JAC Advance Access originally published online on January 6, 2003
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Journal of Antimicrobial Chemotherapy (2003) 51, 423-426
© 2003 The British Society for Antimicrobial Chemotherapy
Ex vivo synergy of arachidonate-enriched serum with ceftazidime and amikacin on multidrug-resistant Pseudomonas aeruginosa
1 4th Department of Internal Medicine, Sismanoglio General Hospital, 1 Sismanogliou Str., 151 26 Maroussi Attikis; 2 Laboratory for Experimental Surgery and Surgical Research; 3 Department of Biological Chemistry, University of Athens, Medical School, Athens, Greece
Received 2 April 2002; returned 8 August 2002; revised 11 September 2002; accepted 27 September 2002
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Abstract |
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Three multidrug-resistant strains of Pseudomonas aeruginosa were incubated ex vivo with sera sampled after a 10 min intravenous infusion of 25 mg/kg of arachidonic acid (AA) in 10 rabbits in the presence of ceftazidime and amikacin. Lipid peroxidation was assessed during bacterial growth. A statistically significant decrease in bacterial cells was found by the interaction of antimicrobials and serum sampled in the middle of infusion and 15 and 30 min after infusion of AA and was accompanied by elevated levels of malonodialdehyde. This effect of AA is probably attributed to lipid peroxidation and raises the possibility of its application in experimental infections.
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Introduction |
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TopAbstractIntroductionAnimals and methodsResults and discussionReferences |
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N-6 polyunsaturated fatty acids (PUFAs) such as gamma-linolenic acid (GLA, C18:3) and arachidonic acid (AA, C20:4) are potent immunomodulators when administered orally.1 In order to be useful in the management of infections, they would have to be administered intravenously. In vitro experiments have shown that n-6 PUFAs may render ceftazidime and amikacin active for multidrug-resistant Pseudomonas aeruginosa.2 This effect occurs in the presence of albumin, which is the carrier of n-6 PUFAs in serum. In order to test the in vitro results in conditions similar to the in vivo setting, an emulsion of AA was administered intravenously in rabbits and serum was sampled at various time intervals before and after infusion. Three multidrug-resistant P. aeruginosa isolates were exposed ex vivo to these samples in the presence of ceftazidime and amikacin.
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Animals and methods |
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TopAbstractIntroductionAnimals and methodsResults and discussionReferences |
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Preparation of the dilution of AA
AA ethyl ester (Sigma Co, St Louis, MO, USA) was dissolved in 99% ethanol (Merck, Darmstadt, Germany) to an initial dilution of 10 mg/mL and kept at –70°C in a nitrogen atmosphere. Respective amounts were diluted into sterile and endotoxin-free water (Difco Laboratories, London, UK) to a final volume of 50 mL for administration.
Animals
Fourteen white New Zealand male rabbits weighing 3.9–4.2 kg were used. The study received permission from the Veterinary Directorate of the Perfecture of Athens according to Greek legislation in conformance with the Council Directive of the EEC.
Study design
The rabbits were sedated by administration of an intramuscular injection of 25 mg/kg of ketamine and 5 mg/kg of xylazine. After a mid-line neck incision, a tracheostomy was carried out and the animals were intubated and mechanically ventilated at 16 breaths/min. Anaesthesia was maintained by the intravenous administration of 20 mg/kg of sodium thiopental. The right jugular vein and the left common carotid artery were then dissected and catheterized by an 18G gauge catheter. AA was administered intravenously by the right jugular vein in 10 animals at a dose of 25 mg/kg within 10 min with a continuous infusion pump, as already described.3 Ethanol 99% dissolved in water was infused in four animals used as controls.
Blood samples (6 mL aliquots) were collected via the left carotid artery before, in the middle and at the end of the infusion and 15, 30, 45 and 60 min afterwards. Samples were collected into sterile and pyrogen-free tubes (Difco Laboratories) and centrifuged at 12 000g and 4°C for 10 min. Serum was stored at –70°C in a nitrogen atmosphere until application.
Bacterial isolates
Three multidrug-resistant strains of P. aeruginosa (A, B and C) isolated from blood cultures collected from three different patients with nosocomial sepsis were studied. MICs of ticarcillin–clavulanate, piperacillin, ceftazidime, imipenem, meropenem, amikacin and ciprofloxacin were determined by a microdilution technique using a 0.1 mL final volume. The respective MICs for strain A were 256/2, 512, 16, 64, 32, 128 and 256 mg/L, for strain B 128/2, >512, 128, 8, 16, 32 and >512 mg/L and for strain C 256/2, >512, >512, >512, 256, >512 and 128 mg/L.
Time–kill curve assays
Time–kill assays were carried out so that each isolate would be exposed to each serum sample derived from each animal at different time intervals in the presence of antimicrobials. Briefly, 1 mL of serum was added to tubes containing Mueller–Hinton broth (BBL Becton Dickinson, Cockeysville, MD, USA), ceftazidime and amikacin and a 5 x 106 cfu/mL log-phase inoculum of each isolate to a final volume of 10 mL. Concentrations of ceftazidime and amikacin were 16 and 16 mg/L, respectively, so as to approach their mean serum level after the administration of their conventional dose.2 Tubes with 1 mL of serum sampled from animals given only 99% ethanol and antimicrobials and tubes containing only antimicrobials served as controls. A growth control tube was also used.
