Hypercholesterolemia has been reported to be the main cause of cardiovascular diseases and the leading cause of death. Therefore, decreasing serum cholesterol level is very important for preventing the cardiovascular diseases. It has been supposed that probiotics in human gastrointestinal tract have the ability to decrease serum cholesterol level by reducing the absorption of cholesterol from the intestinal tract and the bile salt deconjugation. In this study, 28 strains of Lactobacillus spp., isolated from breast-fed infant's feces, were identified and investigated for their bile salt deconjugation ability. The deconjugation ability of the strains was determined by the release of cholic acid resulting from the deconjugation of conjugated bile salts. Research results showed that four of the strains had bile salt deconjugation ability. The strains with deconjugation ability have been identified in species level by using biochemical test, and molecular techniques, API 50CHL test and 16S rRNA gene sequence analysis respectively. LP1, E3, and E9 strains with deconjugation activity were identified as Lactobacillus rhamnosus and GD2 strain as Lactobacillus plantarum. Even if oxgall decreases the viability of bacteria, the highest amount of cholesterol precipitation (42%) was performed by GD2 strain in the presence of 0.3% (w/v) bile. This study demonstrated that the identified Lactobacillus strains had an excellent ability to survive at low pH, a high bile deconjugation ability, and hypocholesterolemic effect in in vitro conditions.
In this study, determination of enterococcus species that were isolated from mastitic milk samples, investigation of their susceptibilities to antibiotics and identifi cation of the existence of resistance genes in resistant strains were conducted. The specimens consist of 600 mastitic milk samples that were collected from 242 cows. Isolation of enterococcus was carried out in selective media and 94 (15.6%) Enterococcus spp. were isolated. A total of 94 species of Enterococci were identifi ed using both sequencing and polymerase chain reaction (PCR). Enterococcus spp. isolates belong to 5 different species (E. faecalis, E. faecium, E. durans, E. hirae, E. mundtii) in sequence analysis and 4 different species (E. faecalis, E. faecium, E. durans, E. hirae) were identify by PCR method with specifi c primers. Analyzing 94 enterococcus strains by antibiotic sensitiveness test a high rate of resistance to tetracycline in 77 (81.9%) isolates was shown. The tet resistance genes were identifi ed as follows: 54 were tetM positive, 23 were tetK positive and 17 were positive on tetM and tetK. Resistance to erythromycin was established in 27 (28.7%) isolates (25 ermB) while the chloramphenicol resistance gene was found in 10 (10.7%) of isolates and the cat gene was identifi ed in nine samples and one isolate was resistant to vancomycin (1.06%) with the VanA gene confi rmed. In conclusion, it was shown that E. faecalis has the biggest role in enterococcus originated mastitis and these strains were found to be mostly resistant to tetracycline. One vancomycine resistant isolate that had the VanA gene was also determined.
Probiotics are gaining popularity and increasing the importance of their accurate speciation. Lactobacillus species are commonly used as probiotic strains mostly of clinical importance. Present knowledge indicates that at least 14 Lactobacillus species are associated with the human intestinal tract. Currently, researchers are interested in developing efficient techniques for screening and selecting probiotics bacteria, but unfortunately most of these methods are time-consuming, labor-intensive and costly. The aim of this study is to develop reliable, rapid and accurate method to identify 14 references Lactobacillus species that could have been found in the human alimentary tract by 16S ribosomal DNA restriction analysis. In this study, to develop an effective method for the genotype-based identification of the reference Lactobacillus species, 1.5 kb of 16S rRNA nucleotide sequences of 14 Lactobacillus were collected from the Gene Bank aligned, in silico restricted and analyzed in respect to their 16S-rRNA restriction fragment polymorphism. In silico restriction profiles of 16S-rRNA indicated that FspBI, HinfI and DraI restriction enzymes (RE) are convenient for differentiation of 14 Lactobacillus species in human intestinal tract except Lb. casei and Lb. paracasei. The patterns of our experimental findings obtained from 16S PCR-ARDRA completely confirmed our in silico patterns. The present work demonstrated that 16S PCR-ARDRA method with FspBI, HinfI and DraI RE is a rapid, accurate and reliable method for the identification of Lactobacillus species from human alimentary tract, especially during the identification of large numbers of isolates and any laboratory equipped with a thermo cycler for probiotic use.
Cytochrome cbb (3) oxidase, a member of the heme-copper oxidase superfamily, catalyses the reduction of oxygen to water and generates a proton gradient. Cytochrome c oxidases are characterized by a catalytic subunit (subunit I) containing two hemes and one copper ion ligated by six invariant histidine residues, which are diagnostic of heme-copper oxidases in all type of the heme-copper oxidase superfamily. Alignments of the amino acid sequences of subunit I (FixN or CcoN) of the cbb (3)-type oxidases show that catalytic subunit also contains six non-canonical histidine residues that are conserved in all CcoN subunits of the cbb (3) oxidase, but not the catalytic subunits of other members of heme-copper oxidases superfamily. The function of these six CcoN-specific conserved histidines of cbb (3)-type oxidase in R. capsulatus is unknown. To analyze the contribution of the two invariant histidines of CcoN, H300 and H394, in activity and assembly of the Rhodobacter capsulatus cbb (3)-type oxidase, they were substituted for valine and alanine, respectively by site-directed mutagenesis. H300V and H394A mutations were analyzed with respect to their activity and assembly. It was found that H394A mutation led to a defect in the assembly of both CcoP and CcoO in the membrane, which results in almost complete loss of activity and that although the H300V mutant is normally assembled in the membrane and retain their stability, its catalytic activity is significantly reduced when compared with wild-type oxidase.
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