Lack of correlation between phenotypic techniques and PCR-based genotypic methods for identification of Enterococcus spp.

Velasco, David; Pérez, Sonia González; Peña, F. J. García; Domínguez, M. Ángeles; Cartelle, Mónica; Molina, Francisca; Moure, Raquel; Villanueva, Rosa; Bou, Germán

doi:10.1016/j.diagmicrobio.2004.03.012

Cited by 30 publications

(23 citation statements)

References 34 publications

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“…PCR with primers complementary to vanA and vanB revealed that 75 of 75 of these strains had plasmids with the vanA gene cluster, and 7 of 75 also possessed plasmids harboring the vanB genes. Through strain typing using a PCR-based species identification method (15)(16)(17)(18)(19), it was determined that eight of the strains were Enterococcus faecalis, and 61 were E. faecium; six strains were not able to be typed by this method and are denoted as Enterococcus spp. A complete list of all strains and the features of their plasmids is in supporting information (SI) Tables 1-3.…”

Section: Plasmid-encoded Vancomycinmentioning

confidence: 99%

“…For all clinical VRE isolates, PCR amplification was performed from purified total DNA or from purified plasmid DNA. Oligodeoxynucleotide primer pairs were synthesized (Integrated DNA Technologies, Coralville, IA) that amplify the ddl E. faecalis and ddl E. faecium genes, the tuf gene, the groES gene, and a strongly conserved sequence in E. faecium, to identify clinical enterococci isolates at the genus and species level, as described (15)(16)(17)(18)(19). A primer pair derived from the vanA gene sequence was used to amplify specifically the vancomycin-resistance determinant in Enterococcus, as well as a pair of PCR primers specific for the vanB gene sequence (70).…”

Section: Isolation Of Plasmids Encoding Vancomycinmentioning

confidence: 99%

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Toxin–antitoxin systems are ubiquitous and plasmid-encoded in vancomycin-resistant enterococci

Moritz¹,

Hergenrother

2007

Proc. Natl. Acad. Sci. U.S.A.

124

127

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Vancomycin-resistant enterococci (VRE) are common hospital pathogens that are resistant to most major classes of antibiotics. The incidence of VRE is increasing rapidly, to the point where over one-quarter of enterococcal infections in intensive care units are now resistant to vancomycin. The exact mechanism by which VRE maintains its plasmid-encoded resistance genes is ill-defined, and novel targets for the treatment of VRE are lacking. In an effort to identify novel protein targets for the treatment of VRE infections, we probed the plasmids obtained from 75 VRE isolates for the presence of toxin-antitoxin (TA) gene systems. Remarkably, genes for one particular TA pair, the mazEF system (originally identified on the Escherichia coli chromosome), were present on plasmids from 75/75 (100%) of the isolates. Furthermore, mazEF was on the same plasmid as vanA in the vast majority of cases (>90%). Plasmid stability tests and RT-PCR raise the possibility that this plasmidencoded mazEF is indeed functional in enterococci. Given this ubiquity of mazEF in VRE and the deleterious activity of the MazF toxin, disruption of mazEF with pharmacological agents is an attractive strategy for tailored antimicrobial therapy.antibiotics ͉ mazEF ͉ axe-txe ͉ relbE ͉ VRE E nterococci are the leading cause of surgical-site infections and the third leading cause of urinary tract and bloodstream infections (1, 2) and are implicated in bacterial endocarditis, intraabdominal infections, bacteremia, and meningitis (3). The intrinsic resistance of enterococci to cephalosporin and aminoglycoside antibiotics complicates treatment strategies. Historically, vancomycin has been effective in the management of enterococcal infections; however, after nearly 30 years of use, vancomycin-resistant enterococci (VRE) emerged in the mid-1980s. In a short time span, intensive care units in the U.S. have seen a dramatic increase in the percentage of enterococcal infections that are resistant to vancomycin, from 0. 4% in 1989 to 25% in 1999 (3). In fact, in a recent study, 60% of Enterococcus faecium clinical isolates were resistant to vancomycin (4).The genes encoding the elaborate protein systems that mediate vancomycin resistance generally reside on mobile genetic elements within the enterococci. In some cases, plasmids with the various vancomycin resistance gene clusters have been recovered from enterococci (5-8), although the prevalence of plasmid-encoded resistance in VRE has not been defined. Large plasmids often have intricate mechanisms by which they maintain themselves in the bacterial population. Some plasmids contain genes encoding postsegregational killing ''plasmid addiction'' systems (9, 10), in which the plasmid encodes a potent toxin and a labile antitoxin. Provided the plasmid is present, the antitoxin binds to and sequesters the toxin. However, if a plasmid-free daughter cell arises, the unstable antitoxin is degraded, and the toxic protein kills the bacterial cell from within.If toxin-antitoxin (TA) systems are prevalent and functional on ...

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Section: Plasmid-encoded Vancomycinmentioning

confidence: 99%

Section: Isolation Of Plasmids Encoding Vancomycinmentioning

confidence: 99%

Toxin–antitoxin systems are ubiquitous and plasmid-encoded in vancomycin-resistant enterococci

Moritz¹,

Hergenrother

2007

Proc. Natl. Acad. Sci. U.S.A.

124

127

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“…correctly (3,7,12,14). Robredo et al (11) used the API20 STREP to identify enterococci obtained from foods of animal origin and compared the results with those obtained using colony hybridization.…”

Section: Introductionmentioning

confidence: 99%

“…However, failure in the identification of other species is a matter of concern specially due to the emergence of antibiotic resistant enterococcal strains (12).…”

mentioning

confidence: 99%

Correlation between API 20 STREP and multiplex PCR for identification of Enterococcus spp. isolated from Brazilian foods

Gomes

Esteves

Palazzo

et al. 2007

Braz. J. Microbiol.

