Streptococcus macedonicus strains isolated from traditional fermented milks: resistance to gastrointestinal environment and adhesion ability.

In this study, Streptococcus macedonicus (S. macedonicus) strains were identified from Algerian traditional fermented milks (Lben and Rayeb). Important prerequisites of probiotic interest such as acidity, bile salts tolerance, and adhesion ability to epithelial cells were investigated. A combination of phenotypic (ability to grow on Bile Esculin Azide medium, BEA; on high salt content medium NaCl 6.5%; on alkaline medium pH 9.6) and genotypic approaches (16S rRNA, ITS genes sequencing and MLST technique) allowed to identify four genetically distinct strains of S. macedonicus. These four strains and two references, Streptococcus thermophilus LMD-9 and Lactobacillus rhamnosus GG (LGG), were tested for their capacity to survive at low pH values, and at different concentrations of an equimolar bile salts mixture (BSM). Two different cell lines, Caco-2 TC7 and HT29-MTX, were used for the adhesion study. The results show that S. macedonicus strains selected constitute a distinct genetic entity from the Greek strain S. macedonicus ACA-DC-198. They were able to survive up to pH 3 and could tolerate high concentrations of bile salts (10 mM), unlike LMD-9 and LGG strains. Our strains also display in vitro adhesion similar to the LGG strain on Caco-2 TC7 and higher adhesion than the LMD-9 strain to Caco-2 TC7 and HT29-MTX cell models. This first characterization allows considering S. macedonicus as a potential candidate for possible probiotic effects that need to be investigated.

A Mixture of Five Bacterial Strains Attenuates Skin Inflammation in Mice.

BACKGROUND: There is a growing interest in the effects of probiotics for the prevention and treatment of skin diseases due to their immunomodulatory and antiinflammatory properties. OBJECTIVE: To assess a mixture of five bacterial strains in the prevention of chronic skin inflammation in mice. METHODS: Hairless SKH-1 mice received daily oral treatment with the probiotic mixture at the dose of 1x109 Colony-Forming Unit (CFU)/day (or vehicle) for three weeks. Chronic skin inflammation was induced by repeated applications of 12-O-tetradecanoylphorbol-13- acetate (TPA; control mice received acetone). Macroscopic and microscopic evaluations of skin lesions were performed and serum levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, IL-17, IL-22, IL-10 and IL-4 measured at the end of the study. RESULTS: Treatment with the probiotic mixture significantly limited the induced chronic skin inflammation at both the macroscopic and microscopic levels. This limitation was consistent with downregulated levels of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α, IL- 17 and IL-22) and up-regulated levels of the anti-inflammatory cytokines, IL-10 and IL-4. CONCLUSION: The results suggest that the probiotic mixture tested could help in preserving skin integrity and homeostasis and that its use could be beneficial in dermatological conditions such as atopic dermatitis and psoriasis.

In vitro and in vivo antagonistic activity of new probiotic culture against Clostridium difficile and Clostridium perfringens.

BACKGROUND: Genus Clostridium accompanies more than 200 known species and at least 30 among them are associated with human and animal diseases. At the moment, the treatment of clostridial infections is based on use of antibiotics. However, due to the European ban on the use of antibiotics in livestock production, novel therapeutic strategies for treatment of these hardly curable infections have been evaluated. Hence, in this study the antimicrobial effect of newly designed probiotic culture consisted of natural isolates Lactobacillus helveticus BGRA43, Lactobacillus fermentum BGHI14 and Streptococcus thermophilus BGVLJ1-44 against Clostridium difficile and Clostridium perfringens was analyzed. RESULTS: The probiotic culture showed strong in vitro antimicrobial effect on C. difficile (human clinical isolate). In addition, individual strains and the probiotic combination exhibited immunomodulatory activity. The probiotic combination significantly increased the proliferation of GALT lymphocytes. At the other hand, none of the bacterial treatments (individual strains and the combination) induced the production of proinflammatory cytokines IL-6 and IL-1ß by intestinal epithelial cells, Caco-2. Interestingly, Caco-2 cells exposed to the probiotic combination produced significantly elevated amount of TGFß pointing to potential protecting effect of the probiotic. In addition, the results of field trial on spontaneously infected goats revealed reduction of C. perfringens in goats (below the detection threshold) after the probiotic treatment. CONCLUSIONS: The results of this study indicated that the novel probiotic deserves to be further investigated as a promising antimicrobial agent against C. difficile and C. perfringens.

