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JAC Advance Access originally published online on March 20, 2006
Journal of Antimicrobial Chemotherapy 2006 57(5):937-944; doi:10.1093/jac/dkl084
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Clarithromycin is an effective immunomodulator in experimental pyelonephritis caused by pan-resistant Klebsiella pneumoniae

Evangelos J. Giamarellos-Bourboulis*, Vassiliki Tziortzioti, Pantelis Koutoukas, Fotini Baziaka, Maria Raftogiannis, Anastasia Antonopoulou, Theodoros Adamis, Labros Sabracos and Helen Giamarellou

4th Department of Internal Medicine, University of Athens, Medical School, Athens, Greece

* Corresponding author. Tel: +30-210-58-31-994; Fax: +30-210-53-26-446; E-mail: giamarel{at}ath.forthnet.gr

Received 5 November 2005; returned 4 January 2006; revised 22 January 2006; accepted 22 February 2006


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Objectives: To apply clarithromycin as an immunomodulatory treatment in experimental infection caused by pan-resistant Klebsiella pneumoniae.

Methods: Acute pyelonephritis was induced in 80 rabbits after inoculation of the test isolate in the renal pelvis. Rabbits were divided into eight groups, with 10 animals in each group. In groups A–D, therapy was administered simultaneously with bacterial challenge as follows: A, controls; B, intravenous clarithromycin; C, amikacin; and D, both agents. In groups E–H, therapy was administered 24 h after bacterial challenge as follows: E, controls; F, intravenous clarithromycin; G, amikacin; and H, both agents. Blood was sampled for estimation of tumour necrosis factor-{alpha} (TNF-{alpha}) and malondialdehyde (MDA); monocytes were isolated for determination of intracellular activity of caspase-3 and ex vivo TNF-{alpha} secretion. Four days after bacterial challenge, animals were sacrificed for quantitative cultures and biopsies of organs.

Results: Serum TNF-{alpha} at 48 h was lower in groups B, C and D compared with group A. Activity of caspase-3 of monocytes was lower at 48 h in group D compared with group A. Bacterial loads of liver and spleen were decreased in group D compared with those of group A. The numbers of inflammatory cells of spleen of group B were lower compared with those of group A; those of kidney and mesenteric lymph nodes of group D were lower than those of group A. Serum MDA of group H was lower than that of group E and serum TNF-{alpha} of group F was lower compared with that of group E. TNF-{alpha} of monocyte supernatants and activity of caspase-3 of monocytes of group F were lower than those of group E. Bacterial tissue loads did not differ among groups E, F, G and H. The numbers of inflammatory cells of liver of groups F and H were lower compared with those of group E; those of kidney of groups F, G and H were lower compared with those of group E.

Conclusions: Clarithromycin administered intravenously in experimental infection caused by pan-resistant K. pneumoniae attenuated systemic inflammatory response and local tissue damage. This effect is probably attributed to immunomodulatory intervention on blood monocytes.

Keywords: K. pneumoniae , immunomodulation , apoptosis , resistance


   Introduction
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Nowadays nosocomial practice is facing the emerging multidrug resistance of isolates, creating questions about what is the best available antimicrobial agent to control an infectious disease.1 Although immunomodulators might be considered an alternative solution, particularly in patients with severe infections, their application in clinical practice has been problematic thus far.2 Newer macrolides might play a significant part in immunointervention, particularly in patients with chronic infections of the respiratory tract where they inhibit oxygen burst and secretion of interleukin-8 by inflammatory cells.3 Intravenously administered clarithromycin extended survival and attenuated systemic inflammatory response in experimental sepsis by multidrug-resistant Pseudomonas aeruginosa.4

The present experimental study was designed to simulate the everyday clinical difficulty. Infection was induced by one pan-resistant isolate of Klebsiella pneumoniae that created several outbreaks in intensive care units (ICUs) of hospitals in Athens during the last trimester of 2003. Animals were treated with clarithromycin in parallel with bacterial challenge in order to test its effect on a spreading infection; other animals were treated with clarithromycin 24 h after induction of the infection. Amikacin was co-administered as occurring in clinical practice, where antimicrobials are often prescribed despite the status of pan-resistance.


