Interaction between legionella pneumophila and biofilm forming organism pseudomonas aeruginosa

INTERACTION BETWEEN LEGIONELLA PNEUMOPHILA AND BIOFILM FORMING ORGANISM PSEUDOMONAS AERUGINOSA WON CHOONG YUN (B.Sc (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2006 Acknowledgements Acknowledgements I would like to express my heartfelt gratitude to the following people who have made a difference in my life during the course of this study: A/Prof Lee Yuan Kun for his invaluable guidance, constant encouragement and patience throughout the course of this study Dr Gamini Kumarasinghe from the Department of Laboratory Medicine, National University Hospital, A/Prof Zhang Lian Hui from Institute of Molecular and Cell Biology, and A/Prof Tim Tolker-Nielsen from BioCentrum-DTU, The Technical University of Denmark, for kindly providing bacterial strains for this study Mr Ma Xi from Nalco Company for his invaluable advice, generous assistance and constant concern Dr Chen Hui and Mr Tim Lim, also from Nalco Company, for their generous sharing of experiences and gracious assistance Mr Low Chin Seng for his precious technical assistance and for being a fatherlyfigure in a laboratory setting Mdm Chew Lai Meng for her encouragement and warm friendship Ho Phui San, Lee Hui Cheng, Wang Shugui and especially Chow Wai Ling and Janice Yong Jing Ying for their generous help, precious friendship and incredible understanding when absentmindedness get the better of me Post-graduate life has never been better without them! Department of Microbiology, NUS i Acknowledgements Toh Yi Er and Lee Kong Heng from Confocal Microscopy Unit, and Toh Kok Tee from Flow Cytometry Unit for their invaluable technical assistance My family and husband, Clement Choo, for their generous love, unwavering support and relentless encouragement through difficult time of my life Especially my father, for his thought-provoking discussions and tremendous help in software improvements for this study My son for sharing his precious life with me Department of Microbiology, NUS ii Table of Contents Table of Contents Acknowledgements i Table of Contents iii List of Tables x List of Figures xi List of Abbreviations xv Summary xvii Chapter 1: Introduction Chapter 2: Literature Review 2.1 Legionella 2.1.1 Introduction to Legionella 2.1.2 General characteristics of Legionella 2.1.3 Taxonomy of Legionella 2.1.4 Legionella and Diseases 2.1.4.1 Clinical presentation 2.1.4.2 Diagnosis 2.1.4.3 Epidemiology 10 2.1.4.4 Epidemiology in Singapore 13 2.1.4.5 Treatment 15 2.1.5 Ecology of Legionella 16 2.1.5.1 Natural and man-made habitats 16 2.1.5.2 Distribution of Legionella in Singapore 18 2.1.5.3 Association of Legionella with protozoa 19 2.1.5.4 Association of Legionella with biofilm 21 Department of Microbiology, NUS iii Table of Contents 2.1.5.5 Interaction of Legionella with Pseudomonas spp 2.2 Biofilm 24 24 2.2.1 Introduction to biofilm 24 2.2.2 General characteristics of biofilm 25 2.2.3 Biofilm development 26 2.2.4 Stages of biofilm development 27 2.2.4.1 Stage 1: Reversible attachment 27 2.2.4.2 Stage 2: Irreversible attachment 28 2.2.4.3 Stage 3: Maturation-1 29 2.2.4.4 Stage 4: Maturation-2 29 2.2.4.5 Stage 5: Dispersion 30 2.2.5 Determinants of biofilm structure 31 2.2.6 Microbial diversity of biofilms 33 2.2.7 Microbial positioning in biofilm 34 2.3 Prevention of legionellosis 35 2.3.1 Control of legionellosis 35 2.3.2 Detection of Legionella 36 2.3.3 Risk assessment of cooling tower for Legionnaires’ disease outbreaks 2.3.4 Water treatment in cooling towers 37 38 Chapter 3: Materials and Methods 41 3.1 Bacterial strains and culture 41 3.1.1 