Identification Of Bifidobacteria Research Articles

Studies on the identification of Bifidobacteria isolated from Human Breast milk of Indian women.

Studies on the identification of Bifidobacteria isolated from Human Breast milk of Indian women.

Contemporary nucleic acid-based molecular techniques for detection, identification, and characterization of Bifidobacterium

ABSTRACT Bifidobacteria are one of the most important bacterial groups found in the gastrointestinal tract of humans. Medical and food industry researchers have focused on bifidobacteria because of their health-promoting properties. Researchers have historically relied on classic phenotypic approaches (culture and biochemical tests) for detection and identification of bifidobacteria. Those approaches still have values for the identification and detection of some bifidobacterial species, but they are often labor-intensive and time-consuming and can be problematic in differentiating closely related species. Rapid, accurate, and reliable methods for detection, identification, and characterization of bifidobacteria in a mixed bacterial population have become a major challenge. The advent of nucleic acid-based molecular techniques has significantly advanced isolation and detection of bifidobacteria. Diverse nucleic acid-based molecular techniques have been employed, including hybridization, target amplification, and fingerprinting. Certain techniques enable the detection, characterization, and identification at genus-, species-, and strains-levels, whereas others allow typing of species or strains of bifidobacteria. In this review, an overview of methodological principle, technique complexity, and application of various nucleic acid-based molecular techniques for detection, identification, and characterization of bifidobacteria is presented. Advantages and limitations of each technique are discussed, and significant findings based on particular techniques are also highlighted.

Identification of bifidobacteria isolated from Asian elephant (Elephas maximus)

Bifidobacteria are considered as one of the key genera in intestinal tracts of animals, and their species composition vary depending on the host. The aim of this study was to identify faecal bifidobacteria from Asian elephants (Elephas maximus), housed in Zoological gardens (Ostrava, Czech Republic). Using culturing, bifidobacteria were found in counts 7.60+/-0.56 log CFU/g. Twenty-six pure strains were isolated from faeces of Asian elephant. The isolates were clustered into two groups according to fingerprinting profiles and fermentation characteristic. Bacteria were identified by a combination of MALDI-TOF MS, PCR methods and sequencing as B. boum (12 isolates) and B. adolescentis (14 isolates). Elephant strains showed different fingerprinting profiles than type and collection strains. Since these two species are frequently isolated from gastrointestinal tract of herbivores, they seem to be typical of animals fed plant diets.

Application of molecular methods to classification and identification of bacteria of the genus Bifidobacterium

The molecular methods currently used in the classification and identification of bifidobacteria are reviewed. The sequencing of the 16S rRNA gene and some other genes considered to be phylogenetic markers is a universal and effective approach for taxonomic characterization of members of the genus Bifidobacterium and to reliable identification of new isolates. Various techniques of obtaining DNA fingerprints (PFGE, RAPD, rep-PCR) are widely used for solving particular problems in identifying bifidobacteria. Bacteria of the genus Bifidobacterium are important organisms in biotechnology and medicine. The research in the field of molecular systematics of bifidobacteria provides a basis not only for the solution of taxonomic problems, but also for monitoring of individual species in the environment and for more detailed study of the genetics and ecology of this group of microorganisms.

The effect of composition of the core region of Escherichia coli K-12 lipopolysaccharides on the surface properties of cells

The methods of molecular systematics currently used in the classification and identification of bifidobacteria are reviewed. The sequencing of the 16S rRNA gene and some other genes considered to be phylogenetic markers is a universal and effective approach to taxonomic characterization of members of the genus Bifidobacterium and to reliable identification of new isolates. Various techniques of obtaining DNA fingerprints (PFGE, RAPD, rep-PCR) are widely used for solving particular problems in identifying bifidobacteria. Bacteria of the genus Bifidobacterium are important organisms in biotechnology and medicine. The research into molecular systematics of bifidobacteria provides a basis not only for the solution of taxonomic problems, but also for monitoring of individual species in the environment and for more detailed study of the genetics and ecology of this group of microorganisms.