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Bacterial isolate

One blood isolate of K. pneumoniae derived from a female patient with acute pyelonephritis and severe sepsis was used. The patient was hospitalized in an ICU and the same pathogen was isolated from five more patients hospitalized with her in the same period. Infections by the same isolate have been reported within 1 month from the first isolation in three more ICUs of three different hospitals in Athens; they were considered to originate from one patient being transferred from the hospital of the first isolation. MICs of ticarcillin/clavulanate, piperacillin, ceftazidime, imipenem, meropenem, ciprofloxacin, clarithromycin, amikacin and colistin determined by a microdilution technique were >256/2, >512, >512, >256, >256, >512, >512, >512 and 256 mg/L, respectively.

The isolate was stored as multiple aliquots in skimmed milk (Oxoid Ltd, London, UK) at –70°C.5 Before each experiment, one aliquot was thawed and cultured onto MacConkey agar plates (Becton Dickinson, Cockeysville, MD, USA). Single colonies were suspended in Mueller–Hinton broth (Oxoid) and incubated for 12 h at 37°C in a shaking water bath. The resulting inoculum was washed three times with 0.9% NaCl to remove any free endotoxins.

Animals

A total of 80 white New Zealand male rabbits with a mean (±SD) weight of 3.31 ± 0.69 kg were used. The study received a permit from the Veterinary Directorate of the Perfecture of Athens according to Greek legislation conforming to the 160/1991 Council Directive of the EU. Animals were housed in single metal cages and had access to tap water and standard balanced rabbit chow that is antibiotic-free ad libitum. Room temperature ranged between 18 and 22°C, relative humidity between 55 and 65% and the light/dark cycle was 6 am/6 pm.

Study design

Acute pyelonephritis was induced as previously described.4 Animals were initially sedated by an intramuscular injection of 25 mg/kg ketamine and 5 mg/kg xylazine. Anaesthesia was maintained by the intramuscular administration of 15 mg/kg xylazine at 30 min time intervals. Through an upper midline abdominal incision, the peritoneal cavity was entered and the intestines were displaced to the left. The right ureter was recognized and ligated with a 3.0 suture just below the pelvis. A total of 1 x 107 cfu of the pathogen, in a volume of 0.1 mL, was injected by a 26-gauge needle into the renal pelvis, proximal to the suture. The peritoneal cavity and the abdominal wall were closed in layers.

Forty of the animals were divided into four study groups (A–D). In these animals clarithromycin and amikacin were administered in parallel to the induction of the infection. These groups are described in detail, as follows:

  • Group A (n = 10); controls were administered 30 mL of normal saline intravenously.
  • Group B (n = 10); animals were administered two doses of clarithromycin. It was provided as a pyrogen-free amorphous powder (Abbott, Chicago, USA). After reconstitution with 10 mL of 5% glucose, appropriate amounts were added into 0.9% NaCl at a final volume of 30 mL that was infused by a pump within 30 min. The first dose, equal to 80 mg/kg, was administered concomitantly to the bacterial challenge; the second dose, equal to 30 mg/kg, 2 h after bacterial challenge. Doses of clarithromycin were selected in analogy to former studies where they achieved serum levels close to 10 mg/L enhancing immunointervention;4,6 each dose was infused within 30 min.
  • Group C (n = 10): animals were administered intravenous bolus 15 mg/kg amikacin, 30 min after bacterial challenge, as proposed elsewhere.7
  • Group D (n = 10): animals were administered both clarithromycin and amikacin; clarithromycin was given as in group B, and the first dose of clarithromycin was followed by amikacin. The interaction of clarithromycin and amikacin is indifferent on bacteria,8 so their co-administration allowed for any effect to be attributed to the immunomodulatory properties of clarithromycin.

In the above groups, 3 mL of blood was sampled from the vein of the left ear of each animal before the operation, and after 1, 2, 24 and 48 h, and collected into pyrogen-free tubes (Vacutainer, Becton Dickinson). After centrifugation, serum was kept refrigerated at –70°C until assayed. At 2, 24 and 48 h, two more millilitres was collected into heparin-coated syringes for the isolation of monocytes.