Bacterial Strains 41 3.1.2 Culture Media 41 Department of Microbiology, NUS iv Table of Contents 3.1.3 Maintenance of stock cultures 3.2 Growth kinetic studies 42 42 3.2.1 Growth kinetics of L pneumophila 42 3.2.2 Growth kinetics of P aeruginosa PAO1 43 3.2.3 Growth kinetics of P aeruginosa PAO1-CFP 43 3.3 Determination of the influent flow rate (Q) for continuous culture in CDC Biofilm Reactor (CBR) 3.4 Optimization of labelling processes 3.4.1 Optimization of L pneumophila labelling with CFDA-SE 43 44 44 3.4.2 Optimization of planktonic P aeruginosa PAO1-CFP labelling with PI 3.4.3 Flow cytometry 44 45 3.4.4 Optimization of P aeruginosa PAO1-CFP biofilm labelling with PI 45 3.5 P aeruginosa PAO1-CFP biofilm formation in CDC Biofilm Reactor (CBR) 46 3.5.1 CDC Biofilm Reactor 46 3.5.2 Setup of CDC Biofilm Reactor assembly 47 3.5.3 P aeruginosa PAO1-CFP biofilm formation 48 3.6 Introduction of L pneumophila into P aeruginosa PAO1-CFP biofilms 50 3.7 Introduction of NALCO 7320 into developing and mature P aeruginosa PAO1-CFP biofilms containing L pneumophila 3.8 Monitoring of each organism in CBR continuous flow system 51 52 3.8.1 Preparation for sampling 52 3.8.2 Taking samples 52 Department of Microbiology, NUS v Table of Contents 3.8.2.1 Sampling bulk fluid 52 3.8.2.2 Sampling biofilm 53 3.8.3 Preparation of coupons 3.8.3.1 Preparation of coupons intended for enumeration 53 53 3.8.3.2 Preparation of coupons intended for visualization by CLSM 53 3.8.4 Disaggregation by homogenization 54 3.8.5 Enumeration of each organism 55 3.8.5.1 Enumeration of P aeruginosa PAO1-CFP by culture 55 3.8.5.2 Enumeration of L pneumophila by immunofluorescence 56 3.8.6 Detection of exogenous contaminants 58 3.8.7 Visualization and image acquisition by CLSM 59 3.8.8 Application of COMSTAT image analysis software package 60 3.8.8.1 Preparation of image stacks 60 3.8.8.2 Thresholding of images 61 3.8.8.3 COMSTAT image analysis for P aeruginosa PAO1-CFP biofilm structure 61 3.8.8.4 COMSTAT image analysis for porosity of P aeruginosa PAO1-CFP biofilm 63 3.8.8.5 COMSTAT image analysis for L pneumophila distribution 64 3.8.9 Statistical analysis 3.9 Screening for effective P aeruginosa PAO1 biofilm-removing agent 65 65 3.9.1 Kinetics of P aeruginosa PAO1 biofilm formation in microtiter plate 3.9.2 Quantification of biofilm Department of Microbiology, NUS 65 66 vi Table of Contents 3.9.3 Biofilm-removing agents used 67 3.9.4 P aeruginosa PAO1 biofilm removal screening 68 3.10 Antimicrobial susceptibility testing of NALCO 7320 69 Chapter 4: Results 70 4.1 Growth kinetics 70 4.2 Determination of the influent flow rate (Q) for continuous culture in CDC Biofilm Reactor (CBR) 4.3 Optimization of labelling processes 4.3.1 Optimization of L pneumophila labelling with CFDA-SE 72 74 74 4.3.2 Optimization of planktonic P aeruginosa PAO1-CFP labelling with PI 75 4.3.3 Optimization of P aeruginosa PAO1-CFP biofilm labelling with PI 77 4.4 Kinetics of P aeruginosa PAO1-CFP biofilm formation in CDC Biofilm Reactor (CBR) 80 4.4.1 Kinetics of biofilm formation 80 4.4.2 Structure of biofilm by image analysis 81 4.4.3 Detachment of biofilm 85 4.5 Introduction of L pneumophila to developing and mature P aeruginosa PAO1-CFP biofilms 87 4.5.1 Adhesion and persistence of L pneumophila in developing and mature biofilms 87 4.5.2 Distributions of L pneumophila cells in developing and mature biofilms 4.5.3 Bio-volume distributions of developing and mature biofilms Department of Microbiology, NUS 90 95 vii Table of Contents 4.5.4 Surface-to-biovolume ratio distributions of developing and mature biofilms 97 4.5.5 Porosity distributions of developing