Louise Suleman, BSc, MSc!!

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

Leahurst Campus, Neston, CH64 7TE.!!

sulemanl@liverpool.ac.uk!

Chronic Wound-Derived Bacteria in Biofilm Form Show High Levels of

Proteolytic Activity."

Background and Methods!

•  Bacterial Biofilms have been associated with chronic wound progression."

•  Bacterial Metalloproteases have been associated with disease processes such as keratitis and burn infections.""•  Research Aim: Assess the proteolytic activity of human and equine chronic wound-derived bacteria in planktonic and biofilm form. "

"

!""

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"

Results: Proteolytic activity of human- and equine-derived isolates in planktonic and biofilm

form."

Refer

ence

PCM

Refer

ence

BCM

hPCM

hBCM

eqPCM

eqBCM

0.0

0.2

0.4

0.6

0.8

Conditioned medium

Avera

ge a

bso

rban

ce a

t 550n

m

S. aureus

Refer

ence

PCM

Refer

ence

BCM

hPCM

hBCM

eqPCM

eqBCM

0.0

0.2

0.4

0.6

0.8

Conditioned medium

Avera

ge a

bso

rban

ce a

t 550n

m

**

****

P. aeruginosa

P ≤ 0.0001

P ≤ 0.01

Figure 1. Bacterial-conditioned medium from reference strains, human- and equine-derived clinical isolates assessed for proteolytic activity using the azocoll assay. Statistical analysis: Kruskel-Wallis ANOVA with Dunn’s multiple comparison test."

•  P. aeruginosa display higher levels of proteolytic activity when compared with S. aureus."

"•  Significant differences were found between biofilm and

planktonic form in human-derived P. aeruginosa.""

Results: Collagen zymography of P. aeruginosa conditioned medium."

Equine!

PCM! PCM! PCM!BCM!

Human!Reference!

BCM! BCM!

55!

34!

kDa!

Las B?!

Alkaline protease?!

Figure 2. Collagen zymography of P. aeruginosa reference strains, human- and equine-derived isolates. "

•  Bands of proteolytic activity were not identified in S. aureus-conditioned medium."

"•  Human-derived P. aeruginosa secrete a 55kDa protease when

in biofilm form.""•  Equine-derived P. aeruginosa secrete this 55kDa protease in

planktonic and biofilm form."

Conclusions!"•  Human-derived bacteria show significantly higher levels

of proteolytic activity in biofilm form when compared with planktonic."

•  The presence of a 55kDa protease is associated with the biofilm form in human-derived P. aeruginosa and both the planktonic and biofilm states of equine P. aeruginosa. "

•  Differences between human- and equine-derived bacteria may be due to the environment of the wound and the influences of resident microorganisms."

•  Whether this protease acts as a virulence factor in chronic wound progression remains to be determined."

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.""