Insight into the Postbiotic Potential of the Autochthonous Bacteriocin-Producing Enterococcus faecium BGZLM1-5 in the Reduction in the Abundance of Listeria monocytogenes ATCC19111 in a Milk Model.

This study aimed to explore the probiogenomic characteristics of artisanal bacteriocin-producing Enterococcus faecium BGZLM1-5 and its potential application in reducing Listeria monocytogenes in a milk model. The BGZLM1-5 strain was isolated from raw cow's milk from households in the Zlatar Mountain region. The whole genome sequencing approach and bioinformatics analyses reveal that the strain BGZLM1-5 is non-pathogenic to humans. Bacteriocin-containing supernatant was thermally stable and antimicrobial activity retained 75% of the initial activity compared with that of the control after treatment at 90 °C for 30 min. Antimicrobial activity maintained relative stability at pH 3-11 and retained 62.5% of the initial activity compared with that of the control after treatment at pH 1, 2, and 12. The highest activity of the partially purified bacteriocin was obtained after precipitation at 40% saturation with ammonium sulfate and further purification by mixing with chloroform. Applying 3% and 5% (v/v) of the bacteriocin-containing supernatant and 0.5% (v/v) of the partially purified bacteriocin decreased the viable number of L. monocytogenes ATCC19111 after three days of milk storage by 23.5%, 63.5%, and 58.9%, respectively.

Antioxidant effect of lactic acid bacteria in human bronchial epithelial cells exposed to cigarette smoke.

AIMS: Chronic lung diseases are a major and increasing global health problem, commonly caused by cigarette smoke. We aimed to explore the antioxidant effects of lactic acid bacteria (LAB) against cigarette smoke in bronchial epithelial cells. METHODS AND RESULTS: The antioxidant effects of 21 heat-killed (HK) LAB strains were tested in cigarette smoke-stimulated BEAS-2B cells and 3-D bronchospheres organoids. We showed that HK Lactiplantibacillus plantarum BGPKM22 possesses antioxidant activity against cigarette smoke, resistance to hydrogen peroxide, and free radical neutralizing activity. We demonstrated that HK BGPKM22 inhibited cigarette smoke-induced expression of the Aryl hydrocarbon receptor (AhR) and Nuclear factor erythroid 2 related factor 2 (Nrf2) genes. The cell-free supernatant (SN) of BGPKM22 fully confirmed the effects of HK BGPKM22. CONCLUSIONS: For the first time, we revealed that HK and SN of Lactip. plantarum BGPKM22 possess antioxidant activity and modulate AhR and Nrf2 gene expression in bronchial epithelial cells exposed to cigarette smoke.

Assessment of stability and bioactive compounds in yogurt containing novel natural starter cultures with the ability to promote longevity in Caenorhabditis elegans.

Yogurt represent one of the oldest fermented foods containing viable lactic acid bacteria and many bioactive compounds that could exhibit beneficial effects on human health and train our immune system to better respond to invading pathogens. Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus are commonly used for yogurt preparation under controlled temperature and environmental conditions. In this study, we investigated probiotic features of S. thermophilus BGKMJ1-36 and L. bulgaricus BGVLJ1-21 strains isolated from artisanal sour milk and yogurt by using Caenorhabditis elegans as an in vivo model system. Further, we evaluated content of total fat, saturated fatty acids, proteins, and lactose, as well as vitamins and AA of yogurt prepared from above-mentioned starter cultures during 21 d of storage at 4°C to get insights of final product stability. We showed that S. thermophilus BGKMJ1-36 and L. bulgaricus BGVLJ1-21 strains applied in combination upregulated the expression of autophagy-related genes in C. elegans. Beside autophagy, we observed activation of TIR-1-dependent transcription of lysozyme-like antimicrobial genes involved in the immune defense of C. elegans. Upregulation of these genes strongly correlates with an increase in the longevity of the worms fed with yogurt culture bacteria. Further, we showed that yogurt prepared with S. thermophilus BGKMJ1-36 and L. bulgaricus BGVLJ1-21, as a final product, is rich with vitamin B2 and dominant AA known by their prolongevity properties. Taken together, our study pointed to the beneficial features of the tested starter cultures and yogurt and highlighted their potential to be used as a fermented food with added-value properties.

The Impact of Synbiotic Treatment on the Levels of Gut-Derived Uremic Toxins, Inflammation, and Gut Microbiome of Chronic Kidney Disease Patients-A Randomized Trial.