and mature biofilms 100 4.5.6 Correlation between SBR and porosity 103 4.5.7 Correlation between legionellae adhesion and parameters of P aeruginosa PAO1-CFP biofilm 104 4.5.8 Localization of L pneumophila in P aeruginosa PAO1-CFP biofilms 4.6 Screening for effective P aeruginosa PAO1 biofilm removing agent 105 108 4.6.1 Kinetics of P aeruginosa PAO1 biofilm formation in microtiter plate 4.6.2 P aeruginosa PAO1 biofilm removal screening 4.7 Characterization of NALCO 7320 108 109 111 4.7.1 Kinetics of P aeruginosa PAO1 biofilm removal 111 4.7.2 Antimicrobial susceptibility testing 112 4.8 Introduction of NALCO 7320 into developing and mature P aeruginosa PAO1-CFP biofilms containing L pneumophila 114 4.8.1 Persistence of P aeruginosa PAO1-CFP in CBR 114 4.8.2 Structure of P aeruginosa PAO1-CFP biofilms treated by NALCO 7320 115 4.8.3 Persistence of L pneumophila in P aeruginosa PAO1-CFP biofilms treated with NALCO 7320 120 4.8.4 Distribution of L pneumophila in P aeruginosa PAO1-CFP biofilms treated with NALCO 7320 Department of Microbiology, NUS 123 viii Table of Contents 4.8.5 Bio-volume distributions of developing and mature biofilms treated with NALCO 7320 125 4.8.6 Porosity distributions of P aeruginosa PAO1-CFP biofilms treated with NALCO 7320 127 Chapter 5: Discussion 130 References 147 Appendix 175 Department of Microbiology, NUS ix References Appl Environ Microbiol 54(10):2492-9 Leoni E, Legnani PP 2001 Comparison of selective procedures for isolation and enumeration of Legionella species from hot water systems J Appl Microbiol 90(1):27-33 Lewandowski Z, Beyenal H, Stookey D 2004 Reproducibility of biofilm processes and the meaning of steady state in biofilm reactors Water Sci Technol 49(11-12):359-64 Lewandowski Z 2000 Notes on biofilm porosity Water Research 34(9):2620-4 Lim HS, Tham MK, Chua KL.1986 Legionnaires' disease report of Singapore's first local case Ann Acad Med Singapore 15(4):610-6 Liu Y, Tay JH 2001 Metabolic response of biofilm to shear stress in fixed-film culture J Appl Microbiol 90(3):337-42 Mah TF, O'Toole GA 2001 Mechanisms of biofilm resistance to antimicrobial agents Trends Microbiol 9(1):34-9 Maiwald M, Helbig JH, Luck PC 1998 Laboratory methods for diagnosis of Legionella infections J Microbiol Methods 33:59-79 Department of Microbiology, NUS 162 References Mampel J, Spirig T, Weber SS, Haagensen JA, Molin S, Hilbi H 2006 Planktonic replication is essential for biofilm formation by Legionella pneumophila in a complex medium under static and dynamic flow conditions Appl Environ Microbiol 72(4):2885-95 Marrão G, Veríssimo A, Bowker RG, da Costa MS 1993 Biofilms as major sources of Legionella spp in hydrothermal areas and their dispersion into stream water FEMS Microbiology Ecology 12(1):25-33 Marston BJ, Plouffe JF, File TM Jr, Hackman BA, Salstrom SJ, Lipman HB, Kolczak MS, Breiman RF 1997 Incidence of community-acquired pneumonia requiring hospitalization Results of a population-based active surveillance study in Ohio The Community-Based Pneumonia Incidence Study Group Arch Intern Med 157(15):1709-18 Marston BJ, Lipman HB, Breiman RF 1994 Surveillance for Legionnaires' disease Risk factors for morbidity and mortality Arch Intern Med 154(21):241722 Massol-Deya AA, Whallon J, Hickey RF, Tiedje JM 1995 Channel structures in aerobic biofilms of fixed-film reactors treating contaminated groundwater Appl Environ Microbiol 61(2):769-77 Department of Microbiology, NUS 163 References McDade JE, Brenner DJ, Bozeman FM 1979 Legionnaires' disease bacterium isolated in 1947 Ann Intern Med 90(4):659-61 McDade JE, Shepard CC, Fraser DW, Tsai TR, Redus MA, Dowdle WR 1977 