The remaining 40 animals were divided into another four study groups (E–H). In these animals clarithromycin and amikacin were administered as treatment, 1 day after induction of the infection. These groups are described in detail, as follows:

  • Group E (n = 10): controls were administered 30 mL of normal saline intravenously 24 h after bacterial challenge.
  • Group F (n = 10): animals were administered two doses of clarithromycin. The first dose of 80 mg/kg was administered 24 h after bacterial challenge and the second dose of 30 mg/kg 26 h after bacterial challenge.
  • Group G (n = 10): animals were administered intravenous bolus 15 mg/kg amikacin 24.5 h after bacterial challenge.
  • Group H (n = 10): animals were administered both clarithromycin and amikacin; clarithromycin was given as in group F, and the first dose of clarithromycin was followed by amikacin.

In the above groups, 3 mL of blood was sampled from the vein of the left ear of each animal before the operation, and after 24, 26, 28 and 48 h. After centrifugation, serum was kept refrigerated at –70°C until assayed. At 24, 28 and 48 h, two more millilitres was collected into heparin-coated syringes for the isolation of monocytes.

All 80 animals were sacrificed 96 h after bacterial challenge by the intravenous administration of sodium thiopental. Under sterile conditions, segments from the right kidney, liver, spleen, lower lobe of the right lung and mesenteric lymph nodes were taken and placed into separate sterile plastic containers for quantitative cultures and biopsy.

Assays for malondialdehyde (MDA) and tumour necrosis factor-{alpha} (TNF-{alpha})

TNF-{alpha} was measured by a bioassay on the L929 fibrosarcoma cell line, as previously described.4,9 Briefly, confluent cells were thoroughly washed with Hank's solution and harvested with 0.25% trypsin/0.02% EDTA (Biochrom AG, Berlin, Germany). Cells were centrifuged, resuspended in RMPI 1640 supplemented with 10% fetal bovine serum and 2 mM glutamine (Biochrom AG) and distributed into a 96-well cell culture plate at a density of 1 x 105 cells/well. The final volume of fluid in each well was 0.05 mL. After incubation for 2–3 h at 37°C in 5% CO2, 0.06 mL of serum or of standard dilutions of known concentrations of human TNF-{alpha} (Sigma, range 5.75–375.00 pg/mL) were added into each well followed by 0.05 mL of a 0.3 mg/mL dilution of cycloheximide (Sigma). Incubation continued overnight; then the supernatant of each well was discarded by aspiration and 0.1 mL of a 0.5 mg/mL Methylene Blue solution in methanol 99% was added. After 10 min, the dye was removed and the wells were thoroughly washed three times with 0.9% NaCl. Wells were left to dry and remnants of the dye in each well were made soluble by the addition of 0.1 mL of 50% glacial acetic acid (Merck, Darmstadt, Germany). Optical density in each well was read at 495 nm (Hitachi Spectophotometer, Tokyo, Japan) against blank wells and control wells without added serum. Concentrations of TNF-{alpha} were estimated by the reduction of the optical density of control wells by unknown samples applying a standard curve generated by standard concentrations. All determinations were performed in quadruplicate. The inter-day variation of the assay was 13.75%.

Lipid peroxidation was estimated by the concentration of MDA, as previously described.10 Briefly, a 0.1 mL aliquot of each sample was mixed with 0.9 mL of 20% trichloroacetic acid (Merck) and centrifuged at 12 000 g and 4°C for 10 min. The supernatant was removed and incubated with 2 mL of 0.2% thiobarbituric acid (Merck) for 60 min at 90°C. After centrifugation, a volume of 10 µL of the supernatant was injected into a high-performance liquid chromatography system (HPLC, Agilent 1100 Series, Waldbronn, Germany) with the following characteristics of elution: Zorbax Eclipse XDB-C18 (4.6 x 150 mm, 5 µm) column under 37°C; mobile phase consisting of a 50 mM K3PO4 (pH 6.8) buffer and 99% methanol at a 60/40 ratio with a flow rate of 1 mL/min; fluorometric detection with signals of excitation at 515 nm and emission at 535 nm. The retention time of MDA was 3.5 min and it was estimated as µmol/L by a standard curve created with 1,1,3,3-tetramethoxy-propane (Merck). All determinations were performed in duplicate. The lowest limit of detection was 0.01 µmol/L and the inter-day variation of the assay was 1.01%.

