SUMMARY: By repeated subcultivation of the parent strain ATCC8081 in the presence of methicillin, a substrain 8081 CRD was developed which grew only when the partly defined medium was supplemented with methicillin or certain other penicillins. The methicillin-dependent strain was not highly resistant to methicillin and grew only in the presence of a limited range of concentrations of it (about 10-300 µg./ml.). Even with an optimal growth concentration of methicillin (50-100 µg./ml.), the dependent organisms grew less well than did the parent strain without methicillin and showed a longer lag period before growth became visible.

Although other derivatives of 6-aminopenicillanic acid (but not of 7-aminocephalosporanic acid) supported moderate growth of 8081 CRD, none was as effective as methicillin, nor was there marked cross-resistance to any of these other derivatives. The more potent the penicillin as inhibitor of growth of the parent strain, the smaller was the optimal concentration needed to support growth of the dependent sub-strain. Several other antibiotics were ineffective as growth factors.

When methicillin was hydrolysed with acid, alkali or pencillinase, activity as a growth factor was lost. During growth of the parent strain and of the methicillin-dependent strain at pH 6.5 material was produced in the medium which was able to destroy the antibiotic potency of methicillin or other penicillins. The substance was not an enzyme, and the presence of methicillin was not necessary to induce its formation by the parent strain.

The methicillin-dependent strain did not grow, with or without methicillin, when sodium acetate was omitted from the medium. No substance of known chemical structure was found which could replace acetate for growth. 8081 CRD grew rapidly in the absence of both acetate and methicillin when the medium was supplemented with yeast extract However, when acetate was present as well as yeast extract, methicillin again became necessary for growth of the dependent organisms.


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