Rapid detection of methicillin-resistant Staphylococcus aureus directly from sterile or nonsterile clinical samples by a new molecular assay

Abstract

A rapid procedure was developed for detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) directly from sterile sites or mixed flora samples (e.g., nose or inguinal swabs). After a rapid conditioning of samples, the method consists of two main steps: (i) immunomagnetic enrichment in S. aureus and (ii) amplification-detection profile on DNA extracts using multiplex quantitative PCR (5'-exonuclease qPCR, TaqMan). The triplex qPCR assay measures simultaneously the following targets: (i) mecA gene, conferring methicillin resistance, common to both S. aureus and Staphylococcus epidermidis; (ii) femA gene from S. aureus; and (iii) femA gene from S. epidermidis. This quantitative approach allows discrimination of the origin of the measured mecA signal. qPCR data were calibrated using two reference strains (MRSA and methicillin-resistant S. epidermidis) processed in parallel to clinical samples. This 96-well format assay allowed analysis of 30 swab samples per run and detection of the presence of MRSA with exquisite sensitivity compared to optimal culture-based techniques. The complete protocol may provide results in less than 6 h (while standard procedure needs 2 to 3 days), thus allowing prompt and cost-effective implementation of contact precautions.

FIG. 1.

Effect of antibody concentration on S. aureus recovery rate. Cowan I strain (NCTC8530; 10³ CFU) was used to evaluate the optimal concentration of anti-spa antibodies required for the enrichment step. (A) Recovery was determined by CFU counts of immunocaptured colonies. The curve showed maximal recovery at an antibody dilution of 1:666. (B). Using this optimized anti-spa dilution, recovery was assessed across >7 orders of magnitude in inoculum size. Average values ± standard errors of the means for four experiments performed in duplicate are given.

FIG. 2.

Immunocapture of MRSA from mixed cultures. 5 (A), 60 (B), or 1,000 (C) CFU of MRSA (ATCC 33591) was mixed with increasing titers of MRSE, yielding ratios ranging from 1:1 to 1:1,000 for S. aureus (black bars) versus S. epidermidis (grey bars). Immunocapture was evaluated by viable CFU counts performed on MH agar. Means ± ranges for two experiments performed in duplicate are shown. ∗, <10 CFU of MRSE.

FIG. 3.

Linearity and limits of detection of the qPCR assay. The linear response of the qPCR mecA assay as a function of input genomic DNA (2 fg to 10 ng) purified from strain ATCC33591 is shown. Correlation coefficient, >0.99; slope, −3.59. Values are means ± standard errors of the means of 4 to 16 determinations. ∗, value excluded from linear regression. ∗∗, amounts of input DNA leading to irregular signal detection.

FIG. 4.

Effect of the immunocapture procedure on the qPCR assay. S. aureus (ATCC 33591; 10³ CFU) was mixed with various S. epidermidis titers (MRSE) and analyzed by qPCR following (○) or not following (•) immunocapture enrichment. MRSA in the presence of >100-fold-excess amounts of MRSE was not detected (NA). Values are means ± standard errors of the means for three experiments performed in triplicate.