MICROBIAL FORENSICS THIRD EDITION Edited by Bruce Budowle Steven Schutzer Stephen Morse AcademicPressisanimprintofElsevier 125LondonWall,LondonEC2Y5AS,UnitedKingdom 525BStreet,Suite1650,SanDiego,CA92101,UnitedStates 50HampshireStreet,5thFloor,Cambridge,MA02139,UnitedStates TheBoulevard,LangfordLane,Kidlington,OxfordOX51GB,UnitedKingdom Copyright©2020ElsevierInc.Allrightsreserved. Nopartofthispublicationmaybereproducedortransmittedinanyformorbyanymeans,electronicormechanical,including photocopying,recording,oranyinformationstorageandretrievalsystem,withoutpermissioninwritingfromthepublisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements withorganizationssuchastheCopyrightClearanceCenterandtheCopyrightLicensingAgency,canbefoundatourwebsite: www.elsevier.com/permissions. ThisbookandtheindividualcontributionscontainedinitareprotectedundercopyrightbythePublisher(otherthanasmaybe notedherein). Notices Knowledgeandbestpracticeinthisfieldareconstantlychanging.Asnewresearchandexperiencebroadenourunderstanding, changesinresearchmethods,professionalpractices,ormedicaltreatmentmaybecomenecessary. Practitionersandresearchersmustalwaysrelyontheirownexperienceandknowledgeinevaluatingandusinganyinformation, methods,compounds,orexperimentsdescribedherein.Inusingsuchinformationormethodstheyshouldbemindfuloftheir ownsafetyandthesafetyofothers,includingpartiesforwhomtheyhaveaprofessionalresponsibility. Tothefullestextentofthelaw,neitherthePublishernortheauthors,contributors,oreditors,assumeanyliabilityforanyinjury and/ordamagetopersonsorpropertyasamatterofproductsliability,negligenceorotherwise,orfromanyuseoroperationof anymethods,products,instructions,orideascontainedinthematerialherein. LibraryofCongressCataloging-in-PublicationData AcatalogrecordforthisbookisavailablefromtheLibraryofCongress BritishLibraryCataloguing-in-PublicationData AcataloguerecordforthisbookisavailablefromtheBritishLibrary ISBN:978-0-12-815379-6 ForinformationonallAcademicPresspublicationsvisitour websiteathttps://www.elsevier.com/books-and-journals Publisher:StacyMasucci AcquisitionEditor:ElizabethBrown EditorialProjectManager:PatGonzalez ProductionProjectManager:SuryaNarayananJayachandran CoverDesigner:MarkRogers TypesetbyTNQTechnologies Contributors M.W. Allard Division of Microbiology, Office of Robert L. Bull FBI Laboratory, Quantico, VA, Regulatory Science, CFSAN, US FDA, College UnitedStates Park,MD,UnitedStates James Burans National Bioforensic and Analysis JonathanAllen LawrenceLivermore NationalLab- Center,Frederick,MD,UnitedStates oratory,Livermore,CA,UnitedStates David O. Carter Laboratory of Forensic Philip S. Amara College of Public Health, Univer- Taphonomy, Forensic Sciences Unit, Division of sity of Nebraska Medical Center, Omaha, NE, Natural Sciences and Mathematics, Chaminade UnitedStates University of Honolulu, Honolulu, HI, United Neel G. Barnaby FBI Laboratory, Quantico, VA, States UnitedStates Angela Choi Department of Microbiology, Icahn John R. Barr Centers for Disease Control and Pre- School of Medicine at Mount Sinai, New York, NY,UnitedStates vention,NationalCenterforEnvironmentalHealth, Division of Laboratory Sciences, Atlanta, GA, Brooke L. Deatherage Kaiser Pacific Northwest UnitedStates NationalLaboratory,Richland,WA,UnitedStates Aeriel Belk Animal Sciences, Colorado State Uni- R.ScottDecker Bio-LogicSecurity,LLC,LasVegas, versity,FortCollins,CO,UnitedStates NV,UnitedStates Nicholas H. Bergman National Bioforensic and HeatherDeel AnimalSciences,ColoradoStateUni- AnalysisCenter,Frederick,MD,UnitedStates versity,FortCollins,CO,UnitedStates Christopher A. Bidwell Federation of American David M. Engelthaler TGen North, Translational Scientists,Washington,DC,UnitedStates GenomicsResearchInstitute,Flagstaff,AZ,United DawnBirdsell ThePathogenandMicrobiomeInsti- States tute, Northern Arizona University, Flagstaff, AZ, Hector F. Espitia-Navarro