Assay for blood monocytes

For the isolation of blood monocytes, heparinized venous blood was layered over Ficoll Hypaque (Biochrom, Berlin, Germany) and centrifuged. The separated mononuclear cells were washed three times with PBS (pH 7.2) and resuspended in RPMI 1640 supplemented with 10% FBS and 2 mM glutamine in the presence of 100 U/mL of penicillin G and 0.1 mg/mL of streptomycin (Sigma). After incubation for 1 h at 37°C in 5% CO2, non-adherent cells were removed while adherent monocytes were washed three times with Hank's solution. Cells were then harvested by 0.25% trypsin/0.02% EDTA and counted. Their purity was more than 90% as assessed after Giemsa staining. Half of them were treated with an ice-cold cell lysis buffer (50 mM HEPES/0.1% CHAPS/5 mM DTT/0.1 mM EDTA, pH 7.4). After centrifugation for 10 min at 10 000 g at 4°C, the activity of caspase-3 was estimated in the cytosolic extract by an enzymatic chromogenic assay (BIOMOL Research Laboratories, Plymouth, PA, USA). It was based on the rate of hydrolysis at 37°C of a substrate releasing p-nitroaniline over time, as assessed by sequential photometry at 410 nm. The assay was also performed in the presence of a caspase-3 inhibitor. The activity of caspase-3 in cell extracts was expressed as pmol/min/103 cells.

The remaining half of monocytes was added into 12-well plates at a volume of 2.4 mL per well with RPMI 1640 supplemented with 10% FBS and 2 mM glutamine. They were incubated for 18 h at 37°C in 5% CO2. TNF-{alpha} of supernatants was estimated, as described above, and expressed in pg/104 cells.

Determination of serum levels of clarithromycin and amikacin

Concentrations of each antimicrobial agent were estimated by a microbiological assay. In order to differentiate clarithromycin and amikacin two different reference strains were required, as proposed elsewhere;11 one resistant to amikacin and susceptible to clarithromycin and another resistant to clarithromycin and susceptible to amikacin. Mutants of Bacillus subtilis ICB6633 resistant to amikacin (MIC > 1028 mg/L) were selected after serial passages of the parent strain onto MacConkey agar (BBL, Becton Dickinson, Cockeysville, MD, USA) impregnated with increasing concentrations of amikacin. MIC determination was repeated before each day of the experiment. Escherichia coli ICB4004 that is resistant to clarithromycin and susceptible to amikacin was used for the determination of amikacin. All determinations were performed in triplicate and the mean was determined. Concentrations were estimated after designing a standard curve with known concentrations of antimicrobials into semi-logarithmic climax. The lower detection limit was 0.25 mg/L for clarithromycin and 0.7 mg/L for amikacin. The standardized coefficient of the inter-day variation for the assay was 5.8% for clarithromycin and 1% for amikacin.

Tissue culture and pathology

Tissue segments were weighed and homogenized; one aliquot of 0.1 mL was diluted 1:10 into sterile NaCl four consecutive times. Another aliquot of 0.1 mL of each dilution was plated onto MacConkey agar and incubated at 35°C for a total period of 3 days. The number of viable cells was estimated after multiplying by the appropriate factor of dilution. Identification of colonies was performed by the API20E system (BioMérieux, Paris, France). The number of viable cells was expressed as its log10 value in cfu/g. The lower detection limit was 10 cfu/g.

Tissue segments were fixed with formalin, sliced appropriately, embedded in paraffin and stained with haematoxylin and eosin. A semi-quantitative scoring system was used where segments of liver, kidney and lung were separately scored for acute and chronic inflammation. Both acute inflammation, characterized by infiltration by neutrophils, and chronic inflammation, characterized by the presence of mononuclear cells, were scored as 0 (absent), 1 (sparse), 2 (moderate) and 3 (intense). For liver sections the latter scores corresponded to 0, 1–3, 4–6 and >6 cells/hpf, respectively; for sections of kidney and lung they corresponded to 0, 1–10, 11–20 and >20 cells/hpf, respectively. Segments of spleen and mesenteric lymph nodes were separately scored for the enlargement of B- and T-lymphocyte areas (0, absent; 1, slight; 2, moderate; and 3, pronounced) and for the presence of germinal centres (0, absent; and 1, present). One single value was used for each organ segment after addition of separate scores (lowest value, 0; highest value, 6). Scoring of each tissue sample represented the mean score of five different high microscopic power fields.

