Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Tue, 18 Dec 2018 17:32:43 Rapid identification of Gram-positive pathogens and their resistance genes from positive blood culture broth using a multiplex tandem RT-PCR assay Briony J. Hazelton, 1 Lee C. Thomas, 1 Tuba Unver 1 and Jonathan R. Iredell 1,2,3 Correspondence Briony Hazelton b.onaidh@gmail.com Received 30 July 2012 Accepted 4 November 2012 1 Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, New South Wales, Australia 2 Centre for Research Excellence in Critical Infection, Westmead Millennium Institute, Westmead Hospital, Westmead, New South Wales, Australia 3 Sydney Emerging Infectious Diseases Institute, University of Sydney, Westmead Hospital, Westmead, New South Wales, Australia The early initiation of targeted antibiotic therapy in patients with bacteraemia and septic shock impacts favourably on outcomes. Rapid methods are therefore increasingly employed for bacterial identification directly from positive blood culture bottles, but with variable success. We evaluated the performance of the Gram Positive 12 multiplex tandem PCR (MT-PCR) assay (AusDiagnostics; catalogue no. 6202, version 07) containing targets for the identification of staphylococci including Staphylococcus aureus, streptococci including Streptococcus pneumoniae, enterococci including Enterococcus faecalis and Enterococcus faecium and their common antibiotic resistance genes (mecA, vanA, vanB). A total of 673 aerobic and anaerobic blood culture broths demonstrating Gram-positive cocci on microscopy were analysed in parallel with traditional phenotypic methods. Amplification of the internal control was inhibited in 79/673 (11.7 %) samples; however, MT-PCR identification was in concordance with phenotypic identification to the genus level in 96.6 % (537/556) of the remaining monomicrobial specimens and to the species level, where applicable, in 100 % (172/172) of samples. MT-PCR identification for 94.7 % (36/38) of polymicrobial samples matched traditional phenotypic identification. Meticillin and vancomycin susceptibility results determined by MT-PCR in blood culture broths demonstrated complete agreement with those determined by phenotypic methods in all 143 Staphylococcus aureus isolates and eight E. faecium isolates, respectively. Gram-positive pathogens and their key antibiotic resistance markers were reliably identified with the MT-PCR assay within 3 h of a positive blood culture result. INTRODUCTION Bacterial blood stream infections (BSIs) remain an important cause of morbidity and mortality worldwide (Bearman & Wenzel, 2005). The time taken to initiate appropriate antibiotic therapy is an independent risk factor for mortality in association with a BSI (Retamar et al., 2012), including BSIs due to Gram-positive organisms such as Staphylococcus aureus (Khatib et al., 2006; Paul et al., 2010), Enterococcus species (Suppli et al., 2011) and Streptococcus pneumoniae (Garnacho-Montero et al., 2010). Each hour of delay in the commencement of effective antibiotic therapy after the onset of hypotension in septic shock is associated with a decrease in survival, up to 7.6 % h 21 over the first 6 h (Kumar et al., 2006). The presence of the mecA gene reliably indicates high-level antibiotic resistance to meticillin and other beta-lactam antibiotics in Staphylococcus aureus (Deurenberg et al., 2007). Six types of vancomycin resistance in enterococci have been characterized on a phenotypic and genotypic basis, with five of these types (VanA, B, D, E and G) corresponding to acquired resistance. Of the genes involved in resistance, only two (vanA and vanB) are common in clinically important species (Courvalin, 2006). In Australia, Abbreviations: CNS, coagulase-negative staphylococci; C q , quantifica- tion cycle; MSSA, meticillin-sensitive Staphylococcus aureus; MRSA, meticillin-resistant Staphylococcus aureus; MT-PCR, multiplex tandem PCR; VRE, vancomycin-resistant enterococci. Journal of Medical Microbiology (2013), 62, 223–231 DOI 10.1099/jmm.0.050385-0 050385 G 2013 SGM Printed in Great Britain 223