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Viscosity on Biofilm Phenotypic Expression University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies Legacy Theses 1999 Effect of viscosity on biofilm phenotypic expression Chan, Wendy Wing Yan Chan, W. W. (1999). Effect of viscosity on biofilm phenotypic expression (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/12502 http://hdl.handle.net/1880/25429 master thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca Wet of Viscosity on Biofilm Phenotypic Expression by Wendy Wing Yan Chan A THESIS SUBbWTO THE FACULTY OF GWUATE STUDIES IN PARTIAL FULFlLEiENT OF THE REQ~~TSFOR THE DEGREE OF bUSTER OF SCIENCE DEP-ARTMENT OF BIOLOGICAL SCIENCES CALGAELY? ALBERTA DECEMBm I999 OWendy Wing Yan Chan I999 National Library Bibliothe ue nationale du Cana1 a Acquisitions and Acquisitions et Bibliographic Services setvices bibliographiques 395 Wellington Street 395, rue Wellington Ortawa ON K1AON4 Ottawa ON KIA ON4 camdm Canada The author has granted a non- L'auteur a accorde me licence non exc1usive licence allowing the exclusive pennettant a la National h'brary of Canada to Bbliotheque nationale du Canada de reproduce, loan, distribute or sell reproduire, prster, distniuer ou copies of this thesis m microform, vendre des copies de cette these sous paper or electronic formats. la fome de microfiche/^ de reproduction sm papier ou sur format eIectroniqne. The author retains ownership of the L'antem conserve la propriete du copyright m this thesis. Neither the droit d'auteur qui protege cette these. thesis nor substantial extracts fiom it Ni Ia these ni des extraits substantieIs may be printed or otherwise de ceIIe-ci ne doivent &re imprimes reproduced without the author's on autrement reproduits sans son perm*ssion. autorisation. &QBSTIWCT Biofilms are adherent colonies of microorganisms growing within a poly saccharide cell surface @ycocaiyx. Antibiotics arid biocides used at concentrations capabie of killing planktonic organisms have littie effect against biofiirns. Bacterial and hngd growth has been obsenred in various viscous environments. It is hypothesized that a viscous environment could act as a mechanism responsible for the phenotypic expression of physio1ogica.I factors characteristic of biofilms. The effect of viscosity on growth characteristics, morphology and antibiotic sensitivity of Pseudomonar aerugrnou and Ctmdida albzcm was evaiuated. Growth curves demonstrated that bacteria and fungus pwbetter at higher viscosity. P. aenigirzosa in viscous solutions were larger than the planktonic cells. C albtcans was reduced in size at higher viscosity. P. aemginosa andC. aibicm grown in viscous solutions demonstrated decreased susceptibility to antibiotics similar to the biofilrn organisms indicating that viscosity of the environment is a possible mechanism for biotiIm phenotypic expression. ACKNOWLEDGEMENTS I would like to thank Dr. Merle Olson for allowing me to pursue my Pul-Sc. degree in his laboratory. I appreciate ail the opportunities he has given me over the years. I would also like to thank Dr. Rob Burrell for his advice and guidance. This project could not have been completed without the technical assistance and support of Liz ~Middlerniss,Carol Stremick and Wayne Jarisen. In addition, thanks to all the people in the Bio& Research Group lab for keeping things entertaining. Lady, I would like to thank Westaim Biomedical for funding my research. To my mom and dad for dl their support TABLE OF CONTENTS APPROVAL PAGE ABSTRACT DEDICATION TABLE OF CONTENTS LIST OF TABLES LIST OF FIGC'RES LIST OF ABBREVLATIONS CEWTER 1: INTRODUCTION Bioflms I. Structure 11Distribution 1.1 Resistance 1 In vitro models 1.1.5 ControI 1.1.6 Psesicloonronus aerugtCnosa 1.1.7 Cattdida albicans 1.2 Viscosity 2 Organisms in viscous environments 1.23 Vicosity experiments 1.3 Research hypothesis and objectives CEIMlXR 2: LMETHODSAND MATERIALS 2.1 Organisms 2.2 Viscous media 2.2.1 PVP 2-22Viscosity 2.23 hmoiality 2.2.4 Accurate delivery 2.3 Growth curves 2.1 klicrosco py 2.41 Negative staining 2.42 TEBL 2.4.3 Light microscopy 2.5 ,Antibiotic susceptibility tests 2.5.1 Drugs 2.52 3IIC (NCCLS method) 2.5.2.I Bacteria 2.5.2.1 Fungi 2.5.2.3 Reference strain 2.53 BIBEC 2.5.2.1 Bacteria 2.5.2.3 Fungi 2.5.4 3UC and hlBCm(fFC in viscous solutions 2.5.3. I Bacteria 3.5.3.3 Fungi 2.6 Statistical analysis CHAPTER 3: RESULTS 3.1 Viscosity 3.3 Accuracy of pipettes 3.4 Growth Curves 3.5 Microscopy 3.5.1 Negative staining 3.5.2 m1 3.53 Light microscopy 3.6 Antibiotic susceptibility 3.6.1 P. aerupnosa 3.6.2 Calbicans 3.7 NIIC and MBC/h(lFC in viscous solutions 3.7.1 P. aerugr0nosa 3.7.1.1 6.5 hourinoculum 3.7.1.2 7-0 hour inocuium followed by 1: 100 dilution 3-72 C alibicans CHAPTER 4: DISCUSSION REFERENCES LIST OF FIGURES Figure Page 1. Viscosity of poiyvinyIpy nolidone solutions 2. Osrnolality of polyvinyIpyrroIidone soiutions 3. Accuracy of pipettes 1. Growth curves of P. uemginosa 5. Growth curves OF C. albicm 6. Negative stabs of P. aemginosa to visualize flagella 7. Morphomeaic measurements of P. amginosa 8. Transmission ei ectron micrographs of P. aemginosa 9. Light micrographs of C. albicm LO. ~Morphometricmeasurements of C. ahtcmtr LIST OF TABLES Table Page I. Antibiotic susceptibility of P. aerugmosa as a planktonic population 67 and biofilm population 2. hphotericin B susceptibiiity of C. albicmrs as a piankfonic population and biomrn population. 