School of Biological Sci- UnitedStates ences, Georgia Institute of Technology, Atlanta, Thomas A. Blake Centers for Disease Control and GA, United States; PanAmerican Bioinformatics Institute,Cali,ValledelCauca,Colombia Prevention, National Center for Environmental Health, Division of Laboratory Sciences, Atlanta, Jacqueline Fletcher Department of Entomology & GA,UnitedStates Plant Pathology, National Institute for Microbial E.W.Brown DivisionofMicrobiology,OfficeofReg- Forensics & Food and Agricultural Biosecurity, OklahomaStateUniversity,Stillwater,OK,United ulatory Science, CFSAN, US FDA, College Park, MD,UnitedStates States Sibyl Bucheli Department of Biological Sciences, Jeffrey T. Foster The Pathogen and Microbiome Institute, Northern Arizona University, Flagstaff, Sam Houston State University, Huntsville, TX, AZ,UnitedStates UnitedStates Bruce Budowle Center for Human Identification, Adolfo García-Sastre Department of Microbiology, Department of Microbiology, Immunology and Icahn School of Medicine at Mount Sinai, New York,NY,UnitedStates; DepartmentofMedicine, Genetics,UniversityofNorthTexasHealthScience Center,FortWorth,TX,UnitedStates IcahnSchoolofMedicineatMountSinai,NewYork, xi xii Contributors NY, United States; Global Health and Emerging Rob Knight Department of Pediatrics, Department Pathogens Institute, Icahn School of Medicine at ofComputerScienceandEngineering,Microbiome Mount Sinai, New York, NY, United States; The Innovation Center, University of California, San Tisch Cancer Institute, Icahn School of Medicine at Diego,CA,UnitedStates MountSinai,NewYork,NY,UnitedStates Asja Korajkic U.S. Environmental Protection Jennifer S. Goodrich National Bioforensic and Agency, Office of Research and Development, AnalysisCenter,Frederick,MD,UnitedStates Cincinnati,OH,UnitedStates BradleyHart LawrenceLivermoreNationalLabora- StevenB.Lee InternationalForensicResearchInsti- tory,Livermore,CA,UnitedStates tute, Florida International University, Miami, FL, DavidR.Hodge DepartmentofHomelandSecurity, UnitedStates; ForensicScienceProgram,Depart- ment of Justice Studies, San Jose State University, Washington,DC,UnitedStates SanJose,CA,UnitedStates PaulJ.Jackson AdjunctProfessor,Nonproliferation and Terrorism Studies, James Martin Center for Anastasia P. Litvintseva Mycotic Diseases Branch, Centers for Disease Control and Prevention, Nonproliferation Studies, Middlebury Institute of Atlanta,GA,UnitedStates International Studies at Monterey, Monterey, Cali- fornia,UnitedStates Douglas G. Luster USDA ARS, Foreign Disease - CrystalJaing LawrenceLivermoreNationalLabora- Weed Science Research Unit, Fort Detrick, MD, UnitedStates tory,Livermore,CA,UnitedStates Rudolph C. Johnson Centers for Disease Control Aaron Lynne Department of Biological Sciences, Sam Houston State University, Huntsville, TX, andPrevention,NationalCenterforEnvironmental UnitedStates Health, Division of Laboratory Sciences, Atlanta, GA,UnitedStates Leonard W. Mayer PanAmerican Bioinformatics I.KingJordan Schoolof BiologicalSciences,Geor- Institute,Cali,ValledelCauca,Colombia; Applied Bioinformatics Laboratory, Atlanta, GA, United gia Institute of Technology, Atlanta, GA, United States States; PanAmerican Bioinformatics Institute, Cali,ValledelCauca,Colombia; AppliedBioin- UlrichMelcher OklahomaStateUniversity,Depart- formaticsLaboratory,Atlanta,GA,UnitedStates ment of Biochemistry & Molecular Biology, Still- Suzanne R. Kalb Centers for Disease Control and water,OK,UnitedStates Prevention, National Center for Environmental EricD.Merkley PacificNorthwestNationalLabora- Health, Division of Laboratory Sciences, Atlanta, tory,Richland,WA,UnitedStates GA,UnitedStates Jessica L. Metcalf Animal Sciences, Colorado State Arnold F. Kaufmann United States Public Health University,FortCollins,CO,UnitedStates Service,StoneMountain,GA,UnitedStates DeEtta K. Mills International Forensic