Rapid detection of non-multidrug-resistant and multidrug-resistant methicillin-resistant Staphylococcus aureus using cycling probe technology for the mecA gene.

Abstract

In clinical laboratories rapid and accurate detection and identification of methicillin/oxacillin resistance in Staphylococcus aureus (MRSA) is essential for the management and treatment of both colonised and infected community and hospitalised patients [1, 2, 3]. In this study, we evaluated a new DNA probe assay, the Velogene-Cycling Probe Technology (CPT) assay (ID Biomedical; Australian Laboratory Services Sydney, Australia), for its ability to detect rapidly methicillin or oxacillin resistance in Staphylococcus aureus isolates showing multidrug resistance (MDR) with homogeneous high-level resistance to methicillin/oxacillin and nonmultidrug resistance (NMDR) with heterogeneous lowlevel methicillin/oxacillin resistance. Infections with heterogeneous NMDR MRSA are sometimes difficult to detect phenotypically due to growth-dependent factors characteristic of certain strains and geographical diversity imposed by selective antibiotic pressures [2, 3]. A total of 90 clinical Staphylococcus aureus isolates were tested. The types of isolates and their minimum inhibitory concentration (MIC) values for oxacillin and methicillin were as follows: 30 methicillin-susceptible Staphylococcus aureus (MSSA) with oxacillin MICs of 0.19 �g/ml–1.5 �g/ml and methicillin MICs of 0.75 �g/ ml–1.5 �g/ml; 30 MDR MRSA with oxacillin and methicillin MIC values greater than or equal to 256 �g/ ml; and 30 NMDR MRSA with oxacillin MIC values of 1.5 �g/ml to greater than 256 �g/ml and methicillin MIC values of 4 �g/ml to greater than 256 �g/ml. Reference strains MSSA ATCC 25923 (mecA negative) and MRSA ATCC 43300 (mecA positive) were used as controls. MICs were determined using E test strips (AB Biodisk, Sweden) and broth dilutions as previously described [3]. All isolates were obtained from separate patients and checked for genetic diversity by pulsed-field gel electrophoresis/restriction fragment length polymorphism typing [3]. The multiplex polymerase chain reaction (PCR) for the mecA and nuc genes was the gold standard method used for MSSA and MRSA identification [3, 4, 5]. The Velogene-CPT assay was performed in accordance with the manufacturer’s instructions, except the Staphylococcus aureus colonies were taken from a 5% horse blood