OBJECTIVE: Altering dysbiotic gut flora through synbiotic supplementation has recently been recognized as a potential treatment strategy to reduce the levels of gut-derived uremic toxins and decrease inflammation. Assessing its efficacy and safety has been the main goal of our randomized, double-blind, placebo-controlled study. METHODS: A total of 34 nondialyzed chronic kidney disease patients, aged ≥18 years, with an estimated glomerular filtration rate between 15 and 45 mL/minute, were randomized either to an intervention group (n = 17), receiving synbiotic (Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium lactis, 32 billion colony forming units per day plus 3.2 g of inulin), or control group (n = 17), receiving placebo during 12 weeks. The impact of treatment on the dynamic of serum levels of gut-derived uremic toxins, total serum indoxyl sulfate, p-cresyl sulfate, and trimethylamine N-oxide, was defined as the primary outcome of the study. Secondary outcomes included changes in the stool microbiome, serum interleukin-6 levels, high-sensitivity C-reactive protein, estimated glomerular filtration rate, albuminuria, diet, gastrointestinal symptom dynamics, and safety. Serum levels of uremic toxins were determined using ultraperformance liquid chromatography. The stool microbiome analysis was performed using the 16S ribosomal ribonucleic acid gene sequencing approach. RESULTS: Synbiotic treatment significantly modified gut microbiome with Bifidobacteria, Lactobacillus, and Subdoligranulum genera enrichment and consequently reduced serum level of indoxyl sulfate (ΔIS -21.5% vs. 5.3%, P < .001), improved estimated glomerular filtration rate (ΔeGFR 12% vs. 8%, P = .029), and decreased level of high-sensitivity C-reactive protein (-39.5 vs. -8.5%, P < .001) in treated patients. Two patients of the intervention arm complained of increased flatulence. No other safety issues were noted. CONCLUSION: Synbiotics could be available, safe, and an effective therapeutic strategy we could use in daily practice in order to decrease levels of uremic toxins and microinflammation in chronic kidney disease patients.

Lactobacillus brevis BGZLS10-17 and Lb. plantarum BGPKM22 Exhibit Anti-Inflammatory Effect by Attenuation of NF-κB and MAPK Signaling in Human Bronchial Epithelial Cells.

Bronchial epithelial cells are exposed to environmental influences, microbiota, and pathogens and also serve as a powerful effector that initiate and propagate inflammation by the release of pro-inflammatory mediators. Recent studies suggested that lung microbiota differ between inflammatory lung diseases and healthy lungs implicating their contribution in the modulation of lung immunity. Lactic acid bacteria (LAB) are natural inhabitants of healthy human lungs and also possess immunomodulatory effects, but so far, there are no studies investigating their anti-inflammatory potential in respiratory cells. In this study, we investigated immunomodulatory features of 21 natural LAB strains in lipopolysaccharide (LPS)-stimulated human bronchial epithelial cells (BEAS-2B). Our results show that several LAB strains reduced the expression of pro-inflammatory cytokine and chemokine genes. We also demonstrated that two LAB strains, Lactobacillus brevis BGZLS10-17 and Lb. plantarum BGPKM22, effectively attenuated LPS-induced nuclear factor-κB (NF-κB) nuclear translocation. Moreover, BGZLS10-17 and BGPKM22 reduced the activation of p38, extracellular signal-related kinase (ERK), and c-Jun amino-terminal kinase (JNK) signaling cascade resulting in a reduction of pro-inflammatory mediator expressions in BEAS-2B cells. Collectively, the LAB strains BGZLS10-17 and BGPKM22 exhibited anti-inflammatory effects in BEAS-2B cells and could be employed to balance immune response in lungs and replenish diminished lung microbiota in chronic lung diseases.

Enterococci from Raw-Milk Cheeses: Current Knowledge on Safety, Technological, and Probiotic Concerns.

