains. Fecal L. murinus DPC6003 counts were also significantly higher than both Lactobacillus salivarius DPC6005 and Lactobacillus pentosus DPC6004 (~10⁶ CFU/g). The L. murinus strains persisted for at least 9 days postadministration in both the feces and the cecum. Animals fed a combination of all five strains excreted ~10⁷ CFU of the administered strains/g, with L. murinus predominating, as determined by randomly amplified polymorphic DNA PCR. Postadministration, variation was observed between animals fed the strain combination, but in general, L. murinus DPC6002 and DPC6003 and L. pentosus DPC6004 predominated in the feces and the cecum while P. pentosaceus DPC6006 was detected only in the cecum. Fifteen days after the start of culture administration, mean fecal Enterobacteriaceae counts were significantly lower in some of the treatment groups. In addition, when mean preadministration counts were compared with those obtained after 21 days of culture administration, Enterobacteriaceae counts were reduced by ~87 to 98% in pigs fed L. salivarius DPC6005, P. pentosaceus DPC6006, L. pentosus DPC6004, and the culture mix. In conclusion, the porcine intestinal isolates have potential as probiotic feed additives for pigs, with differences in strain performance highlighting the advantages of using culture combinations.     

Evaluation of Phytate-Degrading Lactobacillus Culture Administration to Broiler Chickens

Probiotics have been demonstrated to promote growth, stimulate immune responses, and improve food safety of poultry. While widely used, their effectiveness is mixed, and the mechanisms through which they contribute to poultry production are not well understood. Microbial phytases are increasingly supplemented in feed to improve digestibility and reduce antinutritive effects of phytate. The microbial origin of these exogenous enzymes suggests a potentially important mechanism of probiotic functionality. We investigated phytate degradation as a novel probiotic mechanism using recombinant Lactobacillus cultures expressing Bacillus subtilis phytase. B. subtilis phyA was codon optimized for expression in Lactobacillus and cloned into the expression vector pTRK882. The resulting plasmid, pTD003, was transformed into Lactobacillus acidophilus, Lactobacillus gallinarum, and Lactobacillus gasseri. SDS-PAGE revealed a protein in the culture supernatants of Lactobacillus pTD003 transformants with a molecular weight similar to that of the B. subtilis phytase. Expression of B. subtilis phytase increased phytate degradation of L. acidophilus, L. gasseri, and L. gallinarum approximately 4-, 10-, and 18-fold over the background activity of empty-vector transformants, respectively. Phytase-expressing L. gallinarum and L. gasseri were administered to broiler chicks fed a phosphorus-deficient diet. Phytase-expressing L. gasseri improved weight gain of broiler chickens to a level comparable to that for chickens fed a control diet adequate in phosphorus, demonstrating proof of principle that administration of phytate-degrading probiotic cultures can improve performance of livestock animals. This will inform future studies investigating whether probiotic cultures are able to provide both the performance benefits of feed enzymes and the animal health and food safety benefits traditionally associated with probiotics.     

Characterization of Lactobacillus salivarius CECT 5713, a strain isolated from human milk: from genotype to phenotype

Lactobacillus salivarius CECT 5713, isolated from human milk, has immunomodulatory, anti-inflammatory and antiinfectious properties, as revealed by several in vitro and in vivo assays, which suggests a strong potential as a probiotic strain. In this work, the relationships between several genetic features of L. salivarius CECT 5713 and the corresponding phenotypes were evaluated. Although it contains a plasmid-encoded bacteriocin cluster, no bacteriocin biosynthesis was observed, possibly due to a 4-bp deletion at the beginning of the histidine kinase determinant abpK. The genome of L. salivarius CECT 5713 harbours two apparently complete prophages of 39.6 and 48?kbp. Upon induction, the 48-kbp prophage became liberated from the bacterial genome, but no DNA replication took place, which resulted in lysis of the cultures but not in phage progeny generation. The strain was sensitive to most antibiotics tested and no transmissible genes potentially involved in antibiotic resistance were detected. Finally, the genome of L. salivarius CECT 5713 contained four ORFs potentially involved in human molecular mimetism. Among them, protein 1230 was considered of particular relevance because of its similarity with dendritic cell-related proteins. Subsequently, in vitro assays revealed the ability of L. salivarius CECT 5713 to stimulate the maturation of immature dendritic cells and to inhibit the in vitro infectivity of HIV-1.     