Biofilm Formation on Stainless Steel by Streptococcus thermophilus UC8547 in Milk Environments Is Mediated by the Proteinase PrtS.

In Streptococcus thermophilus, gene transfer events and loss of ancestral traits over the years contribute to its high level of adaptation to milk environments. Biofilm formation capacity, a phenotype that is lost in the majority of strains, plays a role in persistence in dairy environments, such as milk pasteurization and cheese manufacturing plants. To investigate this property, we have studied S. thermophilus UC8547, a fast-acidifying dairy starter culture selected for its high capacity to form biofilm on stainless steel under environmental conditions resembling the dairy environment. Using a dynamic flow cell apparatus, it was shown that S. thermophilus UC8547 biofilm formation on stainless steel depends on the presence of milk proteins. From this strain, which harbors the prtS gene for the cell wall protease and shows an aggregative phenotype, spontaneous mutants with impaired biofilm capacity can be isolated at high frequency. These mutants lack the PrtS expendable island, as confirmed by comparison of the genome sequence of UC8547Δ3 with that of the parent strain. The prtS island excision occurs between two 26-bp direct repeats located in the two copies of the ISSth1 flanking this genomic island. The central role of PrtS was confirmed by analyzing the derivative strain UC8547Δ16, whose prtS gene was interrupted by an insertional mutation, thereby making it incapable of biofilm formation. PrtS, acting as a binding substance between the milk proteins adhered to stainless steel and S. thermophilus cell envelopes, mediates biofilm formation in dairy environments. This feature provides S. thermophilus with an ecological benefit for its survival and persistence in this environment.IMPORTANCE The increased persistence of S. thermophilus biofilm has consequences in the dairy environment: if, on the one hand, the release of this microorganism from biofilm can promote the fermentation of artisanal cheeses, under industrial conditions it may lead to undesirable contamination of dairy products. The study of the molecular mechanism driving S. thermophilus biofilm formation provides increased knowledge on how an ancestral trait affects relevant phenotypes, such as persistence in the environment and efficiency of growth in milk. This study provides insight into the genetic factors affecting biofilm formation at dairy plants.

Probiotic strains modulate cytokine production and the immune interplay between human peripheral blood mononucear cells and colon cancer cells.

Human health is tightly connected with a great number of gut microbial cells designated as microbiome or microbiota. We have examined the effect of six microbial strains (MS) included in a commercial probiotic on cytokine production by human peripheral blood mononuclear cells (PBMC) and on their immune dialog with colon carcinoma cells. Non-stimulated and lipopolysaccharide (LPS)-stimulated PBMC were incubated for 24 h with MS. The secretion of tumor necrosis factor alpha (TNFα), IL-1ß, IL-6, interferon gamma, IL-10 and IL-1ra and the effect of MS on the immune interplay between PBMC and cells from HT-29 and RKO colon carcinoma lines were evaluated. MS incubated with non-stimulated PBMC induced high expression of all cytokines examined, whereas in those stimulated by LPS, variability in the results was observed. MS added to PBMC co-cultivated with HT-29 or RKO colon cancer cells resulted in downregulation of most cytokines except IL-6 and TNFα, and enhanced production of TNFα and IL-1ß. The MS incorporated in the probiotic affected PBMC' cytokine production and altered the cross-talk between immune and colon cancer cells. The results may clarify the way by which probiotics modify the intestinal environment resulting in a decline of colon cancer development.

Acid production and conversion of konjac glucomannan during in vitro colonic fermentation affected by exogenous microorganisms and tea polyphenols.

