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
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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
Department of Microbiology, NUS i
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 fatherly-
figure 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!
Acknowledgements
Department of Microbiology, NUS ii
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.
Table of Contents
Department of Microbiology, NUS iii
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 1
Chapter 2: Literature Review 5
2.1 Legionella 5
2.1.1 Introduction to Legionella 5
2.1.2 General characteristics of Legionella 5
2.1.3 Taxonomy of Legionella 7
2.1.4 Legionella and Diseases 8
2.1.4.1 Clinical presentation 8
2.1.4.2 Diagnosis 9
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
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2.1.5.5 Interaction of Legionella with Pseudomonas spp. 24
2.2 Biofilm 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 37
2.3.4 Water treatment in cooling towers 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
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3.1.3 Maintenance of stock cultures 42
3.2 Growth kinetic studies 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) 43
3.4 Optimization of labelling processes 44
3.4.1 Optimization of L. pneumophila labelling with CFDA-SE 44
3.4.2 Optimization of planktonic P. aeruginosa PAO1-CFP labelling
with PI 44
3.4.3 Flow cytometry 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 51
3.8 Monitoring of each organism in CBR continuous flow system 52
3.8.1 Preparation for sampling 52
3.8.2 Taking samples 52
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3.8.2.1 Sampling bulk fluid 52
3.8.2.2 Sampling biofilm 53
3.8.3 Preparation of coupons 53
3.8.3.1 Preparation of coupons intended for enumeration 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 65
3.9 Screening for effective P. aeruginosa PAO1 biofilm-removing agent 65
3.9.1 Kinetics of P. aeruginosa PAO1 biofilm formation in microtiter
plate 65
3.9.2 Quantification of biofilm 66
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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) 72
4.3 Optimization of labelling processes 74
4.3.1 Optimization of L. pneumophila labelling with CFDA-SE 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 90
4.5.3 Bio-volume distributions of developing and mature biofilms 95
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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 105
4.6 Screening for effective P. aeruginosa PAO1 biofilm removing agent 108
4.6.1 Kinetics of P. aeruginosa PAO1 biofilm formation in microtiter
plate 108
4.6.2 P. aeruginosa PAO1 biofilm removal screening 109
4.7 Characterization of NALCO 7320 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 123
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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
Sampling points of 6 independent experiments for the study of P. aeruginosa PAO1-CFP biofilm formation. List of biofilm-removing agents used. Effect of treatment duration on staining and viability of L. pneumophila cells. Effect of treatment duration on staining of P. aeruginosa PAO1-CFP cells. Table showing Pearson’s correlation between Log (Number of L. pneumophila cells) and Log (Number of CFDA pixels per µm3). The ratio of SBR at the bottom 20% versus the top 20% of developing and mature biofilm. Comparing means of porosity over time. Table showing Pearson’s correlation between porosity and SBR. Table showing Pearson’s correlation between legionellae 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. Efficacy of biofilm removing agents. Table showing Pearson’s correlation between bio-volume and legionellae loss.
Schematic diagram of the CDC Biofilm Reactor assembly. Growth curve of L. pneumophila cultured in BCYE broth at 37°C with shaking at 120rpm. Growth curve of P. aeruginosa PAO1 cultured in MM liquid media at 30°C with shaking at 120rpm. Growth curve of P. aeruginosa PAO1-CFP cultured in MM liquid media at 30°C with shaking at 120rpm. Graph of Ln(OD600nm) against time (hr) plotted for the exponential growth phase of P. aeruginosa PAO1-CFP. Histograms illustrating the number of events (cells) plotted against FL1-H (representing green fluorescence of CFDA-stained cells) for L. pneumophila cells that were (A) mock treated, or treated with CFDA-SE for (B) 20mins, (C) 30mins, or (D) 40mins. Histograms illustrating the number of events (cells) plotted against PMT4 Log (representing red fluorescence of PI-stained cells) for P. aeruginosa PAO1-CFP cells that were (A) mock treated, or treated with 1.0mg/ml PI for (B) 5mins, (C) 10mins, or (D) 15mins. Histograms illustrating the number of events (cells) plotted against PMT4 Log (representing red fluorescence of PI-stained cells) for P. aeruginosa PAO1-CFP cells that were (A) mock treated, or treated with 0.1mg/ml PI for (B) 5mins, (C) 10mins, (D) 15mins, or (E) 30mins. CLSM images of a 7 days old P. aeruginosa PAO1-CFP 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. CLSM images of a 7 days old P. aeruginosa PAO1-CFP biofilm and adhered L. pneumophila, stained with 0.1mg/ml PI for 15mins: (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. CLSM images of a 7 days old P. aeruginosa PAO1-CFP biofilm and adhered L. pneumophila, stained with 0.1mg/ml PI for 30mins: (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. Viable cell counts of P. aeruginosa PAO1-CFP biofilm formed in CBR at 30°C with stirring at 120rpm. Bio-volume of P. aeruginosa PAO1-CFP biofilm formed in CBR at 30°C with stirring at 120rpm. Average thickness of P. aeruginosa PAO1-CFP biofilm formed in CBR at 30°C with stirring at 120rpm. Maximum thickness of P. aeruginosa PAO1-CFP biofilm formed in CBR at 30°C with stirring at 120rpm. Substratum coverage of P. aeruginosa PAO1-CFP biofilm formed in CBR at 30°C with stirring at 120rpm. Surface-to-biovolume ratio (SBR) of P. aeruginosa PAO1-CFP biofilm formed in CBR at 30°C with stirring at 120rpm. Roughness coefficient of P. aeruginosa PAO1-CFP biofilm formed in CBR at 30°C with stirring at 120rpm. Viable cell counts of planktonic P. aeruginosa PAO1-CFP in the bulk fluid of CBR at 30°C with stirring at 120rpm. CLSM image of a P. aeruginosa PAO1-CFP biofilm (blue) structure indicative of dispersion stage of biofilm development, with adhered L. pneumophila (green). Adhesion of L. pneumophila to different developmental stages of P. aeruginosa PAO1-CFP biofilm. Status of L. pneumophila in our continuous flow CBR system. Persistence of L. pneumophila in P. aeruginosa PAO1-CFP biofilm.
Distribution of L. pneumophila in (A) developing, and (B) mature P. aeruginosa PAO1-CFP biofilms. Percentage loss of L. pneumophila in developing P. aeruginosa PAO1-CFP biofilm. Percentage loss of L. pneumophila in mature P. aeruginosa PAO1-CFP biofilm. Bio-volume distribution of (A) developing, and (B) mature P. aeruginosa PAO1-CFP biofilms. Surface-to-biovolume ratio (SBR) distribution of (A) developing, and (B) mature P. aeruginosa PAO1-CFP biofilms. Porosity of P. aeruginosa PAO1-CFP biofilm. Porosity distribution of (A) developing, and (B) mature P. aeruginosa PAO1-CFP biofilms. Scatterplot of porosity and SBR both obtained from all data of 6 independent experiments. CLSM images of P. aeruginosa PAO1-CFP biofilm (blue) 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 introduction to developing biofilm, (C) 3hrs after legionellae introduction to mature biofilm (7-days-old), and (D) 4 days after legionellae introduction to mature biofilm. Kinetics of P. aeruginosa PAO1 biofilm formation in microtitre plate at 30°C. Highest percentage biofilm removal of various biofilm-removing agents. Kinetics of biofilm removal by NALCO 7320. Visual determination of minimum inhibitory concentration (MIC). Determination of minimum bactericidal concentration (MBC) of NALCO 7320. Viable cell counts of P. aeruginosa PAO1-CFP biofilms
treated with NALCO 7320. Viable cell counts of planktonic P. aeruginosa PAO1-CFP in CBR treated with NALCO 7320. Bio-volume of P. aeruginosa PAO1-CFP biofilm in CBR treated with NALCO 7320. Average thickness of P. aeruginosa PAO1-CFP biofilm in CBR treated with NALCO 7320. Maximum thickness of P. aeruginosa PAO1-CFP biofilm in CBR treated with NALCO 7320. Substratum coverage of P. aeruginosa PAO1-CFP biofilm in CBR treated with NALCO 7320. Surface-to-biovolume ratio of P. aeruginosa PAO1-CFP biofilm in CBR treated with NALCO 7320. Roughness coefficient of P. aeruginosa PAO1-CFP biofilm in CBR treated with NALCO 7320. Persistence of L. pneumophila in P. aeruginosa PAO1-CFP biofilms treated with NALCO 7320. Cell counts of planktonic L. pneumophila in CBR treated with NALCO 7320. Scatterplot of bio-volume and legionellae loss, obtained from 4 independent experiments. Effect of NALCO 7320 on the distribution of L. pneumophila in (A) developing, and (B) mature P. aeruginosa PAO1-CFP biofilms. Effect of NALCO 7320 on the distribution of bio-volume in (A) developing, and (B) mature P. aeruginosa PAO1-CFP biofilms. Porosity of P. aeruginosa PAO1-CFP biofilm in CBR treated with NALCO 7320. Effect of NALCO 7320 on porosity distribution of (A) developing, and (B) mature P. aeruginosa PAO1-CFP biofilms.
Molecular Probes Inc., U.S.A.; Appendix V) was added. The mixture was mixed
well, dispensed into 3 tubes and incubated at 37°C in the dark with shaking at
120rpm for 20, 30 and 40 mins respectively. To terminate the labelling process,
the L. pneumophila cells were centrifuged at 5,000g for 10 mins and washed twice
with PBS to remove residual CFDA-SE. A portion of the labelled cells were
plated onto BCYE agar using Miles and Misra method (Harrigan, 1998) to ensure
that the cells remained viable after the labelling process, while the remaining
portion were fixed with 1% formaldehyde (Merck, Germany) at 4°C overnight,
before analysis with flow cytometry. A tube of L. pneumophila cells that were
mock-treated with PBS instead of CFDA-SE served as a negative control and
blank.
3.4.2 Optimization of planktonic P. aeruginosa PAO1-CFP labelling with PI
P. aeruginosa PAO1-CFP cells at late-log phase were harvested at 28 hrs of
growth, fixed with 1% formaldehyde at 4°C overnight and washed with PBS.
Using PBS, cell concentration was adjusted to approximately 107 CFU/ml
(corresponding to P. aeruginosa PAO1-CFP cell suspension at OD600nm = 0.5) and
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separated into 2 portions. Final concentrations of freshly prepared 0.1 and
1.0mg/ml propidium iodide (PI; Sigma-Aldrich, U.S.A.) in PBS were then added
into each portion respectively. After vortexing, the mixtures were incubated at
room temperature in the dark and immediately analyzed using flow cytometry at 5
mins interval each. A tube of P. aeruginosa PAO1-CFP that were similarly
processed but mock-treated with PBS instead of PI served as a negative control
and blank.
3.4.3 Flow cytometry
A total of 10,000 cells were analyzed using flow cytometry, FACSVantageTM SE
(Becton Dickinson, U.S.A.) operated on CellQuest program. The WinMDI
Version 2.8 software was used to plot histograms with number of events against
green CFDA-SE fluorescence in 4 decade log (as FL1-H) or red PI fluorescence in
4 decade log (as PMT4 Log).
3.4.4 Optimization of P. aeruginosa PAO1-CFP biofilm labelling with PI
Approximately 107 CFU/ml CFDA-labelled L. pneumophila was inoculated into 7
days old P. aeruginosa PAO1-CFP biofilms (grown in CDC Biofilm Reactor
continous culture system) and allowed to adhere without continuous flow for 1hr.
After allowing for re-stabilization of the continuous system for 3 hours, a coupon
(on which the biofilm was formed) was harvested and soaked in 6ml 4%
paraformaldehyde (PFA) for 30mins in the dark. Then, freshly prepared 600µl of
1mg/ml PI was added into the 4% PFA, and mixed gentle (taking care not to
disturb the biofilm), and incubated at room temperature for 5, 15 and 30 mins in
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the dark. The staining process was terminated by transferring the coupon into
60ml sterile PBS contained in a standard petri dish, with the biofilm surface facing
upwards. A biofilm mock-treated with PBS served as a negative control.
These coupons were then viewed using confocal laser scanning microscope
(CLSM). For PI stains, image scanning was carried out with 543nm laser line
from a HeNe-G laser. To reduce background, emission filter BA-610IF was used.
Similarly, for CFP and CFDA detection, image scanning was carried out with the
405nm laser line from a LD405 laser and 488nm laser line from an M-Ar laser,
respectively. Background was also reduced using BA430-460 and BA505-525
emission filters, respectively. Images of the biofilm in the x-y plane or sections
through the biofilm were generated using Olympus FLUOVIEW Ver.1.3 Viewer.
