Microbiology, Biofilms and Factors affecting Wound Healing. Professor Val Edwards-Jones Director of Research Manchester Metropolitan University

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1 Microbiology, Biofilms and Factors affecting Wound Healing Professor Val Edwards-Jones Director of Research Manchester Metropolitan University

2 Lecture overview Types of chronic wounds New techniques Typical pathogens Infection/colonisation Biofilms MRSA

3 Chronic wounds Venous ulcer Diabetic foot Pressure sore

4 Acute wounds

5 Chronic wounds- why do they not heal?? Static Matrix metalloproteases Endogenous cellular Exogenous bacterial Inhibition of growth factors critically colonised?? what does this really mean? Role of the Biofilm

6 Log phase (Exponential Multiplication) Production of competitive factors Stationary phase production of toxins and invasive enzymes Quorum sensing 10^6 cfu/ml Lag phase (Adaptation) production of adhesins CONTAMINATION COLONISATION CRITICAL COLONISATION INFECTION /INFECTION This is a dynamic process and a number of factors can alter the process

7 Biofilm

8 Role of the Biofilm? Definition A biofilm is a structured community of microorganisms encapsulated within a self-developed polymeric matrix and adherent to a living or inert surface. A complex aggregation of microorganisms marked by the excretion of a protective and adhesive matrix. A coating or covering on the surface of a living or nonliving substrate composed of organisms like bacteria, protozoa, algae, and invertebrate. Adherent layer of bacteria and/or other microorganisms on a solid surface bound together in a bacterially-derived polysaccharide matrix. A cooperative community of single-cell organisms (bacteria) that builds an extracellular matrix of slime or fibrous material. An aggregate of microbes with a distinct architecture.

9 Facts about biofilms Biofilms form on the surface of catheter lines, pacemakers, heart valve replacements, artifical joints and other implants. Major cause of HAI s Bacteria growing in a biofilm are up to 1,000 times more resistant to antibiotics and antiseptics. Standard antibiotic therapy often useless and necessitates the removal of the implant. Fungal biofilms also frequently contaminate medical devices. Biofilms are involved in numerous diseases e.g. Pseudomonas sp. infections in CF lungs- often result in antibiotic resistant biofilms protected by a alginate slime layer.

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12 Strategies for biofilm management James, Swogger and Wolcott et al (2007) Biofilms in Chronic wounds Wound Repair and Regeneration 60% chronic wounds characterised as containing biofilms Chronic wound specimens revealed diverse polymicrobial communities including strict anaerobes Many chronic bacterial infections which are not easily eradicated by conventional antibiotic therapy, involve biofilms. There are recurring symptoms, after cycles of antibiotic therapy, until the sessile population is surgically removed from the body. Significantly greater proportion of biofilms in chronic than in acute wounds

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16 Normal wound sampling procedures using a moist swab

17 Sample the surface layers For example MRSA +++

18 Should we just sample the infected looking areas? Perhaps using curettage or biopsy?

19 Tissue culture mixed coliforms ++ mixed anaerobes++

20 Conventional Techniques

21 Baird-Parker Ciprofloxacin agar Mannitol Oxacillin salt agar ORSAB medium CHROMagar MRSA

22 Typical wound pathogens Staphylococcus aureus (MRSA GRSA) Pseudomonas aeruginosa Gram-negative bacilli (Acinetobacter sp, Klebsiella sp, E.coli) Streptococcus pyogenes other streptococci Enterococci (VRE) Candida sp and Aspergillus sp Anaerobes (dependant upon site)

23 Typical Normal Skin Flora highly diverse hundreds of bacterial species amongst 6 individuals only a few bacteria are common among the individuals. These included Propionibacteria, Corynebacteria, Staphylococcus spp, Streptococcus spp. Davies CE et al Wound Repair Regen 2001, 9: ; Dekio I et al J Med Microbiol 2005, 54:

