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Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2012-08-31.

Permanent Link: http://ufdc.ufl.edu/UFE0042205/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2012-08-31.
Physical Description: Book
Language: english
Creator: Hamad, Afifa
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: Medicine -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Afifa Hamad.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Schultz, Gregory S.
Electronic Access: INACCESSIBLE UNTIL 2012-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042205:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042205/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2012-08-31.
Physical Description: Book
Language: english
Creator: Hamad, Afifa
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: Medicine -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Afifa Hamad.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Schultz, Gregory S.
Electronic Access: INACCESSIBLE UNTIL 2012-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042205:00001


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1 DEVELOPMENT OF A CLINICAL MICROBIOLOGY LABORATORY ASSAY FOR THE PRESENCE OF BACTERIAL BIOFILMS ON CHRONIC WOUNDS By AFIFA HAMAD A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010

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2 2010 Afifa Hamad

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3 To my family

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4 ACKNOWLEDGMENTS I would like to sincere ly thank my advisor Dr. Gregory Schultz for his advice and support I also appreciate the guidance provided by my mast of Dr Ann Progulske Fox and Dr. Patrick Antonelli I would also like to thank Dr. Antonelli for providing me with laboratory work space and Dr. Progulske Fox for allowing me to use the anaerobic chamber in her lab I must also acknowledge an d thank Dr. Priscilla Phillips, Qingping Yang and Edith Sampson for their support and I must also thank Dr. Joyce Stechm iller and Dr. Linda Cowan for thei r assistance with collecting clinical samples and for the ir insight regarding the clinical aspect of my research project I also acknowledge Joyce Conners for her administrative assistance throughout degree. I extend a special acknowledgement to my mother, father, siblings and extended family for their endless encouragement, support and assistance

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 ABSTRACT ................................ ................................ ................................ ................... 11 CHAPTER 1 LITERATURE REVIEW : PRESENCE AND DETECTION OF BIOFILMS IN CHRONIC WOUNDS ................................ ................................ .............................. 13 Normal Healing Process ................................ ................................ ......................... 13 Chronic Wounds ................................ ................................ ................................ ..... 13 Treatment Strategies for Uninfected Wounds ................................ ......................... 15 Bacterial Colonization of Chronic Wounds ................................ .............................. 17 Bacterial Patterns of Growth ................................ ................................ ............. 17 Planktonic vs. biofilm pattern of growth ................................ ...................... 17 Dynamics of biofilm colonization and dispersion ................................ ........ 19 Mechanisms of resistance to antimicrobial agents ................................ ..... 20 Biofilms in medicine ................................ ................................ ................... 22 Bacterial Presence in Wounds ................................ ................................ ......... 23 General: bacterial invasion and colonization ................................ .............. 23 Bacteria in chronic wounds ................................ ................................ ........ 23 Biofilms Delay Wound Healing ................................ ................................ ......... 25 Clinical Identification of Chronic Wounds ................................ ................................ 26 Clinical Signs and Symptoms ................................ ................................ ........... 26 Quan titative Analysis ................................ ................................ ........................ 27 Gold standard ................................ ................................ ............................ 27 Swab vs. deep tissue biopsies ................................ ................................ ... 27 Need for a Clinical Assay to Detect Bacterial Biofilms in Chronic Wounds ...... 29 2 THE UNDERLYING PRINCIPLE OF A DEVELOPING A BIOFILM DETECTING ASSAY: ELIMNATION OF PLANKTONIC BACTERIA ................................ ........... 31 Introduction ................................ ................................ ................................ ............. 31 Principle behind an Assay to Detect Bacterial Biofilms ................................ .... 31 Approach for Detection of Bacterial Biofilms ................................ .................... 31 Selecting the Appropriate Antimicrobial Agent ................................ ................. 33 General Material and Methods ................................ ................................ ................ 34 Lab Strains ................................ ................................ ................................ ....... 34 Culturing Bacteria ................................ ................................ ............................. 34 Serial Dilutio ns and Plating ................................ ................................ ............... 34

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6 Sonication ................................ ................................ ................................ ......... 35 Developing a Representative Biofilm Model ................................ ........................... 36 Introduction ................................ ................................ ................................ ....... 36 Materials and Methods ................................ ................................ ..................... 37 Results ................................ ................................ ................................ ............. 39 Conclusion ................................ ................................ ................................ ........ 39 Calcium Hypochlorite Treatment ................................ ................................ ............. 39 Ma terials and Methods ................................ ................................ ..................... 39 Preparation of treatment solution ................................ ............................... 39 Ca(ClO) 2 concentration necessary to kill early log phase planktonic bacteria in suspension culture ................................ ................................ 40 Inclusion of stationary phase culture ................................ .......................... 41 Complete neutralization of calcium hypochlorite by sodium thiosulfate ..... 43 Results ................................ ................................ ................................ ............. 45 Ca(ClO) 2 concentration necessary to kill early log phase bacteria in suspension culture ................................ ................................ .................. 45 Inclusion of stationary phase culture ................................ .......................... 45 Complete neutralization of calcium hypochlorite by sodium thiosulfate ..... 46 Conclusion ................................ ................................ ................................ ........ 46 3 OPTIMIZATION OF THE ASSAY IN THE LABORATORY ................................ ..... 54 What Makes a Good Clinical Assay? ................................ ................................ ...... 54 Optimizing the Collection Method ................................ ................................ ........... 55 Introduction ................................ ................................ ................................ ....... 55 Materials and Methods ................................ ................................ ..................... 55 Ca(ClO) 2 c oncentration necessary to kill planktonic bacteria collected by cotton swabs ................................ ................................ ...................... 55 Comparing antibiotic treatment to 8 hr exposure at l ow concentrations of Ca(ClO) 2 ................................ ................................ ............................. 58 Alternative collection method ................................ ................................ ..... 59 Concentrations of Ca(ClO) 2 necessary to eliminate planktonic bacteria collected by the cytology brush and the flocked swab ............................ 59 Treatment of PAO1 biofilm collected on flocked swabs ............................. 60 Comparing levels of biofilm pick up by different collection devices ............ 60 Scanning electron microscopy images of different swab materials ............ 62 Results ................................ ................................ ................................ ............. 63 Ca(ClO) 2 concentration necessary to kill planktonic bacteria collected by cotton swabs ................................ ................................ ...................... 63 Comparing antibiotic treatment to 8 hr exposure of low concentrations of Ca(ClO) 2 ................................ ................................ ............................. 64 Concentrations of Ca(ClO) 2 necessary to eliminate planktonic bacteria collected by cytology brush and flocked swab ................................ ........ 64 Treatment of PAO1 biofilm collected on flocked swabs ............................. 65 Comparing levels of biofilm pick up by different collection devices ............ 65 Scanning electron microscopy ima ges of different swab materials ............ 66 Conclusion ................................ ................................ ................................ ........ 67

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7 Optimizing the Transport Method ................................ ................................ ............ 67 Introduction ................................ ................................ ................................ ....... 67 Materials and Method s ................................ ................................ ..................... 68 Concentrations of Ca(ClO) 2 necessary to kill planktonic bacteria in Amies transport media ................................ ................................ ............ 68 P aeruginosa, PAO1 biofilm treatment in different transportation solutions ................................ ................................ ................................ 69 Results ................................ ................................ ................................ ............. 70 Concentrations of Ca(ClO ) 2 necessary to kill planktonic bacteria in Amies transport media ................................ ................................ ............ 70 P aeruginosa, PAO1 biofilm treatment in different transportation solutions ................................ ................................ ................................ 70 Conclusion ................................ ................................ ................................ ........ 70 Optimizing Biofilm Dispersal ................................ ................................ ................... 71 Introduction ................................ ................................ ................................ ....... 71 Materials and Method s ................................ ................................ ..................... 71 Results ................................ ................................ ................................ ............. 72 Conclusion ................................ ................................ ................................ ........ 72 4 INITIAL ASSESSMENT OF THE CLINICAL MICROBIOLOGY BIOFILM ASSAY USING CLINICAL SAMPLES ................................ ................................ ................. 86 Introduction ................................ ................................ ................................ ............. 86 Antibiotic Treated Samples ................................ ................................ ..................... 86 Materials and Methods ................................ ................................ ..................... 86 Results ................................ ................................ ................................ ............. 88 Conclusion ................................ ................................ ................................ ........ 88 Calcium Hypochlorite Treated Samples ................................ ................................ .. 88 Introduction ................................ ................................ ................................ ....... 88 Materials and Methods ................................ ................................ ..................... 89 Standardization of amount of tissue collection ................................ ........... 89 Processing samples ................................ ................................ ................... 90 Results ................................ ................................ ................................ ............. 92 Standardization of sample weight ................................ .............................. 92 Processing samples ................................ ................................ ................... 92 Conclusion ................................ ................................ ................................ ........ 92 LIST OF REFERENCES ................................ ................................ ............................... 99 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 105

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8 LIST OF TABLES Table page 2 1 Treatment of suspension of early log phase cultures with calcium hypochlorite ................................ ................................ ................................ ........ 47 2 2 Observation of growth and optical density measurements for early log phase culture treated with calcium hypochlorite ................................ ............................ 49 2 3 Complete neutralization of calcium hypochlorite by sodium thiosulfate .............. 50 3 1 Treatment of P aeruginosa PAO1 early log phase culture collected on cotton swabs with 0.15% and 0.2% calcium hypochlorite ................................ ... 73 3 2 Exposure of planktonic P aeruginosa, PAO1 bacteria to gentamicin and calcium hypochlorite after collection on cotton swabs ................................ ........ 74 3 3 Treatment of P aeruginosa, PAO1 biofilm collected on flocked swabs .............. 74 3 4 Comparison of P aeruginosa, PAO1 biofilm pickup levels using different collection devices ................................ ................................ ............................... 75 3 5 Treatment of planktonic bacteria with Ca(ClO) 2 in different transport solutions .. 75 3 6 Treatment of P aeruginosa, PAO1 biofilm in different transport solutions ......... 76 4 1 Clinical wound samples treated with antibiotic mixture ................................ ....... 94 4 2 Standardization of sample weight ................................ ................................ ....... 95 4 3 Treatment of chronic wound curette samples from VA in Lake City, FL ............. 96

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9 LIST OF FIGURES Figure page 2 1 I n vitro porcine skin PAO1 biofilm model. A) Removal of 0.9 mm of epidermis layer. B) Padgett B dermatome. C) Explant after wound bed preparation. D) Mature 3 day PAO1 biofilm cultured on the explant. ................................ ........... 5 1 2 2 Protocol for culturing and processing biofilm samples using the in vitro porcine skin PAO1 biofilm model. ................................ ................................ ....... 52 2 3 Treatment of early log phase culture in suspension with calcium hypochlorite ... 53 3 1 Treatment of stationary and early log phase bacteria on swabs with Ca(ClO) 2 ................................ ................................ ................................ ............ 77 3 2 Ca(ClO) 2 necessary to kill planktonic bacteria of different species after collection using cotton swabs. ELG: early log phage, ST: stationary phase. ...... 77 3 3 Exposure of planktonic P. aeruginosa PAO1 bacteria to gentamicin and Ca(ClO) 2 after collection on a cotton swab. ................................ ........................ 78 3 4 Different collection methods. A) Cotton swab. B) Flocked swab. C) Cytology brush. D) 4mm loop curette. ................................ ................................ ............... 78 3 5 Treatment of P aeruginosa, PAO1 biofilm collected on flocked swabs. ............. 79 3 6 Comparison of P aeruginosa PAO1 biofilm pickup levels using different collection devices. A) Collection devices were treated with Ca(ClO) 2 B) Collection devices were treated with 200 g/mL gentamicin. ............................. 80 3 7 Planktonic and biofilm PAO1 collected on cotton swabs. A) Early log phase PAO1 collected on a cotton swab. B) 3 day mature PAO1 biofilm cultured on porcine skin and collected on a cotton swab. Each line represents 6 m. .......... 81 3 8 SEM images of P aeruginosa, PAO1 biofilm collected on different swabs. A) cotton swab. B) cytology brush. C) flocked swab. D) Sponge swab. Each line represents 600 m. ................................ ................................ ............................. 81 3 9 SEM images of porcine skin biopsies after P aeruginosa, PAO1 biofilm collection using different collection methods. A) Cytology brush B) 4 mm loop curette C) Cotton swab. Each line represents 12 m. ................................ ........ 82 3 10 P aeruginosa, PAO1 biofilm treatment in different transportation solutions. ...... 83 3 11 Optimization of PAO1 biofilm dispersion using 8 mm porcine skin explants. A) Pulsed sonications were separated by 1 minute intervals. B) Pulsed vortexing was separated by 1 minute intervals. ................................ .................. 84

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10 3 12 Optimization of PAO1 biofilm dispersion using curetted samples. A) Pulsed sonications were separated by 1 minute intervals. B). Pulsed vortexing was separated by 1 minute intervals. ................................ ................................ ......... 85

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11 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science DEVELOPMENT OF A CLINICAL MICROBIOLOGY LABORATORY ASSAY FOR THE PRESENCE OF BACTERIAL BIOFILMS ON CHRONIC WOUNDS By Afifa Hamad August 2010 Chair: Gregory Schultz Major: Medical Sciences Reports have shown that the majority of chronic wounds contain microbial biofilm: Attached structured microbial communities embedded in a self secreted polymeric matrix. Evidence suggests that bacterial biofilm prolongs inflammation and contributes to the non healing of wounds. Consequently, there is a great need in the clinical wound care field for a standardized clinical microbiology laboratory assay that can be used to detect the presence of functional biofilms to improve clinical eva luation and treatment of chronic wounds. The approach to detect bacterial biofilms is based on the functional definition of a microbial biofilm: The extreme tolerance of mature biofilm to antimicrobial agents that the same microorganisms are susceptible t o when planktonic. The assay was developed using laboratory strains of S. aureus P. aeuriginosa and E.coli grown planktonically in suspension culture or as mature biofilms cultured on porcine skin explants. Various collection devices and treatments method s were assessed. The final assay employs curette collected specimens treated wi th calcium hypochlorite (Ca(ClO) 2 ) to kill planktonic bacteria during a spe cific exposure time followed by neutralization with sodium thiosulfate. The advantage of this method is the use of a nonspecific

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12 antimicrobial agent that is faster and less expensive than a 24 hour antibiotic The method was validated using clinical specimens obtained from chronic wounds Development of a clinical microbiology laboratory assay to generat e a more comprehensive evaluation of the state of a wound w ill help wound care providers determine the correct and necessary treatment strategies to promote wound healing.

