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

Abstract

Selection of resistance to cefamandole has been observed, and the drug has failed to protect animals lethally infected with certain Enterobacteriaceae that appeared to be highly susceptible in vitro. Using spectrophotometric assays, some of these organisms were found to produce beta-lactamases highly active against cefamandole. Cefoxitin, a poor enzyme substrate, was found to be superior to both cephalothin and cefamandole in induction of these enzymes. A simple disk induction test was developed and used to examine 147 Enterobacteriaceae for production of these beta-lactamases. The enzymes were found in 69% of cephalothin-resistant, cefamandole-susceptible strains and in only 3% of strains susceptible to both cephalothin and cefamandole. They were most prevalent among isolates of Enterobacter, indole-positive Proteus, and Serratia. Since selection of resistance and therapeutic failures have occurred most often among these genera, the relationship between presence of inducible enzymes and outcome of therapy should be examined further in humans.Cefamandole (CM;6,11,26) and cefoxitin (CX; 3, 24) possess a greater spectrum of antibacterial activity than previously marketed cephalosporins. Possible bases for the expanded spectrum of these new compounds include their resistance to a variety of beta-lactamases and/ or greater intrinsic activity, i.e., better penetration into the cell and attachment to target proteins (3,5,11,20,22,26). However, results of a recent study with these new drugs showed significant discrepancies between in vitro and in vivo tests, especially with CM (9). Animals infected with organisms appearing to be susceptible to CM in vitro failed to respond to therapy with the drug. Furthermore, in vivo selection of resistant mutants was observed in several instances. The purpose of this investigation was to analyze strains used in the previous study to determine if beta-lactamase production might be responsible for the discrepancies observed. MATERIALS AND METHODSAntibiotics. All drug solutions (weight compensated for purity) were prepared the day of use. Working standards were prepared from the following anti- Beta-lactamase assays. Beta-lactamase activity was evaluated by direct spectrophotometric methods (19) on whole cells. Cells from a 24-h Mueller-Hinton agar culture (Difco) were suspended in phosphate buffer (pH 7) so as to give 109 colony-forming units per ml. Fresh drug solution was added to the cell suspension to give a final concentration of 100 ,M, and, after incubation for 4 h at 37°C in air, the cells were removed by filtration (Millipore membrane filter). Plate counts of the drug-cell suspensions indicated no loss in viability during the 4-h incubation. The concentration of active drug remaining in the filtrate was determined by reading the optical density of the solution at the wavelength of maximal absorption associated with the beta-lactam ring (Beckman DB-GT). This wavelength varied between 255 and 270 nm for the different drugs tested. Drug solutions incubated similarly without cell...

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