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The aim of the study was to apply macro-restriction analysis in order to differentiate selected strains of the Bacillus cereus group. Three different restriction enzymes (SfiI, NotI and SmaI) were used during the research process and it was confirmed that SmaI is the best restriction enzyme applied in macrorestriction analysis of the Bacillus cereus group. The study used 89 Bacillus cereus group strains, and an identical restriction pattern generated by restriction enzymes was confirmed among 23 of the studied Bacillus anthracis strains. Two groups of strains (four and two strains) among 19 Bacillus sp. Ba 813 and three groups (three, two and two strains) among 44 strains of Bacillus thuringiensis showed identical restriction profiles. The similarity of studied strains was ascertained on the basis of the Dice coefficient, and confirmed that strains showing the same restriction profiles were identical (100% similarity). The similarity between the remaining strains of the Bacillus cereus group and the Bacillus anthracis strains oscillated between 20% to 78.6%, while the similarity of these strains to each other oscillated from 15.4 % to 100%. Dendrograms were also constructed using the UPGMA method.
W artykule omówiono wpływ różnych zakresów temperatury przechowywania mleka pasteryzowanego na rozwój w nim Bacillus cereus oraz zagadnienia aktywacji, kiełkowania i niszczenia przetrwalników za pomocą zróżnicowanej obróbki termicznej mleka.
The aim of this work was to determine the survival of spores and vegetative cells of enterotoxic strains of B. cereus in a medium (GM-milk) simulating human stomach environment after consumption of milk or dairy products. Gastric medium (GM-milk) was prepared by mixing equal volumes of a gastric electrolyte solution (4.8 g/l NaCl, 1.56 g/l NaHCO₃, 2.2 g/l KCl, 0.22 g/l CaCl₂, 500 U/l pepsin solution [Sigma-Aldrich Chemie GmbH, nr kat. P6887]) with UHT milk (0,5% fat content). The total count of B. cereus after 6-hours’ incubation in GM-milk (pH~2 and pH~4,5) was determined in a MYP [Merck nr kat. 1.05267.]. After incubating the spores or vegetative cells in a pH~4,5 medium, the count remained unchanged in 70% and 30% strains, decreased by about 1.0 log cfu/ml in 20% and 40% strains or decreased by about 2.0 log cfu/cm³ in 10% and 30% strains, respectively. After incubating the spores in a pH~2 medium, the count remained unchanged in 10% strains, decreased by about 1.0 log cfu/cm³ and 2.0 log cfu/cm3 in 40% and 50% of the strains, respectively. In the same medium no survival of any tested strains of vegetative cells in 1 ml was ascertained after 1 hour incubation. The study demonstrated that the survival of B. cereus depended on the pH of the medium simulating the stomach environment and the physiological state of the cells (spores or vegetative forms).
Two PCR-based methods for identification of emetic toxin producing Bacillus cereus strains were developed. The first set of primers cesBF and cesBR allowed for the amplification of cesBI 838 bp long fragment of cereulide biosynthesis operon for cereulide producing strains, or 421 bp long fragment of nonribosomal peptide synthetase (nrps) for emetic toxin non producing strains. Detection of both genes cesBI and nrps was possible in one PCR reaction. Among 24 strains of Cereus group tested, only one named 19W-cesB contained cesBI gene fragment. Bacillus cereus isolate 19W-cesB did not contain of any other genes of nonribosomal peptide synthetases responsible for the synthesis of other low molecular weight peptide toxins. The shdR and shdF second primer set allowed for specific amplification of the other 690 bp long fragment of cesBII gene. Only strain 19W-cesB allowed for the PCR synthesis of appropriate amplicon from all tested strains. Proposed methods may be fast and reliable techniques for detection of Bacillus cereus strains producing cereulide.
Repetitive element polymorphism-PCR (REP-PCR) is one of the tools that has been used to elucidate genetic diversity of related microorganisms. Using the MB1 primer, REP-PCR fingerprints from 110 Bacillus strains within the "B.cereus group" have identified eighteen distinct categories, while other more distantly related bacterial species fell within six additional categories. All Bacillus anthracis strains tested were found to be monomorphic by fluorophore-enhanced REP-PCR (FERP) fingerprinting using the MB1 primer. In contrast, other non- B.anthracis isolates displayed a high degree of polymorphism. Dendrogramic analysis revealed that the non- B.anthracis strains possessing the Ba813 chromosomal marker were divided into two clusters. One of the clusters shared identity with the B.cereus strains examined.
