Characterization of Airborne Bacteria at a Subway Station

189 Pages·2014·8.62 MB·English

by  Dybwad, Marius

Marius Dybwad

Characterization of Airborne Bacteria at a Subway
Station: Implications for Testing and Evaluation of
Biological Detection, Identification, and Monitoring
Systems

Thesis for the degree of Philosophiae Doctor (PhD)

Norwegian University of Science and Technology
Faculty of Natural Sciences and Technology
Department of Biotechnology

Norwegian Defence Research Establishment
Protection and Societal Security Division

Oslo 2014

Acknowledgements

This PhD study was financed by and performed at the Norwegian Defence Research
Establishment (FFI) in collaboration with the Department of Biotechnology at the
Norwegian University of Science and Technology (NTNU). The study was
supervised by Dr. Janet Martha Blatny (FFI) and Prof. Svein Valla (NTNU), and
co-supervised by Prof. Per Einar Granum at the Norwegian School of Veterinary
Sciences.
This study involved laboratory work at FFI and field work at the Nationaltheatret
subway station in Oslo, as well as nearly six months of laboratory work at NTNU in
collaboration with Ass. Prof. Per Bruheim at the Department of Biotechnology. The
study also consisted of an eight-month stay at the Netherlands Organization for
Applied Scientific Research (TNO) in Rijswijk, The Netherlands, involving
biosafety level-3 laboratory work and scientific collaboration with Dr. Armand
Paauw at TNO.
I would like to thank my supervisors, co-authors and everyone else who have
contributed to the success of this study. My dear friends and colleagues at FFI
deserve a special thank, and especially those in the microbiology group spearheaded
by my inspiring, demanding and knowledgeable supervisor Janet. I have also had
the privilege of supervising several MSc students: Anja Valen, Maria Hedenstad,
and Hanne Grydeland, all of whom have contributed greatly to inspire, challenge
and help me throughout the study. Last but not least I must thank those closest to
me, my beloved parents for their unconditional support and for providing me with a
solid foundation to start from, my beautiful girlfriend for being there through a
rollercoaster ride of ups and downs, and several others that have taken part in
shaping me into the man and scientist I am today.
I started at FFI in April 2009, completely inexperienced in the field of aerobiology
and bioaerosol research, but extremely motivated to dive deep and follow through.
Now, almost five years later I hand in my thesis. These past years have opened my
eyes to previously unappreciated aspects of both my scientific and personal life, and
hopefully awarded me with the tools needed to succeed in both.
This is hopefully just the beginning. Many pages are still left blank. The sky‟s the
limit.

Marius Dybwad, Kjeller, December 2013
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“The difficult is what takes a little time. The impossible is what takes a little longer.”
~ Fridtjof Nansen