Legionnaires' disease: isolation of a bacterium and demonstration of its role in other respiratory disease N Engl J Med 297(22):1197-203 Meers PD, Goh KT, Lim EW.1989 Legionella species, serogroups and subgroups found in the environment in Singapore Ann Acad Med Singapore 18(4):375-8 Montagna MT, Napoli C, Tato D, Spilotros G, Barbuti G, Barbuti S 2006 Clinical-environmental surveillance of legionellosis: an experience in southern Italy Eur J Epidemiol 21(4):325-31 Muder RR, Yu VL 2002 Infection due to Legionella species other than L pneumophila Clin Infect Dis 35(8):990-8 Murga R, Forster TS, Brown E, Pruckler JM, Fields BS, Donlan RM 2001 Role of biofilms in the survival of Legionella pneumophila in a model potable-water system Microbiology 147(11):3121-6 Nadarajah M, Singam S, Jalil HA 1987 Sero-survey for Legionella pneumophila antibodies Singapore experience Ann Acad Med Singapore 16(4):583-5 Department of Microbiology, NUS 164 References Nadarajah M, Goh KT 1986 Isolation of Legionella pneumophila from hospital cooling towers Ann Acad Med Singapore 15(1):6-8 Negron-Alvira A, Perez-Suarez I, Hazen TC 1988 Legionella spp in Puerto Rico cooling towers Appl Environ Microbiol 54(10):2331-4 Okabe S, Yasuda T, Watanabe Y 1996 Uptake and release of inert fluorescence particles by mixed population biofilms Biotechnology and Bioengineering 53(5):459-69 Ong KC, Eng P 1995 Community-acquired pneumonia Singapore Med J 36(5):539-44 O’Toole GA, Pratt LA, Watnick PI, Newman DK, Weaver VB, Kolter R 1999 Genetic approaches to study of biofilms Methods Enzymol 310: 91-109 O'Toole GA, Kolter R 1998 Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development Mol Microbiol 30(2):295-304 Ott M, Messner P, Heesemann J, Marre R, Hacker J 1991 Temperaturedependent expression of flagella in Legionella J Gen Microbiol 137(8):1955-61 Pace NR 1997 A molecular view of microbial diversity and the biosphere Science 276(5313):734-40 Department of Microbiology, NUS 165 References Paszko-Kolva C, Shahmat M, Keiser J and Colwell RR 1993 Prevalence of antibodies against Legionella species in healthy and patient populations pp 24-5 In: Barbaree JM et al (eds.), Legionella: Current Status and Emerging Perspectives Washington, D.C.: American Society for Microbiology Palmer CJ, Tsai YL, Paszko-Kolva C, Mayer C, Sangermano LR 1993 Detection of Legionella species in sewage and ocean water by polymerase chain reaction, direct fluorescent-antibody, and plate culture methods Appl Environ Microbiol 59(11):3618-24 Pearson JP, Passador L, Iglewski BH, Greenberg EP 1995 A second Nacylhomoserine lactone signal produced by Pseudomonas aeruginosa Proc Natl Acad Sci U S A 92(5):1490-4 Pearson JP, Gray KM, Passador L, Tucker KD, Eberhard A, Iglewski BH, Greenberg EP 1994 Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes Proc Natl Acad Sci U S A 91(1):197201 Picioreanu C, multidimensional Kreft JU, Van multispecies Loosdrecht biofilm model MC Appl 2004 Particle-based Environ Microbiol 70(5):3024-40 Department of Microbiology, NUS 166 References Prince EL, Muir AV, Thomas WM, Stollard RJ, Sampson M, Lewis JA 2002 An evaluation of the efficacy of Aqualox for microbiological control of industrial cooling tower systems J Hosp Infect 52(4):243-9 Purevdorj B, Costerton JW, Stoodley P 2002 Influence of hydrodynamics and cell signaling on the structure and behavior of Pseudomonas aeruginosa biofilms Appl Environ Microbiol 68(9):4457-64 Reisner A, Haagensen JA, Schembri MA, Zechner EL, Molin S 2003 Development and maturation of Escherichia coli K-12 biofilms Mol Microbiol 48(4):933-46 Rickard AH, McBain AJ, Stead AT, Gilbert P 2004 Shear rate