Species of Enterococcus faecalis associated with free-living rodents.

Enterococci from different free-living rodents were isolated and selected. The strains were genotyped and their antibiotic sensitivity and /or resistance, production of lactic acid, urease activity, the presence of enterocin structural genes, plasmid detection, and their binding ability to proteins were determined. Among 24 enterococcal strains, 17 strains were allotted to the species Enterococcus faecalis by tDNA-PCR, 7 strains being not yet identified. Only Enterococcus sp. ES66 possessed the structural genes for the production of enterocins (Ent) A and P. E. faecalis EE61 had the gene for EntL50B. The other isolates were Ent-gene-free. Enterococci were mostly sensitive to antibiotic treatment . The plasmid DNA was detected only in the strain EE97. The average value of lactic acid production reached 896 +/- 90 micromol/L. Most of the strains possessed low ureolytic activity. Enterococci bound very well sub-epithelial proteins, reaching the value of 3 by the particle agglutination assay, especially concerning the heparin, bovine lactoferrin and porcine fibronectin.

Partial characterization of Enterococcus faecalis bacteriophage F4.

Large Enterococcus faecalis F4 bacteriophage (described earlier) consisting of double-stranded linear DNA of approximately 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.

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. METHODS AND RESULTS: ECM molecules were immobilized in microtitre plates (mucin and fetuin) or on the surface of latex beads. Porcine mucin was bound by all 13 probiotic strains tested with important inter-strain differences; however, fetuin binding was similar (weak) for all 14 strains tested. Strongly positive (three) binding of bovine fibrinogen was expressed by strains from fermented food (Lactobacillus rhamnosus GG, L. casei Shirota and L. johnsonii La1) as well as by L. casei L.c., Lactobacillus sp. 2I3 and by L. plantarum LP. The other strains expressed moderate (2) or weakly positive (1) binding of bovine fibrinogen. Strongly positive (3) binding of porcine fibronectin was observed only with two strains; however, all other strains also bound this molecule. Bovine lactoferrin was bound to a higher extent than transferrins. SIGNIFICANCE AND IMPACT OF THE STUDY: Some animal strains (at least L. casei L.c. and Lactobacillus sp. 2I3) are comparable with the commercially used strains with respect to their ECM binding ability. As this feature is important for probiotic bacteria to be able to colonize intestine, these strains should be considered for their wider use in fermented feed (or probiotic preparations) for animals.

Binding of extracellular matrix proteins by lactobacilli.

Ten gut and ten vaginal Lactobacillus strains were investigated for their ability to bind type I collagen (Cn-I) and four selected gut lactobacilli were investigated for their binding to other extracellular matrix (ECM) molecules. Immobilized Cn-I (100 mg/L) in wells of microtitre plates was bound by all 10 autoaggregating vaginal strains and by 3 strains of gut lactobacilli from piglets in the range of A570 readings 0.114-1.806. L. acidophilus strain SV31 was much more adherent than the rest of strains. All four gut lactobacilli tested for binding to other ECM molecules displayed good binding to porcine fibronectin and heparin and some of them bound weakly to fetuin and porcine mucin. No binding of these strains was observed to bovine mucin, bovine fibrinogen and bovine lactoferrin.

Binding of E. coli isolates from pigs with postweaning diarrhea or edema disease to crude intestinal mucin of a weaned pig.

The presence of the fedA (gene coding F18 fimbriae) and genes coding STa and LTI enterotoxins and verotoxin Stx2v was determined in 30 E. coli strains isolated from weaned pigs with postweaning diarrhea (PWD) and edema disease (ED). The fedA gene was detected in 22 strains (73.3%). It was mostly associated with the presence of ST gene determinant (14 from 22 fedA positive strains, 63.6%). Two strains possessed ST/Stx2v or LTI/Stx2v combination of genes for both toxins and two strains were negative for investigated toxin determinants. Among 8 fedA-negative strains, five strains without gene determinants for toxins were detected. All 30 E. coli strains were investigated for their binding to crude intestinal mucin of a weaned pig fixed in wells of microtitre plates. Positive mucin binding was observed in most of strains, however, great differences were shown between individual strains. Nineteen strains were classified as strongly adherent, 10 strains as weakly adherent, and only one nonadherent strain was found. Three E. coli strains, selected among the best mucin binders, bound to mucin in a concentration-dependent manner. A high mucin binding by E. coli strains was observed only after their cultivation on blood agar plates. Their cultivation in LB broth or on McConkey agar plates had negative effect on the mucin binding by these strains. The mucin binding is not restricted by the presence of fedA gene because the strains displaying very good binding are found either among fedA positive (1, 602/2, 4/3, 576/6) or fedA negative (DK 6, DK 8) E. coli strains. E. coli strains with the highest mucin binding ability belong to potential ST producents (strains 1, 602/2, 4/3, 6/2, 602/4) while the strains without genes coding toxin production displayed lower binding to mucin substratum with exception of the strains ZV5 and 13.

The use of yeast for microbial degradation of some selected mycotoxins.

