Effect of the passage and storage of strains, and inoculum size on the change of zone pattern types and sizes produced by protease of methicillin resistant Staphylococcus aureus

Abstract

Protease produced by Staphylococcus aureus precipitates digests of casein in standard methods caseinate agar (SMCA) plate, and the various sizes of A to E precipitation types were produced depending on strains of inoculated Staphylococcus aureus, as previously reported. To evaluate these phenomena for epidemiological use, we investigated the effects of bacterial passage, storage and dosage on zonal pattern and the size of precipitation rings. Methicillin resistant Staphylococcus aureus (MRSA) was found to produce variable types and sizes of precipitation rings depending upon the inoculated strains. After 5, 30 and 50 passages in heart infusion broth (HIB), each of the strains randomly selected from MRSA strains showed that the precipitation pattern of A to E produced the same types and sizes of precipitation rings. The storage effects were assessed by comparing those strains in different storage time. The strain stored for 1 and 3 years at room temperature in HIB contained 0.8% agar produced the same pattern of precipitation rings at that of the freshly isolated one. Finally, the dosage of inoculated bacteria was examined. Overnight broth (HIB) cultures of MRSA were directly inoculated in SMCA plates, and were found to produce almost the same size of precipitation rings (reproducible within 3 mm). These findings indicated that protease digestion on SMCA plate provides a rapid, sensitive, and specific assay that can supplement the currently available modalities for typing of MRSA strains.