Comparative Survival Rates of Human-Derived Probiotic Lactobacillus paracasei and L. salivarius Strains during Heat Treatment and Spray Drying

Spray drying of skim milk was evaluated as a means of preserving Lactobacillus paracasei NFBC 338 and Lactobacillus salivarius UCC 118, which are human-derived strains with probiotic potential. Our initial experiments revealed that NFBC 338 is considerably more heat resistant in 20% (wt/vol) skim milk than UCC 118 is; the comparable decimal reduction times were 11.1 and 1.1 min, respectively, at 59°C. An air outlet temperature of 80 to 85°C was optimal for spray drying; these conditions resulted in powders with moisture contents of 4.1 to 4.2% and viable counts of 3.2 × 10⁹ CFU/g for NFBC 338 and 5.2 × 10⁷ CFU/g for UCC 118. Thus, L. paracasei NFBC 338 survived better than L. salivarius UCC 118 during spray drying; similar results were obtained when we used confocal scanning laser microscopy and LIVE/DEAD BacLight viability staining. In addition, confocal scanning laser microscopy revealed that the probiotic lactobacilli were located primarily in the powder particles. Although both spray-dried cultures appeared to be stressed, as shown by increased sensitivity to NaCl, bacteriocin production by UCC 118 was not affected by the process, nor was the activity of the bacteriocin peptide. The level of survival of NFBC 338 remained constant at ~1 × 10⁹ CFU/g during 2 months of powder storage at 4°C, while a decline in the level of survival of approximately 1 log (from 7.2 × 10⁷ to 9.5 × 10⁶ CFU/g) was observed for UCC 118 stored under the same conditions. However, survival of both Lactobacillus strains during powder storage was inversely related to the storage temperature. Our data demonstrate that spray drying may be a cost-effective way to produce large quantities of some probiotic cultures.     

Metabolism of prebiotic products containing beta(2-1) fructan mixtures by two Lactobacillus strains

The capacity of two probiotic strains, isolated from human breast milk, to use several beta(2-1) fructan mixtures as carbon and energy source in in vitro cultures has been tested. Results showed that both strains, Lactobacillus gasseri CECT5714 and Lactobacillus fermentum CECT5716, reached higher growth levels on culture media containing fructooligosaccharide mixtures produced by enzymatic synthesis, compared to those obtained by inulin hydrolysis. Furthermore, the shortest beta(2-1) fructan, kestose, was the only prebiotic compound in the mixtures significantly metabolized in all growth media tested. Analysis of short-chain fatty acid production showed no correlation between the fatty acid profile produced and the carbon source used in each experiment. These data could serve to select appropriate beta(2-1) fructans to be used as prebiotics for L. gasseri CECT5714 and L. fermentum CECT5716 and to design suitable symbiotic food products containing the mentioned lactobacilli.     

Spontaneously Induced Prophages in Lactobacillus gasseri Contribute to Horizontal Gene Transfer

Lactobacillus gasseri is an endogenous species of the human gastrointestinal tract and vagina. With recent advances in microbial taxonomy, phylogenetics, and genomics, L. gasseri is recognized as an important commensal and is increasingly being used in probiotic formulations. L. gasseri strain ADH is lysogenic and harbors two inducible prophages. In this study, prophage ϕadh was found to spontaneously induce in broth cultures to populations of ∼10⁷ PFU/ml by stationary phase. The ϕadh prophage-cured ADH derivative NCK102 was found to harbor a new, second inducible phage, vB_Lga_jlb1 (jlb1). Phage jlb1 was sequenced and found to be highly similar to the closely related phage LgaI, which resides as two tandem prophages in the neotype strain L. gasseri ATCC 33323. The common occurrence of multiple prophages in L. gasseri genomes, their propensity for spontaneous induction, and the high degree of homology among phages within multiple species of Lactobacillus suggest that temperate bacteriophages likely contribute to horizontal gene transfer (HGT) in commensal lactobacilli. In this study, the host ranges of phages ϕadh and jlb1 were determined against 16 L. gasseri strains. The transduction range and the rate of spontaneous transduction were investigated in coculture experiments to ascertain the degree to which prophages can promote HGT among a variety of commensal and probiotic lactobacilli. Both ϕadh and jlb1 particles were confirmed to mediate plasmid transfer. As many as ∼10³ spontaneous transductants/ml were obtained. HGT by transducing phages of commensal lactobacilli may have a significant impact on the evolution of bacteria within the human microbiota.     

