Journal of Postgraduate Medicine Medknow Publications and Staff Society of Seth GS Medical College and KEM Hospital, Mumbai, India ISSN: 0022-3859 EISSN: 0972-2823 Vol. 49, Num. 1, 2003, pp. 11-16

Journal of Postgraduate Medicine, Vol. 49, No. 1, Jan-March, 2003, pp. 11-16

Brief Report

Lipid Peroxidation by Pseudomonas Aeruginosa in the Pathogenesis of Nosocomial Sepsis

Giamarellos-Bourboulis EJ, Skiathitis S*, Dionyssiou-Asteriou A,** Hatziantoniou S,*** Demetzos K,**** Dontas I,* Papaioannou GT,*** Karatzas G,* Helen G

4^th Department of Internal Medicine, and Departments of Experimental Surgery and *Surgical Research, and **Biological Chemistry, University of Athens, Medical School and Departments of ***Pharmaceutical Technology and ****Pharmacognosy, University of Athens, School of Pharmacy, Greece.
Address for Correspondence: E. J. Giamarellos-Bourboulis, MD, 4th Department of Internal Medicine, Sismanoglion General Hospital, 151 26 Maroussi Attikis, Greece E-mail: giamarel@internet.gr

Code Number: jp03003

Abstract:

BACKGROUND: To study whether Pseudomonas aeruginosa may directly trigger peroxidation of polyunsaturated fatty acids, since lipid peroxidation is a mechanism involved in the pathogenesis of sepsis. METHODS: Gamma-linolenic acid (GLA) was administered intravenously at a dose of 25mg/kg in an infusion time of 10 minutes to seven male rabbits. Blood samples were collected from the hepatic veins and from the carotid artery at regular time intervals. One clinical isolate was ex vivo incubated with the serum derived from the latter samples and concentrations of malondialdehyde (MDA) were determined during incubation in the growth medium by the thiobarbiturate assay. RESULTS: Elevated concentrations of MDA compared to their basal levels were found over the first three hours of incubation in the presence of samples collected 30 to 60 minutes after the end of the infusion of GLA. After infusion of GLA concentrations of arachidonic acid in the serum increased to concentrations comparable to those detected in sepsis. CONCLUSION: Direct triggering of lipid peroxidation by nosocomial isolates might be proposed as a pathogenetic mechanism of sepsis. (J Postgrad Med 2003;49:11-16)

Key Words: Sepsis, Pseudomonas aeruginosa, peroxidation.

The peroxidation of polyunsaturated fatty acids (PUFAs) and the subsequent production of malondialdehyde (MDA) have been implicated as a common pathogenetic mechanism involved in the septic inflammatory process. Elevated levels of MDA have been detected in the blood of patients with sepsis and organ failure,¹ a phenomenon accompanied by a decreased antioxidant status.^{1, 2} Although the generation of MDA is believed to originate in the inflammatory cells, it has been shown that peroxidation of two different PUFAs, of gamma-linolenic acid (GLA, C_18:3) and of arachidonic acid (AA, C_20:4) may occur in vitro in the presence of nosocomial Pseudomonas aeruginosa isolates.³ In an attempt to document whether nosocomial isolates may per se trigger lipid peroxidation so as to promote nosocomial sepsis, one P. aeruginosa isolate was incubated in the presence of sera of rabbits supplemented with PUFAs after the intravenous administration of GLA.

Animals and Methods

The emulsion of GLA for intravenous administration was prepared as already described.⁴ Briefly, GLA ethyl ester (Sigma CO, St. Louis, USA) was dissolved in ethanol 99% (Merck, Darmstadt, Germany) to an initial dilution of 10mg/ml and kept refrigerated at -70°C under a nitrogen atmosphere. Respective amounts of that dilution were further diluted into sterile and endotoxin-free water (Difco Laboratories, London, UK) to a final volume of 50ml that was finally administered.