3.5 P. aeruginosa PAO1-CFP biofilm formation in CDC Biofilm
Reactor (CBR)
3.5.1 CDC Biofilm Reactor
The CBR (BioSurface Technologies Corp., U.S.A.) is a one litre glass vessel with
an effluent spout at approximately 400ml. Continuous mixing of the reactor’s bulk
fluid was provided by a Teflon baffled stir bar that was magnetically driven by a
CERAMAG Midi magnetic stirrer (Ika®, U.S.A.). An UHMW (ultra high
molecular weight) polyethylene lid supports 8 independent polypropylene coupon
holders. Each coupon holder houses 3 removable stainless steel coupons
(diameter: 12.7mm), which served as the biofilm growth surfaces, for a total of 24
sampling opportunities. The coupons experienced a consistent high shear from the
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rotation of the baffled stir bar at 120rpm. The CBR operates as a continuous flow
stirred tank reactor (CFSTR), meaning that nutrients are continuously pumped
into and flow out of the reactor at the same rate.
3.5.2 Setup of CDC Biofilm Reactor assembly
Connector
Air vent
Baffled stir bar
Stainless steel
coupon
Coupon holder
Connector
Influent tap
Effluent tap
Air vent
Magnetic stirrer (120rpm)
Nutrient carboy
CBR
Air vent Peristaltic pump
Flow break
Incubator (30°C)
Waste carboy
Figure 3.1. Schematic diagram of the CDC Biofilm Reactor assembly used in this study. The arrow head indicated the direction of continuous flow when the peristaltic pump was switched on. Biofilm was formed on the surface of stainless steel coupons which was facing the baffled stir bar indicated by the striped coupons.
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An autoclavable 10L carboy (Nalge Nunc International, U.S.A.) was used to
contain fresh media to feed into CBR. The carboy top was equipped with 2 barbed
fittings to accommodate the tubings for nutrient and air vent, HEPA-VENTTM
(Whatman, U.K.) attachment. Incorporation of an autoclavable connector before
peristaltic pump facilitated the changing of emptied nutrient carboy with another
that was filled with sterile 10L fresh media. Peristaltic pump, Masterflex® Digital
Console Pump (Cole-Parmer Instrument Company, U.S.A.), was only switched on
for pumping media into CBR during continuous flow phase and the Masterflex®
precision tubing (Cole-Parmer Instrument Company, U.S.A.) passing through the
pump head had an internal diameter of 14mm. Before the fresh media entered
CBR, a flow break (BioSurface Technologies Corp., U.S.A.) prevented backward
contamination of media carboy from CBR. The bulk fluid in CBR was well mixed
by the baffled magnetic stir bar and extra fluid in CBR was drained into the waste
carboy. Positioning of a connector after the spout of CBR allowed changing of a
filled waste carboy with an autoclaved empty carboy.
3.5.3 P. aeruginosa PAO1-CFP biofilm formation
An overnight culture of P. aeruginosa PAO1-CFP (grown in MM+gen at 37°C
with shaking at 120rpm) was inoculated into 400ml fresh MM medium in CBR at
a ratio of 1:100 under sterile conditions. The inoculated CBR was then operated as
a batch culture system at 30°C with 120rpm stirring for 24 hours, with closed
influent and effluent taps. After which, the CBR was switched to continuous
culture phase where both influent and effluent taps were released and a continuous
flow of fresh MM medium was pumped into the CBR at a constant influent flow
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rate of 2.5ml/min (Refer to Chapter 4.2 for the determination of influent flow
rate). Kinetics of P. aeruginosa PAO1-CFP biofilm formation was monitored by:
• Taking planktonic and biofilm samples,
• Enumerating P. aeruginosa PAO1-CFP by plating onto LB+gen plates,
• Detecting contamination from exogenous source(s) by plating onto LB
plates,
• Visualizing biofilm structure using confocal laser scanning microscopy
(CLSM), and
• Image analysis using COMSTAT image analysis software package
(Heydorn A. et al., 2000)
For the study of P. aeruginosa PAO1-CFP biofilm formation, six independent
experiments were performed. For each experiment, there were 6 sampling points
as shown in table 3.1. For image data acquisition, at least 3 image stacks were
taken from 1 or 2 coupon samples at each time point.
Table 3.1. Sampling points of 6 independent experiments for the study of P. aeruginosa PAO1-CFP biofilm formation.
Days from start of continuous culture
Experiment 1 to 3 Experiment 4 to 6
2 3 4 5 6 7 8 9 10 11
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3.6 Introduction of L. pneumophila into P. aeruginosa PAO1-CFP
biofilms
Approximately 109 CFU/ml L. pneumophila grown to late log phase, was stained
with 10µM CFDA-SE for 30 mins and washed with PBS twice, before a ratio of
1:100 was inoculated into the CBR containing developing (Day 3) or mature
biofilm (Day 7). A portion of the legionellae inoculum was serially diluted and
enumerated by immunofluorescence. The continuous flow was stopped for the
adhesion of L. pneumophila onto pre-formed P. aeruginosa PAO1-CFP biofilm
and restarted 1 hr later. Samples of bulk fluid and biofilm were taken immediately
before the addition of L. pneumophila, 3 hrs after the continuous flow was
restarted (to allow for re-stabilization of the continuous flow system) and
everyday for up to 5 days after inoculation. Adhesion and persistence of L.
pneumophila was monitored by:
• Enumerating L. pneumophila by immunofluorescence method,
• Visualizing legionellae distribution using confocal laser scanning
microscopy (CLSM), and
• Image analysis using COMSTATWCY image analysis software package.
At the same time, the surface-to-biovolume ratio and porosity distributions of the
biofilm were also monitored by applying COMSTATLAYER and
COMSTATWCY image analysis software package respectively. At least 3
independent experiments were carried out for each developing stage of biofilm
development. For image data acquisition, at least 3 image stacks were taken from
1 or 2 coupon samples at each time point.
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3.7 Introduction of NALCO 7320 into developing and mature P.
aeruginosa PAO1-CFP biofilms containing L. pneumophila
According to the procedure mentioned in Chapter 3.6, L. pneumophila was
introduced into developing and mature biofilms on day 3 and day 7 respectively.
Twenty four hours later, the peristaltic pump was stopped and fresh culture media
with a final concentration of 100ppm of NALCO 7320 was connected to CBR
continuous flow system. At time zero, 315µl of 100,000ppm NALCO 7320 was
added into the CBR, yielding a final concentration of 100ppm NALCO 7320
within the CBR with the constant mixing by baffle. At the same time, the
peristaltic pump was switched on again, bringing in fresh supply of nutrients and
biofilm removing agent.
Samples of bulk fluid and biofilm were taken before and immediately after the
addition of NALCO 7320. Samples were also taken in the subsequent 4, 8, 12 and
24hrs. Persistence of P. aeruginosa PAO1-CFP and L. pneumophila was
monitored by:
• Enumerating P. aeruginosa PAO1-CFP and L. pneumophila by culture and
immunofluorescence methods respectively,
• Visualizing biofilm structure, biofilm porosity and legionellae distribution
using confocal laser scanning microscopy (CLSM), and
• Image analysis using COMSTAT and COMSTATWCY image analysis
software package respectively.
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Two independent biofilm experiments were carried out for each developing stage
of biofilm development. However, at least 3 image stacks were taken from each of
the 2 coupon samples at each time point.
3.8 Monitoring of each organism in CBR continuous flow system
3.8.1 Preparation for sampling
In preparation of sampling under sterile conditions, 75% denatured ethanol was
sprayed on and around the CBR lid and allowed to air dry for approximately 2
mins. Disturbance to the air within the 30°C incubator was minimized to prevent
contamination of the continuous culture.
3.8.2 Taking samples
3.8.2.1 Sampling bulk fluid
For every sampling point, two 1ml planktonic samples were taken and processed
in parallel. To take planktonic samples, one of the 8 coupon holders was removed
and placed inside a 1L sterile glass bottle (for biofilm sampling) before inserting a
1ml pipette into the CBR to sample the bulk fluid repeatedly. Once removed, the
coupon holder with biofilm covered coupons cannot be replaced back into CBR
because air-water interval of the bulk fluid can detach the biofilm significantly.
For every coupon holders removed, a sterile rubber bung was used to stopper the
hole in the CBR lid to prevent the entry of exogenous contaminants.
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3.8.2.2 Sampling biofilm
For every sampling point, one coupon holder accommodating 3 coupons was
removed. One out of the 3 coupons was used for enumeration by plating and the
rest were prepared for visualization by CLSM. The coupon holder was held
straight up and removed from the 1L bottle. Any drips were collected in a sterile
petri dish placed beneath the rod. All 3 set screws were loosen with sterile set
screw driver (BioSurface Technologies Corp., U.S.A.) to release the coupons,
which were then removed using sterile haemostat, being careful not to disturb the
biofilm on coupon surface that was facing the interior of CBR. Once the coupons
were removed, the coupon holder was re-inserted back to the CBR so as to
minimize any changes in the final volume of bulk fluid in the CBR.
3.8.3 Preparation of coupons
3.8.3.1 Preparation of coupons intended for enumeration
A coupon was held in place on a petri dish with a sterile haemostat and scraped on
the side that faced the baffle with a sterile toothpick. The loosened biofilm was
washed into an empty eppendorf tube using 1ml PBS and then the toothpick was
rinsed by swirling on the bottom of the tube.
3.8.3.2 Preparation of coupons intended for visualization by CLSM
A coupon was immersed in 6ml 4% paraformaldehyde (PFA; Appendix VI)
contained in small sterile tissue culture plates (35×10mm; NuncTM, Denmark) at
room temperature for 30 mins in the dark. Slow immersion of coupon into liquid
resulted in significant biofilm sloughing off, thus each coupon was held near to
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the liquid surface using a haemostat with the biofilm surface facing upwards and
dropped directly into the liquid below. This was done as soon as the coupon was
removed from CBR so as to prevent drying up of the biofilm. Then, freshly
prepared 600µl of 1mg/ml PI was added into 6ml 4% PFA, pipetted up and down
for gentle mixing, and incubated at room temperature for 5mins in the dark. The
staining process was promptly terminated by dropping the coupon into 60ml
sterile PBS contained in a standard petri dish, with the biofilm surface facing
upwards.
Taking care not to touch the top surface of the coupon where the biofilm was to be
visualized, the bottom of coupon was dried using a tissue paper and then placed in
a humid chamber with the biofilm surface facing upwards. To preserve the
fluorescence in the samples, a 20×60mm coverslip with 20µl of FluorSaveTM
Reagent (Merck, Germany) dropped in the middle was inverted and mounted onto
the biofilm surface of the coupon. The mounted coupons were then stored at 4°C
in a humid chamber in the dark for not more than 1 week. These mounted coupons
were air dried at room temperature in the dark overnight before viewing under
CLSM.
3.8.4 Disaggregation by homogenization
For a more accurate enumeration of each organism in either planktonic or biofilm
samples, the samples were subjected to disaggregation by homogenization before
serial dilution. Firstly, 750µl of planktonic and biofilm samples were transferred
into sterilized disposable culture tubes (Asahi Techno Glass Corporation, Japan)
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and homogenized at 20,500rpm for 30 secs using a sterile homogenizer probe, T
25 basic ULTRA-TURRAX® (Ika®, U.S.A.). The probe was cleaned between
samples by firstly rinsing for 30 secs at 20,500rpm in 10ml of sterile PBS,
followed by rinsing at 20,500rpm for 15 secs in 10ml of 75% ethanol. The probe
was then soaked in the ethanol for 1 min. Finally, the probe was rinsed two more
times with 10ml PBS at 20,500rpm for 30 secs each. Any excess liquid on the
probe was removed by gently tapping the tip of the probe against the inner wall of
the last tube containing PBS before inserting the probe into the sample.
3.8.5 Enumeration of each organism
3.8.5.1 Enumeration of P. aeruginosa PAO1-CFP by culture
The disaggregated planktonic and biofilm samples were serially diluted using PBS
as diluent and plated onto LB+gen plates respectively, using Miles and Misra
method (Harrigan, 1998) in duplicates. The plates were then incubated at 37°C for
24 hrs. Density of planktonic P. aeruginosa PAO1-CFP was expressed as Log10
[colony-forming units (CFU)/ml of bulk fluid] while that of biofilm P. aeruginosa
PAO1-CFP was expressed as Log10 (CFU/mm2). Respective calculations were
was applied to each well and the slides were incubated in a moist chamber for 30
mins at 37°C in the dark. The slides were then rinsed with PBS to remove the
conjugates and rinsed with dH2O before air dried. Lastly the slides were mounted
and examined using a fluorescence microscope (Olympus, Japan) within 24 hrs.
FITC-labelled antibody-antigen complex was detected by exposing the slide to
ultraviolet light and L. pneumophila cells appeared as bright yellow-green bacilli
under a 40× objective. At least 3 fields were examined for legionellae count.