Statistical analysis

Results for MDA, TNF-{alpha} and caspase-3 are expressed as their median and range; those of drug levels, tissue bacterial counts and total histology score as their mean ± SE. Comparisons between groups were performed by Mann–Whitney; values were adjusted according to Bonferroni to avoid any random correlation. SPSS for Windows (version 11.0) was used for statistical analysis. The number of animals used per tested group was based on the hypothesis to achieve at least similar results to those of former studies.4,6,12

Any value P < 0.05 was considered as significant (a levels hypothesis).


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Concentrations of serum MDA and TNF-{alpha}, TNF-{alpha} of monocyte supernatants and cytoplasmic activity of caspase-3 of monocytes of groups A, B, C and D are shown in Table 1. Serum TNF-{alpha} was significantly increased at 48 h in group A compared with the baseline (P = 0.007), a difference that was not found in the other three groups. Serum TNF-{alpha} at 48 h was significantly lower in group B compared with group A (P = 0.002), as it also was in group C compared with group A (P = 0.0001) and in group D compared with group A (P = 0.015). Activity of caspase-3 of monocytes was significantly lower at 48 h in group D compared with group A (P = 0.015).


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  		Table 1.. Comparative concentrations of MDA and TNF-{alpha} in serum, of TNF-{alpha} of monocyte supernatants and of the cytoplasmic activity of caspase-3 of monocytes in groups of treatment (therapy was administered in parallel with bacterial challenge)

 
Bacterial loads and pathology scores of the infected kidney, liver, spleen, lower lobe of the right lung and mesenteric lymph nodes on sacrifice of animals of groups A, B, C and D are shown in Figure 1. Sections of liver and kidney are shown in Figure 2. The bacterial load of liver of group D was significantly lower compared with that of group A (P = 0.047) and the bacterial load of spleen of group D compared with that of group A (P = 0.043). The total spleen histology score of group B was significantly lower compared with that of group A (P = 0.046). Similar results were found comparing groups A and D for kidney (P = 0.049) and mesenteric lymph nodes (P = 0.049).


Figure 1
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  		Figure 1.. Bacterial loads and histology scores of liver, spleen, kidney, lung and mesenteric lymph nodes (MLN) of animals with acute pyelonephritis caused by pan-resistant Klebsiella pneumoniae. A, controls; B, clarithromycin; C, amikacin; D, both agents. Therapy was started in parallel with bacterial challenge. aP < 0.05 compared with group A.

 

Figure 2
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  		Figure 2.. (a) Section of kidney of one animal of group A (controls) showing intense infiltration by mononuclear cells with accumulating necrotic foci. (b) Section of kidney of one animal of group D (parallel treatment with clarithromycin and amikacin) showing moderate infiltration by mononuclear cells with scarce areas of necrosis.

 
Mean ± SE concentrations of clarithromycin of group B at 1 and 2 h were 10.08 ± 1.53 and 4.28 ± 1.07 mg/L, respectively and those of group D were 10.10 ± 0.86 and 4.70 ± 1.38 mg/L, respectively [P = not significant (NS) compared with group B]. Mean ± SE concentrations of amikacin of group C at 1 and 2 h were 19.30 ± 1.25 and 5.30 ± 3.62 mg/L, respectively. Respective concentrations of group D were 19.80 ± 1.73 and 7.60 ± 3.26 mg/L (P = NS compared with group C).

Concentrations of serum MDA and TNF-{alpha}, TNF-{alpha} of monocyte supernatants and cytoplasmic activity of caspase-3 of monocytes of groups E, F, G and H are shown in Table 2. Serum MDA of group H was significantly lower than that of group E at 26 and 28 h (P = 0.048 and 0.038, respectively). Serum TNF-{alpha} at 28 h was significantly lower in group F compared with that of group E (P = 0.007). TNF-{alpha} of monocyte supernatants was significantly increased at 28 h compared with 24 h in group E (P = 0.024); similar findings were not observed in groups F, G and H. Activity of caspase-3 of monocytes at 48 h in group H was significantly lower compared with its respective value at 24 h when therapy started (P = 0.012).


View this table:
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  		Table 2.. Comparative concentrations of MDA and TNF-{alpha} in serum, of TNF-{alpha} of monocyte supernatants and of the cytoplasmic activity of caspase-3 of monocytes in groups of treatment (therapy was administered 24 h after bacterial challenge)

 
Bacterial loads and pathology scores of the infected kidney, liver, spleen, lower lobe of the right lung and mesenteric lymph nodes on sacrifice of animals of groups E, F, G and H are shown in Figure 3. Sections of liver and kidney are shown in Figure 4. Bacterial loads were equal between study groups. The total histology score of liver of groups F and H was significantly lower compared with that of group E (P = 0.009 and 0.018, respectively). The total histology scores of kidney of groups F, G and H were significantly lower compared with that of group E (P = 0.032, 0.032 and 0.004, respectively).


