Title: Treatment of Pseudomonas Aeruginosa Infected Burn Wound with AntiFlagellin-A Antibodies in a Murine 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 a leading cause of morbidity and
mortality in patients with infected burns.1-3 Immunotherapy and vaccination (active or
passive) against PA are desirable options in an era of increasing drug resistance. PA
flagellum is an important virulence factor, and therefore targeted for immune therapy.
We evaluated the efficacy of treatment of PA infection with monoclonal antibodies
raised against PA flagellin type-a (anti-aFla), using a murine-infected burn model.
Methods: Groups of 12 mice (CBL57, 18-20 gram) were studied. A full thickness
scald burn involving 6-8% of the total body surface area was induced. Infection was
induced by an immediate post-burn sub-eschar injection of PA clinical strain number
409 (100 l). Study groups included: 1. Immune therapy: 1a. Monoclonal anti-aFla,
1b. Non-specific monoclonal antibodies (NS Ab); 2. Conventional antibiotic:
Imipenem (IMP); 3. Model control: Untreated infected-burn.
Anti-aFla was given intra-peritoneal once daily for 4 days (50 g x 4), and NS Ab
treatment was given similarly. IMP treatment was given intra-peritoneal twice daily
for 4 days (0.5mg x 2). Mortality and morbidity (weight change and functional score)
follow-up was 2 weeks. Bacterial counts from the burn and spleen were performed 6,
24, 48 and 72 hours after infection was induced. Burn histopathology was examined
on days 2 and 14 after infection induction.
Results: In our murine infected burn wound model, mice infected with PA409 (3x105
CFU), mortality rate was 92%. Treatment with anti-aFla reduced the mortality rate to
none (P<0.0001), which was similar to the IMP treated group (Figure 1). The
mortality rate in the NS Ab treated group was 100%. Uninfected burn injured mice or
PA infected mice alone displayed 100% survival.
Morbidity, as expressed by weight loss and functional activity score, paralleled
survival results. In groups treated with anti-aFla or IMP, the weight loss was of lesser
magnitude than in the untreated and NS Ab treated groups (P<0.05) (Figure 2).
Bacterial load in the spleen was significantly lower in mice treated with anti aFla
compared to the non-treated group (P=0.01) (Figure 3).
Burn histology in the untreated infected burn group demonstrated full-thickness
necrosis with intramuscular abscesses, as opposed to mild inflammation and fibrosis
in the dermis and regenerative epidermal changes in the anti-aFla treated group.
Conclusion: Anti-aFla Ab is effective in reducing mortality and morbidity in murine
PA infected burn model. In this era of increasing antibiotic resistance, specific
immune therapy is a promising modality for prevention and treatment of infected
burns.
100
100
Survival %
80
100
60
40
20
8
0
0
Non-treated
infected burn
Imipenem
Non specific IgG Anti-flagellin A Ab
Figure 1. Survival rate in the PA409 3x105 CFU infected burn model, with different
treatment modalities. Treatment with anti-aFla or Imipenem increased survival from
8% to 100%.
100
infected burn
infected burn+IMP
Weight loss
80
infected burn+anti Fla-A
60
infected burn+non specific m-Ab
40
20
0
0
2
4
6
8
10
12
14
16
Days from infection
Figure 2. 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-aFla IgG and Imipenem demonstrated less weight loss than the
untreated and non-specific IgG-treated groups.
9
infected burn: burn
8
infected burn: spleen
Log CFU/g
7
infected burn+Ab: burn
6
infected burn+Ab: spleen
5
4
3
2
1
0
0
0.5
1
1.5
2
2.5
Time from infection (days)
Figure 3. Bacterial load in the burn wound and in the spleen in untreated and anti aFla
treated groups. Bacterial load in the spleen was significantly lower in mice treated
with anti aFla compared to the non-treated group
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.