title: A new approach to determine the susceptibility of bacteria to antibiotics directly from positive blood culture bottles in two hours.
abstract: The rapid identification and antibiotic susceptibility test of bacteria causing bloodstream infections are given a very high priority by clinical laboratories. In an effort to reduce the time required for performing antibiotic susceptibility test (AST), we have developed a new method to be applied from positive blood culture bottles. The design of method was performed using blood culture bottles prepared artificially with five strains which have a known susceptibility. An aliquot of the blood culture was subcultured in the presence of specific antibiotics and bacterial counts were monitored using the Sysmex UF-1000i flow cytometer at different times up to 180min. Receiver operating curve (ROC) analysis allowed us to find out the cut-off point for differentiating between sensitive and resistant strains to the tested antibiotic. This procedure was then validated against standard commercial methods on a total of 100 positive blood culture bottles from patients. First, bacterial identification was performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) directly from positive blood culture bottles as we have previously reported. Secondly, antibiotic susceptibility test was performed in the same way that was carried out in artificially prepared blood culture bottles. Our results indicate that antibiotic susceptibility test can be determined as early as 120min since a blood culture bottle is flagged as positive. The essential agreement between our susceptibility test and commercial methods (E-test, MicroScan and Vitek) was 99%. In summary, we conclude that reliable results on bacterial identification and antibiotic susceptibility test performed directly from positive blood culture bottles can be obtained within 3h.
authors.affiliation: Department of Microbiology, Faculty of Medicine, University of Valladolid, Av. Ramón y Cajal No. 7, 47005 Valladolid, Spain; Service of Microbiology and Immunology, University Clinic Hospital of Valladolid, Ramón y Cajal Avenue No. 3, 47003 Valladolid, Spain. Electronic address: gmr810@hotmail.com.
authors.forename: Gabriel A
authors.initials: GA
authors.lastname: March
authors.affiliation: Service of Microbiology and Parasitology, Medina del Campo Hospital, C/Peñaranda No. 4, 47400 Medina del Campo, Spain. Electronic address: cgarcialoygorri@saludcastillayleon.es.
authors.forename: María C
authors.initials: MC
authors.lastname: García-Loygorri
authors.affiliation: Department of Microbiology, Faculty of Medicine, University of Valladolid, Av. Ramón y Cajal No. 7, 47005 Valladolid, Spain. Electronic address: msimarrogrande@gmail.com.
authors.forename: María
authors.initials: M
authors.lastname: Simarro
authors.affiliation: Department of Microbiology, Faculty of Medicine, University of Valladolid, Av. Ramón y Cajal No. 7, 47005 Valladolid, Spain. Electronic address: gutierre@med.uva.es.
authors.forename: María P
authors.initials: MP
authors.lastname: Gutiérrez
authors.affiliation: Department of Microbiology, Faculty of Medicine, University of Valladolid, Av. Ramón y Cajal No. 7, 47005 Valladolid, Spain; Service of Microbiology and Immunology, University Clinic Hospital of Valladolid, Ramón y Cajal Avenue No. 3, 47003 Valladolid, Spain. Electronic address: orduna@med.uva.es.
authors.forename: Antonio
authors.initials: A
authors.lastname: Orduña
authors.affiliation: Department of Microbiology, Faculty of Medicine, University of Valladolid, Av. Ramón y Cajal No. 7, 47005 Valladolid, Spain; Service of Microbiology and Immunology, University Clinic Hospital of Valladolid, Ramón y Cajal Avenue No. 3, 47003 Valladolid, Spain. Electronic address: bratos@med.uva.es.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Bratos
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001419:Bacteria / Q000145:classification / Q000187:drug effects* / Q000302:isolation & purification; D058491:Bacterial Load; D001769:Blood / Q000382:microbiology*; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013997:Time Factors
chemical_list: D000900:Anti-Bacterial Agents
keywords: Blood culture bottles; Flow cytometry; Rapid antibiotic susceptibility test