Tubes were incubated at 37°C in a shaking water bath and after 3, 5 and 24 h, aliquots were withdrawn to determine viable bacterial counts and lipid peroxidation. Bacterial counts were estimated after five consecutive 1:10 dilutions of a 0.1 mL aliquot into sterile water and after plating another 0.1 mL aliquot of each dilution on to MacConkey agar (BBL Becton Dickinson) so as to avoid any possible antimicrobial carry-over effect. The lowest detection limit was 30 cfu/mL. Growth controls and controls with antimicrobials were assayed eight times each. A total of 310 time–kill curves were carried out, all in duplicate. Bacterial growth in the presence of sera sampled after administration of AA was expressed as the log10 changes of the viable cell counts from baseline minus log10 changes found after growth in the presence of sera sampled after administration of 99% ethanol; they were presented as means (±S.E.).
Determination of lipid peroxides
Lipid peroxidation was determined by the thiobarbiturate assay.4,5 Briefly, a 0.5 mL aliquot from each tube was mixed 1:1 with trichloroacetic acid (TCA) 20% (Merck, Darmstadt, Germany) and centrifuged at 12 000g and 4°C for 10 min. The supernatant was removed and incubated with 1 mL of PBS pH 7.0 (Merck) and 1 mL of thiobarbituric acid (TBA) 0.6% (Merck) for 20 min at 90°C. Absorbance was read at 535 nm (Hitachi Spectrophotometer, Tokyo, Japan). Concentrations of malonodialdehyde (MDA) were determined by a standard curve created with 1,1,3,3-tetramethoxy-propane (Merck) and expressed by their mean (±S.E.). A 0.5 mL water sample treated in the same way was applied as a blank.
Statistical analysis
Comparison of results before and after infusion of AA were carried out by ANOVA (P < 0.05). Bonferroni correction was applied to avoid random correlations.
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Results and discussion |
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TopAbstractIntroductionAnimals and methodsResults and discussionReferences |
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Changes of bacterial growth of each isolate in the presence of sera sampled after the infusion of AA and of their controls are shown in Table 1. Concentrations of MDA determined during incubation of P. aeruginosa with sera sampled before and after the administration of AA in the presence of antimicrobials are shown in Figure 1.
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In vitro results revealed that multidrug-resistant P. aeruginosa becomes susceptible in vitro to the combination of ceftazidime and amikacin in the presence of n-6 PUFAs.2 These results are referred to the present ex vivo setting; AA was administered intravenously in 10 rabbits, and sera sampled before and after its infusion were added to growth medium with ceftazidime and amikacin to evaluate their effect on growth of three multidrug-resistant P. aeruginosa isolates. Antimicrobials were used at concentrations similar to those achieved after administration of their conventional dose.2 The intravenous infusion of single n-6 PUFAs is achieved with difficulty due to their lipophilicity. Although GLA has also been administered intravenously by others,3,6,7 our study group is the first applying one lipid emulsion of AA in experimental animals.3
Test isolates differed in MICs of ceftazidime and amikacin. Isolate A was moderately resistant to ceftazidime and highly resistant to amikacin, isolate B was highly resistant to ceftazidime and moderately resistant to amikacin and isolate C was highly resistant to both antimicrobials. The interaction of ceftazidime and amikacin with serum sampled before the infusion of AA produced a limited decrease of viable cells sometimes reaching 2 log10, which is the criterion applied for synergy.8 That was expected to occur since ceftazidime and amikacin have been shown to interact on multidrug-resistant isolates.9 In the presence of sera drawn after the infusion of AA, the latter decrease of viable cells was statistically enhanced to >3.5 log10. That phenomenon was observed over the first 5 h of bacterial growth with sera collected either at the mid-infusion time or 15 and 30 min after the infusion of AA. It was more pronounced with isolate C since it was found even after 24 h of growth (Table 1).
One mechanism that might explain the ex vivo effect of sera supplemented with AA on multidrug-resistant P. aeruginosa is lipid peroxidation. A gradual increase in the concentrations of malonodialdehyde was shown during bacterial growth (Figure 1) reaching a statistically significant peak in sera collected 15 min after the infusion of AA. Since growth of multidrug-resistant P. aeruginosa was affected by samples drawn at the same time interval, lipid peroxidation might be implicated as a mechanism responsible for the observed ex vivo effect. Lipid peroxidation might also explain the considerable interactive effect of arachidonate-supplemented sera and antimicrobials on the highly resistant isolate C. Cell wall impermeability is a common mechanism of multidrug resistance of these isolates. Lipid peroxides might attack the cell wall facilitating the entrance of antimicrobials into the bacterial cell.
The present study revealed that ex vivo incubation of sera from rabbits intravenously administered AA interacts synergically with ceftazidime and amikacin on multidrug-resistant P. aeruginosa. These data raise future possibilities for the therapeutic management of infections by multidrug-resistant nosocomial isolates in animal models.
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Footnotes |
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* Corresponding author. Tel: +30-1-80-39-542; Fax: +30-1-80-39-543; E-mail: giamarel{at}internet.gr
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References |
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