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Evaluation of the antimicrobial activity of a dialysed cranberry extract against selected isolates of… Leusink, Gwendolyn Jill 2009

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EVALUATION OF THE ANTIMICROBIAL ACTIVITY OF A DIALYSED CRANBERRY EXTRACT AGAINST SELECTED ISOLATES OF STAPHYLOCOCCUS AUREUS  by Gwendolyn Jill Leusink  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science in The Faculty of Graduate Studies (Food Science)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) October 2009 © Gwendolyn Jill Leusink, 2009  ABSTRACT This research examined the antimicrobial activities of a dialysed commercial cranberry extract (CE90), by determining the minimum inhibitory concentration against Staphylococcus aureus and the effect of CE90 on morphology, adhesion to fibronectin, biofilm formation and removal and salt tolerance of Staph. aureus. The minimum inhibitory concentration was 16 mg CE90 ml-1 for 10 of the 24 Staph. aureus isolates and 32 mg CE90 ml-1 for the remaining 14 isolates. Phase contrast microscopy of treated Staph. aureus revealed a dose-dependent bacterial clumping effect, with higher CE90 concentrations resulting in larger clumps. There were no significant differences between CE90 concentrations (0, 2, 4, 8 and 16 mg CE90 ml-1) in Staph. aureus binding to fibronectin, for a parental strain and two mutant strains deficient in either fibronectinbinding proteins A and B or clumping factor A, with the exception of reduced adhesion for the fnbA-/fnbB- mutant at 4 mg CE90 ml-1. Biofilm formation by four isolates of Staph. aureus (PC1, SA 113, 8325-4, 2076) incubated with CE90 generally decreased as CE90 concentration increased (0 – 16 mg CE90 ml-1). The amount of pre-formed biofilm removed by CE90 for Staph. aureus PC1 was not significantly different from the control. Staph. aureus SA 113 and 2076 showed a greater (P < 0.05) level of residual biofilm after exposure to CE90 (SA 113, 2 – 16 mg CE90 ml-1; 2076, 4 – 16 mg CE90 ml-1) than the negative control. There was more (P < 0.05) residual biofilm in the 2 mg CE90 ml-1 treatment versus the 16 mg CE90 ml-1 treatment for Staph. aureus 8325-4. Biofilm index at 8 and 24 h showed a significant interaction between isolates and CE90 concentrations (P < 0.05), and biofilm index of Staph. aureus PC1 and SA 113 showed a significant interaction between time intervals and CE90 concentrations (P < 0.05). There was a  ii  reduction in colony forming units with increasing CE90 concentration on both tryptic soy agar and TSA plus 7.5% NaCl (TSAS), with a greater reduction on TSAS, though this difference between media was significant in only four of the 16 isolate-treatment combinations.  iii  TABLE OF CONTENTS ABSTRACT ....................................................................................................................... ii TABLE OF CONTENTS .................................................................................................. iv LIST OF TABLES............................................................................................................ vii LIST OF FIGURES ......................................................................................................... viii LIST OF ABBREVIATIONS ............................................................................................x ACKNOWLEDGEMENTS.............................................................................................. xii DEDICATION................................................................................................................. xiv 1. INTRODUCTION ..........................................................................................................1 2. LITERATURE REVIEW ...............................................................................................4 2.1 Staph. aureus .............................................................................................................4 2.1.1 Physical and chemical characteristics ................................................................4 2.1.2 Staph. aureus growth .........................................................................................4 2.1.3 Virulence factors ................................................................................................5 2.1.3.1 Staph. aureus toxins ................................................................................... 5 2.1.3.2 Adhesins ..................................................................................................... 7 2.1.3.3 Biofilm production ..................................................................................... 7 2.1.4 Staph. aureus infections in livestock and poultry .............................................. 9 2.2 Antibiotic resistance ................................................................................................10 2.2.1 Staph. aureus antibiotic resistance ...................................................................10 2.2.2 Antibiotic use and development of antibiotic resistance ..................................11 2.2.3 Reduction of antibiotic resistance following restriction of antibiotic use ........12 2.2.4 Multidrug-resistant bacteria .............................................................................13 2.3 Cranberry ................................................................................................................14 2.3.1 Health benefits and antimicrobial activity .......................................................14 2.3.2 Ability of cranberries and cranberry extracts to reduce the incidence of urinary tract infections ...............................................................................................15 2.3.3 Bacterial anti-adhesion mechanism of cranberries and cranberry extracts ......16  iv  2.3.4 Effect of cranberries on bacterial biofilm formation ........................................19 2.3.5 Cranberry proanthocyanidin structure..............................................................19 2.4 Literature review pertaining to methodology...........................................................20 2.4.1 Minimum Inhibitory Concentration (MIC) ......................................................20 2.4.2 Bacterial morphology .......................................................................................22 2.4.3 Extracellular matrix (ECM) binding ................................................................22 2.4.4 Biofilm formation and removal ........................................................................23 2.4.5 Biofilm enumeration ........................................................................................25 2.4.6 Salt tolerance ....................................................................................................26 3. MATERIALS AND METHODS .................................................................................28 3.1 Cranberry extract.....................................................................................................28 3.2 Staph. aureus isolates ..............................................................................................28 3.3 Determination of MIC ............................................................................................29 3.4 Staph. aureus morphology ......................................................................................30 3.5 Attachment to ECM protein (fibronectin) ...............................................................31 3.6 Biofilm formation ...................................................................................................32 3.7 Biofilm removal ......................................................................................................32 3.8 Biofilm index ..........................................................................................................33 3.9 Salt tolerance assay .................................................................................................34 3.10 Statistical analyses ................................................................................................35 4. RESULTS & DISCUSSION ........................................................................................37 4.1 Determination of MIC .............................................................................................37 4.2 Staph. aureus morphology ......................................................................................37 4.3 Attachment to ECM protein (fibronectin) ...............................................................39 4.4 Biofilm formation, removal & index ......................................................................45 4.4.1 Biofilm formation.............................................................................................45 4.4.2 Biofilm removal ...............................................................................................46 4.4.3 Biofilm index ...................................................................................................48 4.5 Salt tolerance assay .................................................................................................50  v  5. CONCLUSION ............................................................................................................54 6. RECOMMENDATIONS FOR FURTHER RESEARCH ............................................56 REFERENCES ..................................................................................................................74 APPENDICES ...................................................................................................................97  vi  LIST OF TABLES Table 1: MICs of CE90 against 24 different isolates of Staph. aureus .............................59 Table 2: Interactions between CE90 concentrations and isolate (Staph. aureus PC1, SA 113, 8325-4 and 2076) at different time intervals ...............................................60 Table 3: Interactions between CE90 concentrations and time for different Staph. aureus isolates .................................................................................................................60 Appendix B: Ingredient specifications of the undialysed cranberry extract......................98 Appendix C: Biofilm formation of 24 Staph. aureus isolates in the absence of CE90 .....99  vii  LIST OF FIGURES Figure 1a: Effect of CE90 on Staph. aureus PC1 morphology: 0 mg CE90 ml-1 .............61 Figure 1b: Effect of CE90 on Staph. aureus PC1 morphology: 4 mg CE90 ml-1 .............61 Figure 1c: Effect of CE90 on Staph. aureus PC1 morphology: 8 mg CE90 ml-1 .............61 Figure 1d: Effect of CE90 on Staph. aureus PC1 morphology: 16 mg CE90 ml-1 ...........61 Figure 2: Effect of CE90 on Staph. aureus binding to fibronectin....................................62 Figure 3: Effect of CE90 on Staph. aureus biofilm formation ..........................................63 Figure 4: Effect of CE90 on removal of biofilm formed by Staph. aureus .......................64 Figure 5a: Effect of CE90 on biofilm index of Staph. aureus after 2 h ............................65 Figure 5b: Effect of CE90 on biofilm index of Staph. aureus after 4 h ............................66 Figure 5c: Effect of CE90 on biofilm index of Staph. aureus after 6 h ............................67 Figure 5d: Effect of CE90 on biofilm index of Staph. aureus after 8 h ............................68 Figure 5e: Effect of CE90 on biofilm index of Staph. aureus after 24 h ..........................69 Figure 6a: Effect of CE90 on Staph. aureus PC1 salt tolerance........................................70 Figure 6b: Effect of CE90 on Staph. aureus SA 113 salt tolerance ..................................71 Figure 6c: Effect of CE90 on Staph. aureus 8325-4 salt tolerance ...................................72 Figure 6d: Effect of CE90 on Staph. aureus 2076 salt tolerance ......................................73 Appendix A: Structure of a typical cranberry proanthocyanidin tetramer composed of epicatechin units with one A-type linkage and two B-type linkages...............97 Appendix D.1: Effect of CE90 on planktonic Staph. aureus PC1 growth ......................100 Appendix D.2: Effect of CE90 on Staph. aureus PC1 biofilm formation .......................101 Appendix D.3: Effect of CE90 on planktonic Staph. aureus SA 113 growth .................102 Appendix D.4: Effect of CE90 on Staph. aureus SA 113 biofilm formation..................103  viii  Appendix D.5: Effect of CE90 on planktonic Staph. aureus 8325-4 growth ..................104 Appendix D.6: Effect of CE90 on Staph. aureus 8325-4 biofilm formation ..................105 Appendix D.7: Effect of CE90 on planktonic Staph. aureus 2076 growth .....................106 Appendix D.8: Effect of CE90 on Staph. aureus 2076 biofilm formation ......................107  ix  LIST OF ABBREVIATIONS ATCC  American Type Culture Collection  AFM  Atomic force microscopy  BSA  Bovine serum albumin  CLSI  Clinical and Laboratory Standards Institute  ClfA  Clumping factor A  clfA  Clumping factor A gene  ClfB  Clumping factor B  clfB  Clumping factor B gene  cfu  Colony forming units  CE90  Cranberry extract 90  DIN  Deutsche Industrie Norm-Medizinische Mikrobiologie  ELISA  Enzyme-linked immunosorbent assay  E. coli  Escherichia coli  ECM  Extracellular matrix  FnbpA  Fibronectin-binding protein A  fnbA  Fibronectin-binding protein A gene  FnbpB  Fibronectin-binding protein B  fnbB  Fibronectin-binding protein B gene  HBSS  Hanks’ balanced salt solution  H. pylori  Helicobacter pylori  h  Hours  MRSA  Methicillin-resistant Staph. aureus  x  MSCRAMMs  Microbial surface components recognizing adhesive matrix molecules  µl  Microlitres  MIC  Minimum inhibitory concentration  min  Minutes  MHA  Mueller Hinton agar  MHB  Mueller Hinton broth  NCCLS  National Committee for Clinical Laboratory Standards  NDM  Non-dialysable material  PBS  Phosphate buffered saline  P. gingivalis  Porphyromonas gingivalis  PAC  Proanthocyanidin  Staph. aureus  Staphylococcus aureus  S. sobrinus  Streptococcus sobrinus  TSA  Tryptic soy agar  TSAS  Tryptic soy agar plus salt (7.5% NaCl)  TSB  Tryptic soy broth  UTI  Urinary tract infection  vanA  Vancomycin A gene  VanA  Vancomycin A plasmid  VRE  Vancomycin resistant enterococci  xi  ACKNOWLEDGEMENTS I would like to express my everlasting gratitude to my UBC supervisor, Dr. Brent Skura, for his outstanding example of excellence in academia and scientific research, his unending patience and the willingness to share his vast knowledge and experience, which have provided such a valuable contribution to my research and scientific training. His integrity, encouragement, cheerful attitude and genuine concern for the professional and personal well-being of his students have greatly enriched my life during these past few years. Thank you. I would also like to extend sincere thanks to my project supervisor, Dr. Moussa Diarra, who provided me with the opportunity to perform this research in the microbiology laboratory at Agriculture and Agri-Food Canada; without him, this project would not have been possible. His scientific expertise, guidance and willingness to perform the SAS statistical analysis are greatly appreciated. I am also indebted to the other members of my committee, namely Dr. David Kitts and Dr. Eduardo Jovel. I am extremely grateful to Dr. Kitts for his patient willingness to provide valuable advice throughout the duration of my time here at UBC, and for the ability to condense his extensive knowledge of statistical principles and procedures into comprehensible explanations. Heartfelt thanks goes to Dr. Jovel for his willingness to be involved in this project by serving on my committee, and the valuable suggestions he has provided therein. This study was funded through Agriculture and Agri-Food Canada and