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Forensic Microbiology focuses on newly emerging areas of microbiology relevant to medicolegal and criminal investigations: postmortem changes, establishing cause of death, estimating postmortem interval, and trace evidence analysis. Recent developments in sequencing technology allow researchers, and potentially practitioners, to examine microbial communities at unprecedented resolution and in multidisciplinary contexts. This detailed study of microbes facilitates the development of new forensic tools that use the structure and function of microbial communities as physical evidence. Chapters cover: * Experiment design * Data analysis * Sample preservation * The influence of microbes on results from autopsy, toxicology, and histology * Decomposition ecology * Trace evidence This diverse, rapidly evolving field of study has the potential to provide high quality microbial evidence which can be replicated across laboratories, providing spatial and temporal evidence which could be crucial in a broad range of investigative contexts. This book is intended as a resource for students, microbiologists, investigators, pathologists, and other forensic science professionals.

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Table of Contents

Cover

Title Page

About the editors

List of contributors

Foreword

Preface

References

CHAPTER 1: A primer on microbiology

1.1 Introduction

1.2 Microbial characteristics

1.3 Microorganisms and their habitats

1.4 Competition for resources

1.5 The ecology of some forensically relevant bacteria

1.6 Archaea and microbial eukaryotes

1.7 Conclusions

Acknowledgments

References

CHAPTER 2: History, current, and future use of microorganisms as physical evidence

2.1 Introduction

2.2 Methods for identification

2.3 Estimating PMI

2.4 Cause of death

2.5 Trace evidence

2.6 Other medicolegal aspects

2.7 Needs that must be met for use in chain of custody

2.8 Summary

Acknowledgments

References

CHAPTER 3: Approaches and considerations for forensic microbiology decomposition research

3.1 Introduction

3.2 Challenges of human remains research

3.3 Human remains research during death investigations

3.4 Human surrogates in research

3.5 Considerations for field studies

3.6 Descriptive and hypothesis‐driven research

3.7 Experiment design

3.8 Validation studies

Acknowledgments

References

CHAPTER 4: Sampling methods and data generation

4.1 Introduction

4.2 Materials

4.3 Sample collection techniques

4.4 Sample preservation, storage, and handling techniques

4.5 Data considerations

4.6 Conclusions

Acknowledgments

References

CHAPTER 5: An introduction to metagenomic data generation, analysis, visualization, and interpretation

5.1 Introduction

5.2 DNA extraction

5.3 DNA sequencing

5.4 Marker gene data analysis, visualization, and interpretation

5.5 Multi‐omics data analysis, visualization, and interpretation

5.6 Statistical analysis

5.7 Major challenges and future directions

References

CHAPTER 6: Culture and long‐term storage of microorganisms for forensic science

6.1 Introduction

6.2 The value of culturing microorganisms

6.3 Collection and handling of samples

6.4 Protocols

6.5 Conclusions

Acknowledgments

References

CHAPTER 7: Clinical microbiology and virology in the context of the autopsy

7.1 Introduction

7.2 The historical view of autopsy microbiology

7.3 Which samples should you collect and how?

7.4 Which methods are available for the diagnosis of infection?

7.5 How do you put the results into context?

7.6 What are the risks of transmission of infection in the postmortem room?

7.7 How does autopsy microbiology contribute to the diagnosis of specific conditions?

7.8 Conclusion

References

CHAPTER 8: Postmortem bacterial translocation

8.1 Introduction

8.2 Bacterial translocation in health and disease

8.3 Bacterial translocation in humans

8.4 Physiological changes after death influencing the selection of commensal bacteria

8.5 Consequences of bacterial translocation

8.6 Conclusion

References

CHAPTER 9: Microbial impacts in postmortem toxicology

9.1 Introduction

9.2 Microbial factors complicating postmortem toxicological analyses

9.3 Precautions taken to limit microbial impacts

9.4 Experimental protocols used to investigate postmortem drug and metabolite degradation due to microbial activity

9.5 Examples of microbially mediated drug degradation

9.6 Concluding remarks

References

CHAPTER 10: Microbial communities associated with decomposing corpses

10.1 Introduction

10.2 The soil microbiology of decomposition

10.3 Freshwater and marine decomposition

10.4 The microbiology of nonhuman models of terrestrial decomposition

10.5 The microbiology of terrestrial human decomposition

10.6 Is there a universal decomposition signature?

10.7 Using microbial signatures to estimate PMI

10.8 Conclusions

Acknowledgments

References

CHAPTER 11: Arthropod–microbe interactions on vertebrate remains:

11.1 Introduction

11.2 Framework for understanding microbe–arthropod interactions on vertebrate remains

11.3 Postcolonization interval

11.4 Future directions and conclusion

Acknowledgments

References

CHAPTER 12: Microbes, anthropology, and bones

12.1 Introduction

12.2 Bone microstructure

12.3 Microbially mediated decomposition

12.4 Bone bioerosion

12.5 Reconstructing postmortem histories

12.6 Conclusions

References

CHAPTER 13: Forensic microbiology in built environments

13.1 Introduction

13.2 The human skin microbiome

13.3 The microbiota of the built environment

13.4 Tools for the forensic classification of the built environment microbiome

13.5 Forensic microbiology of the built environment

13.6 Conclusion

References

CHAPTER 14: Soil bacteria as trace evidence

14.1 The forensic analysis of soil

14.2 Assessing the biological components of soil

14.3 Bacteria in soil

14.4 Molecular techniques for the forensic analysis of soil

14.5 Soil microbial profile data analysis methods

14.6 Feasibility of next‐generation sequencing for forensic soil analysis

14.7 Consensus on methodologies for soil collection and analysis

Acknowledgments

References

CHAPTER 15: DNA profiling of bacteria from human hair

15.1 An introduction to human hair as a forensic substrate

15.2 Current research into hair microbiomes

15.3 Importance of hair sample collection, storage, and isolation of microbial DNA

15.4 DNA sequencing of hair microbiomes

15.5 Conclusions and future directions

Acknowledgments

References

Index

End User License Agreement

List of Tables

Chapter 01

Table 1.1 Phyla of domain Bacteria included in the list of prokaryotic names with standing in nomenclature, http://www.bacterio.net

Table 1.2 Glossary of terms commonly used to describe the habitat preferences and metabolic strategies of microorganisms

Table 1.3 General classification of enzymes commonly associated with microorganisms

Table 1.4 General characteristics of two genera recognized as coryneform bacteria

Table 1.5 Some general characteristics of the classes that comprise phylum Proteobacteria classes

Table 1.6 Grouping of enteric bacteria by metabolic product

Chapter 02

Table 2.1 Sequencing methods and applications

Table 2.2 Advantages and limitations of culture‐based versus molecular‐based microbiological methods

Table 2.3 Examples of human and zoonotic bacterial, parasitic, and viral pathogens transmitted through environmental contamination

Chapter 03

Table 3.1 Animal surrogates from publications that have been used in global research for making inferences of human remains decomposition

Chapter 04

Table 4.1 Metadata information for submission of resulting sequencing data for each sample as determined by the MIMARKS‐compliant metadata template

Table 4.2 Metadata information for submission of resulting sequencing data for each sample as determined by the MIMARKS‐compliant metadata template

Chapter 05

Table 5.1 Commonly used DNA extraction methods for microbiome studies

Table 5.2 Comparison of next‐generation sequencing platforms

Table 5.3 List of commonly used software for multi‐omics data analyses

Chapter 06

Table 6.1 Subset of postmortem bacteria isolated from the skin of a decomposing swine (

Sus scrofa domesticus

) carcass on Oahu, Hawaii (E. Junkins, Chaminade University of Honolulu, unpublished data), where + indicates the presence of a taxon

Table 6.2 Subset of Illumina 16S taxa significantly correlated with oxidation–reduction potential isolated from the larval mass on decomposing swine (

Sus scrofa domesticus

) (Junkins, unpublished data)

Table 6.3 Overview of agar and broth media that are commonly used to culture microorganisms

Table 6.4 A non‐inclusive list of media designed for the growth of anaerobes

Chapter 07

Table 7.1 Standard procedure to ensure optimal quality in microbiological sampling at autopsy

Table 7.2 Techniques routinely available for microbiological analysis of autopsy specimens

Table 7.3 Guide to interpretation of culture results from different anatomical sites

Table 7.4 Characteristics of procalcitonin and C‐reactive protein as biochemical markers of sepsis

Table 7.5 Identification of the risk factors for transmission of infection when undertaking a risk assessment

