Inactivation of beta-lactam antibiotics by Legionella pneumophila

Abstract

Beta-lactam-inactivating activity has been found in all sero-groups of Legionella pneumophila. The ,B-lactamase activity could be detected in intact cells and released by ethylenediaminetetraacetic acid treatment, indicating that it is located in the periplasmic space. The enzyme acted primarily as a cephalosporinase hydrolyzing cefamandole, cephalothin, cephaloridine, and also penicillin G and ampicillin. Cefoxitin and cefuroxime were not hydrolyzed. Clavulanic acid and CP-45,899, f)-lactamase inhibitors, prevented the hydrolysis of cephalosporins and penicillins. The fl-lactamase activity appears to be different from that found in Enterobacteriaceae and Pseudomonas.The reported in vitro resistance of Legionella pneumophila to penicillins and cephalosporins (7,8) as well as the clinical failure of these agents in the treatment of patients with Legionnaire's pneumonia (1) prompted us to evaluate the ability of this organism to destroy fi-lactam antibiotics and to clarify whether the inactivating activity was due to the presence of a f8-lactamase. MATERIALS AND METHODSOrganisms and antibiotics. Isolates of L. pneumophila were provided by the Center for Disease Control, Atlanta, Ga. The following isolates were examined: Seattle, Vermont, Flint, Knoxville, Philadelphia, Pontiac, Bloomington, Togus, and Los Angeles.Organisms were maintained on Mueller-Hinton agar plates supplemented with 0.04% cysteine and 0.025% ferric phosphate. The antibiotics studied were gifts from their respective manufacturers. Clavulanic acid was provided by Beecham Laboratories and CP-45,899, a penicillanic acid sulfone, was provided by Pfizer Inc. Susceptibility tests. Susceptibility tests were performed in Mueller-Hinton broth containing 0.04% cysteine and 0.1% ferric phosphate. The inoculum used was 105 colony-forming units. This was prepared by making a suspension in broth of a 48-h growth removed from agar plates. Tubes were incubated at 35°C for 72 h in the presence of 5% CO2.Osmotic shockates. Osmotic shockates were prepared by the technique of Neu and Chou, using 0.1 g of bacteria to 10 ml of 1 mM ethylenediaminetetraacetic acid in 10 mM tris(hydroxymethyl)aminomethanehydrochloride buffer, pH 7.0.Enzyme preparation. Three-day-old cultures were removed from agar plates by means of a glass spatula, using potassium phosphate buffer (0.05 M, pH 7). The cells were washed twice in the same buffer and then suspended in buffer at a concentration of 1010 cells per ml. The cells were sonically disrupted by a Branson Sonifier, using 30-s pulses for 3 min with the vessel cooled in an ice bath. All procedures were performed in a hood which incinerated the exhaust. The sonic extracts were centrifuged for 2 h at 4°C and 40,000 x g. The cellular debris was discarded, and the supernatant was dialyzed for 24 h at 4°C against the phosphate buffer. This material was used for the assays.f8-Lactamase assays. Intact organisms, material from osmotically shocked organisms, and the crude extract were tested for f,-lactamase activity with the nitrocefin (5), ...

The effect of antibiotics on the cell morphology of Legionella pneumophila

“…Eventually these bubble-like structures would burst, leaving behind an opening in the cell surface. The documented presence of a peptidoglycan layer in the cell envelope of L. pneumophila (Flesher et al, 1979) and the ability of this species to produce p-lactamase (Fu and Neu, 1979) may explain both the observed effects on the cell wall and why only a high concentration (1000 pg/ml) of penicillin is capable of producing these bubble-like structures, whereas lower concentrations (25 and 100 pg/ml) do not. This would be due to the inactivation of penicillin at lower concentrations by the p-lactamase produced by L. pneumophila.…”

Susceptibility of Legionella pneumophila to twenty antimicrobial agents

“…There was no consistent difference in susceptibility between Center for Disease Control-supplied stock strains and recent clinical isolates, but there were marked differences with some agents. Susceptibility testing needs to be standardized in view of the influence of inoculum size, strain variation, and the medium used.Susceptibility testing of Legionella pneumophila, the etiological agent of Legionnaires disease, has been previously reported only with small numbers of isolates (8,9,12,14,16,17) and not with charcoal-yeast extract (CYE) agar, which has become the standard medium for L. pneumophila (6). In addition, most strains tested have been passaged multiple times after isolation on media which do not optimally support their growth.…”

“…Susceptibility testing of Legionella pneumophila, the etiological agent of Legionnaires disease, has been previously reported only with small numbers of isolates (8,9,12,14,16,17) and not with charcoal-yeast extract (CYE) agar, which has become the standard medium for L. pneumophila (6). In addition, most strains tested have been passaged multiple times after isolation on media which do not optimally support their growth.…”

Bactericidal effects of ticarcillin-clavulanic acid against Legionella pneumophila pneumonia in immunocompromised weanling rats

“…Clavulanic acid inhibits the P-lactamase produced by L. pneumophila (2,8), but it is not established whether this is responsible for the synergy or whether the interaction between two antibacterial agents binding to different penicillin-binding proteins is responsible (15). In vivo, the combination of ticarcillin plus clavulanic acid was significantly more effective than clavulanic acid alone.…”