Research PaulKeim ThePathogenandMicrobiomeInstitute, Institute, Florida International University, Miami, NorthernArizonaUniversity,Flagstaff,AZ,United FL,UnitedStates States StephenA.Morse IHRC,Inc.,Atlanta,GA,United TerryL.Kerns Bio-LogicSecurity,LLC,LasVegas, States NV,UnitedStates Ted D. Mullins American Type Culture Collection AliS.Khan CollegeofPublicHealth,Universityof (ATCC),Manassas,VA,UnitedStates Nebraska Medical Center, Omaha, NE, United Randall Murch Virginia Polytechnic Institute and States StateUniversity,Arlington,VA,UnitedStates Rachel E. Kieser Center for Human Identification, ForrestW.Nutter,Jr. IowaStateUniversity,Depart- GraduateSchoolofBiomedicalSciences,University ment of Plant Pathology and Microbiology, Ames, ofNorthTexasHealthScienceCenter,FortWorth, IA,UnitedStates TX,UnitedStates xiii Contributors Francisco M. Ochoa Corona Department of Ento- Orin C. Shanks U.S. Environmental Protection mology & Plant Pathology, National Institute for Agency, Office of Research and Development, Microbial Forensics & Food and Agricultural Bio- Cincinnati,OH,UnitedStates security, Oklahoma State University, Stillwater, Frank P. Simione American Type Culture Collec- OK,UnitedStates tion(ATCC),Manassas,VA,UnitedStates Sam Ogden Animal Sciences, Colorado State Uni- Tom Slezak Lawrence Livermore National Labora- versity,FortCollins,CO,UnitedStates tory,Livermore,CA,UnitedStates RichardT.Okinaka ThePathogenandMicrobiome JeniferA.L.Smith DistrictofColumbiaDepartment Institute, Northern Arizona University, Flagstaff, ofForensicSciences,Washington,DC,UnitedStates AZ,UnitedStates Carla S. Thomas University of California, Plant Talima Pearson The Pathogen and Microbiome PathologyDepartment,Davis,CA,UnitedStates Institute, Northern Arizona University, Flagstaff, Stephan P. Velsko Lawrence Livermore National AZ,UnitedStates Laboratory,Livermore,CA,UnitedStates BernardR.Quigley IHRC,Inc.,Atlanta,GA,United RichardR.Vines AmericanTypeCultureCollection States (ATCC),Manassas,VA,UnitedStates Lavanya Rishishwar School of Biological Sciences, Amy Vogler The Pathogen and Microbiome Insti- Georgia Institute of Technology, Atlanta, GA, tute, Northern Arizona University, Flagstaff, AZ, UnitedStates; PanAmericanBioinformaticsInsti- UnitedStates tute, Cali, Valle del Cauca, Colombia; Applied Bioinformatics Laboratory, Atlanta, GA, United David M. Wagner The Pathogen and Microbiome States Institute, Northern Arizona University, Flagstaff, AZ,UnitedStates JasonW.Sahl ThePathogenandMicrobiomeInsti- tute, Northern Arizona University, Flagstaff, AZ, Karen L. Wahl Pacific Northwest National Labora- UnitedStates tory,Richland,WA,UnitedStates StephenA.Saltzburg WallaceandBeverleyUniver- Charles H. Williamson The Pathogen and Micro- sity Professor, The George Washington University biome Institute, Northern Arizona University, LawSchool,UnitedStates Flagstaff,AZ,UnitedStates Harald Scherm University of Georgia, Department Mark Wilson MRW Analytics, LLC, Fredericks- ofPlantPathology,Athens,GA,UnitedStates burg,VA,UnitedStates David G. Schmale, III Virginia Tech University, August Woerner Center for Human Identification, SchoolofPlantandEnvironmentalSciences,Blacks- University of North Texas Health Science Center, burg,VA,UnitedStates Fort Worth, TX, United States; Department of Microbiology, Immunology and Genetics, Univer- Sarah E. Schmedes Association of Public Health sity of North Texas Health Science Center, Fort Laboratories,SilverSpring,MD,UnitedStates Worth,TX,UnitedStates Steven E. Schutzer Department of Medicine, David S. Wunschel Pacific Northwest National Rutgers New Jersey Medical School, Newark, NJ, Laboratory,Richland,WA,UnitedStates UnitedStates Foreword Iamhonoredthattheauthorshaveaskedme Banburyalreadyhadheldaseriesofmeetings tocontributetheforewordtothethirdeditionof on what was still a “newly emerged” disease, Microbial Forensics. My