the Cranberry Institute. I greatly acknowledge both organizations for their support. Numerous people have contributed to this project through their professional expertise or technical assistance, which is genuinely appreciated. In particular I would like to thank Heidi Rempel, Val Skura and Pedro Aloise, the microbiology laboratory supervisor at Agriculture Canada and the senior technicians of the UBC Food, Nutrition and Health building, respectively, for their efficient organization and laboratory management skills, which have greatly facilitated my work. Sincere thanks go to Garnet Martens and Kevin Hodgson, the research manager and light microscopy technician, respectively, for providing access to equipment in the UBC BioImaging Facility, and for their extremely helpful assistance with microscopy. I thank Doug Klaiber of Decas Botanical Synergies for his generous donations of cranberry extract. Sheila Fitzpatrick and the librarians at UBC are gratefully acknowledged for sharing extensive knowledge of cranberries and assisting in the location of scientific articles and other educational resources, respectively. I thank Jane Zhao and Cynthia Lopez for providing valuable technical assistance. Sincere thanks go to Terry Rigby and the computer technicians at UBC for fixing my computer and network problems, and to Andrew Larkin for his assistance with statistics and excel and for lending me his computer when mine finally died its last death. I thank Monica Purnama for helping me learn how to program SAS.  xii  I would also like to thank those who have contributed to my professional development through their teaching and training, which has increased my interest in science and fascination with biology. My high school and undergraduate biology teachers and professors and my graduate food science professors have provided the education upon which my scientific research career is based. Todd Kabaluk and Bob Vernon provided invaluable scientific training in their entomology research laboratories at Agriculture Canada, and Michael Lyon expanded my scientific experience to include clinical trials in his research laboratory at the Canadian Centre for Functional Medicine. Genuine thanks go to all my friends who have encouraged and inspired me along the way. Thank you for being there for me in daily life and when I needed you most. Your shared laughter, conversations and companionship fill my life with joy. Though it would be impossible to mention everyone who comes to mind with those words, I’d particularly like to thank Fatou Diarrassouba, a constant companion during our time at Agriculture Canada and here at UBC; Carrie Vanderwal, a lifelong friend; and Michelle Contant, my favourite hiking buddy. My greatest debt of gratitude is to my family, for their unending support, encouragement and love. Thank you, Mom, for showing me how to live out my beliefs in daily life, and for always believing in me and my ability to overcome all the obstacles I’ve faced. Thank you, Dad, for working so hard to provide us with the opportunity to do whatever we set our minds to, and for setting an example of achieving excellence with humility. Janna, Dan, Mark, Steph, Laura, Zach and Bev, thank you for being not only my family but also my best friends.  xiii  DEDICATION To my parents, Henry and Helen Leusink. With loving thanks for inspiring me to always do my best, and for your daily prayers that sustain me along the way.  xiv  1. INTRODUCTION Staph. aureus is an important human and animal pathogen (Smeltzer and Gillaspy, 2000; Ben Zakour et al., 2008) that produces several virulence factors including adhesins and the ability to form biofilm. This bacterium is also one of the most common causes of bovine mastitis, a disease that causes important economic losses in milk production (Vasudevan et al., 2003; Bannerman et al., 2004). The widespread use of antibiotics has led to a dramatic increase in antibiotic resistant Staph. aureus strains (Diarra et al., 2002; Tacconelli, 2009). Therefore, we have an urgent need for alternative methods of controlling Staph. aureus infections.  The ability of Staph. aureus to colonise epithelial cells is associated with surface extracellular matrix (ECM) binding proteins called adhesins (Navarre and Schneewind, 1999; Yarwood et al., 2004). In order to establish an infection, bacteria must first adhere to host tissues. Fibronectin-binding proteins, a type of bacterial adhesin, are required for the internalization of Staph. aureus by epithelial cells (Dziewanowska et al., 1999). Thus, fibronectin-binding proteins may be potential targets for protection against Staph. aureus infection. Biofilms are adherent bacterial communities enveloped in an ECM; these bacteria may be 1,000 to 1,500 times more resistant to antibiotics than planktonic bacteria (Ceri et al., 1999; Wu et al., 2003). The ability to form biofilm is another vital virulence factor for Staph. aureus (Fox et al., 2005). The persistence of biofilmassociated bacteria can result in chronic cases of disease. In addition, the formation of adherent bacterial biofilms is the most important factor in the pathogenesis of biomaterial-associated infections (Stewart and Costerton, 2001; Mack et al., 2004).  1  Administration of antibiotics may eliminate planktonic bacteria and therefore the symptoms of infection, but symptoms often recur after removal of treatment due to residual biofilm-associated bacteria (Stewart and Costerton, 2001). Bacteria that are able to survive within a biofilm despite exposure to antibiotics may develop antibiotic resistance (Eydelnant and Tufenkji, 2008). Therefore, alternative methods of controlling Staph. aureus infections should focus on inhibiting bacterial adhesion and biofilm formation.  Nogueira et al. (2003) demonstrated that cranberry juice concentrates possess intrinsic antimicrobial properties against bacterial pathogens. Cranberry products can reduce the incidence of urinary tract infections (Howell, 2002; Gormley, 2003) and inhibit bacterial adhesion (Burger et al., 2002; Turner et al., 2005; Johnson-White et al., 2006; Eydelnant and Tufenkji, 2008). Because there is currently no evidence within the literature to suggest that the adhesion inhibitors present in cranberries are bactericidal, the selection of strains that are resistant to these adhesion inhibitors is unlikely to occur. In addition to its anti-adhesion potential, cranberry has been shown to reduce biofilm formation (Reid et al., 2001; Yamanaka et al., 2004; Steinberg et al., 2004, 2005; Duarte et al., 2006; Koo et al., 2006; Labrecque et al., 2006; Yamanaka et al., 2007), which may be crucial to the efficacy of Staph. aureus control. The cranberry anti-adhesion and biofilm inhibition agent(s) may lead to new therapeutic strategies that are urgently needed because of the world-wide increase in antibiotic resistant bacteria (Sharon and Ofek, 2002).  2  The purpose of the present work was to determine the effect of a freeze-dried highmolecular-weight non-dialysable extract of a spray-dried cranberry juice concentrate powder (CE90) on fibronectin binding, biofilm formation and removal, morphology and salt tolerance of selected Staph. aureus isolates.  Experiments were designed to test the following hypotheses: 1. CE90 (4 – 16 mg ml-1) will have no effect on the clumping behaviour of Staph. aureus PC1, SA 113, 8325-4, 8325-4 fnbA-/fnbB-, 8325-4 clfA-, 2076, SHY97-4320, ABB032487, ATCC 6538 and ATCC 29213. 2. CE90 (4 – 16 mg ml-1) will have no effect on the fibronectin binding ability of Staph. aureus 8325-4 and two mutant strains, 8325-4 fnbA-/fnbB- and 8325-4 clfA-. 3. CE90 (2 – 16 mg ml-1) will have no effect on the extent of biofilm formed by Staph. aureus PC1, SA 113, 8325-4 and 2076. 4. CE90 (2 – 16 mg ml-1) will have no effect on the extent of removal of biofilm produced by Staph. aureus PC1, SA 113, 8325-4 and 2076. 5. CE90 (2 – 16 mg ml-1) will have no effect on the biofilm index of Staph. aureus PC1, SA 113, 8325-4 and 2076. 6. CE90 (4 – 16 mg ml-1) will have no effect on the salt tolerance of Staph. aureus PC1, SA 113, 8325-4 and 2076.  3  2. LITERATURE REVIEW 2.1 Staph. aureus 2.1.1 Physical and chemical characteristics Staph. aureus is a Gram positive, facultatively anaerobic coccus that tends to grow in pairs, short chains, or grape-like clusters. This organism is catalase positive, enabling it to convert hydrogen peroxide to oxygen and water (Varnam and Evans, 1991; Tortora et al., 2001; Jay, 2005; Doyle and Beuchat, 2007). Staph. aureus produces carotenoid pigments that likely provide protection from phagocytic killing and the antimicrobial effects of sunlight through their antioxidant properties (Tortora et al., 2001; Liu et al., 2005). These pigments result in characteristic golden yellow colonies when the bacterium is grown on agar. Bacterial cells are approximately 0.5 - 1 µm in diameter, are non-motile, and do not form capsules or spores. Fermentation of glucose by Staph. aureus produces primarily lactic acid. This bacterium is coagulase positive and oxidase negative (Varnam and Evans, 1991; Tortora et al., 2001; Jay, 2005; Doyle and Beuchat, 2007). Coagulase is able to clot fibrin in blood; the resulting clots of fibrin may protect Staph. aureus from phagocytosis and other host defenses (Tortora et al., 2001). Staph. aureus is sensitive to lysostaphin and resistant to lysozyme (Varnam and Evans, 1991).  2.1.2 Staph. aureus growth A wide range of environmental conditions permit growth of Staph. aureus: a) temperature 6.7 – 47.8oC (optimum 37oC); b) pH 4.0 – 9.8 (optimum 6 - 7); c) aw 0.83 - > 0.99 (optimum > 0.99). Staph. aureus is very salt tolerant, growing well at NaCl  4  concentrations between 0 and 10%; some strains can grow in NaCl concentrations up to 20% (Varnam and Evans, 1991; Jay, 2005).  2.1.3 Virulence factors 2.1.3.1 Staph. aureus toxins Staph. aureus produces a number of toxins, including alpha-toxin, which forms pores in cell membranes resulting in cell lysis. Alpha-toxin possesses hemolytic activity along with several other Staph. aureus hemolysins (Bhakdi and Tranum-Jensen, 1991; Lowy, 1998; Giese et al., 2009).  Exfoliative toxins cause staphylococcal scalded skin syndrome, which reduces cell adhesion within the epidermis, resulting in blisters and the sloughing off of skin. This syndrome occurs most frequently in children and infants (Lowy, 1998; Tortora et al., 2001; Castellano et al., 2008).  Toxic shock syndrome toxin causes a cytokine storm through release of a large amount of cytokines from immune T cells and macrophages. This results in symptoms such as fever and low blood pressure. Toxic shock syndrome may progress to coma, multiple organ failure and even death (Lowy, 1998; Seishima et al., 2009).  Consumption of enterotoxins present in food results in gastroenteritis (Varnam and Evans, 1991; Jay, 2005). Enterotoxins are far more heat resistant than Staph. aureus cells, and are unlikely to be inactivated by cooking and commercially applied heat  5  treatments (Jay, 2005; Doyle and Beuchat, 2007). The fact that enterotoxins are able to exert their effects even in the absence of viable Staph. aureus cells means that food can still cause gastroenteritis even after proper cooking or pasteurization, if the food had a high level of Staph. aureus before heat treatment. Re-contamination after heat treatment and improper refrigeration allowing the production of enterotoxin can also result in illness. The minimum amount of enterotoxin needed to produce symptoms of gastroenteritis in humans is approximately 1 ng/g of food, or a total of 20 ng (Jay, 2005).  Conditions favouring enterotoxin production are more limited than for Staph. aureus growth: a) temperature 10 – 46oC (optimum 40 – 45oC); b) aw 0.86 - > 0.99 (optimum > 0.99); c) NaCl 0 – 10% (Varnam and Evans, 1991; Jay, 2005). The pH range in which enterotoxins are produced varies widely according to the type of toxin, oxygen availability and acidulant. In aerobic conditions, the minimum pH for production of most enterotoxins is approximately 5, whereas in anaerobic conditions, the minimum pH is approximately 6. However enterotoxin A was produced at pH 4.5 when hydrochloric acid was used as the acidulant, and pH 5.0 when lactic acid was used (Jay, 2005). Interactions between temperature, pH, aw, NaCl concentration, acidulant and oxygen availability mean that the range in which each of these factors permits Staph. aureus growth and enterotoxin production can be restricted when the other factors are not ideal (Jay, 2005). This is the basic concept behind hurdle technology, which combines multiple inhibitory factors for food preservation (Leistner, 2000; Doyle and Beuchat, 2007).  6  2.1.3.2 Adhesins Staph. aureus possesses several other important virulence factors, such as adhesins and the ability to form a biofilm. Adhesins are surface proteins embedded in bacterial cell walls, which enable bacterial attachment to a surface thus creating the opportunity for colonization and infection. Microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) (Beenken et al., 2004) are an important class of adhesins. Adhesins such as fibronectin-binding proteins A and B (FnbpA, FnbpB), collagenbinding protein and the fibrinogen-binding proteins clumping factors A and B (ClfA and ClfB) are MSCRAMMs (Gotz, 2002) and play a primary role in the initial attachment of bacteria to cells and biomaterials (Patti et al., 1994; Gotz, 2002; Beenken et al., 2004; Yarwood et al., 2004).  2.1.3.3 Biofilm production After Staph. aureus have become established on a cell surface they may begin to produce a biofilm, which is a complex bacterial community contained within a polysaccharide matrix. The production of multiple cell layers requires polysaccharide adhesins that promote adhesion between bacterial cells (Gotz, 2002). The polysaccharide intercellular adhesin, a component of the ECM, seems to be the primary adhesin responsible for this phenomenon (Heilmann et al., 1996; Yarwood et al., 2004). Adhesion between bacteria allows the formation of densely packed bacterial clusters, with channels running through for delivery of water and nutrients and to facilitate the removal of waste (Annous et al., 2009). Biofilms confer bacterial protection against host defenses (Shiau and Wu, 1998; Stewart, 2002), physical and chemical stresses (Annous et al., 2009) and antibiotics, by  7  hindering delivery of antimicrobial agents (Stewart, 2002) and as a result of the intrinsically antibiotic resistant phenotype that is characteristic of biofilm-associated bacteria (Ceri et al., 1999; Beenken et al., 2004). Since bacteria within a biofilm typically have a slower growth rate than planktonic bacteria, this reduces the impact of antibiotics that target cell wall synthesis; in addition, the decreased oxidative metabolism of biofilm-associated bacteria limits their uptake of aminoglycosides (Lewis, 2001; Stewart and Costerton, 2001; Stewart, 2002). The frequency of genetic transfer within biofilms is typically higher than between planktonic bacteria (Annous et al., 2009), facilitating the spread of antibiotic resistance genes (Licht et al., 1999; Maeda et al., 2006). The ensuing tendency of biofilm-associated infections to resist antibiotics often leads to chronic disease, which may necessitate surgery to remove the source of the biofilm. This applies particularly to cases of indwelling biomedical devices such as prosthetic heart valves, central venous catheters, urinary catheters and prosthetic joints (Gander, 1996; Cirioni et al., 2003).  The genetic regulation of biofilm formation is complex and not yet fully elucidated (Lim et al., 2004). A cell to cell signalling system known as quorum sensing, which allows bacteria to communicate with each other, is involved in the formation of biofilm. Bacteria produce molecules that, when the bacterial cell density and therefore the signalling molecule concentration are high enough, bind to receptors that either activate or repress the transcription of certain genes. This stimulates or represses biofilm formation and allows bacteria to behave as an integrated community, producing a unified response to environmental stress (Annous et al., 2009).  