RAPID DETECTION OF THE STAPHYLOCOCCAL MECA GENE FROM BACTEC BLOOD CULTURE BATTLES BY THE POLYMERASE CHAIN-REACTION

A rapid polymerase chain reaction (PCR) method fur tile direct detecti on of the staphylococcal mecA gene from BACTEC blood culture bottles ( Becton Dickinson, Sparks, MD) was developed. Published primer sequence s and sample preparation using Achromopeptidase for fell lysis were ad apted to the use of tile Idaho Technology Air Thermocycler 1605 (Idaho Technologies, Idaho Fails. ID). The method was validated with 80 stra ins of coagulase-positive and coagulase-negative geographically divers e methicillin-resistant and susceptible isolates of staphylococci. The re was a 100% correlation between the PCR results and the results of s tandard susceptibility testing methods. From BACTEC 9240 blood culture s, mixed aliquots of blood and broth containing gram-positive cocci in clusters mere centrifuged al low speed to sediment red blood cells, A fter additional centrifugation and wash steps, PCR was performed on th e resuspended pellet. The turn around time from initial Gram stain det ection of positive BACTEC bottles to PCR amplicon detection by agarose gel electrophoresis is less than 3 hours. In a clinical evaluation of 181 blood culture isolates, there was a 99% correlation,vith standard susceptibility results for Staphylococcus aureus. Discrepant results for Staphlococcus aureus isolates were verified by a Mueller Hinton pl ate supplemented with 6 mu g/mL of oxacillin and 2% sodium chloride. F or coagulase-negative staphylococci, the PCR method detected an additi onal seven resistant isolates that were reported by the Vitek as susce ptible. Coagulase-negative staphylococcal susceptibility results that were in disagreement with the PCR assay were confirmed by the disk-dif fusion method. This procedure is accurate, rapid and fits well into la boratory work flow, Rapid detection of the mecA gene on positive blood culture vials has become a routine test in the authors' clinical micr obiology laboratory.