connection to the book Lyme disease. The first meeting was held in and to the field in general was as a facilitator 1991, only nine years after Willy Burgdorfer rather than as a researcher, and it was in the had identified the previously unknown path- distant past, 20 years ago! ogen, the spirochete, Borrelia burgdorferi. I put MicrobialForensicsreceivedinputanddirection Suzanne in touch with the organizers of those inaseriesofmeetingsheldattheBanburyCenter, meetings, Steve Schutzer and Ben Luft, and the aconferencecenteratColdSpringHarborLabo- three organized the April 2000 meeting “Strate- ratory. Indeed, some meetings held at the Ban- giesforDetectionandIdentificationofUnknown bury, which predate the emergence of Microbial Pathogens.” Forensics as a formalized discipline, laid some The primary focus of this meeting was ofthegroundworkforthismust-neededforensic health, and the word “forensic” did not appear. field.Banburyhasaninternationalreputationfor However, one of the participants was Roger intensivediscussionmeetingswithfewerthan30 Breeze,thenwiththeUSDepartmentofAgricul- participants of topics in the life sciences that ture.Whilethetitleofthemeetingheorganized would benefit from critical review, topics that in October 2000 had the rather innocuous title are frequently controversial and often involve “The Challenge of Infectious Diseases in the issuesofpolicyaswellasscience. 21st Century,” his view of unrecognized infec- In 1999, Suzanne Vernon and Bill Reeves of tious disease was quite definitely forensic; the the Centers for Disease Control (CDC) invitation referred to the need for strategies to approached me about a meeting to be funded dealwithnewandemergingthreats“.ofwhich by CDC to discuss how it would be possible to biological terrorism is the latest.” detect and develop tests for unknown patho- Finally, in November 2002, Schutzer and gens. CDC was interested because it had been Breeze with Bruce Budowle (then with the FBI proposed that chronic fatigue syndrome (CFS) LaboratoryinQuantico)organizedthefirst“Mi- was an infectious disease, but the pathogen crobial Forensics” meeting, followed by addi- detection techniques used by CDCdserology, tional four meetings. The final meeting in 2010 culture, PCRdhad failed to identify an infec- wason“MicrobialForensicsintheEraofGeno- tious agent associated with CFS. By 1999, new mics,” which reviewed techniques for DNA techniques were coming online, and CDC sequencingandbioinformaticsthatwereunfath- wanted to review how these might be used to omable mere eightyearsearlier. findnovel orpreviouslyuncharacterizedpatho- Despitetheseandotherdramaticadvancesin gens associatedwith CFS. technology, it is not surprising that the same xvi Foreword broad issues remain, some of which are identi- it becameapowerful toolforthe justicesystem. fied in the authors’ introduction, for example, I am proud to recall that a Banbury Center thedifferingneedsoftheworldsofpublichealth meeting in 1988 (attended by Bruce Budowle) and forensics; the latter required meeting the contributed to cementing this technology as the legal standards for evidence that might be used gold standard of the forensicsciences. in the court. Finally, I fully endorse the authors’ closing The authors refer to the issue of whether mi- remarks.Scientificevidencecarriesgreatauthor- crobialforensicswillbecentralized,forexample, ity over and above its evidentiary value. This at the National Bioforensics Analysis Center, or responsibility requires that those employing become distributed. If the latter, adequate scientifictechniquesintheserviceofforensicsci- resourcesegraining and infrastructureewill be ence do so to the highest standards possible. It needed, but this may be hard to justify for took several years for DNA fingerprinting to what one hopes will berare events. reach that position, and it will take the same In some ways, I am