8  2.1.4 Staph. aureus infections in livestock and poultry The pathogenicity of Staph. aureus has serious consequences for human and animal health (Trivier and Courcol, 1996; Bayles et al., 1998; Smeltzer and Gillaspy, 2000; Zadoks et al., 2000; Fitzgerald et al., 2001a, b; Diarra et al., 2002; Hazariwala et al., 2002; Oliveira et al., 2002; Sifri et al., 2003; White et al., 2003; Ben Zakour et al., 2008). Staph. aureus is one of the main pathogens responsible for bovine mastitis (Forsman et al., 1997; Bayles et al., 1998; De Oliveira et al., 2000; Gentilini et al., 2000; Hebert et al., 2000; Zadoks et al., 2000; Fitzgerald et al., 2001a; Vasudevan et al., 2003; Bannerman et al., 2004), which results in a loss of approximately $2 billion per year according to the United States National Mastitis Council (Harmon, 1996). Bacterial chondronecrosis, associated mainly with Staph. aureus, is a common poultry disease (Nairn, 1973; Riddell, 1980; Daum et al., 1990; Cook, 2000; McNamee and Smyth, 2000; Smeltzer and Gillaspy, 2000; White et al., 2003) and was the primary cause of lameness in broiler chicken flocks (Thorp and Waddington, 1997; McNamee et al., 1998). Bacterial chondronecrosis is also known as osteomyelitis, bacterial chondronecrosis with osteomyelitis or femoral head necrosis. Additional poultry diseases caused by Staph. aureus include joint infections such as arthritis (Nairn, 1973; Riddell, 1980; Daum et al., 1990; Smeltzer and Gillaspy, 2000; Jensen and Miller, 2001; Hazariwala et al., 2002) and synovitis (Nairn, 1973; Riddell, 1980; Jensen and Miller, 2001; White et al., 2003); omphalitis, also known as yolk sac infection (Nairn, 1973; Cervantes et al., 1988; Jensen and Miller, 2001; Hazariwala et al., 2002; White et al., 2003; Cortes et al., 2004); blood infections such as bacteremia (Daum et al., 1990) and  9  septicemia (McCullagh et al., 1998; Jensen and Miller, 2001; Hazariwala et al., 2002); wound infections (Cervantes et al., 1988); and skin infections such as dermatitis (Cervantes et al., 1988; McCullagh et al., 1998; Jensen and Miller, 2001; Hazariwala et al., 2002; White et al., 2003), cellulitis (White et al., 2003) and abscesses (McCullagh et al., 1998; Jensen and Miller, 2001).  2.2 Antibiotic resistance 2.2.1 Staph. aureus antibiotic resistance The use of antibiotics for clinical and veterinary medicine, as well as for incorporation into animal feed to control sub-clinical infections and promote animal growth, may contribute to the emergence of bacterial antibiotic resistance by creating a selective pressure favourable for resistant strains. The drastic increase in antibiotic resistant Staph. aureus strains has been attributed to extensive use of antibiotics (Rolinson, 1994; Gruson et al., 2000; Hiramatsu, 2001; Graffunder and Venezia, 2002).  Staph. aureus tends to develop resistance to antibiotics very quickly, which necessitates the continuous development of new drugs. Methicillin was the main antibiotic that was developed in response to penicillin resistant Staph. aureus strains that emerged in the 1940’s (Revazishvili et al., 2006). However Staph. aureus soon became resistant to methicillin in addition to other antibiotics such as erythromycin, tetracycline, rifampicin, aminoglycosides and quinolones (Lilly and Lowbury, 1978; Aubry-Damon et al., 1998; Ito et al., 2003; Revazishvili et al., 2006). Vancomycin and teicoplanin are glycopeptides that are used as last resort antibiotics to treat methicillin-resistant Staph.  10  aureus (MRSA). Staph. aureus are beginning to show resistance to these drugs as well (Revazishvili et al., 2006; Sader et al., 2009; Wohlleben et al., 2009). The rise in multidrug resistant Staph. aureus strains poses a serious concern for public health.  Staph. aureus develops resistance to antibiotics by either the mutation of a gene on its chromosome, or by acquiring an antibiotic resistance gene from another bacterium through conjugation, transduction or transformation (Ito et al., 2003). There is concern that MRSA may obtain the vancomycin resistance gene from enterococci.  2.2.2 Antibiotic use and development of antibiotic resistance The vanA gene confers resistance to vancomycin and teicoplanin, resulting in vancomycin resistant enterococci (VRE). There is a positive correlation between the use of avoparcin as a growth promoter in farm animals and an increased prevalence of VRE in the feces of these animals (Klare et al., 1995; Aarestrup, 1995; Aarestrup et al., 1996; McDonald et al., 1997; van den Bogaard and Stobberingh, 1999). Avoparcin promotes the development of cross-resistance in bacteria to medically important glycopeptides such as vancomycin and teicoplanin (Bates, 1997; Endtz et al., 1997). The same trend was observed in the commensal flora of healthy humans and pets in the Netherlands (McDonald et al., 1997; van den Bogaard et al., 1997), Belgium (Devriese et al., 1996) and Denmark (Aarestrup et al., 1996), even with very limited use of vancomycin in hospitals throughout Europe. This suggests that using antibiotics as growth promoters may increase the prevalence of antibiotic resistant bacteria that can impact human health as well as agriculture. Additionally, administration of teicoplanin, vancomycin or  11  streptomycin to healthy human volunteers or to an experimental mouse model significantly increased VRE colonization of subjects’ intestinal tracts (Van der Auwera et al., 1996; Whitman et al., 1996). Cephalosporins, another important contributor to VRE, also select for resistance to beta-lactams such as penicillins and cephalosporins (Ballow and Schentag, 1992; Jones, 1992; Yates, 1999). Furthermore, use of the fluoroquinolone enrofloxacin as a growth promoter in poultry was linked to quinolone-resistant Campylobacter spp. infections of humans (Endtz et al., 1991); that phenomenon supports the hypothesis that agricultural use of antibiotics has important implications for human health. Neuhauser et al. (2003) found that gram-negative aerobic bacteria, isolated from intensive care unit patients in hospitals across the USA between 1994 and 2000, had increased frequency of resistance to antibiotics, particularly to ciprofloxacin, over the study period. This may be linked to a concurrent national increase in the use of fluoroquinolones.  2.2.3 Reduction of antibiotic resistance following restriction of antibiotic use Studies have shown that restricting antibiotic use may effectively decrease antibiotic resistance. After avoparcin was banned in Europe in 1997, numerous studies reported a reduction in the prevalence of VRE in the fecal matter of farm animals (Bager et al., 1999; Klare et al., 1999; Pantosti et al., 1999; van den Bogaard et al., 2000) and healthy humans (Klare et al., 1999; van den Bogaard et al., 2000). Similarly, decreased prevalence of dalfopristin/quinupristin-resistant enterococci was observed in farm animals and humans during a time of limited virginiamycin availability (van den Bogaard et al., 2000). Studies also show that restricting cephalosporin use may lead to a decrease  12  in cephalosporin resistance (Ballow and Schentag, 1992; Rice et al., 1996; Rahal et al., 1998).  2.2.4 Multidrug-resistant bacteria The main concern regarding VRE is their potential to transfer the vanA gene to more pathogenic bacteria (Bates, 1997). The VanA plasmid carries resistance genes not only for vancomycin and teicoplanin, but also for tetracycline, streptomycin and erythromycin. Therefore, a variety of growth promoters, such as tylosin, which shows cross-resistance to erythromycin, may non-specifically select for the vanA gene (Bates, 1997). Another implication is that transmission of the VanA plasmid increases resistance to additional antibiotics besides vancomycin. E. coli, which is part of the normal flora of humans and many animals, can easily exchange DNA with other members of Enterobacteriaceae and may act as a reservoir of antibiotic resistance genes for these bacteria (Diarrassouba et al., 2007; Diarra et al., 2007; Costa et al., 2008). Staph. aureus, Listeria monocytogenes and several Streptococcus spp. have also been shown to develop vancomycin resistance through obtaining the vanA gene from Enterococcus faecium or Enterococcus faecalis (Leclercq et al., 1989; Noble et al., 1992, respectively). If this transfer would occur in MRSA, the resulting methicillin- and vancomycin-resistant Staph. aureus could cause infections that would be nearly untreatable (Bates, 1997).  Conjugative transposons, DNA transfer elements that have characteristics similar to plasmids, transposons and phages, contribute to the spread of antibiotic resistance genes (Salyers et al., 1995). The frequency that conjugative transposons are transferred by is  13  increased 10- to 1000-fold by pre-exposure to tetracycline (Valentine et al., 1988; Torres et al., 1991; Stevens et al., 1993). Therefore, the use of tetracycline as a growth promoter in livestock feed may select for antibiotic resistant bacterial strains and also enhance the transmission of DNA that contains resistance genes. Some of these transmissible elements have a wide range of bacterial hosts and therefore, can be spread among many different types of bacteria (Bertram et al., 1991). Similar to the VanA plasmid, conjugative transposons carrying resistance genes to tetracycline may also possess genes encoding resistance to a variety of antibiotics such as aminoglycosides, macrolides, lincosamides, streptogramins and chloramphenicol (Courvalin and Carlier, 1986; Ayoubi et al., 1991). This may lead to increased selective pressure for these conjugative transposons. Additionally, it means that the spread of these transmissible elements may result in increased resistance to not only tetracycline but to other clinically significant antibiotics as well (Speer et al., 1992).  2.3 Cranberry 2.3.1 Health benefits and antimicrobial activity Cranberries have been associated with a wide array of health benefits such as maintaining cardiovascular health (Wilson et al., 1998; Porter et al., 2001; Reed et al., 2001; Reed 2002; Yan et al., 2002; Ruel et al., 2005; Vattem et al., 2005a; Ruel et al., 2006; Neto, 2007a) and protecting against cancer (Bomser et al., 1996; Guthrie, 2000; Sun et al., 2002; Ferguson et al., 2004; Vattem et al., 2005a; Neto, 2007a; Kresty et al., 2008; Neto et al., 2008; Liu et al., 2009; Singh et al., 2009). Perhaps the most well-known health benefit of cranberries is their ability to reduce the incidence of urinary tract infections  14  (UTIs). In addition to their effect on urinary tract health, cranberries possess antimicrobial activity against a variety of bacteria (Puupponen-Pimia et al., 2001; Vattem et al., 2004a,b; Weiss et al., 2004; Lin et al., 2004; Lin et al., 2005; Puupponen-Pimia et al., 2005; Vattem et al., 2005b; Nohynek et al., 2006; Apostolidis et al., 2008; Magarinos et al., 2008; Wu et al., 2009). Daily use of a cranberry NDM-containing mouthwash over a period of six weeks significantly reduced the level of salivary mutans streptococci as well as total bacterial counts, compared to a placebo mouthwash. However, the NDMcontaining mouthwash had no effect on the plaque or gingival indices (Weiss et al., 2004). Cranberries were used for food and medicine among North American First Nation societies (Henig and Leahy, 2000; Carson and Riley, 2003; Lynch, 2004; Raz et al., 2004; Nowack, 2007). The fact that cranberries have a long history of use in preventing wound infections and treating sepsis suggests that there is minimal potential for the development of bacterial resistance against cranberry.  2.3.2 Ability of cranberries and cranberry extracts to reduce the incidence of UTIs Cranberries have long been used for the prevention of UTIs, and there is increasing scientific evidence to support this use (Bodel et al., 1959; Moen, 1962; Sternlieb, 1963; Papas et al., 1968; Bate-Smith, 1973; Gibson et al., 1991; Rogers, 1991; Avorn et al., 1994; Haverkorn and Mandigers, 1994; Walker et al., 1997; Dignam et al., 1998; Henig and Leahy, 2000; Kontiokari et al., 2001; Howell, 2002; Stothers, 2002; Griffiths, 2003; Jepson et al., 2004; Raz et al., 2004; Bailey et al., 2007; Jepson and Craig, 2007; Nowack, 2007; Hess et al., 2008). Avorn et al. (1994) conducted a randomized, doubleblind, placebo-controlled trial to determine the effect of cranberry juice on bacteriuria  15  and pyuria in elderly women. They found that the group that drank cranberry juice had fewer cases of bacteriuria with pyuria than the control group that drank a placebo drink without cranberry. Furthermore, subjects in the group that consumed cranberry were able to clear these infections within a month more often than subjects in the placebo group.  It was previously thought that the ability of cranberry to help reduce the incidence of UTIs was either due to acidification of the urine (Bodel et al., 1959; Kinney and Blount, 1979) or to hippuric acid, a strong antibacterial agent and metabolite of quinic and benzoic acids found in cranberries (Howell, 2002). However Kahn et al. (1967) found that at least 1500 ml of cranberry juice should be consumed each day in order to achieve the urinary pH and hippuric acid concentration necessary for this effect. Considering that cranberry juice exerts an antibacterial effect at much lower doses, there must be another mechanism of action (Raz et al., 2004).  2.3.3 Bacterial anti-adhesion mechanism of cranberries and cranberry extracts The ability of cranberry to prevent bacterial adhesion to cell surfaces is now regarded as the likely mechanism of preventing UTIs (Sobota, 1984; Schmidt and Sobota, 1988; Zafriri et al., 1989; Ofek et al., 1991; Howell et al., 1998; Howell and Foxman, 2002; Raz et al., 2004; Howell et al., 2005; McMurdo et al., 2005; Di Martino et al., 2006; Liu et al., 2006). In order to cause a UTI, bacteria, typically uropathogenic P-fimbriated E. coli (Nowack, 2007), must first adhere to uroepithelial cells (Howell et al., 2005) by attachment of their fimbrial adhesins to carbohydrate receptors on the cell surface (Beachey, 1981; Foo et al., 2000a,b; Johnson et al., 2003). Cranberry proanthocyanidins  16  (PACs) have unique A-type linkages (Foo et al., 2000a,b; Howell et al., 2005) (Appendix A) and possess bacterial anti-adhesion ability (Howell et al., 1998; Foo et al., 2000a; Howell et al., 2005); these A-type PACs are thought to be the active ingredient in cranberry that helps prevent UTIs. Cranberry juice and A-type PACs isolated from cranberry have been shown to prevent adhesion of P-fimbriated uropathogenic E. coli to in vitro bladder epithelial cells (Gupta et al., 2007), uroepithelial cells (Sobota, 1984; Schmidt and Sobota, 1988; Zafriri et al., 1989; Ofek et al., 1991; Ofek et al., 1996; Howell et al., 1998; Foo et al., 2000a,b; Howell et al., 2005; Turner et al., 2005; Di Martino et al., 2006; Liu et al., 2006), biomaterials commonly used to make urinary catheters (Eydelnant and Tufenkji, 2008) and cell surfaces with receptor sequences similar to those found on uroepithelial cells (Foo et al., 2000a,b). Cranberry PACs may also affect colonic bacteria (Zafriri et al., 1989), inhibiting their ability to bind to the uroepithelium if they were to travel up the urinary tract (Howell, 2002).  In addition to the effect of cranberry on the urinary tract, a high-molecular-weight component of cranberry juice inhibited the adhesion of H. pylori to human mucus, erythrocytes and cultured gastric epithelial cells (Burger et al., 2002), and suppressed H. pylori infection of humans (Zhang et al., 2005; Gotteland et al., 2008). Cranberry juice and its non-dialysable component inhibited adhesion of oral bacteria such as Streptococcus sobrinus to saliva-coated hydroxyapatite beads (Steinberg et al., 2004; Weiss et al., 2004; Yamanaka et al., 2004).  