Igor Štyriak

Probiotics in prevention and treatment of obesity: a critical view.

The worldwide prevalence of obesity more than doubled between 1980 and 2014. The obesity pandemic is tightly linked to an increase in energy availability, sedentariness and greater control of ambient temperature that have paralleled the socioeconomic development of the past decades. The most frequent cause which leads to the obesity development is a dysbalance between energy intake and energy expenditure. The gut microbiota as an environmental factor which influence whole-body metabolism by affecting energy balance but also inflammation and gut barrier function, integrate peripheral and central food intake regulatory signals and thereby increase body weight. Probiotics have physiologic functions that contribute to the health of gut microbiota, can affect food intake and appetite, body weight and composition and metabolic functions through gastrointestinal pathways and modulation of the gut bacterial community.

Binding of extracellular matrix molecules by probiotic bacteria.

Aims: The aim of this study was to investigate extracellular matrix (ECM) and mucin binding of selected bacterial isolates with probiotic features in comparison with commercially used probiotic bacteria.

Binding of selected extracellular matrix proteins to enterococci and Streptococcus bovis of animal origin

Abstract. Thirty-three enterococcal strains and 10 Streptococcus bovis strains were investigated for their protein-binding cell surface components. Seven extracellular matrix (ECM) proteins were immobilized on Difco latex beads to detect these components on the surface of all enterococcal strains and eight non-autoaggregating S. bovis strains by a particle agglutination assay (PAA). Twenty-three selected strains were also examined in microtiter plate assays. According to the absorbance readings (A570nm), 11 strains were classified as nonadherent (A570nm < 0.1), 10 strains as weakly adherent (0.1 < A570nm > 0.3), and 2 strains as strongly adherent (A570nm > 0.3) in these assays. A direct correlation was found between the values obtained in PAA and A570nm readings of microtiter plate assays. Binding of 125I-labeled bovine lactoferrin to enterococci and streptococci was in the range of 6%–30% and of 125I-labeled human vitronectin in the range of 9%–33% to streptococci. The binding of 125I-labeled ECM proteins to selected strains was much more effectively inhibited by sulfated carbohydrates than by non-sulfated hyaluronic acid, indicating the importance of the sulfate groups of these inhibitors. An inhibition effect of heparin on bLf binding to four selected strains was higher in comparison with fucoidan in the microtiter plates. Thirty-five out of 44 strains had agglutinated rabbit erythrocytes. However, these strains showed no ability to agglutinate bovine or sheep erythrocytes.

Potential of enterococci isolated from horses.

Faecal samples of 122 horses (from farms in Slovakia) were examined to select enterococci to study their probiotic potential for their further use as additives. Each gram of faeces contained 1.0-5.0 cfu (log 10) of enterococci. Of the 43 isolates, 25 (58.1%) were identified as Enterococcus faecium, 3 strains were (6.9%) Enterococcus mundtii and one strain was identified as E. faecalis. Fourteen isolates were not characterized further. A significant proportion of the isolates were resistant to kanamycin, vancomycin and gentamicin. Low urease activity of enterococci dominated. The values of lactic acid ranged from 0.98 to 1.91 mmol/L. Porcine fibronectectin and bovine lactoferrin were bound weakly by tested enterococci, while bovine fibrinogen was bound more strongly. Enterococci from horses did not bind bovine apotransferrin. The isolates adhered with the same ability to human as well as to canine mucus. At least one enterocin gene was detected among 16 analyzed isolates. Ent B gene was detected in all strains tested (16, 100%), followed by the genes ent A, ent P and ent L50B. Three suitable candidates-the strains of E. faecium EF 412, EF 462 and EF 491 were selected for further detail studies and possibilities to be used as additives.

Biodestruction and deferritization of quartz sands by Bacillus species

Abstract Quartz sand containing mineral impurities was subjected to bioleaching with Bacillus spp. strains and subsequent elutriation. Bioleaching of the sample removed visible Fe-bearing minerals that coated quartz grains. During the bioleaching, elements were dissolved from silicate minerals. Poorly crystalline Fe-oxides that sealed siderite nodules were released from intergranular spaces and formed a fine-grained ( 3+ . However, chemical analysis showed a slight increase in Fe 2 O 3 content and a decrease in the FeO content following three months bioleaching in the Bacillus culture. X-ray diffraction analyses showed the loss of Fe-bearing minerals and mica following the bioleaching and the elutriation of the fine-grained fraction. Transmission electron microscopy and energy-dispersive analysis of X-rays showed nucleation of fine-grained Fe,Al-silicates on bacterial surface after three months of bioleaching.

Binding of extracellular matrix proteins by animal strains of staphylococcal species

