title: A novel flow cytometric assay for rapid detection of extended-spectrum beta-lactamases.
abstract: The rapid detection of extended-spectrum beta-lactamases (ESBLs) is a challenge for most clinical microbiology laboratories because inaccurate identification of ESBL producers has important clinical implications for both antibiotic treatment and infection control. The aim of our study was to develop a rapid detection assay of ESBL producers based upon flow cytometric analysis. Antimicrobial susceptibility testing followed by molecular characterization of bla(TEM) , bla(SHV) or bla(CTX-M) genes was performed on clinical isolates (41 ESBL positive and 20 ESBL negative) and isolates expressing well-characterized beta-lactamases, including ESBLs (n = 13), plasmid AmpCs (n = 3), oxacillinases (n = 5) and carbapenemases (n = 3). Additionally, two ATCC strains recommended by CLSI for susceptibility testing were used as controls. The flow cytometry analysis protocol involved an incubation of bacterial cells with different concentrations of ceftazidime (1, 2 and 4 mg/L) and cefotaxime (4, 8 and 16 mg/L) for 1 and 2 hours, in the presence and absence of clavulanic acid; subsequently, cells were stained with the fluorescent dye Bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC(4) (3)], a lipophilic anion able to diffuse across depolarized membranes. Additionally, CFU counts were performed. Susceptible isolates displayed increased fluorescence after 1 hour of incubation; conversely, the increase of the depolarized population was only observed after incubation with clavulanic acid associated with ceftazidime or cefotaxime in ESBL producers. An excellent correlation was obtained between the number of non-depolarized bacteria quantified by flow cytometry and by conventional CFU assays. A novel, accurate and fast flow cytometric assay is available to detect the presence of ESBLs.
authors.affiliation: Department of Microbiology, Faculty of Medicine, University of Porto, Porto. Electronic address: cpinavaz@med.up.pt.
authors.forename: I
authors.initials: I
authors.lastname: Faria-Ramos
authors.affiliation: Department of Microbiology, Faculty of Medicine, University of Porto, Porto; Cardiovascular Research and Development Unit, Faculty of Medicine, University of Porto, Porto; Department of Microbiology, Hospital S. João, Porto, Portugal.
authors.forename: M J
authors.initials: MJ
authors.lastname: Espinar
authors.affiliation: Department of Microbiology, Faculty of Medicine, University of Porto, Porto.
authors.forename: R
authors.initials: R
authors.lastname: Rocha
authors.affiliation: Department of Microbiology, Faculty of Medicine, University of Porto, Porto; Gastroenterology Department, Hospital S. João, Porto, Portugal.
authors.forename: J
authors.initials: J
authors.lastname: Santos-Antunes
authors.affiliation: Department of Microbiology, Faculty of Medicine, University of Porto, Porto; Cardiovascular Research and Development Unit, Faculty of Medicine, University of Porto, Porto.
authors.forename: A G
authors.initials: AG
authors.lastname: Rodrigues
authors.affiliation: Servicio de Microbiología and CIBER en Epidemiología y Salud Pública (CIBERESP), Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS) and Hospital Universitario Ramón y Cajal, Madrid; Unidad de Resistencia a Antibióticos y Virulencia Bacteriana asociada al Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain.
authors.forename: R
authors.initials: R
authors.lastname: Cantón
authors.affiliation: Department of Microbiology, Faculty of Medicine, University of Porto, Porto; Cardiovascular Research and Development Unit, Faculty of Medicine, University of Porto, Porto; Department of Microbiology, Hospital S. João, Porto, Portugal.
authors.forename: C
authors.initials: C
authors.lastname: Pina-Vaz
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002439:Cefotaxime / Q000494:pharmacology; D002442:Ceftazidime / Q000494:pharmacology; D015169:Colony Count, Microbial; D024881:Drug Resistance, Bacterial / Q000235:genetics / Q000502:physiology; D004755:Enterobacteriaceae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification*; D004756:Enterobacteriaceae Infections / Q000382:microbiology; D005434:Flow Cytometry / Q000379:methods*; D005798:Genes, Bacterial; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods*; D050296:Microbial Viability / Q000187:drug effects; D010641:Phenotype; D001618:beta-Lactamases / Q000032:analysis* / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D002442:Ceftazidime; D001618:beta-Lactamases; D002439:Cefotaxime