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Polyunsaturated fatty acids (PUFAs), which may alter eicosanoid biosynthesis and tumor necrosis factor alpha secretion in experimental bacterial infection models (1, 8, 11), are appropriate candidates for immunomodulation of infections caused by multidrug-resistant isolates. Gamma-linolenic acid (GLA) and arachidonic acid (AA), which are n-6 PUFAs, suppress the growth of gram-negative isolates, and when used in combination they possess a considerable killing effect on Pseudomonas aeruginosa isolates susceptible to antipseudomonal agents (3-5), a phenomenon probably attributed to their peroxidation. The present study is interested in their effects on multidrug-resistant P. aeruginosa and on their interactions with antipseudomonal agents.

Twenty-six P. aeruginosa isolates derived from patients with nosocomial infections were tested. Their epidemiological characteristics and resistance patterns are given in Table 1. GLA and AA ethyl esters (Sigma Chemical Co., St. Louis, Mo.) were dissolved in 99% ethanol (Merck, Darmstadt, Germany) to an initial dilution of 10 mg/ml. Appropriate amounts of the latter dilution were added to 10-ml tubes that contained Mueller-Hinton broth (Oxoid Ltd., London, United Kingdom) and a log-phase P. aeruginosa inoculum (10⁶ CFU/ml). GLA and AA were present at final concentrations of 150 µg/ml (0.55 mM GLA, 0.49 mM AA) and 300 µg/ml (1.1 mM GLA, 0.98 mM AA), and 99% ethanol was present at a concentration of 3% (vol/vol) or 6% (vol/vol). Ceftazidime (Glaxo, Macclsfield, United Kingdom) or amikacin (Bristol, Syracuse, N.Y.), or both, were added to tubes with 300 µg of either acid per ml or to tubes with 150 µg of each acid per ml. Antibiotics were provided as white amorphous powders, and they were applied at a concentration of 16 µg/ml, which is equal to the mean level achieved in serum after administration of conventional doses (6). All experiments were repeated after the addition of albumin (Sigma) at the level at which it is found in serum (4 g%). Tubes that contained 99% ethanol, albumin, ceftazidime, or amikacin or their respective combinations served as controls.

All prepared tubes and one growth control tube were left to incubate at 37°C in a shaking water bath, and bacterial growth was determined at standard time intervals (time zero and after 3, 5, and 24 h of incubation) by removing a 0.1-ml aliquot from each tube. The aliquot was serially diluted 1:10 five times in sterile 0.9% NaCl. Another 0.1-ml aliquot of each dilution was diluted onto MacConkey agar (Becton Dickinson, Cockeysville, Md.). These serial dilutions permitted the avoidance of any antimicrobial carryover effect. At each time interval the change in the log₁₀ number of viable cells compared to the number in the starting inoculum was determined. The activity of a combination of a PUFA and an antibiotic was synergistic when it fulfilled the following criteria, in analogy to the definition applied to antimicrobial agents (7): (i) it was bactericidal, achieving a greater than or equal to 3-log₁₀ decrease in viable cell counts from the baseline count, (ii) it was superior to the activity of ceftazidime combined with amikacin, and (iii) it achieved a greater than 2-log₁₀ decrease in viable cell counts compared to that achieved with the combination of one PUFA with ceftazidime and/or amikacin. A total of 1,092 killing curves were prepared for determination of viable cell counts in duplicate. The change in the log₁₀ cell counts was expressed as the median value, and the values were compared by Wilcoxon's rank sum test. The synergy achieved by the combinations tested was compared by Fisher's exact test. Any P value of <0.05 was considered significant after a Bonferroni correction.

The effects of the combinations tested against multidrug-resistant P. aeruginosa are shown in Table 2. Single acids and antimicrobial agents did not exert any considerable effect on the growth of P. aeruginosa, a phenomenon not influenced by the presence of albumin.

The strains included in the present study differed from each other since they were isolated in different time periods and at different hospitals and the MICs of 10 antimicrobial agents for the isolates were different (Table 1). A significant alteration of the growth of multidrug-resistant P. aeruginosa is achieved by the combination of PUFAs, with which synergistic activity against 30.8% of isolates was found (Table 2), but not by a single acid or by the interaction of a single acid with either ceftazidime or amikacin, with which synergistic activity was not found against more than 19.2% of isolates. However, when both ceftazidime and amikacin interact with GLA or AA at 300 µg/ml, synergistic activity was found against 38.5% of isolates after 24 h of growth, raising up to 50% the proportion of isolates against which each PUFA at 150 µg/ml used in combination interacted with both antibiotics. Even earlier synergist activity, after 5 h of growth, is obtained in the presence of albumin, which is extremely important, since PUFAs are transported in blood bound to albumin (10). Although few data on the levels of PUFAs in plasma exist, the concentration of eicosapentaenoic acid under conditions of stress like sepsis reaches 140 µM (2). Since eicosapentaenoate is usually found at much lower concentrations than AA and both GLA and AA coexist in plasma (although it is presumed that the 150-µg/ml concentration should be more easily achievable in plasma than the 300-µg/ml one), the results presented here should be of clinical significance. That assumption is further supported by the application of ceftazidime, amikacin, and albumin at the levels achieved in serum. The bactericidal effect of GLA in combination with AA has also been demonstrated in studies by our group with isolates susceptible to antipseudomonal agents (5).

It has been shown that the action of PUFAs on the bacterial cell is regulated through their peroxidation (5), leading to cell killing, a process reversed in the presence of the antioxidant vitamin E. For all isolates tested, the MICs of antimicrobial agents with diverse chemical structures were elevated, and synergy was irrespective of the MICs of ceftazidime for the isolates (Table 1). Since the multidrug-resistance of these isolates might result in defective permeation of antibiotics through the bacterial outer membrane (9), the products of the peroxidation of PUFAs attack the cell wall, thus making it more permeable, and this might be a suitable hypothesis of the mechanism of the synergistic activities of these acids with ceftazidime and amikacin. It should be emphasized that the effect of PUFAs is not affected by albumin, since binding of PUFAs to albumin does not involve bonds in which the peroxidation process occurs (10).

The present study indicates that n-6 PUFAs might render ceftazidime and amikacin active against multidrug-resistant P. aeruginosa acids. Since the intravenous administration of the lithium salt of GLA to patients with end-stage human immunodeficiency virus infection has been well tolerated and has been accompanied by a transient increase in CD4⁺ cell counts (12), PUFAs might be applied as adjuvants of antimicrobial chemotherapy in experimental infection models.