title: Double triplex real-time PCR assay for simultaneous detection of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus haemolyticus and determination of their methicillin resistance directly from positive blood culture bottles.
abstract: We developed and validated here a double triplex real-time PCR assay to simultaneously detect and identify Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus haemolyticus and their methicillin resistance in a single reaction directly from Gram-positive cocci-in-clusters (GPCs)-positive blood culture bottles. From August 15, 2009 through February 15, 2010, 238 GPC-positive samples were collected and identified by conventional methods as 11 methicillin-resistant S. aureus (MRSA), 28 methicillin-susceptible S. aureus (MSSA), 176 MR coagulase-negative staphylococci (MRCoNS), 21 MSCoNS and two Enterococcus faecalis. The double triplex real-time PCR assay was targeted and detected tuf, nuc and mecA genes in the first tube and atlE, gap and mvaA genes in the second tube which could be run simultaneously. The detection limit of the assay was found at 10(3) CFU/ml for the atleE gene, 10(4) CFU/ml for the mva gene and 10(5) CFU/ml for gap, nuc, mecA and tuf genes based on seeding experiments. All Staphylococcus species except two S. epidermidis were correctly identified by the assay. The double triplex real-time PCR assay quickly and accurately detects S. aureus, S. epidermidis, S. hominis and S. haemolyticus and their methicillin resistance in a single reaction directly from positive blood culture bottles within 83 min.
authors.affiliation: Department of Microbiology and Clinical Microbiology, Gulhane Military Medical Academy and School of Medicine, 06018 Ankara, Turkey. abkilic@gata.edu.tr
authors.forename: Abdullah
authors.initials: A
authors.lastname: Kilic
authors.forename: A Celal
authors.initials: AC
authors.lastname: Basustaoglu
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015373:Bacterial Typing Techniques; D001769:Blood / Q000382:microbiology*; D003470:Culture Media; D017931:DNA Primers; D006801:Humans; D008712:Methicillin / Q000494:pharmacology; D016106:Methicillin Resistance; D008826:Microbial Sensitivity Tests; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D010641:Phenotype; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013203:Staphylococcal Infections / Q000382:microbiology; D013211:Staphylococcus aureus / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D013212:Staphylococcus epidermidis / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D041162:Staphylococcus haemolyticus / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D000900:Anti-Bacterial Agents; D003470:Culture Media; D017931:DNA Primers; D008712:Methicillin