17  Additionally, consumption of cranberry products has been shown to induce bacterial antiadhesion activity of urine in both mice and humans (Sobota, 1984; Howell et al., 2001; Howell and Foxman, 2002; Greenberg et al., 2005; Howell et al., 2005; Di Martino et al., 2006), peaking between four and six h after consumption, and remaining for at least eight h (Howell et al., 2005). This suggests that bioactive PACs were absorbed and likely remained functional throughout metabolism. The detection of PACs in plasma of rats after they were fed grape seed extract supports the hypothesis that PACs are absorbed after ingestion (Prasain et al., 2009).  Conversely, B-type PACs isolated from cranberry (Foo et al., 2000a), apple juice, purple grape juice, green tea and dark chocolate (Howell et al., 2005) did not prevent adherence of P-fimbriated E. coli to cell surfaces with uroepithelial cell-like receptors, with the exception of purple grape juice, which showed very slight activity at a much higher concentration. None of these food products containing B-type PACs resulted in urinary anti-adhesion activity after consumption (Howell et al., 2005). The ability of cranberry juice to reduce non-specific adhesion of several foodborne pathogens to borosilicate glass microscope slides was not reproduced with white cranberry juice (Johnson-White et al., 2006); however, the level of A-type versus B-type PACs in the white cranberry juice was not reported in that study.  Fructose has also been shown to have anti-adhesion activity, specifically towards E. coli with type 1 fimbrial adhesins (Zafriri et al., 1989; Ofek et al., 1996). However, there have been no clinical studies supporting the use of fructose in maintaining urinary tract  18  health (Howell, 2002; Gupta et al., 2007). This may be due to the fact that fructose does not display anti-adhesive activity against P-fimbriae, which most E. coli isolates causing pyelonephritis possess (Vaisanen-Rhen et al., 1984; Arthur et al., 1989; Johnson, 1991; Stapleton, 2005; Di Martino et al., 2006; Lane and Mobley, 2007).  2.3.4 Effect of cranberries on bacterial biofilm formation Cranberry compounds inhibited biofilm formation in uropathogens, the causative agents of urinary tract infections (Reid et al., 2001), and oral pathogens, present in dental biofilm with a primary role in causing dental caries (Steinberg et al., 2004; Yamanaka et al., 2004; Steinberg et al., 2005; Duarte et al., 2006; Koo et al., 2006; Labrecque et al., 2006; Yamanaka et al., 2007). Cranberry juice and its non-dialysable component, as well as other PACs and flavonols derived from cranberry, significantly inhibited the activity of enzymes involved in oral biofilm formation (Steinberg et al., 2004; Duarte et al., 2006; Koo et al., 2006). Furthermore, cranberry non-dialysable material (NDM) promoted desorption of S. sobrinus from biofilm (Steinberg et al., 2005). Biofilm inhibition may be related to inhibition of extracellular polysaccharide synthesis (Steinberg et al., 2004), as well as to reduction of hydrophobicity (Yamanaka et al., 2004).  2.3.5 Cranberry PAC structure Cranberry PACs are composed primarily of epicatechin subunits of 4-10 degrees of polymerization, with at least one A-type interflavan linkage but often multiple A-type linkages, at each degree of polymerization (Foo et al., 2000b; Howell et al., 2005).  19  Cranberry PAC trimers had greater anti-adhesion activity than dimers, suggesting that a higher molecular mass may result in greater activity (Foo et al., 2000a). However, whether or not larger oligomers remain intact and are effective in vivo is not yet known (Howell, 2002).  2.4 Literature review pertaining to methodology 2.4.1 Minimum Inhibitory Concentration (MIC) Antibiotic susceptibility testing may be performed by several different methods, including agar dilution, E-test, disk diffusion, broth macrodilution and microdilution. The agar dilution method (Baker et al., 1991; Wust and Hardegger, 1992; de Castillo et al., 1998; Kirkpatrick et al., 1998; Simpson et al., 1998; Kelly et al., 1999; Reynolds et al., 2003) consists of finding the minimum inhibitory concentrations (MICs) of antibiotic against bacteria by dipping a sterile replicator into broth suspensions of the test strains, and touching the replicator to sterile agar plates containing doubling dilutions of antibiotic. Plates are incubated, and the MIC is determined as the lowest concentration of antibiotic that completely inhibits bacterial growth. The E-test (Baker et al., 1991; Wust and Hardegger, 1992; Kelly et al., 1999) determines MIC values by dipping a cotton swab into bacterial suspensions and streaking to cover the entire surface of an agar plate. After the surface has dried, the E-test strip is placed onto the agar surface and incubated. The MIC is recorded as the point where the zone of inhibition intersects the MIC scale on the strip. Disk diffusion (Baker et al., 1991; de Castillo et al., 1998; Kelly et al., 1999) involves using a cotton swab to cover the entire surface of an agar plate after the swab has been dipped in bacterial inoculum. When the surface of the agar has dried, antibiotic  20  disks are aseptically placed on the agar plate followed by incubation under prescribed conditions. The diameters of the zones of inhibition are measured to the nearest millimetre. Broth macrodilution (Kirkpatrick et al., 1998) and microdilution (Baker et al., 1991; Kelly et al., 1999; Reynolds et al., 2003) determine MIC values by inoculating sterile broth containing doubling dilutions of antibiotic with bacterial suspensions followed by incubation under prescribed conditions. Broth macrodilution uses test tubes, while broth microdilution uses microwell plates. There is generally very good agreement between results obtained by agar dilution, E-test, disk diffusion, broth macrodilution and microdilution (de Castillo et al., 1998; Kirkpatrick et al., 1998; Kelly et al., 1999; Reynolds et al., 2003).  Agar dilution testing in North America follows the Clinical and Laboratory Standards Institute (CLSI) (formerly the National Committee for Clinical Laboratory Standards (NCCLS)) methods, while the CLSI guidelines or those recommended by the British Society for Antimicrobial Chemotherapy are used in Europe (Simpson et al., 1998; Louie and Cockerill, 2001). These methods are similar, with the choice of test media typically being the only difference between them. However laboratories in France and Germany follow testing guidelines recommended by the Societe Francais de Microbiologie and the Deutsche Industrie Norm-Medizinische Mikrobiologie (DIN), respectively (Simpson et al., 1998). A comparison of antibiotic susceptibilities of E. coli strains analysed by the NCCLS and DIN guidelines showed significant differences between the methods (Simpson et al., 1998). This was attributed to methodological variation as well as to a  21  difference in breakpoints categorizing strains as susceptible, intermediate or resistant, with NCCLS permitting higher MICs than DIN for a particular category.  2.4.2 Bacterial morphology Many different types of microscopes are available to visualize bacterial morphology, including bright field (Nandy et al., 2007), dark field (Merrell et al., 2002), phase contrast (Kahl et al., 2000), differential interference contrast (Kahl et al., 2000) and fluorescence microscopes (Kahl et al., 2000; Leevy et al., 2006; Nandy et al., 2007). In addition, transmission electron microscopes (Shimoda et al., 1995; Kahl et al., 2000; Bobin-Dubreux et al., 2001; Diarra et al., 2002) and scanning electron microscopes (Kahl et al., 2003) have been used to provide greatly magnified images with excellent resolution.  2.4.3 ECM binding The ability of bacteria to bind to ECM proteins may be measured by their adherence to protein-coated wells of a microplate (Dziewanowska et al., 1999). Wells are coated with protein solution or bovine serum albumin (BSA) to estimate nonspecific binding and rinsed. Residual binding sites are blocked with heat-denatured BSA in Hanks’ balanced salt solution (HBSS). Bacterial inoculum is added and allowed to incubate, and the wells are rinsed to remove nonadherent bacteria. Adherent bacteria are detached with trypsin and quantified by plating serial dilutions.  22  Bauer and Spinola (1999) developed an enzyme-linked immunosorbent assay (ELISA)based method to assess bacterial binding to ECM proteins. Similar to the microplate method described above, wells are coated with ECM proteins, BSA or fetuin to estimate nonspecific binding, rinsed, and residual binding sites are blocked. Bacteria are added to wells and incubated, and the wells are rinsed to remove unbound bacteria. Adherent bacteria are detected by incubating with rabbit antiserum against the bacterial cells, followed by horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. ELISA plates are incubated with horseradish peroxidase substrate, and the absorbance at 450 nm is measured for each well.  Alternatively, the ECM-binding ability of bacteria may be measured by their ability to bind to protein-coated latex beads. ECM protein is adsorbed onto latex beads and used in a particle agglutination assay, where the beads are mixed with an equal volume of bacterial inoculum on a glass slide and examined for agglutination (Naidu et al., 1988; Styriak et al., 1999, 2002).  2.4.4 Biofilm formation and removal The extent of biofilm formation or removal may be measured by a number of different methods, such as the test tube method, the Congo red agar plate test, microscopy and the microwell plate test (Arciola et al., 2002; Stepanovic et al., 2007). The test tube method involves inoculating broth in a test tube with an isolated colony followed by incubation at an appropriate temperature. The inoculum is removed, tubes are rinsed and dried, and culture tubes stained with an appropriate dye such as trypan blue, safranin or crystal  23  violet. Excess stain is removed; tubes are rinsed and dried upside down. Biofilm production is considered positive when a visible film remains on the test tube walls (Christensen et al., 1985; Mathur et al., 2006). Alternatively, crystal violet that has stained adherent biofilm may be extracted with a solvent such as dimethylsulphoxide, and the absorbance measured by spectrophotometry at 585 nm (Henoun Loukili et al., 2004).  Congo red agar is used to measure biofilm formation by the appearance of colonies. Bacteria are streaked onto congo red agar and plates are incubated. Biofilm producing bacteria form black or very dark colonies, generally with a dry crystalline consistency, whereas biofilm negative bacteria form pink or red colonies (Arciola et al., 2002; Mathur et al., 2006).  Fluorescence microscopy and confocal laser scanning microscopy provide highresolution images of biofilm, and can serve as a measure of biofilm formation or removal (Hyde et al., 1997; Marion-Ferey et al., 2003). In fluorescence microscopy, biofilms are stained with a dye and examined at a wavelength appropriate for the selected dye. The extent of biofilm formation is detected visually, through use of a digital camera or a photomultiplier. Image analysis software such as Scion Image can be used to determine the amount of surface area that is covered by biofilm (Marion-Ferey et al., 2003). Biofilm samples may also be examined by confocal laser scanning microscopy, which provides three dimensional images and a measurement of the biofilm thickness.  24  The microwell plate assay is the most common method used for the analysis of biofilm formation (Stepanovic et al., 2007). In this method, bacterial broth suspensions with or without treatments are incubated in a 96-well microplate to form a biofilm. The inoculum is removed, wells are rinsed, and adherent organisms may or may not be fixed with a solution such as Bouin fixative or 2% sodium acetate. Biofilm is stained with crystal violet. At this point either the biofilm can be measured in the original wells (Christensen et al., 1985; Vasudevan et al., 2003; Kaplan et al., 2004; Mathur et al., 2006), or the crystal violet can be extracted and transferred to a new microwell plate for a more homogenous sample (Narisawa et al., 2005; Yoshida et al., 2009). If the biofilm is left in the original wells, the crystal violet solution is removed and the wells are rinsed to remove unbound crystal violet and the biofilm is dried at room temperature. Biofilm absorbance is measured at 570 nm with a microplate reader. Alternately, the crystal violet solution is removed and the wells are rinsed. The remaining crystal violet that has stained the biofilm is then extracted with 70 - 95% ethanol and transferred to a new microwell plate and the absorbance is measured at 590 nm with a microplate reader.  The microwell plate assay may also be used to examine the ability of a particular treatment to remove pre-existing biofilm (Pitts et al., 2003; Kaplan et al., 2004). The method is essentially the same as when examining biofilm formation, except that treatments are applied after bacteria have formed biofilm, rather than being incubated along with the bacterial suspension. After removing the treatment solutions and rinsing wells, biofilm is stained with crystal violet and measured as described above.  25  2.4.5 Biofilm enumeration Viable plate counts and direct counts are common assays used to enumerate the bacteria present in a biofilm. For viable plate counts, a bacterial suspension is incubated in a tube or microplate to form a biofilm, after which the inoculum is removed. Tubes or wells are then rinsed to remove non-adherent bacteria. At this point several different methods may be used to quantify bacteria. Biofilm-associated bacteria may be resuspended by adding sterile solution to tubes or wells and sonicating (Kadouri and O’Toole, 2005; Tre-Hardy et al., 2008). Alternatively, biofilm can be removed from wells by scraping with a sterile spatula or cell scraper and vigorously vortexing in sterile saline or phosphate buffered saline (PBS) to disperse cells (Di Bonaventura et al., 2004; Kaplan et al., 2004). Serial dilutions of resuspended biofilm bacteria are enumerated by plating onto agar.  Biofilm-associated bacteria may also be enumerated through direct counts (Huang and Stewart, 1999; Ahmad et al., 2008). A biofilm is allowed to form as described above, wells are rinsed to remove planktonic bacteria, and the biofilm is stained with acridine orange. Bacteria are examined with a fluorescence microscope and visually counted.  2.4.6 Salt tolerance Staph. aureus has a particularly high tolerance to salt, and uninjured cells remain viable in the presence of NaCl (Chapman, 1945). However bacterial injury results in a loss of salt tolerance (Hurst et al., 1973; Wyber et al., 1994; Carson et al., 2002). The degree of injury caused by a treatment is determined by the ability of treated Staph. aureus to produce colonies on salt-containing media, and compared with its viability on regular  26  media. The concentration of salt used in the assay is typically about 7.5% (Hurst et al., 1973), as usually only staphylococci are able to grow on media containing 7.5% NaCl (Chapman, 1945). Media such as nutrient agar or trypticase soy agar are generally used for either spot plating or pour plating (Hurst et al., 1973; Tomlinson and Palombo, 2005). However Bruins et al. (2007) used mannitol broth with NaCl concentrations ranging from 1.5 to 12.5%, from which they determined the maximal non-inhibiting concentrations and the MICs.  27  3. MATERIALS AND METHODS 3.1 Cranberry extract Cranberry extract (spray-dried cranberry juice concentrate powder standardized to a minimum of 90% cranberry fruit solids and 35% organic acids, containing 0.6 – 0.95% PACs) was kindly provided by Decas Botanical Synergies (Wareham, MA, USA) (Appendix B). A high-molecular-weight NDM was prepared from this cranberry extract by the method of Zafriri et al., 1989. Briefly, cranberry extract was dialysed at 4oC for three days against six changes of water that had been treated through the Barnstead nanopure water system, using Spectrum Spectra/Por 7 dialysis bags (Spectrum Laboratories Inc., Rancho Dominguez, CA, USA) with a 15,000-molecular-weight cutoff. The NDM was freeze-dried to a powder, and the resulting extract (CE90) was stored at 4oC in the dark. For each experiment, CE90 was reconstituted in nanopure water the same day of the experiment. Fresh CE90 stock solutions (128 mg ml-1) were filter sterilised with a 0.2 µm nylon syringe filter (Fisher Scientific, Ottawa, ON, Canada) and diluted to the desired concentration (2, 4, 8, 16 mg ml-1) in media. CE90 concentrations were chosen based on the MIC of CE90 for the tested Staph. aureus isolates, to analyse the bioactivity of inhibitory and sub-inhibitory concentrations of CE90 against Staph. aureus.  