The present study is focused on the safety, technological characteristics, and probiotic evaluation of Enterococcus species from different artisanal raw milk dairy products, mainly cheeses with ripening. Apart from proteolytic and lipolytic activities, most enterococci show the ability to metabolize citrate and convert it to various aromatic compounds. Long-ripened cheeses therefore have a specific flavor that makes them different from cheeses produced from thermally treated milk with commercial starter cultures. In addition, enterococci are producers of bacteriocins effective against spoilage and pathogenic bacteria, so they can be used as food preservatives. However, the use of enterococci in the dairy industry should be approached with caution. Although originating from food, enterococci strains may carry various virulence factors and antibiotic-resistance genes and can have many adverse effects on human health. Still, despite their controversial status, the use of enterococci in the food industry is not strictly regulated since the existence of these so-called desirable and undesirable traits in enterococci is a strain-dependent characteristic. To be specific, the results of many studies showed that there are some enterococci strains that are safe for use as starter cultures or as probiotics since they do not carry virulence factors and antibiotic-resistance genes. These strains even exhibit strong health-promoting effects such as stimulation of the immune response, anti-inflammatory activity, hypocholesterolemic action, and usefulness in prevention/treatment of some diseases.

Yogurt Produced by Novel Natural Starter Cultures Improves Gut Epithelial Barrier In Vitro.

Yogurt is a traditional fermented dairy product, prepared with starter cultures containing Streptococcus thermophilus and Lactobacillus bulgaricus that has gained widespread consumer acceptance as a healthy food. It is widely accepted that yogurt cultures have been recognized as probiotics, due to their beneficial effects on human health. In this study, we have characterized technological and health-promoting properties of autochthonous strains S. thermophilus BGKMJ1-36 and L. bulgaricus BGVLJ1-21 isolated from artisanal sour milk and yogurt, respectively, in order to be used as functional yogurt starter cultures. Both BGKMJ1-36 and BGVLJ1-21 strains have the ability to form curd after five hours at 42 °C, hydrolyze αs1-, ß-, and κ- casein, and to show antimicrobial activity toward Listeria monocytogenes. The strain BGKMJ1-36 produces exopolysaccharides important for rheological properties of the yogurt. The colonies of BGKMJ1-36 and BGVLJ1-21 strains that successfully survived transit of the yogurt through simulated gastrointestinal tract conditions have been tested for adhesion to intestinal epithelial Caco-2 cells. The results reveal that both strains adhere to Caco-2 cells and significantly upregulate the expression of autophagy-, tight junction proteins-, and anti-microbial peptides-related genes. Hence, both strains may be interesting for use as a novel functional starter culture for production of added-value yogurt with health-promoting properties.

Diversity of non-starter lactic acid bacteria in autochthonous dairy products from Western Balkan Countries - Technological and probiotic properties.

The aim of this review was to summarize the data regarding diversity of non-starter lactic acid bacteria (NSLAB) isolated from various artisanal dairy products manufactured in Western Balkan Countries. The dairy products examined were manufactured from raw cow's, sheep's or goat's milk or mixed milk, in the traditional way without the addition of commercial starter cultures. Dairy products such as white brined cheese, fresh cheese, hard cheese, yogurt, sour cream and kajmak were sampled in the households of Serbia, Croatia, Slovenia, Bosnia and Herzegovina, Montenegro, and North Macedonia. It has been established that the diversity of lactic acid bacteria (LAB) from raw milk artisanal dairy products is extensive. In the reviewed literature, 28 LAB species and a large number of strains belonging to the Lactobacillus, Lactococcus, Enterococcus, Streptococcus, Pediococcus, Leuconostoc and Weissella genera were isolated from various dairy products. Over 3000 LAB strains were obtained and characterized for their technological and probiotic properties including: acidification and coagulation of milk, production of aromatic compounds, proteolytic activity, bacteriocins production and competitive exclusion of pathogens, production of exopolysaccharides, aggregation ability and immunomodulatory effect. Results show that many of the isolated NSLAB strains had one, two or more of the properties mentioned. The data presented emphasize the importance of artisanal products as a valuable source of NSLAB with unique technological and probiotic features important both as a base for scientific research as well as for designing novel starter cultures for functional dairy food.

GABA-Producing Natural Dairy Isolate From Artisanal Zlatar Cheese Attenuates Gut Inflammation and Strengthens Gut Epithelial Barrier in vitro.

