DETECTION OF AEROBIC BACTERIAL ENDOSPORES: FROM AIR

449 Pages·2008·15.05 MB·English

by  Douglas Yung

DETECTION OF AEROBIC BACTERIAL ENDOSPORES:
FROM AIR SAMPLING, STERILIZATION VALIDATION TO ASTROBIOLOGY

Thesis by

Pun To (Douglas) Yung

In Partial Fulfillment of the Requirements

for the Degree of

Doctor of Philosophy

California Institute of Technology

Pasadena, California

2008

(Defended May 9, 2008)
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© 2008

Pun To (Douglas) Yung

All Rights Reserved
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Acknowledgment
I would like to express my deepest gratitude to my thesis adviser, Dr. Adrian Ponce,
for his guidance and support. It is due to his scholastic guidance and encouragement that
ultimately made this work possible. I appreciate the time and attention of my committee
members: Professor Morteza Gharib, Professor Scott Fraser, and Professor Changhuei
Yang. Given their busy schedules, it has been kind of them to play a role in my course of
study.
I owe my gratitude to all members of the Ponce Group who have made this
dissertation possible, and because of them my graduate experience has been one that I
will cherish forever. Elizabeth Lester has kindly introduced me to techniques in
microbiology. Alan Fung has taught me principles in mechanical engineering and
instrument design. I have enjoyed working with Elizabeth Lester, Alan Fung, Morgan
Cable, Hannah Shafaat and Wanwan Yang. I would like express thanks to my summer
undergraduate mentees, Wilson Sung, Christine Tarleton, and William Fan, who have
contributed important pieces to my thesis work. I would like to acknowledge the other
individuals who have contributed to the completion of this thesis: Dr. James Kirby, Dr.
Xenia Amashukeli, Dr. Christine Pelletier, Raymond Lam, Dr. Michael Kempf, Michael
Lee, Dr. Fei Chen, Dr. Anita Fisher, Dr. Steve Monacos, Dr. Stephanie Connon, and Dr.
Donald Obenhuber.
I appreciate the support from the many reliable staff members working at JPL and
Caltech, including Stacey Klinger, Robert Downer, Icy Ma, Steve Gould, Lillian Kremar,
Joe Drew, Cora Carriedo, and Carlos Hernandez. Last but not least, I have to thank Linda
Scott for being the most efficient professional in bioengineering. I would like to thank
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Ray Pini, Amir Ettehdieh and Universal Detection Technology for helpful discussions. I
thank Fei Chen for her help in a preliminary vaporized hydrogen peroxide experiment
and access to the spacecraft assembly clean room. I thank Steris Corporation for
preparing vaporized hydrogen peroxide treated-endospore strips.
I am also indebted to my friends and family for their continuous support, love and
patience. Particularly, I would like to express my heartfelt gratitude to Jerry Ruiz and
Karen Chan. I warmly appreciate the encouragement from Dr. Stephen Shum and Dr.
James Tong from high school, college, to graduate school.
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“The most beautiful and most profound emotion we can experience is the sensation
of the mystical. It is the source of all true science. He to whom the emotion is a
stranger, who can no longer wonder and stand rapt in awe, is as good as dead.”
Albert Einstein
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Abstract
Bacterial endospores are formed in genera such as Bacillus and Clostridium in times
of incipient stresses. Derivative of their remarkable resistance and ubiquity, endospores
are delivery vehicles for anthrax attack, biological indicators for checking sterilization
efficacy, and candidates for Panspermia and potential extraterrestrial life, thereby
underscoring the significance of their rapid detection. In this thesis project, spectroscopy
and microscopy methods are studied to measure the release of a unique constituent,
dipicolinic acid (DPA), via germination as a proxy for endospore viability. In particular, a
luminescence time-gated microscopy technique (called microscopy endospore viability
assay, acronym: µEVA) has been developed to enumerate germination-capable aerobic
endospores rapidly based on energy transfer from DPA to terbium ions doped on a solid
matrix upon UV excitation. The distinctive emission and millisecond lifetime enable
time-resolved imaging to achieve a sensitivity of one endospore.
Effective air sampling of endospores is crucial in view of the potential catastrophe
caused by the dissemination of airborne anthrax endospores. Based on time-gated
spectroscopy of terbium-DPA luminescence, the Anthrax Smoke Detector has been built
to provide real-time surveillance of air quality for timely mitigation and decontamination.
This technology also finds application in the monitoring of airborne endospore bioburden
as an indicator of total biomass in a closed spacecraft system in order to safeguard the
health of astronauts.
Sterilization validation is of prime concern in the medical field and planetary
protection to prevent cross-contaminations among patients and planets. µEVA has yielded
faster and comparable results compared with the culture-based NASA standard assay in
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assessing surface endospore bioburden on spacecraft materials and clean rooms surfaces.
The current analysis time has been expedited from 3 days to within an hour in
compliance with planetary protection requirements imposed on landers and probes
designed for life detection missions.
From the perspective of astrobiology, endospores are time capsules preserving
geological history and may exist as dormant lives in analogous extraterrestrial
environments. µEVA has successfully recovered ancient endospores in cold biospheres
(Greenland ice core, Antarctic Lake Vida, polar permafrost) and hyper-arid biospheres
(Atacama Desert) on Earth as templates for determining life longevity and the search of
extinct or extant life on Mars and other icy celestial bodies. Result authenticity has been
validated by a comprehensive suite of experiments encompassing culture-based and
culture-independent techniques such as epifluorescence microscopy, flow cytometry,
fluorometry, bioluminescence and 16s rRNA analysis. In conclusion, µEVA is a sensitive
analytical tool that opens a new realm in microbiology to provide insights into air
sampling, sterility assessment and exobiology.
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Table of contents
ACKNOWLEDGMENT………………………………………………………………………………………iii 
ABSTRACT ..................................................................................................................................................vi
TABLE OF CONTENTS ............................................................................................................................... viii
