kiewiczd
a Institute of Infectious Diseases and Public Health, University of Ancona, Ancona; b Department of General Surgery 1, INRCA IRRCS, University of Ancona, Ancona; c Biotechnology Centre, Research Department, INRCA IRRCS, Ancona, Italy; d Faculty of Chemistry, University of Gdansk, Gdansk, Poland
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Polymyxins are peptides produced by Paenibacillus polymyxa. Among polymyxins, only polymyxin B and E (colistin) are used therapeutically. They cross the Gram-negative outer membrane and act on the cytoplasmic membrane, leading to permeability changes and cell death. They also bind LPS and provide protection against LPS-associated mortality.4
KFFKFFKFF and IKFLKFLKFL are novel synthetic cationic peptides that have antibacterial properties. They sensitize Gram-negative bacteria to hydrophobic and amphipathic antibiotics such as rifampicin, erythromycin, novobiocin and fusidic acid.5
Fluoroquinolones have been shown to have an immunomodulatory effect on cytokine production by LPS-treated human monocytes, and have a protective effect in LPS-treated mice.6
The aim of the present study was to investigate the efficacy of ip polymyxin B and polymyxin-like peptides, alone or in combination with levofloxacin, in a rat model of septic shock.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Synthetic peptides
Peptides were synthesized using an automatic plus pep synthesizer (Milligen 9050; Millipore Corporation, Burlington, VT, USA) and were purified by reversed-phase high-pressure liquid chromatography (HPLC) on a Knauer two-pump Well-Chrom K1001 system. The products were analysed by HPLC, chemical analysis and fast atom bombardment mass spectrometry.
Drugs
Polymyxin B (SigmaAldrich, Milan, Italy), KFFKFFKFF and IKFLKFLKFL (Faculty of Chemistry, University of Gdansk, Poland) were dissolved in distilled water and diluted in physiological saline. Levofloxacin was obtained from Aventis Pharma S.p.A., Milan, Italy.
Animals
Adult male Wistar rats (weight range 250330g) were used. All animals had access to food and water ad libitum throughout the study. The study was approved by the animal research ethics committee of the INRCA IRRCS, University of Ancona, Ancona, Italy.
Preparation of the inoculum
E. coli ATCC 25922 in the logarithmic phase of growth was centrifuged at 1000g for 15 min. The supernatant was discarded and the bacteria were resuspended in sterile saline to achieve a concentration of 2 x 1010 cfu/mL.
Implantation of inoculum
All animals were anaesthetized by an im injection of ketamine (30 mg/kg) and received an ip inoculum of 1 mL saline containing 2 x 1010 cfu of E. coli ATCC 25922, with the exception of the uninfected control group (C0).
Antibiotic therapy
Four groups of animals were given levofloxacin 7 mg/kg ip, polymyxin B 1 mg/kg, KFFKFFKFF 1 mg/kg and IKFLKFLKFL 1 mg/kg, respectively, immediately after bacterial challenge. In addition, three groups received 1 mg/kg polymyxin B, KFFKFFKFF and IKFLKFLKFL in combination with 7 mg/kg levofloxacin. The study included an untreated control group (C1) and the uninfected group C0.
Plasma endotoxin and TNF- levels
Blood samples were collected from the jugular vein 0, 60, 120 and 240 min post-injection. Endotoxin concentrations were measured by a Limulus amoebocyte lysate test (E-TOXATE; SigmaAldrich). Endotoxin standards (0, 0.015, 0.03, 0.06, 0.125, 0.25 and 0.5 EU/mL) were tested in each run and the concentration of endotoxin in the test samples was calculated by comparison with the standard curve.
TNF- levels were measured with a solid phase sandwich enzyme-linked immunosorbent assay (Nuclear Laser Medicine, S.r.l., Settala, Italy). Samples were compared with the standard curve. All samples were run in duplicate. The lower limit of sensitivity for TNF-
by this assay was 4 pg/mL.
Evaluation of treatment
Surviving animals were killed with chloroform 48 h post-injection, and blood samples for culture were obtained by aseptic percutaneous transthoracic cardiac puncture. To perform quantitative bacterial evaluation of the intraabdominal fluid, 10 mL of sterile saline was injected ip, samples of the peritoneal lavage fluid were serially diluted and a 0.1 mL volume of each dilution was spread on blood agar plates. Plates were incubated in air at 35°C for 48 h. The limit of detection was <1 log10 cfu/mL.