Probiotic bacteria are recognized for their health-promoting properties, including maintenance of gut epithelial integrity and host immune system homeostasis. Taking into account the beneficial health-promoting effects of GABA, the presence of the gadB gene, encoding glutamate decarboxylase that converts L-glutamate to GABA, was analyzed in Lactic Acid Bacteria (LAB) natural isolates from Zlatar cheese. The results revealed that 52% of tested Lactobacillus spp. and 8% of Lactococcus spp. isolates harbor the gadB gene. Qualitative and quantitative analysis of GABA production performed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) revealed the highest GABA production by Lactobacillus brevis BGZLS10-17. Since high GABA-producing LAB natural isolates are the most valuable source of naturally produced GABA, the probiotic properties of BGZLS10-17 were characterized. This study demonstrated high adhesion of BGZLS10-17 strain to Caco-2 cells and the ability to decrease the adhesion of Escherichia coli ATCC25922 and Salmonella enterica C29039. Treatment of differentiated Caco-2 cells monolayer with BGZLS10-17 supernatant containing GABA alleviated inflammation (production of IL-8) caused by IL-1ß and significantly stimulated the expression of tight junction proteins (zonulin, occludin, and claudin 4), as well as the expression of TGF-ß cytokine leading to the conclusion that immunosuppression and strengthening the tight junctions can have significant role in the maintenance of intestinal epithelial barrier integrity. Taken together the results obtained in this study support the idea that using of GABA producing BGZLS10-17 probiotic strain could be a good strategy to modulate immunological response in various inflammatory diseases, and at the same time, it could be a good candidate for adjunct starter culture for production of GABA-enriched dairy foods and beverages offering new perspectives in designing the novel functional foods.

The Influence of Heat-Killed Enterococcus faecium BGPAS1-3 on the Tight Junction Protein Expression and Immune Function in Differentiated Caco-2 Cells Infected With Listeria monocytogenes ATCC 19111.

Listeria monocytogenes, the common foodborne pathogenic bacteria species, compromises the intestinal epithelial barrier, leading to development of the listeriosis, a severe disease especially among immunocompromised individuals. L. monocytogenes infection usually requires antibiotic treatment, however, excessive use of antibiotics promotes emergence of antibiotic resistance and the destruction of gut microbiota. Probiotics, including lactic acid bacteria (LAB), have been repeatedly proven as an alternative approach for the treatment of various infections. We have analyzed the potential of Enterococcus faecium BGPAS1-3, a dairy isolate exhibiting strong direct antilisterial effect, to modulate the response of differentiated Caco-2 intestinal epithelial cells to L. monocytogenes ATCC 19111 infection. We showed that the molecule with antilisterial effect is a bacterial cell-wall protein that is highly resistant to the high-temperature treatment. When we tested the antilisterial potential of heat-killed BGPAS1-3, we found that it could prevent tight junction disruption in differentiated Caco-2 monolayer infected with L. monocytogenes ATCC 19111, induce antilisterial host response mechanisms, and stimulate the production of protective TGF-ß in intestinal epithelial cells. We also showed that the modulation of MyD88 dependent TLR2 and TLR4 pathways by BGPAS1-3 are involved in host response against L. monocytogenes ATCC 19111. Since heat-killed BGPAS1-3 possess strong antilisterial effects, such postbiotic could be used as a controllable and safe therapeutic.

Oral neonatal antibiotic treatment perturbs gut microbiota and aggravates central nervous system autoimmunity in Dark Agouti rats.

Gut microbiota dysbiosis has been considered the essential element in the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Antibiotics were administered orally to Dark Agouti (DA) rats early in their life with the aim of perturbing gut microbiota and investigating the effects of such intervention on the course of EAE. As a result, the diversity of the gut microbiota was reduced under the influence of antibiotics. Mainly, Firmicutes and Actinobacteria were replaced by Proteobacteria and Bacteroidetes, while decreased proportions of Clostridia and Bacilli classes were accompanied by an increase in Gamma-Proteobacteria in antibiotic-treated animals. Interestingly, a notable decrease in the Helicobacteraceae, Spirochaetaceae and Turicibacteriaceae was scored in antibiotic-treated groups. Also, levels of short chain fatty acids were reduced in the faeces of antibiotic-treated rats. Consequently, aggravation of EAE, paralleled with stronger immune response in lymph nodes draining the site of immunization, and increased inflammation within the CNS, were observed in antibiotic-treated DA rats. Thus, the alteration of gut microbiota leads to an escalation of CNS-directed autoimmunity in DA rats. The results of this study indicate that antibiotic use in early life may have subsequent unfavourable effects on the regulation of the immune system.

Pulmonary Aspergillus fumigatus infection in rats affects gastrointestinal homeostasis.

