Objetives:

(1) To use a real-time PCR (LightCycler, Roche Diagnostics, Spain) to differentiate Staphylococcus aureus from coagulase negative Staphylococcus (CNS) and (2) to use a real-time PCR to detect the presence of mecA gene in S. aureus clinical isolates.

Methods:

Seventy-three strains obtained from clinical specimens were identified by MicroScan (Dade Behring) and coagulase test (28 S. aureus, 15 S. epidermidis and 30 other coagulase negative staphylococci). In vitro susceptibility was determined by MicroScan and disc diffusion. A total of 39 S. aureus strains were classified according to methicillin susceptibility: 21 resistant and 18 susceptible to methicillin. DNA was obtained by incubation at 100°C in lysis buffer. Real-time PCR was performed in a LightCycler instrument (Roche Diagnostics, Spain) using two commercially available kits: (1) LightCycler Staphylococcus kit Mgrade: PCR was positive in all staphylococci and they were differentiated according to melting temperature (62.1 ± 2°C for S. aureus, and 43.4–59°C for CNS) and (2) LightCycler MRSA detection kit: PCR was positive in mecA positive S. aureus. An internal control excludes the presence of inhibition. Once the DNA was extracted the whole process takes 1 h.

Results:

Twenty-seven out of 28 S. aureus strains were clearly identified by real-time PCR due to the melting temperature (range from 60.2 to 63.4°C). One S. aureus showed melting temperature of 59.8°C. All S. epidermidis strains showed melting temperature from 50.1 to 53.4°C. S. lugdunensis showed melting temperature of 58.6, 54.6 and 53.1°C. Other CNS showed melting temperature from 51 to 57.1°C. Twenty-five out of 39 (64.1%) strains tested were mecA positive by using this LightCycler MRSA kit and real-time PCR. Among the 25 mecA positive, 21 were fenotipically methicillin resistant (84%) whilst four were methicillin susceptible (16%). All 14 mecA negative strains were susceptible to methicillin by phenotypic methods.

Conclusions:

Real-time PCR (LightCycler) seems to be an accurate method to identify S. aureus and differentiate it from different CNS and to detect resistance to methicillin in S. aureus. Both reactions could be done simultaneously and the whole process takes less than 2 h (DNA extraction plus real-time PCR).