ATTENUATED MUTANTS OF STAPHYLOCOCCUS-AUREUS WITH 2 TEMPERATURE-SENSITIVE LESIONS - ISOLATION AND CHARACTERIZATION

The feasibility of constructing attenuated mutants of Staphylococcus a ureus with two temperature-sensitive (ts) lesions for ultimate develop ment of a live-attenuated strain was investigated. Temperature-sensiti ve S. aureus strain G/1/2, which grows well at 31 degrees C but does n ot replicate at 37 degrees C, was subjected to chemical mutagenesis. A fter two enrichment cycles, fifteen mutants able to grow at 25 degrees C but unable to grow at 31 degrees C, were identified. Growth curves with temperature shifts from 25 to 31 degrees C, and from 31 to 37 deg rees C confirmed that these were mutants with two lesions (dts), each with a different cut-off temperature. The reversion frequency of mutan t G/1/2 at 37 degrees C was 2 x 10(-6) whereas those of several dts mu tants were much fewer (dts7: 7 x 10(-9) and dts12: 1 x 10(-9)). There was no increase in ts mutation reversion rate in response to prolonged incubation at 37 degrees C. The data support the further development of these mutants for use as a stable attenuated vaccine.