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We evaluated the suitability of API 20 STREP and multiplex PCR to speciate 52 Enterococcus spp. obtained from Brazilian foods. A high percentage of isolates (78.9%) presented discrepant results between evaluated tests. Similar results were obtained for six E. faecalis and five E. faecium. The PCR multiplex was more effective than API 20 STREP for complete identification of the isolates. 3633-1936. E-mail: edemarti@usp.br Enterococci are commonly found in soil, water, plants, vegetables and foods (6,9).This genus includes more than 20 species, being E. faecium and E. faecalis the most commonly found (7,8). However, failure in the identification of other species is a matter of concern specially due to the emergence of antibiotic resistant enterococcal strains (12).Identification of enterococci species is generally done by conventional phenotypic and biochemical tests even so molecular methods are becoming the choice tests for bacterial identification (1-3).There are some kits available for a rapid identification of Enterococcus spp. such as ID 32 STREP, API 20 STREP, API 50CH, Api zym (BioMérieux, France) and Phene Plate PhP Plate System (PhPlate Microplate Techniques, Sweden). However, according to Facklam et al. (7) these tests should be carefully used since most of them only correctly identify E. faecalis. The API 20 STREP strip contains 20 microtubes with dehydrated substrates necessary to detect enzymatic activity and fermentation of sugars. The results are obtained within 24 -48 hours, and the identification is based on numerical analysis after a visual interpretation of color changes (BioMérieux, France).The purpose of the present work was to compare the biochemical test -API 20 STREP system and a multiplex Polymerase Chain Reaction (PCR) using specific primers for identification of Enterococcus spp. obtained from Brazilian foods.Fifty two Enterococcus spp. isolates were initialy identified in our lab at genus level based on Gram stain, catalase reaction, growth in de Man Rogosa Sharpe broth -MRS (Oxoid, UK) at 10ºC and 45ºC, MRS broth with 6.5% NaCl, MRS broth pH 9.6 and production of PYR (L-pyrrolidonyl-β-naphthylamide). The presumptive enterococcal isolates were speciated by API 20 STREP (BioMérieux, France), used for identification of streptococci and enterococci, according to the protocol provided by the manufacturer.Multiplex PCR was performed according to protocol described by . Primers were used to amplify the genes ddl of E. faecalis, ddl of E. faecium, vanC-1 of E. gallinarum, vanC-2 and vanC-3 of E. casseliflavus. Overnight cultures of Enterococcus spp. were harvested from trypticase soy agar with 0.6% yeast extract (TSAYE) and transferred to tubes containing 100 µl of PCR water (w-3500-Sigma, USA). This suspension was vortexed, centrifuged for ca. 15 seconds and the supernatant was used as template. The reaction was performed on a DNA thermal cycler (Mastercycler -Eppendorf, Germany) in a final volume of 25 µl containing 60 618 Gomes, B.C. et al.ng of DNA as template, 25 pmol of eac...

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“…Simple identification techniques, such as PCR-RFLP and species-specific PCR, are also available (Arias et al, 2006;Harwood et al, 2004;Velasco et al, 2004;Teng et al, 2001;Tyrrell et al, 1997), but these have either been used only for identification of a few Enterococcus spp. or their results are difficult to interpret.…”

Section: Introductionmentioning

confidence: 99%

Modified 16S–23S rRNA intergenic region restriction endonuclease analysis for species identification of Enterococcus strains isolated from pigs, compared with identification using classical methods and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Nowakiewicz

Ziółkowska

Zięba³

et al. 2015

Journal of Medical Microbiology

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Fast and reliable identification of bacteria to at least the species level is currently the basis for correct diagnosis and appropriate treatment of infections. This is particularly important in the case of bacteria of the genus Enterococcus, whose resistance profile is often correlated with their species (e.g. resistance to vancomycin). In this study, we evaluated restriction endonuclease analysis of the 16S-23S rRNA gene intergenic transcribed spacer (ITS) region for species identification of Enterococcus. The utility of the method was compared with that of phenotypic methods [biochemical profile evaluation and matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS)]. Identification was based on 21 Enterococcus reference strains, of the species E. faecalis, E. faecium, E. hirae, E. durans, E. casseliflavus, E. gallinarum, E. avium, E. cecorum and E. columbae, and 47 Enterococcus field strains isolated from pigs. Restriction endonuclease analysis of the ITS-PCR product using HinfI, RsaI and MboI, in the order specified, enabled species differentiation of the Enterococcus reference and field strains, and in the case of the latter, the results of species identification were identical (47/47) to those obtained by MALDI-TOF MS. Moreover, as a result of digestion with MboI, a unique restriction profile was also obtained for the strains (3/3) identified by MALDI-TOF MS as E. thailandicus. In our opinion, restriction endonuclease analysis of the 16S-23S rRNA gene ITS region of Enterococcus may be a simple and relatively fast (less than 4 h) alternative method for identifying the species occurring most frequently in humans and animals.

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Lack of correlation between phenotypic techniques and PCR-based genotypic methods for identification of Enterococcus spp.

Cited by 30 publications

References 34 publications

Toxin–antitoxin systems are ubiquitous and plasmid-encoded in vancomycin-resistant enterococci

Toxin–antitoxin systems are ubiquitous and plasmid-encoded in vancomycin-resistant enterococci

Correlation between API 20 STREP and multiplex PCR for identification of Enterococcus spp. isolated from Brazilian foods

Modified 16S–23S rRNA intergenic region restriction endonuclease analysis for species identification of Enterococcus strains isolated from pigs, compared with identification using classical methods and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

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