Shortening of the Lactobacillus paracasei subsp. paracasei BGNJ1-64 AggLb Protein Switches Its Activity from Auto-aggregation to Biofilm Formation

AggLb is the largest (318.6 kDa) aggregation-promoting protein of Lactobacillus paracasei subsp. paracasei BGNJ1-64 responsible for forming large cell aggregates, which causes auto-aggregation, collagen binding and pathogen exclusion in vitro. It contains an N-terminus leader peptide, followed by six successive collagen binding domains, 20 successive repeats (CnaB-like domains) and an LPXTG sorting signal at the C-terminus for cell wall anchoring. Experimental information about the roles of the domains of AggLb is currently unknown. To define the domain that confers cell aggregation and the key domains for interactions of specific affinity between AggLb and components of the extracellular matrix (ECM), we constructed a series of variants of the aggLb gene and expressed them in Lactococcus lactis subsp. lactis BGKP1-20 using a lactococcal promoter. All of the variants contained a leader peptide, an inter collagen binding-CnaB domain region (used to raise an anti-AggLb antibody), an anchor domain and a different number of collagen binding and CnaB-like domains. The role of the collagen binding repeats of the N-terminus in auto-aggregation and binding to collagen and fibronectin was confirmed. Deletion of the collagen binding repeats II, III and IV resulted in a loss of the strong auto-aggregation, collagen and fibronectin binding abilities whereas the biofilm formation capability was increased. The strong auto-aggregation, collagen and fibronectin binding abilities of AggLb were negatively correlated to biofilm formation

Piacentinu Ennese PDO cheese as reservoir of promising probiotic bacteria

Piacentinu Ennese is a protected designation of origin (PDO) cheese produced in the surrounding area of Enna (Sicily, Italy), using raw ewe’s milk without the addition of any starter cultures. In the present study, the Lactobacillus population of Piacentinu Ennese PDO cheese was in vitro screened in order to select promising probiotic strains to be further used in humans. One hundred and sixty-nine lactic acid bacteria (LAB) were isolated from 90 days ripened cheeses and identified by Rep-PCR genomic fingerprinting, using the (GTG)5-primer, and by MALDI-TOF MS. One hundred and thirteen (113) isolates belonging to QPS-list species were characterized for both safety and functional properties. All tested isolates were considered safe because none showed either gelatinase, DNase, mucinase, or hemolytic activity. Tolerance to lysozyme, bile salts, and acidic conditions, along with ability to survive under simulated gastrointestinal digestion, were observed. In addition, based on antimicrobial activity against pathogens, cell surface characteristics, Caco-2 adhesion abilities, and anti-inflammatory potential, it was possible to confirm the strain-dependent functional aptitude, suggesting that Piacentinu Ennese PDO cheese may be considered a precious source of probiotic candidates

"In vitro" evaluation of some properties in spore former bacteria isolated from calves faeces

Aims: To isolate and evaluate spore-former bacteria for being used as probiotic additives in animal nutrition by their technological features. Study Design: The study was experimental, by using calves’ faeces for spore-forming identification and further evaluation of their “in vitro” probiotic-related properties. Place and Duration of Study: Laboratory of Preventive Microbiology, Centro de Referencia para Lactobacilos (CERELA-CONICET), between June 2013 and November 2013. Methodology: In this work, some Bacillus strains were isolated from calves’ faeces and evaluated for their “in vitro” beneficial characteristics: Surface properties, biosurfactant and emulsification production, and inhibition of pathogens. The antibiotic sensibility was also assayed. Results: Two Bacillus strains were selected, identified by phenotypic and molecular techniques as Bacillus subtilis strains M14 and M12. Spores resulted to be more hydrophobic than vegetative cells. The M14 strain showed biosurfactant and emulsifying properties. Inhibition assays against pathogenic bacteria indicated they inhibit gram-positive microorganisms. The antibiotic susceptibility showed that the two strains were sensitive to the antibiotics assayed, except Bacillus M12 that was resistant to Kanamycin. Conclusion: The results indicate these strains can be further studied for their inclusion in the design of a probiotic product for newborn calves.Fil: Maldonado, Natalia Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Silva de Ruíz, Clara. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; ArgentinaFil: Nader, Maria Elena Fatima. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentin

High mannose-specific lectin Msl mediates key interactions of the vaginal Lactobacillus plantarum isolate CMPG5300