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1 3 CHAPTER 1 LITERATURE REVIEW: PR ESENCE AND DETECTION OF BIOIFLM S IN CHRONIC WOUNDS Normal Healing Process A normal and complete wound healing process can be divided into four consecutive stages including coagulation inflammation, cell proliferation and matrix repair, and finally remodeling ( Bjarnsholt et al ., 2008 ; Falanga, 2002 ) For successful healing, various classes of host factors must be involved in the healing process including inflammatory cells, platel ets, leukocytes, fibroblasts, keratinocytes endothelial cells, growth factors and various enzymes ( Jones et al ., 2004 ; Blakytny and Jude 2006 ) Platelets promote homeostasis and the release of growth factors, leukocytes are associated with the inflammatory response while fibroblasts and keratinocytes facilitate remodeling and wound closure by epithelization ( Jones et al ., 2004 ) Cytokines and chemokines also play a crucial role in regulating cellular response mechanisms including the activation of fibroblasts and keratino cytes ( Jones et al ., 2004 ) Chronic Wounds Wounds that take more than four to eight weeks to advance through the normal process of healing are usually considered chronic wounds ( Izadi and Ganchi, 2005) Non healing wounds are characterized by p rolonged i nflammation as well as impairment in re epithelialization and matrix remodeling ( Edwards and Harding, 2004 ). T he prolonged pr esence of neutrophils and other inflammatory mediators like prostaglandin E2 and thomboxane in the wound bed can be detrimental to healing process as they release cytotoxic enzymes and free oxygen radicals that are damaging to host tissue (Schultz et al ., 2003; Jones et al ., 2004) Some factors like arterial insufficiency, venous hypertension, trauma, diabetes mellitus and rheumat oid arthritis

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14 are usually present in individuals with chronic wounds as they are more susceptible to bacterial invasion ( Edwards and Harding, 2004) Bacterial invasion in wounds can progress from contamination, to local colonization and may proceed to an i nfection causing cel lulitis or septic emia ( Edwards and Harding, 2004 ). Chronic wounds are a critical health issue around the world and include diabetic foot ulcers (DFU), pressure ulcers (PU), venous leg ulcers (VLU) and ischemic ulcers (James et al ., 2008 ; Izadi and Ganchi, 2005). Chronic wound patients undergo many challenges in their everyday life as they usually live with open wounds for a long period of time. Usually these patients have other health conditions, have visited several physicians and have been treated with more than one wound dressing or medication (Pozez et al 2007). M any of the chronic wound patients are elders which becomes troublesome for the society in terms of the increasing health expenses as well as the loss of efficiency and mobi lity of these indivi duals (Izadi and Ganchi, 2005). As a consequence, billions of dollars are spent on health care for chronic wounds in the U S each year (Mustoe et al ., 2006). In the U S more than 4 million patients have chronic wounds and the estimat ed treatment cost is about 9 billion dollars per year ( Izadi and Ganchi, 2005) Diabetes patients are especially at risk of developing chronic wounds. Diabetic foot ulcers are caused by the continuous load on the neurophatic and ischemic foot which can be treated by offloading and increased circulation (Bjarnsholt et al ., 2008). Estimates show that 7.7% of diabetes patients have a history of a foo t ulcer ( Reiber and McFarland 2006) and 14 24% of diabetic patients with foot ulcers will eventually undergo amputation ( American Diabetes Association, 1999). In the U.S. alone, i t is

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15 estimated that about 100,000 diabetic patients undergo limb amputations each y ear (James et al ., 2008). Venous leg ulcers, which population, are very painful and distressing to patients ( Trent et al ., 2005 ). These patients develop venous hypertension in the crural veins, increased pressure in the capi llaries and edema as a result of the failure of venous valves ( Bjarnsholt et al ., 2008 ) Venous pressure higher than 45 mmHg indicates the development of a venous leg ulcer which is usually treated by compression (Bjarnsholt et al ., 2008). Pressure ulcers also constitute a widespread and costly problem in various clinical settings reha bilitation units, nursing homes as well as amongst patients receiving home care (James et al ., 2008). Pressure ulcers are caused by continuous or recurring pressure on areas like sciartic tuberculum, sacral regions, heels and shoulders usually in immobile patients ( Bjarnsholt et al ., 2008 ) Pressure relief by cushioning and movement is usually the appropriate method to prevent and treat such ulcers ( Bjarnsholt et al ., 2008 ) D espite treatment, some pressure ulcers become chronic and will not heal due to a number of hindering factors such as immunological defects, malnutrition, obesity, drug abuse, alcoholism or smoking (Bjarnsholt et al ., 2008). Treatment Strategies for Uninfe cted Wounds A successful outcome for wound care providers is to direct the wound to complete and permanent closure. This is usually accomplished through one or more wound care strategies including topical, surgical or tissue engineering therapies. Usually it is not simple to predict the outcomes of a treatment plan or why a specific strategy did not yield the desir ed or predicted results making wound care a complicated and prolonged process (Pozez et al 2007).

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16 Topical treatments may serve several functio ns includ ing covering the wound, providing a moist environment, promoting debridement, preventing bacterial infections and absorbing excess flui d discharge to promote healing ( Pozez et al 2007 ) One type of topical treatment is the application of hydrogels which pro vide moisture to the wound to p romote epithelializtion and angiogenesis and also reduces the frequency of dressing changes ( Bowler et al ., 2001; Pozez et al ., 2007 ) Dressings designed to absorb exudates may conta in a lginates obta ined from brown seaweed that can take up excess moisture and swell into a gel like material that can be removed easily ( Pozez et al ., 2007). This is especially important since chronic cludes necrotic tissue as well as exudates (Falanga, 2002) Exudate in the wound bed contains proteases that cleave extracellular matrix proteins and inhibit proliferation and function of cells, like fibroblasts and keratinocytes, which are important facto rs in the wound healing process (Falanga, 2002). Absorptive foam dressings which are usually coated with hydrophilic polyurethane polymer or gel also protect the wound bed from severe temperature variations ( Pozez et al ., 2007 ) Hydrocolloid dressings are mostly made from pectin but also from carboxymethylcellulose and gelatins which serve to facilitate autolytic debridement especially if the wound has been surgically debrided prior to dressing application ( Pozez et al ., 2007 ; Barr et al ., 1995 ) Proteolyt ic enzyme based treatments using papain urea based combinations and collagenase can also be used to digest necrotic tissue and reduce b acterial burden (Falanga, 2002).

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17 Surgical intervention may be necessary during treatment of chronic wounds, particularly in cases of deep infect ion Debridement is usually performed to remove necrotic tissue and debris to allow for fresh tissue expansion in the wound bed and to facilitate wound closure ( Pozez et al ., 2007 ) Also a negative pressure vacuum may be applied to the wound bed after debridement which promotes wound closure by expanding granulation tissue and eliminating excess space and not necessarily by reducing the microbial load ( Pozez et al ., 2007 ; Moue ¨ s et al ., 2004). If necessary, after debridement, other surgical procedures such as grafts, direct closure or other forms of tissue reconstruction may be used (Pozez et al 2007). Bacterial Colonization of Chronic Wounds Bacterial Patterns of Growth Planktonic vs. biofilm pattern of growth In contrast to the commonly perceived idea that most bacteria are planktonic free floating cells, many bacterial species tend to irreversibly attach to surfaces ( Donlan and Costerton, 2002). The attachment of bacteria to surfaces is dependent on the nutrit ional signals as well as on the number of bacteria present (Serralta et al ., 2001). When these bacteria attach and aggregate, they start to recruit other microorganism s like bacteria from the same or from different species, as well as fungi and protozoa (S erralta et al ., 2001). These aggregates, called biofilms, are encased in a self secreted three dimensional extracellular polymeric substance (EPS) matrix ( Donlan and Costerton, 2002; Percival and Bowler, 2004). The EPS is generally composed of secreted pol ysaccharides, proteins glycoproteins glyco lipids and extracellular DNA (Flemming et al ., 2007). Unlike planktonic bacteria, most cells in biofilm colonies have low metabolic activity and are resistant to most antibiotics, biocides and host defense

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18 mechan isms (Lewis, 2001; Nadell et al ., 2008 ) The EPS matrix surrounding biofilms serves many protective roles including shielding the bacteria from aggressive surroundings, acting as a barrier to the penetration of antimicrobial agents a nd encasing the bacteria into an environment in which they can communicate through quorum sensing molecules (Hurlow and Bowler, 2009). Biofilms form three dimensional porous structures separated by voids forming water channels that deliver nutrients and remove waste products ( Lewandowski et al ., 1993 ; Serralta, 2001 ). These voids appear to provide bacterial cells with about 50% of the total needed oxygen by promoting oxygen delivery from the surrounding liquid to the cells (DeBeer et al ., 1994). Plankto nic and biofilm bacteria also have differential gene expression patterns supporting the specific growth pattern ( Resch et al ., 2005 ; Beenken et al ., 2004 ; Cho and Caparon, 2005 ) For example, Resch et al (2005) found that i n Staphylococcus aureus biofilms genes encoding for enzymes involved in cell envelope synthesis and function as well as proteins involved in the synthesis of murein and glucosaminoglycan polysa ccharide intercellular adhesion were significantly up regulated. It was also found that f ormat e fermentatio n, urease activity, oxidative stress response acid and ammonium production and other process that contribute to biofilm survival were also up regulated ( Resch et al ., 2005 ) Biofilms colonization is an ordered process that is divided into a number of stages (Phillips et al 2010; Hall Stoodley et al ., 2004). Fo r example, Sauer et al (2002) divided the stages of P. aeruginosa biofilm development into five stages including reversible attachment, irreversible attachment, maturation 1, maturation 2, and

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19 dispersion They found that t he average difference in protein regulation between each of the five stages was 35% which is about 525 proteins, and therefore contributing to changes in the regulation of motility, alginate production, and q uorum sensing ( Sauer et al 2002). Dynamics of biofilm colonization and dispers ion Although most biofilms are multispecies and diverse, studies have shown that in some cases, there is one or more species that become dominant depending on the growth rates of the species ( Banks and Bryers, 1991 ) When two species are cultured together, usually the organism that grows more rapidly becomes dominant while the organism growing slowly continues to multiply in the biofilm ( Banks and Bryers, 1991 ) However, introdu cing a new organism to pre existing single species biofilm depends more on the relative growth rates of the two bacterial species (Banks and Bryers, 1991). Also, simple genome mutations in one species can allow it to adapt to the presence of another organ ism forming an intimate and specialized association resulting in a more stable and productive community (Hanse n et al ., 2007). Other research indicates that biofilms containing mixed strains show competitive rather than cooperative behavior where polymer producing cells drive later generations towards a more oxygen rich environment while harming other non polymer producing cells ( Xavier and Foster, 2007). Biofilms can disperse and spread to form new colonies on distant surfaces by different strategies. In dividual cells or daughter cells, can be released from the colony into the surrounding environment which is called ( Hall Stoodley et al ., 2004 ; Donlan, 2002 ) Bacterial aggregates may also shed off as clumps

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20 Hall Stoodley et al ., 2004) Mechanisms of resistance to antimicrobial agents When bacteria attach to surfaces and form biofilm, they become approximately 1 0 1,000 times less susceptible to antimicrobial agents when compared to planktonic cells ( Davies, 2003 ; Mah et al ., 2003 ). For example, Staphylococcus aureus biofilms require a 600 fold increase in the concentration of sodium hypochlorite to achieve the same reduction in cell numbers as planktonic bacteria ( Luppens et al 2002 ). Resistance factors for P seudomonas aeruginosa biofilms cultured using an artifici al model were found to be increased 120 fold for chlorine, 34 fold for glutaraldehyde, 29 fold for 2,2 dibromo 3 nitrilopropionamide, and 1900 fold for an alkyl dime thyl benzyl ammonium compound ( Grobe et al ., 2002 ). Biofilms tolerance is a result of sever al factors including the difficulty of penetration of antimicrobia l agents into biofilm colonies, increased activity of multidrug efflux pumps, involvement of quorum sensing systems, stress responses, and genetic switches that turn susceptible planktonic c ells into antibiotic resistant persisters (Leid, 2009). Kim et al (2009) found that dormant P aeruginosa cells found at the bottom layer within a biofilm culture showed different tolerance rates to some antimicrobial agents when compared to the active cells found at the upper layer of the biofilm near the air interface Active cells showed a higher tolerance to chlorine since the high intracellular density allowed cellular components to rapidly react with the chlorine, a reactive oxidant, and therefore reduce the concentration of available chlorine ( Kim et al ., 2009). On the other hand, dormant cells showed more resistance to other antimicrobial agents like tobramy cin and silver ( Kim et al 2009).

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21 It has been demonstrated that bacteria within the bio film environments are in a dormant state and have a slow growth rate due to limited nutrition ( Sternberg et al ., 1999 ). In this case, biofilm bacteria are believed to become tolerant to antibiotics which specifically act on dividing and metabolically active cells ( Davies, 2003; Lewis, 2001 ) Ashby et al (1994) supported this hypothesis by showing that antibiotics like imipenem and ciprofloxacin, th at act on non growing bacteria result in increased kill of E scherichia coli biofilm bacteria when compar ed with use of other antibiotics like lactams which act on dividing bacteria. However the activity of these antibiotics was not enough to completely eradicate E scherichia coli biofilms and their activity on biofilms was less than the activity against pla nktonic bacteria ( Ashby et al ., 199 4 ) Mah et al (2003) showed that the mechanism of biofilm tolerance to antibiotics involves periplasmic glucans that bind to and sequester antibiotics in P aeruginosa This mechanism prevents the entrance of antibiotics to the cytoplasm which is the site of action for many antibiotics ( Mah et al ., 2003) This binding slows the diffusion of antibiotics into the cytoplasm, therefore allowing more time for the bacteria to ada pt to the antibiotics presence ( Mah et al 2003). Also, the negative charge of the EPS matrix plays a role in preventing aminoglycoside antibiotics, which are positively charged, from penetrating into the biofilm colonies (Lewis, 2001 ). It has also been show n that sub inhibitory levels of aminoglycoside antibiotics induce biofilm formation in P. aeruginosa and E. coli by a defensive mechanism based on alterations in the levels of c di GMP (Hoffman et al 2005).

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22 Biofilms in medicine Biofilms may form on teeth, respiratory tract tissues intestinal mucosa and surgical implants and are inherently resistant to immune defenses and most antimicrobials and antibiotics (Donlan and Costerton, 2002; Hall Stoodley et al ., 2004 ; Davies, 2003 ) Estimates report that 65% of nosocomial infections are related to biofilms with a treatment cost of over one billion dollars each year in the U S (Percival and Bowler, 2004). Biofilm associated infections include periodontal disease, endocarditis and otitis media as well as i nfections related to catheters, sutures and contact lenses (Hurlow and Bowler., 2009 ; Donlan and Costerton, 2002 ). Microcolonies surrounded by exopolymeric matrix have also been show n to be present in bacterial prostatitis and biliary tract infec tions (Bja rnsholt et al ., 2008). Biofilms can attach to and establish colonies on medical devices, like central venous catheters and needleless connectors, endotracheal tubes, intrauterine devices, mechanical heart valves, pacemakers, peritoneal dialysis catheters, prosthetic joints, tympanostomy tubes, urinary catheters, and voice prostheses ( Donlan, 2001) The species that are commonly isolated from such medical devices include G ram positive bacteria like Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus viridans ; a s well as G ram negative bacteria like Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa (Donlan, 2001) Sometimes bacterial species that are considered normal ski n microflora may form harmful biofilms such S taphylococci spp. biofilms forming on intravenous catheters, hip and knee joint prostheses and other surgical implants ( Percival and Bowler, 2004 ). Pseudomonas aeruginosa an environmental bacterium that forms biofilms, is especially

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23 dangerous for immunosupressed patients and is usually associated with skin burn infections as well as lung infections in cystic fibrosis patients (Drenkard, 2003; Percival and Bowler, 2004 ) Although a few species may be cultured from a biofilm colony most biofilms are polymicrobial and may contain many uncultivable species ( i.e. more than 350 various organisms in some dental plaques ) (Percival and Bowler, 2004). Bacterial Presence in Wounds General: bacterial invasio n and colonization Intact human skin harbors many harmless and in some cases beneficial bacterial species such as Staphylococcus epidermidis which plays a role in preventing colonization by other pathogenic bacteria ( Bowler, 2003 ) Wounded skin is prone to contamination by bacteria either normally found on the skin or bacteria from external flora. However, most wound contaminating bacteria are endogenous flora derived from the mucosal surfaces of the host ( Bowler, 2003 ) Wound bed infections are usually a complex colonization of multiple bacterial organisms which may lead to competition between species ( Bowler, 2003 ) Although the wound environment contains an excess of moisture, warmth and nutrients which promote microb ial diversity, some harmless symbiotic bacteria become pathogenic in order to compete for resources ( Bowler, 2003 ) One of the ways bacteria avoid clearance by the host immune system and dislodgement by host secreted exudates is by attaching to host cells and secreting cell protecting components such as the EPS matrix surrounding biofilm microcolonies (Bowler, 2003). Bacteria in chronic wounds Although it has generally been acce pted that a bacterial load of 10 5 or more colonies forming units per gram of t issue (CFU/g) is sufficient to diagnose an infection,

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24 this rule may apply differently to acute wounds as compared to chronic wounds ( Bowler, 2003 ; Serena et al ., 2006 ) In acute wounds, bacterial load is very important in diagnosing infection and predicting the progression of healing, but for chronic wounds other factors must be considered including the extreme tolerance to antimicrobial treatment, the effect bacterial EP S and other secreted factors, and the synergetic effect of two or more bacteria to ( Bowler, 2003 ). Therefore, this general 10 5 CFU/g guideline is not reliable when referring to chronic wound infections since it does not take i nto account the bacterial species biofilm microcolonies, or the complexity and adaptability of the bacteria in such cases (Bowler, 2003). In a study performed by Frankel et al (2009) in which curettage was performed on 29 chronic wounds persisting for 6 weeks or longer showed an average of 2.1 different species of bacteria with a total of 104 different isolates. The data also show ed that 83% of the isolates had 10 5 or more CFU/g and 21% had cell counts of 1 0 8 o r more CFU/g ( Frankel et al ., 2009) Furthermore, six out of the seven patients whose wounds were assessed before and after antibiotic treatment showed that there was still significant presence of bacteria after treatment ( Frankel et al 2009). Also, i n a prospe ctive study of 52 chronic wounds in which the presence of aerobic bacteria were assessed, P aeruginosa was found to be the most prevalent organism followed by E.coli (Basu et al ., 2009). Chronic wounds can be colonized by a diverse population of bacteria l species including both aerobes and anaerobes ( Nadell et al ., 2008; Dowd et al ., 2008 ) This is especially true for biofilms as most of them are multi species colonies that are present

