Substrate Inhibition of Beta-Lactamases, a Method for Predicting Enzymatic Stability of Cephalosporins

Abstract

Selected cephalosporins, including cefamandole, cephaloridine, cephaloglycin, and cefoxitin, were examined for their ability to inhibit the enzymatic activity of and act as substrates for beta-lactamases produced by Enterobacter cloacae and Staphylococcus aureus. Enzyme inhibition was determined by Michaelis-Menten kinetic measurements and by a spot plate assay using a chromogenic substrate (Glaxo compound 87/312). These two methods provide comparable estimates of kinetic parameters. Inhibition of beta-lactamase, as measured by these two methods, was generally found to correlate with resistance to hydrolysis and is proposed as a preliminary method of assessing susceptibility of cephalosporins to beta-lactamase hydrolysis. Four 7-aOCH3, 7-aH cephalosporin analogue pairs were also examined. The presence of the 7-aOCH3 substituent invariably resulted in reduced susceptibility to enzymatic hydrolysis, regardless of the other C7 substituent. The 7-aOCH3 compounds were also better inhibitors than were their 7-aH analogues, with the exception that 7-aOCH3 compounds having C7 adipic acid substituents were less inhibitory to the S. aureus enzyme than were the corresponding 7-aH analogues. Response of these two enzymes to 7-aOCH3 and 7-aH cephalosporins suggests that beta-lactamase hydrolysis of these compounds involves attack at the alpha side of the betalactam ring.Two naturally occurring 7-a-methoxycephalosporins, 7-a-methoxycephalosporin C and deacetyl-3-O-carbamoyl-7-a-methoxycephalosporin C, were originally isolated from two species of Streptomyces by Nagarajan et al. (9). Both antibiotics exhibited greater activity against gram-negative organisms than did cephalosporin C or deacetyl-3-O-carbamoylcephalosporin C, their 7-hydrogen analogues, and the carbamoyloxymethyl derivative was reported (18) to be more active against gramnegative than gram-positive bacteria. Cefoxitin, a thienylacetyl derivative of deacetyl-3-0-carbamoyl-7-a-methoxycephalosporin C, has been reported to have activity against grampositive and gram-negative organisms (21). This 7-methoxylated cephalosporin generally showed resistance to the action of beta-lactamases from both gram-positive and gram-negative bacteria (14), and it was suggested that this characteristic contributed to the broad antimicrobial spectrum observed. There is considerable disagreement as to the relative importance of beta-lactamases in bacterial resistance to beta-lactam antibiotics (20). Because of the lack of uniform correlation between in vitro minimal inhibitory concentration values and lability of these compounds to enzymatic hydrolysis (1, 10, 11), this lability, by itself, is not sufficient evidence to predict bacterial susceptibility. Despite this, it is probable that the action of these enzymes does contribute to resistance although not always as the limiting factor. Given this assumption, the affinity of a betalactamase for a given cephalosporin should play a role in overall resistance of a beta-lactamaseproducing organism. The affinity (Ki) can be measured directl...

Emergence of Resistance to Cefamandole: Possible Role of Cefoxitin-Inducible Beta-Lactamases

“…Although the ability of CX to act as an inducer of beta-lactamases has been reported previously (4,10,12,20,25), its superiority over similar drugs as an inducer and the enhanced activity of the induced enzyme for CM has not been reported. This phenomenon resembles methicillin induction of penicillinase in Staphy- (5,14,18). As such, it may prove to be a very useful agent in vitro for the study of beta-lactamases from gram-negative bacilli.…”

Lack of Correlation Between β-Lactamase Production and Susceptibility to Cefamandole or Cefoxitin Among Spontaneous Mutants of Enterobacteriaceae

“…Quantitation of B8-lactamase in crude extracts was determined spectrophotometrically with compound 87/312 (14). Specific activities of the /3-lactamase using cefamandole and cephaloridine as substrates were determined as previously described (11 15) were selected from colonies growing on the antibiotic-containing plates. These isolates were inoculated into brain heart infusion broth and incubated overnight at 37°C.…”

Transition state structures of a dipeptide related to the mode of action of beta-lactam antibiotics.

“…The implication that the a face is attacked finds support in recent experiments (52)(53)(54). The 6a-substituted penicillins have been found to be less reactive and also poorer inhibitors of transpeptidase than the 6a-H analogs (52,53).…”

“…The 6a-substituted penicillins have been found to be less reactive and also poorer inhibitors of transpeptidase than the 6a-H analogs (52,53). Substitution of methoxyl groups at the 7a position of cephalosporins results in reduced susceptibility to hydrolysis by j3-lactamases and also generally produces better inhibitors of these enzymes (54). Both of these findings suggest that nucleophilic attack at the receptor sites could involve the a-face of the fl-lactam ring.…”