Detection of clinical vancomycin‐resistant enterococci in Denmark by multiplex PCR and sandwich hybridization

Poulsen, Rikke Lykke; Pallesen, Lars; Frimodt‐Møller, Niels; Espersen, Frank

doi:10.1111/j.1699-0463.1999.tb01573.x

Cited by 16 publications

(9 citation statements)

References 19 publications

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“…Specificity was very high. A few other studies reported PCR assays with 100% sensitivity (253,260). The discrepancies between these studies may reflect differences in the number of isolates tested, the type of sample used, and the need to carefully control the composition of multiplex PCR incubations.…”

Section: Detection Of Resistancementioning

confidence: 79%

Molecular Detection of Antimicrobial Resistance

2001

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The determination of antimicrobial susceptibility of a clinical isolate, especially with increasing resistance, is often crucial for the optimal antimicrobial therapy of infected patients. Nucleic acid-based assays for the detection of resistance may offer advantages over phenotypic assays. Examples are the detection of the methicillin resistance-encoding mecA gene in staphylococci, rifampin resistance in Mycobacterium tuberculosis, and the spread of resistance determinants across the globe. However, molecular assays for the detection of resistance have a number of limitations. New resistance mechanisms may be missed, and in some cases the number of different genes makes generating an assay too costly to compete with phenotypic assays. In addition, proper quality control for molecular assays poses a problem for many laboratories, and this results in questionable results at best. The development of new molecular techniques, e.g., PCR using molecular beacons and DNA chips, expands the possibilities for monitoring resistance. Although molecular techniques for the detection of antimicrobial resistance clearly are winning a place in routine diagnostics, phenotypic assays are still the method of choice for most resistance determinations. In this review, we describe the applications of molecular techniques for the detection of antimicrobial resistance and the current state of the art

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Section: Detection Of Resistancementioning

confidence: 79%

Molecular Detection of Antimicrobial Resistance

2001

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“…For instance, in order to improve gene identification, PCR is often followed by an additional hybridisation step with internal probes such as routinely performed in the Danish surveillance programme of clinical Vanomycin-resistant enterococci (VRE) isolates [51]. In other procedures the PCR protocol has been adapted to improve sensitivity and/or specificity in which the initial PCR is followed by a second PCR using a primer combination located within the amplified fragment.…”

Section: Pcr-based Detection Of Antibiotic Resistance Genesmentioning

confidence: 99%

Molecular tools for the characterisation of antibiotic-resistant bacteria

Aarts¹,

Sidi-Boumedine

Nesme

et al. 2001

Vet. Res.

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-This review will discuss a number of molecular tools which are currently used as well as some innovative approaches for the characterisation of antibiotic-resistant bacterial strains. Various methods involved in the detection and characterisation of genes and mutations associated with antibiotic resistance and that are used for strain typing as part of epidemiological studies, are described. Furthermore, a few examples are discussed in which the results of both gene and strain characterisation are combined to investigate the underlying mechanism of the spread of antibiotic resistance. Some of the available molecular techniques are heavily supported by the existence of databases on the Internet. These databases either contain a fast growing amount of sequence information or a large number of allelic or fingerprint profiles. The current progress in applied DNA technology and the ongoing projects on the elucidation of the whole genomic sequence of bacterial species have lead and will further lead to the development and application of sophisticated new strategies for the analysis of antibiotic resistant bacterial strains.antibiotic resistance / molecular detection / molecular typing / horizontal gene transfer / new approaches Résumé -Outils moléculaires pour la caractérisation de bactéries résistantes aux antibiotiques. Cette revue présente les outils moléculaires utilisés actuellement ainsi que des nouvelles approches pour la caractérisation de souches bactériennes résistantes aux antibiotiques. Différentes méthodes de détection et de caractérisation des gènes et des mutations impliqués dans la résistance aux antibiotiques, utilisées dans des études épidémiologiques, sont décrites. Quelques exemples sont présentés où sont associées les caractérisations du gène et de la souche bactérienne dans l'étude des mécanismes de diffusion de résistances aux antibiotiques. Certaines techniques moléculaires font

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“…We have found two VanA-and three VanB-positive isolates by phenotypic susceptibility tests; by multiplex PCR the two vanApositive and only 1 of 3 vanB-positive strains were identified (Table 1). Also, the second set of primers (31) failed to amplify the same two vanB-positive strains. Notwithstanding the fact that the alignment of the chosen primers shows no mismatch with the published sequences, the vanB primers may fail to amplify some vanB genes because of variability of the sequences in the primer regions (16,29).…”