Wells containing only L. pneumophila cells and only P. aeruginosa PAO1-CFP
cells served as positive and negative control respectively.
Density of planktonic L. pneumophila was expressed as Log10 (cells/ml of bulk
fluid) while that of biofilm L. pneumophila was expressed as Log10 (cells/mm2).
Calculations were shown below:
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Planktonic L. pneumophila count
Log10 (cells/ml)
= Log10 [(Average number of cells per field) × (Area of each well/Area of each
field*) × (50/concentration factor)]
= Log10 [(Average number of cells per field) × (π(3)2 / π(0.2)2) ×
(50/concentration factor)]
Biofilm L. pneumophila count
Log10 (cells/mm2)
= Log10 {[(Average number of cells per field) × (Area of each well/Area of each
field*) × (50/concentration factor)] / (Area of coupon)}
= Log10 {[(Average number of cells per field) × (π(3)2 / π(0.2)2) ×
(50/concentration factor)] / (π(12.7/2)2)}
* Diameter of each field of 40× objective was measured using a stage micrometer.
L. pneumophila adhesion to P. aeruginosa PAO1-CFP biofilm
Number of legionellae adhering to biofilm per coupon per 106 legionellae
inoculated into CBR
= (Number of legionellae per coupon/ Total number of legionellae inoculated into
CBR) × 106
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L. pneumophila persistence in P. aeruginosa PAO1-CFP biofilm
Percentage legionellae remaining in biofilm
= (Biofilm legionellae count at day n/ Biofilm legionellae count on day of
inoculation) × 100%
where, n = number of days following legionellae inoculation
Loss of L. pneumophila from biofilm
Amount of legionellae loss from biofilm
= (Biofilm legionellae count before the addition of biocide - Biofilm legionellae
count at 24hrs of exposure to biocide)
Loss of L. pneumophila per unit biomass lost
Legionellae loss per unit biomass lost
= (Biofilm legionellae count before the addition of biocide - Biofilm legionellae
count at 24hrs of exposure to biocide)*1000 / (Bio-volume before addition of
biocide – Bio-volume at 24hrs of exposure to biocide)
= (cells/mm3)
3.8.6 Detection of exogenous contaminants
In addition to plating on LB+gen for P. aeruginosa PAO1-CFP counts, serially
diluted samples were also plated on LB for detection of exogenous contaminants
using Miles and Misra method. Similarly, these plates were incubated at 37°C for
24 hrs before observation.
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3.8.7 Visualization and image acquisition by CLSM
At each sampling point, two coupons were processed for visualization by CLSM.
From each coupon, at least three image stacks were acquired from random
positions using Olympus FV500 confocal scanning laser microscope (Olympus
Corporation, Japan).
Images were acquired at 1.0µm intervals down through the biofilm, thus the
number of images in each stack varied according to the thickness of the biofilm.
The 512 pixels × 512 pixels images were obtained with a PlanApo 60× /1.40 oil
immersion objective. Together, each pixel was considered as a box (voxel) with
the dimensions 0.41432µm3 (x-axis) × 0.41432µm3 (y-axis) × 1.000µm3 (z-axis).
Since each image had a coverage of 45,000µm2, thus a total of 3 images per
coupon reflected the coverage of >100,000µm2 per coupon, sufficient to obtain a
representative data of the Pseudomonas biofilm (Korber et al., 1993). For CFP, PI
and CFDA, image scanning was carried out using the 405nm laser line from a
LD405 laser, 543nm laser line from a HeNe-G laser and 488nm laser line from an
M-Ar laser respectively. To reduce background, either emission filter BA430-460,
BA-610IF and BA505-525 was used respectively. Variables that may influence
the quality of the images for each fluorescence, like the photomultiplier tube
(PMT), confocal aperture (CA) and laser power, were kept constant for all
experiments.
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3.8.8 Application of COMSTAT image analysis software package
COMSTAT (Heydorn et al., 2000) was written as a script in MATLAB 5.1 (The
MathWorks Inc., Natick, Massachusetts), equipped with Image Processing
Toolbox. The COMSTAT package contained 5 programs (COMSTAT,
CHECKALL, LOOK, LOOKTIF and CONVERT000) and a number of functions
used by the programs.
3.8.8.1 Preparation of image stacks
In order to store image data in a format that can be analyzed by COMSTAT, the
images were prepared as follows:
• Images of a stack, of each fluorescence type, acquired by CLSM were
extracted and saved as individual ‘.tif’ files in the MATLABR11/work
folder using Olympus FLUOVIEW Ver.1.3 Viewer.
• An ‘.info’ file was created using Notepad by writing a text file with the
extension ‘.info’ and the ‘.info’ file contained vital information of the
image stack arranged in the following order:
Range #1# #13# Pixelsize (x) #0.41432# Pixelsize (y) #0.41432# Pixelsize (z) #1.000# ‘Range’ reflected the number of images in this stack. The ‘pixelsize’
represented the distance between 2 neighboring pixels and was given in
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micrometer. The name of the ‘.info’ file was also changed according to the
name of the images in the stack.
• The original MATBLAB script of the CONVERT000 program was
improved to allow renaming and reversing the order of the images of a
stack in a way that COMSTAT can analyze. The improved program was
named “CONVERTWKL”.
• CHECKALL program was run to check that all the stacks of images were
intact.
3.8.8.2 Thresholding of images
After the image preparations, LOOK program was run to allow manual
determination of the threshold value for each stack of images of different
fluorescence type. LOOKTIF program allowed closer view of individual images.
Such thresholding of a stack of images in COMSTAT resulted in the formation of
a 3-dimensional matrix with a value of ONE in positions where pixel values in the
original image were above or equal to the threshold value (representing biomass
or data point of biofilm) and ZERO where the pixel values were below the
threshold value (representing background).
3.8.8.3 COMSTAT image analysis for P. aeruginosa PAO1-CFP biofilm
structure
To study P. aeruginosa PAO1-CFP biofilm structure, stacks of CFP images were
analyzed by COMSTAT program running on ‘connected volume filtered images’.
Connected volume filtration of the stacks of images removed noise from images
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by removing biomass that is not in some way connected to the substratum. The
following COMSTAT image analysis features were run:
• Bio-volume (µm3/µm2) - volume of the biofilm normalized by the surface
area of the field of view.
• Average thickness (µm) – average biofilm height taken over the entire
field of view.
• Maximum thickness (µm) – maximum distance from the substratum that
the biofilm reaches.
• Surface to bio-volume ratio (SBR) (µm2/µm3) – the area summation of all
biomass voxel surfaces exposed to the background per unit bio-volume,
thus reflects the fraction of biofilm apparently exposed to nutrient flow.
• Substratum coverage (%) – the area coverage in the first image of the
stack, i.e. at the substratum, thus reflects how efficiently the substratum is
colonized by bacteria of the population.
• Roughness coefficient – a measure of variability in biofilm thickness and
consequently, an indicator of biofilm heterogeneity.
To obtain the distribution of bio-volume, the image analysis program COMSTAT
was improved to report the number of CFP pixels in each layer of the biofilm. The
improved program was named “COMSTATWCY” and run with connected
volume filtration. For each stacks of images, the number of CFP pixels belonging
to each sections of the biofilm were summed up. Bio-volume of the biofilm was
then calculated using the following formula:
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Bio-volume
Bio-volume (µm3µm-2) = [(Number of CFP pixels × voxel size) / (area of the field
of view)]
Loss of bio-volume
Amount of bio-volume loss
= (Bio-volume before the addition of biocide – Bio-volume at 24hrs of exposure
to biocide)
To obtain the distribution of SBR, the image analysis program COMSTAT was
improved to report the SBR in each layer of the biofilm. The improved program
was named “COMSTATLAYER” and run with connected volume filtration.
Surface area of each layer of the biofilm was then obtained by dividing SBR with
corresponding bio-volume. For each stack of images, both the surface area and
bio-volume belonging to each sections of the biofilm were summed up separately
before calculating the sectional SBR by dividing sectional surface area with
corresponding sectional bio-volume.
3.8.8.4 COMSTAT image analysis for porosity of P. aeruginosa PAO1-CFP
biofilm
To study the porosity of the biofilms, COMSTATWCY was used to analyze the
stacks of PI and CFP images, and run without connected volume filtration. The
report of the analysis detailed the number of PI and CFP pixels in each images of
a stack, that is, in each layer of the biofilm. For each stack of images, the numbers
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of PI and CFP pixels belonging to each sections of the biofilm were summed up.
Porosity of the biofilm was then calculated using the following formula:
Porosity
Porosity = [Number of PI pixels / (Number of CFP pixels × voxel size)] = Number
of PI pixels per µm3 of biomass
3.8.8.5 COMSTAT image analysis for L. pneumophila distribution
COMSTATWCY was also used to analyze the stacks of CFDA images, without
connected volume filtration. The report stated the number of CFDA pixels in each
layer of the biofilm. For each stack of images, the numbers of CFDA pixels
belonging to each sections of the biofilm were summed up while the CFP data was
the same as that in the above section. Subsequently, the following calculations
were performed:
L. pneumophila concentration
Log (Legionellae concentration)
= Log [Number of CFDA pixels / (Number of CFP pixels × voxel size)]
= Log (Number of CFDA pixels per µm3 of biomass)
L. pneumophila loss from P. aeruginosa PAO1-CFP biofilm
% Loss of legionellae
= {[(legionellae concentration at day of inoculation) – (legionellae concentration
at day n)] / (legionellae concentration at day of inoculation)} × 100%
where, n = number of days following legionellae inoculation
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3.8.9 Statistical analysis
All statistical analyses in this study were carried out using SPSS 13.0.
Independent samples t-test was used to compare the means for 2 groups of cases.
If the significance value for the Levene test was high (typically greater than 0.05),
equal variances for both groups were assumed. A low significance value for the t-
test (typically less than 0.05) indicated significant difference between the 2 group
means. In addition, if the confidence interval for the mean difference did not
contain zero, this indicated that the difference was significant.
Pearson correlation was used to determine if there is a linear association between
variables on the assumption that the data are normally distributed. The values of
the correlation coefficient ranged from -1 to 1. The sign of the correlation
coefficient indicated the direction of the relationship while the absolute value of
the correlation coefficient indicated the strength, with larger absolute values
indicating stronger relationships. The significance level (or p-value) was the
probability of obtaining results as extreme as the one observed.
3.9 Screening for effective P. aeruginosa PAO1 biofilm-removing
agent
3.9.1 Kinetics of P. aeruginosa PAO1 biofilm formation in microtiter plate
An overnight culture of P. aeruginosa PAO1 (grown in MM at 37°C with shaking
at 120rpm) was inoculated into fresh MM medium at a ratio of 1:100. The freshly
inoculated medium was dispensed into each of the 8 wells (100µl per well) of the
first column of non-tissue culture treated, flat bottom, 96-well polystyrene plates
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(BD FalconTM, U.S.A.). The inoculated 96-well plate was then incubated at 30°C
with shaking at 120rpm, while the remaining inoculated medium was stored at
4°C to prevent any growth. At every 2 hr interval, the inoculated medium was
mixed well and dispensed in the same way, into the subsequent column of 8 wells.
One column of every 96-well plate contained only MM (blank and negative
control). After 40 hrs from the first inoculation, the biofilm formed on the walls of
each wells were quantified as described below. Three independent experiments
were conducted for this study.
3.9.2 Quantification of biofilm (O’Toole et al., 1999)
After biofilm was formed in 96-well plates, optical density reflecting total
bacterial growth (OD600nm) of the 96-well plates were first taken using ELISA
Touch Screen plate reader (Tecan, Austria) operated on Magellan2 software. The
spent culture medium, together with unattached bacteria, were then carefully
removed from each wells and replaced with 100µl of 1% (w/v) crystal violet in
deionized water (dH2O). After 10 mins of incubation at room temperature, excess
crystal violet was washed away by rinsing the plate in several basins of dH2O.
These washed plates were then tapped to remove excess water and air-dried.
Biofilm-bound crystal violet (reflecting the amount of biofilm formed) was
solubilized by adding 100µl of 95% ethanol to each well, incubated at room
temperature with shaking at 100rpm for 10 mins and then quantified by measuring
OD470nm of the 96-well plates.
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3.9.3 Biofilm-removing agents used
To obtain the most effective biofilm-removing agent, commercially available
products of a variety of nature were screened. All 8 biofilm-removing agents used
in this study were listed in table 3.2.
Table 3.2. All biofilm-removing agents used. * represents active ingredients of the various biofilm-removing agents.
