spatial heterogeneity in pseudomonas aeruginosa biofilms and how it affects antibiotic tolerance

144 Pages·2006·0.79 MB·English

by  Lee Richards

SPATIAL HETEROGENEITY IN PSEUDOMONAS AERUGINOSA BIOFILMS
AND HOW IT AFFECTS ANTIBIOTIC TOLERANCE

By
Lee Alexander Richards

A dissertation submitted in partial fulfillment
of the requirements for the degree

of

Doctor of Philosophy

in

Engineering

MONTANA STATE UNIVERSITY
Bozeman, Montana

July 2006
© COPYRIGHT
by
Lee Alexander Richards
2006
All Rights Reserved
ii

APPROVAL
of a dissertation submitted by
Lee Alexander Richards
This dissertation has been read by each member of the dissertation committee
and has been found to be satisfactory regarding content, English usage, format,
citations, bibliographic style, and consistency, and is ready for submission to the
Division of Graduate Education.
Philip S. Stewart
Approved for the Department of Chemical and Biological Engineering
Ron Larsen
Approved for the Division of Graduate Education
Joseph J. Fedock
iii

STATEMENT OF PERMISSION TO USE

In presenting this dissertation in partial fulfillment of the requirements for a
doctoral degree at Montana State University, I agree that the library shall make it
available ot borrowers under the rules of the library. I further agree that copying of this
dissertation is allowable only for scholarly purposes, consistent with “fair use” a
prescribed in the U.S. Copyright Law. Requests for extensive copying or reproduction of
the dissertation should be referred to Bell & Howell Information and Learning, 300 North
Zeeb Road, Ann Arbor, Michigan 48106, to whom I have granted “the exclusive right to
reproduce and distribute my dissertation in and from microform along with the non-
exclusive right to reproduce and distribute my abstract in any format in whole or in part”

Lee Alexander Richards
July 2006
iv

ACKNOWLEDGEMENTS

I would like to thank Frank Roe, Betsey Pitts, Garth Ehrlich, and Phil Stewart for
their contributions to chapter 1 of this dissertation. Frank Roe and Betsey Pitts
both contributed to the microelectrode data presented in Figure 2.1.
v

TABLE OF CONTENTS

ACKNOWLEDGEMENTS...................................................................................iv

LIST OF TABLES...............................................................................................viii

LIST OF FIGURES...............................................................................................ix

ABSTRACT.........................................................................................................xiv

1. INTRODUCTION..........................................................................................1

Scope of This Dissertation............................................................................11
References.....................................................................................................13

2. CONTRIBUTIONS OF OXYGEN AND GLUCOSE LIMITATION TO
PSEUDOMONAS AERUGINOSA BIOFILM TOLERANCE TO
CIPROFLOXACIN AND TOBRAMYCIN.................................................22

Abstract.........................................................................................................22
Introduction...................................................................................................23
Materials and Methods.........................................................................24
Strains Media and Antibiotics..............................................................24
Biofilm Preparation..............................................................................25
Biofilm Growth Patterns......................................................................26
Oxygen Concentrations in Biofilms.....................................................26
Biofilms Susceptibility.........................................................................26
Planktonic Susceptibility.....................................................................27
Results...........................................................................................................29
Reduced Antibiotic Susceptibility of Biofilm Bacteria.......................29
Chemical and Physiological Heterogeneity in Biofilms......................29
Test of the Importance of Oxygen Availability for
Antibiotic Susceptibility......................................................................34
Tests of Importance of Glucose Availability for
Antibiotic Susceptibility......................................................................39
Discussion.....................................................................................................40
Acknowledgements.......................................................................................44
References.....................................................................................................45
vi

TABLE OF CONTENTS CONTINUED

3. DORMANT CELLS WITHIN PSEUDOMONAS AERUGINOSA
BIOILFMS ARE PROTECTED FROM KILLING BY ANTIBIOTIC
TREATMENT...........................................................................................47

