title: High-resolution melting-curve analysis of ligation-mediated real-time PCR for rapid evaluation of an epidemiological outbreak of extended-spectrum-beta-lactamase-producing Escherichia coli.
abstract: Methods for the confirmation of nosocomial outbreaks of bacterial pathogens are complex, expensive, and time-consuming. Recently, a method based on ligation-mediated PCR (LM/PCR) using a low denaturation temperature which produces specific melting-profile patterns of DNA products has been described. Our objective was to further develop this method for real-time PCR and high-resolution melting analysis (HRM) in a single-tube system optimized in order to achieve results within 1 day. Following the optimization of LM/PCR for real-time PCR and HRM (LM/HRM), the method was applied for a nosocomial outbreak of extended-spectrum-beta-lactamase (ESBL)-producing and ST131-associated Escherichia coli isolates (n = 15) and control isolates (n = 29), including four previous clusters. The results from LM/HRM were compared to results from pulsed-field gel electrophoresis (PFGE), which served as the gold standard. All isolates from the nosocomial outbreak clustered by LM/HRM, which was confirmed by gel electrophoresis of the LM/PCR products and PFGE. Control isolates that clustered by LM/PCR (n = 4) but not by PFGE were resolved by confirmatory gel electrophoresis. We conclude that LM/HRM is a rapid method for the detection of nosocomial outbreaks of bacterial infections caused by ESBL-producing E. coli strains. It allows the analysis of isolates in a single-tube system within a day, and the discriminatory power is comparable to that of PFGE.
authors.affiliation: Department of Clinical Microbiology, Kalmar County Hospital, SE-391 85 Kalmar, Sweden.
authors.forename: Hanna
authors.initials: H
authors.lastname: Woksepp
authors.forename: Cecilia
authors.initials: C
authors.lastname: Jernberg
authors.forename: Maria
authors.initials: M
authors.lastname: Tärnberg
authors.forename: Anna
authors.initials: A
authors.lastname: Ryberg
authors.forename: Alma
authors.initials: A
authors.lastname: Brolund
authors.forename: Michaela
authors.initials: M
authors.lastname: Nordvall
authors.forename: Barbro
authors.initials: B
authors.lastname: Olsson-Liljequist
authors.forename: Karin Tegmark
authors.initials: KT
authors.lastname: Wisell
authors.forename: Hans-Jürg
authors.initials: HJ
authors.lastname: Monstein
authors.forename: Lennart E
authors.initials: LE
authors.lastname: Nilsson
authors.forename: Thomas
authors.initials: T
authors.lastname: Schön
mesh_terms: D000368:Aged; D016000:Cluster Analysis; D003428:Cross Infection / Q000453:epidemiology / Q000382:microbiology; D004196:Disease Outbreaks; D004926:Escherichia coli / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D004927:Escherichia coli Infections / Q000453:epidemiology* / Q000382:microbiology; D005838:Genotype; D006801:Humans; D017720:Molecular Epidemiology / Q000379:methods; D058889:Molecular Typing / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D044366:Transition Temperature; D001618:beta-Lactamases / Q000378:metabolism*
chemical_list: D001618:beta-Lactamases