Simple assay of beta-lactamase with agar medium containing a chromogenic cephalosporin, pyridinium-2-azo-p-dimethylaniline chromophore (PADAC)

A new beta-lactamase assay method with agar plates containing pyridinium-2-azo-p-dimethylaniline chromophore (PADAC) (50 microM), a beta-lactamase-labile, chromogenic cephalosporin, was examined. On the PADAC plates inoculated with beta-lactamase-producing gram-negative bacteria (10(4) CFU per spot) and incubated at 37 degrees C, a yellow zone showing hydrolysis of PADAC by beta-lactamase was formed around the colony. The zone diameter increased with incubation time. Examination with Enterobacter cloacae GN7471 revealed that beta-lactamase activity was present in the agar around the colony, decreasing exponentially with increasing distance from the colonial margin; this suggests that the PADAC hydrolysis zone is formed by an extracellular enzyme. At 18 h, significant correlations were obtained between the zone diameters of the 10 species (clinical isolates) examined and their periplasmic beta-lactamase activities determined spectrophotometrically. The addition of clavulanic acid (0.5 to 10 micrograms/ml) inhibited zone formation on the PADAC plates inoculated with type IIIa, Va, Vb, PSE-1, and Ic beta-lactamase producers. When the clinical isolates were tested on plates with clavulanic acid (2 micrograms/ml), inhibition was observed in 41 to 58% of the Escherichia coli, Serratia marcescens, and Pseudomonas aeruginosa isolates and in all isolates of Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus vulgaris. Thus, the use of the inhibitor made it possible to detect penicillinase or type Ic cephalosporinase producers. These results proved that the PADAC plate might be a useful tool permitting easy, semiquantitative determination of beta-lactamase activity.