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25 in chronic wounds and can retard wound healing ( Basu et al ., 2009). Dowd et al (2008) found that the major species found in chronic wound biofilms are Staphylococcus, Pseudomonas, Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia and Serratia. In addition, the study found that different types of chronic wounds ha ve different bacterial population profiles in terms of the identity of the major bacterial species found within the biofilms For example, 62% of the bacterial populations in pressure ulcers were found to be obligate anaerobes ( Dowd et al 2008). Biofilms Delay Wound Healing Microscopy studies indicated that bacterial biofilms are present in 60% of chronic wounds and only in 6% of acute wounds (James et al 2008). This suggests that b iofilm presence in wounds contributes to impaired healing and increases the chances that a wound will become chronic. A wound infection prolongs the inflammatory stage therefore prolonging the secretion of proteases and free oxygen radicals consequently causing degradation of fresh granulation tissue vital for normal wound healing (Knox et al ., 2007 ; Percival and Bowler, 2004 ; Jones et al ., 2004 ) Bacteria embedded within the biofilm matrix are likely to be resistant to both immunological and non specific defense mechanisms of the body (Percival and Bowler, 2004). The structure of biofilms is such that immune responses may be directed only to those antigens found on the outer surface of the biofilm while antibodies and other serum proteins often fail to penetrate into the biofilm ( Percival and Bowler, 2004 ) Bacteria may produce endotoxins that may attack and kill heal thy cells and therefore create a significant mass of necrotic tissue in the wound bed (Jones et al ., 2004). Also, h ost defenses like phagocytic cells can easily penetrate into the biofilm matrix and ing est the bacteria and therefore continue to release lysozymes which

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26 damage nearby tissue ( Percival and Bowler, 2004 ; Jones et al ., 2004 ; Leid, 2009 ) Complement activation can also be blocked by the polysaccharide component of the surrounding bacterial EPS matrix if specific antibodies are not present or rendered ineffective by the bacterial polymeric matrix ( Percival and Bowler, 2004 ) The biofilm matrix also prevents chemotaxis and degranulation by polymorphonucleocytes (PMNs) and macrophages and inhibits the lymphoproliferative response of mono cytes to polyclonal activators ( Percival and Bowler, 2004 ) Not only are host defenses unable to deal effectively with biofilms, but their persistence can cause tissue damage ( Jones et al ., 2004; Leid, 2009 ). Clinica l Identification o f Chronic Wounds Clinical Signs and Symptoms Many wound care providers rely on clinical signs and symptoms to identify chronic wound infections. Gardner et al (2001) showed that signs specific to secondary wounds (exudates, delayed healing, discoloration of granulation tissue, friable granulation tissue, pocketing at the base of the wound, foul odor and wound breakdown) are better purulence). This group also reported that s ome signs and symptoms have a higher validity than others according to four parameters: sensitivity, specificity, discriminatory power and positive predictive power. Increas ing pain, friable granulation tissue, wound breakdown and foul odor showed validity based on all four parameters ( Gardner et al ., 2001) Edema, exudates and concurrent inflammation showed validity based on three parameters while heat and delayed healing we re valid based on two parameters ( Gardner et al ., 2001) Erythema, purulent exudates and discoloration of granulation tissue showed validity based on one parameter ( Gardner et al ., 2001) Furthermore

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27 increasing pain and wound breakdown were shown to be su fficient signs for wound infection with 100% specificity therefore the bes t indicators of would infection ( Gardner et al 2001). Since these signs are subjective and require prolonged observation of the s of infection and observation of these signs is not a very reliable way to diagnose infected wounds (Serena et al ., 2006). Quantitative Analysis Gold standard Relying on quantitative analysis is necessary and more reliable for identifying infected wounds and treating them accordingly. The gold standard for i dentifying wound infections is the presence of more than 10 5 cultivable colony forming units (CFU) per gram of tissue ( Serena et al ., 2006 ; Bowler, 2003 ). Clinical studies indicate that successful healing of burns, pressure ulcers and skin grafts depends on maintaining a bacterial load that is below 10 5 CFU per gram of tissue (Edwarks and Harding, 2004). However, most wound care providers still rely on physical observation and subjective signs and s ymptoms to diagnose infected wounds ( Bamberg et al ., 2002 ; Gardner et al ., 2001 ). Swab vs. deep tissue biopsies Swabs have been used to collect samples for microbiology analysis for over 100 years; however, there is still a lot of controversy about the me thodology of swabbing technique and reliabil ity of the results ( Dow, 2003 ; Bowler, 2003; Gardner et al ., 2006 ). Although various techniques have been described there is no standard swabbing technique that is followed across wound care clinics in the U.S. (Serena et al ., 2006). A cotton swab is the most commonly used device to collect wound fluid or tissue d ebris from the wound bed to be used for semi quantitative and qualitative analysis of the

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28 microbial flora (Bowler et al ., 2001) Many argue that swab cu ltures reflect the bacterial flora colonizing the surface of the wound and miss invasive bacteria especially if practitioners do not remove non vital tissue from the wound surface before swabbing ( Bowler et al ., 2001; Dow, 2003 ) Since the collection metho d can affect the data recovered from a clinical sample, the gold standard for dete ctin g the presence of bacteria and identifying the bacterial species is using a biopsy tissue sample collected from the wound bed ( Bamberg et al ., 2002 ; Bowler et al 2001 ). It is hard to meaningfully quantify the bacterial presence from swab samples since bacteri al counts are reported as CFU/mL of elution solution and not CFU/g of tissue. To complicate matters, t here are conflicting reports on the accuracy of the quantitative correlation between colony counts obtained from swabs and tissue biopsies. Some studies have suggested that that both methods are equally reliable in determinin g bacterial load and diversity. In a study using an acute wound rat model, results show ed that the one point quantitative swab culture method using rayon tipped swabs is able to detect the same bacterial isolates as tissue biopsies although it underestimates bacterial counts by a factor of two logs when co mpared to tissue biopsies (Sullivan et al ., 2004). Another s tud y show ed that there is no statistical difference between results obtained from swabbing and tissue biopsies in terms of either the types of isolated species or the frequency of isolation in dia betic foot ulcers after antimicrobial therapy and that both appear to be reliable for initial screening of bacterial presence ( Pellizzer et al ., 2001 ) However, deep tissue sampling appears to be more sensitive and accurate that swabbing in detecting antib iotic resistant strains which are found to be present after 30 days of treatment (Pellizzer et al ., 2001). In the case of diabetic foot osteomyelitis,

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29 percutaneous bone biopsies are found to be a more reliable detection method than superficial swabb ing (Se nneville et al ., 2006). Despite the fact that quantitative biopsies are generally considered a more reliable assessment of wound infections, they are painful, invasive, and expensive and usually not performed regularly in most wound care clinics in the U. S. (Bamberg et al ., 2002). Thus, s wab cultures using a defined technique and collection protocol can be a valuable method to initially detect infection and bacterial species and semi quantitatively determine the bacterial load in chronic wounds of similar type. Swabbing has been shown to reliably identify pathogenic bacteria in diabetic foot wounds when the bone is not involved ( Slater et al ., 2004 ) Swab culturing methods can be especially useful in administering the appropriate antibiotic treatme nt in the case that surgical debridement cannot be performed (Slater et al ., 2004). Overall, s wabbing provides initial and general information about wound infection s and detects organisms on the wound surface while tissue biopsies target restricted areas o f the wound which may be problematic especially in large wound beds (Bowler, 2003). Need for a Clinical Assay to Detect Bacterial Biofilms in Chronic Wounds N one of the commonly used methods of detecting bacteria in chronic wounds distin guish between pla nktonic free floating a nd biofilm protected bacteria which require different treatment strategies. Since bacterial biofilms are a major contributor to the delay in healing in a colonized wound, it is necessary to develop a clinical assay that can be used t o accurately detect biofilm as opposed to planktonic bacteria. This will provide the wound care provider with information necessary to administer the appropriate treatment to patients. This is especially important since antibiotic and

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30 antiseptic susceptibl e planktonic bacteria and highly tolerant biofilm protected bacteria have very different characteristics and need to be targeted in different ways

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31 CHAPTER 2 THE UNDERLYING PRINC IPLE OF A DEVELOPING A BIOFILM DETECTING ASSAY: ELIMNATION OF PLANKTONIC BACTERIA Introduction Principle b ehind an Assay to Detect Bacterial Biofilms Evidence suggests that the presence of bacterial biofilm s in chronic wounds prolongs inflammation and contributes to the non healing of some wounds ( Edwards and Harding 2004 ; Knox et al ., 2007 ) Currently, all clinical microbiology culturing techniques do not distinguish between planktonic bacteria and biofilm bacteria Consequently, there is a great need in the clinical wound care field for a standardized clinical micr obiology laboratory assay that can be reliably used to detect the presence of functional biofilms. Such an assay can be used by wound care providers to administer the necessary treatment, such as aggressive surgical debridement, in the case of biofilm colo nization. It will also allow for the correlation of treatment with biofilm levels and with wound healing Development of a clinical microbiology laboratory assay to generate a more comprehen sive evaluation of the state of chronic wound s w ill greatly facili tate and advance the field of wound care. Approach for Detection of Bacterial Biofilms In selecting an approach to detect bacterial biofilms, we used the functional definition of a microbial biofilm which is the extreme tolerance of sessile biofilm to antimicrobial agents that the same microorganisms are susceptible to when planktonic, or fr ee floating ( Lewis, 2001; Davies, 2003; ) The underlying principle of this assay is to expose a clinical sample to a treatment that will kill all the planktonic bacteria and allow for the detection of only protected biofilm bacteria by routine culturing techniques. This treatment must include exposure to an antimicrobial

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32 agent that planktonic bacteria are susceptibl e to but one that does not harm the biofilm protected bacteria. This assay must be one that can be easily adapted in cli nical microbiology labs and yields valid and accurate results in a reasonable time frame. The use of a known and widely used antimicrobial agent can be adapted in a protocol to detect biofilms One option is the use of a mixture of broad spectrum antibiot ics which kill and inhibit the growth of gram positive and gram negative planktonic bacteria However, a clinical sample may contain a wide variety of bacterial species which may include antibiotic resistant strains. In this case, antibiotic resistant bact eria will survive the treatment and give a fa lse positive result for biofilm presence. Since, a prospective study of 52 chronic wounds showed that 18.8% of the isolated pathogens were antibiotic resistant (Basu et al ., 2009) ; the use of an antibiotic treat ment canno t be used to reliably detect biofilms. Another option is the use of a widely available chemical agent that can eliminate planktonic bacteria and not harm most of the bacteria in biofilms Assay conditions could be optimized to determine a speci fic exposure time to a known concentration. A potential antimicrobial agent for the development of this type of assay is calcium hypochlorite (Ca(ClO) 2 ) an effective, stable, affordable and commercially available age nt. It is also non specific and targets all bacterial species (except for endospores) which eliminates the problem of false positive results ( Rutala and Weber, 1997 ). The protocol for the assay includes exposing a clinical sample to a treatment solution at a specific concentration for a set period of time that is known to eliminate all the planktonic bacteria but not compromise the levels of biofilm protected bacteria Also, the action of the antimicrobial agent must be neutralized at the end of exposure period

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33 to avoid any detrimen tal effects to the biofilm bacteria in the sample due to prolonged exposure. Selecting the Appropriate Antimicrobial Agent Chlorine releasing agents are widely used as disinfectants in households, public places as well as in clinical and research settings ( Bernard, 2007; Rutala and Weber, 1997 ) Active chlorine compounds with antimicrobial activity include sodium hypochlorite, calcium hypochlorite, chlorine dioxide, liquid chlorine and organic and inorganic chloramines ( Rutala and Weber 1997). Hypochlorite solutions are widely used in hospitals as disinfectants for medical equipments and other surfaces to prevent nosocomial infections ( Rutala and Weber 1997) They are also used as irrigations solutions in some of health care professions includ ing endodontics (Estrela et al ., 2002) and in rare cases as antiseptics ( Rutala and Weber 1997). Sodium hypochlorite (aqueous solution) and calcium hypochlorite (salt) contain the active antimicrobial agent of bleach which is hypochlorous acid (HOCl) Hypochlorous acid is also secreted by polymorphonuclear cells in the body as a mechanisms of the immune system to kill bacteria (Bernard, 2007). The antimicrobial activity of this agent depends on the dissociation of hypochlorous acid to the hypochlorite i on (OCl ) which is less active against microorganisms ( Rutala and Weber ,1997) The impaired ability of the hypochlorite ions to diffuse across the membrane of bacteria is believed to be due to the negative charge whereas hypochlorous acid is able to diffu se and oxidize important cellular components (Bernard, 2007). This dissociation is directly proportionate to the pH where a higher pH correlates to a higher dissociation to hypochlorite ions and therefo re less antimicrobial activity ( Rutala and Weber 1997 ). A pH of 6 corresponds to optimal antimicrobial activity ( Rutala and Weber 1997 ).

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34 General Material a nd Methods Lab S trains The bacterial strains used were lab strains of Pseudomonas aeruginosa (PAO1), Staphylococcus aureus (SA35556 and SH1000) and Esc herichia coli ( These organisms are biofilm forming bacteria commonly found in infected chronic wounds. Culturing B acteria Lab strains of Psuedomonas aeruginosa (PAO1), Staphylococcus aureus (SA 35556 and SH1000) and Escherichia coli cultured from froz en stocks by streak plating on to tryptic soy agar (TSA) followed by incubation for 16 18 hours at 3 7 C. A single isolated colony of each strain was used to inoculate 5 mL of tryptic soy broth ( TSB) and was incubated overnight at 37 C while rotating at 150 rpm ( Queue Orbital 4730 Shaker) The following day, 150 L of the overnight stationary phase cultu re was used to inoculate 25 50 mL of TSB and incubated at 37 C with 150 rpm rotation ( Queue Orbital 4730 Shaker) for about 3 hours to obtain an early log phase culture Early log phase is reached when the culture has an optical density of 0. 2 0. 4 at 640 nm (UNICO 1100 Uniten Products & Instrument INC) or 600 nm (Bio Rad SmartSpec 3000) Serial D ilutions and P lating The generally used and accepted method for quantifying biofi lms is viable plate counts where the resuspended and dispersed biofilm cells are plated onto a solid growth medium, incubated at 37 C and counted ( Donlan and C osterton 2002) Therefore, s erial dilutions and plati ng were performed for quantification of colony forming units present in one m illiliter of each sample (CFU/mL ) For each sample, 0.5

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35 mL was serially diluted in 4.5 mL of phosphate buffered saline (PBS) with vortexing for 10 seconds before each dilution for serial 10 fold dilution s Plating was performed on tryptic so y agar (TSA) by spreading 100 L of each dilution using 6 10 sterile beads and followed by shaking of the plates to completely spread the sample volume Three replicas of each dilution were plat ed for accuracy of the final bacterial quantification Plates were incubated overnight at 37 C without CO 2 ( Forma Scientific 3326 ) and colonies were counted the next day. The final number of CFU/mL was calculated by taking the average of the 3 plate readi ngs for each dilution and using it in t he following formula: [(# CFU/mL )/(0.1 m L )] [(1)/(dilution factor)]. The final CFU/mL value refers to 1 mL of sonication solution. Sonication Before culturing, biofilm bacteria must be removed from the surface on wh ich they are growing on by mechanical forces like vortexing or sonication (Donlan and C osterton 2002 ). Biofilm samples were sonicate d before culturing in order to liberate and disperse the bacteria from the biofilm matrix and allow for accurate bacterial quantification in each sample. Samples were sonicated 5 times for 1.5 minute s with 1 minute waiting intervals between each sonication in a Fisher Scientific B2200R 1 Bransonic ultrasonic cleaner (50/60Hz, 117 Volts, 1.0 Amps) unless otherwise stated The sonication solution was 5 mL of PBS + 5ppm Tween 80 unless otherwise stated. Tween 80 which is a detergent and surfactant was added to the sonication so lution in order to optimally dislodge bacteria from the biofilm matrix.