Abstract......................................................................................................47
Introduction................................................................................................49
Materials and Methods...............................................................................51
Bacterial Strains, Media, and Antibiotics............................................51
Biofilm Preparation..............................................................................51
Visualization of GFP Patterns in Biofilms...........................................52
Flow Cytometry...................................................................................53
Antibiotic Treatment of Shifted Populations.......................................54
Results and Discussion..............................................................................55
Conclusion.................................................................................................70
References..................................................................................................73

4. CONCLUSIONS........................................................................................75

Implications................................................................................................77
Future Work...............................................................................................78

APPENDICES.......................................................................................................81

APPENDIX A: STAIN UPTAKE OF PSEUDOMONAS
AERUGINOSA BIOFILMS.......................................................................82

Introduction................................................................................................83
Materials and Methods...............................................................................85
Bacterial Strain and Media...................................................................85
Staining................................................................................................85
Visualization of Biofilms and Image Analysis....................................86
Results and Discussion..............................................................................87
Conclusion...............................................................................................103
References................................................................................................105
vii

TABLE OF CONTENTS CONTINUED

APPENDIX B: MATHMATICAL MODEL OF OXYGEN
DIFFUSION INTO PSEUDOMONAS AERUGINOSA DRIP FLOW
BIOFILM.................................................................................................106

Introduction..............................................................................................107
Discussion................................................................................................108
Conclusion...............................................................................................126
References................................................................................................128
viii

LIST OF TABLES

Table Page

2.1. Biofilm thickness and dimension of zone in which GFP was
expressed..............................................................................................33

2.2. Antibiotic susceptibility of P. aeruginosa under various conditions
of nutrient and oxygen availability......................................................35

2.3. Summary of the effects of oxygen limitation and growth rate to
the protection of biofilm killing with tobramycin and ciprofloxacin..36

2.4. Summary of tests of the contribution of glucose limitation to
antibiotic killing in P. aeruginosa biofilms.........................................39

3.1 Differential antibiotic susceptibility of putative dormant and active
P. aeruginosa in colony and drip-flow biofilms in downshift
experiments..........................................................................................68

3.2 Differential antibiotic susceptibility of putative dormant and active
P. aeruginosa in colony and biofilms in upshift experiments.............69
ix

LIST OF FIGURES

Figure Page

2.1 Oxygen concentrations in Pseudomonas aeruginosa biofilms.
Panel A shows a representative oxygen concentration profile with
depth in the biofilm. Zero on the x-axis corresponds to the bulk
fluid-air interface. Negative positions are located in the headspace
above the biofilm and positive positions are located inside the fluid
film and biomass. Panel B shows the coupling between oxygen
and glucose utilization. The oxygen microelectrode was
positioned at a location within the biofilm where the oxygen
concentration was low. The medium flowing over the biofilm was
switched between one containing glucose and ammonium ion
(C,N) and a medium lacking these constituents (no C,N) as
indicated by the arrows. Data for this figure generated by Frank
Roe and Betsey Pitts............................................................................31

2.2 Antibiotic susceptibility of P. aeruginosa under various conditions
of nutrient and oxygen availability. All tests were performed on
planktonic cells, except for the intact biofilm result included for
comparison. The uncertainty indicated is the standard error of the
mean. Negative log reductions reported for the untreated control
result from cell growth during the test period......................................33

2.3 Antibiotic killing of P. aeruginosa in intact biofilms as a function
of the oxygen concentration in the bulk fluid after the medium has
exited the reactor. Data are for 12 h of ciprofloxacin (") or
tobramycin (!) treatment. In all cases the biofilm was grown in
the presence of air for 72 h prior to antibiotic treatment. Only
during the antibiotic treatment phase was the oxygen composition
in the gas phase changed. Error bars represent standard deviations
of at least 3 replicates...........................................................................38

3.1 Dynamics of induction of GFP expression in P. aeruginosa cells in
planktonic exponential phase (●), planktonic stationary phase (■),
and intact colony biofilms (▲). Arabinose was added to the
system at time zero. Vertical axis represents total percent of
overall events that register as bright by flow cytometric analysis.
Error bars represent standard deviations of at least 3 experiments......57