Nine strains belonging to Bacillus cereus group has been isolated from food and environmental samples. Their taxonomic position was confirmed by RFLP analysis of 16S rRNA gene digested with Taq\. The detection of DNA sequences encoding the hemolysin BL complex and enterotoxin NHE, was studied in Bacillus sp. isolates. Set of primers was used to amplify fragment of hblD gene by PCR. For the detection of nheB gene a new primer set was developed which allowed to amplify 273 bp fragment from wide number of strains belonging to B. cereus group. The hblD gene was present in 7 out of 9 isolates whereas nheB gene occurred in all of them. Reference strains of B. cereus LOCK 0807, and B. thuringiensis NCAIM 01262 contained both genes. Strains of B. subtilis ATCC 6633 and B. pumilus LOCK 0814 do not contain both genes. Obtained results showed that B. thuringiensis NCAIM 01262 contains both genes and therefore may be harmful for human beings.
Określono występowanie В. cereus (ogólną liczbę oraz NPL przetrwalników) w mleku surowym, pobieranym od pojedynczych krów (108 prób) z dwóch gospodarstw wielkostadnych o różnym poziomie higieny pozyskiwania mleka oraz w mleku zbiorczym (19 prób) z jednego z tych gospodarstw. W mleku od pojedynczych krów (20 prób) i mleku zbiorczym (19 prób) pozyskiwanym w gospodarstwie o wysokim poziomie higieny doju ogólna liczba B. cereus jak i NPL jego przetrwalników nie przekraczały 2 w 1 cm3. W mleku pozyskiwanym w gospodarstwie o niższym poziomie higieny doju (88 prób od pojedynczych krów) ogólna liczba komórek B. cereus zawierała się w granicach 5,0-6,0-102 j.t.k., a NPL przetrwalników - w granicach 0,018 - > 1,6-102 w 1 cm3.
W pracy badano tworzenie egzopolimerycznych składników biofilmu (EPS) przez trzy szczepy bakterii z gatunku Bacillus cereus. Egzopolimery wydzielane przez po­szczególne szczepy różniły się jakościowo i ilościowo. Wzajemne proporcje polisachary­dów i białek EPS zależały w najwyższym stopniu od dostępności źródła węgla w środo­wisku oraz od obecności dodatkowych powierzchni przylegania bakterii. Biologiczne po­wierzchnie w postaci piór drobiowych stymulowały produkcję polisacharydów przez bakte­rie, natomiast abiotyczne w postaci płytek szklanych indukowały zwiększone wydzielanie białek. W białkowej części biofilmu elektroforetycznie wykazano obecność dodatkowych frakcji białkowych, nieobecnych w warunkach kontrolnych. Tworzenie egzopolimerycznej macierzy przez bakterie uwarunkowane jest genetycznie, ale również w wysokim stopniu zależało od dostępności składników odżywczych, a także od czasu trwania hodowli.
The aim of this study has been to determine the survival ability of B. cereus vegetative cells and spores in media simulating human stomach content after the ingestion of dairy or meat products contaminated with this species. The enterotoxic strain Bacillus cereus 228 isolated from raw milk was the subject of examination in this study. The gastric media (GM) simulating human stomach content used in the study were prepared by mixing: 4.80 g NaCl, 1.56 g NaHCO₃, 2.20 g KCl, 0.22 g CaCl₂ and 25 U/l pepsin solution with sterile UHT milk (0% and 3.2% fat content) or chicken broth in a 1:1 ratio. The pH values of the media were then adjusted and ranged from 2.0 to 4.5. Spore or vegetative cell suspensions of Bacillus cereus were added to GM to obtain the target initial populations of about 6.0 log CFU/ml. Incubation was carried out for 6 h at 37°C under aerobic conditions. The study revealed that B. cereus bacteria entering the human gastrointestinal tract have the ability to survive in the conditions prevailing in the human stomach environment. The survival ability in such conditions depends on the form of the ingested cells (vegetative cells or spores), on pH values, and on the kind of food with which they enter the gastrointestinal tract. B. cereus spores are highly resistant to the acidity of the human stomach environment both in the presence of chyme (at pH ~4.5) and in the absence of chyme (at pH ~2.0), regardless of the kind of food the spores were ingested with. On the other hand, B. cereus vegetative cells show diversified resistance to the conditions in the human stomach environment, depending on the pH (the quantity of chyme) and on what food the bacteria were ingested with. After inoculation with vegetative cells no survival of the vegetative cells was observed in 1 ml of each of the applied gastric media having pH 2.0 and 2.5 in the second hour of incubation. The survival rate of the vegetative cells at pH 4.5 was: 85.4% in GM-milk (3.2% fat content), 34.5% in GM-milk (0% fat content) and 4.5% in GM-chicken.
W artykule omówiono zagadnienia występowania Bacillus cereus w mleku pasteryzowanym w różnych krajach. Scharakteryzowano wady, jakie gatunek ten może powodować podczas dłuższego przetrzymywania mleka spożywczego.
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