A total of 10 white New Zealand male rabbits weighing 3.9-4.2 kg were chosen for the study. The study received permit from the Veterinary Directorate of the Perfecture of Athens according to the Greek legislation conforming to the Council Directive of the EU. Animals were housed in single IFFA Credo metal cages and had access to tap water and standard balanced rabbit chow ad libitum. Temperature ranged between 18°C and 22°C, relative humidity between 55 and 65% and the light/dark cycle was 6am/6pm.

Animals were initially sedated by the intramuscular injection of 25mg/kg of ketamine and 5mg/kg of xylozine. They were then intubated after incision of their trachea. Anaesthesia was maintained by the intravenous administration of 20mg/kg of sodium thiopental. Mechanical ventilation was adjusted to 16 breaths/min. After a midline neck incision, the left jugular vein and the common carotid arteries were dissected and catheterised by a 20G catheter whereas another catheter was inserted through the right jugular vein to lodge in the hepatic veins. GLA was administered intravenously by the left jugular vein in seven rabbits. Its dose had already been adjusted to 25mg/kg⁴ and an infusion pump was applied for a total time of 10 minutes; the respective amount of ethanol 99% diluted in water was administered intravenously by the same route in three rabbits in order to be applied as controls.

Blood samples of five ml each were collected through catheters inserted in the left carotid artery and in the hepatic veins; sampling was performed just before the infusion, at the midpoint, at the end of the infusion and at 15, 30, 45 and 60 minutes after the infusion. Samples were collected into sterile and pyrogen-free tubes (Difco Laboratories, London, UK) and they were centrifuged at 12,000g and 4°C for 10 minutes. Serum samples were kept refrigerated at -70°C under a nitrogen atmosphere until processing. The volume of blood sampled was chosen as the maximum that could be applied for the peroxidation assay and for lipid analysis without any haemodynamic derangement of the animal.

One P.aeruginosa blood isolate from a patient dying from sepsis and multiple organ dysfunction was collected. The isolate was resistant to piperacillin / tazobactam, ceftazidime, imipenem, meropenem, ciprofloxacin and amikacin as defined after determination of the Minimal Inhibitory Concentrations of these antimicrobials by a microdilution technique.

A 5x10⁵ CFU/ml log-phase inoculum was added to tubes with Mueller Hinton broth (Becton Dickinson, Cockeysville, Md) containing already one ml of each serum sample derived to a final volume of 10ml. One tube without any added serum sample served as control. Tubes were left to incubate at 37°C in a shaking water bath and at 3, 5 and 24 hours of growth, aliquots of 0.1ml were taken for the determination of viable bacterial counts and aliquots of 0.5ml for the determination of lipid peroxidation by the thiobarbiturate assay. Time intervals for sampling were selected in order to be representative of the phases of growth of P.aeruginosa.⁵ Bacterial cells were estimated after consecutive 1:10 dilutions of the aliquots into sterile and pyrogen-free water and after plating another 0.1ml aliquot of each dilution onto McConkey agar (BBL Becton Dickinson, Cockeysville, Md). The lowest detection limit was 30 CFU/ml. A total of 150 time-growth curves were performed; all in duplicate.

Lipid peroxidation was estimated by the thiobarbiturate assay.^{3, 6} Briefly, a 0.5ml aliquot of each sample was mixed by a 1:1 ratio to trichloroacetic acid 20% (Merck, Darmstadt, Germany) and centrifuged at 12,000g and 4°C for 10 minutes. The supernatant was then removed and incubated with 1ml of PBS (pH: 7.0, Merck, Darmstadt, Germany) and 1ml of thiobarbituric acid 0.6% (Merck, Darmstadt, Germany) for 20 minutes at 90°C. Optical density was read at 535nm (Hitachi Spectophotometer, Tokyo, Japan). Malondialdehyde was then determined at mM by a standard curve created with 1, 1, 3, 3-tetramethoxy-propane (Merck, Darmstadt, Germany). A 0.5ml water sample treated in the same way was applied as a blank. All determinations were performed in duplicate and their mean was applied. The intra-day coefficient of variation of the assay was 5.45% and the inter-day coefficient of variation of the assay 13.89%.