Development of a Probiotic Cheddar Cheese Containing Human-Derived Lactobacillus paracasei Strains

Cheddar cheese was manufactured with either Lactobacillus salivarius NFBC 310, NFBC 321, or NFBC 348 or L. paracasei NFBC 338 or NFBC 364 as the dairy starter adjunct. These five strains had previously been isolated from the human small intestine and have been characterized extensively with respect to their probiotic potential. Enumeration of these strains in mature Cheddar cheese, however, was complicated by the presence of high numbers (>10⁷ CFU/g of cheese) of nonstarter lactic acid bacteria, principally composed of lactobacilli which proliferate as the cheese ripens. Attempts to differentiate the adjunct lactobacilli from the nonstarter lactobacilli based on bile tolerance and growth temperature were unsuccessful. In contrast, the randomly amplified polymorphic DNA method allowed the generation of discrete DNA fingerprints for each strain which were clearly distinguishable from those generated from the natural flora of the cheeses. Using this approach, it was found that both L. paracasei strains grew and sustained high viability in cheese during ripening, while each of the L. salivarius species declined over the ripening period. These data demonstrate that Cheddar cheese can be an effective vehicle for delivery of some probiotic organisms to the consumer.     

Two Lactobacillus strains, isolated from breast milk, differently modulate the immune response

AIMS: The ability of two different Lactobacillus strains (Lactobacillus salivarius CECT5713 and Lactobacillus fermentum CECT5716), isolated from human breast milk, to modulate the immune response was examined. METHODS AND RESULTS: In rodent bone-marrow-derived macrophages (BMDM), the presence of Lact. fermentum CECT5716 induced pro-inflammatory cytokines, in contrast to the activation of IL-10 induced by Lact. salivarius CECT5713. Although both strains reduced the lipopolysaccharide (LPS)-induced inflammatory response in BMDM, the effect of Lact. salivarius CECT5713 was more efficient, probably because of the production of higher amounts of IL-10 cytokine. In vivo assays in mice showed similar results; the consumption of Lact. fermentum CECT5716 enhanced the production of Th1 cytokines by spleen cells and increased the IgA concentration in faeces. However, the consumption of Lact. salivarius CECT5713 induced IL-10 production by spleen cells. CONCLUSION: Therefore, in general, the effect of Lact. fermentum CECT5716 is immunostimulatory in contrast to the anti-inflammatory effect of Lact. salivarius CECT5713. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study show that two Lactobacillus strains isolated from breast milk can exert different and even opposing effects on immune response demonstrating the specificity of each strain.     

<Emphasis Type="Italic">Lactobacillus salivarius</Emphasis> NK02: a Potent Probiotic for Clinical Application in Mouthwash