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confidence: 99%

“…It has been succesfully used to identify vancomycin-resistant enterococci (8,13,18,21,22,25,26,30,31), but it may also be used broadly to identify all enterococci; nevertheless, this approach certainly fails to identify some enterococcal species outside the reach of the primers, and for this reason it is also necessary to include among the proposed molecular methods for enterococcal species identification the amplification and sequencing of the 16S ribosomal DNA (rDNA) gene (25,28,38). Two hundred seventy-nine clinical strains consecutively identified as enterococci by three hospital laboratories in Rome were studied ( Table 1 All strains submitted as glycopeptide resistant were verified with the E-test method (AB BIODISK, Solna, Sweden) in our laboratory.…”

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confidence: 99%

Routine Molecular Identification of Enterococci by Gene-Specific PCR and 16S Ribosomal DNA Sequencing

et al. 2001

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For 279 clinically isolated specimens identified by commercial kits as enterococci, genotypic identification was performed by two multiplex PCRs, one with ddl E. faecalis and ddl E. faecium primers and another with vanC-1 and vanC-2/3 primers, and by 16S ribosomal DNA (rDNA) sequencing. For 253 strains, phenotypic and genotypic results were the same. Multiplex PCR allowed for the identification of 13 discordant results. Six strains were not enterococci and were identified by 16S rDNA sequencing. For 5 discordant and 10 concordant enterococcal strains, 16S rDNA sequencing was needed. Because many supplementary tests are frequently necessary for phenotypic identification, the molecular approach is a good alternative.Identification at the species level of enterococci isolated from clinical specimens is considered necessary, as is quantitative evaluation of their resistance to penicillin, ampicillin, vancomycin, and teicoplanin and high-level resistance to gentamicin and streptomycin (11). It is also necessary to distinguish the low-virulence motile enterococcal species with constitutive low-level resistance to vancomycin from the species that are more frequently isolated from clinical specimens, such as Enterococcus faecalis and E. faecium, which in some countries can often show high-level inducible and transmissible resistance to glycopeptides (37).Commercially available kits are often used by clinical laboratories as an alternative to the numerous physiological tests needed to identify enterococcal species (6, 9, 10, 11, 37); nevertheless, all commercial kits vary in their performance and persistently show many drawbacks, especially in cases of atypical strains, and at best need supplementary manual tests, which somewhat impair their usefulness (14, 17, 23, 33, 34, 36; P. A. d'Azevedo, C. G. Dias, A. L. S. Gonçalves, F. Rowe, and L. M. Teixeira, Abstr. 100th Gen. Meet. Am. Soc. Microbiol., abstr. C-242, 2000). In some cases identification of atypical E. faecium strains may be problematic, as is distinguishing E. gallinarum from E. faecium and also identifying E. durans, E. avium, E. raffinosus, E. hirae, and E. mundtii (1, 2, 6, 7, 35, 37).In 1995 a multiplex PCR was devised for the identification of E. faecium and E. faecalis by primers targeted at specific sequences in the ddl (D-Ala-D-Ala ligase) chromosomal genes of the two species and in the glycopeptide resistance ligase genes vanA, vanB, and vanC (8). The vanC gene is present in the motile, low-level constitutive glycopeptide-resistant species E. gallinarum (vanC-1) and E. casseliflavus and E. flavescens (vanC-2/3); thus, demonstration of its presence indicates the presence of one of the aforementioned species (4,24,27,29,30).The use of a PCR with primers for ddl E. faecalis and ddl E. faecium , together with primers for vanC-1, -2, and -3, may be the most simple molecular approach for both rapid and precise identification of enterococci while avoiding the drawbacks of commercial kits. It has been succesfully used to identify vancomycin-resistant enteroco...

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Detection of clinical vancomycin‐resistant enterococci in Denmark by multiplex PCR and sandwich hybridization

Cited by 16 publications

References 19 publications

Molecular Detection of Antimicrobial Resistance

Molecular Detection of Antimicrobial Resistance

Molecular tools for the characterisation of antibiotic-resistant bacteria

Routine Molecular Identification of Enterococci by Gene-Specific PCR and 16S Ribosomal DNA Sequencing

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