Several biodegradation experiments were carried out using 10 different yeast strains.Saccharomyces spp., Kluyveromyces spp. andRhodotorula spp. were tested for biodegradation of selected mycotoxins (ochratoxin A, nivalenol, deoxynivalenol and fumonisin B1) standardsin vitro. There was confirmed that some yeast strains are able to degrade some mycotoxins. However, great differences between individual strains were observed. Moreover, 12Saccharomyces cerevisiae strains were tested for their potential capability to degrade zearalenone and fumonisins in Sabouraud broth. Two strains were capable to degrade zearalenone totally, one strain decreased the mycotoxin concentration up to 25%, and one strain up to 75% of original amount. Two strains were capable to degrade fumonisins partially.

Lack of GATC sites in the genome of Streptococcus bovis bacteriophage F4.

A strong bias against GATC sites was observed in the genome of phage F4, a lytic Streptococcus bovis bacteriophage. Only three GATC sites were found within the 60.4-kbp genome of this phage. The comparative lack of GATC sequences within the F4 genome was probably not due to dam methylation, as no modification within this site was detected using methylation-sensitive isoschizomer pair restriction endonuclease analysis. The short oligonucleotide composition of available S. bovis DNA sequences suggested the existence of an unknown mechanism for counterselection of GATC sites in S. bovis bacteriophages.

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

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 (A(570nm)), 11 strains were classified as nonadherent (A(570nm) < 0.1), 10 strains as weakly adherent (0.1 < A(570nm) > 0.3), and 2 strains as strongly adherent (A(570nm) > 0.3) in these assays. A direct correlation was found between the values obtained in PAA and A(570nm) readings of microtiter plate assays. Binding of (125)I-labeled bovine lactoferrin to enterococci and streptococci was in the range of 6%-30% and of (125)I-labeled human vitronectin in the range of 9%-33% to streptococci. The binding of(125)I-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.

Collagen (Cn-I) binding by gut lactobacilli.

Four gut Lactobacillus strains displaying the features which make them particularly promising for the preparation of probiotic products were investigated together with 5 fresh isolates and one collection strain of Lactobacillus plantarum for their ability to bind type I collagen (Cn-I). Immobilised Cn-I in microtitre plates was bound only by 3 strains of gut lactobacilli from piglets and the collection strain Lactobacillus plantarum LHI 10 from Prague in range of A570nm readings 0.114-0.221. Six strains (isolates from turkeys and a calf) did not bind Cn-I (A570nm < 0.1) in this assay. An influence of cultivation medium on Cn-I binding was significant (P < 0.001) in all four adherent strains. Significantly higher (P < 0.001) binding of Cn-I was observed for Lactobacillus casei L 81 and Lactobacillus plantarum LHI 10 grown on solid medium (MRS agar) than for MRS broth-grown cells, however, Lactobacillus plantarum L 5 and Lactobacillus fermentum L 435 expressed significantly (P < 0.001) higher Cn-I binding during cultivation in MRS broth. The specificity of the binding was confirmed because the Cn-I binding by lactobacilli after their preincubation with this protein was completely abolished. Three selected inhibitors (fucoidan, heparan sulphate and hyaluronic acid) significantly (P < 0.001) reduced Cn-I binding by the Lactobacillus plantarum L 5 strain. Following up on some earlier strain characteristics, these results suggest that the selected piglets lactobacilli are also able to bind Cn-I and therefore should antagonize collagen niche colonization by various enteropathogens when used for probiotic purposes.

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.

Lack of surface receptors not restriction-modification system determines F4 phage resistance in Streptococcus bovis II/1.

The resistance of Streptococcus bovis strain II/1, the producer of SbvI restriction endonuclease, to F4 phage infection was demonstrated by the double-agar-layer method. Despite the presence of restriction endonuclease SbvI which can cleave F4 phage DNA to numerous fragments in vitro, the evidence that adsorption inhibition is the most important defence mechanism in phage resistance of S. bovis II/1 strain was obtained by adhesion experiments in vivo. Electron microscopy of phage-host mixtures showed many phage particles on the bacterial surface of phage-sensitive S. bovis 47/3 control strain in comparison with no phage particles seen on S. bovis II/1 (phage-resistant) strain surface.

The adherence of three Streptococcus bovis strains to cells of rumen epithelium primoculture under various conditions.

Three Streptococcus bovis strains were tested in biotype assay and examined for the adherence to cells of rumen epithelium primoculture. The adherence pattern of ruminal streptococci in phosphate buffered saline at pH values ranging from 4.1 to 8.5 was determined. Our isolates of Streptococcus bovis strains adhered best at pH 7.0-7.3. To characterize the adhesive determinants, the bacterial cells were exposed to various treatments. Protease treatment dramatically decreased the adherence of all Streptococcus bovis strains, thus suggesting that the determinants responsible for the adherence are largely proteinaceous. Carbohydrates could be also significantly involved in the active sites of bacterial surface because metaperiodate-treated cells adhered much more poorly than control, sodium iodate-treated cells. Addition of carbohydrates (lactose, maltose and saccharose) had no significant effect on the adherence of Streptococcus bovis strains although a slight decrease in the adhesion was detected.