reminded of the early commitment for microbial forensics to attain days of forensic DNA fingerprinting. The tech- the same level of credibility and acceptance. niques and associated population genetic ana- But without a doubt, it will be well worthwhile lyses had been developed in academic as this burgeoning discipline continues to fulfill laboratories, and there were many obstacles to its promise. be overcome before DNA fingerprinting was accepted. It required training in techniques and Jan AWitkowski PhD theory, as well as an acceptance by the public, Professor law enforcement, and defense attorneys, before Cold Spring Harbor Laboratory Introduction Stephen A. Morse1, Bruce Budowle2, Steven E. Schutzer3 1IHRC,Inc., Atlanta, GA, UnitedStates; 2Center forHuman Identification,University of North Texas Health Sciences Center, FortWorth, TX,UnitedStates; 3Department ofMedicine, RutgersNew Jersey Medical School, Newark,NJ, UnitedStates Deliberatedisseminationofabiologicalagent Microbialforensicinvestigationsandmolecu- viaseveraldifferentroutes,includingair,water, lar epidemiologic investigations share many food, infected vectors, and manmade avenues, aspects but are performed for different reasons. presents a challenge to global health and secu- An epidemiologic investigation aims to identify rity. The ability to identify the perpetrator(s) in the source of an outbreak and to understand an expeditious and effective manner is of para- routesoftransmissioninordertopreventfurther mount importance as both a deterrent and for spread and reduce the risk of future outbreaks gauging an appropriate response. Eliminating througheffectivepreventivemeasures.Amicro- those individuals who are falsely associated bialforensicinvestigationisundertakentoiden- withevidenceoracrimeisofequalimportance. tifytheperpetratorofacrimeandtogatherhigh The past decade has seen major advances in qualityevidencethatcanbeusedforindictment the scientific discipline of microbial forensics, and conviction of perpetrators or to help deci- whose core objective is attribution: the inves- sion makers prepare an appropriate response tigative process aimed at identifying the perpe- to an attack. A microbial forensic investigation trators of a biowarfare, bioterrorism, or hasbeendescribedasconsistingofthreeinterre- biocrime event and bringing them to justice. lated stages: identification of the biological This highly interdisciplinary field, which has agent(s) responsible for an event; characteriza- largely emerged through US initiatives (Tucker tion of the event as intentional, unintentional and Koblentz, 2009; U.S., 2010), encompasses (e.g.,duetonegligenceorcarelessness),ornatu- traditionallawenforcementandforensicinvesti- rally occurring; and, if the event is deemed gativemethodsaswellasthedetailedcharacter- illegitimate (i.e., intentional or unintentional), ization of samples containing biological agents. attribution of use to a specific perpetrator(s) Moreover, with the advent of high throughput (Koblentz and Tucker, 2010). Additionally, technologies, such as massively parallel analyses may be used to determine the pro- sequencing, microbial forensics as a discipline cess(es) used to produce a bioweapon, which has expanded beyond its primary focus on can provide additional forensic evidence and microbes and their products involved in bioter- insight into the sophistication of the production rorism and biocrimes to other applications such process. Regardless if an event was intentional asexploiting thehumanmicrobiomeforhuman or accidental, an immediate goal is to prevent identification, human geolocation, and deter- additional attacks and protect the public. mining post mortem interval. xviii Introduction Many of the questions asked during the first detection and identification and did not include two stages of a microbial forensic investigation detailed characterization and