Concentrated enzymatic formulation for digestion of
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- Enzyme protein Lipase triacylglycerol*
proprietary
- Enzyme protein Cellulase* proprietary - Enzyme protein Cellulase* proprietary - Enzyme protein Amylase* proprietary - Enzyme protein Alpha-Amylase*
proprietary
- 5-Chloro-2-methyl-4-isothiazolin-3-one*
<0.09
- 2-Methyl-4-isothiazolin-3-one*
<0.09
- Linear alkyl pyrrolidone <5 - Polyethylene oxide derivative of synthetic alcohols
<20
- Polyoxyethylene, polyoxypropylene, polyoxybuthylene ether of a mixture of aliphatic alcohols
<10
- Glycerine <10 - Borax <10
biological debris, biofilm, protozoa and reducing bacterial resistance to common biocides
3.9.4 P. aeruginosa PAO1 biofilm removal screening
P. aeruginosa PAO1 biofilm was formed in 96-well plates for 12 hrs at 30°C with
shaking at 120rpm before biofilm-removing agents, diluted with dH2O to varying
concentrations (in terms of parts per million, ppm), were added into each column
of 8 wells and their efficiency of biofilm removal were monitored over the
subsequent 8 hrs (with 2 hrs interval). The concentrations of biofilm-removing
agents used were 10, 50, 100, 500 and 1000ppm. One column of every 96-well
plate was mock-treated with dH2O (negative control) and similarly, one column of
every 96-well plate contained only MM (blank). Three independent experiments
were conducted for this study.
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3.10 Antimicrobial susceptibility testing of NALCO 7320
Standard macrodilution method (Ferraro, 2003) was used to ascertain the
minimum inhibitory concentration (MIC) and minimum bactericidal concentration
(MBC) of NALCO 7320. P. aeruginosa PAO1 and L. pneumophila cells grown to
late-log phase were harvested at 28 and 24 hrs of growth respectively, and re-
suspended in PBS. Cell concentrations were adjusted to OD600nm = 0.5 and
enumerated by Miles and Misra method on appropriate agar. 3ml of culture media,
MM for P. aeruginosa PAO1 and 20% BCYE broth for L. pneumophila,
containing different concentrations of biofilm-removing agents (1,000ppm,
500ppm, 100ppm, 50ppm and 10ppm) were inoculated with 100µl of the cell
suspension and incubated at 30°C for 24 hrs. One tube was mock-treated with
dH2O (negative control) and similarly, one tube contained culture media only
(blank).
The MIC was recorded as the lowest concentration of biofilm-removing agent that
completely inhibited visible growth. The MBC was determined by spread plating
100µl from the tubes with no visible growth onto appropriate agar. Three
independent experiments were conducted for this study.
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Chapter 4: Results
4.1 Growth kinetics
Figure 4.1, 4.2 and 4.3 illustrated the growth kinetics and time taken to reach late
log phase of L. pneumophila ATCC 33152, P. aeruginosa PAO1 and P.
aeruginosa PAO1-CFP respectively. All Legionella, P. aeruginosa PAO1 and P.
aeruginosa PAO1-CFP cells used in the subsequent experiments were harvested at
late log phase, unless otherwise stated.
Growth Curve of L. pneumophila ATCC 33152
0.0
1.0
2.0
3.0
4.0
5.0
6.0
0 5 10 15 20 25 30 35
Time (hr)
OD
(600
nm)
Late Log Phase (24hr)
Figure 4.1. Growth curve of L. pneumophila cultured in BCYE broth at 37°C with shaking at 120rpm. The error bars represent standard deviation of 3 independent experiments.
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Growth Curve of Pseudomonas aeruginosa PAO1
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
0 5 10 15 20 25 30 35
Time (hr)
OD
(600
nm)
Late Log Phase (28hr)
Figure 4.2. Growth curve of P. aeruginosa PAO1 cultured in MM liquid media at 30°C with shaking at 120rpm. The error bars represent standard deviation of 3 independent experiments.
Growth Curve of Pseudomonas aeruginosa PAO1-CFP
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
0 5 10 15 20 25 30 35
Time (hr)
OD
(600
nm)
Late Log Phase (28hr)
Exponential Phase
Figure 4.3. Growth curve of P. aeruginosa PAO1-CFP cultured in MM liquid media at 30°C with shaking at 120rpm. The error bars represent standard deviation of 3 independent experiments.
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4.2 Determination of the influent flow rate (Q) for continuous
culture in CDC Biofilm Reactor (CBR)
Exponential growth phase of Pseudomonas aeruginosa PAO1-CFP
y = 0.2505x - 6.5649R2 = 0.9951
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
18 20 22 24 26 28 30
Time (hr)
Ln [O
D (6
00nm
)]
Figure 4.4. Graph of Ln(OD600nm) against time (hr) was plotted for the exponential growth phase of P. aeruginosa PAO1-CFP, so as to obtain the maximum specific growth rate, which was reflected by the gradient of the best straight line plotted. The error bars represent standard deviation of 3 independent experiments.
From figure 4.4, the maximum specific growth rate (µmax) = 0.2505 hr-1
= 4.18 × 10-3 min-1.
Doubling time (td) = ln2 / µmax = ln2 / (4.18 × 10-3) = 165.8 mins = 2.76 hr.
In order to select for biofilm growth in the CBR, the hydraulic residence time (θ)
must be less than the doubling time for the suspended cells. This will result in the
suspended cells washing out of the reactor, leaving only biofilm.
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To determine the nutrient influent flow rate (Q) such that θ < td, the following
calculations were performed:
Since θ = V/Q,
V/Q < 165.8
Q > V/165.8
Q > 400 / 165.8
∴Q > 2.41 ml/min
Where, V is maximum volume of bulk fluid in CBR during continuous flow (with
all coupons and coupon holders removed) = 400ml.
In conclusion, continuous culture were conducted with Q = 2.5ml/min.
As such, highest possible θ = V/Q = 400 / 2.5 = 160mins = 2.6hr.
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4.3 Optimization of labelling processes
4.3.1 Optimization of L. pneumophila labelling with CFDA-SE
Together, figure 4.5 and table 4.1, show that 30mins was the longest treatment
duration (with 10µM CFDA-SE) that resulted in more than 95% of Legionella
cells being labeled with CFDA without compromising viability.
(A) (B) (C) (D)
FL1-H FL1-H FL1-H FL1-H
Figure 4.5. Histograms illustrating the number of events (cells) plotted against FL1-H (representing green fluorescence of CFDA-stained cells) for L. pneumophila cells that were (A) mock treated, or treated with CFDA-SE for (B) 20mins, (C) 30mins, or (D) 40mins. Table 4.1. Effect of treatment duration on staining and viability of L. pneumophila cells. * represents % Cells stained represented the area of graph (see above) under Marker 1 (M1). Duration of treatment with CFDA-SE Mock-treated 20mins 30mins 40mins % Cells stained*
1.90% 97.0% 97.0% 97.5%
% Viable cells after staining process
- 100% 100% 86.8%
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4.3.2 Optimization of planktonic P. aeruginosa PAO1-CFP labelling with PI
Figure 4.6 and table 4.2 below, show that within 5mins of 1.0mg/ml PI treatment,
majority (up to 88.1%) of formaldehyde fixed P. aeruginosa PAO1-CFP cells
picked up PI stain. Unsurprisingly, fewer cells attained the comparable level of
fluorescence for the same treatment duration when 0.1mg/ml PI was used instead
(figure 4.7 and table 4.2). In addition, even after 30mins of treatment with
0.1mg/ml PI, only 64.2% of the cells attained comparable high level of
fluorescence. These imply that the PI concentration of 0.1mg/ml was limited for
substantial staining of a 107 CFU/ml P. aeruginosa PAO1-CFP cell suspension.
igure 4.6. Histograms illustrating the number of events (cells) plotted against
(A) (B) (D) (C)
FPMT4 Log (representing red fluorescence of PI-stained cells) for P. aeruginosa PAO1-CFP cells that were (A) mock treated, or treated with 1.0mg/ml PI for (B) 5mins, (C) 10mins, or (D) 15mins.
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(A) (B) (C)
(D) (E)
Figure 4.7. Histograms illustrating the number of events (cells) plotted against PMT4 Log (representing red fluorescence of PI-stained cells) for P. aeruginosa PAO1-CFP cells that were (A) mock treated, or treated with 0.1mg/ml PI for (B) 5mins, (C) 10mins, (D) 15mins, or (E) 30mins. Table 4.2. Effect of treatment duration on staining of P. aeruginosa PAO1-CFP cells. * represents % Cells stained represented the area of graph (see above) under Marker 1 (M1).
Duration of treatment in 1.0mg/ml PI
Duration of treatment in 0.1mg/ml PI
Negative control
5mins 10mins 15mins 5mins 10mins 15mins 30mins
% Cells stained*
9.91% 88.7% 91.8% 92.8% 55.0% 53.3% 60.2% 64.2%
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4.3.3 Optimization of P. aeruginosa PAO1-CFP biofilm labelling with PI
When a low concentration of 0.1mg/ml PI was applied to formaldehyde-fixed 7
days old P. aeruginosa PAO1-CFP biofilm with L. pneumophila for merely
5mins, regions of the biofilm with the greatest access to the external dye was
observed to be stained with a higher intensity of redness (figure 4.8 (C)). Since
freshly introduced L. pneumophila co-localized with these regions as seen in
figure 4.8 (D), this further affirms the proposition that the more PI pixels per unit
biomass, the higher the porosity of the biofilm.
(A) (B)
(C) (D)
Figure 4.8. CLSM images of a 7 day old P. aeruginosa PAO1-CFP 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. The scale represents 30µm in each image. Arrow indicates the porous flow channel within cell clusters of biofilm.
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However, when the PI treatment duration was increased to 15mins (figure 4.9) and
30mins (figure 4.10), the biofilms were over-stained and regions of higher
porosity were not discernible. Henceforth, a concentration of 0.1mg/ml PI and
treatment duration of 5mins were applied to all biofilm samples intended for
CLSM examination.
(C) (D)
(A) (B)
Figure 4.9. CLSM images of a 7 day old P. aeruginosa PAO1-CFP biofilm and adhered L. pneumophila, stained with 0.1mg/ml PI for 15mins: (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. The scale represents 30µm in each image.
Results
Department of Microbiology, NUS 79
(C) (D)
(A) (B)
Figure 4.10. CLSM images of a 7 day old P. aeruginosa PAO1-CFP biofilm and adhered L. pneumophila, stained with 0.1mg/ml PI for 30mins: (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. The scale represents 30µm in each image.
Results
Department of Microbiology, NUS 80
4.4 Kinetics of P. aeruginosa PAO1-CFP biofilm formation in
CDC Biofilm Reactor (CBR)
4.4.1 Kinetics of biofilm formation
Figure 4.11 illustrated the steady increase in the average number of viable P.
aeruginosa PAO1-CFP cells in biofilm until it became relatively levelled off (at
7.04 ×104 CFU/mm2) after 6 days of growth in the continuous flow CBR system.
This developmental plateau was observed in at least 3 independent experiments,
thus was reproducible in this system. Henceforth, mature biofilm was demarcated
by the 6th day of development in this system, while developing biofilm
corresponded to the days before the plateau was reached. Even so, there was a
slight but insignificant increase (independent sample t-test, p > 0.1; assuming
equal variance) in the average viable cell counts on Day 10 and 11 of biofilm
Figure 4.11. Viable cell counts of P. aeruginosa PAO1-CFP biofilm formed in
development to 1.18 × 105 CFU/mm2 and 1.64 × 105 CFU/mm2 respectively.
CBR at 30°C with stirring at 120rpm. The error bars represent standard deviation of at least 3 independent experiments.
Pseudomonas aeruginosa PAO1-CFP counts in biofilm
0.0
1.0
2.0
3.0
4.0
5.0
6.0
2 3 4 5 6 7 8 9 10 11Days
Log
PAO
1-C
FP c
once
ntra
tion
[Log
(C
FU/m
m2 )]
Mature biofilmDeveloping biofilm
Results
Department of Microbiology, NUS 81
4.4.2 Structure of biofilm by image analysis
Image analysis revealed that the profile of biofilm bio-volume (figure 4.12),
average thickness (figure 4.13) and maximum thickness (figure 4.14)
corresponded with that of P. aeruginosa PAO1-CFP viable counts up to day 9.
Despite the relatively constant maximum thickness, there was a noticeable but not
significant increase in bio-volume and average thickness on day 10 and 11. Figure
4.15 illustrated yet another perspective of the biofilm where average substratum
coverage peaked on day 3 at 74.5% ± 10.2% and dropped drastically to reach a
low of 13.1% ± 8.88% on day 6. On subsequent days, the average substratum
coverage remained low between 20.0%-30.0% as compared to >50.0% for early
developing biofilm.
urface-to-biovolume ratio (SBR) remained comparable (between 0.400 – 0.650
µm2/µm3) throughout biofilm development, as shown in figure 4.16. However,
figure 4.17 demonstrated that the roughness coefficient started off high at 0.483 ±
0.145 and decreased gradually to 0.319 ± 0.220 on day 8, before dropping
noticeably to between 0.110 – 0.130 on day 9 onwards.