To characterize the interaction potential of the human vaginal isolate Lactobacillus plantarum CMPG5300, its genome was mined for genes encoding lectin-like proteins. cmpg5300.05_29 was identified as the gene encoding a putative mannose-binding lectin. Phenotypic analysis of a gene knock-out mutant of cmpg5300.05_29 showed that expression of this gene is important for auto-aggregation, adhesion to the vaginal epithelial cells, biofilm formation and binding to mannosylated glycans. Purification of the predicted lectin domain of Cmpg5300.05_29 and characterization of its sugar binding capacity confirmed the specificity of the lectin for high-mannose glycans. Therefore, we renamed Cmpg5300.05_29 as a mannose-specific lectin (Msl). The purified lectin domain of Msl could efficiently bind to HIV-1 glycoprotein gp120 and Candida albicans, and showed an inhibitory activity against biofilm formation of uropathogenic Escherichia coli, Staphylococcus aureus and Salmonella Typhimurium. Thus, using a combination of molecular lectin characterization and functional assays, we could show that lectin-sugar interactions play a key role in host and pathogen interactions of a prototype isolate of the vaginal Lactobacillus microbiota

Death and transfiguration in static staphylococcus epidermidis cultures

The overwhelming majority of bacteria live in slime embedded microbial communities termed biofilms, which are typically adherent to a surface. However, when several Staphylococcus epidermidis strains were cultivated in static liquid cultures, macroscopic aggregates were seen floating within the broth and also sedimented at the test tube bottom. Light- and electron microscopy revealed that early-stage aggregates consisted of bacteria and extracellular matrix, organized in sheetlike structures. Perpendicular under the sheets hung a network of periodically arranged, bacteria-associated strands. During the extended cultivation, the strands of a subpopulation of aggregates developed into cross-connected wall-like structures, in which aligned bacteria formed the walls. The resulting architecture had a compartmentalized appearance. In late-stage cultures, the wall-associated bacteria disintegrated so that, henceforth, the walls were made of the coalescing remnants of lysed bacteria, while the compartment-like organization remained intact. At the same time, the majority of strand containing aggregates with associated culturable bacteria continued to exist. These observations indicate that some strains of Staphylococcus epidermidis are able to build highly sophisticated structures, in which a subpopulation undergoes cell lysis, presumably to provide continued access to nutrients in a nutrient-limited environment, whilst maintaining structural integrity

Mobilization of Pollutant-Degrading Bacteria by Eukaryotic Zoospores

This study was supported by the Spanish Ministry of Science and Innovation (CGL2010-22068-C02-01 and CGL2013- 44554-R), the Andalusian Government (RNM 2337), and the CSIC JAE Program (RS). PvW has funding support from the BBSRC and NERC. Thanks are also given to Sara Hosseini of the Uppsala BioCenter, SLU, Uppsala, Sweden for a useful discussion on oomycete zoospores.Peer reviewedPostprin

Chytridiomycosis of marine diatoms : the role of stress physiology and resistance in parasite-host recognition and accumulation of defense molecules

Little is known about the role of chemotaxis in the location and attachment of chytrid zoospores to potential diatom hosts. Hypothesizing that environmental stress parameters affect parasite-host recognition, four chytrid-diatom tandem cultures (Chytridium sp./Navicula sp., Rhizophydium type I/Nitzschia sp., Rhizophydium type IIa/Rhizosolenia sp., Rhizophydium type IIb/Chaetoceros sp.) were used to test the chemotaxis of chytrid zoospores and the presence of potential defense molecules in a non-contact-co-culturing approach. As potential triggers in the chemotaxis experiments, standards of eight carbohydrates, six amino acids, five fatty acids, and three compounds known as compatible solutes were used in individual and mixed solutions, respectively. In all tested cases, the whole-cell extracts of the light-stressed (continuous light exposure combined with 6 h UV radiation) hosts attracted the highest numbers of zoospores (86%), followed by the combined carbohydrate standard solution (76%), while all other compounds acted as weak triggers only. The results of the phytochemical screening, using biomass and supernatant extracts of susceptible and resistant host-diatom cultures, indicated in most of the tested extracts the presence of polyunsaturated fatty acids, phenols, and aldehydes, whereas the bioactivity screenings showed that the zoospores of the chytrid parasites were only significantly affected by the ethanolic supernatant extract of the resistant hosts