Rapid Identification of Bifidobacteria in Dairy Products by Gene-targeted Species-specific PCR Technique and DGGE

In this paper, a rapid and reliable gene-targeted species-specific polymerase chain reaction (PCR) technique based on a two-step process was established to identify bifidobacteria in dairy products. The first step was the PCR assay for genus Bifidobacterium with genus specific primers followed by the second step, which identified the species level with species-specific primer mixtures. Ten specific primer pairs, designed from nucleotide sequences of the 16-23S rRNA region, were developed for the Bifidobacterium species including B. angulatum, B. animalis, B. bifidum, B. breve, B. catenulatum, B. infantis, B. longum, B. minimum, B. subtile, and B. thermophilum. This technique was applied to the identification of Bifidobacterium species isolated from 6 probiotic products, and four different Bifidobacterium spp. (B. bifidum, B. longum, B. infantis, and B. breve) were identified. The findings indicated that the 16S-23S rDNA gene-targeted species-specific PCR technique is a simple and reliable method for identification of bifidobacteria in probiotic products. PCR combined with Denaturing Gradient Gel Electrophoresis (DGGE) for identification of the bifidobacteria was also evaluated and compared with the gene-targeted species-specific technique. Results indicated that for fermented milk products consistency was found for both species-specific PCR and PCR-DGGE in detecting species. However, in some lyophilized products, the bands corresponding to these species were not visualized in the DGGE profile but the specific PCR gave a positive result.

Bifidobacteria as indicators of faecal contamination along a sheep meat production chain

The potential use of bifidobacteria as indicators for faecal contamination was studied along a sheep meat production and processing chain. The levels of bifidobacteria were compared with those of Escherichia coli. Total viable counts were followed along the chain (244 samples). Forty-three per cent of the samples contained bifidobacteria, of which 15% were solely detected using a PCR method based on the hsp60 gene and not by a culture-based method. Bifidobacteria were detected in only three of nine sheep faeces samples using one or the other method. However, carcasses (types C and E) were highly contaminated. These sample types (30% and 28%, respectively) were positive for bifidobacteria and negative for E. coli. The species Bifidobacterium pseudolongum and Bif. thermophilum, isolated from faecal samples, were predominant. Bifidobacterium choerinum were found in C, D, E and F sample types. Bifidobacteria were shown more efficient than E. coli in carcasses samples. The presence of Bif. choerinum suggested a faecal pork contamination. Detection and identification of bifidobacteria, in correlation with E. coli counting, should improve hygiene quality of mutton processing chains.

Enumeration, isolation, and identification of bifidobacteria from dairy products

Six dairy products were tested. Bifidobacteria were enumerated and isolated using TPY agar modified by the addition of mupirocin (100 mg l–1). Isolates were identified to the genus level by the detection of fructose-6-phosphate phosphoketolase (F6PPK) and by the FISH method. Bifidobacteria were characterised using API 50 CHL and API ID 32 A Rapid tests. In addition, grow at 46 °C was tested. Subsequently, all strains were identified to the species level using computer program Bacter. The identification was also carried out by PCR method using genus- and species-specific primers. The bifidobacterial counts in products tested varied from 2.37 to 7.17 log CFU/ml. High selectivity was seen for modified TPY agar from which all isolates were identified as bifidobacteria. While most strains were identified as B. animalis using Bacter program, the same isolates had positive reaction with B. lactis-specific primers. Strain isolated from Rajo yogurt was identified as B. longum by Bacter as well as using PCR, but its bacterial counts were too low. Our results showed, that most of the bifidobacterial strains currently used in food products could be of animal origin.

Enumeration, isolation, and identification of bifidobacteria from infant feces.

Thirty-three fully breast-fed infants aged between 1 and 12 weeks were screened for bifidobacteria in feces. Bifidobacteria counts in most fecal samples determined both by TPY agar and FISH procedure ranged from 10(8) to 10(11) CFU/g. Three infants did not contain any bifidobacteria in their fecal samples. One child was delivered by caesarean section and the other two by normal vaginal delivery. All bifidobacteria-free infants possessed Gram-positive regular rods as a major group of their fecal flora. These bacteria were identified as clostridia using genus-specific FISH probe. In bifidobacteria-positive samples, B. longum (57.9% of the samples) was the most frequently found species, followed by B. adolescentis (31.6%), B. bifidum (21.0%), B. breve (10.5%), B. pseudocatenulatum (5.3%), and B. dentium (5.3%).