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36 Developing a Representative Biofilm Model Introduction To ensure that the assay developed in the lab is applicable in a clinical setting, it is necessary to use an appropriate and representative in vitro model that mimics biofilm presence in a wound bed. It is also imp ortant to determine the level of tolerance of the in vitro cultured biofilm to biocides and antimicrobial agents to ensu re the maturity of the biofilm and therefore meet the functional biofilm definition of protected, tolerant and resistant bacteria. Previous in vivo and in vitro models to study biofilm growth and behavior include culturing bacteria on plastic, metals, or organic surfaces like skin explants ( Gottrup et al ., 2000 ). Partial thickness wound models have also been developed in live animal s, such as pigs or mice to better represent the wound bed (Serralta et al ., 2001 ; Braiman Wiksman et al ., 2007 ). It has been shown that bacteria behave differently depending on the substratum (Luppens and ten Cate, 2005; Richter et al ., 1999 ) Proteomic ana lysis of Streptococcus mutans indicated that protein expression patterns are dependent on the strain that is used and also on the model used to culture biofilm (Luppens and ten Cate, 2005). For this reason, it is crucial to use a model that accurately repr esents wound bed environmental factors affecting biofilm growth and behavior In vitro models are usually rapid, simple, affordable and involve minimal ethical considerations when compared to in vivo wound models ( Gottrup et al ., 2000) Also, in vitro model s allow for the assessment of single compounds or agents on the tissue without the complexity of other factors (Gottrup et al ., 2000) However, it is sometime difficult to use the data from an in vitro model to predict similar results in a complete o rganism.

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37 Materials and Methods An in vitro biofilm model was recently developed in our lab and was used to test the effects of different antimicrobial dressings on P aeruginosa biofilms ( Yang, 2008; Phillips et al ., 2010) In this model, 8 mm explants of porcine skin were prepared and sterilized by chlorine gas. A borehole ( 3 mm in diameter and approximately 1.0 mm in depth ) in the center of each e xplant was inoculated with 10 L of early log phase PAO1 culture. The explants were transported to a plate wi th soft agar ( 0.5% agar and 3% TSB) containing 50 g/mL of gentamicin to inhibit planktonic growth. The explants were incubated for three days at 37 C and 5% C O 2 ( Forma Scientific 3326 ) Explants were transported to a fresh plate of soft agar (0.5% agar) every 24 hours. For the development of the clinical assay, some modifications were introduced to this model to make it more representative of wound beds and to increase the ease of use for the necessary experiments. Porcine skin was obtained from a commer cial abattoir, Chiefland Custom Meat 14053 NW HWY129, Gainesville, FL, cleaned, shaved and stored in a 20 C refrigerator. Larger rectangular explants were prepared with a wound bed that is approximately 3 cm by 4 cm at the center of each explant. Wound beds were made using an electric Badgett B dermatome (Figure 2 1B) to remove 0.9 mm of the epidermis of the skin and expose the dermis layer (Figure 2 1 A,C ) After preparation of wound beds explants were washed for 10 minutes in 10% bleach + PBS + 5ppm tween 80. They were then sterilized by chlorine gas in a sealed chamber (BEL ART Desiccat or Shields) inside the chemical hood A mixture of 40 mL of household bleach and 80 mL of acetic acid (Acros Organics, Acetic acid, 99.5% pure, No. 124040010) w as used to produce enough chlorine gas to sterilize about 6 8

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38 explant s after 60 minutes of exposure. The reaction was stopped by adding sodium carbonate to the chlorine gas reaction. The explants w ere then transported to the laminar flow hood in a sterile container and washed with 10% bleach + PBS + 5 ppm tween 80 for 10 minutes and rinse d 3 times (5 minutes each) with sterile distilled water Any remaining liquid on t he wound beds was aspirated by vacuum using sterile glass pipettes. After sterilization and drying, the explants were transported to plates containing soft agar (0.5% agar) and 50 g/mL gentamicin. Each woun d bed was inoculated with 150 L of PAO1 early log phase culture by dispensing the volume on the wound bed and spreading it using a sterile pipette tip. The explants were incubated at 37 C and 5% CO 2 Each day, the explants were transferred to a fresh soft agar (0.5% agar) + 50 g/mL gentamicin plate and were incubated f or three da ys unless otherwise stated. After three days, mature biofilm was present in the wound bed and could be visually seen as slime (Figure 2 1D) The protocol for the preparation and culturing of PAO1 biofilm on porcine skin explants is described in Figure 2 2 The figure also includes the protocol for collecting, treating, dispersing and plating of biofilm samples to optimize the bi ofilm clinical assay. To ensure PAO1 biofilm maturity 3 day mature 8 mm biopsies were taken and treated with 200 g/mL of gentami cin for 24 hours then sonicated, serially diluted, and spread on TSA plates The used concentration ( 200 g/mL ) is 100 minimum inhibitory concentration (MIC) and was previously determined to kill all planktonic PAO1 bacteria after a 24 hour incubation at 37 C ( Yang, 2008 ) PAO1 b iofilm biopsies were also

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39 treated with different concentrations of calcium hypochlorite to ensure antimicrobial resistance of the biofilm. Results PAO1 3 day biofilm maturity was validated by QingPing Yang as a part of her und to be tolerant to a number of antimicrobial agents ( Yang, 2008 ) Also, previous data from this model show ed that a 24 hour antibiotic treatment of biopsies taken from the PAO1 biofilm kill s only 1 2 logs out of 6 or 7 logs of total bacteria l load In addition, treatment with 0.2% Ca(ClO ) 2 which completely killed all planktonic bacteria in suspension culture (Figure 2 3) did not significantly reduce the levels of biofilm bacteria cultured on porcine skin Conclusion PAO1 biofilm cultured o n an organic substratum like porcine skin closely resembles biofilm tolerance to antimicrob ial agents observed in chronic wounds ( Phillips et al ., 2010 ) T he 3 day in vitro porcine skin model was chosen as a source of bacterial biofi lm for the purpose of developing and optimizing a clinical assay to detect bacterial biofilm in the laboratory. Calcium Hypochlorite Treatment Materials and Methods Preparation of t reatment s olution A f resh 5% calcium hypochlorite (Ca(ClO) 2 ) solution was prepared daily by using 0.5 g of the s tock calcium hypochlorite salt ( Acros Organics AC300340010; 65% chlorine) and dissolving it in 10 mL of saline ( 0.85% NaCl in dd H 2 O; pH 5.3 6.7 ). Fifteen to twenty dr ops of 12 N hydrochloric acid was added to the solu tion until the pH was in the range of 5.3 6.7 ( the range hypochlorous acid is most effective as an

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40 antimicrobial agent ) The solution was filtered using a syringe driven filter unit (filter: Millex GP, PES 0.22 m; syringe : BD disposable needleless 10 mL syringe ). Aliquots of the 5% solution were diluted in saline A f resh 5% sodium thiosulfate (Na 2 (S 2 O 3 )) stock solution was prepared by dissolving 0.5 g of sodium thiosulfate penthydrate crystals (Sigma Aldrich, 217247) in sa line The solution was filtered using a syringe driven filter unit. Ca(ClO) 2 c oncentration n ecessary to k ill early log phase planktonic b acteria in s uspension c ulture It was important to determine the minimum concentration of Ca(ClO) 2 necessary to kill planktonic bacteria because the concentration and exposure time will ensure that any bacteria detected by the clinical assay is biofilm protected bacteria and not free floating planktonic bacteria. The objective of this experiment was to determine the mini mum concentration of Ca(ClO) 2 necessary to kill all planktonic bacteria of different species that are usually found in chronic wound beds. A range of concentrations was tested to ensure that all planktonic bacteria were killed and also that the levels of b iofilm protected bacteria were not compromised. Lab strains of Pseudomonas aeruginosa (PAO1), Staphylococcus aureus (SA 35556) and Escherichia coli Early log phase bacteria was cultured and 0.5 mL was serially diluted and plated on TSA to obtain a refer ence count as to how many CFU/mL were present in the original culture. A fresh stock solution of 5% Ca(ClO) 2 was prepared and lower concentrations were obtained by diluting appropriate amounts of th e stock solution in sterile saline for final concentrations of 0.05%, 0.1%, 0.15% and 0.2%. Also a control condition of 0%

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41 Ca(ClO) 2 was included The experiment included 3 replicates (n=3) of the control and each of the experimental conditions. Equal vol umes (0.75 mL) of early log phase culture s and the appropriate Ca(ClO) 2 solution were mixed in sterile 2 mL microcentrifuge tubes. The tubes were briefly vortexed and incubated at room temperature for 5 minutes. After the exposure the total solution was t ransferred to a sterile syringe with a filter unit attached ( Millipore Swinnex Filter Holder, 13 mm No. SX0001300; Millipore MF membrane filters, 0.4 5 L HA No. HAWP01300). The filter units were used to collect any remaining bacteria that were not killed by the Ca(ClO) 2 by dispensing the solution through the filter unit using a syringe and washing with 1.5 m L of sterile saline. The filters were the n removed and t ransferred to 5mL of TSB and incubated overnight at 37 C to detect the growth of surviving bacteria Before the tubes were incubated 0.5 m L of the TSB was used for a serial dil u tion in 4.5 mL of PBS and 100 L of each di lution was plated in triplicate on TSA plates. Growth in each 5 m L TSB tube contain ing the filter was ranked as +, ++, +++ and optical density measurements were taken at 640 nm. Also, the colonies on TSA plates were counted to calculate CFU/mL. Inclusion of s tationary p hase c ulture In a clinical assay where all planktonic or non biofilm bacteria must be eliminated to allow tion of a biofilm which is the extreme tolerance to antimicrobial agents that the same microorganisms are susceptible to when planktonic ( Lewis, 2001; Davies, 2003; Mah )

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42 Previous research indicated that fresh and aged (6 days) planktoni c cultures as well as dispersed and resuspended biofilm bacteria are all equally susceptible to antimicrobial agents (Stewart et al ., 2001). Also, gene expression analysis of S. aureus indicated that sessile bacteria in a biofilm environment have different gene expression patterns when compared to planktonic bacteria that are in the expo nential and post exponential phas e ( i.e. stationary phase) (Beenken et al ., 2004). However, it is also known that due to limited nutritional availability cells in the stationary phase have altered gene expression patterns, cell morphology and activate multiple stress response mechanisms ( Hengge Aronis, 1996) and therefore have higher levels of tolerance to antimicrobial agents Therefore, a higher concentration or exposure time might be required to kill all bacteria in the stationary phase. To ensure that stationary phase bacteria fall under the appropriate category of planktonic bacteria, their susceptibility to antibiotics was tested and compared to that of lo g phase culture. 50 L of early log phase and stationary phase PAO1 was added to 3 mL of TSB containing 20 0 g/mL of gentamicin ( 100 minimum inhibitory concentration; MIC ) and incubated at 37 C with 150 rpm rotation for 24 hours. The culture was then filtered and the filter s were transferred to 5 mL of TSB and incubated overnight at 37 C for 24 hours. After incubation growth was recorded and 100 L of the TSB was spread on TSA plates to ensure sterility. T o determine if stationary phase bacte ria are more tolerant to Ca(ClO) 2 100 L of PAO1 and SH1000 stationary phase cultures was added to 2 mL of 0.2 % Ca(ClO) 2 The bacteria were exposed for 5 minutes, 10 minutes and 30 minutes and sodium thiosulfate was added to neutralize the solution. 100 L aliquots were used to inoculate 5 mL of

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43 TSB and also spread on TSA plates. Observable growth in the TSB and presence of colonies on TSA plates were recorded. Complete n eutralization of c alcium h ypochlorite by s odium t hiosulfate After Ca(ClO) 2 trea tment sodium thiosul fate ( Na 2 (S 2 O 3 ) ) was added to the treatment solution to neutralize the hypochlorite ions and to stop any antimicrobial activity. Exposing bacteria to Ca(ClO) 2 for a specified time period require d stopping the reaction quickly after the specified time ha d elapsed. This is especially important if a large number of samples are processed at the same time to ensure consistency and accuracy of the assay Thus, it was necessary to determine the ratio of (Na 2 (S 2 O 3 )) to Ca(ClO) 2 for complete neutralization of the hypochlorite ions. Hypochlorite ions are neutralized by thiosulfate by two different reactions depending on the environment in which the reaction is occurring. If the environment is basic and hydroxide ions (OH ) are presen t, the following reaction occurs: 4 ClO + (S 2 O 3 ) 2 + 2 OH 2 (SO 4 ) 2 + 4 Cl + H 2 O By using our calcium hypochlorite stock powder (65% free chlorine) the reaction becomes: 2 Ca(ClO) 2 + 2 Na + (S 2 O 3 ) 2 + 2 OH 2 Ca(SO 4 ) + 2 Na + + 4 Cl + H 2 O According to the balanced reac tion and the molecular weights of each of the reagents, 1 g of Ca(ClO) 2 is neutralized by 0.87 g of Na 2 (S 2 O 3 ) However, accounting for the fact that only 65% of chlorine ions are available in the stock powder, 0.56 g of Na 2 ( S 2 O 3 ) will be sufficient to neutralize 1 g of Ca(ClO) 2

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44 However, if the environment in which the reaction is occurring is neutral, the reaction becomes: ClO + 2 (S 2 O 3 ) 2 + H 2 O (S 4 O 6 ) 2 + C l + 2 OH By using our Ca(ClO) 2 stock powder (65% free chlorine) the reaction becomes: Ca(ClO) 2 + 2 Na + + 2 ( S 2 O 3 ) 2 + H 2 O Ca( S 4 O 6 ) + 2 N a + + Cl + 2 O H According to the balanced reaction and the molecular weights for each of the reagents, 1 g of Ca(ClO) 2 is neutralized by 3.47 g of Na(S 2 O 3 ) However, accounting for the fact that only 65% of chlorine ions are available in the stock powder, 2.26 g of Na 2 (S 2 O 3 ) will be sufficient to neutralize 1 g of Ca(ClO) 2 The second reaction is the one expected to occur since all Ca(ClO) 2 and Na 2 (S 2 O 3 ) solutions were not prepared using a basic solution. In order to confirm that the ratios calculated from this reaction are applicable in vitro the neutralization ratios were experimentally tested. Fresh solutions of 5% Ca(ClO) 2 and Na 2 (S 2 O 3 ) were prepared by adding 0.5 g to 10 mL of saline and filter sterilized (no HCl was added) Sterile 15 mL tubes were used and 1 mL of 5% Ca(ClO) 2 was added to each tube. In order to have a final volume of 4 mL, appropriate volumes of saline were added to each tube. Then the appropriate volume of 5% Na 2 (S 2 O 3 ) w as added according to the two calcu lated ratios (1:0.6 and 1:2.3) for a final volume of 4 mL. The tubes were briefly vortexed and incubated at room temperature for 5 minutes to allow the thiosulfate to neutralize the hypochlorite ions. Then 1 mL of early log phase SA35556 culture was added to each tube for a final volume of 5 mL and the tubes were briefly vortexed and incubated for an additional 5 minutes. A control was also includ ed in the reaction in which 4 mL of saline and 1 mL of

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45 early log phase c ulture were mixed. The reaction tubes we re serially diluted in PBS and spread on TSA plates Results Ca(ClO) 2 concentration necessary to kill early log phase bacteria in suspension culture Results from colony counts on TSA plat e s show that 0.15% was enough to kill log phase PAO1 SA35556 after 5 minutes (Table 2 1, Figure 2 3 ). However, observations of growth in the TSB tubes indicate that there was growth in 2 of the 3 tubes corresponding to 0.15% treatment of SA35556. O ptical density readings indicated consistent resul ts (Table 2 2). For further experiments, 0.2% Ca(ClO) 2 can be used as the minimum concentration necessary to completely kill all planktonic bacteria of the three tested bacterial species in suspension culture. Inclusion of stationary phase culture For cult ures treated with gentamicin, t here was no growth in the TSB culture tubes containing the filter s or on the TSA plates for both early log phase and stationary phase culture s For the cultures treated with 0.2% Ca(ClO) 2 there was no growth in the culture tubes corresponding to 10 minute and 30 minute treatment for both PAO1 and SH1000. Previous data indicated that mature PAO1 biofilm cultured on porcine skin survived a 10 and 30 minute exposure to 0.2% Ca(ClO) 2 A lthough a slightly longer exposure time was necessary to eliminate all stationary phase bacteria when compared to early log phase bacteria, they were still susceptible to low concentrations of Ca(ClO) 2 These results show that both early log phase and stationary PAO1 fall under the definition of p lanktonic since they are both susceptible to antibiotics and antimicrobial

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46 agents Therefore, bacteria in both phases of growth should be eliminated in an assay designed to detect only biofilm protected bacteria. Complete neutralization of calcium hypochlorite by sodium thiosulfate The plates corresponding to the 1:0.6 ratio of Ca(ClO) 2 to Na 2 (S 2 O 3 ) did not have any viable colonies while the plates corresponding to the 1:2.3 ratio had an average of 1.9 9 10 7 CFU/mL Colony counts and calcula ted CFU/mL numbers showed that the number of CFU /mL from the control tube and the tubes c orresponding to 1:2.3 ratio are within the same log of CFU/mL counts This indicates that all the hypochlorite ions were neutralized by adding 0.23 mL of thiosu lfate w hich is why there was no reduction in the number of recovered bacteria when compared to the control. However, the 1:0.6 ratio only neutralized part of the hypochlorite ions which allowed the remaining hypochlorous acid to kill the planktonic bacteria (Table 2 3 ) For all later experiments, the added Na 2 (S 2 O 3 ) volume was 2.3 times the volume of added Ca(ClO) 2 ( 0.2% Ca(ClO) 2 final concentration) to ensure complete hypochlorite ion neutralization. Conclusion Treatment of early log phase culture s with 0.2% Ca(ClO) 2 for 5 minutes completely kill ed all S. aureus P. aeuriginosa and E.coli in early log phase growth The exposure time was increased to 10 minutes to include elimination of stationary phase culture. The Ca(ClO) 2 was effectively neutraliz ed by adding Na 2 (S 2 O 3 ) according the ratio of 1g Ca(ClO) 2 : 2.3g Na 2 (S 2 O 3 ).

