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 i “The difficult is what takes a little time. The impossible is what takes a little longer.” ~ Fridtjof Nansen ii 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 iii 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. iv 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 v vi 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. vii viii