Effect of hydrolyzed milk on the adhesion of Lactobacilli to intestinal cells

Milk is an essential part of the human diet and is undoubtedly a major calcium source in human nutrition, accepted well by most individuals. Knowledge on how the components from dairy products support or reduce the adherence of probiotics to the intestinal epithelium is limited. The purpose of this study was to investigate the effect of acid-hydrolyzed milk on the adhesion ability of two potentially probiotic strains (Lactobacillus plantarum S2, Lactobacillus gasseri R) to in vitro human intestinal epithelial model consisting of Caco-2 and mucus-secreting HT29-MTX co-culture. The adhesion of our tested strains L. gasseri and L. plantarum was 4.74 and 7.16%, respectively, when using inoculum of 2 × 108 CFU ml–1. Addition of acid-hydrolyzed milk to co-culture decreased the adherence by 53.7% for L. gasseri R and by 62.2% for L. plantarum S2. The results of this study evidently indicate the potential importance of the food matrix as a factor influencing probiotic colonization of the gut

Protective Yeasts Control V. anguillarum Pathogenicity and Modulate the Innate Immune Response of Challenged Zebrafish (Danio rerio) Larvae

Indexación: Web of ScienceWe investigated mechanisms involved in the protection of zebrafish (Danio rerio) larvae by two probiotic candidate yeasts, Debaryornyces hansenii 97 (Dh97) and Yarrowia Iypolitica 242 (YI242), against a Vibrio anguillarum challenge. We determined the effect of different yeast concentrations (10(4)-10(7) CFU/mL) to: (i) protect larvae from the challenge, (ii) reduce the in vivo pathogen concentration and (iii) modulate the innate immune response of the host. To evaluate the role of zebrafish microbiota in protection, the experiments were performed in conventionally raised and germ free larvae. In vitro co-aggregation assays were performed to determine a direct yeast-pathogen interaction. Results showed that both yeasts significantly increased the survival rate of conventionally raised larvae challenged with V. anguillarum. The concentration of yeasts in larvae tended to increase with yeast inoculum, which was more pronounced for Dh97. Better protection was observed with Dh97 at a concentration of 106 CFU/mL compared to 104 CFU/mL. In germ-free conditions V anguillarum reached higher concentrations in larvae and provoked significantly more mortality than in conventional conditions, revealing the protective role of the host microbiota. Interestingly, yeasts were equally (Dh97) or more effective (YI242) in protecting germ-free than conventionally-raised larvae, showing that protection can be exerted only by yeasts and is not necessarily related to modulation of the host microbiota. Although none of the yeasts co aggregated with V anguillarum, they were able to reduce its proliferation in conventionally raised larvae, reduce initial pathogen concentration in germ-free larvae and prevent the upregulation of key components of the inflammatory/anti-inflammatory response (il1b, tnfa, c3, mpx, and il10, respectively). These results show that protection by yeasts of zebrafish larvae challenged with V anguillarum relates to an in vivo anti-pathogen effect, the modulation of the innate immune system, and suggests that yeasts avoid the host-pathogen interaction through mechanisms independent of co-aggregation. This study shows, for the first time, the protective role of zebrafish microbiota against V. anguillarum infection, and reveals mechanisms involved in protection by two non-Saccharomyces yeasts against this pathogen.http://journal.frontiersin.org/article/10.3389/fcimb.2016.00127/ful

Bacterial Biofilm and its Clinical Implications

Microbial biofilm created huge burden in treatment of both community and hospital infections. A biofilm is complex communities of bacteria attached to a surface or interface enclosed in an exopolysaccharide matrix and protected from unfavorable conditions such as presence of antibiotics, host defense or oxidative stresses. Biofilms are often considered hot spot for horizontal gene transfer among same or different bacterial species. Furthermore, bacteria with increased hydrophobicity facilitate biofilm formation by reducing repulsion between the extracellular matrix and the bacterium. There is a marked increase in the rate of persons nonresponsive to antibiotic therapy for infections of the Urinary Tract (UTIs), burns and upper respiratory tract due to biofilm formations. It is estimated that 90% of nosocomial infections are mediated by biofilm. The role of biofilm in infections has become so great that the treatment of such antibiotic resistance infections is proving difficult and costly to health care systems. The biofilm related infections varied from dental plaque, destruction of prosthetic valve to death of cystic fibrosis patients. This review aims to provide a summary of role of bacterial biofilm and its clinical implications for the patients