Three Pseudomonas aeruginosa strains which constitutively produced chromosomal (Id, or Sabath and Abraham) /Mactamase in large amounts were resistant to latamoxef (moxalactam) MICs, 128-256 mg/1). Their 0-lactamase-basal mutants, which produced 1200-18,000-fold less enzyme, were latamoxef-sensitive (MICs, 4-16 mg/1), suggesting that the enzyme caused the resistance of the parent organisms. Latamoxef was a feeble substrate of the enzyme (kM <0-5/min) but reacted to form a stable complex that lacked catalytic activity against benzylpenicillin. The complex was isolated by gel filtration and was shown to be stable to isoelcctric focusing, suggesting a covalent link between the enzyme and latamoxef. During incubation the complex underwent a slow breakdown, regenerating active enzyme. This breakdown obeyed first-order kinetics, and the half-life of the inactivated form was 19±lmin at 37°C. Binding of antibiotic molecules in this complex may contribute to the latamoxef-resistance observed in the /Mactamase-dereprcsscd strains. This 'covalent trapping ' should be distinguished from the 'non-covalent trapping ' proposed elsewhere as a general mechanism of /J-lactamase-mediated resistance to reversibly-bound weak-substrate /Mactams.

Summary. The activities of P-lactam antibiotics were compared against Enterobacter cloacae clinical isolates and mutants which had inducible, stably-derepressed, and basal expression of a PI 8.4 subtype of the Ia chromosomal P-lactamase. These activities were correlated with the results of studies of the P-lactamase-lability and /?lactamase-inducer-power of the antibiotics. Cefoxitin and ampicillin were labile, and induced p-lactamase production strongly at concentrations below their MIC values. Consequently, /?-lactamase-inducible and p-lactamase-stably-derepressed organisms were highly resistant (MIC> 256 mg/L) to these antibiotics, whereas enzyme-basal strains and mutants were much more susceptible (MIC 1-16 mg/L). Imipenem also induced p-lactamase production strongly at concentrations below its MIC, but was more stable than ampicillin and cefoxitin. It was active against enzyme-inducible and stably-derepressed organisms at 0-25-0.5 mg/L and against P-lactamase-basal organisms at 0.06-0-25 mg/L. Thus the P-lactamase afforded only very low-level protection against imipenem; this appeared to be by a non-hydrolytic mechanism, with the enzyme binding to the antibiotic in a relatively stable complex. This complex, which probably was an intermediate in a hydrolytic pathway, was isolated by gelfiltration chromatography and shown to have a breakdown half-life of 47 f 2 min. Cefotaxime, ceftriaxone and mezlocillin were labile to the PI 8.4 P-lactamase but induced p-lactamase production weakly at concentrations below their MIC values. Consequently, P-lactamase-inducible and P-lactamase-basal organisms remained equally susceptible (MIC 0.06-4 mg/L), but stably-derepressed organisms were considerably more resistant (MIC> 64 mg/L) to these antibiotics.

complex to HT-29 intestinal mucosal cells. inhibit binding of Mycobacterium avium Rifabutin and sparfloxacin but not azithromycin

A method is reported for automating measurements of Km and specific Kml values from single progress curves of hydrolysis of /Mactam antibiotics by crude (or purified) /f-lactamase preparations. The method is based on the half-time analysis procedure of Wharton & Szawelski (1982). The specific Kmai is obtained in units of mols of substrate hydrolysed /min/mg dry wt of cells, but could be expressed as easily in terms of mols of enzyme or mg of protein. We propose that the method will allow reporting of Km and specific Kmal values, rather than relative rates of hydrolysis, in surveys of bacterial /Mactamases.

Pseadomonas aeruginosa fi-\actamase as a defence against azlocillin,