In vitro evaluation of pyridine-2-azo-p-dimethylaniline cephalosporin, a new diagnostic chromogenic reagent, and comparison with nitrocefin, cephacetrile, and other beta-lactam compounds

Abstract

azo-p-dimethylanaline cephalosporin (PADAC), a chromogenic reagent which is purple and changes to yellow upon cleavage of its beta-lactam ring, was evaluated in comparison with other chromogenic cephalosporins. PADAC exhibited little antimicrobial activity against gram-negative bacteria, but did have good activity (minimum inhibitory concentration, 0.12 to 0.5 ,ug/ml) against Staphylococcus aureus, a quality comparable to nitrocefin. Nitrocefin, however, demonstrated an unexpected and uniquely potent activity against Streptococculs faecalis (minimum inhibitory concentration,-0.06 to 0.12 p.g/ml). The relative hydrolysis rate of PADAC when subjected to six different beta-lactamases was substantially greater than that of cephacetrile, but less than that of nitrocefin. The relative hydrolysis rates of PADAC and nitrocefin were comparable with type Illa beta-lactamase and that derived from Bacillus cereus. The inhibition of betalactamase hydrolysis of the chromogenic cephalosporin substrates by six enzymestable inhibitors was generally greater with PADAC than with nitrocefin. Unlike nitrocefin, PADAC mixed with 50% human serum or various broth culture media showed no evidence of color change or degradation over several hours. The subsequent enzyme hydrolysis rates of such mixtures were the same as in phosphate buffer. Beta-lactamase-containing bacterial suspensions and clinical specimens containing such bacteria produced positive visual and spectrophotometric color changes when mixed with PADAC or nitrocefin. Although color changes occurred more slowly with PADAC than with nitrocefin, PADAC was not adversely influenced (non-enzyme-related color change) by the protein content of specimens. PADAC appears to be a promising alternative for betalactamase diagnostic testing in the clinical and research microbiology laboratory.

Enterobacter gergoviae membrane modifications are involved in the adaptive response to preservatives used in cosmetic industry

“…In this assay, nitrocefin is hydrolysed by the periplasmic ß‐lactamase of Gram‐negative bacteria, and the detection of chromogenic product is an indicator of the outer membrane (OM) permeability (Jones et al . ).…”

In vitro evaluation of CENTA, a new beta-lactamase-susceptible chromogenic cephalosporin reagent

“…types I and IV, both commonly found in gramnegative clinical isolates. CENTA, PADAC, and cephacetrile (5,12) all had slower relative hydrolysis rates than nitrocefin. The RHR inhibition profiles ( Fig.…”

“…434, 1981). Like PADAC, cephacetrile, and nitrocefin, CENTA seems to offer promise as a diagnostic reagent in the clinical microbiology laboratory, particularly in the early rapid recognition of beta-lactamase production by clinical bacterial isolates (5,9,11). Research applications of such substances have also been accelerated in the quest for betalactamase-stable antimicrobial agents (3,4,13).…”

Differential regulation of fasA and fasH expression of Escherichia coli 987P fimbriae by environmental cues

“…Beta-lactamase assays were performed using the chromogenic reagent PADAC (pyridine-2-azo-p-dimethylaniline cephalosporin; Calbiochem, La Jolla, CA, USA), which is routinely used for diagnostic purposes (Kobayashi et al, 1988) and is more stable than other reagents used for ␤-lactamase analysis (Jones et al, 1982). PADAC was dissolved as described by the supplier in methanol-acetic acid, and then diluted to 1 mg ml ¹1 in PADAC buffer [10 mM NaH 2 PO 4 (pH 7)].…”