Table 7.6 Clinical and statutory guidelines on biosafety in the work environment

Table 7.7 Agents with bioterrorism potential as categorized by US Centers for Disease Control and Prevention (Centers for Disease Control and Prevention, 2006)

Table 7.8 Diagnostic samples and microbiology tests for category A bioterrorism agents

Chapter 08

Table 8.1 Bacteria isolated from blood samples on different remains, in relation to the postmortem interval (Mesli, 2013)

Chapter 09

Table 9.1 Some functionalities found susceptible to microbial metabolism

Chapter 10

Table 10.1 Presence of several bacterial taxa in three decomposition models: mouse, swine, and human

List of Illustrations

preface

Figure P.1 Forensic microbiology and microbial ecology is a broad field that contributes to investigations ranging from pathology and bioterrorism to the quality of food and the environment. This text will focus on newly emerging areas of forensic microbiology related to postmortem microbiology and trace evidence (indicated in gray). The text will also serve to direct readers to established texts on other aspects of forensic microbiology, particularly bioterrorism and food microbiology

Chapter 01

Figure 1.1 Scanning electron micrograph showing some of the contrasting morphologies observed in microbial cells including the widely observed coccus (a:

Staphylococcus cohnii

) and rod (b:

Bacillus subtilis

) shapes, which can vary and occur in association with other morphologies (c). Microbial cells can be observed as several other shapes including spiral (spirilla), curved rods (vibrio), and club shaped (coryneform), the latter of which is observed in

Sporosarcina contaminans

(d)

Figure 1.2 The growth rate of microorganisms is regulated, in part, by the availability of resources. Zymogenous () microbes are able to use resources and multiply rapidly so they can dominate a habitat in which resources are abundant. In contrast, autochthonous () microbes tend to represent the basal, resting community that uses resources and multiplies slowly and forms the foundation of a microbial community

Figure 1.3 Scanning electron micrograph of endospore‐forming Firmicutes

Bacillus subtilis

(a) and

Sporosarcina contaminans

(b) collected from the skin of a decomposing swine carcass on Oahu, Hawaii

Figure 1.4 Scanning electron micrographs of enteric Proteobacteria

Ignatzschineria indica

collected from the skin of a decomposing swine carcass on Oahu, Hawaii presented as a colony (a) and as a single cell on a filter (b)

Chapter 02

Figure 2.1 Examples of bacteria isolated on agar (a) and broth (b) growth media

Figure 2.2 Mouse model displaying stages of decomposition. Fresh stage immediately after sacrifice (a), 3–4 hours after death showing signs of discolorization and livor mortis (b), and 5–7 hours after death showing rigor mortis (c). The remaining stages include bloat (d), active decay, shown here with purging of body fluids (e), and drying of tissues later in the stage (f), and natural mummification (g) or skeletonization (not shown). Mouse mass and length varied between 16–22 g and 7.5–8.5 cm, respectively

Figure 2.3 Enactment of collecting microbial trace evidence from a cadaver with a sterile cotton swab

Figure 2.4 Enactment of proper aseptic conditions to ensure samples avoid contamination and meet the proper chain of custody needs

Chapter 03

Figure 3.1 Pechal

et al.

(2013) conducted a manipulation experiment where necrophagous arthropods were either allowed to (a) naturally interact with and colonize the carcasses or (b) were excluded from accessing and interacting with the carcasses for 5 days of decomposition using mesh netting that was sealed at the bottom edges with plastic and soil. The exclusion treatments were 99% effective at preventing any arthropods from accessing the treatment (excluded) carcasses. Inadvertent arthropod access into the exclusion treatments was monitored by small glue traps (i.e., white rectangle attached to the wire case in panel (b) placed inside the mesh cages. The small cages made of chicken wire in both (a) and (b) were antivertebrate scavenging cages to prevent access of vertebrates to the carcasses. Both worked at 100% efficiency in preventing vertebrate scavenging

Figure 3.2 Examples of common types of pseudoreplication: (a) simple, (b) sacrificial, and (c) temporal. The larger rectangles represent the experimental unit with the circles denoted as one set of rodent remains that is sampled. In the case of temporal pseudoreplication, the same set of remains is sampled repeatedly over time and each sampling event is incorrectly considered a replicate sample from that carcass

