Early effects of beta-lactams on aminoglycoside uptake, bactericidal rates, and turbidimetrically measured growth inhibition in Pseudomonas aeruginosa

Abstract

In vitro studies of tircarcillin or cefsulodin combined with [3H]tobramycin were performed with Pseudomonas aeruginosa. The rate of bacterial killing, the uptake of tobramycin, and the effects on optical density were measured. Both ,I-lactams increased the uptake of subinhibitory concentrations of tobramypin. This result was quantitatively associated with a 2-to 4-h time-kill potentiation and confirmed earlier studies on the mechanism of J3-lactam-aminoglycoside synergy in Escherichia coil (P. H. Plotz and B. D. Davis, Science 135:1067-1068, 1962 (17,23). This finding suggests that this mechanism of drug potentiation is strain or species specific.The purpose of this study was to examine the effects of 1-lactam antibiotics on the stimulation of increased aminoglycoside uptake in Pseudomonas aeruginosa. We also wanted to confirm in gram-negative bacilli our findings with gram-positive cocci that increased uptake correlates temporally with enhanced killing by P-lactam-aminoglycoside combinations and that these effects are apparent after 2 to 4 h of incubation (17; 20 .Ci/mg. Samples were counted in a liquid scintillation counter by using a toluene-based scintillant (16). All studies were done at least in duplicate. Figure 1A shows the effects of ticarcillin at one-quarter of the MIC on the uptake of tobramycin at one-half of the MIC. This figure shows the results of a representative experiment in which ticarcillin was added 30 min before tobramycin was added. The results were similar when the antibiotics were added simultaneously. Exposure of the cells to ticarcillin was associated with a marked stimulation of tobramycin uptake. Neither ticarcillin nor tobramycin alone inhibited cell growth, whereas the combination was associated with a bactericidal effect at 2 to 4 h (Fig. 1B). Figure 1C shows the effects of antibiotics on growth (as measured by OD), which increased without antibiotics and with either ticarcillin or tobramycin alone. The ticargillin-tobramycin combination showed no effect from 0 to 2 h, after which an inhibition of turbidimetrically measured growth was observed. Experiments with ticarcillin at one-quarter of the MIC and tobramycin at one or two times the MIC also showed an excellent correlation among ,-lactam-induced tobramycin uptake, bactericidal effect, and growth inhibition (data not shown). In these experiments, however, ,B-lactam-enhanced uptake and bacterial killing were apparent only before 1 h. Thereafter, the rates of tobramycin uptake and bacterial killing were similar to those with and without ticarcillin.Experiments with cefsulodin and tobramycin each at one-quarter to one-half of the MIC showed no increased uptake of tobramycin, increased bacterial killing, or growth inhibition (as measured by OD). However, when cells were exposed to cefsulodin at the MIC in combination with tobramycin at one-half of the MIC ( Fig. 2A), there was stimulation of tobramycin uptake. Cell viability was similar in cells not exposed to antibiotics and in cells exposed to cefsulodin (Fig. 2B). In contrast, ...

Once-daily aminoglycoside therapy

“…Bryan demonstrated that aminoglycoside-selected small-colony resistant mutants had respiratory transport enzyme changes that resulted in a marked reduction in the transmembrane potential (9). In a manner not yet understood, in the presence of an active beta-lactam there is a preservation of the high transmembrane potential in small-colony variants of P. aeruginosa (42).…”

Combination Therapy for Treatment of Infections with Gram-Negative Bacteria

“…The combination of a ␤-lactam and an aminoglycoside allows for different mechanisms of bacterial killing (8,66,98,237). ␤-Lactam-mediated disturbance of the cell walls of Gram-negative bacilli facilitates passage of aminoglycosides into the periplasmic space (105,169).…”

Activities of Antibiotic Combinations against Resistant Strains of Pseudomonas aeruginosa in a Model of Infected THP-1 Monocytes

“…Most of these studies concluded that there was synergy but to an extent that proved to be highly strain dependent. While the exact mechanism of this synergy often remains uncertain, a number of pharmacological reasons have been proposed, such as the enhancement of the uptake of the companion antibiotics by colistin through its destabilizing effects on the outer membrane (15) or the increased diffusion of entry of aminoglycosides through the peptidoglycan when altered by ␤-lactams (16).…”