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

Background and objective: Staphylococci are widespread pathogens and are fr equently associated with nosocomial infections. Many hospitals struggle wit h increasing amounts of methicillin-resistant Staphylococcus aureus (MRSA) which are >> multiresistant << against all betalactam antibiotics. Often, a pplicable antibiotics for treatment are only glycopeptides like vancomycin and teicoplanin. In addition, MRSA infected patients require expensive inte nsive isolation measures and strict hygiene. To efficiently prevent dissemi nation of these pathogens rapid and reliable identification and a close col laboration between clinicians and microbiologists ate required. The purpose of our study was to set up a rapid and reliable identification procedure f or 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 hosp ital of the Justus-Liebig University of Gie ss en were examined. The femB g ene was used to differentiate between Staphylococcus aureus (S. aureus) and coagulase-negative staphylococci (CNS), a gene which allows the species-sp ecific identification of methicillin-resistant (MRSA) and -susceptible S. a ureus (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 oligonucleo tides MRSA strains were unequivocally detected within 3 hours. The femB gen e was detected in all 102 strains of S. aureus but in none of the 51 CN5. T he mecA determinant was detected in 12 S. aureus. Among these, 11 strains w ere phenotypically methicillin-resistant and one strain was susceptible. Th e methicillin-resistance of this particular mecA-positive/methicillin-susce ptible strain (cryptic MRSA) was inducible by cultivation on agar plates su pplemented with flucloxacillin. Conclusions: The described method specifically detects S. aureus and identi fies phenotypical and cryptic MRSA. These cryptic MRSA are of particular re levance since they are undetectable using common phenotypically based detec tion methods. It is conceivable that the methicillin resistance of these st rains is induced under antibiotic therapy with flucloxacillin and that the mec-encoded feature of methicillin-resistance can be transferred to previou sly methicillin-susceptible strains. Using the reliable detection of these strains by PCR, failure of flucloxacillin therapy is avoidable.