Characterization of beta-lactam antagonist in Supplement C

In the course of using Mueller-Hinton agar with 1% Supplement C (SC) (Difco, Detroit, MI) as a susceptibility test medium for Haemophilus influenzae, one lot of SC was encountered whose use was associated with markedly increased ampicillin MICs. Acidimetric and chromogenic cephalosporin filter paper disc tests of SC failed to detect beta-lactamase activity. Macrobroth dilution MIC tests to determine substrate specificity showed SC to antagonize benzylpenicillin and ampicillin but not cephalothin, cefazolin, or cefaclor, with the antagonism being prevented by the addition of clavulanic acid. High pressure liquid chromatographic analysis of reference and reaction solutions of benzylpenicillin with SC showed almost complete degradation of benzylpenicillin to benzylpenicilloic acid after 24 hr at 37 degrees C. For two other lots of SC that had passed MIC quality control testing, similar high pressure liquid chromatographic studies demonstrated slow conversion of small amounts of benzylpenicillin to benzylpenicilloic acid. These findings indicate that the beta-lactam antagonism by SC was due to the presence of a contaminating beta-lactamase directed primarily toward the penicillins.