S
Results
Department of Microbiology, NUS 82
Bio-volume
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
2 3 4 5 6 7 8 9 10 11Days
Bio
-vol
ume
(µm
3 /µm
2 )Developing biofilm Mature biofilm
Figure 4.12. Bio-volume of P. aeruginosa PAO1-CFP biofilm formed in CBR at 30°C with stirring at 120rpm. The bio-volume of each experiment was obtained from 3-8 image stacks from 1 or 2 coupons. The error bars represent standard deviation of at least 3 independent experiments.
Average thickness
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
2 3 4 5 6 7 8 9 10 11Days
Ave
rage
thic
knes
s (µ
m)
Developing biofilm Mature biofilm
Figure 4.13. Average thickness of P. aeruginosa PAO1-CFP biofilm formed in CBR at 30°C with stirring at 120rpm. The average thickness of each experiment was obtained from 3-8 image stacks from 1 or 2 coupons. The error bars represent standard deviation of at least 3 independent experiments.
Results
Department of Microbiology, NUS 83
Maximum thickness
0.0
10.0
20.0
30.0
40.0
50.0
60.0
2 3 4 5 6 7 8 9 10 11Days
Max
imum
thic
knes
s (µ
m)
Developing biofilm Mature biofilm
Figure 4.14. Maximum thickness of P. aeruginosa PAO1-CFP biofilm formed in CBR at 30°C with stirring at 120rpm. The maximum thickness of each experiment was obtained from 3-8 image stacks from 1 or 2 coupons. The error bars represent standard deviation of at least 3 independent experiments.
Substratum coverage
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
2 3 4 5 6 7 8 9 10 11Days
Subs
trat
um c
over
age
(%)
Developing biofilm Mature biofilm
Figure 4.15. Substratum coverage of P. aeruginosa PAO1-CFP biofilm formed in CBR at 30°C with stirring at 120rpm. The substratum coverage of each experiment was obtained from 3-8 image stacks from 1 or 2 coupons. The error bars represent standard deviation of at least 3 independent experiments.
Results
Department of Microbiology, NUS 84
Surface-to-biovolume ratio0.90
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
2 3 4 5 6 7 8 9 10 11Days
Surf
ace-
to-b
iovo
lum
e ra
tio (µ
m2 /µ
m3 )
Developing biofilm Mature biofilm
Figure 4.16. Surface-to-biovolume ratio (SBR) of P. aeruginosa PAO1-CFP iofilm formed in CBR at 30°C with stirring at 120rpm. The SBR of each
CBR at 30°C with stirring at 120rpm. The roughness coefficient of each
bexperiment was obtained from 3-8 image stacks from 1 or 2 coupons. The error bars represent standard deviation of at least 3 independent experiments.
Roughness coefficient
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.700
2 3 4 5 6 7 8 9 10 11Days
Rou
ghne
ss c
oeffi
cien
t
Developing biofilm Mature biofilm
Figure 4.17. Roughness coefficient of P. aeruginosa PAO1-CFP biofilm formed inexperiment was obtained from 3-8 image stacks from 1 or 2 coupons. The error bars represent standard deviation of at least 3 independent experiments
Results
Department of Microbiology, NUS 85
4.4.3 Detachment of biofilm
Figure 4.18 illustrated a gradual increase in the average number of P. aeruginosa
om a low of 1.01 × 105 CFU/ml on day 2 to 2.26 ×
andard deviation of at least 3 independent experiments .
PAO1-CFP in the bulk fluid fr
105 CFU/ml on day 5, followed by a significant increase (independent sample t-
test, p = 0.006, assuming equal variance) to 6.58 × 105 CFU/ml on day 6. After
which, the average planktonic viable count remained relatively constant but
increased slightly from day 10 onwards to a high of 1.65 × 106 CFU/ml on day 11.
Such viable counts of P. aeruginosa PAO1-CFP in the bulk fluid reflected
instantaneous detachment of biofilm because the high nutrient influent flow rate
applied to the CBR would have resulted in the washout of planktonic P.
aeruginosa PAO1-CFP before these cells could replicate (Chapter 4.2).
Planktonic P. aeruginosa PAO1-CFP counts
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
2 3 4 5 6 7 8 9 10 11Days
Log
PAO
1-C
FP c
once
ntra
tion
[Log
(C
FU/m
l)]
Developing biofilm Mature biofilm
Figure 4.18. Viable cell counts of planktonic P. aeruginosa PAO1-CFP in the bulk fluid of CBR at 30°C with stirring at 120rpm. The error bars represent st
Results
Department of Microbiology, NUS 86
Biofilm structures indicative of the final stage in Pseudomonas biofilm
development, namely the dispersion stage (Tolker-Nielsen et al., 2000; Sauer et
(blue) structure L.
neumophila (green). Biofilm stained with PI (red) reflected porous regions. The -y view of the biofilm (main view) is flanked by y-z (right) and x-z (bottom)
al., 2002), were observed occasionally and earliest seen on day 7 of P. aeruginosa
PAO1-CFP biofilm development (Figure 4.19). The “wall” of P. aeruginosa
PAO1-CFP cells that encompassed the void space containing sparse amount of P.
aeruginosa PAO1-CFP cells were low in porosity, thereby preventing PI staining
of the P. aeruginosa PAO1-CFP cells within the void. It is also worth noting that
no legionellae has been found within such voids.
Void
Figure 4.19. CLSM image of a P. aeruginosa PAO1-CFP biofilmindicative of dispersion stage of biofilm development, with adhered pxsections of the biofilm, with red arrows pointed towards the top of biofilm. The scale represents 30µm in each image.
Results
Department of Microbiology, NUS 87
4.5 Introduction of L. pneumophila to developing and mature P.
eruginosa PAO1-CFP biofilmsa
4.5.1 Adhesion and persistence of L. pneumophila in developing and mature
biofilms
per coupon per 106 inoculated legionellae and 38.7 ± 26.4 cells per
The amount of legionellae adhering to developing and mature biofilm were 10.8 ±
9.0 cells
coupon per 106 inoculated legionellae respectively, as shown in figure 4.20. Using
SPSS, independent samples t-test was calculated. It was found that p = 0.056
(assuming equal variances), thus we cannot conclude that there was significant
difference between the adhesion of L. pneumophila to each coupon of developing
and mature biofilms.
Adhesion of L. pneumophila to P. aeruginosa PAO1-CFP biofilm
0.000
10.000
20.000
30.000
40.000
50.000
60.000
Developing Mature
Num
ber o
f leg
ione
llae
per c
oupo
n pe
r 106
legi
onel
lae
inoc
ulat
ed in
to C
BR
70.000
Figure 4.20. Adhesion of L. pneumophila to different developmental stages of P. aeruginosa PAO1-CFP biofilm. The error bars represent standard deviation of 5 independent experiments.
Results
Department of Microbiology, NUS 88
With regards to the persistence of L. pneumophila in P. aeruginosa PAO1-CFP
biofilm, figure 4.21 illustrated an approximately 1 log decrease in legionellae
counts over 5 and 4 days following its introduction to developing and mature
biofilm respectively. Interestingly, in developing biofilms, L. pneumophila cell
counts only started to decrease noticeably on day 6 of the biofilm development,
which corresponds to the maturation of the P. aeruginosa PAO1-CFP biofilm.
Figure 4.21 also showed that the average planktonic L. pneumophila cell counts
remained high at >1.00 × 10 cells/ml, even after 3 hrs of allowance for washout
following legionellae introduction into both types of biofilms, revealing the
instability of initial legionellae adhesion to the biofilms. Nevertheless, on
subsequent days after L. pneumophila introduction into both types of biofilm,
planktonic legionellae dropped drastically and remained low at <100 cells/ml.
When the persistence of L. pneumophila in P. aeruginosa PAO1-CFP biofilms
was examined more closely in figure 4.22, only 35.2% ± 16.8% of legionellae
remained in mature biofilm 1 day after its introduction into CBR while 98.8% ±
1.0% of legionellae remained in developing biofilm. It was 3 days after
legionellae introduction, which corresponded to the maturation of the developing
biofilm, when the biofilm legionellae started to decrease, leaving 47.9% ± 9.8% in
the biofilm. The release of legionellae from matured biofilms was fastest initially
and slowed down within the next 3 days. Finally, legionellae loss from mature
biofilm tended to stabilize with slightly >10% of legionellae remaining in the
mature biofilm 4 days after the introduction of exogenous legionellae into CBR.
4
Results
Department of Microbiology, NUS 89
Status of L. pneumophila in continuous flow CBR system
0.0
1.0
2.0
3.0
4.0
5.0
6.0
1 2 3 4 5 6 7 8 9 10 11 12
Days
Log
Legi
onel
la c
once
ntra
tion
in
plan
kton
ic p
hase
[Log
(c
ells
/ml)]
0.0
1.0
2.0
3.0
4.0
5.0
6.0Log Legionella concentration in
biofilm [Log (cells/m
m2)]
Figure 4.21. Status of L. pneumophila in our continuous flow CBR system. The error bars represent standard deviation of 3 independent experiments.
Figure 4.22. Persistence of L. pneumophila in P. aeruginosa PAO1-CFP biofilm. The error bars represent standard deviation of 3 independent experiments.
(I-J) Std. Error Sig. Lower Bound Upper Bound95% Confidence Interval
4567891011
The mean difference is significant at the .05 level.*.
Results
Department of Microbiology, NUS 102
Figu
re
4.29
. Po
rosi
ty
dist
ribut
ion
of
(A)
deve
lopi
ng,
and
(B)
mat
ure
P.
aeru
gino
sa
PAO
1-C
FP b
iofil
ms.
Day
0
deno
ted
the
day
of
intro
duct
ion
of
legi
onel
lae
into
re
spec
tive
biof
ilms.
The
num
ber
of P
I pi
xels
of
each
ex
perim
ent
was
ob
tain
ed f
rom
3-8
im
age
stac
ks
from
1
or
2 co
upon
s. Th
e er
ror
bars
re
pres
ent
stan
dard
er
ror
of
3 in
depe
nden
t ex
perim
ents
.
Poro
sity
dis
trib
utio
n in
dev
elop
ing
biof
ilm
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
20% (b
ottom
)
x<
Sect
ions
of b
iofil
m
Porosity (Number of PI pixels per µm3) D
ay 0
Day
1D
ay 2
Day
3D
ay 4
Day
5
Poro
sity
dis
trib
utio
n in
mat
ure
biof
ilm
x<
(bott
om)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
20%
Day
4
Sect
ions
of b
iofil
m
Porosity (Number of PI pixels per µm3)
Day
3D
ay 2
Day
1D
ay 0
B)
(
A)
(
Results
Department of Microbiology, NUS 103
4.5
Sta significant linear correlation
bet lack of obvious relationship
bet e scatterplot below (Figure
4.3
Table 4.6. Table showing Pearson’s correlation between porosity and SBR. Each ontributing porosity and SBR data was obtained from 3-8 image stacks from 1 or
2 coupons of each experiment. The correlation between the 2 variables was btained from all data of 6 independent experiments.
Figure 4.30. Scatterplot of porosity and SBR both obtained from all data of 6 independent experiments.
Correlation is significant at the 0.05 level (2-tailed).*.
esion and parameters of P.
amount of legionellae adhering to P. aeruginosa PAO1-CFP biofilm was
gionellae adhesion and the rest of the biofilm parameters.
Table 4.7. Table showing Pearson’s correlation between legionellae adhesion to . aeruginosa PAO1-CFP biofilm (representing the number of legionellae per
coupon per 106 legionellae inoculated into CBR) and parameters of the biofilm. or each experiments, 3-8 image stacks from 1 or 2 coupons were used. The
correlations between the variables were obtained from 10 independent xperiments.
aeruginosa PAO1-CFP biofilm
Pearson’s correlations between the number of legionellae per coupon per 106
legionellae inoculated into CBR and biofilm parameters are presented in Table
4.6. The
significantly positively correlated (at 0.05 level) to the overall SBR of biofilm.
Otherwise, there was no significant linear relationship between the amount of
le
P
F
e
Results
Department of Microbiology, NUS 105
4.5.8 Localization of L. pneumophila in P. aeruginosa PAO1-CFP biofilms
Figure 4.31(A) and (C) revealed that L. pneumophila adhered to regions of high
porosity in both developing and mature P. aeruginosa PAO1-CFP biofilms, and
could also be found near the substratum. Four days later, majority of the
remaining legionellae were found “hidden” within the biofilms, away from
regions stained by PI (figure 4.31(B) and (D)).