LIST OF FIGURES ........................................................................................................................................ xv
LIST OF TABLES ...................................................................................................................................... xviii
CHAPTER 1: INTRODUCTION ....................................................................................... 1
1.1 INTRODUCTION ...................................................................................................................................... 1
1.2 LIFE CYCLE OF AN SPORE-FORMING BACTERIA ....................................................................................... 5
1.3 DPA-TRIGGERED TERBIUM PHOTOLUMINESCENCE ASSAY ...................................................................... 7
1.4 OUTLINE OF THESIS .............................................................................................................................. 10
1.5 REFERENCES ........................................................................................................................................ 16
CHAPTER 2: REVIEW OF ENDOSPORE DETECTION TECHNOLOGY ................. 21
2.1 ABSTRACT ........................................................................................................................................... 21
2.2 INTRODUCTION .................................................................................................................................... 21
2.3 BIOMARKER METHODS ......................................................................................................................... 24
2.3.1 Calcium dipicolinate ................................................................................................................... 25
2.3.2 Dipicolinic acid ........................................................................................................................... 27
2.3.3 DNA ............................................................................................................................................. 31
2.4 OPTICAL PROPERTY AND STAINABILITY ............................................................................................... 34
2.4.1 Direct epifluorescent filtration technique .................................................................................... 34
2.4.2 Coulter counter ........................................................................................................................... 35
2.4.3 Method of Schaeffer and Fulton .................................................................................................. 35
2.4.4 Phase contrast microscopy .......................................................................................................... 36
2.4.5 Electron microscopy .................................................................................................................... 38
2.4.6 Flow cytometry ............................................................................................................................ 38
2.5 METABOLISM ....................................................................................................................................... 39
2.5.1 Impedance measurement ............................................................................................................. 39
2.5.2 Microcalorimetry ......................................................................................................................... 40
2.5.3 ATP firefly luciferin-luciferase assay .......................................................................................... 40
2.6 BIOCHEMICAL TESTS ............................................................................................................................ 44
2.7 COLONY FORMATION ........................................................................................................................... 46
2.7.1 Culturing (plate count and most probable number) .................................................................... 47
2.7.2 Turbidity measurement ................................................................................................................ 47
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2.8 SPORE COAT ......................................................................................................................................... 48
2.8.1 Immunoassay + Flow cytometry ................................................................................................. 49
2.8.2 Immunofluorescence resonance energy transfer ......................................................................... 49
2.9 SPORE DETECTION INSTRUMENTS ........................................................................................................ 50
2.10 CONCLUSION ..................................................................................................................................... 54
2.11 REFERENCES ...................................................................................................................................... 55
CHAPTER 3: AN AUTOMATED FRONT-END MONITOR FOR ANTHRAX
SURVEILLANCE SYSTEMS BASED ON THE RAPID DETECTION OF AIRBORNE
ENDOSPORES ................................................................................................................. 61
3.1 ABSTRACT ........................................................................................................................................... 61
3.2 INTRODUCTION .................................................................................................................................... 62
3.3 MATERIALS AND METHODS ................................................................................................................. 64
3.3.1 Chemicals .................................................................................................................................... 64
3.3.2 Biological samples ...................................................................................................................... 65
3.3.3 Anthrax Smoke Detector .............................................................................................................. 65
3.3.4 Spectrometry ................................................................................................................................ 66
3.3.5 Simulated anthrax attack ............................................................................................................. 67
3.4 RESULTS .............................................................................................................................................. 68
