[Detection of methicillin-resistant Staphylococcus aureus using PCR and non-radioactive DNA probes (II)]

Methicillin resistance in staphylococci is primarily due to the presence of a mec A gene which encodes the novel penicillin-binding protein 2'. Some chromosomal factors, fem A and fem B, also participate in the expression of methicillin resistance in S. aureus. This study was designed to detect mec A, fem A and fem B genes for identification of staphylococcal species and for discrimination of methicillin-resistant cells. Three different pairs of DNA primers (PBP2'AF-PBP2'AR, fem AF-fem AR and fem BF-fem BR) complementary to unique regions of mec A, fem A and fem B genes were synthesized for use in polymerase chain reaction with DNAs of methicillin-sensitive S. aureus (MSSA), S. epidermidis, methicillin-resistant S. aureus (MRSA) and S. epidermidis. Amplified target DNAs of 630, 509, and 651 bp were resolved on ethidium bromide-stained gel, and hybridized to DNA probes conjugated to alkaline phosphatase. When applied to pure cultures on the MRSA screen agar, all three DNA probes tested detected MRSA in 47 of 61 culture-positive specimens (77.1%); the detection ratio of MRSA with mec A and either fem A or fem B probes was increased to 95.9%. By contrast, the fem A and fem B probes did not detect S. epidermidis. The result of detecting these species streaked on mannitol-salt agar were similar, while the detection of MSSA with the fem A and fem B probes was incomplete irrespective of the presence or absence of mec A. These findings suggest a good correlation between cultivation and DNA probe assay with respect to MRSA detection.