3.2 Staph. aureus isolates Twenty-four Staph. aureus isolates were used in the in vitro evaluation of CE90. These included bovine and poultry isolates and Staph. aureus PC1, SA 113, 8325-4, 8325-4 fnbA-/fnbB-, 8325-4 clfA- and 2076 from the collection at Agriculture and Agri-Food  28  Canada (Agassiz, BC, Canada), as well as Staph. aureus ATCC 25923 and 29213 from the American Type Culture Collection that were used as reference strains. Staph. aureus SA 113 and 2076 were chosen as the biofilm positive and negative isolates, respectively, based on the literature (Cucarella et al., 2001) and a preliminary study that confirmed a high level of biofilm production by Staph. aureus SA 113 and a low level of biofilm production by Staph. aureus 2076 (Appendix C). Staph. aureus 8325-4, 8325-4 fnbA/fnbB- and 8325-4 clfA- were chosen to test the effect of CE90 on fibronectin binding ability in a strain that possesses fibronectin binding proteins as well as clumping factor A, which allows Staph. aureus to bind to fibrinogen, and the two mutant strains that each possess one of these adhesins but not the other. All 24 Staph. aureus isolates from the collection at Agriculture and Agri-Food Canada were used in order to evaluate the MIC of CE90 against a range of different Staph. aureus isolates. All culture media were from Becton, Dickinson and Company (Sparks, MD, USA). Staph. aureus from frozen stock at -80°C in tryptic soy broth (TSB) containing 25% glycerol were streaked onto TSA plates or TSB and then incubated for 18-24 h at 37°C. For most experiments, the isolates were sub-cultured in TSB, on agar plates (TSA), Mueller Hinton agar plates (MHA) or in Mueller Hinton broth (MHB) for an additional 18-24 h.  3.3 Determination of MIC The MIC of CE90 against 24 Staph. aureus isolates was determined by the agar dilution method according to the NCCLS (1999). A 48-pin replicator was sterilized by dipping in 95% ethanol and flaming with a bunsen burner and then cooled by touching the surface of a sterile TSA plate. The sterility of the TSA plate and the replicator pins was  29  confirmed by incubating the TSA plate for 24 h at 37oC. The replicator was dipped into a 96-well plate containing 100 µl well-1 of Staph. aureus (1.5 x 108 cfu ml-1 in PBS) (one isolate per well). MHA plates containing doubling dilutions of CE90 ranging from 64 mg CE90 ml-1 – 1 mg CE90 ml-1 were inoculated with approximately 300 colony forming units (cfu) spot-1 by touching the replicator to the surface of the agar. Plates were incubated at 37oC, and the MIC of CE90 against each isolate was determined at 24 h. The MIC was defined as the lowest concentration of CE90 that completely inhibited Staph. aureus after 24 h incubation at 37oC.  3.4 Staph. aureus morphology The effect of CE90 on Staph. aureus morphology was examined by exposing 10 Staph. aureus isolates to 0, 4, 8 or 16 mg CE90 ml-1 in TSB, TSB + 7.5% NaCl, or TSB + 7.5% EDTA at 37oC. Isolates used were PC1, SA 113, 8325-4, 8325-4 fnbA-/fnbB-, 8325-4 clfA-, 2076, SHY97-4320, ABB03-2487, ATCC 6538 and ATCC 29213. Ten µl samples of an isolate suspended in each treatment (CE90 in TSB with or without NaCl or EDTA) were placed on glass slides and covered with a coverslip. The edges of coverslips were sealed onto the glass slides with fingernail polish to reduce the evaporation of samples. Samples of Staph. aureus in this CE90-TSB solution were observed at time 0 and after 5, 10, 15, 20 and 60 min incubation at 37oC without shaking. Glass slides were examined by phase contrast microscopy (National 163 PH, Microscope World, Carlsbad, CA, USA) at 400x magnification and digitally photographed.  30  3.5 Attachment to ECM protein (fibronectin) Staph. aureus adherence to fibronectin was evaluated by the method described by Dziewanowska et al., 1999 using three Staph. aureus strains (8325-4 parental strain and two mutant strains, 8325-4 fnbA-/fnbB- deficient in FnbpA and FnbpB, and 8325-4 clfAdeficient in ClfA). Briefly, wells in a 96-well microtiter plate (Costar Inc., Corning, NY, USA) were coated with 100 µl of fibronectin solution from bovine plasma (lyophilized powder, cell culture tested, Sigma, Oakville, Ontario, Canada, 50 µg ml-1 in HBSS (10 x, liquid, cell culture tested, Sigma) with bivalent ions (0.35 g L-1 sodium bicarbonate)) and incubated overnight at room temperature. The wells were then rinsed three times with HBSS with bivalent ions, and residual binding sites were blocked with 100 µl of heatdenatured 0.1% BSA (powder, cell culture tested, Sigma) in HBSS (without bivalent ions) for 60 min at room temperature. The wells were rinsed with TSB containing 0.25% glucose with 0.1% heat-denatured (90oC water bath, 10 min) BSA. Staph. aureus were incubated with 0, 4, 8 or 16 mg CE90 ml-1 TSB for 2 h at 37oC on an orbital platform shaker at 190 rpm. Samples were centrifuged at 12,850 x g for 12 min at 20oC. The Staph. aureus pellets were resuspended in PBS and centrifuged at the same settings two more times. Pellets were resuspended in TSB containing 0.25% glucose with 0.1% heatdenatured BSA. Then 100 µl of Staph. aureus cell suspension (5 × 105 cfu ml-1 in this TSB-glucose-BSA solution) were added to each well. The plates were incubated for 1 h at room temperature and rinsed four times with PBS to remove non-adherent Staph. aureus. Adherent Staph. aureus were detached from the wells by incubating with 0.25% porcine trypsin in an HBSS with phenol red solution containing 0.02% EDTA (Sigma) for five min at 37oC. Detached Staph. aureus were serial diluted in PBS to the 10-6  31  dilution, and 20 µl were drop plated onto TSA. Plates were incubated at 37oC for 18 – 24 h, and colonies were counted. The results were recorded as the protein adherence index = (cfu ml-1 recovered / cfu ml-1 added)*100.  3.6 Biofilm formation The effect of CE90 on biofilm formation in Staph. aureus PC1, SA 113, 8325-4 and 2076 was evaluated according to the modified method of Vasudevan et al. (2003). TSB containing 0.25% glucose and 0, 2, 4, 8 or 16 mg CE90 ml-1 (195 µl) was added to sterile 96-well flat bottom polystyrene tissue culture plates along with 5 µl of inoculum (approximately 1.5 x 108 cfu ml-1) and incubated at 37oC for 18 – 24 h. The wells were rinsed three times with sterile PBS to remove planktonic cells, dried at room temperature and stained with 1% (w/v) crystal violet (microscopy grade, Sigma) (200 µl well-1) for 15 min at room temperature. After removing the crystal violet and rinsing the wells with distilled water, the plate was thoroughly dried upside down at room temperature, and the absorbance was measured at 570 nm with a microplate reader. Staph. aureus SA 113 and Staph. aureus 2076 served as the biofilm positive and negative control isolates, respectively.  3.7 Biofilm removal The effect of CE90 on removal of Staph. aureus PC1, SA 113, 8325-4 and 2076 biofilms was evaluated according to the modified method of Vasudevan et al. (2003). TSB containing 0.25% glucose (195 µl) was added to sterile 96-well flat bottom polystyrene tissue culture plates along with 5 µl of inoculum (approximately 1.5 x 108 cfu ml-1) and  32  incubated at 37oC for 18 – 24 h. Inoculum was then removed by pipetting and wells were rinsed twice with sterile PBS to remove planktonic cells. Sterile PBS containing CE90 (0, 2, 4, 8 or 16 mg ml-1) were added to each well (200 µl) and left at ambient temperature for another 24 h. The CE90-PBS solutions were removed by pipetting, and wells were rinsed three times with sterile PBS. Wells were dried at room temperature and stained with 1% crystal violet (200 µl well-1) for 15 min at room temperature. After removing the crystal violet and rinsing the wells with distilled water three times, the plate was thoroughly dried upside down at room temperature, and the absorbance was measured at 570 nm with a microplate reader. Staph. aureus SA 113 and Staph. aureus 2076 served as the biofilm positive and negative control isolates, respectively.  3.8 Biofilm index The amount of Staph. aureus PC1, SA 113, 8325-4 and 2076 forming a biofilm versus remaining in a planktonic state during exposure to CE90 over a 24 h period was determined as follows. Sterile 24-well flat bottom polystyrene tissue culture plates (Cellstar TC-Plate with lid, DNase, RNase free) (Greiner Bio-One, Monroe, North Carolina, USA) containing 1.95 ml of TSB with 0.25% glucose and 0, 2, 4, 8 or 16 mg CE90 ml-1 were inoculated with 500 µl of Staph. aureus broth suspension (approximately 3.0 x 107 cfu ml-1). Tissue culture plates were incubated at 37oC for 2, 4, 6, 8 or 24 h. One 24-well plate was used for each incubation time, with each plate containing every CE90 concentration and the control. At each time interval, the specified tissue culture plate was removed from the incubator, and supernatant was removed from each well and stored at 4oC until the preparation of planktonic Staph. aureus serial dilutions (within the  33  hour). Wells were rinsed three times with sterile PBS to remove unattached Staph. aureus. Biofilm was removed by swabbing each well with a dry, sterile cotton swab. Each swab was transferred to 1 ml of 0.85% sterile saline where it was swirled and shaken vigorously to suspend biofilm-associated Staph. aureus. Cotton swabs were discarded and Staph. aureus suspensions in the tubes were vortexed for one min. Samples of supernatant containing planktonic Staph. aureus were removed from the fridge, and planktonic and biofilm Staph. aureus were each serial diluted in sterile saline to the 10-5 dilution and drop plated (10 µl) onto TSA. Plates were incubated at 37oC for 18 – 24 h, and planktonic and biofilm-associated Staph. aureus were enumerated as total planktonic cfu per well or total biofilm cfu per well. The biofilm index was calculated as: Biofilm index = (total cfu biofilm Staph. aureus / (total cfu biofilm Staph. aureus + total cfu planktonic Staph. aureus))*100.  3.9 Salt tolerance assay The modified method of Tomlinson and Palombo (2005) was used to evaluate the effect of CE90 in terms of the proportion of Staph. aureus killed versus injured. Four Staph. aureus isolates (PC1, SA 113, 8325-4 and 2076) were used in the assay. This was evaluated by examining the ability of treated Staph. aureus to grow on TSA supplemented with 7.5% NaCl. Briefly, Staph. aureus (3.0 x 107 cfu ml-1 final) in 96well polystyrene tissue culture plates were treated for two h with 0, 4, 8 or 16 mg CE90 ml-1 in TSB (222 µl final volume) at 37oC with shaking (175 rpm). Samples were centrifuged in 1.5 ml polypropylene microcentrifuge tubes (VWR, Edmonton, Alberta, Canada) with an Eppendorf 5810R centrifuge (VWR) for 10 min at 25,200 x g at room  34  temperature and resuspended in PBS twice to remove CE90. Samples were serial diluted in PBS to the 10-6 dilution, drop plated (10 µl) onto TSA and on TSA supplemented with 7.5% NaCl (TSAS) and incubated for 24 h at 37oC. For each Staph. aureus isolate and CE90 concentration, growth on either normal (TSA) or salt-containing media (TSAS) was calculated as a percentage of that isolate’s growth on TSA without CE90. Percent growth on TSA corresponds to the proportion of live Staph. aureus, while percent growth on TSAS corresponds to the proportion of uninjured Staph. aureus for each CE90 concentration. The number of Staph. aureus presumed to be killed is the difference between the TSA count at 0 mg CE90 ml-1 and other concentrations of CE90. The proportion of injured Staph. aureus was calculated as the difference between counts on TSA and TSAS for each concentration of CE90. % surviving = (cfu ml-1 on TSA / cfu ml-1 on TSA of isolate at 0 mg ml-1)*100 % killed = 100 – % surviving % healthy = (cfu ml-1 on TSAS / cfu ml-1 on TSA of isolate at 0mg ml-1)*100 % injured = % surviving - % healthy  3.10 Statistical analyses All data on bacterial numbers were log transformed before statistical analysis. For each experiment, the effects of different concentrations of CE90 were assessed on two different days in duplicate (biofilm formation and removal in quadruplicate) and were analysed using the General Linear Model procedure of SAS (Statistical Analysis System Institute Inc., 2000). Biofilm index data was analysed using the repeated measurements  35  option. Least significant difference was used to separate the means whenever the ANOVA F value was significant. The 0.05 P-value was used to declare significance.  36  4. RESULTS & DISCUSSION 4.1 Determination of MIC Ten of the 24 Staph. aureus isolates were completely inhibited by 16 mg CE90 ml-1 after 24 h incubation. The MIC of CE90 against the remaining 14 Staph. aureus isolates after 24 h was 32 mg CE90 ml-1 (Table 1).  The MIC values found in the current study were lower than the MIC of dried cranberry juice reported by Valentova et al. (2007) (179-357 mg ml-1), indicating that CE90 has a greater ability to inhibit Staph. aureus growth than the dried cranberry juice used in their study. However, other studies have reported biological activities of cranberry components against bacteria at much lower concentrations. Wu et al. (2008) found that 5 µl ml-1 of cranberry concentrate caused morphological damage to Staph. aureus, E. coli, L. monocytogenes and S. typhimurium, including breakage of cell walls and membranes. The wide range of cranberry concentrations capable of inhibiting bacterial growth may be due to differences in the source and variety of cranberries used between studies, variations in the susceptibility to cranberry compounds between different bacteria, as well as differences in experimental methods used to measure growth inhibition.  4.2 Staph. aureus morphology An examination of Staph. aureus morphology after treatment with CE90 permitted visualization of the effect on Staph. aureus. Staph. aureus treated with CE90 (4 – 16 mg ml-1) appeared as relatively large, dose-dependent clumps of Staph. aureus for all 10 test isolates (PC1, SA 113, 8325-4, 8325-4 fnbA-/fnbB-, 8325-4 clfA-, 2076, SHY97-4320,  37  ABB03-2487, ATCC 6538 and ATCC 29213) at each time interval tested (0, 5, 10, 15, 20 and 60 min exposure to CE90) (Figure 1a,b,c,d). The same phenomenon was found with Staph. aureus incubated in CE90-TSB solutions containing either 7.5% NaCl or 7.5% EDTA. There were no observable differences in Staph. aureus clumping in response to CE90 with the presence or absence of NaCl or EDTA. No clumping was found in the 0 mg ml-1 control for any isolate.  Staph. aureus clumping in the presence and absence of either NaCl or EDTA suggests that the observed clumping effect is not due to electrostatic interactions or to positive cations. The lack of clumping in the 0 mg ml-1 control suggests that the clumping was not due to autoaggregation of Staph. aureus.  