Isolation and species delineation of genus Bifidobacterium using PCR-RFLP of partial hsp60 gene fragment

Jena, Rajashree; Choudhury, Prasanta Kumar; Puniya, Anil Kumar; Tomar, Sudhir Kumar

doi:10.1016/j.lwt.2017.02.032

Cited by 11 publications

(3 citation statements)

References 63 publications

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“…The antibacterial activity of probiotics against pathogenic bacteria has many different modes, including cell adhesion, nutrient competition, ecological niche competition and the secretion of antibacterial substances (Barbosa et al, 2015; Gonzalez-Ochoa et al, 2017). Bifidobacterium species, as representative probiotics, are widely used in the fields of food fermentation and biomedicine (Jena et al, 2017). In this study, the antibacterial activity of YH68 mainly originated from the CFCS.…”

Section: Discussionmentioning

confidence: 99%

Antibacterial Activity of Bifidobacterium breve Against Clostridioides difficile

Yang

2019

Front. Cell. Infect. Microbiol.

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Bifidobacterium breve (YH68) is widely used in the fields of food fermentation and biomedicine. In this study, we explored the antibacterial activity of the cell free culture supernatant (CFCS) of YH68 against Clostridioides difficile ATCC 9689 (CD) by measuring multiple indexes, including the growth, spores production, toxin A/B production, and the expression levels of the tcdA and tcdB genes of CD. In addition, we examined the changes in major cellular functional groups, structures, permeability, integrity, and the proton motive force (PMF) of the cytoplasmic membrane. The results showed that double-dilution ratio of YH68-CFCS (3 × 10 9 CFU/mL) was the MIC value. The cell density, spores production, and the toxin production of CD treated with YH68-CFCS were lower than that of the control ( p < 0.05). In addition, the gene expression levels of tcdA and tcdB in CD treated with YH68-CFCS were significant downregulated ( p < 0.05). Marked differences were observed in the cell membrane and cell wall by a FT-IR spectroscopy and SEM. Analysis of the cell membrane permeability and integrity of the CD cells revealed that YH68-CFCS induced the leakage of a large amount of intracellular K + , inorganic phosphate, ATP, nucleic acids and proteinaceous substances. Furthermore, PMF analysis indicated that there was a significant change in Δψ and ΔpH. These findings demonstrated that the antibacterial activity of YH68-CFCS against CD involved the inhibition of growth, spore production, toxin production, and virulence genes expression; a consumption of PMF in the cytoplasmic membrane, the formation of pore in the cell membrane, together with the enhanced cell membrane permeability; and, eventually, cell completely disintegration.

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Section: Discussionmentioning

confidence: 99%

Antibacterial Activity of Bifidobacterium breve Against Clostridioides difficile

Yang

2019

Front. Cell. Infect. Microbiol.

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“…Bacterial strains were grown in Bifidobacterium medium (DSMZ-specification) and conditions as described in our previous study. 30 After incubation, the bacterial pellets were collected by centrifugation at 10,000 rpm for15 min, and to it 800 µL of CTAB buffer (Cetyl trimethylammonium bromide) was added and mixed thoroughly. The suspension was kept for 1 h at 70°C and then mixed thoroughly.…”

Section: Genomic Dna Extractionmentioning

confidence: 99%

Applicability of rpoB Gene for PCR-RFLP based Discrimination of Bifidobacterial Species Isolated from Human and Animal Sources

Jena¹,

Choudhury²,

Puniya³

et al. 2022

J. Pure Appl. Microbiol.

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Bifidobacteria are widely used as probiotics for their application in the development of functional food and prophylactic therapy. This has necessitated the development of a molecular approach for the genera to be widely identified up to species and subspecies level. In the current study, PCR-RFLP of the partial RNA polymerase β-subunit (rpoB) gene fragment was evaluated for differential identification of Bifidobacterium species. The rpoB gene partial sequences of 575 bp were amplified from 93 previously identified isolates collected from various sources of human and animal origin along with 12 standard reference strains. The PCR amplified products were digested with three restriction endonucleases HhaI, HinfI and BanI separately. Dendrograms constructed from the patterns of HhaI, were found to be more discriminatory and successfully differentiated all the twelve species and also at sub-species level in between B. longum subsp. longum and B. longum subsp. infantis. However, B. adolescentis and B. pseudocatenulatum group clusters were not separated and represented by one group. The groups were further discriminated by HinfI restriction digestion. A separate combination thereof may be used for inferring the classification of bifidobacterial species targeted on rpoB PCR-RFLP analysis. To our knowledge, this work is the first report based on use of rpoB PCR-RFLP for discrimination of the isolates of genus Bifidobacterium and also provides insights into specific advantages of this method over hsp60 PCR-RFLP in differentiating B. longum subsp. longum and B. longum subsp. infantis.

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“…Molecular confirmation of the isolates as lactobacilli was done after DNA isolation followed by polymerase chain reaction (PCR) amplification using genus specific primers. Genomic DNA was isolated as per the modified methods of Jena et al [14] from MRS broth of respective isolates. Briefly, the bacterial cell suspension was transferred to a 2 mL microcentrifuge tube and pelleted by centrifugation at 8,000×g for 10 min.…”

Section: Genomic Dna Isloationmentioning

confidence: 99%

Conjugated linoleic acid producing potential of lactobacilli isolated from goat (AXB) rumen fluid samples

Tyagi

Kumar

Choudhury

et al. 2020

Asian-Australas J Anim Sci

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Objective: The present investigation was aimed to explore the potential of lactobacilli for conjugated linoleic acid (CLA) production, isolated from rumen fluid samples of lactating goats.Methods: A total of 64 isolates of lactobacilli were obtained using deMan-Rogosa-Sharpe (MRS) agar from rumen fluid of goats and further subjected to morphological and biochemical characterizations. Isolates found as gram-positive, catalase negative rods were presumptively identified as Lactobacillus species and further confirmed by genus specific polymerase chain reaction (PCR). The phylogenetic tree was constructed from the nucleotide sequences using MEGA6.Results: Out of the 64 isolates, 23 isolates were observed positive for CLA production by linoleate isomerase gene-based amplification and quantitatively by UV-spectrophotometric assay for the conversion of linoleic acid to CLA as well as gas chromatography-based assay. In all Lactobacillus species cis9, trans11 isomer was observed as the most predominant CLA isomer. These positive isolates were identified by 16S rRNA gene-based PCR sequencing and identified to be different species of L. ingluviei (2), L.salivarius (2), L. curvatus (15), and L. sakei (4).Conclusion: The findings of the present study concluded that lactic acid bacteria isolated from ruminal fluid samples of goat have the potential to produce bioactive CLA and may be applied as a direct fed microbial to enhance the nutraceutical value of animal food products.

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Isolation and species delineation of genus Bifidobacterium using PCR-RFLP of partial hsp60 gene fragment

Cited by 11 publications

References 63 publications

Antibacterial Activity of Bifidobacterium breve Against Clostridioides difficile

Antibacterial Activity of Bifidobacterium breve Against Clostridioides difficile

Applicability of rpoB Gene for PCR-RFLP based Discrimination of Bifidobacterial Species Isolated from Human and Animal Sources

Conjugated linoleic acid producing potential of lactobacilli isolated from goat (AXB) rumen fluid samples

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