Chapter 04

Figure 4.1 Examples of sampling methodologies for collecting microbial communities from various substrates. (a) Inverted cone trap for collecting insects. A bait resource, in this instance the white arrow pointing to a “stink jar” consisting of decomposed liver and water, is placed at the bottom opening as an attractant for necrophagous insects. (b) An antiscavenging cage placed over a swine carcass the field is an excellent place to attached passive trapping methods, such as glue traps (see (c) for an up close view), for collecting flying invertebrates attracted to carrion. (d) Swabs can be effectively used to collect the epinecrotic microbial communities from cadavers. (e) In aquatic habitats, stable structures placed in an aqueous habitat, such as these unglazed ceramic tiles attached to a brick, create a standardized area for biofilm formation and can be left for weeks to months or up to a year depending on the scope of the study

Chapter 05

Figure 5.1 Flow diagram of a typical multi‐omics workflow

Figure 5.2 Schematic showing barcoded fusion primer design for (a) Ion Torrent PGM/454 and (b) MiSeq

®

sequencing platforms

Chapter 06

Figure 6.1 Generalized methodology for the collection and analysis of microbial samples.

Figure 6.2 Sampling the larval mass in the oral cavity of a decomposing swine (

Sus scrofa domesticus

) carcass (a) and the streaking for growth in a Petri dish containing standard nutrient agar (b) resulting in the growth of several colonies with contrasting morphology (c) at room temperature following incubation in a laminar flow cabinet (d)

Figure 6.3 Aseptic technique of flaming the collection loop (a) to obtain bacterial cells of

Ignatzschineria indica

for broth culture (b). Cells are scraped on the inside of the culture tube (c) to be washed down into broth medium and placed on incubation shaker (d) until turbid. Turbidity can occur within 24 hours at room temperature

Figure 6.4 Turbidity of broth medium in a culture tube (a) indicates growth and readiness for cryostorage in a 2 mL microfuge tube containing 1.5 mL of broth (b) and 0.5 mL of glycerol (c) mixed thoroughly via vortex (d) and placed in cryobox for storage at −80°C

Figure 6.5 Portable anaerobic jars are great options for incubation under anoxic and microaerophilic conditions or for short‐term transportation of anaerobes. Pictured are two versions of the BD GasPak™ (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) incubating at room temperature (a) and in a 37°C incubator (b). The anaerobic jar in (a) contains a sachet of activated charcoal to remove oxygen and five Petri dishes (9 cm diameter, 1.5 cm height)

Figure 6.6 Anaerobic chambers come in a range of specifications including rigid (a) and soft‐sided (b). These chambers can be enhanced with the inclusion of instrumentation. For example, the large Coy Laboratory Products vinyl chamber (b) contains a plate reader and liquid handling robot to facilitate high‐throughput culturing and interrogation of cultures. The pass box is located on the left of the both chambers, which allows for the movement of samples between aerobic and anaerobic conditions

Chapter 08

Figure 8.1 Bacterial species and quantification depending on the location on the gastrointestinal tract

Figure 8.2 Mechanisms and factors involved in bacterial translocation.

Figure 8.3 Running paths of bacteria through enterocytes.

Figure 8.4 Bacterial proliferation of

Escherichia coli

in relation to temperature.

Square root model (full line), Ratkowsky model (slight dotted line), and exponential sum model (heavy dotted line) obtained from nonlinear regression analysis

.

Figure 8.5 Bacterial proliferation of

Clostridium perfringens

in relation to temperature on three different substrates.

Chapter 10

Figure 10.1 Succession of microbial decomposers during human decomposition. The microbial (bacterial, eukaryotic, and fungal) communities associated with two human cadavers at Sam Houston State University’s Southeast Texas Applied Forensic Science Facility were determined using deep marker gene sequencing and analysis of samples collected from the cadavers throughout decomposition. A small subset of taxa associated with specific decomposition stages are highlighted here. Shown here is a photo of a fly larval mass under the skin during active decay and a skull during dry remains.

Chapter 11

Figure 11.1 A framework of the postmortem interval (PMI) divided into the preappearance interval (PAI), development interval (DI), and presence interval (PI) as proposed by Matuszewski (2011).

Figure 11.2 Framework proposed by Tomberlin

et al.

(2011) for entomology‐based phases of the decomposition process.

Figure 11.3 Adult

Chrysomya rufifacies

(Diptera: Calliphoridae) resting on vegetation.

