RFLP analysis of a PCR-amplified fragment of the 16S rRNA gene as a tool to identify Enterococcus strains

Scheidegger, Erica Miranda Damásio; Fracalanzza, Suely Aparecida Pimenta; Teixeira, Lúcia Martins; Cardarelli-Leite, Paola

doi:10.1590/s0074-02762009000700011

Cited by 25 publications

(21 citation statements)

References 27 publications

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“…Conventional phenotypic characterization of enterococci is laborious and may present problems in the interpretation of the results or even cause errors, because many enterococcal species vary by only one phenotypic trait (23,27,38). Several molecular methods have been used to differentiate Enterococcus species.…”

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confidence: 99%

Identification of Enterococci from Broiler Products and a Broiler Processing Plant and Description of Enterococcus viikkiensis sp. nov

Rahkila

Johansson

Säde

et al. 2011

Appl Environ Microbiol

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In two previous studies dealing with lactic acid bacteria (LAB) from modified-atmosphere-packaged (MAP) broiler products and a broiler processing plant, several isolates remained unidentified. According to 16S rRNA gene sequence analysis, 36 isolates were assigned to the genus Enterococcus. Numerical analysis of combined HindIII and EcoRI ribopatterns of these isolates resulted in species-specific clusters that were congruent with the clusters obtained by both DNA-directed RNA polymerase subunit A (rpoA) and phenylalanyl-tRNA synthetase ␣ chain (pheS) housekeeping gene analyses. In the analyses, a group of five isolates distinct from any known enterococcal species clustered together. The five isolates were positioned in the Enterococcus avium group, with E. devriesei being the closest phylogenetic neighbor. The DNA-DNA hybridization levels with E. devriesei ranged from 28.8 to 54.3% and indicated that these strains represented a novel species. The name Enterococcus viikkiensis sp. nov. is proposed, with strain DSM 24043 T (LMG 26075 T ) being the type strain. Our study demonstrated that the identification of enterococci within the E. avium phylogenetic group demands polyphasic taxonomic approaches. The rpoA and pheS gene similarities (99.0 to 99.2% and 94.3 to 95.4%, respectively) between E. viikkiensis and its closest phylogenetic neighbor, E. devriesei, were higher than those previously reported within the enterococci. In addition, the phenotypic profiles of the species in the E. avium group were also highly similar, and some traits were found to be misleading for enterococci, such as E. viikkiensis does not grow at 45°C. The numerical analysis of combined HindIII and EcoRI ribopatterns was of considerable assistance in distinguishing enterococcal species within the E. avium group.Enterococci are Gram-positive, catalase-negative, facultatively anaerobic cocci typically associated with the gastrointestinal tract of humans and animals. Enterococcus strains are also present in many food products, in fermented food, on plants, and in water and soil (1,14,15,17,22). Enterococcus faecium and Enterococcus faecalis are among the leading causes of nosocomial infections, and the recent increase in antibioticresistant strains has caused serious concern (51). The genus was described in 1984 (39), and on the basis of 16S rRNA gene sequence analysis, several phylogenetic groups have been distinguished (E. faecium, E. faecalis, Enterococcus avium, Enterococcus casseliflavus, Enterococcus dispar, Enterococcus saccharolyticus, and Enterococcus cecorum species groups) (16, 22, 52). Several novel species have recently been described (24,30,41,43,44,45), and 40 Enterococcus species are currently recognized (13) (last full update, 8 September 2010).The identification of enterococcal species is often challenging. Conventional phenotypic characterization of enterococci is laborious and may present problems in the interpretation of the results or even cause errors, because many enterococcal species vary by only one phenotypic trait (...

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mentioning

confidence: 99%

Identification of Enterococci from Broiler Products and a Broiler Processing Plant and Description of Enterococcus viikkiensis sp. nov

Rahkila

Johansson

Säde

et al. 2011

Appl Environ Microbiol

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“…Glycerol cultures of all of the isolates were prepared and stored at -80°C for further analysis. All of the isolated clinical bacterial strains were subjected to identification using biochemical tests and a Vitek® 2-C15 automated system for bacterial identification (BioMerieux Inc., France), according to manufacturer's instructions [11,17].…”

Section: Bacterial Isolation and Identificationmentioning

confidence: 99%

“…RFLP analysis of the isolated E. faecalis strains (n = 21) was performed in two steps including (i) amplification and purification of the 16S rDNA genes of all of the strains, followed by (ii) digestion of the purified 16S rDNA amplicons with ApaI restriction enzyme and analysis of the digests using agarose gel electrophoresis [17].…”

Section: Rflp Analysismentioning

confidence: 99%

“…The gel was placed in ethidium bromide solution (1 μg/mL) for 30 min, and then on an ultraviolet transilluminator to visualize the DNA. The amplified 16S rDNA products were removed from the agarose with a sterile razor blade, and the DNA was purified from the agarose using QIAquick gel Extraction Kits (Qiagen, USA) according to the manufacturer's instructions [17,27].…”

Section: Rflp Analysismentioning

confidence: 99%

“…Briefly, 12.8 μL of nuclease-free water was added to sterile PCR tubes, followed by the addition of 2 μL RE 10 × buffer (Promega, USA), 0.2 μL acetylated BSA (Promega, USA), 3 μL DNA, and 2 μL of ApaI (Promega, USA). The mixtures were mixed by pipetting, centrifuged for a few seconds in a micro-centrifuge and incubated in a water bath at 37°C for 4 h. The ApaI digestion products of the 16S rDNA amplicons were analyzed using 2% agarose gel electrophoresis, and the gels were stained with ethidium bromide and photographed [17,27].…”

Section: Rflp Analysismentioning

confidence: 99%

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RFLP analysis of a PCR-amplified fragment of the 16S rRNA gene as a tool to identify Enterococcus strains

Abstract: Restriction fragment length polymorphism (RFLP) analysis of a PCR

Cited by 25 publications

References 27 publications

Identification of Enterococci from Broiler Products and a Broiler Processing Plant and Description of Enterococcus viikkiensis sp. nov

Identification of Enterococci from Broiler Products and a Broiler Processing Plant and Description of Enterococcus viikkiensis sp. nov

Clonal diversity and antimicrobial resistance of Enterococcus faecalis isolated from endodontic infections

Chamomile essential oil improves the growth, immunity, and antioxidant status of Nile tilapia exposed to nanosized alumina

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