Differentiation of bifidobacteria by use of pulsed-field gel electrophoresis and polymerase chain reaction

Roy, Denis; Ward, Pierre; Champagne, Guy

doi:10.1016/0168-1605(95)00013-5

Cited by 83 publications

(60 citation statements)

References 16 publications

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“…Only a limited number of studies have reported the enumeration of bifidobacteria from cecal contents and values ranged from 3 ϫ 10 3 CFU/g for rabbit (35), 5 ϫ 10 6 CFU/ml for human (21), and 4 ϫ 10 9 CFU/g for hen (35). PFGE has previously been found to discriminate between Bifidobacterium strains (29,38), a finding extended in the present study for a wide range of animal derived strains. In addition, PFGE was previously shown to be an effective technique for determining the genomic diversity of bifidobacteria isolated from human feces (17,23).…”

Section: Discussionsupporting

confidence: 65%

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Genomic Diversity and Relatedness of Bifidobacteria Isolated from a Porcine Cecum

et al. 2003

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This study initially involved the isolation of a number of bifidobacteria from either the lumen or the epithelium of a porcine cecum. A total of 160 isolates were selected at random on MRS plates containing cysteine hydrochloride (0.5 g/liter) and mupirocin (50 mg/liter). All were identified as bifidobacteria based on fructose-6-phosphate phosphoketolase activity. Following genomic digestion with the restriction enzyme XbaI and pulsed-field gel electrophoresis (PFGE), the isolates produced 15 distinct macro-restriction patterns. Several of the PFGE patterns differed by only 1, 2, or 3 DNA fragments and were grouped as related patterns into seven PFGE types, termed A through G. The related patterns appeared to show genomic plasticity within the isolates arising from chromosomal mutations or possibly horizontal transfer of plasmids. The relative frequency of each PFGE type was maintained within each cecal sample, with PFGE type E representing approximately 50% of the isolates. Randomly amplified polymorphic DNA PCR, cell morphology, whole-cell protein profiling, 16S ribosomal DNA sequencing, and DNA-DNA hybridization were used to determine if the seven apparently unrelated PFGE types represented genetically distinct isolates. Four groups were identified: PFGE types A, C/D/G, B/E, and F, and these appeared to represent Bifidobacterium minimum, Bifidobacterium pseudolongum subsp. pseudolongum, and Bifidobacterium pseudolongum subsp. globosum and two new species, respectively. The data demonstrate the presence of considerable genomic diversity within a relatively simple bifidobacteria population, consisting of 15 distinct strains representing four groups, which was maintained throughout the porcine cecal contents and epithelial layer.

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Section: Discussionsupporting

confidence: 65%

“…PFGE strain discrimination. Previous studies have reported that PFGE following genomic digestion with the restriction enzyme XbaI can effectively discriminate strains from a number of Bifidobacterium species (29,38). However, no strains of animal origin were included.…”

Section: Resultsmentioning

confidence: 99%

Genomic Diversity and Relatedness of Bifidobacteria Isolated from a Porcine Cecum

et al. 2003

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“…By using PFGE technique the following distinctions have been made among probiotic bacteria species: Bifidobacterium longum and B. animalis (Roy et al, 1996), Lb. casei and Lb.…”

Section: Pulsed-field Gel Electrophoresis (Pfge)mentioning

confidence: 99%

Analyses of Dynamics in Dairy Products and Identification of Lactic Acid Bacteria Population by Molecular Methods

Sofu

2017

Turkish JAF Sci.Tech.

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Lactic acid bacteria (LAB) with different ecological niches are widely seen in fermented meat, vegetables, dairy products and cereals as well as in fermented beverages. Lactic acid bacteria are the most important group of bacteria in dairy industry due to their probiotic characteristics and fermentation agents as starter culture. In the taxonomy of the lactic acid bacteria; by means of rep-PCR, which is the analysis of repetitive sequences that are based on 16S ribosomal RNA (rRNA) gene sequence, it is possible to conduct structural microbial community analyses such as Restriction Fragment Length Polymorphism (RFLP) analysis of DNA fragments of different sizes cut with enzymes, Random Amplified Polymorphic DNA (RAPD) polymorphic DNA amplified randomly at low temperatures and Amplified Fragment-Length Polymorphism (AFLP)-PCR of cut genomic DNA. Besides, in the recent years, non-culture-based molecular methods such as Pulse Field Gel Electrophoresis (PFGE), Denaturing Gradient Gel Electrophoresis (DGGE), Thermal Gradient Gel Electrophoresis (TGGE), and Fluorescence In-situ Hybridization (FISH) have replaced classical methods once used for the identification of LAB. Identification of lactic acid bacteria culture independent regardless of the method will be one of the most important methods used in the future pyrosequencing as a Next Generation Sequencing (NGS) techniques. This paper reviews molecular-method based studies conducted on the identification of LAB species in dairy products.