moderates community diversity in freshwater biofilms Appl Environ Microbiol 70(12):7426-35 Rickard AH, McBain AJ, Ledder RG, Handley PS, Gilbert P 2003 Coaggregation between freshwater bacteria within biofilm and planktonic communities FEMS Microbiol Lett 220(1):133-40 Rickard AH, Leach SA, Hall LS, Buswell CM, High NJ, Handley PS 2002 Phylogenetic relationships and coaggregation ability of freshwater biofilm bacteria Appl Environ Microbiol 68(7):3644-50 Department of Microbiology, NUS 167 References Rickard AH, Leach SA, Buswell CM, High NJ, Handley PS 2000 Coaggregation between aquatic bacteria is mediated by specific-growth-phase-dependent lectinsaccharide interactions Appl Environ Microbiol 66(1):431-4 Ricketts K, Joseph C 2005 Legionnaires' disease in Europe 2003-2004 Euro Surveill 10(12): 256-9 Rogers J, Dowsett AB, Keevil CW 1995 A paint incorporating silver to control mixed biofilms containing Legionella pneumophila J Ind Microbiol 15(4):37783 Rogers J, Dowsett AB, Dennis PJ, Lee JV, Keevil CW 1994 Influence of temperature and plumbing material selection on biofilm formation and growth of Legionella pneumophila in a model potable water system containing complex microbial flora Appl Environ Microbiol 60(5):1585-92 Rogers J, Keevil CW 1992 Immunogold and fluorescein immunolabelling of Legionella pneumophila within an aquatic biofilm visualized by using episcopic differential interference contrast microscopy Appl Environ Microbiol 58(7):2326-30 Rowbotham TJ 1986 Current views on the relationships between amoebae, legionellae and man Isr J Med Sci 22(9):678-89 Department of Microbiology, NUS 168 References Ruehlemann SA, Crawford GR 1996 Panic in the potting shed: The association between Legionella longbeachae serogroup and potting soils in Australia Med J Aust 164(1):36-8 Erratum in: Med J Aust 1996 164(11):704 Sauer K, Camper AK, Ehrlich GD, Costerton JW, Davies DG 2002 Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm J Bacteriol 184(4):1140-54 Schmid T, Panne U, Adams J, Niessner R 2004 Investigation of biocide efficacy by photoacoustic biofilm monitoring Water Res 38(5):1189-96 Shapiro HM, Nebe-von-Caron G 2004 Multiparameter flow cytometry of bacteria pp33-43 In: Hawley TS and Hawley RG (eds.) Flow cytometry protocols, 2nd Edition Humana Press, Totowa, N.J Shelton BG, Flanders WD, Morris GK 1994 Legionnaires’ disease outbreaks and cooling towers with amplified Legionella concentrations Curr Microbiol 28:359363 Srikanth S, Berk SG 1993 Stimulatory effect of cooling tower biocides on amoebae Appl Environ Microbiol 59(10):3245-9 Steele TW, Lanser J, Sangster N 1990 Isolation of Legionella longbeachae serogroup from potting mixes Appl Environ Microbiol 56(1):49-53 Department of Microbiology, NUS 169 References Steinert M, Hentschel U, Hacker J 2002 Legionella pneumophila: an aquatic microbe goes astray FEMS Microbiol Rev 26(2):149-62 Steinert M, Emody L, Amann R, Hacker J 1997 Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii Appl Environ Microbiol 63(5):2047-53 Stoodley P, Sauer K, Davies DG, Costerton JW 2002 Biofilms as complex differentiated communities Annu Rev Microbiol 56:187-209 Stoodley P, Wilson S, Hall-Stoodley L, Boyle JD, Lappin-Scott HM, Costerton JW 2001 Growth and detachment of cell clusters from mature mixed-species biofilms Appl Environ Microbiol 67(12):5608-13 Stoodley P, Lewandowski Z, Boyle JD, Lappin-Scott HM 1999 Structural deformation of bacterial biofilms caused by short-term fluctuations in fluid shear: an in situ investigation of biofilm rheology Biotechnol Bioeng 65(1):83-92 Stoodley P, deBeer D and Lewandowski Z 1994 Liquid flow in biofilm systems