To the author’s knowledge, the dose-dependent clumping observed upon exposure of Staph. aureus to CE90 has not been previously reported for CE90 or other cranberry components. However there have been several studies that report a morphological change of bacteria after treatment with cranberry products. Matsushima et al. (2008) found that H. pylori treated with cranberry extract changed from a spiral-shaped bacterium into a coccoid form, while Ahuja et al. (1998) showed that P-fimbriated E. coli grown on cranberry agar had an elongated morphology compared to the control. Wu et al. (2008) found that S. aureus, E. coli, S. typhimurium and L. monocytogenes treated with cranberry concentrate experienced breakage of cell walls and membranes, with lysate and cytoplasmic material surrounding bacterial cells.  38  The clumping effect may help to explain the ability of cranberry to reduce the incidence of UTIs. Large clumps of causative bacteria such as E. coli would have greater mass and therefore momentum when travelling along the urinary tract, and therefore likely would not attach to uroepithelial cells as easily as single bacteria in the absence of cranberry.  The tendency of Staph. aureus to agglutinate to form increasingly larger and therefore fewer clumps with increasing concentrations of CE90 has important implications for the interpretation of the results in the present study. Experiments using colony counts to determine the effect of CE90 on Staph. aureus may give misleading results, if one fails to account for the fact that a clump of bacteria will give rise to only a single colony, which is indistinguishable from a colony formed from a single cell. Since the clumping effect was not discovered until after the other experiments had already been performed, it was impossible to determine, after the fact, the extent to which clumping was responsible for a reduction in colony counts, versus the extent to which CE90 was having a real effect. The implications for the interpretation of results for each experiment will be discussed in more detail below.  4.3 Attachment to ECM protein (fibronectin) Considering the role of fibronectin-binding proteins in bacterial adhesion to cultured mammalian cells, airway epithelium, endothelium, biomaterials and intravascular catheters (Menzies, 2003), and the findings that cranberry can reduce the incidence of UTIs (Howell, 2002; Gormley, 2003) and inhibit bacterial binding to cultured uroepithelial (Turner et al., 2005) and gastric cells, erythrocytes and immobilized human  39  mucus (Burger et al., 2002), I examined the impact of CE90 on Staph. aureus adhesion to fibronectin-coated plates, using a strain deficient in FnbpA and FnbpB, a strain deficient in ClfA, and the parental strain (8325-4). CE90 (4 – 16 mg ml-1) did not show any significant effect (P > 0.05) on fibronectin binding in these strains of Staph. aureus, with the exception of the fibronectin-binding protein double-mutant strain fnbA-/fnbB-, which showed less fibronectin binding in the 4 mg CE90 ml-1 concentration than either the untreated control or the higher CE90 concentrations (8 – 16 mg ml-1) (P < 0.05) (Figure 2). The lack of any significant effect on Staph. aureus binding to fibronectin seems to support the conclusion that the anti-adhesion activity of CE90 was not due to interference with fibronectin binding. This would make sense if the anti-adhesion activity was due to Staph. aureus clumping rather than any effect on Staph. aureus fibronectin adhesins, in particular. However, the fact that CE90 causes Staph. aureus to clump complicates the interpretation of these results. Rather than having no effect, it is possible that CE90 inhibited Staph. aureus from binding to fibronectin, but that the few Staph. aureus that were able to bind were associated with a large clump of Staph. aureus. Since Staph. aureus that had bound to fibronectin were detached with a 0.25% trypsin-EDTA solution before enumeration, trypsin may also break the cell to cell adhesions within the clumps, if trypsin would digest Staph. aureus proteins that interact either with CE90 or with other Staph. aureus proteins that promote cell to cell adhesion, such as extracellular adherence protein or intercellular adhesin. Therefore, when serial dilutions and colony counts were made, the number of colonies formed would be much greater than the number of Staph. aureus that had actually bound to fibronectin. In this case, the ability of CE90 to inhibit Staph. aureus from binding to fibronectin would be masked by the clumping effect. It is  40  impossible to determine from this study whether the lack of significant differences was due to CE90 having no effect on fibronectin binding, or to inhibition of fibronectin binding that was masked by the clumping effect. Furthermore, CE90 could inhibit Staph. aureus binding to fibronectin as a consequence of clumping, in the sense that clumping would cause many of the Staph. aureus to be physically held apart from the fibronectin. Even if the number of Staph. aureus that had bound to fibronectin could somehow be determined, this still would not reveal whether the Staph. aureus within the clumps would have been able to bind to fibronectin, if they had been able to come in contact with it. Further research should be done to clarify the effect of CE90 on fibronectin binding, apart from the clumping effect. There were no significant differences in fibronectin binding between strains (P > 0.05) (Figure 2). It is unclear why the fnbA-/fnbB- strain has as much fibronectin binding as the parental strain. Further research should be done to clarify the reasons for this finding.  Several studies show that cranberry products have an effect on bacterial adhesion. Labrecque et al. (2006) found that 50 µg ml-1 of cranberry NDM inhibited Porphyromonas gingivalis adhesion to surfaces coated with collagen, fibrinogen or human serum. Koo et al. (2006) reported that treating saliva- or glucan-coated hydroxyapatite with cranberry juice resulted in a 40 – 85% reduction in adherence of Streptococcus mutans compared to the control. There was no pH effect reported in that study. Yet when S. mutans rather than hydroxyapatite was treated with cranberry juice, bacteria retained their ability to attach to these untreated surfaces. This finding suggests that cranberry compounds may have an anti-adhesive mechanism apart from directly  41  affecting bacterial adhesins. Eydelnant and Tufenkji (2008) found that there was less attachment of uropathogenic bacteria to biomaterials that urinary catheters are made from when either the bacteria or biomaterials were coated with high-molecular-weight PACs derived from cranberry. They found similar results for non-biological latex microspheres substituted for bacteria, which suggests a non-biospecific anti-adhesion mechanism of cranberry PACs. This supports the suggestion that the anti-adhesion activity of CE90 may be due to physical clumping of Staph. aureus. The results of Eydelnant and Tufenkji (2008) did not correlate with theoretical predictions of adhesion based on the sum of the predominant physicochemical forces between the bacteria and a solid surface. However, a model accounting for steric hindrance provided theoretical predictions of adhesion that agreed with experimental results. Therefore the anti-adhesion activity of cranberry PACs may be at least partially explained by steric interactions.  Furthermore, several studies support the hypothesis that cranberry compounds exert an effect on bacterial fimbriae as the mechanism of inhibiting bacterial adhesion. Liu et al. (2006) demonstrated that P-fimbriated E. coli exposed to cranberry juice adjusted to pH 7 have shorter fimbriae and higher densities of biopolymer around the bacterial cell than the control, as measured by atomic force microscopy (AFM). They postulated this was due to cranberry juice inducing a conformational change in the fimbriae, causing them to go from an extended to a compressed state. These changes were completely reversible when the cranberry juice was replaced with water. Increasing concentrations of cranberry juice were correlated with lower adhesion forces between the P-fimbriated E. coli and the AFM silicon nitride tip. Liu et al. (2006) hypothesized that the cranberry  42  compounds responsible for inhibiting bacterial adhesion were hydrophilic PACs, so their binding to the hydrophobic fimbriae decreased adhesion between the fimbriae and the non-polar AFM tip. This hypothesis was supported by Yamanaka et al. (2004), who found that cranberry juice reduced the adhesion and biofilm formation, as well as the cell surface hydrophobicity of oral streptococci, presumably by binding to or masking hydrophobic cell surface proteins. The effects of cranberry on bacterial fimbriae length, biopolymer density and adhesion found by Liu et al. (2006) were not seen with nonfimbriated bacteria. The mass of surface biomolecules in their study was constant between treated and control bacteria, suggesting that cranberry juice does not cause removal of fimbriae, and that a loss of fimbriae is not required for inhibiting bacterial adhesion. Conversely, Ahuja et al. (1998) found that treatment with cranberry juice resulted in the loss of E. coli P- and Type 1 fimbriae, as well as decreased adhesion. This apparent discrepancy may be due to the fact that the study conducted by Liu et al. (2006) did not allow sufficient time for bacterial growth to occur, whereas Ahuja et al. (1998) used four subsequent 48 h incubations. A plausible explanation could be that cranberry does not cause fimbriae removal in mature bacteria, but may affect growing bacteria by down-regulating genes involved in fimbriae expression. Lin et al. (manuscript in preparation; as cited in Johnson et al., 2008) performed gene expression analysis on E. coli grown in 10% cranberry juice. They found that flagellar basal body rod and motor proteins were down regulated compared to the control. In support of these genotypic results, scanning electron microscope images showed fewer visible P-fimbriae for E. coli grown in media containing cranberry PACs than E. coli grown in media without cranberry components (Johnson et al., 2008).  43  The fact that the results of the present study did not corroborate those of Liu et al. (2006) and Ahuja et al. (1998), who demonstrated the effect of cranberry on bacterial fimbriae, may be due to the choice of Staph. aureus as the test bacteria, rather than E. coli possessing P- or Type 1 fimbriae. Perhaps the effect that cranberry has on these fimbriae was not apparent in the current study because Staph. aureus has different adhesins that may not be affected in the same way.  Taken together, these results suggest that the anti-adhesive activity of cranberry components is likely due to several different mechanisms of action. Cranberry compounds may affect fimbrial structure and therefore, adhesive ability; alternatively, cranberry components may result in the loss of bacterial fimbriae or may prevent their expression. Bacterial strain or length of exposure may determine the predominant mechanism. The finding that cranberry PACs inhibit the adhesion of latex microspheres to biomaterials indicates an additional non-biological mechanism of action, perhaps related to steric hindrance. Certain bacterial strains may not be affected by the antiadhesion activity of cranberry compounds, possibly due to different adhesins that are not affected in the same way. Although CE90 did not demonstate a significant effect on Staph. aureus adhesion to fibronectin, it may have shown an effect on Staph. aureus adhesion had this been examined with different ECM proteins or surfaces. Further research should be done to analyse the effect of CE90 on Staph. aureus adhesion and the initial attachment stage of biofilm formation.  44  4.4 Biofilm formation, removal & index The ability of biofilm-associated bacteria to resist antibiotic treatment and contribute to persistence of infection, and the results of previous research indicating that cranberry can inhibit biofilm formation, made the potential for CE90 to inhibit biofilm formation or remove pre-existing biofilm an important question to address.  4.4.1 Biofilm formation There was a significant interaction between isolate and CE90 concentration (P < 0.05) (Figure 3), indicating that the CE90 concentrations had a different effect on the biofilm formation of different isolates. Staph. aureus PC 1 and 8325-4 produced similar amounts of biofilm in the 0 mg CE90 ml-1 control (OD 0.813 and 0.727, respectively) (Figure 3), and both these isolates produced more biofilm with low levels of CE90 (2 mg ml-1) compared to the control (P > 0.05) and higher levels of CE90 (8 and 16 mg ml-1) (P < 0.05) (Figure 3). Biofilm formation by Staph. aureus SA 113 was not stimulated by any concentration of CE90 compared to the control, and this isolate had the largest decrease in biofilm formation with CE90 treatment compared to the control (OD 2.21 difference between 0 and 16 mg CE90 ml-1) (Figure 3). The varying effects of CE90 treatments on the biofilm formation of different isolates may be due to potentially different genotypic or phenotypic characteristics of the isolates.  CE90 treatments had a significant effect on Staph. aureus biofilm formation, showing a general trend of less biofilm formation at higher concentrations of CE90, which is supported by the finding that cranberry compounds inhibit biofilm production by  45  uropathogens such as E. coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Proteus mirabilis (Reid et al., 2001) and oral pathogens such as streptococci (Yamanaka et al., 2004; Steinberg et al., 2004, 2005; Duarte et al., 2006; Koo et al., 2006), P. gingivalis (Labrecque et al., 2006) and synergistic P. gingivalis and Fusobacterium nucleatum (Yamanaka et al., 2007), as well as the activity of enzymes involved in biofilm formation (Steinberg et al., 2004; Duarte et al., 2006; Koo et al., 2006). The clumping effect may contribute to the inhibition of biofilm formation, as the large, seemingly unorganized clumps of Staph. aureus may be less capable of forming biofilm because biofilms are complex, organized structures of bacteria, which would be difficult to develop with large bacterial clumps randomly packed together.  Staph. aureus PC 1 and SA 113 formed more biofilm in the presence of CE90 than Staph. aureus 8325-4 and 2076 (P < 0.05) (Figure 3). The greater amount of biofilm formed by Staph. aureus SA 113 than Staph. aureus 2076 is in accordance with my expectations, as these isolates were used as the biofilm positive and negative control isolates, respectively. Further research should be performed to analyse the effect of CE90 on additional Staph. aureus isolates, as the varying effects of CE90 on these four isolates show that isolates react differently to the presence of CE90.  4.4.2 Biofilm removal There was a significant interaction between isolate and CE90 concentration (P < 0.05) (Figure 4), indicating that CE90 concentrations had specific effects on the biofilm removal of different Staph. aureus isolates. The most noticeable difference in the effect  46  of CE90 between different isolates is that Staph. aureus SA 113 showed greater levels of residual biofilm after exposure to CE90 (2, 4, 8 and 16 mg ml-1) compared to the control (P < 0.05) (Figure 4). The varying effects of CE90 concentrations on the biofilm removal of different isolates may be due to either genotypic or phenotypic differences between the isolates.  