(A)
Results
Department of Microbiology, NUS 106
(C)
(B)
Results
Department of Microbiology, NUS 107
(D)
Fi (blue) with
gionellae introduction to developing biofilm (3-days-old), (B) 4 days after legionellae introduction to developing biofilm, (C) 3hrs after legionellae
troduction to mature biofilm (7-days-old), and (D) 4 days after legionellae introduction to mature biofilm. Biofilm stained with PI (red) reflected porous
gions. The x-y view of the biofilm (main view) is flanked by y-z (right) and x-z (bottom) sections of the biofilm, with red arrows pointed towards the top of
iofilm. The scale represents 30µm in each image.
gure 4.31. CLSM images of P. aeruginosa PAO1-CFP biofilmadhered L. pneumophila (green) taken on different occasions: (A) 3hrs after le
in
re
b
Results
Department of Microbiology, NUS 108
4
Kinetics of P. aeruginosa PAO1 Biofilm Formation at 30°C
0.000
0.010
0.020
0.030
0.040
0.050
30282624222018161412
OD
470nm
0.000
0.100
0.200
0.300
0.400
OD
600n
m
0.060
0.070
80
4038363432Time (hr)
0.500
0.600 0.0
Amount of biofilm (OD470nm) Amount of total bacteria (OD600nm)
.6 Screening for effective P. aeruginosa PAO1 biofilm removing
gent
P. aeruginosa PAO1 biofilm formation in microtiter plate
P. aeruginosa PAO1 biofilm formation reached a
ximum on the 18th hour, detached drastically after 20th hour and subsequently
ained low with OD470nm at approximately 0.010. The detachment corresponded
rise and high level of total bacteria in the well. Therefore biofilm removal
hour intervals starting from the 12th hour of
formation.
Figure 4.32. Kinetics of P. aeruginosa PAO1 biofilm formation in microtitre plate at 30°C. The error bars represent standard deviations of 3 independent experiments.
a
4.6.1 Kinetics of
As shown in figure 4.32,
ma
rem
to the
assays were conducted at every 2
biofilm
Results
Department of Microbiology, NUS 109
4.6.2 P. aeruginosa PAO1 biofilm removal screening
Figure 4.33 revealed that NALCO 7330, NALCO 7320 and ACTI-PLUS 2818
had the highest comparable P. aeruginosa PAO1 biofilm removing efficiency. But
table 4.7 showed that NALCO 7320 had the highest efficacy since only 50ppm
was required to yield such a high percentage biofilm removal of 71.8% ± 16.8%.
Hence, NALCO 7320 was chosen for further characterization in P. aeruginosa
biofilm removal.
Figure 4.33. Highest percentage biofilm removal of various biofilm removing agents. The error bars represent standard deviations of 3 independent experiments.
Highest percentage biofilm removal of each biofilm removing agents
100.0%
(
-60.0%-40.0%-20.0%
0.0%20.0%40.0%60.0%80.0%
NALCO 73
30
NALCO 73
20
ACTI-PLU
S 2818
CT quart
erly c
leane
r
NALCO 90
001
NALSPERSE® 73
48
NALCO 73
38
NALCO 73
550Pe
rcen
tage
bio
film
rem
oval
%)
Results
Department of Microbiology, NUS 110
Table 4.8. Efficacy of biofilm removing agents. The standard deviations were obtained from 3 independent experiments.
Biofilm removing agent Percentage biofilm Concentration Time
4.7.1 Kinetics of P. aeruginosa PAO1 biofilm removal
As shown in figure 4.34, biofil NALCO s time dependent
but no dependent ency of lowering removal efficacy
with increasing concentration abo Nevertheless, at 10ppm, no biofilm
rem
Figure 4.34. Kinetics of biofilm removal by NALCO 7320. The error bars represent standard deviations of 3 independent experiments.
m removal by 7320 wa
t concentration , with a tend
ve 50ppm.
oval was observed.
Kinetics of biofilm removal by NALCO 7320
-250.0%
-200.0%
-150.0%
-100.0%
-50.0%
0.0%
50.0%
100.0%
0 2 4 6 8Exposure time (hr)
Perc
enta
ge b
iofil
m re
mov
al
(%)
1000ppm500ppm100ppm50ppm10ppmMock treated
Department of Microbiology, NUS 111
Results
Department of Microbiology, NUS 112
4.7.2 Antimicrobial susceptibility testing
Minimum inhibitory concentration (MIC) is defined the lowest concentration of
spread plating.
Figure 4.35 demonstrated that MIC of NALCO 7320 on P. aeruginosa PAO1 and
L. pneumophila were 50ppm and 10ppm respectively. In addition, figure 4.36
revealed that MBC of NALCO 7320 on P. aeruginosa PAO1 and L. pneumophila
were 100ppm and 50ppm respectively. Hence, to ensure maximum biofilm
removal with bactericidal effect on planktonic P. aeruginosa PAO1, a final
concentration of 100ppm of NALCO 7320 was chosen for further characterization
in P. aeruginosa biofilm removal.
antimicrobial agent that completely inhibits the growth of the organism as
detected by the unaided eye while minimum bactericidal concentration (MBC) is
the lowest concentration of antimicrobial agent that completely eradicated the
organism as detected by
Results
Department of Microbiology, NUS 113
NALCO 7320
Figure 4.35. Visual determination of minimum inhibitory concentration (MIC).
Figure 4.36. Determination of minimum bactericidal concentration (MBC) of NALCO 7320. The error bars represent standard deviations of 3 independent experiments. The dashed line denotes the detection limit of spread plating technique. ‘to’ represent the initial bacterial concentration before the addition of NALCO 7320.
L. pneumophila
P. aeruginosa
Bla
nk
PAO1
Moc
k tre
ated
500p
pm
100p
pm
50pp
m
10pp
m
1,00
0ppm
Determination of minimum bactericidal concentration (MBC)
9.0010.00
0.001.002.003.004.005.006.007.008.00
to
Mock t
reated
1k pp
m
500 p
pm
100 p
pm
50 pp
m
10 pp
m
Log
(CFU
/ml)
P. aeruginosa L. pneumophila
Results
Department of Microbiology, NUS 114
Persistence of P. aeruginosa PAO1-CFP biofilms treated with NALCO 7320
treated with NALCO 7320. The dashed line represented the detection limit of the
Figure 4.38. Viable cell counts of planktonic P. aeruginosa PAO1-CFP in CBR
plating technique used.
4.8.2 Structure of P. aeruginosa PAO1-CFP biofilms treated by NALCO 7320
Upon addition of NALCO 7320, bio-volume (Figure 4.39), average thickness
(Figure 4.40) and maximum thickness (Figure 4.41) of developing biofilm
immediately dropped and subsequently recovered by the 4th hr. Next, bio-volume
(Figure 4.39) and average thickness (Figure 4.40) dropped even lower than before
and remained low within the range of 6.00-10.0µm3µm-2 and 6.00-11.0µm
respectively, from the 8th hr of exposure to NALCO 7320 onwards. However, the
maximum thickness (Figure 4.41) dropped slightly and remain within the range of
15.0-22.0µm until 24th hr.
Results
Department of Microbiology, NUS 116
In mature biofilm, by the 4th hr of exposure to NALCO 7320, bio-volume (Figure
ple t-test, p = 0.015, assuming equal variance) 4hrs
fter the addition of NALCO 7320 and remained at <65% thereafter. Substratum
ALCO 7320. Figure 4.43 demonstrated that the surface-to-biovolume ratio of
dependent sample t-test, p = 0.01, assuming equal
ariance) from <0.20 to 0.612 ± 0.220.
4.39) and average thickness (Figure 4.40) decreased significantly (independent
sample t-test, p = 0.002 and 0.007 respectively, assuming equal variance) and
remained within the range of 18.0-22.0µm3µm-2 and 21.0-26.0µm respectively, for
the subsequent 8hrs. However, maximum thickness (Figure 4.41) of mature
biofilm merely exhibited a decreasing trend. Nevertheless, at the end of 24hrs, the
bio-volume, average thickness and maximum thickness in both developing and
mature biofilm were comparable.
Figure 4.42 showed that substratum coverage of developing biofilm dropped
significantly (independent sam
a
coverage of mature biofilm remained comparable with or without exposure to
N
developing biofilm increased to >0.800µm2µm-3 after 24hrs of exposure. In
contrast, NALCO 7320 had no apparent effect on the surface-to-biovolume ratio
of mature biofilm.
Roughness coefficient of developing biofilm increased significantly (independent
sample t-test, p = 0.031, assuming equal variance) from <0.15 to >0.55 after 8hrs
of exposure to NALCO 7320 (figure 4.44). However, it was only after 24hrs of
exposure to NALCO 7320, when the roughness coefficient of mature biofilm
increased significantly (in
v
Results
Department of Microbiology, NUS 117
with NALCO 7320. The bio-volume was obtained from at least 3 image stacks per coupon. The error bars represent standard deviation of 4 coupons from 2 independent experiments.
Figure 4.39. Bio-volume of P. aeruginosa PAO1-CFP biofilm in CBR treated
represent standard deviation of 4 coupons
Figure 4.40. Average thickness of P. aeruginosa PAO1-CFP biofilm in CBR treated with NALCO 7320. The average thickness was obtained from at least 3 image stacks per coupon. The error barsfrom 2 independent experiments.
Bio-volume
25.0
30.0
35.0B
o-vo
lum
e (µ
m3 /µ
m2
0.0
5.0
10.0
15.0
20.0
Beforetreatment
0hr 4hr 8hr 12hr 24hr
Exposure time
i)
Developing biofilm (day 4) Mature biofilm (day 8)
Average thickness
20.0
25.0
35.0
40.0
ickn
ess
(µ
0.0
5.0
15.0
30.0
Exposure time
e th
m)
10.0
Beforetreatment
0hr 4hr 8hr 12hr 24hr
Ave
rag
Developing biofilm (day 4) Mature biofilm (day 8)
Results
Department of Microbiology, NUS 118
Figure 4.41. Maximum thickness of P. aeruginosa PAO1-CFP biofilm in CBR treated with NALCO 7320. The maximum thickness was obtained from at least 3 image stacks per coupon. The error bars represent standard deviation of 4 coupons from 2 independent experiments.
Maximum thickness
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
Beforetreatment
0hr 4hr 8hr 12hr 24hr
Exposure time
Max
imum
thic
knes
s (µ
m)
Developing biofilm (day 4) Mature biofilm (day 8)
Substratum coverage
0.0%
20.0%
40.0%
60.0%
80.0%
100.0%
120.0%
Beforetreatment
0hr 4hr 8hr 12hr 24hr
Exposure time
Subs
trat
um c
over
age
(%)
Developing biofilm (day 4) Mature biofilm (day 8)
Figure 4.42. Substratum coverage of P. aeruginosa PAO1-CFP biofilm in CBR treated with NALCO 7320. The substratum coverage was obtained from at least 3 image stacks per coupon. The error bars represent standard deviation of 4 coupons from 2 independent experiments.
Results
Department of Microbiology, NUS 119
Figure 4.43. Surface-to-biovolume ratio of P. aeruginosa PAO1-CFP biofilm in CBR treated with NALCO 7320. The surface-to-biovolume ratio was obtained from at least 3 image stacks per coupon. The error bars represent standard deviation of 4 coupons from 2 ind
e error bars represent standard deviation of 4 coupons from 2 independent experiments.
igure 4.44. Roughness coefficient of P. aeruginosa PAO1-CFP biofilm in CBR
ependent experiments.
igure 4.44. Roughness coefficient of P. aeruginosa PAO1-CFP biofilm in CBR
Surface-to-biovolume ratio
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Beforetreatment
0hr 4hr 8hr 12hr 24hr
Exposure time
Surf
ace-
to-b
iovo
lum
e ra
tio (µ
m2 /µ
m3 )
Developing biofilm (day 4) Mature biofilm (day 8)
Roughness coefficient
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Beforetreatment
0hr 4hr 8hr 12hr 24hr
Exposure time
Rou
ghne
ss c
oeffi
cien
t
Developing biofilm (day 4) Mature biofilm (day 8)
FFtreated with NALCO 7320. The roughness coefficient was obtained from at least 3 image stacks per coupon. The error bars represent standard deviation of 4 coupons from 2 independent experiments.
treated with NALCO 7320. The roughness coefficient was obtained from at least 3 image stacks per coupon. The error bars represent standard deviation of 4 coupons from 2 independent experiments.
Results
Department of Microbiology, NUS 120
4.8.3 Persistence of L. pneumophila in P. aeruginosa PAO1-CFP biofilms
o apparent linear correlation was found between legionella and bio-volume loss
treated with NALCO 7320
As shown in figure 4.45, there was a steady and gradual decrease in legionellae
viable cell counts in both developing and mature biofilms. Figure 4.46
demonstrated the presence of legionellae in the bulk fluid of CBR (at between 2.0-
2.5 cells/ml for every experiments) even after 24hrs of exposure to NALCO 7320.