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47 Table 2 1. Treatment of s uspension of e arly l og p hase c ulture s with c alcium h ypochlorite Bacteria % Ca(ClO) 2 Cell c ount (CFU/mL) Average Standard deviation X 1 X 2 X 3 P. aeruginosa (PAO1) Early log p hase 1.77E+09 1.77E+09 0% 2.87E+08 1.30E+08 1.02 E+08 1.73 E+08 9.97E07 0.05% 1.33E+08 2.38E+07 4.20E+07 6.62 E+07 5.85E+07 0.1% 1.11E+07 8.80E+06 4.70E+06 8.20E+06 2.65E+06 0.15% 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.2% 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 S. aureus (SA3556) Early log p hase 5.10E+07 5.10E+07 0% 2.67E+07 3.6E+07 1.59E+07 2.62E+07 1.01E+07 0.05% 9.00E+06 4.30E+06 8.00E+06 7.10E+06 2.48E+06 0.1% 2.23E+05 7.5E+05 1.90E+06 9.60E+05 3.14E+05 0.15% 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.2% 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 E. coli Early log p hase 2.40E+08 2.40E+08 0% 9.30E+07 5.20E+07 2.74E+07 5.70E+07 3.31E+07 0.05% 5.20E+07 1.01E+08 9.10E+07 8.10E+07 2.59E+07 0.1% 0.00E+00 2.00E+01 1.60E+02 6.00E+01 8.70E+01

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48 Table 2 1. Continued Bacteria % Ca(ClO) 2 Cell c ount (CFU/mL) Average Standard deviation E. coli (DH5 ) X 1 X 2 X 3 0.15% 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.2% 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00

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49 Table 2 2. Observation of g rowth and o ptical d ensity m easurements for e arly l og p hase c ulture t reated with c alcium h ypochlorite P.aeuriginosa (PAO1) 0% 0.05% 0.1% 0.15% 0.2% 1 ++ ++ ++ 2 ++ ++ ++ 3 ++ ++ ++ 1 0.8 0.831 0.778 0.09 0.14 2 0.758 0.759 0.792 0.1 0.1 3 0.743 0.762 0.788 0.24 0.22 S.aueus (SA35556) 0% 0.05% 0.1% 0.15% 0.2% 1 ++ ++ + + 2 ++ ++ + ++ 3 ++ ++ + 1 1.027 1.223 0.933 1.027 0.011 2 1.163 1.083 0.936 1.113 0.018 3 1.289 1.238 1.238 0.024 0.001 E.coli 0% 0.05% 0.1% 0.15% 0.2% 1 ++ ++ ++ 2 ++ + ++ 3 ++ ++ ++ 1 1.033 0.977 0.940 0.034 0.030 2 0.975 1.005 0.986 0.006 0.012 3 0.942 0.989 0.983 0.008 0.011

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50 Table 2 3. Complete n eutralization of c alcium h ypochlorite by s odium t hiosulfate Treatment Cell c ount (CFU/mL) Average Standard deviation X 1 X 2 X 3 Control 2.89E+07 2.89 E+07 1:0.6 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 1:2.3 2.36 E+07 6.3 0E+07 3.9 0E+07 4.2 0E+07 1.9 9E+07

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51 Figure 2 1. In vitro por cine skin PAO1 biofilm model. A) Removal of 0.9 mm of epidermis layer. B) Padgett B d ermatome. C) Explant after wound bed prepa ration. D) Mature 3 day PAO1 biofilm cultur ed on the explant.

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52 Figure 2 2. Protocol for culturing and processing biofilm samples using the in vitro porcine skin PAO1 biofilm model.

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53 Figure 2 3 Treatment of e arly l og p hase c ulture in s uspension with calcium hypochlorite

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54 CHAPTER 3 OPTIMIZATION OF THE ASSAY IN THE LABORAT ORY What Makes a Good Clinical Assay? Although it is important to specify the factors that contribute to a valid and reliabl e clinical assay for detecting functional biofilm present in a clinical sample, it is also important to consider what factors make an assay practical, adaptable, and easy to use by wound care providers in a variety of clinical settings. Not all clinical wound care practices ar e similar and may differ in terms of their setting staff and distal proximity from a microbiology lab. Key f actors that must be considered i nclude the collection device validity of the collection method, transportation to a microbiology lab, reasonable p rocessing time and the validity of results. Incorporating a microbiology assay into the clinical field requires optimization of collection, transport and processing of the clinical sample. Also the clinical assay must be one that may be easily adopted by wound care providers in different clinical settings. A n optimal clinical assay should require minimal steps at the bedside, cause minimal pain o r discomfort to the patient include easy transportation of the sample from the clinic to the lab without compromising validity of the results and yield resu lts in a reasonable time frame. In addition, for such an assay to give accurate qualitative and quantitative and semi quantitative results, the treatment must targ et al l bacterial species and ensure that any bacteria detected after treatment are biofilm protected bacteria and not ones that have escaped the treatment due to any physical, chemical or biological reason

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55 Optimizing t he Collection Method Introduction There are many different collect ion and sampling devices used in the clinical care field, depending on the location of the sampled area and na ture of the sample of interest. A commonly used method of collecting clinical samples is swabbing. Although the most commonly used swabs are the cotton fiber swabs, there are many other swabs with different shapes and composed of different materials Traditional fiber s wab tips may be made out of rayon, polyester, alginate and dacron. There are also cloth, sponge, flocked swabs and cytology brushes that have been developed to collect different types of clinical samples. The advantage of optimizing this assay using a swab is that it is non invasive and a ny nurse can collect a clinical sample using a swab This makes swabbing a col lection method that can be easily and readily performed in any clinical setting. On the other hand, the use of a sharp instrument that might cause bleeding, such as a curett e or punch biopsy that would generate more standardizable tissue specimens and allo w deeper sampling can only be performed by a physician, physician assistant, wound, ostomy and continence nurse (WOCN), advanced registered nurse practitioner ( ARNP ) or a registered nurse (RN) who received special training Such specialized personnel m ight not always be available in all wound care facilities especially nursing homes and extended care facilities. Materials and Methods Ca(ClO) 2 concentration necessary to kill planktonic bacteria collected by cotton swabs 5 minute exposure to 0.2% Ca(ClO ) 2 To determine if the same concentration sufficient to kill all planktonic bacteria in suspension will also kill planktonic bacteria

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56 collected by a cotton swab (Fisherbrand small cotton tipped applicators) early log phase was cultured and collected by d ipping a swab into the culture for 15 seconds. The concentrations used to kill S aureus, P. aeuriginosa and E.coli planktonic bacteria collected by swabs are 0.15% and 0.2% Ca(ClO) 2 Also a control condition was added in which the swabs were treated with saline. This experiment was necessary to ensure that 0.2% Ca(ClO) 2 is enough to kill all planktonic bacteria collected by cotton swab s as described above Swabs were gas sterilized using ethylene oxide by the Shands hospital sterilization unit. Early log phase bacteria (OD 640 between 0.2 0.4) were collected by dipping the swab in 10 mL of culture for 15 seconds. The s wabs were then submerged in 3 mL of the appropriate concentration of Ca(ClO) 2 or saline solution and incubated at room temperature for 5 minutes. The swabs were then transferred into 3 mL of Na 2 (S 2 O 3 ) solution and incubated at room temperature for 5 minutes. Each swab was transferred into 5 mL of sterile PBS + 5ppm t ween 80 and vor texte d briefly. Then 0.5 mL of the bacteria suspended in PBS + t ween 80 was serially diluted in PBS and spread on TSA plates The plates were incubated overnight at 37 C and plate counts were used to determine CFU/ mL of the bacterial suspension from each experimental condition. The experiment was also repeated with agitation of the swabs during the 5 minute Ca(ClO) 2 exposure. The volume of treatment solution was also increased to allow for better fluid movement. Keeping all concentrations of Ca(ClO) 2 and Na 2 (S 2 O 3 ) constant, sterile 50 mL conical tubes with 10 mL of the treatment solution were used instead of using 15 mL tubes with 3 m L of treatment solution. T he conical tubes were incubated on a plate shaker (200 300 rpm) during both the Ca(ClO) 2 and Na 2 (S 2 O 3 ) 5 minute

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57 exposures. The swabs were transferred to 5 mL of PBS + tween 80, vortexed, and the bacterial suspension was serially diluted in PBS and spread on TSA plates. Colony counts were reported as CFU/mL of the bacterial suspension from each experime ntal condition A final experiment was performed verify previous results that de term ine d the minimum concentration of Ca(ClO) 2 necessary to kill all planktonic bacteria collected on a cotton swab after 5 minutes of exposure was not incorrect due to inheren t limitations of bacterial detection using dilution plating methodology Planktonic PAO1 was collected by dipping a cotton swab into 1 mL of early log phase suspension culture Swabs were transported to 2 mL of the appropriate concentration of Ca(ClO) 2 (0% 3%, 4%, 5%), vortexed, and incubated for 5 minutes. A n appropriate amount of Na 2 (S 2 O 3 ) was added to each tube after which the tubes were vortexed and incubated for 5 minutes to completely neutralize the reaction. The swabs were then transported to 5 mL of TSB and incubated overnight at 37 C and growth was recorded as positive or negative L ong exposure to low concentrations of Ca(ClO ) 2 Another option is to expose clinical samples collected on swabs to a low concentration of Ca(ClO) 2 for a longer period of time. It was hypothesized that a low er concentration with a longer exposure time will not harm biofilm protected bacteria and will allow the hypochlorous acid to penetrate through the swab fibers and kill all the planktonic bacteria that may have been deeply embedded inside the swab Early log phase and stationary phase P.auriginosa (PAO1) and S.aureus (SH1000) bacteria were collected by cotton swabs and treated with 1.5 mL of different concentrations (0%, 0.1%, 0.2%) of Ca(ClO) 2 for either 4 hours or 8 hours. After the

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58 specified exposure time, the swabs were transferred to 5 mL of T SB and 100 L f rom the original treatment solution s as well as the TSB bacterial suspension w each spread on TSA plates The TSB bacterial suspension tubes were incubated at 37 C overnight and positive or negative growth as well as any colonies formed on TSA plates w ere recorded (Figure 3 1). Mixed culture exposure to low concentrations of Ca(ClO ) 2 Different bacterial species were cultured together to determine the concentration of Ca(ClO) 2 and exposure time necessary to kill all planktonic bacteria in a mixed culture which is more representative of the multi species nature of bacterial colonization in wound beds. Equal volumes (50 L into 50 mL of TSB + 50% calf plasma (Lampire Biolog ical Laboratories Calf Plasma in Na Heparin Catalog No. 8311111). The plasma facilitates the growth of the three species together and mimics the actual wound fluid obtained from clinical samples ( Banda et al ., 1982 ) Mixed culture planktonic bacteria were collected by dipping a cotton swab in 1 mL of culture for 15 seconds and then the swab was transported to 1.5 mL of 0.1% Ca(ClO) 2 and incubated for different time points (1, 2, 4, 7 hrs). Also, a control of 0% Ca(ClO) 2 was paired with each time point. The same procedure previously described (Figure 3 1) was followed to process the samples after the necessary time point elapsed. Comparing a ntibiotic treatment to 8 hr exposure at low concentrations of Ca(ClO) 2 Sterile cotton swabs were used to collect planktonic PAO 1 bacteria and incubated in 2 mL of appropriate treatment solution. These treatment solutions included saline (control), 0.1% Ca(ClO) 2 0.2% Ca(ClO) 2 and T SB containing 200 g/mL of gentamicin.

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59 Swabs treated with saline and Ca(ClO) 2 were incu bated at room temperature for 8 h ours while swabs treated with gentamicin were incubated at 37 C for 24 hours. After the specified incubation period, the swab tips were transferred to TSB and appropriate dilutions of the bacterial suspension were spread o n TSA plates to calculate CFU/mL. Positive or negative growth detected in the bacterial suspension was recorded. Alternative c ollection m ethod Although swabs are the most widely used clinical collection method, there are other collection devices that are a lso used and in some cases yield a more reliable result as to what pathogens are present in a sample if any. Such devices like curettes, biopsy punches and other sharp collection de vices pick up samples from deeper tissue s instead of only sampling the surface. This is especially important in the case of chronic wound s where some bacteria, especially those that are obligate anaerobes, might invade and form biofilms in deeper tissue s It is important to optimize the condition s for this assay according to different collection methods like curettage samples and punch biopsies to make this assay complete and comprehensive. Concentrations of Ca(ClO) 2 necessary to eliminate pl anktonic bacteria collected by the cytology b rush and th e f locked s wab A cotton swab, a flocked swab and a cytology brush (Figure 3 4A,B,C) were used to collect early log phase PAO1 and were then inc ubated in 1.5 mL of 0.2% Ca(ClO) 2 for 8 hours. The collection devices were transferred to 5 mL of TSB and 100 L of the treatment solution wa s spread on TSA plates and both incubated overnight at 37 C. Additionally the cytology brush was used to collect stationary phase PAO1 and incubated in 2 mL of 0.2% Ca(ClO) 2 for 10 minutes, 30 minutes and 1 hour. After the

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60 exposure time, Na 2 (S 2 O 3 ) was added and the brush was transferred to TSB and incubated overnight at 37 C. The flocked swab was also used to collection stationary phase PAO1 and SH1000 incubated in 2 mL of Ca(ClO) 2 for 1 hour, 2 hours and 4 hours. After ea ch time point, Na 2 (S 2 O 3 ) was added and the swab was trans ferred to 5 mL of TSB and incubated overnight at 37 C. Treatment of PAO1 biofilm collected on flocked swabs Mature 3 day PAO1 biofilm was cultured on porcine skin and equal areas of 3 explants wer e sampled using flocked swabs. The swabs were trans ferred to 2 mL of either saline for 1 hour, 0.2% Ca(ClO) 2 for 1 hour or TSB containing 200 g /mL gentamicin for 24 hours at 37 C. After the necessary exposure time Na 2 (S 2 O 3 ) was added and 3 mL of PBS + 5 ppm tween 80 was added to each 15 mL tube for the saline and Ca(ClO) 2 treated swabs The swab s treated with gentamicin were transferred to 5 mL of PBS + 5ppm tween 80 and the remaining antibiotic solution was filte red and the filters were added t o the PB S + tween 80 with the swabs. The samples were sonicated, serially diluted and spread on TSA plates. Comparing l evels of b iofilm pick up by d ifferent c ollection d evices Different collection devices were tested to assess their ability to pick up biofilm prot ected bacteria. A curette loop (Miltex 4 mm dermal curette Figure 3 4D ), a cytology brush (Medical Packaging Cyto Soft Figure 3 4C ) and a cotton swab (Fisherbrand small cotton tipped applicator Figure 3 4A ) were used to collect mature 3 day PAO1 biofilm from large explants. Each collection device was streaked across the length of the wound bed twice to sample a consistent area of the explant. Three explants were used

