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.

A type III-like restriction endonuclease functions as a major barrier to horizontal gene transfer in clinical Staphylococcus aureus strains

“…One of these barriers could be the presence of a capsule that reduces transformation efficiency. Indeed, agglutination assays have shown that the UAMS-1 strain is heavily capsulated (36). Moreover, preparation of competent cells requires the use of B2 medium supplemented with histidine, indicating that the growth conditions affect the constitution of the bacterium.…”

Does Colonization with Methicillin-Susceptible Staphylococcus aureus Protect against Nosocomial Acquisition of Methicillin-Resistant S. aureus?

“…Identification of MSSA and MRSA from colonies suggestive of staphylococci were performed using standard methods 4 according to Clinical and Laboratory Standards Institute recommendations and confirmed with multiplex quantitative polymerase chain reaction (PCR) for the genes femA and mecA. 22 …”

Effect of Screening for Methicillin-Resistant Staphylococcus aureus Carriage by Polymerase Chain Reaction on the Duration of Unnecessary Preemptive Contact Isolation

“…The use of polymerase chain reaction (PCR) for screening can shorten this latency period considerably, more so than the use of culture on chromogenic agar. In our study, we quantified the impact on logistics and costs of replacing the use of culture on chromogenic agar with the use of rapid multiplex quantitative PCR (qPCR) 6,7 for the screening of previous MRSA carriers at hospital admission. Previous MRSA carriers are flagged by a computerized rapid alert system.…”

“…During the intervention period, use of culture on chromogenic agar (MRSA ID; bioMerieux), which was the microbiological method normally used for MRSA screening, was replaced by the use of qPCR. 6 During both periods, samples were obtained on 2 consecutive days from the anterior nares and the groin using a cotton swab moistened with sterile saline solution.…”