Rapid screening and identification of methicillin-resistant Staphylococcus aureus from clinical samples by selective-broth and real-time PCR assay

Abstract

A screening method for methicillin-resistant Staphylococcus aureus (MRSA) by using selective broth and real-time PCR (broth-PCR) was developed and evaluated. The samples (n = 304) were cultured in the broth overnight, followed by nuc gene detection by real-time PCR. nuc-negative samples were further checked for the presence of nuc amplification inhibitors by a PCR internal inhibitor assay. nuc-positive samples and nuc-negative samples with PCR inhibitors were cultured onto plates and processed further. The diagnostic values for this MRSA screening method were 93.3% sensitivity, 89.6% specificity, 31.8% positive predictive value, and 99.6% negative predictive value. The application of the broth-PCR method will be able to report most of the negative samples (258 of 289 [89.3%]) on the next morning and can save as much as 84.9% (258 of 304) of the labor and cost spent on processing the nuc-negative specimens on plates. In the study, all the samples were processed in parallel by the broth enrichment method and the plating method for comparison. To identify MRSA, the isolated oxacillin-resistant S. aureus strains were tested by a duplex real-time PCR targeting the mecA gene and the nuc gene. A collection of MRSA, methicillin-susceptible Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, and methicillin-susceptible Staphylococcus epidermidis strains and a panel of standard strains of 11 bacterial species other than S. aureus were also tested by this method, which was proved to be a valuable tool for MRSA identification in a routine microbiological laboratory.

FIG. 1.

T_m curves in the broth-PCR for detection of the nuc gene (MRSA screening).

FIG. 2.

Flowchart and results of the broth-PCR method in this study. a, the broth, when it turned turbid or after a maximal 5-day incubation, was spread to the agar plates and processed in a conventional way. b, one (sample 12) of the MRSA strains was not detected by the direct plating method. c, an MRSA strain was isolated from sample 13 on the MSA plate by the direct plating method.

FIG. 3.

Sensitivity of the real-time PCR assay for detection of the nuc gene from the overnight-cultured broth.

FIG. 4.

T_m curves for MRSA, MSSA, MRSE, and MSSE in mecA and nuc duplex real-time PCR (MRSA identification).

FIG. 5.

Sensitivity of the mecA and nuc duplex real-time PCR assay, determined through serial dilutions of the template DNA extracted from MRSA strain CCUG 31966.