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61 and each explant was sampled once with each with the three collection devices. Each collection device was used in triplicates (one for each explant). Using Ca(ClO) 2 to detect levels of PAO1 biofilm pickup by different devices. The levels of biofilm pick up and retrieval by the different collection devices were assessed by treating each de vice with 3 mL of 0.2% Ca(ClO) 2 in 15 mL conical tubes Curette and Brush samples were treated for 10 minutes while the cotton swab samples were treated for 8 hours. The different treatment times are consistent with previous findings that cotton swabs requ ire an 8 hour exposure time while curette and brush samples require a 10 minute exposure in order to kill all planktonic bacteria. Curetted material was shaken off into the 0.2% Ca(ClO) 2 solution and the device was not incubated However, the swab and bru sh were incubated along with the collected material. After the necessary incubation time, each device was neutralized with Na 2 (S 2 O 3 ) and incubated for 10 minutes. Then 2 mL of PBS + 5 ppm tween 80 was added for a final volume of 5 mL. The samples were sonicated serially diluted and plated on TSA plates in triplicate. The plates were incubated at 37 C overnight and colony counts were used to determine CFU/m L of sonication solution. Using gentamicin to detect levels of PAO1 biofilm pickup by different devices. The levels of biofilm pick up and retrieval by the different collection devices were assessed by treating each device with 3 mL of TSB containing 200g/mL gentamicin for 24 hours at 37 C. Curetted material was shaken off into the antibiotic solut ion and the device was not incubated However, the swab and brush were incubated along with the collected material. After the necessary incubation time, each device was trans ferred to 5 mL of sonication solution. The remaining antibiotic

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62 incubation soluti on was filtered and the filter was transported to the tube in which the device was incubated. This step was included to collect the biofilm bacteria floatin g in the incubation solution. The samples were sonicated serially diluted and plated on TSA plates in triplicate. The plates were incubated at 37 C overnight and colony coun ts were used to determine CFU/mL of sonication solution. Scanning electron m icroscopy images of different swab materials To compare the ability of differen t collection devices to collect and retain bacterial biofilms, scanning electron microscopy (SEM) images were obtained of swabs composed of different materials. Mature PAO1 biofilm was cultured on porcine skin and the biofilm was collected on different swa bs by streaking across an equal area of the explants for each swab. After the samples were collected, they were treated with Trumps solution (4% formaldehyde, 1% glutaraldehyde 95% water) and were incubated at 4 C. Then the samples were washed with PBS for 10 minutes ( 1X ), ddH 2 O for 10 minutes ( 3X), then with graded ethanol washes (25%, 50%, 75%, 95%, 100%) for 10 minutes each. The samples were then dehydrated with HMDS ( hexamethyldisilazane 99%, Gelest Inc, No. SIH6610.1) by washing twice for 5 minutes each. The samples were then dehyd rated under a vacuum for up to week. Different swab samples were prepared for SEM imaging with different collection and treatment conditions. Cotton, flocked, sponge and rayon swabs were use d to collect 3 day mature PAO1 and were either fixed immediately or treated with 0.2% Ca(ClO) 2 for 6 hrs and then fixed ; the brush was only treated with 0.2% Ca(ClO) 2 for 10 minutes.

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63 Results Ca(ClO) 2 concentration necessary to kill planktonic bacteria collected by cotton swabs 5 minute exposure to 0.2% Ca(ClO ) 2 The r esults show ed that neither 0.15% nor 0.2% Ca(ClO) 2 was sufficient to completely kill planktonic bacteria collected by cotton swabs (Table 3 1) This indicated that the Ca(ClO) 2 concentrations sufficient to kill all planktonic bacteria in suspension culture were not enough to kill all planktonic bacteria collected on a cotton swab. This may be due to a diffusion barrier preventing hyp ochlorite ions from penetrating into the cotton fibers of the saturated cotton swab and hindering the antimicrobial activity of Ca(ClO) 2 I ntroduced changes to the protocol including shaking and increasing the treatment solution v olume did not eliminate th e problem. Long exposure to low concentrations of Ca(ClO ) 2 The minimum concentration to kill all early log phase bacteria collected by a cotton swab is 0.1% after an 8 hour exposure for P.aeruginosa S. aureus and E.coli (Figure 3 2) However, in the case of stationary phase culture, exposure to 0.1% Ca(ClO) 2 for 8 hours was not sufficient to kill all bacteria T herefore the concentration of Ca(ClO) 2 was increased to 0.2%. Therefore, the proposed exposure time for clinical samples collected on cotton swabs is 0.2% for 8 hours Mixed culture exposure to low concentrations of Ca(ClO ) 2 The results show ed that the minimum time exposure to kill all the planktonic bacteria in a mixed culture ( P.aeruginosa S. aureus and E.coli) was 4 hrs (Figure 3 2) For 1 and 2 hour exposure conditions, the plates corresponding to the treatment solution showed minimal or no growth However, the plates corresponding to the TSB culture around the swab

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64 before overnight incubat ion show ed significant growth. These results conf irm that the antimicrobial treatment of bacteria collected on cotton swabs needs more time for the hypochlorous acid to penetrate and diffuse into the swab fibers to access the bacteria. Comparing antibiotic treatment to 8 hr exposure of low concentrations of Ca(ClO) 2 PAO1 bacteria were collected on cotton swabs and treated with 0.2% Ca(ClO) 2 for 8 hours which kill ing all planktonic and stationary phase bacteria. However, exposure to 0.1% Ca(ClO) 2 for 8 hours kill ed all early log phase but on average, 2 logs of stationary phase bacteria survive d the treatment. Although the gentamicin treatment kill ed all log phase and stationary phase bacteria in suspension culture the results show ed that on average, 2 log of early log phase and 3 logs of stationary p hase culture survive d the antibiotic treatment ( Table 3 2 Figure 3 3) These results confirm that it is difficult for antimicrobial agents to diffuse through the swab fibers The concentrations and exposure time to antimicrobial agents should be increased in order to completely kill planktonic bacteria collected on a cotton swab or a similarly tightly woven collection device. Concentrations of Ca(ClO) 2 necessary to eliminate pl anktonic bacteria collected by cytology brush and flocked s wab E xposure to 0.2% Ca(ClO) 2 for 8 hours was enough to kill all planktonic PAO1 bacteria collected on cotton swabs, flocked swabs, and cytology brush. Since the cytology brush was designed differently than the cotton and rayon swabs (not as densely packed) a 10 minute exposure to 0.2% Ca(ClO) 2 was sufficient to kill all planktonic bacteria. For the flocked swab, a 1 hour exposure to 0.2 Ca(ClO) 2 was sufficient to kill all stationary PAO1 and a 2 hour exposu re is sufficient to kill all stationary pha s e SH1000 bacteria.

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65 Treatment of PAO1 biofilm collected on flocked swabs T reatment of PAO1 biofilm on flocked swabs with gentamicin and 0.2% Ca(ClO) 2 for 1 hour result ed in similar levels of PAO1 recovery ( Table 3 3 Figure 3 5 ). Statistical analysis was p erformed using GraphPad Prism 5 and a o ne way ANOVA, followed by a t ukey's m ultiple c omparison t est The results showed no significant difference between the 24 hour gentamicin treatment and the 0.2% Ca(ClO ) 2 treatment for 1 hr. Comparing levels of biofilm pick up by different collection devices Using Ca(ClO ) 2 to detect levels of PAO1 biofilm pickup by different devices. Differences in the levels of recovered biofilm after the collection and treatment with the curette and brush do not vary by more than one log of bacteria for the third explants. The levels of biofilm collected by the brush are highly consistent among the three explants. However, biofilm recovered by the cotton swab was more than a log lower when compared with the brush and curette for the first exp lants. In the second and third explants, there were no colonies detected on the TSA plates even at 10 2 dilution factor. This confirms the inconsistency of cot ton swab collection of biofilm. These results indicate that the cytology brush and the loop curette are both reliable methods for accurately detecting the amount of biofilm in wounds ( Table 3 4 Figure 3 6 A ). Using gentamicin to detect levels of PAO1 biofilm pickup by different devices. The r esults show that the amount of biofilm collected by the cotton swab, cytology brush and 4 mm loop curette is not significantly different (Table 3 4 Figure 3 6 B) Differences in the levels of recovered biofilm after the collection and treatment with the three devices do not vary by more than one log of bacteri a. Statitistical analysis was performed and a o ne way ANOVA, followed by a t ukey's m ultiple c omparison t est showed no significant difference between collection using the three methods (p value

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66 0.5401). This indicates that any of the three collection methods are reliable methods for accurately detecting the amount of biofilm in wounds. Scanning electron m icroscopy images of different swab materials Biofilm and early log phase bacteria collected on cotton swabs images clearly indicated the differences between the two modes of growth (Figure 3 7). Images of different collection devices elucidated the differences in the composition and design of the different swabs. It also emphasized how these differences affect the collection of bacteria but also most important ly the treatment of biofilm coll ected on these swab tips. It was experimentally found that cotton and rayon swabs require a significantly longer exposure time to Ca(ClO) 2 in order to kill all planktonic bacteria as compared to other collection devices. The scanning electron microscopy (SEM) images showed that cotton and sponge tipped swabs are tightly woven allowing bacteria to penetrate deeply inside when a dry swab is dipped in a bacterial culture or used to collect a clinical sample However, when the sa turated sw ab is transported to the Ca(ClO) 2 solution, it will take a long time in order for the hypoch lorous acid to diffuse and act on deeply e mbedded bacteria (Figure 3 8 A,D) The cytology brush and the flocked swab are also very effective at picking up large pieces of the intact biofilm matrix by trapping in between the bristles. It is also designed in a way that allows the hypochlorous acid to easily and rapidly diffuse into the large space s between the bristles theref ore requiring a short amount of time to reach and kill all planktonic bacteria (Figure 3 8 B,C). Images of porcine skin biopsies after collection with different devices indicates how the biofilm matrix attached to the irregular skin surface (Figure 3 9).

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67 Conclusion The best collection method for this assay is either a loop curette or a cytology brush or any other similar device that allows for the diffusion of Ca(ClO) 2 and exposure of the collected sample. The c otton swab is unreliable and continuously gives inconsistent results in terms of complete kill of planktonic bacteria or the amounts of recovered biofilm after treatment. An alternative to using the cotton swab is using the flocked swab or the cytology brush as they require a reasonable treatment time without increas ing the concentration of Ca(ClO) 2 This provides a practical alternative for wound care settings where most of the sample collectors trained to take swab samples only and not curette samples or punch biopsies. Optimizing t he Transport Method Introduction Transport of clinical samples from the clinic to the microbiology lab is not always quick and easy. In some cases, samples must be shipped or transported to a processing lab which might take up to 24 hours or more. Amies transport medi a is usually used in such cases to preserve and stabilize the sample and inhibit growth or death of any bacteria in the sample. Also liquid and gel transport systems have been developed with different swabs in order to make sample collection easy and conve nient for clinicians. There are multiple commercially available transport systems composed of cotton or rayon swabs and a stabilizing transport media tube that are convenient for clinical staff to use and immediately send the sample to the clinical microbiology laboratory. Amies transport medium was designed for the transport of aerobic, anaerobic and fastidious bacteria. This medium allows for the stable shipping and transport of clinical

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68 samples by preventing both growth and death of any bacteria i n the sample which allows for accurate culturing of bacterial numbers from a clinical sample. However, the transport medium contains many salts and stabilizing agents that react with, neutralize and deactivate the Ca(ClO) 2 This becomes problematic when a swab, curetted sample or a punch biopsy is transported in A mies media to the microbiology laboratory where calcium hypochlorite is added to the solution for a specified final concentration. In this case, a higher concentration of Ca(ClO) 2 must be used in o rder to provide enough free hypochlorous acid to kill the planktonic bacteria in the sample after any neutralization reaction occurs. Materials and Methods Concentrations of Ca(ClO) 2 necessary to kill planktonic bacteria in A mies transport media It is nece ssary to adapt the assay in order to allow for samples to be transported in Amies media in situations where samples cannot be delivered to the laboratory for processing within a couple of hours. The concentrations of Ca(ClO) 2 necessary to kill early log ph ase and stationary phase PAO1 and SH1000 were tested in A mies transport media ( C opan diagnostics, part of the ESwab Patented Sample Collection & Delivery System ) as well as in saline. A volume of 100 L of culture was added to 1 mL of A mies transport media and then the necessary volume of 5% Ca(ClO) 2 was added for a final concentration of 0.1%, 0.2%, 0. 5%, 0.8%, 1%, 1.5%. Also, 100 L of culture was added to another set of 2 mL centrifuge tubes containing 1 mL of Ca(ClO) 2 at the same con centrations in saline. Bacteria were exposed for 10 minutes after which Na 2 (S 2 O 3 ) was added to neutralize the reaction. A volume of 100 L was spread on TSA plates to

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69 check for growth of any remainin g bacteria. Also, another 100 L was added to 5 mL of TS B to check growth. P aeruginosa PAO1 biofilm treatment in different transportation solutions Mature, 3 day PAO1 biofilm was cultured on 8 mm bio psies with a 1mm borehole (10 L innoculum) in the center of each biopsy. Different conditions were tested to understand how the transport and treatment medium affects the basic principle of this assay which is to kill all the planktonic bacteria but not harm any of the biofilm protected bacteria. Biopsies were emer s ed in 2mL of treatment solution for a specified period of time after whic h they were processed and CFU/mL was calculated. To compare the effects of different concentrations of Ca(ClO) 2 on the levels of recovered biofilm, biop sies were emer s ed in 2mL of 0.1% or 0.2% Ca(ClO) 2 for 10 minutes after which sodium thiosulfate was added to neutralize the reaction. A set of biopsies was also treated with 0.1% Ca(ClO) 2 for 24 hours to determine levels of biofilm decrease after a prolong ed period of time. Also, to compare the levels of recovered biofilm from samples in A mies transport medi um biopsies were transferred to 2 mL of A mies transport media to which Ca(ClO) 2 added for a final concentration of 1% and 1.5%. After 10 minutes, Na 2 (S 2 O 3 ) was added to neutralize the reaction. A set of biopsies was also imme r sed in 2 mL of TSB containing 200g/mL gentamicin and incubated at 37 C for 24 hours to serve as the standard for the levels of biofilm in a single biopsy. After the necessary treatment time samples were sonicated, serially diluted and spread on TSA plates.