Appl Environ Microbiol 60(8):2711-6 Stout JE, Rihs JD, Yu VL 2003 Legionella Vol.2 pp809-23 In: Murray PR (ed.), Manual of clinical microbiology, 8th Edition ASM Press, Washington, D.C Department of Microbiology, NUS 170 References Stout JE, Yu VL, Best MG 1985 Ecology of Legionella pneumophila within water distribution systems Appl Environ Microbiol 49(1):221-8 Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ, Brinkman FS, Hufnagle WO, Kowalik DJ, Lagrou M, Garber RL, Goltry L, Tolentino E, Westbrock-Wadman S, Yuan Y, Brody LL, Coulter SN, Folger KR, Kas A, Larbig K, Lim R, Smith K, Spencer D, Wong GK, Wu Z, Paulsen IT, Reizer J, Saier MH, Hancock RE, Lory S, Olson MV 2000 Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen Nature 406(6799):959-64 Sutherland IW 2001 The biofilm matrix an immobilized but dynamic microbial environment Trends Microbiol 9(5):222-7 Swanson MS, Hammer BK 2000 Legionella pneumophila pathogenesis: a fateful journey from amoebae to macrophages Annu Rev Microbiol 54:567-613 Tesh MJ, Miller RD 1981 Amino acid requirements for Legionella pneumophila growth J Clin Microbiol 13(5):865-9 Thomas V, Bouchez T, Nicolas V, Robert S, Loret JF, Levi Y 2004 Amoebae in domestic water systems: resistance to disinfection treatments and implication in Legionella persistence J Appl Microbiol 97(5):950-63 Department of Microbiology, NUS 171 References Tison DL, Pope DH, Cherry WB, Fliermans CB 1980 Growth of Legionella pneumophila in association with blue-green algae (cyanobacteria) Appl Environ Microbiol 39(2):456-9 Tolker-Nielsen T, Brinch UC, Ragas PC, Andersen JB, Jacobsen CS, Molin S 2000 Development and dynamics of Pseudomonas sp biofilms J Bacteriol 182(22):6482-9 Tossa P, Deloge-Abarkan M, Zmirou-Navier D, Hartemann P, Mathieu L 2006 Pontiac fever: an operational definition for epidemiological studies BMC Public Health 6:112 Tsai TF, Finn DR, Plikaytis BD, McCauley W, Martin SM, Fraser DW 1979 Legionnaires' disease: clinical features of the epidemic in Philadelphia Ann Intern Med 90(4):509-17 van Loosdrecht MC, Heijnen JJ, Eberl H, Kreft J, Picioreanu C 2002 Mathematical modelling of biofilm structures Antonie Van Leeuwenhoek 81(14):245-56 Verissimo A, Marrao G, da Silva FG, da Costa MS 1991 Distribution of Legionella spp in hydrothermal areas in continental Portugal and the island of Sao Miguel, Azores Appl Environ Microbiol 57(10):2921-7 Department of Microbiology, NUS 172 References Wadowsky RM, Yee RB 1985 Effect of non-Legionellaceae bacteria on the multiplication of Legionella pneumophila in potable water Appl Environ Microbiol 49(5):1206-10 Walker JT, Sonesson A, Keevil CW, White DC 1993 Detection of Legionella pneumophila in biofilms containing a complex microbial consortium by gas chromatography-mass spectrometry analysis of genus-specific hydroxy fatty acids FEMS Microbiol Lett 113(2):139-44 Watnick PI, Kolter R 1999 Steps in the development of a Vibrio cholerae El Tor biofilm Mol Microbiol 34(3):586-95 Webb JS, Thompson LS, James S, Charlton T, Tolker-Nielsen T, Koch B, Givskov M, Kjelleberg S 2003 Cell death in Pseudomonas aeruginosa biofilm development J Bacteriol 185(15):4585-92 Westall F, de Wit MJ, Dann J, van der Gaast S, de Ronde CEJ and Gerneke D 2001 Early Archean fossil bacteria and biofilms in hydrothermally-influenced sediments from the Barberton greenstone belt, South Africa Precambrian Res 106:93-116 Winiecka-Krusnell J, Linder E 1999 Free-living amoebae protecting Legionella in water: the tip of an iceberg? Scand J Infect Dis 31(4):383-5 Department of Microbiology, NUS 173 References Winn WC Jr 1999 Legionella pp572-85 In: Murray PR (ed.), Manual of clinical microbiology, 7th Edition ASM Press, Washington D.C Wright JB 2000 Legionella Biofilms: their Implications, Study and Control pp291-310 In: L.V Evans (ed.), Biofilms: recent advances in their study and control Harwood Academic, Amsterdam Yamamoto H, Ezaki T, Ikedo M, Yabuuchi E 1991 Effects of biocidal treatments to inhibit the growth of legionellae and other microorganisms in cooling towers Microbiol Immunol 35(9):795-802 Yang X, Beyenal H, Harkin G, Lewandowski Z 2000 Quantifying biofilm structure using image analysis Journal of Microbiological Methods 39(2):10919 Yang S, Lewandowski Z 1995 Measurement of local mass transfer coefficient in biofilms Biotechnology and Bioengineering 48:737-44 Yu VL, Plouffe JF, Pastoris MC, Stout JE, Schousboe M, Widmer A, Summersgill J, File T, Heath CM, Paterson DL, Chereshsky A 2002 Distribution of Legionella species and serogroups isolated by culture in patients with sporadic communityacquired legionellosis: an international collaborative survey J Infect Dis 186:127–8 Department of Microbiology, NUS 174 Appendix Appendix Appendix I Edelstein BCYE liquid media: 2.0g Activated charcoal 10.0g Yeast extract 1L Deionized water Autoclaved at 121°C for 15mins The media was allowed to cool before adding Legionella BCYE growth supplement (Oxoid Limited, UK) reconstituted as directed and filter sterilized Appendix II Luria Bertani (LB) broth: 5g Yeast extract 10g Tryptone 10g NaCl 1L Deionized water Autoclaved at 121°C for 15mins LB agar: Additional inclusion of 15g granulated agar in 1L LB broth and autoclaved at 121°C for 15mins Appendix III Minimal media (MM): 10.5g K2HPO4 4.5g KH2PO4 2.0g (NH4)2SO4 2.0g Mannitol 0.2g MgSO4.7H2O 10mg CaCl2 5mg FeSO4.7H2O 2mg MnCl2 1L Deionized water Autoclaved at 121°C for 15mins Appendix IV Phosphate Buffer Saline (PBS): 0.24g KH2PO4 1.44g Na2HPO4 8g NaCl 0.2g KCl 1L Deionized water Adjusted to pH 7.4 with 1N NaOH or 1M HCl, and autoclaved at 121°C for 15mins Department of Microbiology, NUS 175 Appendix Appendix V CFDA-SE stock solution (3.6mM): 1) Dissolve 2mg CFDA-SE (Molecular weight: 557) in 20μl DMSO 2) Top up to 1ml with ethanol (reagent grade) 3) Filter-sterilize & store at -20ºC in the dark 4) Working concentration: 10µM Appendix VI 4% Para-formaldehyde (PFA) solution: 1) Dissolved EM grade PFA in PBS with stir bar (4g to 100ml) 2) Add few drops of 1N NaOH and heat in hood (keep bottle cap loose) at 60°C to dissolve 3) Cool to room temperature and adjust to pH 7.4 with 1M HCl *Prepare fresh prior to use Department of Microbiology, NUS 176 [...]... days old P aeruginosa PAO1-CFP 77 biofilm and adhered L pneumophila, stained with 0.1mg/ml PI for 5mins: (A) P aeruginosa PAO1-CFP biofilm (blue fluorescence), (B) CFDA-stained L pneumophila (green fluorescence), (C) PI-stained P aeruginosa PAO1-CFP biofilm, and (D) overlapping display of the above 3 images Figure 4.9 CLSM images of a 7 days old P aeruginosa PAO1-CFP 78 biofilm and adhered L pneumophila, ... developing and mature biofilm Table 4.5 Comparing means of porosity over time Table 4.6 Table showing Pearson’s correlation between porosity and 103 SBR Table 4.7 Table showing Pearson’s correlation between legionellae 104 adhesion to P aeruginosa PAO1-CFP biofilm (representing the number of legionellae per coupon per 106 legionellae inoculated into CBR) and parameters of the biofilm Table 4.8 Efficacy of biofilm. .. P 94 aeruginosa PAO1-CFP biofilm Figure 4.25 Percentage loss of L pneumophila in mature P aeruginosa 94 PAO1-CFP biofilm Figure 4.26 Bio-volume distribution of (A) developing, and (B) mature 96 P aeruginosa PAO1-CFP biofilms Figure 4.27 