Figure 3
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  		Figure 3.. Bacterial loads and histology scores of liver, spleen, kidney, lung and mesenteric lymph nodes (MLN) of animals with acute pyelonephritis caused by pan-resistant Klebsiella pneumoniae. E, controls; F, clarithromycin; G, amikacin; H, both agents. Therapy was started 24 h after bacterial challenge. aP < 0.05 compared with group E.

 

Figure 4
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  		Figure 4.. (a) Section of liver with intense perivenular inflammation and one area of necrosis of one animal of group E (control). (b) Section of liver with areas of mild inflammation of one animal of group F (treatment with clarithromycin). (c) Section of kidney showing accumulated necrotic foci of one animal of group E (control). (d) Section of kidney showing moderate infiltration by mononuclear cells of the interstitium of one animal of group F (treatment with clarithromycin).

 
Mean ± SE concentrations of clarithromycin of group F at 26 and 28 h were 9.05 ± 1.91 and 13.16 ± 1.97 mg/L, respectively (P = NS between 26 and 28 h); those of group H were 8.00 ± 2.87 and 14.43 ± 2.76 mg/L, respectively (P = NS between 26 and 28 h; P = NS compared with group F). Mean ± SE concentrations of amikacin of group G at 26 and 28 h were 13.55 ± 3.55 and 4.25 ± 2.78 mg/L, respectively. Respective concentrations of group H were 17.88 ± 3.99 and 7.62 ± 2.92 mg/L (P = NS compared with group G).

In isolates of K. pneumoniae grown from tissue segments of two animals of group D and of three animals of group H, the MIC of amikacin was re-estimated. No differences were found compared with the MIC for the parent test isolate.


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Multidrug resistance of bacterial pathogens to antimicrobials is an emerging problem in everyday clinical practice so an urgent solution is required. An attempt to find a novel approach has been described in the present study. Severe sepsis that occurred in three ICUs in Athens by the same spread pan-resistant isolate of K. pneumoniae was simulated by an experimental model of pyelonephritis. Based on previous studies where clarithromycin was an effective immunomodulator,4,6,12 clarithromycin was administered both simultaneously with bacterial challenge and as treatment after induction of the infection. Therapy with clarithromycin concomitant to bacterial challenge aimed to define its effect against an evolving infection; clarithromycin was also given 24 h after bacterial challenge. Co-administration with amikacin was performed to simulate clinical practice.

Treatment with a single course of clarithromycin in parallel to bacterial challenge did not affect bacterial replication since bacterial load in organs was similar to controls after animal sacrifice. When co-administered with amikacin the number of bacterial counts in liver and spleen was significantly decreased (Figure 1). The administration of clarithromycin attenuated local inflammation of spleen where total histology score decreased. Co-administration of clarithromycin and amikacin was accompanied by a lower inflammatory reaction in kidneys and mesenteric lymph nodes without affecting the number of bacterial counts of the pathogen in these organs (Figures 1 and 2), thus indicating the probability of an immunomodulatory effect. Decrease in histology score of kidney affected equally infiltration by neutrophils and mononuclear cells.

Treatment with clarithromycin either alone or in combination with amikacin simultaneously to bacterial challenge decreased serum levels of TNF-{alpha} at 48 h and intracellular activity of caspase-3 of blood monocytes (Table 1). These findings characterize clarithromycin as an agent refraining the burst of the inflammatory cascade when given in parallel to bacterial challenge, since decrease in pro-inflammatory cytokines was accompanied by attenuation of inflammatory reaction in spleen, kidney and mesenteric lymph nodes. Its effect might be exerted on blood monocytes as demonstrated by the lower intracellular activity of caspase-3.