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Abstract

Biological detection, identification, and monitoring (BioDIM) systems that are able to provide
rapid and reliable early-warning in the event of a bioterrorism attack may contribute to reduce
the impact of such incidents. Currently, few if any available BioDIM systems have been able
to meet all the users‟ requirements with respect to reliable, sensitive, and selective detect-to-
warn capabilities in different operational environments. BioDIM efforts at most real life sites
must be accomplished against a naturally occurring biological aerosol (bioaerosol)
background. The bioaerosol background may be both complex and variable, and could
challenge the operational performance of BioDIM systems, potentially resulting in the
triggering of false alarms, or even worse, the failure to respond to a real incident. One way to
improve the operational performance of BioDIM systems is to increase our understanding of
relevant bioaerosol backgrounds. Subway stations are enclosed and crowded public
environments which may be regarded as potential bioterrorism targets, and therefore also as a
relevant operating environment for BioDIM systems.
In order to improve our understanding of the bioaerosol background at subway stations, and
especially how it may challenge the operational performance of BioDIM systems, the airborne
bacterial background at the Nationaltheatret subway station in Oslo, Norway, was
characterized in this study. Information about the concentration level, diversity, size
distribution, and temporal variability of the airborne bacterial background was obtained. In
addition various virulence- and survival-associated airborne bacterial characteristics such as
hemolytic activity, antibiotic resistance, pigmentation, and spore fraction were investigated.
The obtained bioaerosol background characteristics were consolidated with similar and
different types of existing characteristics from other subway stations, and used to define a set
of realistic subway station background characteristics. Such background characteristics may
be valuable when applied in a BioDIM context for several reasons. The information may be
used to improve the operational performance of BioDIM systems (e.g. by optimizing alarm
algorithms), but also to develop more-realistic methods for testing and evaluation (T&E) of
BioDIM systems that take into account the real life background. The defined background
characteristics may be used to guide the construction of realistic synthetic subway station
bioaerosol backgrounds that can be recreated together with a biological threat agent aerosol
challenge during simulated operational T&E of BioDIM systems in aerosol chambers. While
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the airborne bacterial background information mainly was intended for use in a BioDIM
context in this study, it may also be relevant when viewed in the context of public and
occupational health as well as microbial ecology.
An important part of this study also involved testing and implementation of sampling and
analysis methods for airborne bacteria. Based on the recognized need for air samplers with
well-defined performance criteria, comparative T&E of air samplers was performed in an
aerosol chamber to establish their physical and biological sampling efficiencies. The obtained
results revealed significant differences between the samplers, which were used to assess their
suitability for various bioaerosol sampling applications.
A matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF
MS) bacterial identification method (MALDI Biotyper) was evaluated and implemented as a
rapid and cost-effective screening tool for airborne bacterial isolate collections. The
identification results obtained with the MALDI Biotyper were shown to correspond well with
16S rRNA gene sequencing-based results. However, the MALDI Biotyper failed to obtain
reliable identifications when the reference database did not contain library entries at the
corresponding species or genus level, and it was suggested that the coverage of environmental
airborne bacterial taxa in the reference database should be increased.
Another BioDIM-relevant topic addressed as part of this study was rapid identification
methods for Bacillus anthracis spores in suspicious powders (e.g. letters). A MALDI-TOF
MS-based identification method for B. anthracis spores in powders was developed and
validated. The observed performance of the analysis method demonstrated its potential
applicability as a rapid, specific, sensitive, robust, and cost-effective analysis tool for
resolving incidents involving suspicious powders in less than 30 min.
The work presented in this thesis contributes to a deeper understanding of the bioaerosol
background, especially at subway stations. It similarly highlights the potential importance of
the bioaerosol background in a BioDIM context, and emphasizes the need for increased
research efforts to close existing knowledge gaps. This thesis may also serve to highlight the
research efforts that will be needed before real life bioaerosol background information may be
fully exploited in a BioDIM context.

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Abbreviations

ATOFMS Aerosol time-of-flight mass spectrometry
BioDIM Biological DIM
BSE Biological sampling efficiency
BTA Biological threat agent
CAB Cultivable airborne bacteria
CBRN Chemical, biological, radiological, and nuclear
DIM Detection, identification, and monitoring
FCM Flow cytometry
FFI Norwegian Defence Research Establishment
FISH Fluorescence in situ hybridization
FM (Epi-)Fluorescence microscopy
MALDI Matrix-assisted laser desorption ionization
NTNU Norwegian University of Science and Technology
PhD Philosophiae Doctor
PM10 Particulate matter <10 μm
PM2.5 Particulate matter <2.5 μm
PSE Physical sampling efficiency
qPCR Quantitative polymerase chain reaction
R&D Research and development
RH Relative humidity
ROC Receiver operating characteristic
SASP Small acid-soluble protein
T&E Testing and evaluation
TAB Total airborne bacteria
TNO Netherlands Organization for Applied Scientific Research
TSP Total suspended particulates
UV-LIF Ultraviolet laser-induced fluorescence
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List of publications

This thesis is based on the following peer-reviewed journal publications:

Paper I
Dybwad, M, PE Granum, P Bruheim, and JM Blatny. 2012. Characterization of Airborne
Bacteria at an Underground Subway Station. Applied and Environmental
Microbiology 78(6):1917-1929.

Paper II
Dybwad, M, G Skogan, and JM Blatny. 2014. Temporal Variability of the Bioaerosol
Background at a Subway Station: Concentration Level, Size Distribution and Diversity of
Airborne Bacteria. Applied and Environmental Microbiology 80(1):257-270.

Paper III
Dybwad, M, G Skogan, and JM Blatny. 2014. Comparative Testing and Evaluation of Nine
Different Air Samplers: End-to-End Sampling Efficiencies as Specific Performance
Measurements for Bioaerosol Applications. Aerosol Science and Technology 48(3):281–294.

Paper IV
Dybwad, M, AL van der Laaken, JM Blatny, and A Paauw. 2013. Rapid Identification of
Bacillus anthracis Spores in Suspicious Powder Samples by Using Matrix-Assisted Laser
Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS). Applied and
Environmental Microbiology 79(17):5372-5383.

The following peer-reviewed journal publications were contributed to during the PhD study,
but not included in the thesis proper:

Paper V
Stafsnes, MH, M Dybwad, A Brunsvik, and P Bruheim. 2013. Large scale MALDI-TOF MS
based taxa identification to identify novel pigment producers in a marine bacterial culture
collection. Antonie van Leeuwenhoek 103(3):603-615.

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