Louise Suleman, BSc, MSc!!

Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool,

Leahurst Campus, Neston, CH64 7TE.!!

sulemanl@liverpool.ac.uk!

The Effect of Chronic Wound-Derived Bacteria on Wound Closure and Host Protease

Production In Vitro."

Background and Methods!

•  Bacterial Biofilm infection and its involvement in pathogenic processes have come to the forefront in recent years due to its association with antibiotic resistance. Bacterial biofilms have been identified in chronic wounds and have been shown to impede wound closure. ""•  Research Aim: Assess the effect of chronic wound-derived P. aeruginosa and S. aureus, in planktonic and biofilm form, on the wound closure of human dermal fibroblasts in vitro."

"

!""

Chronic wound-derived bacteria!Human (n=36)!

P. aeruginosa (n=17) S. aureus (n=19)"Equine (n=16)!

P. aeruginosa (n=8) S. aureus (n=8)"

Planktonic-conditioned medium (PCM)"

24hr"

Biofilm-conditioned medium (BCM)"

72hr"

Human dermal fibroblast scratch assay"

Results: In vitro wound closure of fibroblasts treated with planktonic and biofilm-conditioned

medium."

"""

0 24 480

50

100

150

Time%(Hours)

Percentage%wound%closure%% Clinical'P.#aeruginosa#PCM

Clinical'P.#aeruginosa#BCM

P.#aeruginosa'ATCC'27863'PCM

P.#aeruginosa#ATCC'27863'BCM

Untreated'control

0 24 480

50

100

150

Time((Hours)

Per

cent

age

wou

nd c

losu

re %

Clinical'S.#aureus#PCMClinical'S.#aureus'BCMS.#aureus#ATCC'25923'PCMS.#aureus'ATCC'25923'BCMUntreated'control

Figure 1. Percentage wound closure using human dermal fibroblasts (adult) after treatment with planktonic and biofilm conditioned medium. Human dermal fibroblasts were grown to confluence before creating a scratch in the monolayer. Fibroblast scratches were then treated with either planktonic- or biofilm-conditioned medium. Images were taken at 0, 24 and 48 hours. Scratch closure was assessed using ImageJ. (n=3) technical replicates.!

Results: Host/bacterial protease production using zymography. "

Controls! Planktonic! Biofilm!

55"34"

Figure 2. Gelatin Zymography of scratch wound-derived medium.. A 2.5% gelatin and 7.5% acrylamide gel was made using the BioRad MiniProtean II system. (n=3 technical repeats). "

•  P. aeruginosa biofilm-conditioned medium alone showed faint expression of a proteolytic band. The addition of this medium to the in vitro scratch wound resulted in the release of a 34kDa band after 48 hours."

Conclusions!

Acknowledgements!!Academic supervisors: Dr C A Cochrane, Prof D Archer, Prof P D Clegg.""Prof David Williams of Cardiff University for the kind donation of human chronic wound-derived bacterial isolates.""Advanced Medical Solutions Group Plc for their financial support.""

"•  P. aeruginosa is a known pathogenic player in numerous disease processes but is also thought to be a key player in the development of chronic wounds, particularly in biofilm form.""•  This study shows that P. aeruginosa in biofilms causes significant inhibition of human dermal fibroblast wound closure when compared to it’s planktonic counterparts."

•  Furthermore, the treatment of human dermal fibroblasts with P. aeruginosa biofilm-conditioned medium induced the release of a 34kDa protease which was not seen in culture medium treated with planktonic-conditioned medium."

•  Whether the release of this protease is indicative of reduced wound closure in the presence of a biofilm, remains to be elucidated."