Rapid and reliable detection of multiresistant Staphylococcus aureus (MRSA) using a multiplex PCR

Background and objective: Staphylococci are widespread pathogens and are frequently associated with nosocomial infections. Many hospitals struggle with increasing amounts of methicillin-resistant Staphylococcus aureus (MRSA) which are >> multiresistant << against all betalactam antibiotics. Often, applicable antibiotics for treatment are only glycopeptides like vancomycin and teicoplanin. In addition, MRSA infected patients require expensive intensive isolation measures and strict hygiene. To efficiently prevent dissemination of these pathogens rapid and reliable identification and a close collaboration between clinicians and microbiologists ate required. The purpose of our study was to set up a rapid and reliable identification procedure for MRSA by the amplification of specific gene determinants by PCR in order to to efficiently support therapy and eradication of the pathogen. Methods: 153 strains of staphylococci isolated from in-patients of the hospital of the Justus-Liebig University of Gie ss en were examined. The femB gene was used to differentiate between Staphylococcus aureus (S. aureus) and coagulase-negative staphylococci (CNS), a gene which allows the species-specific identification of methicillin-resistant (MRSA) and -susceptible S. aureus (MSSA). Additionally, MRSA harbor the mecA gene encoding methicillin-resistance, which is absent in MSSA strains. Results: Using a multiplex PCR with femB and mecA gene-specific oligonucleotides MRSA strains were unequivocally detected within 3 hours. The femB gene was detected in all 102 strains of S. aureus but in none of the 51 CN5. The mecA determinant was detected in 12 S. aureus. Among these, 11 strains were phenotypically methicillin-resistant and one strain was susceptible. The methicillin-resistance of this particular mecA-positive/methicillin-susceptible strain (cryptic MRSA) was inducible by cultivation on agar plates supplemented with flucloxacillin. Conclusions: The described method specifically detects S. aureus and identifies phenotypical and cryptic MRSA. These cryptic MRSA are of particular relevance since they are undetectable using common phenotypically based detection methods. It is conceivable that the methicillin resistance of these strains is induced under antibiotic therapy with flucloxacillin and that the mec-encoded feature of methicillin-resistance can be transferred to previously methicillin-susceptible strains. Using the reliable detection of these strains by PCR, failure of flucloxacillin therapy is avoidable.