A new multiplex PCR-based reverse line-blot hybridization (mPCR/RLB) assay for rapid staphylococcal cassette chromosome mec (SCCmec) typing

Abstract

A new multiplex PCR-based reverse line-blot hybridization (mPCR/RLB) assay for rapid staphylococcal cassette chromosome mec (SCCmec) typing The aim of this study was to develop a new discriminatory method for MRSA SCCmec typing based on multiplex PCR-based reverse line-blot hybridization (mPCR/RLB) assay to enable rapid identification and classification of MRSA SCCmec types in a clinical laboratory. Forty-five primer sets and 49 probes were designed and tested in uniplex PCR (uPCR) and mPCR/RLB. Probes were compared in silico to 14 whole-genome sequences and 18 partial SCCmec gene sequences of Staphylococcus aureus and complete genome and partial SCCmec genes of seven non-MRSA strains, including meticillin-susceptible S. aureus and meticillin-resistant coagulasenegative staphylococci. The method was tested on a set of 42 well-characterized reference MRSA strains. It identified all five epidemiologically relevant SCCmec types and 26 subtypes, including established and new subtypes of SCCmec III, IV (eight subtypes each) and V (three subtypes). The discriminatory power of mPCR/RLB SCCmec typing was similar to that of MLST and spa typing (Simpson indices of diversity of 0.916, 0.926 and 0.882, respectively; differences not statistically significant). The application of mPCR/RLB hybridization assay to MRSA SCCmec typing can improve the specificity, discriminatory power and throughput of the typing procedure. The detection of up to 43 mPCR products in a single hybridization assay transforms MRSA SCCmec typing from passive epidemiological library typing into a potential tool for near-real-time infection control surveillance and tracking of MRSA transmission in hospitals. INTRODUCTIONMorbidity and mortality from meticillin-resistant Staphylococcus aureus (MRSA) infections and the emerging threat of community-associated MRSA highlight the importance of timely surveillance (Aires de Sousa & de Lencastre, 2004;Ma et al., 2002). Meticillin resistance is conferred by the mecA gene, which is carried on a mobile genetic element known as the staphylococcal cassette chromosome mec (SCCmec) (Ito & Hiramatsu, 1998;Ito et al., 1999). The combination of mec complex class and cassette chromosome recombinase (ccr) genes determines the SCCmec type (Ito et al., 2004;Kondo et al., 2007). Currently, seven SCCmec types (I-VII) and a number of subtypes have been described, ranging in size from 20.9 to 66.9 kb (Ito et al., 2001(Ito et al., , 2004Daum et al., 2002;Oliveira et al., 2006a;Takano et al., 2008). SCCmec types I and IV-VII are usually resistant only to b-lactam antibiotics, while types II and III are often resistant to multiple classes of antibiotics due to additional resistance genes integrated into SCCmec (Deurenberg & Stobberingh, 2008 3These authors contributed equally to this work.

Prospective Genotyping of Hospital-Acquired Methicillin-Resistant Staphylococcus aureus Isolates by Use of a Novel, Highly Discriminatory Binary Typing System

“…(i) Reference isolates. Forty-two well-characterized epidemiologically unrelated MRSA reference isolates representing the dominant hospital-and community-associated clones in Australia and a number of important international clones were used; this collection has been described in detail elsewhere (2,3,13) and includes two MRSA isolates with published whole-genome sequences (COL and MW2).…”

“…The primers and probes used in this study are listed in the supplemental material. We used previously published primers and probes (2,3,13), modified as necessary to avoid primer dimer formation and to produce amplicons of 100 to 200 bp which gave strong probe signals. Each target was represented by two probes on the membrane; nuc and mecA probes were used as controls.…”

“…RLB membrane preparation, probe hybridization, and product detection were performed as previously described (2,3,10,13,14). A probe signal was interpreted as positive if it was at least as strong as the control probe for that target.…”

“…The most informative targets from three previous binary typing systems (toxin gene profiling [3], phage-derived open reading frame typing [13], and SCCmec subtyping [2]) were selected and incorporated into a multiplex PCR/reverse line blot (mPCR/RLB) assay platform. We describe the development and assessment of performance characteristics of the system, following established guidelines (19), and present results from 1 year of routine MRSA strain typing in a high-prevalence nosocomial setting.…”

Quantitative Estimation of the Stability of Methicillin-Resistant Staphylococcus aureus Strain-Typing Systems by Use of Kaplan-Meier Survival Analysis

“…spa sequence typing was performed as described previously (8), and spa types were assigned using the SpaServer (Ridom Bioinformatics) (9). Three multiplex PCR/ reverse line blot (mPCR/RLB) binary typing systems were employed (10, 11): (i) toxin gene profiling to target sea, seb, sec, sed, see, seg, seh, sei, eta, etb, etd, tst, and lukS-PV (Panton-Valentine leukocidin) genes (8), (ii) phage-derived open reading frame typing (PDORF) to examine 16 loci derived from integrated prophages (12), and (iii) staphylococcal cassette chromosome mec (SCCmec) subtyping to determine the mec class and ccr type and to interrogate 14 loci in the three junkyard regions (13). Reference strains that, in combination, were positive for each probe and a DNA-free control were used as the positive and negative controls, respectively.…”