A specific strain of naturally occurring oral lactobacilli was isolated and identified based on morphological, biochemical, and 16S rRNA gene sequencing. The phylogenetic affiliation of the isolate confirmed that the NK02 strain had close association with the Lactobacillus salivarius. An effective mouthwash was developed for treatment of periodontitis and suppression of the indicator bacterium Aggregatibacter actinomycetemcomitans which is an obvious pathogen of periodontal disease. The mouthwash containing L. salivarius NK02 was tested at a dose level of 10⁸ (colony forming units (CFU) ml^?1), monitoring over a period of 4 weeks. The study was a randomized double-blind placebo control trial, and the patients were treated in two groups of control and test by using scaling and root planing (SRP) + placebo and scaling and root planing (SRP) + probiotic, respectively. It appeared that the probiotic mouthwash was able to inhibit the bacterial growth on both saliva and sub-gingival crevice and exhibited antibacterial activity against A. actinomycetemcomitans. The results also showed that SRP+ probiotic treatment led to a significant decrease of gingival index (GI) and bleeding on probing (BOP) compared with that of SRP + placebo for the probiotic group. The rate of decrease in pocket depth was displayed in the group with SRP + probiotic treatment equal to 1/2 mm, and probing pocket depth (PPD) value was decreased in the probiotic bacteria treatment group that can explain the decrease in inflammation in gingiva. Our findings suggest that probiotic mouthwash is healthy for daily use as an alternative for maintaining dental and periodontal health.     

In vitro evaluation of the probiotic potential of Lactobacillus salivarius SMXD51

Lactobacillus salivarius SMXD51 was previously isolated from the cecum of a Tunisian poultry and found to produce a bacteriocin-like substance highly active against the foodborne pathogen Campylobacter jejuni. The aim of this study was to examine some probiotic properties of the strain: acid and bile tolerance, capacity of adhesion, stimulation of immune defences (IL-6, IL-8, IL-10 and β-defensin 2), and modulation of the barrier integrity. The results showed that L. salivarius SMXD51 can tolerate gastrointestinal conditions (acid and bile), adhere to intestinal cells and stimulate the immune system. The bacterium strengthened the intestinal barrier functions through the increase of the transepithelial electrical resistance (TEER) and reinforcement of the F-actin cytoskeleton. One hour pretreatment with L. salivarius SMXD51 protected against Pseudomonas aeruginosa PAO1-induced decrease of TEER and damage of the F-actin cytoskeleton. Our results highlight that L. salivarius SMXD51 fulfils the principle requirements of an efficient probiotic and may be seen as a reliable candidate for further validation studies in chicken.     

Characterization of a Bacteriocin-Like Substance Produced by a Vaginal Lactobacillus salivarius Strain

A novel bacteriocin-like substance produced by vaginal Lactobacillus salivarius subsp. salivarius CRL 1328 with activity against Enterococcus faecalis, Enterococcus faecium, and Neisseria gonorrhoeae was characterized. The highest level of production of this heat-resistant peptide or protein occurred during the late exponential phase. Its mode of action was shown to be bactericidal. L. salivarius subsp. salivarius CRL 1328 could be used for the design of a probiotic to prevent urogenital infections.     

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.     

Detection and Specific Enumeration of Multi-Strain Probiotics in the Lumen Contents and Mucus Layers of the Rat Intestine After Oral Administration

Although the detection of viable probiotic bacteria following their ingestion and passage through the gastrointestinal tract (GIT) has been well documented, their mucosal attachment in vivo is more difficult to assess. In this study, we investigated the survival and mucosal attachment of multi-strain probiotics transiting the rat GIT. Rats were administered a commercial mixture of the intestinal probiotics Lactobacillus acidophilus LA742, Lactobacillus rhamnosus L2H and Bifidobacterium lactis HN019 and the oral probiotic Streptococcus salivarius K12 every 12 h for 3 days. Intestinal contents, mucus and faeces were tested 6 h, 3 days and 7 days after the last dose by strain-specific enumeration on selective media and by denaturing gradient gel electrophoresis. At 6 h, viable cells and DNA corresponding to all four probiotics were detected in the faeces and in both the lumen contents and mucus layers of the ileum and colon. Viable probiotic cells of B. lactis and L. rhamnosus were detected for 7 days and L. acidophilus for 3 days after the last dose. B. lactis and L. rhamnosus persisted in the ileal mucus and colon contents, whereas the retention of L. acidophilus appeared to be relatively higher in colonic mucus. No viable cells of S. salivarius K12 were detected in any of the samples at either day 3 or 7. The study demonstrates that probiotic strains of intestinal origin but not of oral origin exhibit temporary colonisation of the rat GIT and that these strains may have differing relative affinities for colonic and ileal mucosa.