Loss of legionellae per unit biomass lost from developing and mature biofilms
were calculated and no significant difference between them were found
(independent sample t-test, p = 0.414, equal variances not assumed). In addition,
n
(Table 4.8). Nevertheless, the scatterplot (Figure 4.47) revealed that 3 out of 4
data points have a linear relationship, thus implying the need for more work to
examine this relationship.
Results
Department of Microbiology, NUS 121
Persistence of L. pneumophila in biofilms treated with NALCO 7320
Table 4.9. Table showing Pearson’s correlation between bio-volume and legionellae loss. Each contributing bio-volume data was obtained from at least 3 image stacks from each of the 2 coupons used per experiment. The correlation between the 2 variables was obtained from 4 independent experiments.
dependent experiments.
Figure 4.47. Scatterplot of bio-volume and legionellae loss, obtained from 4
3000.000 3500.000 4000.000 4500.000
Legionellae loss
20.000
14.000
15.000
16.000
17.000
18.000
19.000
21.000
Bio
-vol
ume
loss
in
Results
Department of Microbiology, NUS 123
4.8.4 Distribution of L. pneumophila in P. aeruginosa PAO1-CFP biofilms
treated with NALCO 7320
Despite increasing exposure time to NALCO 7320, the distribution of L.
ost L.
s while least
biofilm
rema L.
ature
biofilm
bottom
pneumophila in developing biofilm (figure 4.48(A)) remained similar, with the
exception of the fourth hour after NALCO 7320 addition. Generally, m
pneumophila resided in the 20%-60% region of developing biofilm
legionellae were found at the top 20% of the biofilm. But 4hrs after NALCO 7320
addition, the peak was temporarily shifted to 60%-80% of the biofilm.
Figure 4.48(B) showed that the distribution of L. pneumophila in mature
ined similar with increasing exposure time to NALCO 7320. Most
pneumophila resided in the 40%-80%, especially 60%-80% region of m
s while least legionellae were found at comparable levels at both top and
20% of mature biofilms.
Results
Department of Microbiology, NUS 124
Ef
fect
of
L.
in
(A)
P.
aeru
gino
sa
obta
ied
e
ent
Figu
re
4.48
.N
ALC
O
7320
on
th
e di
strib
utio
n of
pn
eum
ophi
la
deve
lopi
ng,
and
(B)
mat
ure
PAO
1-C
FP b
iofil
ms.
The
num
ber
of C
FDA
pix
els
per
µm
3
nfr
om
at
leas
t 3
imag
erro
r ba
rs
repr
es
was
stac
ks p
er c
oupo
n. T
he
stan
dard
dev
iatio
n of
4
coup
ons
from
2
inde
pend
ent e
xper
imen
ts.
(A)
(B)
Effe
ct o
f NA
LCO
732
0 tr
eatm
ent o
n di
strib
utio
n of
L. p
neum
ophi
la i
n de
velo
ping
bio
film
-4.0
0
-3.5
0
-3.0
0
-2.5
0
-2.0
0
-1.5
0
-1.0
0
x<20
% (bott
om)
Sect
ions
of b
iofil
m
Log (Number of CFDA pixels per µm3) B
efor
e tre
atm
ent
0hr
4hr
8hr
12hr
24hr
Effe
ct o
f A
732
dist
ibut
n of
L.m
a b
i
-2.0
0
-1.5
0
-1.0
0
tom)
NLC
O
0 tr
eaen
tr
io
pne
umla
ture
ofilm
-4.0
0
-3.5
0
-3.0
0
-2.5
0
x<20
% (bot
tm o
n op
hi in
Sect
ins
of
o b
iofil
m
Log (Number of CFDA pixels per µm3) B
efor
e tre
atm
ent
0hr
4hr
8hr
12hr
24hr
Results
Department of Microbiology, NUS 125
4.8 and mature biofilms treated
it
ig cell mass was rather uniformly
ist % biofilm, before and immediately
fte 4hrs later, the peak was shifted
.
ubsequently, the peak of bio-volume was progressively shifted towards the 20%-
0% region of the biofilm, maintaining at this spot until the 24th hr of exposure to
ALCO 7320.
igure 4.49(B) illustrated that peak bio-volume was found at 60%-80% region of
ature biofilm, before and up to the 8th hr of exposure to NALCO 7320.
ubsequently, the peak was shifted to 40%-60% region by the 12th hr of exposure
nd e tually to 20%-40% region by the 24th hr.
.5 Bio-volume distributions of developing
h NALCO 7320
ure 4.49(A) revealed that majority of the
ributed in the 20 -80% region of developing
r the addition of NALCO 7320. However,
w
F
d
a
upwards and was more concentrated at the 60%-80% region of the biofilm
S
4
N
F
m
S
a ven
Results
Department of Microbiology, NUS 126
Effe
ct o
f NA
LCO
732
0 tr
eatm
ent o
n bi
o-vo
lum
e di
strib
utio
n in
dev
elop
ing
biof
ilm
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
x<20
% (bott
om)
Sect
ions
of b
iofil
m
Bio-volume (µm3µm
-2) B
efor
e tre
atm
ent
0hr
4hr
8hr
12hr
24hr
Effe
ct
bio-
volu
e di
2.00
3.00
4.00
5.00
6.00
7.00
8.00
tom)
of N
ALC
O 7
320
tm
strib
u i
a
biof
ilm
0.00
1.00
x<20
% (bot
reat
men
t on
tion
n m
ture
Sect
ions
of b
iofi
Bio-volume (µm3µm
-2)
lmB
efor
e tre
atm
ent
0hr
4hr
8hr
12hr
24hr
Figu
re
4.49
.N
ALC
O
7320
on
th
e di
strib
utio
n of
bi
o-vo
lum
e de
velo
ping
, an
d (B
) m
atur
e
aPA
O1-
CFP
bio
film
s. Th
e bi
o-vo
lum
e w
as o
btai
ned
stac
ks p
er c
oupo
n. T
he
stan
dard
dev
iatio
n of
4
coup
ons
from
2
inde
pend
ent e
xper
imen
ts.
Ef
fect
of
in
(A)
ugin
sa e
ent
P.er
o
from
at
le
ast
3 im
ag
erro
r ba
rs
repr
es
Results
Department of Microbiology, NUS 127
4.8.6 treated with
AL
igu osity from 8.80 ± 1.95µm-3 to 14.8
0.4 fter the addition of NALCO 7320.
y t dropped and remained <4.50µm-3
.19µm-3 in mature biofilm was also observed immediately after the addition of
ALCO 7320. The level of porosity also dropped by the 4th hr of exposure and
mained <4.00µm-3 until the 24th hr.
igure 4.51(A) demonstrated that the porosity at the lower 60% of developing
iofilm increased drastically to >3.000µm-3 (with peak porosity at 20%-40%
gion of the biofilm) immediately after the addition of NALCO 7320 into the
BR. However, 4hrs later, the level of peak porosity dropped and remain within
e range of 1.000-1.500µm-3 at 40%-80% of the biofilm until the 24th hr of
enerally, NALCO 7320 had no observable effect on the porosity distribution of
ature biofilm where peak porosity was always found at 60%-80% region of the
iofilm (figure 4.51(B)). However, there was a noticeable increase in the porosity
f mature biofilm from peak porosity between 1.000-1.500µm-3 to >2.000µm-3),
mediately after the addition of NALCO 7320 into the CBR.
Porosity distributions of P. aeruginosa PAO1-CFP biofilms
CO 7320
re 4.50 illustrated a drastic increase of por
µm
N
F
-3 in developing biofilm immediately a
he 4
±
th hr of exposure, the level of porosityB
until the 24th hr. A slight increase of porosity from 3.49 ± 2.59µm-3 to 6.59 ±
1
N
re
F
b
re
C
th
exposure.
G
m
b
o
im
Results
Department of Microbiology, NUS 128
Figure 4.50. P. aeruginosa
Porosity of PAO1-CFP biofilm in CBR treated with NALCO 7320. The porosity was obtained from at least 3 image stacks per coupon. The error bars represent standard deviation of 4 coupons from 2 independent experiments.
Porosity18.0
)
0.0
8.0
16.0
treatmenthr 8hr 12hr 24hr
er p
i p
e3
2.0
4.0
6.0
10.0
12.0
14.0
Before 0hr 4
Poro
sity
(Num
b o
f PI
xels
r µm
Developing biofilm (Day 4) Mature biofilm (Day 8)
Results
Department of Microbiology, NUS 129
Figu
re
4.51
. Ef
fect
of
N
ALC
O
7320
on
po
rosi
ty
dist
ribut
ion
of
(A)
deve
lopi
ng,
and
(B)
mat
ure
P.
aeru
gino
sa
PAO
1-C
FP b
iofil
ms.
The
poro
sity
w
as
obta
ined
fr
om
at
leas
t 3
imag
e st
acks
per
cou
pon.
The
er
ror
bars
re
pres
ent
stan
dard
dev
iatio
n of
4
coup
ons
from
2
inde
pend
ent e
xper
imen
ts.
Effe
ct o
f NA
LCO
732
0 tr
eatm
ent o
n po
rosi
ty d
istr
ibut
ion
in d
evel
opin
g bi
ofilm
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
x<20
% (bott
om)
Sect
ions
of b
iofil
m
Porosity (Number of PI pixels per µm3) B
efor
e tre
atm
ent
0hr
4hr
8hr
12hr
24hr
Effe
ct73
20 tr
eatm
ent o
n po
rbu
tion
in m
atur
e of
ilm
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
x<20
% (bott
om)
24hr
of N
ALC
O
osity
dis
tri
bi
Sect
ions
of b
iofil
m
Porosity (Number of PI pixels per µm3)
12hr
8hr
4hr
t0h
rB
efor
e tre
atm
en
Discussion
Department of Microbiology, NUS 130
Chapter 5: Discussion
Biofilm formation
To date, biofilm development is best studied in P. aeruginosa PAO1. The fusion
of an ecfp gene (encoding for enhanced cyan fluorescent protein) to a constitutive
promoter and subsequent insertion into a neutral intergenic region downstream of
the glmS gene on P. aeruginosa PAO1 genome (Klausen et al., 2003) allowed
CLSM observations of P. aeruginosa PAO1-CFP biofilms in the absence of
exogenous fluorescent dyes. The fluorescently tagged strain did not show any
phenotypic changes compared with the parental strain when tested in liquid
medium or flow chamber biofilms (Klausen et al., 2003). Similarly, the growth of
P. aeruginosa PAO1 and P. aeruginosa PAO1-CFP in minimal media supplied
with mannitol as the sole carbon source yielded indistinguishable growth curves
(Figure 4.2 and 4.3) in the present study.
The P. aeruginosa PAO1-CFP biofilm model was established at 30°C under high
shear, in the CBR system with continuous supply of minimal media containing
mannitol as the sole carbon source. After 6 days of growth under continuous
culture, both viable counts of P. aeruginosa PAO1-CFP in the biofilm (Figure
4.11) and the maximum thickness of the biofilm (Figure 4.14) reached a
reproducible plateau. This finding corroborated with a P. aeruginosa PAO1
biofilm development study, in which minimal medium containing glutamic acid as
the sole carbon source was used (Sauer et al., 2002). Thus in the present study,
mature biofilm is defined as one that has reached its maximum thickness on day 6
Discussion
Department of Microbiology, NUS 131
while developing biofilm is one that has yet to reach its penultimate thickness
(before day 6).
Despite the attainment of maximum thickness on day 6 (Figure 4.14), the slight
increase in viable counts of planktonic (Figure 4.18) and biofilm (Figure 4.11) P.
aeruginosa PAO1-CFP, and in bio-volume (Figure 4.12) and average thickness
(Figure 4.13) of the biofilm on day 10 and 11, provided yet another experimental
evidence to support the hypothesis that biofilms never reached steady state
(Heydorn et al., 2000; Lewandowski et al., 2004). Early study by Bakke et al.
(1989) also demonstrated that older biofilms were continuously increasing their
density, even though their thickness remained constant. However, it is not possible
to differentiate between growth and attachment. Therefore, it is possible that
bacteria in the bulk fluid had been “captured” by mature biofilm. Additionally, no
sloughing event that might jeopardize the reproducibility of the biofilm structure
(Lewandowski et al., 2004), was observed during the course of this study.
As the P. aeruginosa PAO1-CFP biofilm matures, structural changes were
detected. The cell mass of biofilm started to move upwards on day 5, as suggested
by the drastic decrease in substratum coverage (Figure 4.15), the peak shift in
biomass distribution from 20%-40% to 40%-60% region of biofilm (Figure
4.26(A)), and drastic increase in SBR at bottom 20% of biofilm (Figure 4.27(A))
and in corresponding SBR ratio (bottom 20%: top 20% of biofilm) (Table 4.4).
