An evaluation of the necessity of 24-hour incubation for oxacillin minimum inhibitory concentrations

Current standards of the National Committee for Clinical Laboratory Standards (NCCLS) for microtube dilution recommend 24-hour incubation of staphylococci when testing for oxacillin/methicillin resistance. This study was conducted to quantify the need for this requirement. Standard 16-hour readings were compared with subsequent 24-hour readings of 515 fresh clinical isolates (256 Staphylococcus aureus, 259 coagulase-negative staphylococci) that were susceptible to oxacillin (microtube dilution minimum inhibitory concentration [MIC] < or equal to 2 micrograms/mL) after 16 hours. Five hundred two of the susceptible isolates (97.5%) were still susceptible at 24 hours. The remaining 13 isolates were resistant (MIC > 2 micrograms/mL) at 24 hours. Duplicate retesting, alternative method testing (Kirby-Bauer, E-Test) and mec A gene analysis were performed on these 13 isolates. All 13 isolates possessed the mec A gene. Four isolates always tested resistant (including all 16-hour repeat microtube dilution readings), and one isolate always tested susceptible. The remaining eight isolates produced variable results suggestive of an MIC very close to the resistance/susceptible break point. Overall , conversion from susceptible to resistant was entirely dependent upon the 16-hour MIC. There were 53 isolates with 16-hour MICS of 2 micrograms/mL. All 13 converters came from this group. No isolate with an oxacillin MIC < 2 micrograms/mL at 16 hours was resistant at 24 hours. Based on these results, the authors instituted a selective reincubation of only those staphylococcal isolates with oxacillin readings of 2 micrograms/mL at 16 hours.