Microbiota inhabiting mucosal tissues is involved in maintenance of their immune homeostasis. Growing body of evidence indicate that dysbiosis in gut influence immune responses at distal sites including lungs. There are also reports concerning gut involvement with pulmonary injury/inflammation in settings of respiratory viral and bacterial infections. The impact of infections with other microorganisms on gut homeostasis is not explored. In this study, the rat model of sublethal pulmonary infection with Aspergillus fumigatus was used to investigate the effect of fungal respiratory infection on gut immune-mediated homeostasis. Signs of intestinal damage, intestinal and gut-draining lymphoid tissue cytokine responses and gut bacterial microbiota diversity were examined. Intestinal injury, inflammatory cell infiltration, as well as increased levels of intestinal interferon-γ (IFN-γ) and interleukin-17 (IL-17) (as opposed to unchanged levels of anti-inflammatory cytokine IL-10) during the two-week period depict intestinal inflammation in rats with pulmonary A. fumigatus infection. It could not be ascribed to the fungus as it was not detected in the intestine of infected rats. Increased production of pro-inflammatory cytokines by major gut-draining mesenteric lymph nodes point to these lymphoid organs as places of generation of cytokine-producing cells. No changes in spleen or systemic cytokine responses was observed, showing lack of the effects of pulmonary A. fumigatus infection outside mucosal immune system. Drop of intestinal bacterial microbiota diversity (disappearance of several bacterial bands) was noted early in infection with normalization starting from day seven. From day three, appearance of new bacterial bands (unique to infected individuals, not present in controls) was seen, and some of them are pathogens. Alterations in intestinal bacterial community might have affected intestinal immune tolerance contributing to inflammation. Disruption of gut homeostasis during pulmonary infection might render gastrointestinal tract more susceptible to variety of physiological and pathological stimuli. Data which showed for the first time gut involvement with pulmonary infection with A. fumigatus provide the baseline for future studies of the impact of fungal lung infections to gut homeostasis, particularly in individuals susceptible to these infections.

New Insight into Biofilm Formation Ability, the Presence of Virulence Genes and Probiotic Potential of Enterococcus sp. Dairy Isolates.

Enterococci have controversial status due to their emerging role in nosocomial infections and transmission of antibiotic resistance genes, while some enterococci strains are used as probiotics for humans and animals and starter cultures in dairy industry. In order to improve our understanding of factors involved in the safe use of enterococci as potential probiotics, the antibiotic susceptibility, virulence and probiotic traits of 75 dairy enterococci isolates belonging to Enterococcus durans (50), En. faecium (15), En. faecalis (6), En. italicus (3), and En. hirae (1) were evaluated. The results revealed that ciprofloxacin resistance and biofilm formation are correlated with isolates originated from Golija mountain (Serbia), while gelatinase activity was more common in isolates from Prigorje region (Croatia), pointing to uncontrolled use of antibiotics and anthropogenic impact on dairy products' microbiota in these regions. The virulence genes were sporadically present in 13 selected dairy enterococci isolates. Interestingly, biofilm formation was correlated with higher ability of strains to reduce the adhesion of E. coli and Salmonella Enteritidis to HT29-MTX cells. To our knowledge this is the first study reporting the presence of the esp gene (previously correlated with pathogenesis) in dairy enterococci isolates, mostly associated with the genes involved in adhesion property. Hence, the results of this study revealed that the virulence genes are sporadically present in dairy isolates and more correlated to adhesion properties and biofilm formation, implicating their role in gut colonization rather than to the virulence traits.

Promotion of Early Gut Colonization by Probiotic Intervention on Microbiota Diversity in Pregnant Sows.

The aim of this work was to design a novel mixed probiotic culture for piglets and to evaluate its beneficial effect on the piglets' gut health. The possible mechanisms of probiotic activity, such as adhesion, competitive pathogen exclusion and influence on gut microbiota diversity were determined. Mixed probiotic starter culture is composed of three thermophilic lactic acid bacteria (LAB) strains: Lactobacillus helveticus BGRA43, Lactobacillus fermentum BGHI14 and Streptococcus thermophilus BGVLJ1-44. The strains BGVLJ1-44 and BGRA43 showed good technological properties (fast milk curdling, strong proteolytic activity). In addition, the strain BGVLJ1-44 produces exopolysaccharide (EPS), BGHI14 is heterofermentative LAB strain with significant immunomodulatory effect, while the strain BGRA43 showed strong antimicrobial activity against different pathogens and exhibited significantly higher level of adhesion to Caco-2 cells comparing to other two strains. Both lactobacilli strains BGRA43 and BGHI14 (p < 0.05), as well as probiotic combination (p < 0.01) significantly reduced the adhesion of Escherichia coli ATCC25922 to Caco-2 cells, while the strains BGVLJ1-44 (p < 0.01) and BGRA43 (p < 0.05) significantly reduced adhesion of Salmonella 654/7E (veterinary isolate). The results of farm trial revealed that treatment of sows with new fermented dairy probiotic influenced the piglets' gut colonization with beneficial bacteria and reduced the number of enterobacteriaceae in litters from some treated sows (no significant due to high variability among animals). Finally, this is the first study reporting that the treatment of sows with probiotic combination resulted in the improved microbiota diversity in neonatal piglets.