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70 Results Concentrations of Ca(ClO) 2 necessary to kill planktonic bacteria in A mies transport media Although exposure to 0.2% Ca(ClO) 2 in saline for 10 mi nutes is enough to kill planktonic (early log phase and stationary phase) PAO1 and SH1000, it was not enough to reduce the amount of planktonic bacteria in A mies transport media The minimum concentration found to kill early log phase PAO1 was 0.5%, while the minimum concentration to kill stationary phase PAO1 was 0.8%. As for SH1000, the minimum concentration to kill planktonic bacteria was 1% whereas the minimum concentration to kill stationary phase bacteria was 1.5% (Table 3 5) P aeruginosa, PAO1 biofilm treatment in different transportation solutions The r esults the levels of recovered biofilm after the standard 24 hour treatment with gentamicin are slightly higher than those recovered by the Ca(ClO) 2 treatment in either saline or Amies transport media (Table 3 6 Figure 3 10 ) PAO1 biofilm in A mies transport medium treated with 1% Ca(ClO) 2 for 10 minutes produced a greater decrease in the levels of recovered biofilm as compared to treatment with 0.1% and 0.2% in saline solution. Statistical analys is using o ne way ANOVA followed by a d unnett's m ultiple c omparison t est comparing all groups to the gentamicin treated condition showed a significant difference (p value < 0.0001) between the 24 hour gentamicin treatment and all the other experimental cond itions. Conclusion Treating samples in A mies transport medi um requires significantly higher concentrations of Ca(ClO) 2 which might compromise the validity of the results and harm some of the biofilm that is present in the sample. Transportation of the sample in saline

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71 is a valid alternative to Amies transport media especially if the samples are transported to the processin g microbiology laboratory on the same day. Optimizing Biofilm Dispersal Introduction In order to accurately detect the number of biofilm colony forming units in a sample, the biofilm protected bacteria must be dispersed and liberated from the extracellular matrix. This can be done by agitation through delivering of ultrasonic energy or dislodging the bacteria by vortexing. However, excessive sonication or vortexing might kill some bacteria and therefore result in inaccurate readings of colony f orming units. It is necessary to determine if one or both dispersion methods are effective for dislodging the biofilm protected bacteria from the matrix without harming the m. Optimizing the conditions for these two dispersion methods will result in a more accurate quantitative reading of the number of colony forming units in each sample. Also, some clinical microbiology labs do not have the necessary equipment to sonicate samples; therefore optimizing dispersion conditions by vortexing will provide a relia ble alternative. This will allow the assay to be adopted by a larger number of labs without the burden of buying new equipment and altering their laboratory protocols. Materials and Methods The objective of this experiment is to compare dispersion levels of biofilm bacteria by vortexing versus sonication for different time periods. The tested sonication conditions are sonicating in a water bath for 1 min, 3 1.5 minutes with 1 minute waiting intervals, 5 1.5 minutes with 1 minute waiting intervals and 5 minutes of continuous sonication. The tested vortexing conditions are vortexing for 1 minute, 3 1 minutes

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72 with 1 minute waiting intervals, 5 1 minutes with 1 minute waiting intervals and 7 1 minutes with 1 minute waiting intervals. Curette samples (4 mm loop curette) and biopsy punches (8 mm) were taken from PAO1 biofilm cultured on porcine skin explants for 4 days. For each condition, 3 curette and 3 biopsies were used. Each sample was treated with 0.2% Ca(ClO) 2 in 1 mL of saline in a 15 mL conical tube for 10 minutes, neutralized with Na 2 (S 2 O 3 ) and vortexed or sonicated according to the appropriate condition. Samples were serially diluted, spread on TSA plates and incubated overnight at 37 C. Colony counts were performed to calculate CFU/mL values. Results After statistical analysis u sing one way ANOVA, followed by a t ukey's m ultiple c omparison t est results showed no significant difference between th e different sonication or vortexing time points for both the biopsy and curette samples (Figure 3 11, Figure 3 12) This indicates that biofilm bacteria are tolerant to prolonged and continuous sonication and vortexing. Conclusion Sonication of biofilm samples for up to 5 minutes continuously o r 5 1.5 minute pulses with 1 minute waiting intervals will liberate bacteria from the matrix without significantly reducing bacterial counts. Also, vortexing for up to 7 minutes (1 minute pulses) will liberate bacteria from the biofilm matrix without sig nificantly reducing bacterial counts. This indicates that both dispersion methods are reliable for the purposes of this assay.

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73 Table 3 1 Treatment of P aeruginosa PAO1 early log phase culture collected on cotton swabs with 0.15% and 0.2% calcium hypo chlorite Ca(ClO) 2 t reatment CFU/mL X 1 X 2 X 3 Average Standard deviation Early l og p hase >3.00E+10 >3.00E+10 0% 1.41E+07 9.60E+07 1.85E+07 4.29E+07 4.61E+07 0.15% 2.77E+06 8.00E+06 5.00E+06 5.26E+06 2 62 E+06 0.2% 2.63E+06 3.50E+06 6.2E+06 4.11E+06 1 86 E+06 Ca(ClO) 2 Treatment with increased volume and s haking CFU/mL X 1 X 2 X 3 Average Standard deviation Early log p hase 2.12E+09 2.12E+09 0% 4.00E+05 7.20E+05 5.3E+05 5.50E+05 1 6 1E+05 0.15% 2.32E+05 5.80E+05 1.13E+06 6.47E+05 4 5 3E+05 0.2% 1.86E+05 2.26E+05 1.76E+06 7.24E+05 8 97 E+05

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74 Table 3 2 Exposure of planktonic P aeruginosa, PAO1 bacteria to gentamicin and calcium hypochlorite after collection on cotton swab s CFU/mL X 1 X 2 X 3 Average Standard deviation 0% Ca(ClO ) 2 Log phase 1.43E+08 1.21E+08 1.94E+08 1.53E+08 3.74E+07 Stationary 1.58E+08 1.58E+08 1.37E+08 1.51E+08 1.21E+07 200 /mL gentamicin Log phase 3.0E+01 5.3E+02 3.5E+02 3.03E+02 2.53E+0 2 Stationary 4.1E+03 1.00E+03 6.7E+02 1.92E+03 1.89E+03 0.1% Ca(ClO ) 2 Log phase 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 Stationary 3.00E+00 0.00E+00 2.0E+02 6.77E+01 1.14E+02 0.2% Ca(ClO ) 2 Log phase 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 Stationary 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 Table 3 3 Treatment of P aeruginosa, PAO1 b iofilm collected on flocked swabs Treatment Cell c ount (CFU/mL) Average Standard deviation Explant 1 Explant 2 Explant 3 Saline for 1 hr 1.35E+09 7.60E+08 9.50E+08 1.02E+09 3.01E+08 200 g/mL gentamicin for 24 hours (37 C) 4.70E+08 3.10E+08 4.70E+08 4.20E+08 9.24E+07 0.2% Ca(ClO) 2 for 1 hr 2.45E+08 5.70E+07 5.6E+08 2.87E+08 2.54E+08

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75 Table 3 4 Comparison of P aeruginosa, PAO1 biofilm p ickup levels using different collection devices Ca(ClO ) 2 t reatment CFU/mL Explant 1 Explant 2 Explant 3 Average Standard deviation Cotton 1.52E+06 0.00E+00 0.00E+00 5.07E+05 8.78E+05 Brush 8.10E+07 1.40E+08 1.37E+08 1.19E+08 3.32E+07 Curette 7.70E+07 6.80E+08 9.00E+06 2.55E+08 3.69E+08 200 g/mL gentamicin t reatment CFU/mL Explant 1 Explant 2 Explant 3 Average Standard deviation Cotton 1.74E+08 1.00E+09 1.89E+07 3.98E+08 5.27E+08 Brush 1.23E+08 1.14E+08 1.44E+08 1.27E+08 1 .50E+07 Curette 3.12E+08 1.22E+09 1.34E+08 5.55E+08 5.82E+08 Table 3 5. Treatment of p lanktonic b acteria with Ca(ClO ) 2 in different transport solutions Saline Amies Transport Media 0.1% 0.2% 0.5% 0.8% 1% 1.5% P aeruginosa (PAO1) Log p hase Stationary S. aureus (SH1000) Log p hase Stationary E.coli (DH5 ) Log p hase Stationary

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76 Table 3 6 Treatment of P aeruginosa, PAO1 b iofilm in different transport solutions CFU/mL Explant 1 Explant 2 Explant 3 Average Standard Dev Control (saline) 7.10E+08 7.60E+08 7.00E+08 7.23E+08 3 21 E+07 24 hrs in gentamicin 7.90E+08 1.32E+09 7.20E+08 9.43E+08 3 28 E+08 0.1% (10 mins) 3.70E+08 5.50E+08 5.00E+08 4.73E+08 9 29 E+07 0.2% (10 mins) 5.30E+08 2.77E+08 5.40E+08 4.49E+08 1 49 E+08 0.1% (24 hrs) 1.91E+08 3.15E+08 2.73E+08 2.60E+08 6 3 1E+07 1% (TM, 10 mins) 1.05E+08 1.75E+08 1.40E+08 1.40E+08 3 50 E+07 1.5% (TM, 10 minutes) 4.60E+07 2.87E+05 9.80E+06 1.87E+07 2 41 E+07

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77 Figure 3 1. Treatment of stationary and early log phas e bacteria on swabs with Ca(ClO ) 2 Figure 3 2. Ca(C lO) 2 necessary to kill planktonic bacteria of different species a fter collection using cotton swabs ELG: early log phage, ST: stationary phase.

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78 Figure 3 3. Exposure of planktonic P. aeruginosa PAO1 bacteria to gentamicin and Ca(ClO) 2 after collection on a cotton swab Figure 3 4. Different collection methods. A ) C otton swab. B ) F locked swab. C ) C ytology brush. D ) 4mm loop curette

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79 Figure 3 5. Treatment of P aeruginosa PAO1 b iofilm collected on flocked swabs

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80 Figure 3 6 Comparison of P aeruginosa PAO1 biofilm p ickup levels using different collection devices A ) Collection d evices were treated with Ca(ClO) 2 B ) Collection devices were treated with 200 g/mL gentamicin.

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81 Figure 3 7. Planktonic and biofilm PAO1 collected on cotton swabs. A) Early log phase PAO1 co llected on a cotton swab. B) 3 day mature PAO1 biofilm cultured on porcine skin and collecte d on a cotton swab. Each line represents 6 m. Figure 3 8 SEM images of P aeruginosa PAO1 biofilm collected on different swabs. A) cotton swab. B) cyt ology brush. C) flocked swab. D) Sponge s wab. Each line represents 600 m.

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82 Figure 3 9. SEM images of porcine skin biopsies after P aeruginosa PAO1 biofilm collection using different collection methods. A ) Cytology brush B) 4 mm loop curette C ) Cotton swab. Each line represents 12 m.

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83 Figure 3 10 P aeruginosa PAO1 biofilm treatment in different transportation solutions

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84 Figure 3 11 Optimization of PAO1 biofilm dispersion using 8 mm porcine skin explants A ) Pulsed sonications were separated by 1 minute intervals. B ) Pulsed vortexing w as separated by 1 minute intervals.

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85 Figure 3 12. Optimization of PAO1 biofilm dispersion using curetted samples. A ) Pulsed sonications were separated by 1 minute intervals. B ) Pulsed vortexing w as separated by 1 minute intervals.

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86 CHAPTER 4 INITIAL ASSESSMENT OF THE CLINICAL MICR OBIOLOGY BIOFILM ASSAY USING CLINICAL SAMPLES Introduction A clinical assay that is developed in the laboratory must be validated using clinical samples including all the optimized parameters of collecting, transporting and processing the samples. This is an important step to ensure that the assay is practical and reliable using actual clinical specimens containing a wide variety of bacterial species. Initial assessment of the optimized assay using clinical samples will allow for the complete optimi zed assay to be evaluated. Antibiotic Treated Samples Materials and Methods Clinical wound samples were collected by Dr. Linda Cowan from patients in the Gainesville VA hospital using the BBL Culture Swab Plus transport system and transported to the lab ora tory A total of 19 samples were collected from 16 different patients, as some samples were taken from 2 different wounds from the same patient and one was a repeat sample from the same wound. Sa mples were collect ed using the BBL c harcoal (catalog # 220116) to collect and transport the samples to the lab oratory This system consist ed of a sterile peel pouch including a rayon tip swab used to collect the clinical sample and a tube containing 5 m L of A mies transport media gel in which the swab is deposited. The A mies transport media gel does not support bacterial growth and is buffered with phosphate. Also, the sodium thioglycollate formulation provides a reduced environment while the moisture preve nts the sample from drying out. Also some A mies transport media formulations contain charcoal which helps preserve fastidious

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87 In the laboratory, s wab tips were incubated in 5 m L of TSB con tain ing 10 g/mL of rifampicin and 50 g/m L of gentamicin (Gentamicin Sulfate USP Grade, MP No. 190057) for 24 hrs at 37 C. Swab tips were then sonicated in the TS B containing antibiotics serially diluted and 100 L was spread on enriched and se lective agar plates and incubated at 37 C for 10 day s Colony counts were recorded to calclulate CFU/mL of sonication solution. Only aerobic bacteria were cultured for this set of samples The enriched media plates that were used include: Choc o late Agar (GC II Agar with Hemoglobin and IsoVitaleX) for the isolation and cultivation of fastidious microorganisms ( BD Diagnostic Systems No. 221169) Trypticase Soy Agar with 5% Sheep Blood for c ultivating fastidious microorganisms and for the visualization of he molytic reactions produced by many bacterial species The selective media plates that were used include: EMB (Eosin Methylene Blue) Agar, Modified, Holt Harris and Teague for the isolation of gram negative enteric bacilli ( BD Diagnostic Systems No. 221354) Chrom Agar, Staph aureus (isolation of Staphylococcus aureus, BD Diagnostic Systems No. 214982) or Chrom agar for MRSA isolation (BD Diagnostic Systems, No. 215084) Pseudomonas isolation agar (Teknova, No.P0144) Saubaourad Dextrose agar with Chloramphe nicol and Gentamicin for the isolation of dermatophytes and opportunistic fungi (BD Diagnostic Systems No. 296359 ) Mannitol Salt Agar for the isolation and enumeration of staphylococci (BD Diagnostic Systems No. 221173) To confirm if these detected colonie s are biofilm protected bacteria and not antibiotic resistant strains, a single colony from each plate was picked and streaked on both a TSA plate containing 10 g/mL rifampicin and a TSA plate containing 50g/m L gentamicin. These plates were incubated overnight at 37 C and growth of any bacterial

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88 colonies was recorded. This was done for 4 out of the 6 samples for which colonies were detected Results Colonies were detected in plates corresponding to 6 out of th e 19 samples and colonies were detected on multiple growth media plates for 3 of them. Only one sample which came from a healing abdominal surgical wound had a high number of CFU/mL. However, 2 out of the 4 samples tested for antibiotic resistance show tha t the detected colonies were in fact antibiotic resistant strains and not biofilm protected bacteria (Table 4 1) Conclusion Treating samples with a mixture of broad spectrum antibiotics targeting both gram + and gram bacteria is not a reliable method o f detecting biofilms. The main problem with the use of antibiotics in this assay is the presence of antibiotic resistant strains which yield false positive results and thus might be falsely interpreted as the presence of biofilm protected bacteria. The way to solve this issue is to perform replic a plating on antibiotic plates adding another level of complexity to the assay which is intended to be rapid and efficient. Also the use of antibiotics is expensive and 48 hours are needed to obtain preliminary resu lts by observing colony counts and 72 hours to obtain final results after confirming biofilm presence by antibiotic plates. Calcium Hypochlorite Treated Samples Introduction Since the use of an antibiotic mixture is not the best method for detecting biofilms, another set of clinical samples w as collected to validate the clinical assay using the Ca(ClO) 2 treatment. All collection, transport and processing conditions were optimi zed

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89 for the use of Ca(ClO) 2 a s for the elimination of planktonic bacteria and consequently the detection of biofilms. Materials and Methods Standardization of amount of tissue collection The widely used method for wound tissue sampling is aseptically colle cted collecting a sample that is then transported to the microbiology lab, weighed, homogenized, then serially diluted and cultured (Bowler et al 2001). Also, p revious methods to quantifying bacterial counts from chronic burn wounds transferred a biopsy sample to a pre weighed homogenizer bag and re weighed it and the weight difference was used as a way to standardize the amount of tissue in each sample and to allow results to be reported as CFU/g of tissue (Ganatra and Ganatra, 2007) The proposed metho d to calculate the weight of the collected sample is to pre weigh the collection tube containing the solution before the sample collection and then re weigh the tube again after sample collection. The difference between these two values theoretically shoul d be the weight of the sample and can be reported as CFU/g of tissue To test the consistency of this method, 10 curettes and 10 conical cubes (15 mL) contain 1 mL of saline were weighed The curettes were used to collect biofilm samples cultured on porcin e skin explants and were re weighed. The samples were then transferred to the tube contain saline and the curette was shaken off to ensure that the entire sample was transferred to the solution and that no residual saline remained on the loop. The tubes co ntaining the sample were re weighed. Differences in weight were calculated to determine the weight of the sample on the curette and the weight of the sample in the tube.