Serum lipids were extracted by the Bligh-Dyer method⁷ using chloroform/methanol mixtures. Serum lipids were subjected to hydrolysis and trans esterification by KOH 2N in methanol. The resultant methyl esters of the fatty acids were extracted by n-hexane (Merck, Darmstadt, Germany) and well analyzed by Gas Chromatography Mass Spectrometry in a Hewlett Packard (HP-6890) system operating in the EI mode. A HP-5 MS fused silica capillary column of a length of 30m x 0.25mm and a film thickness of 0.25mm was used for analysis. The temperature of the column remained at 170°C for 15 minutes raising steadily by 2°C per minute to 220°C where it remained for 30 minutes. The oven temperature was 250°C and helium was applied as the carrier gas. Mass unit conditions: ion source 230°C, ionization energy 70eV, and electron current 1453A. Identification of the fatty acids was based on their mass spectral fragmentation pattern using the Wiley 138.I/NBS, GC-MS spectrometry library and with authentic samples.

Concentrations of malondialdehyde were expressed as their means (±SD). Comparisons between consecutive time intervals of incubation in the presence of P. aeruginosa as well as between different periods of blood sampling were performed by ANOVA. To avoid random correlation a correction according to Bonferroni was added. Any value of P below or equal to 0.05 was considered as significant.

Results

The addition of serum samples did not produce any effect on the growth of the applied P. aeruginosa strain reaching mean inocula equal to 6x10⁶, 1x10⁸ and 1x10⁹CFU/ml at 3, 5 and 24 hours of growth respectively. The concentrations of MDA after incubation in the presence of serum samples derived from the hepatic veins is shown in Figure 1. Mean concentration of MDA in tubes with samples derived before the infusion of GLA was 1070m. It was then followed by a progressive decline reaching a mean peak of 1450m in tubes containing serum samples derived 30 minutes after the end of the infusion of GLA.

The concentrations of MDA after incubation in the presence of serum samples derived from the carotid artery is shown in Figure 2. Mean concentration of MDA in tubes with samples derived before the infusion of GLA was 933.3mM. It was then followed by a progressive increase up to a mean peak of 2305.7mM in tubes containing serum samples derived 60 minutes after the end of the infusion of GLA.

Concentrations of MDA in controls treated with serum derived from animals administered ethanol 99% ranged between 150 and 300mM at all time of growth.

GLA was not detected in any sample. The only PUFA to be detected was AA. Mean concentrations of AA in samples of the hepatic veins and of the carotid artery are given in Figure 3.

Discussion

Elevated levels of malondialdehyde as a result of lipid peroxidation has been found in the blood of patients with sepsis and organ failure¹ implicating lipid peroxidation as a pathogenetic mechanism involved in sepsis. There is literal debate whether multidrug resistant strains are highly efficient in stimulating the inflammatory cascade.⁸ In the majority of studies in experimental animals lipid peroxidation is induced by endotoxins and not by intact bacterial strains.⁹ In order to study whether lipid peroxidation may be triggered by nosocomial isolates, one clinical isolate was ex vivo exposed to the serum of rabbits supplemented with GLA. The tested hypothesis was based on previous findings that the concentrations of PUFAs in serum are increased in stressed conditions like sepsis.¹⁰ Blood was collected by the hepatic veins so as to select samples indicative of the hepatic metabolism and by the carotid artery so as to select samples of the peripheral circulation. The ex vivo model was preferred instead of an experimental model of sepsis where numerous other factors like pro-inflammatory cytokines and hemodynamic alterations may trigger lipid peroxidation.¹

The ex vivo incubation of serum samples with a clinical isolate of P.aeruginosa revealed that P.aeruginosa represents a biological system in the presence of which peroxidation of PUFAs may occur. Taking as controls the levels of malondialdehyde produced when samples obtained prior infusion of GLA were incubated with P. aeruginosa as well as when samples obtained by animals administered ethanol 99% were incubated with P. aeruginosa, it might be considered that ex vivo lipid peroxidation above basal levels was mainly achieved after three hours of incubation (Figures 1 and 2); lower concentrations of malondialdehyde were found after five and 24 hours. That observation might be of importance since bacterial growth is initiated over the first hours of incubation.