Impacts of exogenous microorganisms and tea polyphenols on acid production and conversion during in vitro colonic fermentation of konjac glucomannan (KGM) were assessed in this study. Colonic fermentation of KGM by the fecal extract of healthy adults resulted in a propionate-rich profile, as acetic, propionic, butyric and lactic acids production were 16.1, 13.0, 3.3 and 20.2 mmol/L, respectively. Inoculation of one of ten exogenous microorganisms in the fermentative systems increased acetic, propionic and butyric acids production by 50-230%, 9-190% and 110-350%, respectively, and also accelerated lactic acid conversion by 14-40%. Tea polyphenols in the fermentative systems showed clear inhibition on both acid production and conversion; however, this inhibition could be partially or mostly antagonised by the inoculated exogenous microorganisms, resulting in improved acid production and conversion. In total, Lactobacillus brevis and Sterptococcus thermophilus were more able to increase acid production, and the propionate-rich profile was not changed in all cases.

Implication of sortase-dependent proteins of Streptococcus thermophilus in adhesion to human intestinal epithelial cell lines and bile salt tolerance.

Streptococcus thermophilus (ST) is a lactic acid bacterium widely used in dairy industry and displays several properties which could be beneficial for host. The objective of this study was to investigate, in vitro, the implication of sortase A (SrtA) and sortase-dependent proteins (SDPs) in the adhesion of ST LMD-9 strain to intestinal epithelial cells (IECs) and resistance to bile salt mixture (BSM; taurocholoate, deoxycholate, and cholate). The effect of mutations in prtS (protease), mucBP (MUCin-Binding Protein), and srtA genes in ST LMD-9 in these mechanisms were examined. The HT29-MTX, HT29-CL.16E, and Caco-2 TC7 cell lines were used. HT29-MTX and HT29-CL.16E cells express different mucins found in the gastro intestinal tract; whereas, Caco-2 TC7 express cell surface proteins found in the small intestine. All mutants showed different adhesion profiles depending on cell lines. The mutation in genes srtA and mucBP leads to a significant decrease in LMD-9 adhesion capacity to Caco-2 TC7 cells. A mutation in mucBP gene has also shown a significant decrease in LMD-9 adhesion capacity to HT29-CL.16E cells. However, no difference was observed using HT29-MTX cells. Furthermore, ST LMD-9 and srtA mutant were resistant to BSM up to 3 mM. Contrariwise, no viable bacteria were detected for prtS and mucBP mutants at this concentration. Two conclusions could be drawn. First, SDPs could be involved in the LMD-9 adhesion depending on the cell lines indicating the importance of eukaryotic-cell surface components in adherence. Second, SDPs could contribute to resistance to bile salts probably by maintaining the cell membrane integrity.

Prophage-mediated modulation of interaction of Streptococcus thermophilus J34 with human intestinal epithelial cells and its competition against human pathogens.

The human intestinal microbiota plays an important role in human health. While adhesion to gastrointestinal mucosa is a prerequisite for colonisation, inhibition of adhesion is a property which may prevent or reduce infections by food borne pathogens. Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus represent the two lactic bacteria constituting the yoghurt culture. These starter cultures have been claimed to be probiotic. In our study we compared two S. thermophilus strains (i.e. lysogenic strain J34 and corresponding non-lysogenic [prophage-cured] strain J34-6), with respect to (1) their in vitro adhesion properties to HT29 cells and (2) their cell surface hydrophobicities. Effects of the two strains on inhibition of adhesion of the pathogens Listeria monocytogenes Scott A, Staphylococcus aureus 6732 and Salmonella enteritidis S489 were studied in vitro with HT29 cell cultures. Lysogenic strain J34 was shown to be considerably more effective than the non-lysogenic derivative strain J34-6.

[Investigation of the effects of probiotic bacteria on bacterial translocation that developed during diagnostic laparoscopy: an experimental study]. / Tanisal Amaçla Uygulanan Laparoskopi Sirasinda Olusan Bakteriyel Translokasyona Probiyotik Bakterilerin Etkilerinin Arastirilmasi: Deneysel Bir Çalisma.

