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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.

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.

Aims:  Isolation, characterization and identification of lactic acid bacteria (LAB) from artisanal Zlatar cheese during the ripening process and selection of strains with good technological characteristics. Methods and Results:  Characterization of LAB was performed based on morphological, physiological and biochemical assays, as well as, by determining proteolytic activity and plasmid profile. rep‐polymerase chain reaction (PCR) analysis and 16S rDNA sequencing were used for the identification of LAB. PCR analysis was performed with specific primers for detection of the gene encoding nisin production. Strains Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Enterococcus faecium and Enterococcus faecalis were the main groups present in the Zlatar cheese during ripening. Conclusions:  Temporal changes in the species were observed during the Zlatar cheese ripening. Mesophilic lactobacilli are predominant microflora in Zlatar cheese. Significance and Impact of the Study:  In this study we determined that Zlatar cheese up to 30 days old could be used as a source of strains for the preparation of potential starter cultures in the process of industrial cheese production. As the Serbian food market is adjusting to European Union regulations, the standardization of Zlatar cheese production by using starter culture(s) based on autochtonous well‐characterized LAB will enable the industrial production of this popular cheese in the future.

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.

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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-1β and significantly stimulated the expression of tight junction proteins (zonulin, occludin, and claudin 4), as well as the expression of TGF-β 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.