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

Abstract

Nine strains of Bacteroides fragilis were cultivated in stirred fermentors and tested for their ability to produce β-lactamase. There was a correlation between formation of β-lactamase and high values of the minimal inhibitory concentration against β-lactam antibiotics. B. fragilis strain B70 was used for optimizing the production of β-lactamase. The highest bacterial yield was obtained in a proteose peptone-yeast extract medium. Optimal conditions for growth and β-lactamase production were obtained at 37 C and pH 7.0. The β-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 β-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 β-lactamases from aerobic gram-negative species with regard to isoelectric points showed no similarities. Also the molecular weight of the β-lactamase from strain B70 of 43,000 indicates that this is a new class of β-lactamase.