Probiotics which are non-pathogenic live microorganisms ingested along with food or as dietary supplements, are thought to be beneficial to the host by supporting the microbial balance in digestive system. Various studies suggest that the effects of probiotics on the intestinal mucosa and immunity are protective against bacterial translocation. We aimed to investigate bacterial translocation related to the amount of CO2 insufflation given during laparoscopy and the effect of probiotic bacteria in an experimental peritonitis model. In this study 60 Wistar rats were used in six groups consisting of 10 rats. Group 1, 3 and 5 consisted of the rats that were fed without probiotics, while the rats in Group 2, 4, and 6 were fed with water containing 5 x 108 cfu/ml probiotic bacteria complex (Bifidobacterium lactis, Lactobacillus bulgaricus, Streptococcus thermophilus) for 15 days. To generate experimental peritonitis, 2 x 107 cfu/ml Escherichia coli ATCC 25922 was inoculated intraperitoneally to all of the rats. Thereafter, laparoscopy was applied in all groups. Application in Group 1 and Group 2 was without CO2; Group 3 and Group 4 with 14 mmHg CO2 insufflation, and Group 5 and Group 6 with 20 mmHg CO2 insufflation. Blood samples were taken in 2nd, 4th, and 6th hours. Mesenteric lymph node, liver and spleen samples were taken at 6th hour when the rats were sacrificed and then these were evaluated microbiologically with qualitative and quantitative methods. Bacterial translocation and bacteremia were found in the rats that were undergone experimental peritonitis during laparoscopy. All positive tissue and blood cultures yielded E.coli. The highest level of bacterial translocation was found to be in mesenteric lymph nodes (in 3/10, 6/10 and 10/10 in groups 1, 3 and 5 fed without probiotics, respectively; in 2/10, 3/10 and 4/10 in groups 2, 4 and 6 fed with probiotics, respectively). The bacterial translocation rates were found to be related to the increased CO2 insufflation. It was found that probiotic bacteria were more effective for decreasing bacterial translocation rates and bacteremia in the groups that were given high CO2 pressure during laparoscopy. It was also found that these results were correlated with bacterial translocation per gram of tissue. As an example, the quantitative bacterial growth values detected in mesenteric lymph node were 5.4 ± 2.9 x 103, 10.6 ± 3.3 x 103 and 21.5 ± 12.4 x 103 cfu/g in groups 1, 3 and 5, fed without probiotics, respectively; and 2.0 ± 1.3 x 103, 3.8 ± 1.9 x 103 and 9.0 ± 3.1 x 103 cfu/g in groups 2, 4 and 6, fed with probiotics, respectively. Our data emphasized that probiotic bacteria may be used as prophylactic agents for the prevention of bacterial translocation during laparoscopy, however comprehensive and clinical studies are needed to support these experimental results.

A newly established bovine intestinal epithelial cell line is effective for in vitro screening of potential antiviral immunobiotic microorganisms for cattle.

We evaluated whether a bovine intestinal epithelial (BIE) cell line could serve as a useful in vitro model system for studying antiviral immune responses in bovine intestinal epithelial cells (IECs) and for the primary screening of immunobiotic microorganisms with antiviral protective capabilities. Immunofluorescent analyses revealed that toll-like receptor 3 (TLR3) was expressed in BIE cells, and the results of real-time quantitative PCR showed that these cells respond to stimulation with poly(I:C) by up-regulating pro-inflammatory cytokines and type I interferons. In addition, we demonstrated that BIE cells are useful for the primary screening of immunobiotic lactic acid bacteria strains which are able to beneficially modulate antiviral immune responses triggered by TLR3 activation in bovine IECs. The characterization of BIE cells performed in the present study represents an important step towards the establishment of a valuable bovine in vitro system that could be used for the development of immunomodulatory feed for bovine hosts.