comparative ana- are identical to those of anepidemiologic inves- lyses. Thus,the first edition was developed as a tigation,andthesamemethodsandtechnologies foundational text to stimulate scientists, legal aregenerallyusedtoanswerthem(Sjodinetal., experts, and decision makers responsible for 2013).However,theattributionstepisuniqueto analyzing and interpreting evidence from an microbial forensics. In addition to traditional actofbiologicalwarfare,bioterrorism,biocrime, forensic analyses of recovered evidence from or inadvertent release of a microorganism or thecrimescene(analysisofhumanDNA,finger- toxinforattributionpurposesandtodescribethe prints, fibers, etc.), detailed analyses are con- discipline. In addition, the initial edition of this ducted of the responsible biologic agent book discussed some of the future opportunities (bacterium,virus,fungus,ortoxin).Itispossible and challenges for the microbial forensic field. thattheresultsofanepidemiologicinvestigation The secondedition of this book,published in maysuggestadeliberateorunintentionalrelease 2011,containedseveralchaptersthatbroughtthe of a biological agent and thus lead to the initia- Amerithrax investigation to a conclusion. The tion of a microbial forensic investigation. The anthrax mail investigation, one of the most intentional release of a biological agent can be intense and expansive investigations to date by done overtly, in which case the perpetrators the Federal Bureau of Investigation and US will announce their responsibility, or covertly, Postal Service (http://www.fbi.gov/antrhrax/ in which case they will not call attention to the amerithraxlinks.htm), is now considered closed. release of a biological agent (Budowle et al., However, the third edition of this book revisits 2005).Itislikelythatmostunintentionalreleases this case and provides a first-hand account of willbecovert-likeinnature(ZimmerandBurke, the investigation as well as lessons learned on 2009). what should be done in microbial forensics to Sincethepublicationofthefirsteditionofthis support an investigation and equally important bookin2005,thefieldofmicrobialforensicshas whatshould be avoided. evolvedsubstantiallyinitsabilitytoattributethe Many of the elements discussed in the first source of microorganisms and toxins that have and second editions of this book are echoed in been used in cases of bioterrorism and in bio- the 2009 US National Research and Develop- crimes. The 2001 anthrax mail attack (often ment Strategy for Microbial Forensics (U.S., referredtoas“Amerithrax”)fortunatelyremains 2010),whichidentifiesthreatawareness,preven- theworstandbest-knownexampleofsuccessful tion and protection, surveillance and detection, bioterrorism on US soil (Chapters 2 and 3). At and response and recovery as the essential pil- the time, the US was ill prepared to investigate lars of US national biodefense policy. the unique types of microbiological evidence “Attribution”dthe investigative process by forattributionthatweregeneratedbythatinves- which the US government links the identity of tigation, although traditional types of forensic a perpetrator or perpetrators of illicit activity evidence could readily be collected, analyzed, andthepathwayleadingtocriminalactivitydis andinterpretedwithintheframeworkofastatis- part of the surveillance and detection pillar. tically sound scientific foundation. The first Making a determination of attribution for a edition of this book had only limited reference planned or actual perpetrated biological attack to the anthrax mail attack because much of the would be the culmination of a complex investi- scientific examination was still in progress and gative process drawing on many different sour- methodologies were still under development. ces of information, including traditional Early capabilities were initially limited to forensic analysis of material evidence collected