Gustaferro, C A

We describe a novel procedure for direct covalent coupling of horseradish peroxidase to rRNA and ribotyping by using enhanced chemiluminescence. Compared with their 32P-end-labeled counterparts, chemiluminescent rRNA probes are stable and easy to synthesize and provide results as good as or superior to those obtained with isotopic labeling. Direct chemiluminescent labeling of Escherichia coli rRNA produces a sensitive, universal probe suitable for clinical laboratory use in the investigation of nosocomial outbreaks.

A multiplex PCR assay for detection of the staphylococcal mecA gene (the structural gene for penicillin-binding protein 2a) was compared with agar dilution and disk diffusion susceptibility test methods for identifying methicillin resistance. The multiplex PCR assay combined two primer sets (mecA and 16S rRNA) in a single reaction. A total of 500 staphylococcal isolates (228 isolates of Staphylococcus aureus and 272 isolates of coagulase-negative staphylococci) from clinical specimens were studied. For S. aureus, 40 of 40 mecA-positive isolates and 4 of 188 mecA-negative isolates were oxacillin resistant (positive and negative predictive values of 100 and 98%, respectively). In 3 of 4 discordant isolates, resistance was due to hyperproduction of beta-lactamase. For coagulase-negative staphylococci, 148 of 159 mecA-positive isolates and 0 of 113 mecA-negative isolates were oxacillin resistant (positive and negative predictive values of 93 and 100%, respectively). Twenty-six isolates were categorized as indeterminate because of the absence of a detectable 16S rRNA product. Four of these 26 isolates contained mecA when retested. The assay is designed to be incorporated into the work flow of the clinical microbiology laboratory and allows for the identification of intrinsic resistance in a timely and reliable manner.