Rapid PCR-Based Identification of Methicillin-Resistant Staphylococcus aureus from Screening Swabs

Abstract

A PCR identification of methicillin-resistant Staphylococcus aureus (MRSA), obviating the need for subculture on agar media, was investigated. The combination of MRSA detection by mecA femB PCR with prior enrichment in selective broth was tested for 439 swabs. PCR identified 36 MRSA-positive samples, in concordance with conventional methods.Accurate and rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) in clinical specimens is essential for timely decisions on isolation procedures and effective antimicrobial chemotherapy. Numerous approaches that improve turnaround time for the identification of MRSA have been described. Fluorescence tests (14), PCR assays (4), or penicillin-binding protein 2a (PBP2a) antibody agglutination tests have been described elsewhere (12). Yet, these require subculture on solid media, and many are unable to determine species and methicillin susceptibility at the same time. A simultaneous test of methicillin resistance and species confirmation by a mecA femB duplex PCR has been proposed elsewhere (16). The mecA gene encodes the extra PBP2a, which is unique to methicillin-resistant staphylococci. The femB gene codes for an enzyme important in cross-linking peptidoglycan in various different Staphylococcus spp. The specificity of the femB PCR primers used for DNA amplification of the species S. aureus has been demonstrated previously (8).This study describes the performance of this technique in a clinical setting of moderate MRSA endemicity where large numbers of screens need to be processed on a daily basis. Moreover, the robustness of the test was investigated by determining the number of false-positive readings due to coamplification of femB and mecA from methicillin-susceptible S. aureus (MSSA) and methicillin-resistant coagulase-negative staphylococci (R-CNS) coexisting at the sample site (C. M.

Antibiotic Resistance Pattern of Methicillin Resistant and Methicillin Sensitive Staphylococcus aureus Isolates in Tehran, Iran

“…PCR primers specific for mecA gene (mecA1: GTAGAAAT-GACTGAACGTCCGATAA and mecA2: CCAATTCCACATT-GTTTCGGTCTAA) were selected from Jonas et al (20) and synthesized by Tib-Molbiol (Berlin, Germany). The PCR mixture containing; 10X PCR buffer, taq DNA polymerase (0.5 U/μl) (HT Biotechnology, Cambridge, United Kingdom), each primer (1.6 μM), MgCl2 (1.2 μM) and each dNTP (0.64 μM).…”

Public computer surfaces are reservoirs for methicillin-resistant staphylococci

“…Biochemical testing showed that all isolates were catalase-positive, while 10 were also coagulase-positive. DNA from each colony was isolated and used in a multiplex PCR analysis ( Figure 1a) that targeted the 16S rRNA gene of Staphylococcus spp (Mason et al, 2001), femB (Jonas et al, 2002) and mecA (Jonas et al, 2002). The targeted femB sequence is S. aureus-specific and encodes a protein crucial for peptidoglycan crosslinking, while mecA has been established as a standard marker for confirming methicillin resistance in staphylococci (Clinical and Laboratory Standards Institute, 2006).…”

“…A control, 'field blank' swab that was briefly exposed to the air in each computer facility was also collected. Swabs were incubated (48 h at 351C, with shaking) in tryptic soy broth (TSB) supplemented with oxacillin (2 mg l À1 ) (Jonas et al, 2002). Oxacillin has replaced methicillin as the current agent of choice for selecting bacteria that are resistant to penicillinase-stable penicillins.…”

Molecular diagnostics and genotyping of MRSA: an update

“…Multiplex PCR (agarose gel) detecting femB and mecA 100 100 3 [56,57] Real-time PCR detecting mecA 100 100 2 [58] High-throughput real-time PCR detecting mecA and nuc 100 100 2.5 [59] Real-time PCR detecting mecA and SA442 100 100 4 [60] Isothermal signal amplification (CytAMP) detecting coa and mecA 83 93 3.5 [61] Real-time PCR detecting nuc and mecA 93 90 2.5 [62] Direct from clinical specimen (swabs)…”