3.4.1 Spectrometer time-gated performance ........................................................................................ 69
3.4.2 Quantification of DPA and spores in water ................................................................................. 69
3.4.3 ASD response to simulated anthrax attack .................................................................................. 70
3.5 DISCUSSION ......................................................................................................................................... 71
3.6 CONCLUSION ....................................................................................................................................... 73
3.7 REFERENCES ........................................................................................................................................ 74
CHAPTER 4: AIRBORNE ENDOSPORE BIOBURDEN AS AN INDICATOR OF
SPACECRAFT CLEANLINESS ...................................................................................... 82
4.1 ABSTRACT ........................................................................................................................................... 82
4.2 INTRODUCTION .................................................................................................................................... 82
4.3 METHODS AND PROCEDURE ................................................................................................................. 85
4.3.1 Chemicals .................................................................................................................................... 85
4.3.2 Microbiological samples ............................................................................................................. 85
4.3.3Microbial Event Monitor .............................................................................................................. 86
4.3.4 Air Sample Collection ................................................................................................................. 87
4.3.5 Determination of total biomass ................................................................................................... 88
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4.3.6 Correlating airborne and total biomass in a laboratory controlled environment ....................... 90
4.3.7 Correlating airborne and total biomass in indoor and outdoor environemnts ............................ 91
4.3.8 Comparison of biofilm-forming environmental isolate with lab-strain B. subtilis ...................... 92
4.3.9 Data Analysis .............................................................................................................................. 93
4.4 RESULTS .............................................................................................................................................. 94
4.4.1 Comparison test of 3 different air samplers ................................................................................ 94
4.4.2 Aerosolized biofilm endospore testing in the laboratory ............................................................. 94
4.4.3 Correlation of airborne and surface endospores in a closed laboratory environment ................ 95
4.4.4 Correlation of AEB and total biomass in indoor environments ................................................... 96
4.4.5 Correlation of AEB and total biomass in outdoor environments ................................................. 97
4.4.6 Comparison of biofilm-forming environmental-strain B. subtilis and lab-strain B. subtilis
endospores ............................................................................................................................................ 98
4.5 DISCUSSION ......................................................................................................................................... 98
4.6 CONCLUSION ..................................................................................................................................... 102
4.7 REFERENCES ...................................................................................................................................... 103
CHAPTER 5: RAPID STERILIZATION ASSESSMENT BY MONITORING
INACTIVATION OF GERMINABLE BACILLUS ENDOSPORES ............................. 114
5.1 ABSTRACT ......................................................................................................................................... 114
5.2 INTRODUCTION .................................................................................................................................. 114
5.3 METHODS .......................................................................................................................................... 117
5.3.1 Chemicals .................................................................................................................................. 117
5.3.2 Preparation of endospore stock suspension .............................................................................. 117
5.3.3 Sample Preparation for µEVA experiments ............................................................................... 118
5.3.4 The µEVA instrument................................................................................................................. 119
5.3.5 Endospore germination and germinable endospore assignment ............................................... 119
5.3.6 Phase contrast microscopy for measuring total endospore concentration ................................ 120
5.3.7 Inactivation experiments ........................................................................................................... 120
5.3.8 Statistical analysis ..................................................................................................................... 121
5.3.9 Spectroscopy .............................................................................................................................. 122
5.4 RESULTS ............................................................................................................................................ 123
5.4.1 Monitoring single endospore germination dynamics ................................................................ 123
5.4.2 Sensitivity, dynamic range, and false positive rate .................................................................... 124
5.4.3 Monitoring thermal and UV sterilization of Bacillus atrophaeus endospores .......................... 125
5.5 DISCUSSION ....................................................................................................................................... 126
5.6 REFERENCES ...................................................................................................................................... 129