Rapid identification and typing of Staphylococcus aureus by PCR-restriction fragment length polymorphism analysis of...

The Staphylococcus aureus aroA gene, which encodes 5-enolpyruvylshikimate-3-phosphate synthase, was used as a target for the amplification of a 1,153-bp DNA fragment by PCR with a pair of primers of 24 and 19 nucleotides. The PCR products, which were detected by agarose gel electrophoresis, were amplified from all S. aureus strains so far analyzed (reference strains and isolates from cows and sheep with mastitis, as well as 59 isolates from humans involved in four confirmed outbreaks). Hybridization with an internal 536-bp DNA fragment probe was positive for all PCR-positive samples. No PCR products were amplified when other Staphylococcus spp. or genera were analyzed by using the same pair of primers. The detection limit for S. aureus cells was 20 CFU when the cells were suspended in saline; however, the sensitivity of the PCR was lower (5 x 10(2) CFU) when S. aureus cells were suspended in sterilized whole milk. TaqI digestion of the PCR-generated products rendered two different restriction fragment length polymorphism patterns with the cow and sheep strains tested, and these patterns corresponded to the two different patterns obtained by antibiotic susceptibility tests. Analysis of the 59 human isolates by our easy and rapid protocol rendered results similar to those of other assays.