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“…Several molecular methods that alleviate this inconvenience have recently been described: PCR-Elisa method based on the 16S rRNA to detect the most common Bifidobacterium species in humans (Malinen et al, 2002); pulsed-field gel electrophoresis (PFGE) and PCR targeting the 16S rRNA (Roy et al, 1996;Bonjoch et al, 2004); PCR in denaturing gradient gel electrophoresis (DGGE) targeting the transaldolase gene for identification, detection and enumeration of human Bifidobacterium species (Requena et al, 2002); PCR-RFLP method based on the 16SrRNA to detect the most common species from animal and human origins (Delcenserie et al, 2004;Roy and Sirois, 2000), and real-time quantitative PCR from the 16S or the transaldolase gene (Requena et al, 2002). They have also been used in the detection of human Bifidobacterium species from feces Requena et al, 2002, Mullié et al, 2003Venema and Maathuis, 2003), of bifidobacteria as probiotics (Brigidi et al, 2003;Fasoli et al, 2003) or as fecal indicators in waters (Bernhard and Field, 2000).…”

Section: Introductionmentioning

confidence: 99%

A PCR method for detection of bifidobacteria in raw milk and raw milk cheese: comparison with culture-based methods

Delcenserie

Bechoux

China

et al. 2005

Journal of Microbiological Methods

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Bifidobacteria are well known for their beneficial effects on health and are used as probiotics in food and pharmaceutical products. As they form one of the most important groups in both human and animal feces, their use as fecal indicator organisms in raw milk products has recently been proposed. Bifidobacteria species isolated in humans are different from those isolated in animals. It should therefore be possible to determine contamination origin (human or animal).A method of detecting the Bifidobacterium genus was developed by PCR targeting the hsp60 gene. The genus Bifidobacterium was identified by PCR amplification of a 217-bp hsp60 gene fragment. The degenerated primer pair specific to the Bifidobacterium genus used was tested for it specificity on 127 strains. Sensitivity was measured on artificially contaminated samples. Food can however be a difficult matrix for PCR testing since it contains PCR inhibitors. So an internal PCR control was used. An artificially created DNA fragment of 315 bp was constructed. The PCR detection method was tested on raw milk and cheese samples and compared with three culture-based methods, which comprised enrichment and isolation steps. The enrichment step used Brain Heart Infusion medium with propionic acid, iron citrate, yeast extract, supplemented with mupirocin (BHMup) or not (BH) and the isolation step used Columbia blood agar medium, supplemented with mupirocin (CMup) or not (C). The method using mupirocin at both enrichment and isolation steps and the PCR method performed from the culture in BHMup enrichment medium were shown to be the most efficient. No significant difference was observed in raw milk samples between PCR from BHMup and the culture-based method BHMup/CMup, while a significant difference was noticed between the same methods in raw milk cheese samples, which would favor using PCR.The results suggested that PCR on the hsp60 gene was convenient for a rapid detection of bifidobacteria in raw milk and raw milk cheese samples and that bifidobacteria always present throughout raw milk cheese production could be efficiently used as fecal indicators.

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Differentiation of bifidobacteria by use of pulsed-field gel electrophoresis and polymerase chain reaction

Cited by 83 publications

References 16 publications

Genomic Diversity and Relatedness of Bifidobacteria Isolated from a Porcine Cecum

Genomic Diversity and Relatedness of Bifidobacteria Isolated from a Porcine Cecum

Analyses of Dynamics in Dairy Products and Identification of Lactic Acid Bacteria Population by Molecular Methods

A PCR method for detection of bifidobacteria in raw milk and raw milk cheese: comparison with culture-based methods

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