title: Duplex real-time PCR assay for rapid detection of ampicillin-resistant Enterococcus faecium.
abstract: Rapid and accurate identification of carriers of resistant microorganisms is an important aspect of efficient infection control in hospitals. Traditional identification methods of antibiotic-resistant bacteria usually take at least 3 to 4 days after sampling. A duplex real-time PCR assay was developed for rapid detection of ampicillin-resistant Enterococcus faecium (ARE). Primers and probes that are used in this assay specifically detected the D-Ala-D-Ala ligase gene of E. faecium and the modified penicillin-binding protein 5 gene (pbp5) carrying the Glu-to-Val substitution at position 629 (Val-629) in a set of 129 tested E. faecium strains with known pbp5 sequence. Presence of the Val-629 in the strain set from 11 different countries was highly correlated with ampicillin resistance. In a screening of hospitalized patients, the real-time PCR assay yielded a sensitivity and a specificity for the detection of ARE colonization of 95% and 100%, respectively. The results were obtained 4 h after samples were harvested from overnight broth of rectal swab samples, identifying both species and the resistance marker mutation in pbp5. This novel assay reliably identifies ARE 2 to 3 days more quickly than traditional culture methods, thereby increasing laboratory throughput, making it useful for rectal screening of ARE. The assay demonstrates the advantages of real-time PCR for detection of nosocomial pathogens.
authors.affiliation: Institute of Medicine, Department of Pharmacology, University of Bergen, Norway. stein.christian.mohn@helse-bergen.no
authors.forename: Stein Christian
authors.initials: SC
authors.lastname: Mohn
authors.forename: Arve
authors.initials: A
authors.lastname: Ulvik
authors.forename: Roland
authors.initials: R
authors.lastname: Jureen
authors.forename: Rob J L
authors.initials: RJ
authors.lastname: Willems
authors.forename: Janetta
authors.initials: J
authors.lastname: Top
authors.forename: Helen
authors.initials: H
authors.lastname: Leavis
authors.forename: Stig
authors.initials: S
authors.lastname: Harthug
authors.forename: Nina
authors.initials: N
authors.lastname: Langeland
mesh_terms: D019943:Amino Acid Substitution; D000668:Ampicillin Resistance / Q000235:genetics*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D016984:Enterococcus faecium / Q000187:drug effects / Q000235:genetics*; D016908:Gram-Positive Bacterial Infections / Q000188:drug therapy / Q000382:microbiology; D006801:Humans; D010453:Peptide Synthases / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D010453:Peptide Synthases; C019638:D-alanylalanine synthetase