A novel pheromone quorum-sensing system controls the development of natural competence in Streptococcus thermophilus and Streptococcus salivarius.

In streptococcal species, the key step of competence development is the transcriptional induction of comX, which encodes the alternative sigma factor sigma(X), which positively regulates genes necessary for DNA transformation. In Streptococcus species belonging to the mitis and mutans groups, induction of comX relies on the activation of a three-component system consisting of a secreted pheromone, a histidine kinase, and a response regulator. In Streptococcus thermophilus, a species belonging to the salivarius group, the oligopeptide transporter Ami is essential for comX expression under competence-inducing conditions. This suggests a different regulation pathway of competence based on the production and reimportation of a signal peptide. The objective of our work was to identify the main actors involved in the early steps of comX induction in S. thermophilus LMD-9. Using a transcriptomic approach, four highly induced early competence operons were identified. Among them, we found a Rgg-like regulator (Ster_0316) associated with a nonannotated gene encoding a 24-amino-acid hydrophobic peptide (Shp0316). Through genetic deletions, we showed that these two genes are essential for comX induction. Moreover, addition to the medium of synthetic peptides derived from the C-terminal part of Shp0316 restored comX induction and transformation of a Shp0316-deficient strain. These peptides also induced competence in S. thermophilus and Streptococcus salivarius strains that are poorly transformable or not transformable. Altogether, our results show that Ster_0316 and Shp0316, renamed ComRS, are the two members of a novel quorum-sensing system responsible for comX induction in species from the salivarius group, which differs from the classical phosphorelay three-component system identified previously in streptococci.

Probiotic, as well as conventional yogurt, can enhance the stimulated production of proinflammatory cytokines.

BACKGROUND: Lactic acid bacteria have been shown to stimulate the secretion of cytokines by lymphocytes and monocytes in a strain-dependent manner. Therefore, in this study, the effect of a daily intake of probiotic yogurt on cytokine production in young healthy women was compared with that of a conventional product. METHODS: For 2 weeks each, subjects consumed 100 g, then 200 g of either a probiotic or a conventional, commercially available yogurt, both containing Lactobacillus bulgaricus and Streptococcus thermophilus with additional Lactobacillus casei DN 114 001 in the probiotic product. Cytokine production in blood culture following stimulation with phytohaemmaglutinin and lipopolysaccharide was measured using Cytometric Bead Array and enzyme-linked immunosorbent assay. RESULTS: Stimulated production of tumour necrosis factor-alpha increased significantly following consumption of conventional or probiotic yogurt (+63% and +24% compared with baseline, respectively, P < 0.001). There was also a significantly higher production of interleukin (IL)-1beta in the conventional (+40%, P = 0.006) and of interferon gamma in the probiotic group (+108%, P < 0.05). IL-10 decreased following consumption of the probiotic product, but increased significantly after intake cessation (+129%, P < 0.001). No significant differences in cytokine responses between the conventional and the probiotic yogurt were observed. CONCLUSION: Both conventional and probiotic yogurt enhanced the stimulated production of pro-inflammatory cytokines.

Bebida fermentada probiótica de soro de leite / Probiotic drink of milk serum

O presente trabalho propôs desenvolver uma bebida composta por soro de leite fermentado com microrganismos probióticos, saborizada com polpa de frutas, caracterizando-a em seus aspectos físico-químicos, microbiológicos e sensoriais. Foram elaborados três tratamentos diferidos apenas pelo tipo de fermento lácteo empregado. Os resultados mostraram a viabilidade tecnológica de apenas um tratamento composto por fermento YC-X11, contendo cepas mistas de L.delbrueckii subsp. bulgaricus e S.salivarius subsp. thermophilus, o qual foi submetida a análise físico-químicas e microbiológicas para caracterização do produto e posterior avaliação sensorial pelo método de escala hedônica. Obteve-se uma bebida probiótica inovadora, de excelente valor nutritivo, dentro dos parâmetros de identidade microbiológica e físico-química e de grande aceitação pelo consumidor final.