Statistical analysis
Survival data were compared using log rank test; qualitative results from blood and intra-abdominal fluid cultures were analysed using 2, Yates's correction and Fisher's exact test, depending on the sample size. Quantitative bacterial evaluation of the intra-abdominal fluid cultures were presented as mean ± S.D. of the mean; statistical comparisons between groups were made using ANOVA. Post hoc comparisons were performed by Bonferroni's test. Plasma endotoxin and TNF-
levels were analysed using the KruskalWallis test; multiple comparisons between groups were performed using the appropriate standard procedure. Each comparison group contained 15 rats. Significance was accepted when the P value was
0.05.
![]() |
Results and discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
All intraperitoneal antibiotic treatments given immediately after challenge were better than no treatment (P < 0.05). There were significant differences in mortality between KFFKFFKFF and IKFLKFLKFL combination groups compared with polymyxin B combination group and the group that received single drugs. Survival rates were 100% for KFFKFFKFF and IKFLKFLKFL combined with levofloxacin, and 86.7% for polymyxin B and levofloxacin. In the single drug group, polymyxin B gave the highest survival rate (80%), whereas with KFFKFFKFF, IKFLKFLKFL and levofloxacin, survival was 66.7, 66.7 and 73.3%, respectively.
Qualitative blood and intra-abdominal fluid cultures showed a higher antimicrobial activity of KFFKFFKFF and IKFLKFLKFL combinations than any other treatment (P < 0.05). Similar results have been shown by quantitative bacterial cultures (P < 0.05). The results are summarized in the Table.
There were significant differences in plasma endotoxin and TNF- levels between the control groups C0 and C1 at 60, 120 and 240 min after the challenge. In the C1 group endotoxin and TNF-
concentrations increased, with mean peak levels at 240 min post-injection (0.125 EU/mL and 25 pg/mL, respectively). All drugs and combinations produced significant reduction in plasma TNF-
levels compared with C1. However, levofloxacin significantly increased endotoxin levels compared with the control group C1. The results at 240 min post-injection are summarized in the Table
.
|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Bone, R. C. (1993). Gram-negative sepsis: A dilemma of modern medicine. Clinical Microbiology Reviews 6, 5768. [Abstract]
3
.
Mira, J. P., Cariou, A., Grall, F., Delclaux, C., Losser, M. R., Heshmati, F. et al. (1999). Association of TNF2, a TNF promoter polymorphism, with septic shock susceptibility and mortality. Journal of the American Medical Association 282, 5618.
4 . Danner, R. L., Joiner, K. A., Rubin, M., Patterson, W. H., Johnson, N., Ayers, K. M. et al. (1989). Purification, toxicity, and antiendotoxin activity of polymyxin B nonapeptide. Antimicrobial Agents and Chemotherapy 33, 142834. [ISI][Medline]
5 . Vaara, M. & Porro, M. (1996). Group of peptides that act synergistically with hydrophobic antibiotics against Gram-negative enteric bacteria. Antimicrobial Agents and Chemotherapy 40, 18015. [Abstract]
6
.
Khan, A. A., Slifer, T. R. & Remington, J. S. (1998). Effect of trovafloxacin on production of cytokines by human monocytes. Antimicrobial Agents and Chemotherapy 42, 17137.
7 . Abraham, E., Anzueto, A., Gutierrez, G., Tessler, S., Pedro, G. S., Wunderink, R. et al. (1998). Double-blind randomised controlled trial of monoclonal antibody to human tumour necrosis factor in treatment of septic shock. Lancet 351, 92933. [ISI][Medline]
8
.
Angus, D. C., Birmingham, M. C., Balk, R. A., Scannon, P. J., Collins, D., Kruse, J. A. et al. (2000). E5 murine monoclonal antiendotoxin antibody in gram-negative sepsis: a randomized controlled trial. E5 Study Investigators. Journal of the American Medical Association 283, 172330.
9 . Prins, J. M., van Deventer, S. J. H., Kuijper, E. J. & Spellman, P. (1994). Clinical relevance of antibiotic-induced endotoxin release. Antimicrobial Agents and Chemotherapy 38, 12118. [ISI][Medline]
Received 9 May 2001; returned 18 August 2001; revised 26 September 2001; accepted 12 October 2001