Another upward movement of the cell mass was detected on day 9 when the
roughness coefficient of the biofilm dropped appreciably (Figure 4.17), a peak
Discussion
Department of Microbiology, NUS 132
shift in biomass distribution from 40%-60% to 60%-80% region of the biofilm
was observed (Figure 4.26(B)), and the SBR at top 20% of biofilm decreased
noticeably (Figure 4.27(B)) resulting in the further increase in SBR ratio (Table
4.4). Eventually, the cell mass congregated at the 40%-80% region of the mature
P. aeruginosa PAO1-CFP biofilm with the least bio-volume found at the bottom
20% (Figure 4.26(B)). Expectedly, the region of biofilm with the lowest SBR
became increasingly prominent and coincided with that of the cell mass core on
day 5 onwards (Figure 4.27(B)). The roughness coefficient of P. aeruginosa
PAO1-CFP biofilm exhibited a general decreasing trend (Figure 4.17) thus
implying decreasing structural heterogeneity of the biofilm structure in the CBR
continuous flow system of this study. The production and redistribution of
biomass has been modeled in several investigations, where each model assumes a
different mechanism for biomass redistribution (Cogan and Keener, 2004;
Picioreanu et al., 2004; Alpkvist et al., 2006). However, in the absence of
empirical investigation, it is not clear how to judge the validity of the
redistribution mechanisms in the models.
Interestingly, despite the structural changes, the overall SBR remained relatively
uniform (Figure 4.16). Few studies applied the SBR function in COMSTAT in
their studies, but observations in this study corroborated with that of a previous
study in which P. aureofaciens, P. fluorescens and P. aeruginosa PAO1 each
exhibited relatively constant SBR throughout respective biofilm development and
structural changes (Heydorn et al., 2000). By extricating the overall SBR into 5
sections along the biofilm thickness, the present study demonstrated the
Discussion
Department of Microbiology, NUS 133
usefulness of SBR distribution in providing insights into biofilm structural
differences.
Yang et al. (2000) first attempted to describe biofilm porosity using areal porosity.
Areal porosity is the ratio of the combined areas of the voids to the total area of
the image. However it is calculated from two-dimensional confocal images when
porosity characterizes three-dimensional space (Lewandowski, 2000). This study
presented an unprecedented method of quantifying porosity of paraformaldehyde
fixed biofilms by limiting the time of staining with PI (Chapter 3.4.4), obtaining
confocal image stacks under constant variables that may affect the quality of the
images (Chapter 3.8.7), applying constant threshold to all image stacks (Chapter
3.8.8.2) and quantifying the number of PI pixels per unit biomass (Chapter
3.8.8.4). PI has specificity for double stranded nucleic acids and bears a double
positive charge, thus readily enters and stains non-viable cells (Shapiro and Nebe-
von-Caron, 2004). Since with increasing incubation with 0.1mg/ml PI beyond 5
minutes resulted in excessive staining of the biofilm (Chapter 4.3.3), the amount
of PI molecules were not limiting. In this study, no viable bacteria were detected
from paraformaldehyde fixed biofilms, hence the possibility that the cells within
the biofilms remained viable (thus not picking up the PI dye) is eliminated.
Furthermore, extracellular DNA comprises <1-2% of the biofilm matrix
(Sutherland, 2001) therefore is not likely to contribute significantly to the number
of PI pixels detected. These imply that any variation in the number of PI pixels per
unit biomass is dependent on the accessibility of the biofilm cells to PI molecule,
thus reflecting the porosity of the biofilm in a three-dimensional context.
Discussion
Department of Microbiology, NUS 134
Knowledge of the way in which substances are transported within biofilm is
essential for control or eradication. Based on the fact that biofilms consist of
microbial cell clusters separated by interstitial “voids”, “channels” or “pores”
(Lawrence et al., 1991; de Beer et al., 1994a and 1994b; Massol-Deya et al.,
1995), mass transport in the interstitial voids is mainly facilitated by convective
flow (Stoodley et al., 1994) and mass transfer inside the microbial clusters is
entirely due to molecular diffusion (de Beer et al., 1994a). On the contrary, Yang
and Lewandowski (1995) demonstrated that mass transfer coefficients were not
only found to vary both horizontally and vertically in the biofilm, they also
fluctuated significantly inside microbial cell clusters. This observation spurred the
proposal of a new conceptual model of biofilm microbial cluster structure, which
assumes the existence of flow channels with variable cross-sectional areas and
irregular orientations inside biofilm clusters. To the best of our knowledge, the
present study provided the first physical evidence of porous flow channels within
biofilm cell cluster (Figure 4.8).
Previous studies applied SBR to reflect the fraction of the biofilm that is exposed
to nutrient flow (Heydorn et al., 2000). Similar fraction of the biofilm has been
experimentally proven to be stained by PI (Figure 4.8) and can be represented by
the parameter “porosity” in the present study. On the contrary, overall SBR does
not correlate to overall porosity of P. aeruginosa PAO1-CFP biofilm (Chapter
4.5.6). Furthermore, there is no obvious correlation between the distribution of
SBR (Figure 4.27) and porosity (Figure 4.29). These observations suggest that the
structure of the biofilm alone is not enough to reflect the porosity of the biofilm.
Discussion
Department of Microbiology, NUS 135
Although no attempts were made to detect EPS in the present study, it is well
established that biofilms comprise microbial cells within a matrix of EPS and
these microcolonies are separated by interstitial voids and channels. EPS, as the
major structural components of the biofilm matrix, has been implicated in the
protection of embedded microbial cells by either neutralizing or binding to toxic
substances, or merely serving as a physical barrier to environmental challenges
(Hall-Stoodley et al., 2004). Therefore, it is highly likely that the changes in the
quantity or nature of EPS had influenced the porosity of the biofilm in this study.
The overall porosity of the biofilm exhibited a general decreasing trend but
dropped significantly (p<0.01) on day 8 of development (Figure 4.28), suggesting
a drastic change in the quantity or property of EPS. However, throughout biofilm
development, the profile of porosity distribution remained comparable (Figure
4.29(A) and (B)). Thus suggesting the change in EPS was rather uniform
throughout the biofilm.
Discussion
Department of Microbiology, NUS 136
Association of Legionella with biofilm
In this study, there was no significant difference (p=0.056) between the number of
legionellae adhering to developing and mature biofilm (Figure 4.20). The amount
of legionellae adhesion was found to be dependent on overall SBR of the biofilm
and independent on other biofilm parameters, especially porosity (Table 4.6). On
the contrary, legionellae adhesion patterns (Figure 4.23) did not emulate the
distribution patterns of SBR (Figure 4.27) for both developing and mature
biofilms. Interestingly, the legionellae adhesion patterns and biofilm porosity
distributions (Figure 4.23 and 4.29 respectively) were comparable, and the
attached legionellae were found co-localized with regions of high porosity even if
it was at the bottom of the biofilm (Figure 4.31(A) and (C)). These results
demonstrated that legionellae adhesion was dependent on the structure of the
biofilm, where biofilms with higher SBR can capture more planktonic legionellae,
but the adhesion might be hindered because legionellae only had access to biofilm
at areas of higher porosity. In a similar study, Langmark et al. (2005) found that
the accumulation of model pathogens (including L. pneumophila) was generally
independent of the biofilm cell density and was shown to be dependent on the
particle surface properties, where hydrophilic spheres accumulated to a larger
extent than hydrophobic ones. Taken together with the current study, the amount
and localization of legionellae adhering to biofilms may be determined by the
interplay of cell surface properties, biofilm structure and porosity.
In this study, figure 4.22 illustrated the 2-days delayed release of legionellae from
developing biofilm, until day 6 of biofilm development (Figure 4.21), which
Discussion
Department of Microbiology, NUS 137
corresponded to biofilm maturation. The significant increase (p<0.01) in P.
aeruginosa PAO1-CFP biofilm detachment on day 6 (Figure 4.18) was likely the
cause of the sudden release of legionellae from P. aeruginosa PAO1-CFP biofilm
after the latter matured. This corroborated with another study which demonstrated
that detachment was one of the primary mechanisms affecting the loss of
microspheres and legionellae from biofilms within a pilot-scale distribution
system, as well as disinfection and biological grazing (Langmark et al., 2005).
Although the transport of particulates in biofilms has been largely neglected, it is
believed that in microbial competition in mixed population biofilms, slow
growing microorganisms are forced towards the biofilm surface and eventually
displaced (Okabe et al., 1996). In present study, legionellae release slowed down
(Figure 4.21 and 4.22) even though the bacteria was unable to replicate in the
continuous flow CBR system (which was fed with minimal media that supported
the growth of P. aeruginosa PAO1-CFP only) and the biofilm detachment
remained high, or even increased slightly on day 10 and 11 (Figure 4.18). In
addition, majority of remaining legionellae were found embedded in the biofilms,
away from porous regions (Figure 4.31(B) and (D)), implying reattachment of
planktonic legionellae to P. aeruginosa PAO1-CFP biofilm was not significant.
These indicate the existence of stable regions within P. aeruginosa PAO1-CFP
biofilm that harbored and protected legionellae from being desorbed. Figure 4.24
and 4.25 revealed that highest legionellae losses were found at the top 40% of the
biofilm while least legionellae losses were located at the bottom 60%, especially
at bottom 20%.
Discussion
Department of Microbiology, NUS 138
The development of bimodal legionellae distribution 4 days after its adhesion to
developing biofilm (corresponding to day 7 of biofilm development) and
occurrence of alternate unimodal and bimodal distributions in mature biofilm
(Figure 4.23) revealed unbalanced advective transport of legionellae towards
biofilm surface took place after biofilm maturation. Similarly, Okabe et al. (1996)
observed that the trapped tracer beads were gradually transferred from the depth
of the biofilm to the surface but this advective transport was unbalanced. Since the
authors concluded that cell growth is an important factor for the entrapment and
release of the tracer beads, they attributed this phenomenon to unbalanced cell
growth. Therefore, it is likely that the concentration of biomass (thus cell growth)
near the substratum in developing biofilms (Figure 4.26(A)) resulted in unimodal
legionellae distributions (Figure 4.23(A)) and the faster loss of legionellae from
bottom 60% of developing biofilm (Figure 4.24) than from mature biofilm (Figure
4.25). On the other hand, the concentration of biomass in 40%-60% region of
mature biofilm (Figure 4.26(B)) was likely to result in bimodal legionellae
distributions (Figure 4.23), where legionellae from 40%-60% region of biofilm
were advected towards the surface of biofilm while legionellae loss at the bottom
slowed down (Figure 4.25). Therefore, the results from present study supported
the proposition of the existence of unbalanced cell growth in mature biofilm.
Discussion
Department of Microbiology, NUS 139
Applications of biofilm-removing agents used in this study
Products from NALCO Company (www.nalco.com) such as NALCO 7320,
NALCO 7330, NALCO 7338, NALSPERSE® 7348 and NALCO 73550 are
registered as water treatment products to the NSF Registration Guidelines for
Proprietary Substances and Nonfood Compounds (www.nsf.org/usda) while
NALCO 90001 is registered to the New Zealand Food Safety Authority
(www.nzfsa.govt.nz). All the above products, except NALCO 73550, are
acceptable for treating boilers, steam lines and/or cooling systems where neither
the treated water nor the steam produced may contact edible products in and
around food processing areas. On the other hand, all the products, except NALCO
7320, 7330 and 90001, are acceptable for treatment of cooling and retort water in
and around food processing areas (www.nsf.org/usda). In addition, ACTI-PLUS
2818 is registered with U.S. Environmental Protection Agency (www.epa.gov),
under the Pest Control Products Act. It is an agent for controlling algal, bacteria
and fungal slime in condensing and cooling equipment to which recirculating
water is used as a cooling media. It can also be used to control bacterial and algal
slime in decorative fountains and brewery pasteurizers. Lastly, COOLING
TOWER QUARTERLY CLEANER was a product by Novapharm Research
(Australia) Pty Ltd., subsequently renamed and patented as Aeris-Guard
Enzymatic Coil Cleaner in 2003 (www.aerisguard.com). In March 2006 Quarterly
Report, Aeris Technologies Ltd. (www.aerisguard.com) reported several
successful applications of this product in both cooling towers and large industrial
water systems, and stated intentions to widen industrial applications in areas such
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.
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Appendix
Department of Microbiology, NUS 175
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 K2HPO44.5g KH2PO42.0g (NH4)2SO42.0g Mannitol 0.2g MgSO4.7H2O 10mg CaCl25mg FeSO4.7H2O 2mg MnCl21L Deionized water Autoclaved at 121°C for 15mins. Appendix IV Phosphate Buffer Saline (PBS): 0.24g KH2PO41.44g Na2HPO48g 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.
Appendix
Department of Microbiology, NUS 176
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.