CE90 concentrations had a significant effect on Staph. aureus biofilm removal, with the exception of PC1. However, the only statistically significant differences between the untreated control biofilms and those exposed to CE90 were that the latter had greater levels of residual biofilm than the untreated controls for some isolates of Staph. aureus (P < 0.05) (Figure 4). These results show that CE90 was not effective in removing preexisting biofilm. Steinberg et al. (2005) found that cranberry NDM (2 mg ml-1) promoted desorption of S. sobrinus from biofilm; however this difference from the results of the current study may be due to the difference in bacterial species, cranberry extract, experimental methods or method of analysis. Labrecque et al. (2006) reported that P. gingivalis biofilm was not desorbed by treatment with cranberry NDM, though they used a much lower concentration (0.25 mg ml-1) and a different bacterial species. The finding that exposure to CE90 results in greater levels of residual biofilm for certain isolates (SA 113) compared to the control suggests that further research should be done to determine if this is a common phenomenon, since this could have negative implications for the use of CE90 in many applications.  47  There was a significant difference in the amount of biofilm remaining after exposure to CE90 between each isolate (SA 113 > 8325-4 > PC1 > 2076) (P < 0.05) (Figure 4). The greater amount of residual biofilm in Staph. aureus SA 113 compared to Staph. aureus 2076 supports the biofilm formation results and the choice of these isolates as the biofilm positive and negative control isolates.  4.4.3 Biofilm index There was a significant interaction between bacterial isolate and CE90 concentration at 8 and 24 h (P < 0.05) (Table 2), indicating that CE90 concentration had specific effects on the biofilm index of different Staph. aureus isolates at these time intervals. At 8 h, Staph. aureus PC1 and SA 113 showed a higher biofilm index at 2, 4 and 8 mg ml-1 compared to 0 and 16 mg ml-1, whereas Staph. aureus 8325-4 showed no significant difference in biofilm index between CE90 concentrations at this time interval (Figure 5d). At 24 h, the different effects of CE90 among Staph. aureus isolates were more pronounced. For Staph. aureus PC1, all the CE90 concentrations showed a lower biofilm index than the 0 mg ml-1 control, and the 8 mg ml-1 treatment resulted in a lower biofilm index than the 16 mg ml-1 treatment (P < 0.05) (Figure 5e). For Staph. aureus SA 113, the 2, 8 and 16 mg ml-1 treatments resulted in a lower biofilm index than the 0 mg ml-1 control, the 8 mg ml-1 resulted in a lower biofilm index than the 4 mg ml-1 treatment, and the 16 mg ml-1 treatment showed a lower biofilm index than all the CE90 concentrations (P < 0.05) (Figure 5e). For Staph. aureus 8325-4, all the CE90 concentrations had a decreased biofilm index compared to the control, and the 8 mg ml-1 treatment resulted in a higher biofilm index compared to the 4 and 16 mg ml-1 treatments (P < 0.05) (Figure 5e).  48  The finding that CE90 concentration had a different effect between Staph. aureus as time progressed may be a result of the Staph. aureus isolates becoming more established over time. As bacteria mature, isolates may become more differentiated from each other regarding their gene expression and cell surface proteins, which may result in the tested CE90 concentrations having a wider range of effects than with less mature bacterial populations.  Staph. aureus PC1 and SA 113 also showed a significant interaction between time and concentration of CE90 (P < 0.05) (Table 3), indicating that the concentration of CE90 had specific effects on these bacteria at different time intervals. These isolates tended to have a higher biofilm index at 2, 4 and 8 mg ml-1 than at 0 and 16 mg ml-1 from 2 h to 8 h (Figure 5a, b, c, d). However, by 24 h, all of the CE90 concentrations resulted in a lower biofilm index than the 0 mg ml-1 control for both isolates (P < 0.05; SA 113 at 4 mg ml-1, P > 0.05) (Figure 5e).  The finding that low to medium concentrations of CE90 resulted in an initial increase in biofilm index does not support the claim that cranberry products inhibit biofilm formation (Reid et al., 2001; Yamanaka et al., 2004; Steinberg et al., 2004, 2005; Duarte et al., 2006; Koo et al., 2006; Labrecque et al., 2006; Yamanaka et al., 2007). However, the finding that by 24 h each concentration of CE90 decreased the biofilm index compared to the 0 mg ml-1 control does support this claim as well as the optical density measurements that showed inhibition of biofilm formation at higher concentrations of  49  CE90 over 24 h. The finding that the concentration of CE90 had a different effect on Staph. aureus PC1 and SA 113 biofilm index at different time intervals may be related to the above explanation that bacteria express different genes and produce different proteins as they mature. This may result in CE90 having a different effect on biofilm index over time.  Biofilm index values were similar between isolates at each time interval, with slightly higher values for Staph. aureus SA 113 at 6 and 24 h (Figure 5c,e) (P < 0.05). This may be explained by the naturally high levels of biofilm produced by this isolate.  Staph. aureus SA 113 at 24 h showed the greatest reduction in biofilm index with increasing CE90 concentration, from 65% at 0 mg CE90 ml-1 to 44% at 16 mg CE90 ml-1 (Figure 5e) (P < 0.05). This 20% reduction in biofilm index is significant, considering the high levels of biofilm produced by Staph. aureus SA 113.  4.5 Salt tolerance assay The question of whether CE90 has a bactericidal effect on Staph. aureus or simply injures these bacteria was important to address, for a number of reasons. First of all, if CE90 is able to prevent infections by inhibiting Staph. aureus adherence or otherwise injuring Staph. aureus without actually killing the bacteria, the development of Staph. aureus resistance to CE90 would be less likely than if CE90 has a bactericidal effect. In the latter case there would be a selective pressure for Staph. aureus that could survive in the presence of CE90, and resistance may develop as it has for antibiotics. However, if  50  the effect of CE90 on Staph. aureus is not bactericidal this selective pressure would not exist and therefore resistance to CE90 would be unlikely.  Another reason for addressing this question is that bacteria present in foods often suffer sublethal injuries (Hernandez et al., 1993), which prevent them from growing in selective media used to detect bacteria in foods. However, these injured bacteria may eventually recover under appropriate environmental conditions (Hernandez et al., 1993), such as the food being left at room temperature to thaw. The resulting potential for high levels of undetected food-associated bacteria to cause foodborne illness poses a serious public health risk (Hernandez et al., 1993). It would be beneficial to know whether CE90 has a similar effect of injuring Staph. aureus that may recover at a later stage to cause illness.  Bacteria that have sustained damage to their cell membranes may become more permeable and are unable to maintain an appropriate osmotic pressure or to exclude toxic materials (Carson et al., 2002). The resulting loss of salt tolerance is suggestive of membrane damage in sublethally injured Staph. aureus (Iandolo and Ordal, 1966; Carson et al., 2002). There was a dose-dependent decrease in Staph. aureus viability on both TSA and TSAS. Isolate PC1 formed fewer colonies on TSA at the higher CE90 concentrations (8 and 16 mg ml-1) than in the absence of CE90 (control) (Figure 6a) (P < 0.05). There was also a significant reduction in colonies formed by PC1 on TSAS at all CE90 concentrations (4, 8 and 16 mg ml-1) compared to the control (P < 0.05), as well as at 16 mg ml-1 compared to 4 mg ml-1 (P < 0.05) (Figure 6a). Staph. aureus SA 113 on TSA and TSAS, and Staph. aureus 8325-4 on TSA, formed fewer colonies at 16 mg ml-1  51  than at 4 and 8 mg ml-1 (P < 0.05), and fewer colonies at 4 and 8 mg ml-1 than the control (P < 0.05) (Figure 6b,c). Staph. aureus 8325-4 showed a decrease in viability on TSAS at 16 mg ml-1 compared to 0 and 4 mg ml-1 (P < 0.05), and at 8 mg ml-1 compared to 0 mg ml-1 (P < 0.05) (Figure 6c). There was a significant reduction in colonies formed by isolate 2076 on TSA and TSAS at 16 mg ml-1 compared to 0 and 4 mg ml-1 (P < 0.05), and at 4 and 8 mg ml-1 compared to 0 mg ml-1 (P < 0.05) (Figure 6d). For a particular concentration, Staph. aureus counts were lower on TSAS than on TSA, with the exception of the controls without CE90, and Staph. aureus 8325-4 at the 4 mg CE90 ml-1 treatment (Figure 6a,b,c,d). However this difference between media was significant only for PC1 at 4 and 16 mg ml-1, 8325-4 at 8 mg ml-1, and 2076 at 16 mg ml-1 CE90 (P < 0.05) (Figure 6a,c and d, respectively). There were no significant interactions between isolate and CE90 concentration (P > 0.05).  Based on the assumption that reduced viability on TSA and TSAS corresponds to Staph. aureus death and injury, these results suggest that CE90 not only injures Staph. aureus but also has a bactericidal effect. In addition, the ability of CE90 to increase Staph. aureus sensitivity to NaCl, which is used as a food preservative, suggests that CE90 may have the potential to be used as an additional barrier in hurdle technology to minimize foodborne illness and food spoilage.  However, the interpretation of these results as an indication of Staph. aureus death and injury may need to be re-examined in light of the effect of CE90 on Staph. aureus morphology. The dose-dependent Staph. aureus clumping effect observed after  52  treatment with CE90 means that even if CE90 neither injures nor kills Staph. aureus, we would still expect to see a dose-dependent reduction in cfu on TSA and TSAS, since each clump of bacteria gives rise to only one colony. In that case the reduced cfu on TSA and TSAS should be interpreted not as an indication of Staph. aureus death or injury, but merely as the result of Staph. aureus clumping in response to treatment with CE90. The fact that most samples of the biofilm index experiment did not show a significant reduction in either planktonic- or biofilm-associated Staph. aureus viability on TSA after treatment with CE90 over 24 h (Appendix D.1 – D.8) suggests that CE90 does not have any significant bactericidal effect on Staph. aureus. Yet the difference in Staph. aureus counts between TSA and TSAS (Figure 6a,b,c,d) suggests that there is a certain amount of injury taking place, and therefore the clumping effect is not solely responsible for the reduced counts. It may be likely that the reduction in colony forming units is partially explained by Staph. aureus clumping, but that CE90 injures and possibly kills Staph. aureus, as well. Further studies are required to separate this confounding variable in order to elucidate the effect of CE90 on Staph. aureus health and survival. To the author’s knowledge there are no previously published studies examining the effect of cranberry or cranberry extracts on the salt tolerance of Staph. aureus.  53  5. CONCLUSION The concentration of CE90 for practical applications should be carefully selected, as certain isolates may actually produce more biofilm in response to low concentrations of CE90, or they may produce biofilm that is more resistant to removal by rinsing after exposure to CE90 than in the absence of CE90. In addition, the observed variations between isolates in the effect of CE90 means that we can’t generalize among all isolates of Staph. aureus, as some isolates react differently to CE90 than others.  Staph. aureus PC1, SA 113, 8325-4 and 2076 showed decreased salt tolerance with increasing CE90 concentration. This increase in bacterial sensitivity to salt may have potential applications to food preservation. However further research must be done to determine whether the salt tolerance results are a true reflection of Staph. aureus injury and killing, or whether they were a result of clumping. If salt tolerance results were due to Staph. aureus clumping in response to treatment with CE90, the clumps may break apart either in the food or within the host after food consumption.  The results of this study show that the clumping effect of CE90 has important implications for the microbiological analysis of cranberry products. Since bacterial counts of samples plated onto agar are a measure of the colony forming units, if large clumps of bacteria are plated, each clump would give rise to only one colony. This would make it appear as though the bacterial counts had decreased, even if the number of viable bacteria remains the same.  54  In summary, CE90 has the potential to cause clumping, reduce biofilm formation (8, 16 mg ml-1) and decrease salt tolerance of Staph. aureus PC1, SA 113, 8325-4 and 2076. CE90 was not effective at removing pre-formed biofilm for these four isolates, and even resulted in higher levels of residual biofilm than the control for Staph. aureus SA 113 and 2076. CE90 did not affect Staph. aureus 8325-4, 8325-4 fnbA-/fnbB- or 8325-4 clfAfibronectin binding, except for 8325-4 fnbA-/fnbB- at 4 mg ml-1, which showed less fibronectin binding than the control.  55  6. RECOMMENDATIONS FOR FURTHER RESEARCH A-type PACs are also found in cinnamon, curry, peanuts, plums and avocados (Gu et al., 2003).  Additional research should be done to determine whether these alternative  sources of A-type PACs also have anti-adhesion activity (Howell et al., 2005). The level of A-type versus B-type PACs present in white cranberries should be analysed, and further research could be done to confirm or contradict the findings of Johnson-White et al. (2006), which claimed that white cranberry juice did not possess anti-adhesion activity. Different sources of cranberry compounds and various methods of extraction and preparation, which would result in different compositions and purities of extracts, should be evaluated.  The effect of CE90 on the normal flora of humans and animals, and other beneficial bacteria such as probiotics, should be studied. Similarly, the effect of CE90 on multidrug resistant bacteria such as MRSA and VRE should be examined.  The extent to which different concentrations of CE90 result in Staph. aureus clumping could be quantified and statistically analysed.  The viability of clumps could be  confirmed by staining Staph. aureus that had been exposed to CE90 with Live/Dead staining and examining the clumps with a microscope. Additionally, further studies could determine whether clumps remain stable over time and in different environments, or whether they tend to break apart.  56  Further studies could examine the effect of CE90 on Staph. aureus adhesion to additional ECM proteins besides fibronectin, such as fibrinogen and collagen, and to epithelial cells.  The observation among the biofilm formation, removal and biofilm index experiments in the present study that low to medium concentrations of CE90 result in higher levels of biofilm with specific isolates requires further study since enhanced biofilm formation and diminished biofilm removal may have negative consequences.  Studies should be  performed to examine the effect of CE90 on quorum sensing and the genetic regulation of biofilm formation. Different methods of quantifying the biofilm index could be tested to ensure the most accurate enumeration of planktonic and biofilm bacteria. Using a sterile spatula or cell scraper to remove biofilm might result in a more accurate measurement, as bacteria may be washed away from these instruments into saline more easily than from a cotton swab. Alternatively, saline could be added to the biofilm coated wells, and biofilm-associated bacteria could be suspended by sonication or glass beads.  The effect of CE90 on the survival and health of Staph. aureus could be clarified by repeating the experiment and examining samples under a microscope to determine the extent of clumping.  This information, along with an assessment of viability using  Live/Dead staining, could assist in estimating whether the reduced colony counts are primarily due to Staph. aureus injury and killing, or are simply a result of bacterial clumping. Alternatively, an attempt could be made to break apart the clumps before plating onto TSA and TSAS, perhaps by adding trypsin or sonicating the samples.  