Production of malondialdehyde started to occur in samples either derived from the hepatic veins or from the carotid artery 30 minutes after the end of the infusion of GLA. It presented its major peak in incubated samples derived from the hepatic veins 30 minutes after the end of the infusion of GLA (Figure 1) and in incubated samples derived from the carotid artery 60 minutes after the end of the infusion of GLA (Figure 2). Although recent experimental studies on sepsis induced after the intraperitoneal injection of living bacteria signify the importance of the interaction between host and bacterium in the induction of lipid peroxidation,¹¹ the presented results imply that P.aeruginosa may per se elicit lipid peroxidation. In spite of the variety of hypothesis that could be proposed, production of enzymes like elastases and phospholipase C by P.aeruginosa might explain its effect.¹²

Differences of malondialdehyde production between samples derived from the hepatic veins and from the carotid artery may reflect the different content of these samples in AA. GLA was not detected in the collected samples. AA was found to increase after the infusion of GLA to a major extent in the hepatic veins and to a lesser extent in the carotid artery (Figure 3). Since the content of the hepatic veins corresponds to the products of the hepatic metabolism, it might be hypothesized that the observed discrepancies of the concentrations of AA may result from the impact of the liver function on the metabolism of the administered GLA.

The presented results propose a model for the initiation of lipid peroxidation involved in nosocomial sepsis by nosocomial isolates. The model implies that when serum is found enriched by PUFAs and mainly of AA, nosocomial isolates found in contingency may trigger their peroxidation. That reaction may take place over the first few hours of rapid bacterial growth. The model is based on the findings of concentrations of AA in the sera of rabbits after the administration of GLA above 10mM that are found in serum in sepsis.¹⁰ The proposed early triggering of lipid peroxidation in the pathogenesis of nosocomial sepsis might render the early application of antioxidants as important, in the management of nosocomial sepsis.

References

1. Goode HF, Cowley HC, Walker BE, Howdle PD, Webster NR. Decreased antioxidant status and increased lipid peroxidation in patients with septic shock and secondary organ dysfunction. Crit Care Med 1995;23:646-51.
2. Basu S, Eriksson M. Vitamin E in relation to lipid peroxidaton in experimental septic shock. Prostaglandins Leukot Essent Fatty Acids 2000;62:195-9.
3. Giamarellos-Bourboulis EJ, Grecka P, Dionyssiou-Asteriou A, Giamarellou H. In vitro activity of polyunsaturated fatty acids on Pseudomonas aeruginosa: relationship to lipid peroxidation. Prostaglandins Leukot Essent Fatty Acids 1998;58:283-7.
4. Giamarellos-Bourboulis EJ, Skiathitis S, Dionyssiou-Asteriou A, Donta I. Hatziantoniou S, Demetzos K, et al. Rapid alterations of serum oxidant and antioxidant status with the intravenous administration of n-6 polyunsaturated fatty acids. Prostaglandins Leukot Essent Fatty Acids 2002;67:57-62.
5. Murray PR, Rosenthal KS, Kobayashi GS, Pfaller MA. Bacterial metabolism and growth. In: Murray PR, Rosenthal KS, Kobayashi GS, Pfaller MA, editors. Medical microbiology. 4^th edn. St Louis: Mosby; 2002. pp. 25-34.
6. Bégin ME, Ells G, Horrobin DF. Polyunsaturated fatty acid-induced cytotoxicity agaist tumour cells and its relationship to lipid peroxidation. J Natl Cancer Inst 1988;80:188-94.
7. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1958;37:911.
8. Kurahashi K, Kajikawa O, Sawa T, Ohara M, Gropper MA, Frank DW, et al. Pathogenesis of septic shock in Pseudomonas aeruginosa pneumonia. J Clin Invest 1999;104:743-50.
9. Clements Jr NC, Habib MP. The early pattern of conjugated dienes in liver and lung after endotoxin exposure. Am J Respir Crit Care Med 1995;151:780-4.
10. Falconer JS, Ross JA, Fearon KC, Hawkins RA, O'Riordain MG, Carter DC. Effect of eicosapentaenoic acid and other fatty acids on the growth in vitro of human pancreatic cell lines. Br J Cancer 1994;69:826-32.
11. Unlu A, Turkozkan N, Cimen B, Karabicak U, Yaman H. The effect of Escherichia coli-derived lipopolysaccharides on plasma levels of malondialdehyde and 3-nitrotyrosine. Clin Chem Lab Med 2001;39:491-3.
12. Murray PR, Rosenthal KS, Kobayashi GS, Pfaller MA. Pseudomonas and related organisms. In: Murray PR, Rosenthal KS, Kobayashi GS, Pfaller MA, editors. Medical microbiology. 4^th edn. St Louis: Mosby; 2002. pp. 297-304.

