1. SYSTEMIC POLYMORPHONUCLEAR COUNT
Copenhagen University Hospital
Department of Clinical Microbiology
Compromised Wound Healing in BALB/c Mice with Local Pseudomonas aeruginosa Biofilm Infection
Hannah Trøstrup 1, Christian Johann Lerche1, Lars Christophersen1, Kim Thomsen1, Peter Østrup Jensen 1, Hans Petter Hougen3, Niels Høiby1,2, Claus Moser1.
1Department of Clinical Microbiology, Copenhagen University Hospital, Rigshospitalet, Denmark
2Institute for Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark
3Department of Forensic Medicine, University of Copenhagen, Copenhagen, Denmark
Background
The impact of Pseudomonas aeruginosa biofilm
infection in chronic wounds and the clinical impact on healing is receiving increasing attention. However, the patophysiology of the host/pathogen interplay is not
fully understood.
AIm
New treatment strategies are necessary for the increasing population of chronic wound patients. Thus, we assessed host response and spontaneous wound closure for a
period of 14 days after wound infliction (10 days after wound infection) in a model of P. aeruginosa biofilm-infected wounds.
Materials and methods
Animals: 54 C3H/HeN, relatively resistant to infection and 54 BALB/c, relatively susceptible to infection, female pathogen-free, weeks.
Wound procedure: Induction a full-thickness skin necrosis by of a stream of hot air (330°C) for 5 seconds.
Chronic biofilm infection, procedure: 106 CFU of P.aeruginosa biofilm was injected subcutaneously 4 days post wounding.
Bacteriology: Aliquots of serial dilutions of wound homogenates were spread on agar plates followed by counting of CFUs following overnight incubation.
Cytokines: Quantitative analysis was performed on supernatants; S100A8/A9 by ELISA (Immundiagnostik, Bensheim Germany) and IL-1β, G-CSF and KC by Luminex (Bio-Rad,Luminex Corp., Austin, TX, USA).
Histopathology: evaluation of hematoxylin and eosin (HE) stained slides of 4 mm punch biopsies.
Count of systemical polymorphonuclear leukocytes was performed by flow cytometry (FACSCanto).
Wound closure was evaluated by digital planimetry (Image J®, version 1.47), assessment carried out blinded to the investigator.
HISTOPATHOLOGY
4 days post infection, significantly more peripheral wound biopsies
from C3H/HeN mice (A) had moderate to severe inflammation than BALB/c (C ), χ2=6, p< 0.02). B and D: non-infected control biopsies
at same time points.
REDUCED NEUTROPHIL-RELATED
MARKERS
S100A8/A9
Keratinocyte-derived chemokine
Granulocyte-Colony Stimulating Factor
RESULTS
• No infection control was achieved in either strain of mice.
• Significantly more peripheral biopsies from C3H/HeN mice had moderate to severe inflammation than the BALB/c counterparts at 4 days post infection(p<0.02).
• Systemic polymorphonuclear count was prolonged in infected BALB/c mice.
 P. aeruginosa biofilm induced IL-1β.
• P. aeruginosa biofilm suppressed neutrophil effector cytokines S100 A8/A9, KC and G-CSF
• Only C3H/HeN wounds showed significant healing.
Table 1. Experimental chronic wound infection in two strains of mice
Bacterial burden (log10 CFU/wound) (mean±SD)
Mouse strain
C3H/HeN
BALB/c
4 DPI
8.06 ± 0.89 (n=6)
8.33 ± 0.39 (n=6)
7 DPI
7.87 ± 0.82 (n=6)
8.30 ± 0.36 (n=6)
10 DPI
8.40 ± 0.39 (n=5)
7.93 ± 0.49 (n=5)
INDUCTION OF PROINFLAMMATORY IL-1β
DPI days post infection; CFU colony-forming units; SD standard deviation. Animals were challenged with 100 µl
1·107 CFU/ml P. aeruginosa (PAO1, Iglewski)
subcutaneously
WOUND CLOSURE
SYSTEMIC POLYMORPHONUCLEAR COUNT
Upper panel: a representative C3H/HeN mouse 4, 7 and 10 days post infection. Lower panel: a representative BALB/c mouse at the same time points. Scale bar = 1 cm.
CONCLUSION
• P. aeruginosa biofilms perturbs host defense
and thereby induce a steady-state of chronic infection
which may impair wound healing.
• Systemic reaction to the infection is pronounced and reduction of polymorphonuclear cells and
wound closure are delayed in the susceptible BALB/c mouse strain.
• These results indicate therapeutic options for
immune modulation of biofilm-infected wounds.
SYSTEMIC WHITE BLOOD CELL COUNT