Molecular Approaches for the Detection and Identification of Bifidobacteria and Lactobacilli in the Human Gastrointestinal Tract

In this review an overview of various molecular techniques and their application for the detection and identification of bifidobacteria and lactobacilli in the gastrointestinal (GI) tract is presented. The techniques include molecular typing techniques such as amplified ribosomal DNA restriction analysis (ARDRA), randomly amplified polymorphic DNA (RAPD), pulsed field gel electrophoresis (PFGE), ribotyping and community profiling techniques such as PCR coupled to temperature and denaturing gradient gel electrophoresis (PCR-TGGE and PCR-DGGE, respectively). Special attention is given to oligonucleotide probes and primers that target the ribosomal RNA (rRNA) sequences and their use in PCR and different hybridisation techniques such as DNA microarrays and fluorescent in situ hybridisation (FISH). In addition, recent findings based on the molecular studies of bifidobacteria and lactobacilli in the GI-tract are reviewed.

Identification and quantification of Bifidobacterium species isolated from food with genus-specific 16S rRNA-targeted probes by colony hybridization and PCR.

A Bifidobacterium genus-specific target sequence in the V9 variable region of the 16S rRNA has been elaborated and was used to develop a hybridization probe. The specificity of this probe, named lm3 (5'-CGGGTGCTI*CCCACTTTCATG-3'), was used to identify all known type strains and distinguish them from other bacteria. All of the 30 type strains of Bifidobacterium which are available at the German culture collection Deutsche Sammlung von Mikroorganismen und Zellkulturen, 6 commercially available production strains, and 34 closely related relevant strains (as negative controls) were tested. All tested bifidobacteria showed distinct positive signals by colony hybridization, whereas all negative controls showed no distinct dots except Gardnerella vaginalis DSM4944 and Propionibacterium freudenreichii subsp. shermanii DSM4902, which gave slight signals. Furthermore, we established a method for isolation and identification of bifidobacteria from food by using a PCR assay without prior isolation of DNA but breaking the cells with proteinase K. By this method, all Bifidobacterium strains lead to a DNA product of the expected size. We also established a quick assay to quantitatively measure Bifidobacterium counts in food and feces by dilution plating and colony hybridization. We were able to demonstrate that 2.1 x 10(6) to 2.3 x 10(7) colonies/g of sour milk containing bifidobacteria hybridized with the specific nucleotide probe. With these two methods, genus-specific colony hybridization and genus-specific PCR, it is now possible to readily and accurately detect any bifidobacteria in food and fecal samples and to discriminate between them and members of other genera.

Identification of bifidobacteria from dairy products and evaluation of a microplate hybridization method

Sixteen strains of Bifidobacterium isolated from 15 dairy products such as yogurt, cultured milk, butter and cheese were characterized on the basis of phenotypic characteristics and DNA similarities were examined by a microplate hybridization method. Three of the strains were identified as Bifidodobacterium longum, one strain was identified as Bifidobacterium bifidm, and one strain was assigned to the species Bifidobacterium breve on the basis of phenotypic characteristics, and this identification was confirmed by the analysis of DNA similarities. The remaining 11 strains could not be identified by examining their phenotypic characteristics and, contrary to the product label information, these strains were identified as Bifudidobacterium animalis on the basis of DNA similarities. The applicability of the colorimetric hybridization method in micro dilution wells to genetic identification of Bifidobacterium species was also studied.

Ribosomal DNA Sequences of Bifidobacteria: Implications for Sequence-based Identification of the Human Colonic Flora

We sequenced 16S ribosomal DNA from eight Bifidobacterium strains to explore the feasibility of using ribosomal RNA sequence to characterise the human colonic flora. The genus was well defined by this method. Closely related species could be readily distinguished. Ribosomal RNA should be an appropriate target for identification of bifidobacteria in the human colonic flora. Keywords - Bifidobacteria; Ribosomal RNA; 16S ribosomal RNA; Ribosomal DNA; lntestines/microbiology.