Importance of extracellular and cell-bound beta-lactamase in mediating resistance of Staphylococcus aureus to mezlocillin

Abstract

The 3-lactamase of Staphylococcus aureus is excreted into the surrounding medium, although to different extents (1, 3). The proportion of extracellular enzyme depends on the strain studied and on the conditions of growth (2). The majority of penicillin-resistant strains causing nosocomial infections are known to produce large amounts of extracellular 3-lactamase (4). There is some evidence that the cellassociated enzyme molecules are covalently bound to phosphatides (3), but it is not yet clear whether they are precursors of the free enzyme, as has been shown to be the case in Bacillus licheniformis (7).Investigators in this laboratory have an interest in the importance of cell-bound and extracellular 1-lactamase in mediating resistance of S. aureus to mezlocillin because of the potential influence on the outcome of susceptibility tests. When minimal inhibitory concentrations are determined by the broth or the microdilution technique, the excreted 13-lactamase accumulates in a small volume of broth and thus may rapidly inactivate the pool of hydrolyzable antibiotic in the test system. In vivo, on the other hand, a considerable portion of exoenzyme may diffuse away from the microbial cells, depending on the site of infection, and therefore may lose its protective effect. Therefore, the question arose whether the enzyme associated with the cell wall alone constitutes a barrier strong enough to inactivate the intruding antibiotic, thus preventing it from reaching the target sites. This was examined by a new in vitro test model designed to eliminate the effect of exoenzyme. MATERIALS AND METHODSBacteria. The test strains were fresh clinical isolates of S. aureus which were stored in liquid nitrogen until use. Strains Va7871 and Va7906 were selected because of their extracellular 3-lactamase activity. Exponentially growing cultures (35°C) were induced with 1 ,i.g of cefoxitin per ml for 3 h (final cell count, 2 x 108 to 5 x 108 CFU/ml), and the extracellular ,-lactamase was measured after centrifugation by iodometric assay (6). The amounts of penicillin G de-125 stroyed by 1 ml of culture supernatant from strains Va7871 and Va7906 were 1.3 and 1.9 mmol/min, respectively. Minimal activity was found without induction. S. aureus Va8143 proved to be 3-lactamase negative under these conditions, even in the presence of subinhibitory concentrations of cefoxitin.In vitro model for elimination of exoenzyme. Iso-Sensitest broth (Oxoid) was used as the nutrient medium throughout all experiments. Inocula were preincubated at 35°C on a rotary shaker for 5 h to obtain cell material of early stationary growth phase. Approximately 105 cells were spread on a sterile cellulose-nitrate filter (Schleicher & Schull Co.; PH 79/1, 0.1-,um-pore size, 76-mm diameter) and were covered by a second membrane. This membrane sandwich containing the bacteria was then mounted in a sterile ultrafiltration chamber (Amicon 402) and incubated at 35°C. During incubation, a constant flow (280 ml/h) of prefiltered nutrient broth containing the antibiotic c...

Extracellular and membrane-bound lactamase of Staphylococcus aureus: their importance for the expression of penicillin resistance

“…In vitro, the importance of extracellular p lactamase in determining resistance predominates but this may not be true in vivo. p Lactamases in the environment of the bacterial cells can be diluted by diffusion (Haller, 1984) or their effectiveness may be reduced by other tissue factors, e.g., low pH values in inflamed tissues, the action of proteases and oxygen radicals formed during phagocytosis, or inactivation by antibodies. Membrane-bound p lactamase may therefore provide a second line of defence which could be highly effective because it is located close to the target sites of penicillin (Bush and Sykes, 1984).…”

“…It has been reported (Haller, 1984) that intracellular p lactamase could not protect staphylococci from higher concentrations of mezlocillin because resistant strains became susceptible to this penicillin when extracellular p lactamase was washed off.…”

Catechol-Vanadate Analogues as β-Lactamase Inhibitors

“…One of the main mechanisms of β -lactam resistance utilizes the β-lactamase enzymes. In gram positive bacteria, these enzymes are excreted into the extracellular matrix, 11 while the enzyme is concentrated in the periplasmic space by gram negative bacteria. 12 These enzymes have the ability to bind to penicillin molecules and hydrolyze their β-lactam core, forming an inactive carboxylic acid (Scheme 2).…”