nitrocefin and pyridine-2-azo-4-dimethylaniline-cephalosporin

Tyr-105 is a conserved residue in the Class A beta-lactamases and is in close proximity to the active-site. Tyr-105 in beta-lactamase from Bacillus licheniformis was converted into Phe by site-directed mutagenesis. This mutation caused no significant effect on the structure of the enzyme and had only small effects on the catalytic properties. In particular, in comparison to the wild-type, kcat. for benzylpenicillin was increased slightly, whereas it was decreased slightly for several other substrates. For each substrate examined, Km increased 3-4-fold in the mutant compared with the wild-type enzyme. Examination of the effect of pH on the catalytic reaction revealed only small perturbations in the pK values for the acidic and basic limbs of the kcat./Km pH profiles due to the mutation. Overall effects of the Y105F substitution on the catalytic efficiency for different penicillin and cephalosporin substrates ranged from 14% to 56% compared with the wild-type activity. We conclude that Tyr-105 is not an essential residue for beta-lactamase catalysis, but does contribute to substrate binding.

Topics: Bacillus; Base Sequence; beta-Lactamases; Cephalosporins; Chromogenic Compounds; Circular Dichroism; Conserved Sequence; DNA Primers; Hydrogen-Ion Concentration; Indicators and Reagents; Molecular Sequence Data; Mutagenesis, Site-Directed; Penicillin G; Penicillin V; Protein Structure, Secondary; Structure-Activity Relationship; Substrate Specificity; Tyrosine

Pyridinium-2-azo-p-dimethylaniline chromophore was evaluated as a test tube, filter paper and spectrophotometric assay for detection of beta-lactamases from gram-positive and gram-negative organisms. Although useful for detection of TEM beta-lactamases in Haemophilus influenzae and Neisseria gonorrhoeae, it was a poor agent for detecting TEM, OXA and PSE enzymes in Enterobacteriaceae. It also proved poor for detecting cephalosporinases in Pseudomonas aeruginosa and Enterobacteriaceae, and penicillinases in Staphylococcus aureus when compared to nitrocefin. As a spectrophotometric substrate it was equivalent to nitrocefin and cephaloridine with various beta-lactamases.

Topics: Anti-Bacterial Agents; Bacteria; beta-Lactamase Inhibitors; beta-Lactamases; Cephaloridine; Cephalosporins; Chromogenic Compounds; Drug Resistance, Microbial; Indicators and Reagents; Spectrophotometry

Four methods for detecting beta-lactamase activity in anaerobic bacteria were evaluated and compared to a nitrocefin saturated filter paper test. The methods studied were Cefinase, PADAC, Beta Lactam, and the slide iodometric technique. Fifty anaerobes were tested including species of Bacteroides, Fusobacterium, and Clostridium. The percentages of agreement with the nitrocefin test are: Cefinase 100%, PADAC 96%, slide iodometric 78%, and Beta Lactam 72%. Positive nitrocefin tests have been shown to correlate with a penicillin MIC greater than 0.78 micrograms/ml.

Topics: Bacteria, Anaerobic; Bacteriological Techniques; Bacteroides; beta-Lactamases; Bromcresol Purple; Cephalosporins; Chromogenic Compounds; Clostridium; Fusobacterium; Indicators and Reagents; Penicillin G

Pyridine-2-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 microgram/ml) against Staphylococcus aureus, a quality comparable to nitrocefin. Nitrocefin, however, demonstrated an unexpected and uniquely potent activity against Streptococcus faecalis (minimum inhibitory concentration, less than or equal to 0.06 to 0.12 microgram/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 IIIa beta lactamase and the derived from Bacillus cereus. The inhibition of beta-lactamase hydrolysis of the chromogenic cephalosporin substrates by six enzyme-stable 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 beta-lactamase diagnostic testing in the clinical and research microbiology laboratory.

Topics: Bacteria; beta-Lactamases; Cephacetrile; Cephalosporins; Indicators and Reagents

CENTA is a newly synthesized, beta-lactamase-labile, chromogenic cephalosporin reagent which changes color from light yellow (lambda maximum ca. 340 nm) to chrome yellow (lambda maximum ca. 405 nm) concomitant with hydrolysis of the beta-lactam ring. This compound offers promise as a diagnostic reagent comparable to other chromogens (PADAC and nitrocefin) for the early detection of beta-lactamase-producing clinical isolates, while retaining some antimicrobial effect against Escherichia coli, Klebsiella spp., Proteus mirabilis, Staphylococcus aureus, and non-enterococcal Streptococcus spp. CENTA is relatively unaffected by commonly used microbiological media and human serum.

Topics: Bacteriological Techniques; beta-Lactamase Inhibitors; beta-Lactamases; Cephalosporins; Citrobacter; Drug Evaluation, Preclinical; Enterobacteriaceae; Escherichia coli; Evaluation Studies as Topic; Indicators and Reagents; Klebsiella; Proteus mirabilis