Expert's Comments

Infection with P. aeruginosa or other bacterial isolates may result in tissue injury, primarily mediated by host-derived intermediates, including cytokines and eicosanoids, resulting in a massive influx of phagocytes to the tissues.^1,2 These inflammatory cells are the first line of defence against invading microorganisms and are known to exert their antimicrobial actions by releasing reactive oxygen species, proteolytic enzymes and other toxic metabolites.^1,3,4 Unfortunately, these toxic metabolites do not discriminate between microbial pathogens and host cells and thus can injure cells that exist in close proximity to activated inflammatory cells.^3,4 This condition can lead to the initiation of membrane lipid peroxidation, and damage to DNA, proteins, nucleic acids and other macromolecules.⁵

Peroxidation of membrane lipids has been described in humans with sepsis or after administration of endotoxin and other bacterial by-products in animal models.^6-8 Generally, lipid peroxidation is an irreversible chain reaction that occurs when polyunsaturated fatty acids react with reactive oxygen species, resulting in changes in the structure and function of cellular membranes, which may lead to increases in permeability and cell injury. In the manuscript entitled "Lipid Peroxidation by P. aeruginosa in the Pathogenesis of Nosocomial Sepsis" the authors have shown that nosocomial isolates of P. aeruginosa may directly trigger lipid peroxidation as well.

The conclusion in this paper can be strengthened by carrying out a dose response where the levels of lipid peroxidation to increasing number of P. aeruginosa isolates is assessed. Moreover, it is recommended that a newer method of assessing lipid peroxidation should be used instead of the older MDA/thiobarbituric acid method. A question that will need to be addressed in the future is whether this effect is unique to P. aeruginosa or is it also a characteristic of other microorganism(s) as well? Nevertheless, to the knowledge of this author, this is the first effort where the ability of P. aeruginosa in stimulating lipid peroxidation has been studied and it would be of great interest to elucidate the mechanism(s) of such an effect.

Zach Suntres

Defence Research and Development Canada, 1133 Sheppard Avenue West, Toronto, Ontario, M3M 3B9, Canada

References

1. Glauser, MP, Zanetti G, Baumgartner JD, Cohen J. Septic shock: pathogenesis. Lancet 1991;338:732-6.
2. Wilson, R, Dowling RB, Jackson AD. The effects of bacterial products on airway cells and their function. Am J Respir Crit Care Med 1996;154:S197-S201.
3. Sibille Y, Reynolds HY. Macrophages and polymorphonuclear neutrophils in lung defence and injury. Am Rev Respir Dis 1990;141:471-501.
4. Weiss, S J. Tissue destruction by neutrophils. N Engl J Med 1989;320:365-76.
5. White, CW, Repine JE. Pulmonary antioxidant defence mechanisms. Exp Lung Res 1985;8:81-96.
6. Goode, HF, Webster NR. Free radicals and antioxidants in sepsis. Crit Care Med 1993;21:1770-6.
7. Goode, HF, Cowley HC, Walker BE, Howdle PD, Webster NR. Decreased antioxidant status and increased lipid peroxidation in patients with septic shock and secondary organ dysfunction. Crit Care Med 1995;23:646-51.
8. Yoshikawa, T, Takano H, Takahasi H, Ichikawa H, Kondo M. Changes in tissue antioxidant enzyme activities and lipid peroxides in endotoxin-induced multiple organ failure. Circ Shock 1994;42:53-8.

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