Novel Aggregation Promoting Factor AggE Contributes to the Probiotic Properties of Enterococcus faecium BGGO9-28.

The understanding of mechanisms of interactions between various bacterial cell surface proteins and host receptors has become imperative for the study of the health promoting features of probiotic enterococci. This study, for the first time, describes a novel enterococcal aggregation protein, AggE, from Enterococcus faecium BGGO9-28, selected from a laboratory collection of enterococcal isolates with auto-aggregation phenotypes. Among them, En. faecium BGGO9-28 showed the strongest auto-aggregation, adhesion to components of ECM and biofilm formation. Novel aggregation promoting factor AggE, a protein of 178.1 kDa, belongs to the collagen-binding superfamily of proteins and shares similar architecture with previously discovered aggregation factors from lactic acid bacteria (LAB). Its expression in heterologous enterococcal and lactococcal hosts demonstrates that the aggE gene is sufficient for cell aggregation. The derivatives carrying aggE exhibited the ten times higher adhesion ability to collagen and fibronectin, possess about two times higher adhesion to mucin and contribute to the increase of biofilm formation, comparing to the control strains. Analysis for the presence of virulence factors (cytolysin and gelatinase production), antibiotic resistance (antibiotic susceptibility) and genes (cylA, agg, gelE, esp, hylN, ace, efaAfs , and efaAfm ) showed that BGGO9-28 was sensitive to all tested antibiotics, without hemolytic or gelatinase activity. This strain does not carry any of the tested genes encoding for known virulence factors. Results showed that BGGO9-28 was resistant to low pH and high concentrations of bile salts. Also, it adhered strongly to the Caco-2 human epithelial cell line. In conclusion, the results of this study indicate that the presence of AggE protein on the cell surface in enterococci is a desirable probiotic feature.

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.

EPS-SJ Exopolisaccharide Produced by the Strain Lactobacillus paracasei subsp. paracasei BGSJ2-8 Is Involved in Adhesion to Epithelial Intestinal Cells and Decrease on E. coli Association to Caco-2 Cells.

The aim of this study was to determine the role of an exopolysaccharide produced by natural dairy isolate Lactobacillus paracasei subsp. paracasei BGSJ2-8, in the adhesion to intestinal epithelial cells and a decrease in Escherichia coli's association with Caco-2 cells. Annotation of the BGSJ2-8 genome showed the presence of a gene cluster, epsSJ, which encodes the biosynthesis of the strain-specific exopolysaccharide EPS-SJ, detected as two fractions (P1 and P2) by size exclusion chromatography (SEC) coupled with multi-angle laser light scattering (MALLS) detection. SEC-MALLS analysis revealed that an EPS-SJ(-) mutant (EPS7, obtained by insertion mutagenesis of the glps_2198 gene encoding primary glycosyltransferase) does not produce the P2 fraction of EPS-SJ. Transmission electron microscopy showed that EPS7 mutant has a thinner cell wall compared to the EPS-SJ(+) strain BGSJ2-83 (a plasmid free-derivative of BGSJ2-8). Interestingly, strain BGSJ2-83 showed higher adhesion to Caco-2 epithelial intestinal cell line than the EPS7 mutant. Accordingly, BGSJ2-83 effectively reduced E. coli ATCC25922's association with Caco-2 cells, while EPS7 did not show statistically significant differences. In addition, the effect of EPS-SJ on the proliferation of lymphocytes in gastrointestinal associated lymphoid tissue (GALT) was tested and the results showed that the reduction of GALT lymphocyte proliferation was higher by BGSJ2-83 than by the mutant. To the best of our knowledge this is the first report indicating that the presence of EPS (EPS-SJ) on the surface of lactobacilli can improve communication between bacteria and intestinal epithelium, implying its possible role in gut colonization.

