Title: Annual Israeli Society of Plastic and Aesthetic Surgery Best Paper 2003:
Efficacy of Anti-Flagellin B Antibodies in Pseudomonas aeruginosa
Murine Infected Burn Wound Model
Authors: Yoav Barnea, MD 1, Eyal Gur, MD 1, Boris Kuzmenko, MA 2, Orly
Hammer-Munz, MSc 2, Ehud Ilan, PhD 3, Rachel Eren, PhD 3,
Yehuda Carmeli, MD 2, Shiri Navon-Venezia, PhD 2
1
Dept. Plastic Surgery, 2 Microbiology Laboratories for Molecular Epidemiology,
Tel-Aviv Medical Center, 3 XTL Biopharmaceuticals, Rehovot, Israel
Background: Pseudomonas aeruginosa (PA) is an important pathogen causing
infections in patients with burn injuries. Among these patients, infection with P.
aeruginosa is a leading cause of morbidity and mortality.1-3 In an era of increasing
drug resistance alternative treatment modalities are sought. Immunotherapy is a
desirable option as it may prevent the adverse outcomes of infections without
increasing the antibiotic pressure. PA flagellum is an important virulence factor, and
therefore may serve as a target for immune therapy.
We evaluated the efficacy of prevention and treatment with polyclonal IgG raised
against N’-terminal PA flagellin type b (anti-bFla), in a murine-infected burn model.
Methods: The burn model included groups of 12 mice, weighing 18-20 gram. A full
thickness scald burn involving 6-8% of the total body surface area was induced.
Infection was caused by sub-eschar injection of PAO1 (2 x 106 and 5 x 106 CFU).
Study groups included: 1) Immune therapy: 1a. Polyclonal anti-bFla, 1b. Nonspecific IgG (NS IgG); 2) Conventional antibiotics: 2a. Imipenem (IMP), 2b. Topical
silver sulfadiazine (SSD); 3) Model control: Untreated infected-burn.
Anti-bFla IgG was given intra-peritoneal as 3 separate regimens: pre-infection (0.5
mg 2 hours prior to PA infection); post-infection (0.5 mg 4 hr after PA infection and
0.3 mg daily for 4 days); or combined (0.5 mg 2 hr before PA infection and 0.3 mg
daily for 4 days). IMP was given daily intra-peritoneal as 2 x 0.5 mg treatment. NS
IgG was given as anti-bFla and SSD was applied twice per day for 4 days. Mortality
and morbidity (weight change and functional activity score) follow-up was 2 week.
Wound healing was determined by the time needed for complete re-epithelialization
of a burn wound. Burn histopathology was performed. Statistics were calculated using
Fisher’s exact test.
Results: In murine infected burn wound model with PA (2 x 106 and 5 x 106 CFU),
mortality rate ranged between 58-83% (Figure 1, 2). Treatment of groups of 12 mice
with IMP or SSD reduced the mortality rate to 8% (Figure 1). The mortality rate in
anti-bFla IgG treated mice ranged between 0-17% (P<0.005) (Figure 1, 2). Treatment
with in anti-bFla IgG before the infection was induced, after, or combined strategy
resulted in similar results (Figure 2).
Morbidity, as expressed by weight loss and functional activity score, paralleled
survival results (Figure 3).
Wound healing with complete re-epithelialization of the burn wound was more
rapidly achieved in mice treated with anti-bFla IgG or with imipenem compared to
mice treated with silver sulfadiazine, non-specific IgG antibodies, or not treated at all.
Burn histology in the control group demonstrated full-thickness necrosis with
intramuscular abscesses, as opposed to mild inflammation and fibrosis in the dermis
and sub-cutis in the anti-bFla IgG treated group.
Conclusion: Anti-bFla IgG is effective in reducing mortality and morbidity in murine
PA infected burn model. It improves infected burn wound healing and can be used to
prevent or treat PA burn infections. The activity of anti bFla IgG was equivalent to
conventional systemic anti-pseudomonal treatment.
Specific immune therapy is a promising treatment modality in an era of increasing
drug resistance.
*
100
*
*
*
Survival %
80
60
40
20
0
Infected
burn
imipenem
silver
non specific
antisulfadiazine
IgG
flagellin Ab
Figure 1. Survival rate in the PA01 2x106 infected burn model, with different
treatment modalities. Asterisks (*) denote statistically difference from the untreated infected burn control group
Survival %
100
80
60
40
20
0
Infected
burn
NS IgG: NS IgG: anti-bFla: anti-bFla: anti-bFla:
precombined
prepostcombined
infection treatment infection infection treatment
Figure 2. Survival rate in the PA01 5x106 infected burn model, with different
antibody regimens. Anti-bFla IgG treatment in all three regimens (pre-infection,
post-infection and combined) had similar efficacy.
infected burn
imipenem
Relative weight (%)
100
non-specific IgG
anti-flagellin Ab
95
90
85
80
0
5
10
15
Days from infection
Figure 3. Daily weight changes over time revealed a rapid weight loss followed by
a steady weight gain, reaching constant weight at 2 weeks post-infection. Groups
treated with anti-bFla IgG and imipenem demonstrated less weight loss than the
untreated and non-specific IgG-treated groups. The latter 2 groups had a residual
weight loss at 14 days post-infection.
References
1. Estahbanati, H. K., Hashani, P. P., Ghanaatpisheh, F. Frequency of
Pseudomonas aeruginosa serotypes in burn wound infections and their
resistance to antibiotics. Burns, 28: 340-348, 2002.
2. Holder, I. A., Naglich, M. S. Experimental studies of the pathogenesis of
infections due to Pseudomonas aeruginosa: Immunization using divalent
flagella preparation. J. Trauma, 26: 118-122, 1986.
3. Pirnay, J. P., De Vos, D., Cochez, C., et al. Molecular epidemiology of
Pseudomonas aeruginosa colonization in a burn unit: Persistence of a
multi-drug-resistance clone and a silver sulfadiazine-resistant clone. J.
Clin. Microbiol., 41:1192-1202, 2003.