57  Preliminary experiments would need to be performed to determine the effectiveness of trypsin or sonication in breaking apart the clumps without negatively affecting viability.  The in vitro nature of these experiments means that the results cannot be extrapolated in vivo to provide an indication of the effect of CE90 in a human or animal model. Results are limited to situations where CE90 has direct contact with bacteria, since the complex environment of a living system and the effects of digestion and absorption on the bioactivity of CE90 is unknown. Testing the effect of CE90 on bacteria incubated with cell lines, and eventually on bacteria within animal models or human subjects would provide an indication of the effect of CE90 in vivo. For example the ability of CE90 to reduce the risk of UTIs may be a valuable research question to address. It may be of interest to determine whether CE90 can control sub-clinical infections and help promote growth and feed efficiency in farm animals. If successful, this may reduce the reliance on feed supplemented with antibiotics by providing the agricultural industry with a more natural alternative that produces minimal, if any, selective pressure for resistance (Howell and Foxman, 2002; Eydelnant and Tufenkji, 2008; McMurdo et al., 2009).  Studies should be done to verify the belief that bacteria do not develop resistance to cranberry compounds.  The sites of action, effective dose, and structure-activity  relationships of CE90 are also important research questions to address.  58  7. TABLES Table 1: MICs of CE90 in mg ml-1 against 24 different isolates of Staph. aureus Origin Reference Isolate MIC (mg CE90 ml-1) SHY97-3906 32 1* Diarra, 2002 SHY97-3923-3 32 1* Diarra, 2002 SHY97-4085 32 2* SHY97-4242 32 2* SHY97-4320 32 1* Diarra, 2002 SHY97-4343 16 1* Diarra, 2002 ATCC 25923 16 3 Diarra, 2002 PC1 16 4 Diarra, 2002 NCTC 9789 32 4 Diarra, 2002 2076 32 4 Diarra, 2002 22260 32 4 Diarra, 2002 ST79/741 32 4 Diarra, 2002 3804 (pII) 32 4 Diarra, 2002 RN9 32 4 Diarra, 2002 FAR 8 32 4 Diarra, 2002 FAR 10 16 4 Diarra, 2002 3906 PGR 32 2 SA 113 16 5 8325-4 16 6 8325-4 fnbA /fnbB 16 6 8325-4 clfA16 6 SHY99-730-2 32 2* MRSA 16 7 ATCC 29213 16 3 Diarra, 2002 1 = Laboratoire Provincial de Pathologie Animale of St-Hyacinthe (QC, Canada) 2 = Dr Diarra (Agassiz, BC, Canada) 3 = American Type Culture Collection (ATCC) (Manassas, VA, USA) 4 = Vanderbilt University School of Medicine (Nashville, TN, USA) (beta-lactam antibiotic-resistant) 5 = Pharmacia 6 = Dr Foster (Ontario, Canada) 7 = Serge Messier (University of Montreal) (St-Hyacinthe, QC, Canada) * = clinical bovine mastitis  59  Table 2: Interactions between CE90 concentrations and isolate (Staph. aureus PC1, SA 113, 8325-4 and 2076) at different time intervals1. Time Concentration*Isolate 2h P > 0.05 4h P > 0.05 6h P > 0.05 8h P < 0.05 24 h P < 0.05 1. Analysis is a two-way ANOVA of the biofilm index from two separate experiments performed in duplicate.  Table 3: Interactions between CE90 concentrations and time for different Staph. aureus isolates1. Isolate Concentration*Time PC1 P < 0.05 SA 113 P < 0.05 8325-4 P > 0.05 1. Analysis is a two-way ANOVA of the biofilm index from two separate experiments performed in duplicate.  60  8. FIGURES  a  b  c  d  Figure 1: Effect of CE90 on Staph. aureus PC1 morphology viewed through a phase contrast microscope: formation of Staph. aureus clumps (magnification: 400x). Image processing steps used: red channel selected; unsharp mask (3x) (Adobe Photoshop 5.5) (Russ, J. C. 2007). a: 0 mg CE90 ml-1, 5 min exposure to CE90 b: 4 mg CE90 ml-1, 5 min exposure to CE90 c: 8 mg CE90 ml-1, 5 min exposure to CE90 d: 16 mg CE90 ml-1, 5 min exposure to CE90  61  100 Adherence index (%)  90 80  a  a a  a  a  a b  a  70  a  a  a  a  60 50 40 30 20 10 0 8325-4  8325-4 fnbA-/fnbB-  8325-4 clfA-  Strain 0 mg/ml  4 mg/ml  8 mg/ml  16 mg/ml  Figure 2: Effect of CE90 on Staph. aureus binding to fibronectin. Treatments within each strain with different letters are significantly different (P < 0.05). There were no significant differences between strains (P > 0.05). Error bars represent the standard error of two repeats performed in duplicate.  62  4  A  A  3.5 3  a  a B  a  2.5 OD 570nm  B  2 1.5 1  ab abc b  a  bc b  b  0.5  ab  ab  bc  a  a b  c  bc  c  0 -0.5  SA 113  2076  PC1  8325-4  -1 Isolate 0 mg/ml  2 mg/ml  4 mg/ml  8 mg/ml  16 mg/ml  Figure 3: Effect of CE90 on Staph. aureus biofilm formation. Treatments within each isolate with different letters (a, b, c) are significantly different (P < 0.05). Isolates with different letters (A, B) are significantly different (P < 0.05). Values represent the least squares mean and standard error of optical density measurements at 570nm from two separate experiments performed in quadruplicate. There was a significant interaction between Staph. aureus isolate and CE90 treatment (P < 0.05).  63  c  A  4  b b  3.5  b b  D  OD 570nm  3 2.5  a  a  2  ab  C  1.5  ab  ab  B  1  a a  0.5  a ab  b  a  a  a  b b  0 SA 113  2076  PC1  8325-4  Isolate 0 mg/ml  2 mg/ml  4 mg/ml  8 mg/ml  16 mg/ml  Figure 4: Effect of CE90 on removal of biofilm formed by Staph. aureus. Treatments within each isolate with different letters (a, b) are significantly different (P < 0.05). Isolates with different letters (A, B, C, D) are significantly different (P < 0.05). Values represent the least squares mean and standard error of optical density measurements at 570nm from two separate experiments performed in quadruplicate. There was a significant interaction between Staph. aureus isolate and CE90 treatment (P < 0.05).  64  b  100 90 Biofilm index (%)  80 70 60 ab  50 40  ab  a  a  b  a  a  a  a b  b b  b  c  a  30 20 10 0 PC1  SA 113  8325-4  Isolate 0 mg/ml  2 mg/ml  4 mg/ml  8 mg/ml  16 mg/ml  Figure 5a: Effect of CE90 on biofilm index of Staph. aureus after 2 h. Biofilm index = (cfu ml-1 biofilm Staph. aureus / (cfu ml-1 biofilm Staph. aureus + cfu ml-1 planktonic Staph. aureus))*100. Different letters (a, b, c) represent significant differences of treatments within each isolate (P < 0.05). There were no significant differences between isolates (P > 0.05). Error bars represent the standard error of two repeats performed in duplicate.  65  100 90 Biofilm index (%)  80 70 60  a  50 40  a  b  a  a b  a  a  a  b  b  a  ab  bc  c  30 20 10 0 PC1  SA 113  8325-4  Isolate 0 mg/ml  2 mg/ml  4 mg/ml  8 mg/ml  16 mg/ml  Figure 5b: Effect of CE90 on biofilm index of Staph. aureus after 4 h. Biofilm index = (cfu ml-1 biofilm Staph. aureus / (cfu ml-1 biofilm Staph. aureus + cfu ml-1 planktonic Staph. aureus))*100. Different letters (a, b, c) represent significant differences of treatments within each isolate (P < 0.05). There were no significant differences between isolates (P > 0.05). Error bars represent the standard error of two repeats performed in duplicate.  66  100 90 Biofilm index (%)  80 A  70 60 50  a  a  a  B a  a a  a  A  ab  b  a b  a  a  a a  40 30 20 10 0 PC1  SA 113  8325-4  Isolate 0 mg/ml  2 mg/ml  4 mg/ml  8 mg/ml  16 mg/ml  Figure 5c: Effect of CE90 on biofilm index of Staph. aureus after 6 h. Biofilm index = (cfu ml-1 biofilm Staph. aureus / (cfu ml-1 biofilm Staph. aureus + cfu ml-1 planktonic Staph. aureus))*100. Different letters (a, b) represent significant differences of treatments within each isolate (P < 0.05). Different letters (A, B) represent significant differences between isolates (P < 0.05). Error bars represent the standard error of two repeats performed in duplicate.  67  100 90 Biofilm index (%)  80 70 60 50  a  a  b  a  a b  a  a  b  a  a  a  a  b  40 30 20 10 0 PC1  SA 113  8325-4  Isolate 0 mg/ml  2 mg/ml  4 mg/ml  8 mg/ml  16 mg/ml  Figure 5d: Effect of CE90 on biofilm index of Staph. aureus after 8 h. Biofilm index = (cfu ml-1 biofilm Staph. aureus / (cfu ml-1 biofilm Staph. aureus + cfu ml-1 planktonic Staph. aureus))*100. Different letters (a, b) represent significant differences of treatments within each isolate (P < 0.05). There were no significant differences between isolates (P > 0.05). Error bars represent the standard error of two repeats performed in duplicate.  68  a  100  B A  90  A  Biofilm index (%)  80  a ab  70 60  a  bc bc  bc  c  c  b  a d  bc  c  b  50 40 30 20 10 0 PC1  SA 113  8325-4  Isolate 0 mg/ml  2 mg/ml  4 mg/ml  8 mg/ml  16 mg/ml  Figure 5e: Effect of CE90 on biofilm index of Staph. aureus after 24 h. Biofilm index = (cfu ml-1 biofilm Staph. aureus / (cfu ml-1 biofilm Staph. aureus + cfu ml-1 planktonic Staph. aureus))*100. Different letters (a, b, c, d) represent significant differences of treatments within each isolate (P < 0.05). Different letters (A, B) represent significant differences between isolates (P < 0.05). Error bars represent the standard error of two repeats performed in duplicate.  69  c  A  X AB a  Log10 cfu/ml  10 9 8 7 6 5 4 3 2 1 0  Y b  B  B a YZ Z  0 mg/ml  4 mg/ml 8 mg/ml Treatment TSA  16 mg/ml  TSAS  Figure 6a: Effect of CE90 on Staph. aureus PC1 salt tolerance. Different letters within each media (A, B = TSA; X, Y, Z = TSAS) represent significant differences between treatments (P < 0.05). Different letters within each treatment (a, b) represent significant differences between media (P < 0.05). Error bars represent the standard error of two repeats performed in duplicate. % surviving = (cfu ml-1 on TSA / cfu ml-1 on TSA of isolate at 0 mg ml-1)*100 % killed = 100 – % surviving % healthy = (cfu ml-1 on TSAS / cfu ml-1 on TSA of isolate at 0mg ml-1)*100 % injured = % surviving - % healthy  70  A A  X  X  Log 10 cfu/ml  10 9 8 7 6 5 4 3 2 1 0  BB  Y Y  BB YY CC  ZZ  0 mg/ml  4 mg/ml 8 mg/ml Treatment TSA  16 mg/ml  TSAS  Figure 6b: Effect of CE90 on Staph. aureus SA 113 salt tolerance. Different letters within each media (A, B, C = TSA; X, Y, Z = TSAS) represent significant differences between treatments (P < 0.05). There were no significant differences between media (P > 0.05). Error bars represent the standard error of two repeats performed in duplicate.  71  A A  X X  B  Log 10 cfu/ml  10 9 8 7 6 5 4 3 2 1 0  B  XY  XY  B B a a  YZ YZ bb  C  C  Z  Z  0 mg/ml  4 mg/ml 8 mg/ml Treatment TSA  16 mg/ml  TSAS  Figure 6c: Effect of CE90 on Staph. aureus 8325-4 salt tolerance. Different letters within each media (A, B, C = TSA; X, Y, Z = TSAS) represent significant differences between treatments (P < 0.05). Different letters within each treatment (a, b) represent significant differences between media (P < 0.05). Error bars represent the standard error of two repeats performed in duplicate.  72  A  X  B  Log10 cfu/ml  10 9 8 7 6 5 4 3 2 1 0  Y BC YZ  C a Z b  0 mg/ml  4 mg/ml 8 mg/ml Treatment TSA  16 mg/ml  TSAS  Figure 6d: Effect of CE90 on Staph. aureus 2076 salt tolerance. Different letters within each media (A, B, C = TSA; X, Y, Z = TSAS) represent significant differences between treatments (P < 0.05). 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Application of pulsed-field gel electrophoresis and binary typing as tools in veterinary clinical microbiology and molecular epidemiologic analysis of bovine and human Staphylococcus aureus isolates. J. Clin. Microbiol. 38:1931-1939. Zafriri, D., I. Ofek, R. Adar, M. Pocino and N. Sharon. 1989. Inhibitory activity of cranberry juice on adherence of Type 1 and Type P fimbriated Escherichia coli to eucaryotic cells. Antimicrob. Agents Chemother. 33:92-98. Zhang, L., J. Ma, K. Pan, V. L. W. Go, J. Chen and W. You. 2005. Efficacy of cranberry juice on Helicobacter pylori infection: a double-blind, randomized placebocontrolled trial. Helicobacter. 10:139-145.  96  APPENDICES Appendix A: Structure of a typical cranberry proanthocyanidin tetramer composed of epicatechin units with one A-type linkage and two B-type linkages. (Neto, 2007b)  97  Appendix B: Ingredient specifications of the undialysed cranberry extract1. Ingredient specification Typical range Cranberry fruit solids (%) 90% minimum Moisture NMT 5% pH (10% solution) 3.7 – 4.3 Titratable acidity (w/w) 8.5 – 13% Bulk density (tapped) 0.5 +/- 0.1 g/ml Organic acids 35% (9% quinic acid typical) Total phenolics 2.00 – 3.80% Total anthocyanins 0.15 – 1.00% Total proanthocyanidins 0.95% maximum Antioxidant (ORAC) 175 – 400 µmol Trolox/g Antioxidant (DPPH) 55 – 70% radical inhibition Ellagic acid 200 – 415 µg/g Quercetin 300 – 435 µg/g Yeast and mould < 50/g Standard plate count < 500/g E. coli Negative/25g Salmonella Negative/25g Coliform < 10/g 1. Undialysed cranberry extract = the cranberry extract provided by Decas Botanical Synergies, which was used in the preparation of a high-molecular-weight NDM (CE90) tested against Staph. aureus.  98  Appendix C: Biofilm formation of 24 Staph. aureus isolates in the absence of CE901. Isolate OD570nm SHY97-3906 0.052 SHY97-3923-3 -0.006 SHY97-4085 0.123 SHY97-4242 0.1215 SHY97-4320 0.083 SHY97-4343 0.152 ATCC 25923 0.1205 PC1 0.262 NCTC 9789 0.0815 2076 0.004 22260 -0.0115 ST79/741 0.01 3804 (pII) 0.0945 RN9 0.276 FAR 8 0.031 FAR 10 0.0855 3906 PGR 0.1895 SA 113 0.784 8325-4 0.262 8325-4 fnbA /fnbB 0.2055 8325-4 clfA0.3035 SHY99-730-2 0.2155 MRSA 0.105 ATCC 29213 0.292 1. Values represent optical density measurements at 570nm, in a preliminary experiment.  99  Appendix D.1: Effect of CE90 on planktonic Staph. aureus PC1 growth1.  12  Log cfu ml  -1  10 8 6 4 2 0 0  2  4  8  16  -1  Treatment (mg CE90 ml ) 0h 1.  2h  4h  6h  8h  24 h  Planktonic cfu ml-1 values of the biofilm index experiment over 24 h. Error bars represent the standard error of two repeats performed in duplicate.  100  Appendix D.2: Effect of CE90 on Staph. aureus PC1 biofilm formation1.  10 9 8  Log cfu ml  -1  7 6 5 4 3 2 1 0 0  2  4  8  16  -1  Treatment (mg CE90 ml ) 0h 1.  2h  4h  6h  8h  24 h  Biofilm cfu ml-1 values of the biofilm index experiment over 24 h. Error bars represent the standard error of two repeats performed in duplicate.  101  Appendix D.3: Effect of CE90 on planktonic Staph. aureus SA 113 growth1.  10 9 8  Log cfu ml  -1  7 6 5 4 3 2 1 0 0  2  4  8  16  -1  Treatment (mg CE90 ml ) 0h 1.  2h  4h  6h  8h  24 h  Planktonic cfu ml-1 values of the biofilm index experiment over 24 h. Error bars represent the standard error of two repeats performed in duplicate.  102  Appendix D.4: Effect of CE90 on Staph. aureus SA 113 biofilm formation1.  12  Log cfu ml  -1  10 8 6 4 2 0 0  2  4  8  16  -1  Treatment (mg CE90 ml ) 0h 1.  2h  4h  6h  8h  24 h  Biofilm cfu ml-1 values of the biofilm index experiment over 24 h. Error bars represent the standard error of two repeats performed in duplicate.  103  Appendix D.5: Effect of CE90 on planktonic Staph. aureus 8325-4 growth1.  12  Log cfu ml  -1  10 8 6 4 2 0 0  2  4  8  16  -1  Treatment (mg CE90 ml ) 0h 1.  2h  4h  6h  8h  24 h  Planktonic cfu ml-1 values of the biofilm index experiment over 24 h. Error bars represent the standard error of two repeats performed in duplicate.  104  Appendix D.6: Effect of CE90 on Staph. aureus 8325-4 biofilm formation1.  10 9 8  Log cfu ml  -1  7 6 5 4 3 2 1 0 0  2  4  8  16  -1  Treatment (mg CE90 ml ) 0h 1.  2h  4h  6h  8h  24 h  Biofilm cfu ml-1 values of the biofilm index experiment over 24 h. Error bars represent the standard error of two repeats performed in duplicate.  105  Appendix D.7: Effect of CE90 on planktonic Staph. aureus 2076 growth1.  12  Log cfu ml  -1  10 8 6 4 2 0 0  2  4  8  16  -1  Treatment (mg CE90 ml ) 0h  1.  2h  4h  6h  8h  24 h  Planktonic cfu ml-1 values of the biofilm index experiment over 24 h. Error bars represent the standard error of two repeats performed in duplicate.  106  Appendix D.8: Effect of CE90 on Staph. aureus 2076 biofilm formation1.  10 9 8  Log cfu ml  -1  7 6 5 4 3 2 1 0 0  2  4  8  16  -1  Treatment (mg CE90 ml ) 0h 1.  2h  4h  6h  8h  24 h  Biofilm cfu ml-1 values of the biofilm index experiment over 24 h. Error bars represent the standard error of two repeats performed in duplicate.  107  

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