Relation of β-Lactamase Activity and Cellular Location to Resistance of Enterobacter to Penicillins and Cephalosporins

Mutants with enhanced P-lactam resistance were selected from strains of Enterobacter cloacae and E. aerogenes by using three antibiotics. High-level ,Blactamase-producing mutants had similar degrees of increased resistance, enzyme substrate profiles, and isoelectric (pl) values irrespective of the selective agent. Reverse mutants from a resistant E. cloacae mutant regained the susceptibility pattern originally exhibited by the wild type, or were of enhanced susceptibility, and no longer expressed increased P-lactamase production. ,-Lactamases of the mutants were similar in pI values to the wild-type enzyme. The increased resistance of the mutants therefore appeared to be accounted for by increased Plactamase production.Previous studies from this laboratory (4, 10) and others (18) have shown that cefamandoleand ampicillin-resistant mutants of Enterobacter species occur at a frequency as great as 10-5.Two classes of resistant mutants were selected by the antibiotics, one of which had markedly enhanced ability to inactivate the antibiotic in vitro. In our studies, the proportions of these two classes of mutants were approximately equal. We suggested that cefamandole resistance of the mutants with increased ,-lactamase production might be explained by a mutational change involving the enzyme which extended its spectrum of activity to include cefamandole. We further demonstrated cross-resistance between ampicillin and cefamandole in strains with enhanced enzyme production. Ott et al. (18) reported a lack of correlation between 3-lactamase production and susceptibility to cefamandole, found no qualitative differences between the substrate specificities of enzymes from a wildtype Enterobacter strain and its enhanced enzyme-producing mutant, and concluded that these mutants may have owed their resistance to genetic determinants other than those coding for ,-lactamase production.This study focused exclusively on mutants with enhanced 1-lactamase production and was designed to resolve these differences in interpretation.

supporting

confidence: 66%

Mutational Enzymatic Resistance of Enterobacter Species to Beta-Lactam Antibiotics

Lampe

Allan

Minshew

et al. 1982

“…A recent report on the outer membrane complex in B. fragilis shows, however, that its cell envelope is typical of gram-negative bacteria (13). It has been stated that R-factor-mediated (3-lactamases in aerobic gram-negative rods are released by osmotic shock treatment (19), whereas chromosomally mediated ones are cell bound (20). One report proposes that the molecular weight of the ,3-lactamase should be a ANTIMICROB.…”

Section: Resultsmentioning

confidence: 99%

Formation of Beta-Lactamase in Bacteroides fragilis : Cell-Bound and Extracellular Activity

Olsson

Nord

Wadström

1976

Nine strains ofBacteroides fragilis were cultivated in stirred fermentors and tested for their ability to produce (3-lactamase. There was a correlation between formation of ,8-lactamase and high values of the minimal inhibitory concentration against 3-lactam antibiotics. B. fragilis strain B70 was used for optimizing the production of /8-lactamase. The highest bacterial yield was obtained in a proteose peptone-yeast extract medium. Optimal conditions for growth and (8-lactamase production were obtained at 37 C and pH 7.0. The 3-lactamase was released into the surrounding medium during the growth period to about 50%. Osmotic shock released about 20% of the total activity, and remaining activity was found in the cytoplasmic fraction. Substrate profile studies on four (3-lactamase-producing strains showed that the enzymes were mainly cephalosporinases. They are inhibited by cloxacillin, p-chloromercuribenzoate, and iodine. Analytical isoelectric focusing in polyacrylamide gel gave an isoelectric point of 4.9 + 0.2 for three of the strains and 5.6 + 0.2 for one. Comparison with 38-lactamases from aerobic gram-negative species with regard to isoelectric points showed no similarities. Also the molecular weight of the 8-lactamase from strain B70 of 43,000 indicates that this is a new class of f-lactamase.

“…These findings suggest that in a number of species resistance is related to hydrolysis of the cefamandole. We did not investigate whether the cefamandole acted to induce a beta-lactamase in the E. cloacae strains, but previous studies would suggest this had occurred (5). Both the S. marcescens and P. morganii have beta-lactamase activity that is not subject to induction.…”

Section: Methodsmentioning

confidence: 99%

Cefamandole, a Cephalosporin Antibiotic with an Unusually Wide Spectrum of Activity

Neu

1974

Self Cite

133

The in vitro activity of cefamandole, 7-D-mandelamido-3-(1 methyl-lH-tetrazol-5 yethiomethyl)-3-cephem-4 carboxylic acid, was investigated. The majority of streptococci and pneumococci were inhibited by 0.1 sg/ml. Activity against Staphylococcus aureus was below that of cephalothin, but most strains were inhibited by 0.4 ,ug/ml. Enterococci were not inhibited by less than 25 'gg/ml.Cefamandole was very active against most members of the Enterobacteriaceae, with 70% of Escherichia coli, 86% of Klebsiella pneumoniae, and 88% of Proteus mirabilis inhibited by 1.6 ,g/ml. A number of Enterobacter aerogenes, Proteus vulgaris, and Serratia marcescens strains were inhibited by less than 25 sg/ml. Pseudomonas species were resistant. Cefamandole was more active than cephalothin, cephaloridine, or cephalexin against members of the Enterobacteriaceae. Inoculum size and type of medium had variable effects on the minimal inhibitory concentration, depending upon the particular strain tested. Strains of Enterobacter, Serratia, indole-positive Proteus species showed the greatest inoculum effect. Similarly, these species showed a several-fold difference of bactericidal and inhibitory levels. Resistance of cefamandole to hydrolysis by gram-negative beta-lactamase played a partial role in its activity, but some strains that hydrolyzed the agent were susceptible.Although the currently available cephalosporin antibiotics, namely cephalothin, cephloridine, cephalexin, and cefazolin, are active against a wide range of bacteria, they show minimal differences in in vitro activity against gram-negative species. In addition, these agents are inactive against a number of the members of the Enterobacteriaceae, namely, Enterobacter species, Serratia, Citrobacter, and Herellae, all of which are seen with increased frequency as nosocomial infections. There is, indeed, suggestive evidence which might link some of the upsurge in Enterobacter and Serratia infections with widespread use of cephalosporin antibiotics. Cefamandole is a 7-D-mandelamido-3-(1 methyl-lH-tetrazol-5 ylthiomethyl) -3-cephem-4 carboxylic acid and has recently become available for investigation (10). This paper reports studies of the overall antibacterial activity of cefamandole in comparison with other cephalosporin antibiotics and investigates its activity in relation to resistance to hydrolysis by beta-lactamase-containing bacteria.