All 81 strains of Staphylococcus species isolated mainly from animals express high surface hydrophobicity as a stable property upon cultivation on blood agar. Bovine lactoferrin, human vitronectin, human fibronectin, heparin, human and bovine serum albumin were immobilized on latex beads to detect protein-binding cell surface components of 67 non-autoaggregating staphylococcal strains by a particle agglutination assay. Bovine lactoferrin was bound well by 22 strains (3 or 2) while 15 strains reacted weakly (1) and 30 did not react (0) with the lactoferrin-coated latex beads. The particle agglutination assay showed similar differences among staphylococcal strains in binding other proteins with the exception of human and bovine serum albumins for which 66 of 67 strains were negative (0). The specificity of the agglutination reaction was confirmed by a particle agglutination inhibition assay by preincubating bacterial cells with the protein (lactoferrin, vitronectin, etc.) used subsequently in particle agglutination assay. Autoaggregating strains together with some non-autoaggregating strains were selected for microtitre plate assay. According to absorbance at 570 nm, 14 strains were classified as non-adherent, 16 strains as weakly adherent and 18 strains as strongly adherent to bovine lactoferrin in microtitre plate assays. A direct correlation was found between the absorbance values at 570 nm of microtitre plate binding assay and test values obtained in particle agglutination assay. Binding of bovine lactoferrin to 81 staphylococcal strains as well as of human vitronectin and human fibronectin to a selected number of these strains was studied with radiolabeled (125I-labeled) proteins. Radiolabeled bovine lactoferrin was bound common by all except four strains (7 to 39%). Staphylococcal strains isolated from diseased pigs commonly bound 125I-labeled vitronectin (21 to 42% of the vitronectin added). Binding of vitronectin and lactoferrin was efficiently inhibited by preincubating of staphylococcal cells with sulphated carbohydrate compounds as heparin, dextran sulphate and fucoidan, but not by other non-sulphated highly charged glycoconjugates such as hyaluronic acid.

Binding of extracellular matrix molecules by enterococci

The bacterial surfaces of enterococci are not uniform. This fact is confirmed by several studies and by our results when great differences between individual strains with regard to their cell surface hydrophobicity, binding of eight ECM (extracellular matrix) molecules immobilized on latex beads and four selected ECM molecules in microtiter plates were observed. The strains expressing high binding of ECM molecules (e.g., HJ 18, HJ 23, HJ 24, HJ 26, HJ 28, HJ 36, etc.) were found among Enterococcus faecalis and E. faecium by PAA (particle agglutination assay). On the other hand, weak ECM binders (e.g., HJ 21, HJ 32, HJ 34, HJ 38, HJ 39, HJ 42, HJ 43) were also found. A direct correlation was found between porcine mucin and fetuin binding ability of eight selected strains tested in microtiter plates and by PAA. Moreover, the influence of tunicamycin treatment was different because significant (P < 0.001) blocking effect of tunicamycin was observed with two selected strains (HJ 26 and HJ 36), whereas two strains (HJ 18 and HJ 22) were not significantly affected in their fetuin binding. The treatment of six enterococcal strains with proteolytic enzymes, pronase P, and trypsin, and with sodium metaperiodate also significantly (P < 0.001) decreased their fetuin binding. This suggests that both protein and carbohydrate moieties are involved in the binding of immobilized fetuin. However, the influence of these chemicals on the fetuin binding by individual strains was different.

Isolation and characterization of a new ruminal bacteriophage lytic toStreptococcus bovis

A new bacteriophage, designated F4, was isolated from the ruminal fluid of a calf. The host range of F4 phage was limited to five strains ofStreptococcus bovis out of ten tested on which clear plaques 0.6–1.2 mm in diameter were found. Bacteriophage F4 had an elongated head 75 nm long and 33 nm wide with a noncontractile flexible tail 100 nm in length on average. This phage is defective in the generation of plaques at low multiplicities of infection. Its genome consists of double-stranded linear DNA of 60.38 kb lacking cohesive ends. The F4 DNA was analyzed with 13 restriction enzymes. The restriction enzymes that did not cleave it wereBamHI,EcoRI,PvuI, andSmaI. The circular restriction map was constructed with four restriction endonucleases (XbaI,EcoI,SalI, andBglI).

Partial characterization of Enterococcus faecalis bacteriophage F4

Large Enterococcus faecalis F4 bacteriophage (described earlier) consisting of double-stranded linear DNA of ≈60 kb was characterized. Library was prepared of its random DNA fragments and selected recombinants were sequenced. Three phage essential genes were characterized: DNA polymerase, replicative DNA helicase and a minor capsid protein, showing only limited homology to other known phage encoded genes. The occurrence of these genes among enterococci was determined by PCR method. Only two out of 40 tested isolates possessed all three genes, another three isolates contained at least one of the genes, demonstrating low frequency F4 lysogens among natural enterococcal isolates.

Mode of binding of fibrinogen, fibronectin and iron-binding proteins by animal enterococci

Sixty-two animal enterococci were examined for their binding of bovine fibrinogen, porcine fibronectin, bovine lactoferrin, bovine apotransferrin and human holotransferrin in the particle agglutination assay (PAA). Individual strains expressed binding of selected glycoproteins to various degrees (0, 1, 2, 3), whereas bovine fibrinogen binding of enterococci from goats, rabbits and rodents was the strongest (3) in general. Porcine fibronectin was bound weakly (1 or 2) by enterococci from horses, dogs, poultry, rabbits and rodents, while most of the goat isolates and half of the dog feed isolates did not bind fibronectin (0). Bovine lactoferrin was bound especially by the isolates from rodents and rabbits. Bovine apotransferrin was bound very weakly (1) by only a few isolates. Human holotransferrin was bound to a greater extent than apotransferrin by some isolates from rabbits and rodents. Since multiresistant strains are preferred in our binding studies, enterococci were also examined for their antibiotic resistance pattern. Almost all investigated isolates were resistant at least to one antibiotic. However, some strains displayed resistance to five or six antibiotics of 10 antibiotics tested. In a study of the inhibitory effect of heparin, porcine mucin and hyaluronic acid, the greatest effect was observed after heparin treatment of bacterial cells. These observations, as well as the expression of heparin binding by most strains, may suggest that at least one mode of enterococcal attachment utilizes glycosaminoglycan chains present on the surface of adherent cells.