Surface-to-biovolume ratio (SBR) distribution of (A) 99 developing, and (B) mature P aeruginosa PAO1-CFP biofilms Figure 4.28 Porosity of P aeruginosa PAO1-CFP biofilm. .. greater insights of biofilm structures and properties Consequently, biofilm structures and development were better described, and the first physical evidence of porous channels within biofilm cell cluster was observed Legionella pneumophila adhesion study revealed that legionellae adhesion to biofilms was independent of developmental stage of the latter Instead, biofilm structure and porosity were found... microbial biofilm communities, and failed to identify all the organisms present and their contribution to the survival and multiplication of legionellae Additionally, Pseudomonas aeruginosa PAO1, a wound isolate (Holloway, 1955), is generally found in the same aquatic environments as L pneumophila (Murga et al., 2001), is the most widely used P aeruginosa laboratory strain (Stover et al., 2000) and its biofilm. .. context was developed Using the model and better descriptive methods of biofilm structure and porosity, it was determined if there is any difference (in numbers and distribution pattern) in accumulation and persistence of L pneumophila in developing and mature biofilm, and if the structure or porosity of biofilm plays a role in the accumulation and persistence of L pneumophila In a bid to deepen the knowledge... 87 stages of P aeruginosa PAO1-CFP biofilm Figure 4.21 Status of L pneumophila in our continuous flow CBR 89 system Figure 4.22 Persistence of L pneumophila in P aeruginosa PAO1- 89 CFP biofilm Department of Microbiology, NUS xii List of Figures Figure 4.23 Distribution of L pneumophila in (A) developing, and (B) 93 mature P aeruginosa PAO1-CFP biofilms Figure 4.24 Percentage loss of L pneumophila in... (A) P aeruginosa PAO1-CFP biofilm (blue fluorescence), (B) CFDA-stained L pneumophila (green fluorescence), (C) PI-stained P aeruginosa PAO1-CFP biofilm, and (D) overlapping Department of Microbiology, NUS xi List of Figures display of the above 3 images Figure 4.10 CLSM images of a 7 days old P aeruginosa PAO1-CFP 79 biofilm and adhered L pneumophila, stained with 0.1mg/ml PI for 30mins: (A) P aeruginosa. .. introduction to developing biofilm, (C) 3hrs after legionellae introduction to mature biofilm (7-daysold), and (D) 4 days after legionellae introduction to mature biofilm Figure 4.32 Kinetics of P aeruginosa PAO1 biofilm formation in 108 microtitre plate at 30°C Figure 4.33 Highest percentage biofilm removal of various biofilm- 109 removing agents Figure 4.34 Kinetics of biofilm removal by NALCO 7320... developing, and (B) mature P 102 aeruginosa PAO1-CFP biofilms Figure 4.30 Scatterplot of porosity and SBR both obtained from all 103 data of 6 independent experiments Figure 4.31 CLSM images of P aeruginosa PAO1-CFP biofilm (blue) 107 with adhered L pneumophila (green) taken on different occasions: (A) 3hrs after legionellae introduction to developing biofilm (3-days-old), (B) 4 days after legionellae ... developing and mature biofilms 97 4.5.5 Porosity distributions of developing and mature biofilms 100 4.5.6 Correlation between SBR and porosity 103 4.5.7 Correlation between legionellae adhesion and. .. of biofilm by image analysis 81 4.4.3 Detachment of biofilm 85 4.5 Introduction of L pneumophila to developing and mature P aeruginosa PAO1-CFP biofilms 87 4.5.1 Adhesion and persistence of L pneumophila. .. L pneumophila into P aeruginosa PAO1-CFP biofilms 50 3.7 Introduction of NALCO 7320 into developing and mature P aeruginosa PAO1-CFP biofilms containing L pneumophila 3.8 Monitoring of each organism