Occurrence of Fusarium sacchari var. subglutinans and its mycotoxin production ability in broiler feed.

Spores of Fusarium sacchari var. subglutinans isolated from broiler feed BR1 were obtained at an average concentration of 1.5/mg in 25% of tested samples. The spore concentration was increased from 1 to 100/mg of solid substrate (BR2; biscuit) or to 1/nL of Sabouraud broth after 3 weeks of cultivation. Mycotoxin analyses of these three substrates showed negative reactions for T-2 toxin and zearalenone but a positive reaction for deoxynivalenol (DON) which was found in concentrations of 5 ppm in Sabouraud broth, 50 ppm in BR2 and 220 ppm in biscuit. Therefore, our F. sacchari isolate appeared to be a DON producer.

Isolation and partial characterization of three rumen Lactobacillus plantarum bacteriophages.

The first isolation of Lactobacillus plantarum bacteriophages from ruminal fluid is reported. Three bacteriophages were characterized on the basis of plaque morphology, host ranges, stability, electron microscopic morphology and DNA restriction endonuclease digestion patterns. They formed clear plaques and are placed in group A of Bradley's scheme and have identical host ranges. Bacteriophages were stable to urea and chloroform. They were relatively thermostable but partially inactivated by rumen fluid and by acetate. DNA restriction analysis showed that phage L20 had different numbers of cleavage sites in comparison with the next two phages.

DAP-decarboxylase activity and lysine production by rumen bacteria.

The last step of pathway of lysine biosynthesis by rumen bacteria was tested. The first measurements of DAP-decarboxylase activity and of lysine production by Megasphera elsdenii, Selenomonas ruminantium, Clostridium spp., Butyrivibrio fibrisolvens and Bacteroides succinogenes as well as the first attempts to increase the lysine production by ruminal streptococci by mutation are described. The highest values were measured in Selenomonas ruminantium (DAP-decarboxylase activity = 146 micrograms DAP.min-1.mg-1 protein and lysine production was 390 micrograms.mg-1 protein) and the lowest values were ascertained in Butyrivibrio fibrisolvens (DAP-decarboxylase activity = 27 micrograms DAP.min-1.mg-1 protein and lysine production was 32 micrograms.mg-1 protein). DAP-decarboxylase activity was increased by mutation especially in Streptococcus bovis, the lysine production in both of tested ruminal streptococci. The potential use of lysine-excreting mutants in calves in future is suggested.

Adherence of ruminal Streptococcus bovis and Lactobacillus strains to primary and secondary cultures of rumen epithelium.

Six strains of rumen Lactobacillus and four Streptococcus bovis strains isolated from rumen wall and fluid samples were examined for the adherence to cells of primary and secondary cultures of ruminal epithelium (REC) prepared from sheep and calf. S. bovis adhered to the keratinized REC. Ruminal lactobacilli did not adhere. The presence of rumen lactobacilli in mixture had no influence on the adherence of S. bovis strains. No difference was observed in the adherence of tested bacteria to epithelial cells of primary or secondary cultures, but adhesion was only detected on keratinized cells.

Preliminary observations of interaction between bacteriophages and Streptococcus bovis bacteria on ruminal epithelium primoculture.

Five Streptococcus bovis strains (47/3, 59/2, 4/1, 46/2 and 44/9) isolated from calf ruminal fluid samples were examined for the adherence to cultured ruminal epithelium cells. Four strains (47/3, 59/2, 4/1 and 46/2) were able to attach to the cultured epithelial cells. However, S. bovis 47/3 strain attached to the target cells in significantly greater numbers than the other strains. Strain 44/9 did not adhere to cells of ruminal epithelium. The adherent bacteria were observed on the surface of differentiated (mainly keratinized) cells of ruminal epithelium primoculture only. The different effect of F4, F5 and F6 bacteriophages was ascertained on S. bovis bacteria adhering to rumen epithelial primoculture. A significant decrease in the number of adherent bacteria was shown after cultivation of strains 47/3 and 4/1 with F6 bacteriophage and of 47/3 strain with F4 phage. The F5 bacteriophage had no significant effect on these bacteria.

Isolation and characterization of two rumen Streptococcus bovis bacteriophages.

A method for the isolation of Streptococcus bovis bacteriophages from ruminal fluid of calves is described. Thirty to 2 x 10(3) phages per ml infecting Streptococcus bovis strains 4/1 and 47/3 were isolated directly from ruminal fluid. Two bacteriophages were characterized on the basis of plaque morphology, host ranges, electron microscopic morphology and DNA restriction endonuclease digestion patterns. The F1 and F3 phages formed clear plaques of different sizes. The plaque size of the F1 phage was about 1-1.5 mm in diameter, while the plaques of the F3 phage were larger (1.5-2.5 mm in diameter). Both phages are placed in group B of Bradley's scheme and have different host ranges. The first isolation of Streptococcus bovis phage DNA is reported. Restriction analysis of their DNAs showed that phages F1 and F3 had different numbers of cleavage sites in their genomes and that they were not identical.