24 Molecular Techniques Population analysis studies PCR- Amplify 16s ribosomal DNA separate using Denaturing Gradient Gel electrophoresis- (DGGE) sequence the gene fragments James et al (2008)- Biofilms in Chronic Wounds Wound Repair and Regeneration 16: Davies CE et al (2004) Use of 16S ribosomal DNA PCR and denaturing gradient gel electrophoresis for analysis of the microfloras of healing and nonhealing chronic venous leg ulcers. J Clin Microbiol.;42:

25 Polymerase chain reaction (PCR) Increase the number of copies of the genes

26 DGGE

27 DNA Sequencing Different coloured bases (ATCG)-capillary sequencing

28 Chronic wounds Study by Citron et al., (2007) Bacteriology of diabetic foot infections Tend to be polymicrobial (83.8%) 48% aerobes only 43.7% aerobes and anaerobes 1.3% anaerobes only

29 Aerobes v Anaerobes MSSA (14.3%) MRSA (4.4%), CNS (14.3%) Streptococcus sp (15.5%) Enterococcus sp (13.5%) Corynebacterium sp (10.1%) Enterobacteriacae (12.8%) Pseudomonas aeruginosa (3.5%) Gram pos cocci (45.2%) Prevotella sp (13.6%) Porphyromonas sp (11.3%) Bacteroides fragilis gp (10.2%)

30 Percentage of Population 100% 80% Aerobes 60% Facultative Anaerobes 40% Strict Anaerobes 20% 0% Venous Diabetic Pressure Dowd et al. BMC Microbiology :43

31 Could debridement help with removal of the biofilm? Biofilms are notorious difficult to remove from surfaces Physical removal with cleaning agents High pressure wash Sonication Debridement will remove dead tissue from which the bacteria gain their nutrition and also disrupt the physical structure of the biofilm.

32 DEBRIDEMENT METHODS Autolytic Sharp Larval Enzymatic Mechanical water knife-versajet

33 Bio-Surgery (Lucilia Sericata) natures little surgeons!

34 Log Colony Counts (Cfu/mL) Qualitative Score (+, ++, +++) Log Colony Counts (Cfu/mg/mL) Qualitative score (+, ++, +++) Bacterial Profile - Patient 1 (Biopsy) 1.E E Comparison of three therapies for treatment of MRSA-infected foot ulcers 1.E+04 1.E+02 1.E Sample Number Qualitative vs. Quantitative MRSA Total count Qualitative MRSA score Patient removed from study MRSA negative; clinically improved wound 1.E+08 1.E+06 Bacterial Profile - Patient 1 (Swab) E E E Sample Number MRSA Total count Qualitative MRSA score

35 Colony counts (Cfu/mg/mL) Colony counts (Cfu/mL) Bacterial Profile - Patient 4 (Swab) 1.E+08 1.E+06 1.E+04 MRSA Total count MRSA still present in both biopsy and swab at week E+02 1.E Sample Number However, wound clinically improved 1.E+08 Bacterial Profile - Patient 4 (Biopsy) 1.E+06 1.E+04 MRSA Total count 1.E+02 1.E Sample Number

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39 In-vitro study Versajet Porcine model Wounds deep, surface, sinus Infected with MRSA, Pseudomonas aeruginosa, E.coli and washings from chronic wound Sampled before, after incubation and after versajet Histology, microbiology, EM

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44 What about topical antimicrobial treatments? Dressings and creams/ointments silver Iodine based dressings and creams Chlorhexidine dressing Stabilised hydrogen peroxide PHMB (polyhexamethylene biguanide) Honey Manuka honey ph alteration VAC THESE COULD CREATE AN IMBALANCE OF THE BACTERIAL BIOFILM AND HELP RESOLVE INFECTION?

45 Thank you Collaborators CMHT Prof Andrew Boulton Mr Frank Bowling (funded Diabetes UK) MMU Monika Stuczen Anne Leahy-Gilmartin Helen Duxberry Allan Surrey Burns SMUHT Mr Ken Dunn Jackie Edwards (Framework 7 EU funded)