Diversity and antibiotic susceptibility of autochthonous dairy enterococci isolates: are they safe candidates for autochthonous starter cultures?

Enterococci represent the most controversial group of dairy bacteria. They are found to be the main constituent of many traditional Mediterranean dairy products and contribute to their characteristic taste and flavor. On the other hand, during the last 50 years antibiotic-resistant enterococci have emerged as leading causes of nosocomial infections worldwide. The aim of this study was to determine the diversity, technological properties, antibiotic susceptibility and virulence traits of 636 enterococci previously isolated from 55 artisan dairy products from 12 locations in the Western Balkan countries (WBC) of Serbia, Croatia and Bosnia and Herzegovina. All strains were identified both by microbiological and molecular methods. The predominant species was Enterococcus durans, followed by Enterococcus faecalis and Enterococcus faecium. Over 44% of the isolates were resistant to ciprofloxacin and erythromycin, while 26.2% of the isolates were multi-resistant to three or more antibiotics belonging to different families. 185 isolates (29.1%) were susceptible to all 13 of the antibiotics tested. The antibiotic-susceptible isolates were further tested for possible virulence genes and the production of biogenic amines. Finally, five enterococci isolates were found to be antibiotic susceptible with good technological characteristics and without virulence traits or the ability to produce biogenic amines, making them possible candidates for biotechnological application as starter cultures in the dairy industry.

Environmental waters as a source of antibiotic-resistant Enterococcus species in Belgrade, Serbia.

Despite the number of studies on antibiotic-resistant enterococci from Serbian clinical settings, there are no data about environmental contamination with these bacteria. Thus, this study investigated the prevalence of antibiotic-resistant enterococci in Belgrade, Serbia. Enterococcus species collected from ten surface water sites, including a lake, two major river systems, and springs, were tested. Among enterococci, we found single (21.7 %), double (17.4 %), and multiple antibiotic resistance patterns (56.3 %). Vancomycin-resistant strains were not found, indicating that their abundance in Belgrade is tightly linked to clinical settings. The multiple drug-resistant strains Enterococcus faecalis, Enterococcus faecium, and Enterococcus mundtii were frequently detected in the lake during the swimming season and in the rivers near industrial zones. We confirmed the presence of ermB, ermC, ant(6)-Ia, tetM, and tetL and mutations in gyrA genes. The phylogenetic analysis of 16S rRNA gene of E. faecium isolates that harbor esp gene classified them into two groups based on high-bootstraps scores in the tree analysis. Pulsed-field gel electrophoresis analysis of antibiotic-resistant enterococci revealed genomic similarity ranging from 75 to 100 %. This study indicates the importance of anthropogenic impact to the spread of antibiotic-resistant enterococci in environmental waters of Belgrade, Serbia.

Characterization of lactic acid bacteria isolated from artisanal Travnik young cheeses, sweet creams and sweet kajmaks over four seasons.

The aim of this study was to investigate the composition of lactic acid bacteria (LAB) in autochthonous young cheeses, sweet creams and sweet kajmaks produced in the Vlasic mountain region of central Bosnia and Herzegovina near the town of Travnik over a four season period. These three products were made from cow's milk by a traditional method without the addition of a starter culture. Preliminary characterization with phenotype-based assays and identification using rep-PCR with a (GTG)5 primer and 16S rDNA sequence analysis were undertaken for 460 LAB isolates obtained from all the examined samples. Fifteen species were identified as follows: Lactococcus lactis, Lactococcus raffinolactis, Lactococcus garviae, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus helveticus, Enterococcus faecium, Enterococcus durans, Enterococcus faecalis, Enterococcus italicus, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Leuconostoc lactis, Streptococcus thermophilus and Streptococcus mitis. A wide genotypic and phenotypic heterogeneity of the species was observed, particularly within the Lc. lactis strains. In all of the tested dairy products across four seasons, a significantly positive correlation (r = 0.690) between the presence of lactococci and enterococci and a negative correlation (r = 0.722) between the presence of lactococci and leuconostocs were recorded. Forty-five percent of the lactobacilli and 54.4% of the lactococci exhibited proteolytic activity, whereas 18.7% of the total LAB isolates exhibited antimicrobial activity.