Treatment of experimental pyelonephritis caused by pan-resistant K. pneumoniae with clarithromycin or by the co-administration of clarithromycin and amikacin did not affect tissue bacterial load. However, histology scores of liver and kidney were lower on sacrifice after therapy with clarithromycin (Figures 3 and 4), equally involving infiltration by either neutrophils or mononuclear cells. The latter effect was accompanied by lower serum MDA (Table 2), which is an indicator of serum oxidant status and subsequently of the propensity of the host for local tissue damage.13 The concomitant findings of attenuation of local inflammation and of decreased serum MDA are compatible with an immunomodulatory intervention by clarithromycin. Monocytes of animals treated with clarithromycin were characterized by lower ex vivo release of TNF-{alpha} and lower intracellular activity of caspase-3, thus indicating blood monocytes as one probable target of immunointervention by clarithromycin.

The presented results for the beneficial effect of clarithromycin in experimental infection caused by pan-resistant K. pneumoniae are unique in the literature. The immunomodulatory effects of macrolides have been presented for chronic infections like bronchiectasis and cystic fibrosis.3,14 Moreover, they are considered as probable candidates for immunointervention in acute inflammatory disorders.14 The addition of macrolides to the antimicrobial regimen significantly decreased mortality in severe acute infections of the lower respiratory tract. Their effect is attributed to their immunomodulatory properties thus signifying their role for acute inflammatory conditions.15 This class of antimicrobials has been found to be potent in decreasing biosynthesis of pro-inflammatory cytokines and to withhold oxygen burst in neutrophils.16–18 The mode of immunointervention by clarithromycin in the presented experimental model is in accordance with these studies. Clarithromycin has been found to act on blood monocytes only at concentrations between 1 and 10 mg/L.19 Intravenous administration of clarithromycin in the described experimental model resulted in concentrations within the desired range so as to explain the findings of decreased ex vivo secretion of TNF-{alpha} and of decreased intracellular activities of caspase-3 of monocytes of animals administered clarithromycin.

The present study showed that clarithromycin administered intravenously in the experimental infection caused by pan-resistant K. pneumoniae attenuated systemic inflammatory response and local tissue damage. Its effect was most probably attributable to immunomodulatory intervention on blood monocytes. The beneficial effect of clarithromycin was proved following its administration either simultaneously with bacterial challenge or as treatment after induction of the infection. These findings indicate that clarithromycin was equally potent in refraining evolution to infection as well as to treat an established infection. These results might render a novel perspective for the management of nosocomial infections caused by pan-resistant isolates.


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None to declare.


   Acknowledgements
 
This study was supported by a grant from Abbott Laboratories, Abbott Park, Chicago, USA.


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1. Blot S, Vandewoude K, De Bacquer D et al. Nosocomial bacteremia caused by antibiotic-resistant Gram-negative bacteria in critically ill patients: clinical outcome and length of hospitalization. Clin Infect Dis 2002; 34: 1600–6.[CrossRef][Medline]

2. Vincent JL, Sun Q, Dubois MJ. Clinical trials of immunomodulatory therapies in severe sepsis and septic shock. Clin Infect Dis 2003; 34: 1084–93.

3. Schultz MJ. Macrolide activities beyond their antimicrobial effects: macrolides in diffuse panbronchiolitis and cystic fibrosis. J Antimicrob Chemother 2004; 54: 21–8.[Abstract/Free Full Text]

4. Giamarellos-Bourboulis EJ, Adamis T, Laoutaris G et al. Immunomodulatory clarithromycin treatment of experimental sepsis and acute pyelonephritis caused by multidrug-resistant Pseudomonas aeruginosa. Antimicrob Agents Chemother 2004; 48: 93–9.[Abstract/Free Full Text]

5. Reimer LG, Caroll KC. Procedures for the storage of microorganisms. In: Murray PR, Baron EJ, Jorgensen JH et al., eds. Manual of Clinical Microbiology, 8th edn. Washington, DC: ASM Press, 2003; 67–73.

6. Giamarellos-Bourboulis EJ, Baziaka F, Antonopoulou A et al. Clarithromycin co-administered with amikacin attenuates systemic inflammation in experimental sepsis by Escherichia coli. Int J Antimicrob Agents 2005; 25: 168–72.[Medline]

7. Robaux MA, Dube L, Caillon J et al. In vivo efficacy of continuous infusion versus intermittent dosing of ceftazidime alone or in combination with amikacin relative to human kinetic profiles in a Pseudomonas aeruginosa rabbit endocarditis model. J Antimicrob Chemother 2001; 47: 617–22.[Abstract/Free Full Text]

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