1. Val Edwards-Jones Clinical Director MelBec Microbiology Ltd

2.  Acute wounds-usually heal within 28 days ◦ Surgical site ◦ Burns ◦ Trauma-degloving, bites etc  If infected:  Usually a single organism (occasionally two or three)  Usually respond to systemic antibiotics

3.  Chronic wounds- greater than 28 days-years ◦ Diabetic foot ulcers ◦ Venous leg ulcers ◦ Pressure sores ◦ Recalcitrant wounds ◦ Dehiscence following surgery  Mixed bacterial flora  Difficult to determine a single causative organism  Often fail to respond to systemic antibiotics

5.  Inflammatory phase  Proliferative phase  Remodelling phase factors that delay healing age social factors poor nutrition reduced oxygen supply to the wound Glycated growth factors Metalloproteases Microbial colonisation

6. Gram positive  Staphylococcus aureus (MRSA…GRSA)  Streptococcus pyogenes  other streptococci  Enterococci (VRE) Gram negative  Pseudomonas aeruginosa  Gram-negative bacilli (Acinetobacter sp, Klebsiella sp, E.coli)- Extended spectrum beta lactamase producers (ESBL’s) Anaerobes (dependant upon site) Gram positive and Gram negative Fungi  Candida sp and Aspergillus sp

7.  MRSA-old news!, now CA-MRSA  VRE-continued problems  Increasing resistance of Gram negative organisms e.g. E. coli and Klebsiella to 3 rd generation cephalosporins reflects the marked spread of extended spectrum b-lactamases (ESBLs).  Imipenem and meropenem resistance in Acinetobacter baumannii – increasing  Other infecting organisms showing resistance eg penicillin resistant gonorhoea, Tb

8.  The Antimicrobial Resistance Strategy 2013-2018. Three major aims  improve the knowledge and understanding of antimicrobial resistance  conserve and steward the effectiveness of existing treatments,  stimulate the development of new antibiotics, diagnostics and novel therapies.

9.  Cost of bringing a new antibiotic to market estimated at $US500m and $US1bn (£300- 550m)  Only 10% of new agents which start clinical trials go to market  Pharma companies are withdrawing from this area of research (more profit in lifestyle drugs – eg statins)  Since 2009, only two new antibiotics have been approved in the United States.

10. PCR-Denaturing Gradient Gel Electrophoresis-Sequencing (DGGE) Amplify ribosomal DNA separate using gel electrophoresis sequence the gene fragments Population analysis studies Demonstrated numerous bacterial species not normally identified by pathology laboratory No single species causing the chronicity 60% chronic wounds have mixed species, only 6% acute wounds - biofilm James et al (2008)- Biofilms in Chronic Wounds Wound Repair and Regeneration 16: 37-44 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.;42:3549-57.

11. Dowd et al. BMC Microbiology 2008 8:43

12. Acute Infection Chronic Infection Biofilm and Chronic Inflammation JWC Vol17(8), 2008

13.  Biofilms are surface-attached communities of microorganisms, encased in an extracellular matrix of carbohydrates, proteins and/or DNA, that assume phenotypes distinct from those of planktonic (free floating) cells  Sessile cells(attached)

14.  Staphylococcus epidermidis (IV lines)  Staphylococcus aureus (dialysis)  Streptococcus mutans (teeth)  Pseudomonas aeruginosa (CF)  Candida albicans (yeast) (teeth, dialysis)

15. Stages:  Initial interaction of cells with a surface or with each other  Aggregation  Production of extracellular matrix (genetically triggered?)  Biofilms can be structurally different due to the specific organisms in the biofilm and environmental conditions  Communication between species using quorum sensing

16.  Quorum sensing (production of auto-inducer molecules at the end of exponential phase) is demonstrated in biofilms ◦ FISH

17.  Antibiotic assays use planktonic cells. ◦ Chronic / device related infections go unresolved even though organisms are susceptible.  Cells protected by the matrix  Cells at base of biofilm reproduce at a slower rate  MICs can be 1000 fold different when tested on sessile cells  B-lactamase accumulates in the biofilm matrix ◦ inactivating the antibiotic and increases potential for resistance to develop.

18.  scanning electron microscopy (SEM)  epifluorescent microscopy (EpiFM)  confocal scanning laser microscopy (CSLM)  detection of quorum sensing molecules (QS)  molecular characterisation of species present

19. James Wound Repair and Regeneration Vol 16, 2008 AcuteChronic

20. Confocal microscopy Green Fluorescent Protein expressing S. aureus strain shown by 3-D reconstruction of a confocal image after several hours of biofilm growth (taken from Analysis of the Genetic Basis of S. aureus Biofilms Fischer and Stenz)

21. Removal of wound biofilms? Removal of wound biofilms?  Biofilms are notorious difficult to remove from surfaces but most treatments reduce numbers of microorganisms  Disruption of the physical structure of the biofilm through cleaning and debridement ◦ Autolytic ◦ Sharp ◦ Larval - Maggots ◦ Enzymatic ◦ Mechanical  Hopefully wound healing process recovers

22. Rhoads JWC Vol 17(11), 2008

23. Appropriate dressings Bacterial Balance

24. Debridement Exudate Management Critical colonisation /Local Infection – consider topical antimicrobials Bacterial Balance

25. Debridement Exudate Management Infection – consider antibiotics if systemic signs of infection topical antimicrobials (Ag +,slow release Iodine, PHMB, honey)

26.  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

30.  Dressings ◦ Slow release nanocrystalline metals, silvers, ?copper ◦ Honey ◦ Incorporation of biocides (e.g PHMB)  New materials ◦ Sorbact - DACC technology  Dialkylcarbamoylchloride (fatty acid) ◦ Kytocel - chitosan  Antimicrobial enzymes ◦ Flaminal – glucose oxidase / lactoperoxidase  Reactive oxygen species

31.  101 year old lady who lives with family  Usually mobile with the aid of a stick. On admission

32. 5 days 14 days 11 days7 days 18 days

33. Female, 64 yrs old. Middle-east origin. No foreign travel in last 6 months. Trip to Iraq within two years. Eaten soft cheeses. recurrent cellulitis of non healing wound on the sternum, SSI. Blood Culture taken on admission -POSITIVE Gram Stain: Small GNB ?Flu / ?Acinetobacter /? Pseudomonad

34.  Gram: GNCB  Growth: Poor, Choc & CLED only  Oxidase: Positive  Stringy Test: Negative  Catalase: Positive  Indole: Negative  Next Step:  Urea Slope: Positive in 30 minutes  Brucella melitensis.

35. Regular debridement is the main tool for maintaining a healthy wound bed in most chronic wounds R.D. Wolcott, J.P. Kennedy, S.E. Dowd Journal of Wound Care 18(2): 54 - 56 (Feb 2009) Biofilm maturity studies indicate sharp debridement opens a time- dependent therapeutic window R.D Wolcott, K.P Rumbaugh, G James, G Schultz, P Phillips, Q Yang, C Watters, P.S. Stewart, S.E. Dowd Journal of Wound Care 19(8): 320 - 328 (Aug 2010)