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90 For cotton swabs, the best way to standardize the amount of sample collected is to de termine the saturation value of the swab and use it as a baseline value. D ifferent volumes of H 2 O were dispensed on a piece of parafilm and using a dry cotton swab to collect absorb the specific volume. The volume that was totally absorbed by the cotton swab is the saturated volume that can be used to standardize results from multiple samples. Processing samples Clinical samples were collected from patients with chronic wounds from the VA hospital in Lake City, Florida. The samples were collected by Dr. J oyce Stechmiller from the UF College of Nursing. Curette s amples were obtained using a 4 mm curette loop and transported to the lab on the same day. The selected transport media was the C opan diagnostics A mies transport media ( part of the ESwab Patented Sa mple Collection & Delivery System ) which was shown to yield best recovery results for fastidious organisms like Neisseria gonorrhoeae ( Thompson and French 1999) A full loop curette sample was collected and shaken off in the appropriate tube containing ei ther saline in order to standardize the amount of tissue in each sample. Samples were transported in a s tyrofoam box with an ice p ack to keep temperature of samples low during transportation. The appropriate amount of f reshly prepared Ca(ClO ) 2 solution was added to each tube to achieve the desired final concentration. Each tube was vortexed briefly and allowed to sit at room temperature for 10 minutes. E nough Na 2 (S 2 O 3 ) was added to neutralize the reaction. Tubes were sonicated and 100 L was sp read on selective and enriched agar plates

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91 For this set of clinical samples, anaerobic bacteria were cultured by spreading 100 L on an anaerobic blood agar plate ( CDC Anaerobe 5% Sheep Blood Agar, BD Diagnostic, No.:221733) for isolation and cultivation of fastidious and slow growing, obligately anaerobic bacteria. The plates were incubated in an anaerobic chamber overnight at 37 C. Replica plating was performed by using TSA + 5% sheep blood plates which were incubated overnight in a regular incubator at 37 C while the anaerobic plates were returned to the anaerobic chamber. The two plates were compared to identify obligate and facul tative anaerobes. Also, mannitol salts agar plates were replaced with BBL ChromAgar plates which were found to be a more reliable way to detect S aureus in heavily contaminated respiratory clinical samples when compared to standard culture media ( Flayhart et al ., 2004). After colony counts and recording colony morphology descriptions, all plates were incubated at 37 C for up to 10 days. The f inal protocol for processing clinical samples for the d etection of functional biofilms is as follows : Pre weigh t ransport tube (containing 9.6 mL of saline) Collect curette, biopsy or flocked swab sample and transfer sample to saline. Transport to lab in a cooler containing an ice pack Weigh each transport tube to determine the weight of the collected sample. Add 40 L of freshly made 5% Ca(ClO)2 to saline containing samples for a final concentration of 0.2%, vortex briefly and allow to sit for 10 minutes Add 92 L of 5% Na2(S2O3) vortex briefly and allow to sit for 10 minutes at room temperature Sonicate in Fisher Scientific B2200R 1 Bransonic ultrasonic cleaner (50/60Hz, 117 Volts, 1.0 Amps) water bath for 5 1.5 minutes with 1 minute waiting interval between each sonication. Vortex briefly before plating Plate 100 L of solution on selective and enriched a gar plates

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92 Centrifuge remaining solution, aspirate fluid, re suspend pellet in 5 mL of growth medium (tryptic soy broth or brain heart Infusion) Incubate plates and tubes at 37 C for 24 hrs and record colony counts for pla tes and + or growth for tubes Leave plates in incubator for 10 days and record number of colonies daily Photograph each plate Results Standardization of sample weight There was a difference in the weight of the biofilm sample on the curette when compared to the weight of the sample on in the tube. However, this error is very reproducible which indicates that this method can potentially be used with a known adjustment that accounts for this error. The volume of fluid collected on cotton swabs was 100 L. Processing samples All curette sa mpl es from chronic wounds were from Lake City indicate d the pre sence of some level of biofilm (Table 4 3 Table 4 4 ) Also, all of the samples indicated the presence of multispecies biofilms including enteric, staphylococci and anaerobic bacteria. This indicates that the proposed assay reliably detects biofilm presence and gives accurate quantitative and qualitative results. Conclusion Validating the assay with clinical information provides significant informat ion and insight about biofilm presence in chronic wounds. All the wound samples processed according to the developed protocol had some level of multi species biofilm presence as determined by the biofilm assay The bacterial load an d the bacterial species provided important information as to how heavily the wound is colonized and what pathogenic

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93 species might be most problematic. This assay can be easily adapted by any wound care setting and will provide wound care providers with crucial information that wi ll help improve clinical practice in the wound care field.

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94 Table 4 1. Clinical wound s amples t reated w ith a ntibiotic m ixture Sample Selective/ enriched media CFU/mL Colony description Gentamicin (50 g/mL) Rifampicin (10 g/mL) Biofilm presence (#1) Abdominal healing surgical wound Chocolate Agar 450 + + No Blood Agar 510 Hemolytic + + No Chrom Agar 550 + + No Manitol Salts Agar 510 + + No (#2) Abdominal wound (1 yr) Chrom Agar 1 green NA NA NA (#3) Chest surgical wound Chrom Agar 3 Yes (#8) Acute Healing wound: sebaceous cyst wound Chochlate Agar 1 purple + Yes Blood Agar 12 Yes Chrom Agar 4 Yes (#12) Ischium pressure ulcer chronic (2 yrs) Chochlate Agar 6 + + No (#17) Abdominal surgical wound (6 weeks) Chochlate 1 NA NA NA Blood Agar 1 NA NA NA Chrom Agar 2 purple NA NA NA S.aureus (SA35556 lab strain) P. aeuriginosa (PAO1 lab strain) +*

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95 Table 4 1. Continued Sample Selective/ enriched media CFU/mL Colony description Gentamicin (50 g/mL) Rifampicin (10 g/mL) Biofilm presence E.coli (DH5 lab strain) P. aeuriginosa is naturally resistant to Rifampicin. Table 4 2. Standardization of sample weight Sample on c urette Sample in transport t ube Difference 1 0.065 0.049 0.016 2 0.035 0.019 0.016 3 0.041 0.019 0.022 4 0.035 0.024 0.011 5 0.032 0.017 0.015 6 0.032 0.022 0.01 7 0.034 0.021 0.013 8 0.044 0.029 0.015 9 0.050 0.034 0.016 10 0.033 0.021 0.012 Mean 0.040 0.026 0.015 Std. d eviation 0.01057 0.009710 0.003406 Std. e rror 0.003341 0.003070 0.001077

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96 Table 4 3 Treatment of chronic wound curette s amples from VA in Lake City FL Sample ID EMB Chrom PIA Saubaourad Chochlate Blood Anaerobe (#1) sacral pressure ulcer stage IV, treated with dakins 1.10E+02 dark pink, smooth 6.50E+02 mauve dark center 2.00E+01 yellow 2.00E+01 large, yellow, irregular TNTC 2.90E+02 brown translucent hemolytic Facultative: 2.00E+01 non hemolytic 1.30E+02 hemolytic 8.0E +0 1 large, translucent 4.80E+02 yellow Obligate: 4.00E+01 non hemolytic (#1) Repeat (3.5 weeks after first sample ) TNTC TNTC navy blue 8.00E+01 light yellow, 5.00E+02 white, smooth, raised 2.00E+02 large, translucent, hemolytic NA TNTC small yellow 2.49E+03 yellow, raised 1.00E+01 large bright yellow 4.60E+03 irregular, raised, light yellow (#1) Repeat (5 weeks after first sample) 1.63E+03 small, irregular, dark center 9.80E+03 mauve 1.00E+01 large NA 2.00E+02 dark, brown, large, irregular NA 2.00E+01 large, pink, irregular, flat 7.00E+02 yellow TNTC small

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97 Table 4 3. Continued Sample ID EMB Chrom PIA Saubaourad Chochlate Blood Anaerobe (#2)foot pressure ulcer stage IV treated with a silver dressing TNTC small, pink TNTC blue, purple 0.00E+00 2.20E+02 yellow, irregular TNTC small, beige TNTC small, beige TNTC (#2) Repeat (3.5 weeks after first sample ) 6.60E+02 small, flat, shiny green 7.30E+02 navy blue/purple raised, 0.00E+00 0.00E+00 1.90E+02 white, smooth NA Facultative: 9.40E+02 white, irregular, flat, hemolytic 2.00E+01 mauve 6.00E+01 large, smooth, raised 9.40E+02 white (#2) Repeat (4.5 weeks after first sample) 4.00E+02 large, smooth, dark purple with pink ring 1.60E+03 large, mauve, yellow ring TNTC small, white/ grey, smooth NA 1.03E+04 small, dark pink 5.00E+02 turquoise blue TNTC small yellow (#3) sacral pressure ulcer, treated with dakins 5.40E+02 big, purple, hairy 3.48E+03 navy blue, yellow 0.00E+00 1.12E+03, light yellow, raised, filamentous 1.80E+03 large, white, filamentous, raised 3.60E+02 big yellow, irregular Facultative: 1.00E+02 big yellow, irregular

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98 Table 4 3. Continued Sample ID EMB Chrom PIA Saubaourad Chochlate Blood Anaerobe 1.52E+03 small, purple 3.50E+03 beige, smooth, flat 3.64E+03 small, hemolytic, white, flat, translucent Facultative: 2.37E+03 small, hemolytic, white, flat, translucent (#3) Repeat ( 4.5 weeks after first sample ) 2.60E+03 dark pink/purple, large, irregular TNTC pink, turquoise 1.80E+02 large, yellow, raised, filamentous TNTC light brown, smooth NA 4.40E+03 large, yellow, flat TNTC grey, hemolytic (#4) Left medial, lower leg, pyoderma gangrenosum, chronic venous insufficiency 1.00E+02 large, maroon, raised TNTC green TNTC green, translucent TNTC yellow, irregular, dark center TNTC grey TNTC dark brown NA TNTC transulcent

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102 Jones, S.G., Edwards, R., and Thomas, D.W. (2004). Inflammation and w ound h ea ling: t he r ole of b acteria in the i mmuno r egulation of w ound h ealing. International Journal of Lower Extremity Wounds 3 201 208. Kim, J., Hahn, J. S ., Franklin, M.J., Stewart, P.S., and Yoon, J. (2009). Tolerance of dormant and active cells in Pseudomonas aeruginosa PA01 biofilm to antimicrobial agents. J Antimicrob Chemother 63 129 35. Knox, K.R., Datiashvili R.O., and Granick M.S. (2007). Surgical wound bed preparation of chronic and acute wounds. Clinics in Plastic Surgery 34 633 664. Leid, J.G. (2009). Bacterial biofilms r esist k ey h ost d efenses Microbe 4 66 70. Lewandowski, Z., Stoodley, P., Altobelli, S., and Fukushima, E. (19 93). Hydrodynamics and kinetics in biofilm systems recent advances and new problems. Proceedings of the Second IAWQ International Specialized Conference on Biofilm Reactors. Paris, France, 313 319 Lewis, L. (2001). Riddle of biofilm resistance. Antimicrobial agents and chemotherapy 45 999 10007. Luppens, S. B., Reij, M. W. van der Heijden, R. W., Rombouts, F. M. and Abee, T. (2002). Development of a standard test to assess the resistance of Staphylococcus aureus biofilm cells to disinfectants. Appl. Environ. Microbiol 68 4194 4200 Luppens S B ., and ten Cate, J.M. (2005). Effect of biofilm model, mode of growth, and strain on Streptococcus mutans p rotein expression as determined by two dimensional difference gel electrophoresis. J Proteome Res 4 232 237 Mah, T F.C., O'Toole, G.A. (2001). Mechanisms of biofilm resistance to antimicrobial agents. Trends in microbiology 9 34 39. M a h, T F., Pitts, B., Pellock, B., W a lker, G.C., Stew a rt, P.S., and O'Toole, G. A (2003). A genetic basis for Pseudomonas a eruginos a biofilm a ntibiotic resistance N a ture 426 306 310. Moue¨s, C.M., Vos M.C., Van Den Bemd C.M. G J., Stijnen T., and H ovius S.E.R. (2004). Bacterial load in relation to vacuum assisted closure wound therapy: A prospective randomized trial Wound Rep Regen 12 11 17. Mustoe, T.A., O'Shaughnessy, K., and Kloeters, O. (2006). Chronic wound pathogenesis and current treatment strategies: a unifying hypothesis. J Plast Reconstr Surg 117 35 41.

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103 Nadell, C.D ., Xavier, J.B ., and Foster, K.R (2008). The sociobiology of biofilms. FEMS Microbiol Rev 33 206 2 24. Percival, S.L., and Bowler, P.G. (2004). Biofilms and their pot entia l role in wound healing. Wounds 16 234 240. Pellizzer, G., Strazzabosco, M., Presi, S., Furlan, F., Lora, L., Benedetti, P., Bonato, M., Erle, G., and de Lalla, F. (2001). Deep tissue biopsy vs superficial swab culture monitoring in the microbiologic al assessment of limb threatening diabetic foot infection. Diabet Med 18 822 827. Phillips, P.L., Yang, Q., Sampson, E., Schultz, G. (2010). Effects of antimicrobial agents on an in vitro biofilm model of skin wounds. Advances in Wound Care 1 299 304. P ozez, A L., Aboutanos, S.Z ., and Lucas, V.S (2007). Diagnosis and t reatment of u ncommon w ounds. Clin Plasic Surg 34 749 764. Reiber, G.E., and McFarland, L.V. (2006). Epidemiology and health care costs for diabetic foot problems. The Diabetic Foot, Second Edition, 39 50. Resch, A., Rosenstein, R., Nerz, C., and Gotz, F. (2005). Diffe rential gene expression profiling of Staphylococcus aureus cultivated under biofilm and planktonic conditions. Appl Environ Microbiol 71 2663 2676. Richter, A., Smith, R., Ries, R., and Wildenauer, F. (1999). Fe atures of biofilm growth on solid surfaces analysed by m onte carlo simulations. Phys Stat Sol 176, 953 967. Rutala, W.A., and Weber, D.J. (1997). Uses of inorganic hypochlorite (bleach) in health care facilities. Clin Microbiol Rev 10 597 610. Sauer, K ., Camper, A.K., Ehrlich, G.D., Costerton, J.W., and Davies D.G. ( 2002 ) Pseudomonas aeruginosa displays multiple phenot ypes as a biofilm. J. Bacteriol 184 1140 1154. Schultz G S Sibbald R G Falanga V Ayello E A Dowsett C Harding K Romanelli M Stacey M C Teot L and Vanscheidt W. (2003). Wound bed preparation : a systematic approach to wound management Wound r epair r egen 11 (Suppl. 1) S1 28 Senneville, E., Melliez, H., Beltrand, E., Legout L., Valette, M., Cazaubiel, M., Cordonnier, M., Caillaux M., Yazdanpanah Y., and Mouton, Y. (2006). Culture of percutaneous bone biopsy specimens for diagnostic of diabetic foot osteomyelitis: concordance with ulcer swab cultures. Clin Infect Dis 42 57 62.

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104 Serena, T., Robson, M.C., Cooper, D.M., and Ignatius, J., (2006). Lac k of reliability of clinical/visual assessment of chronic wound infection: the incidence of biopsy proven infection in venous leg ulcers. Wounds 18 197 202. Serralta V W Harrison Balestra C Cazzaniga A L Davis S C and Mertz P M (2001). Lifestyles of bacteria in wounds: p resence of biofilms? Wounds 13 29 34. Slater, R.A., Lazarovitch. T., Boldur, I., Ramot, Y., Buchs, A., Weiss, M., Hindi, A., and Rapoport M.J. (2004). Swab cultures accurately identify bacterial pathogens in diabetic foot wounds not involving bone. Diabet Med 21 705 9. Stewart, P.S., Rayner, J., Roe, F., and Rees, W.M. (2001). Biofilm penetration and disinfection efficacy of alkaline hypochlorite and chlorosulfamates J Appl Microbiol 91 525 32. Sullivan, P.K., Conner Kerr, T.A., Hamilton, H., Smith, E.P., Tefertiller, C., and Webb, A. (2004). Assessment of wound bioburden development in a rat acute wound model: quantitative swab ver sus tissue biopsy. W ounds 16 115 123. Thompson, D.S., and French, S.A. (1999). Comparison of commercial Amies transport systems with in house Amies medium for recovery of Neisseria gonorrhoeae J Clin Microbiol 37 3020 3021 Trent, J.T., Falabella, A., E aglstein, W.H., and Kirsner, R.S. (2005). Venous ulcers: pathophysiology and treatment options. Ostomy wound manage 51 38 54. Xavier, J.B., and Foster, K.R. (2007). Cooperation and conflict in microbial biofilms. Proc Natl Acad Sci 104 876 81. Yang, Q. (2008). Development and validation o f an in vitro porcine skin model of bacterial biofilms in chronic wounds. Master s Thesis, University of Florida.

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105 BIOGRAPHICAL SKETCH Afifa Hamad was born in Chicago Illinois and grew up between Saudi Arabia, Australia and the Florida She attended the University of Florida in June of 2005 and in neurobiological sciences In May of 2008 degree and graduated with high honors afte r completing a research project on the expression of mller cell promoters in chicken retina in Dr. Sue Semple In August of 2008 she began her graduate education and entered the m f Florida d epartment of m icr obiology and molecular genetics.