3. Gentamitin susceptibility of P. aeniginosa incubated in viscous solutions for 6.5 hours. 4. Piperacillin susceptibility of P. aerugirosa incubated in viscous solutions for 6.5 hours. 5. Genramicin susceptibility ofP. aeruginusa incubated in viscous solutions for 20 horn followed b y 1 : 100 dilution and 3 hour incubation. 6. Piperacillin susceptibility of P. uerugrnosa incubated in viscous soIutions for 20 horn foIIowed b y L :100 diIution and 3 hour incubation, 7. .hphotericin B susceptibility of C. albzconr in viscous solutions LIST OF ABBREVIATIONS AD = .Air-displacement BF = Biofilm CBD = Calgruy biofiIm device CFU = Colony forming units cP = Centipoise dW20= Double distilled water DMSO = Dimethyl sulfoxide MBC = Minimum bactericidal concentration ABEC = AWnimumbiofiim eradicating concentration MFC = Minimum fungicidal concentration MHB = MuelIer Hinton Broth MIC = Minimum inhibitory concentration MIV = Minimum immobolidng viscosity PD = Positive dispIacement PVP = Polyvinylpyrrolidone RP = Reverse pipeaing SGA = Sabouraud-Glucose agar TEM = Tmsmission eIectron microscopy TSA = Tryptic soy agar TSB = Tryptic soy broth wlv = weight per volume CHAPTER 1: INTRODUCTION 1.1 Biofirms Historicallyythe field of microbiology has focused on fieely floating planktonic bacteria grown in batch culture. Studies of microorganisms in sihr and in vivo have shown that they are mote often dace-associated and existing as biofilms (Costerton et al, 1987). Biofilms are adherent colonies of microorganisms growing within a polysaccharide cell daceglycocalyx. Growing as a biofih appears to result in modifications of physiological status as compared to the same organism growing in planktonic mode (Costerton et d, 1987). Adhesion triggers the expression of a large number of genes so that biofih ceUs are clearly phenotypically distinct f?om their planktonic counterparts @eretic et al, 1994; Marshall, 1992). Antibiotics and biocides used at concentrations capable of killing planktonic organisms have littie effect against biofih (Ced et d, 1999; Nickel et al, 1985). Biohorganisms also differ fiom planktonic organisms in growth rate (Brown et aI, 1988), celI wall structure and compositioo, immmogenicity, and enzyme activity (Costerton and Lappin-Scott, 1989). 1.1.1 Structure Bacterial and &gaI celIs appear to attach nonspec5caUy to substrata No plastic or metal dacehas been shown resistant to bacterial colonization (Costerton and Lappin-Scott, 1989). Cells initially adhere to surfaces by reversible association and then irrevefsltble adhesion faciIitated by exopoLysaccharide glycocdyx polymefs (Costerton et aI, 1987). In reversible association, bacteria demonstrate Brownian motion and can be removed fiom daceby moderate shear force. When bacteria are irrevem%Ly adbered, 2 bacteria lack Brownian motion, and cannot be disrupted by moderate shear force (Marshall, 1992). Bacteria divide to produce sister ceMs within glycocalyx matrix, initiating the development of microcolonies. A mature biofilm is achieved by division of cells within the microcolonies and recruitment of planktonic ceils fiom the bulk fluid phase (Costerton et al, 1987; Hoyle et aI, 1990). BiofiIms consist of single cells and microcoIonies of sister cells embedded within a polyanionic matrix (Costerton et ai, 1987; Hoyle et aI, 1990). The biofilm contains channels in which nutrients can circulate. Ceffs in different regions of the biofilm exhibit merent patterns of gene expression (Costerton, 1999). According to Anwar et a1 (1992), bioNms consist of two major areas; surface cells and embedded cells. The dacebiofih cells are found in the upper regions. These cells have access to nutrients incIuding oxygen and have little trouble discharging metabolic waste products. They are metabolically active and large and the ceII enveIopes are permeable to nutrients, much Iike their planktonic counterparts. The embedded biofiIm cells in the gIycocaIyx are less metabolically active due to their poor access to nutrients. The cells are srnd and dormant (Anwar et aI, I992). The restriction to essential nutrients influences the physiology of the celI. However another study stated that direct examination of estabIished biofih demonstrated that more than 70% of cells within biofilms were metaboIicdy active, and very active cells were found deep within the biofih, adjacent to originally coIonized dace(Nickel et al, 1985). Biofilm bacteria are not static popuIations, they exhibit an adaptable and envim~lentdyresponsive phenotype.
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