Figure 11.4 Adult

Nicrophorus marginatus

Fabricius (Coleoptera: Silphidae) present on swine carrion.

Figure 11.5 Swine carrion exhibiting (a) fresh, (b) active, and (c) skeletal stages of decomposition.

Chapter 12

Figure 12.1 Thin section of a human femur. Secondary osteons, Haversian canals (H), a Volkmann’s canal (V), and lamellar bone (L) are visualized under transmitted light and marked accordingly (a). Birefringence of mineralized collagen fibrils arranged in concentric lamellae in osteons is observed in polarized light (b). Several osteons display Maltese cross extinction patterns of birefringence (arrows).

Figure 12.2 Section of a metacarpal from a 64‐year‐old male with disseminated coccidiodomycosis, a fungal infection caused by

Coccidoides immitis

endemic to the San Joaquin Valley in California and the Rio Grande River Valley of Texas and New Mexico. The section shows chronic inflammation with fibrosis and granuloma formation, causing loss of bone (B). The section is decalcified and stained with haematoxylin and eosin (National Museum of Health and Medicine 2009.0004; AFIP Orthopathology Study Set A, MS78.

Figure 12.3 Schematic representation of microbial alteration of bone at the microscopic level displaying linear longitudinal (1), budded (2), lamellate (3), and (4) Wedl tunneling.

Figure 12.4 Bioerosion of bone indicated by arrows in a thin section of human femur displaying bacterial alteration under normal light (a) and polarized light (b). Thin section of cattle bone displaying fungal alteration (c).

Figure 12.5 Complete transverse thin section of a human rib (a) with a magnified image showing adipocere crystalline structures (b).

Chapter 13

Figure 13.1 Predicted relative contributions of different occupants to the microbial communities associated with six home surfaces over the course of a 4‐week sampling window. Microbial sources were inferred through the Source Tracker algorithm. Note that when Person 1 leaves the residence for travel, as indicated by the black bars along the

x

‐axis, their predicted microbial contribution to the home surfaces either vanishes or diminishes, and increases after their return to the home

Chapter 14

Figure 14.1 Hypothetical abundance chart displaying soil microbial profiles from three soil samples. The evidentiary soil profile is visually more similar to the profile produced from the crime scene soil than the alibi location, indicating the latter can be excluded as the possible source of the evidentiary soil

Figure 14.2 Exemplary MDS plot ordinating replicate bacterial profiles from an alibi location (A) and a crime scene (B). An evidentiary sample profile (unknown) is clustering with the profiles from the crime scene; thus, it is more similar to this location than the alibi location

Figure 14.3 Exemplary HCA dendrogram displaying clusters of hypothetical crime scene (A), alibi location (B), and evidentiary (unknown) microbial profiles. The evidentiary profile is clustering with the soil samples collected at the crime scene, indicating it is more similar to those samples than alibi location soils

Chapter 15

Figure 15.1 Morphology of the three growth stages of human hair roots: anagen (a: 100× magnification), catagen (b: 200×), and telogen (c: 200×)

Figure 15.2 Example of an amplicon‐based next‐generation workflow diagram

Figure 15.3 Avenues in which contamination of hair evidence may occur prior to sample collection (blue) and post sample collection (red)

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American Academy of Forensic Sciences

Published and forthcoming titles in the Forensic Science in Focus series

Published

The Global Practice of Forensic ScienceDouglas H. Ubelaker (Editor)

Forensic Chemistry: Fundamentals and ApplicationsJay A. Siegel

Forensic MicrobiologyDavid O. Carter, Jeffery K. Tomberlin, M. Eric Benbow and Jessica L. Metcalf

Forthcoming

The Future of Forensic ScienceDaniel A. Martell

Humanitarian Forensics and Human IdentificationPaul Emanovsky and Shuala M. Drawdy

Forensic Anthropology: Theoretical Framework and Scientific BasisClifford Boyd and Donna Boyd

Forensic Microbiology

 

EDITED BY

David O. CarterChaminade University of Honolulu, USA

Jeffery K. TomberlinTexas A&M University, USA

M. Eric BenbowMichigan State University, USA

Jessica L. MetcalfColorado State University, USA

 

 

 

 

 

 

This edition first published 2017 © 2017 John Wiley & Sons Ltd

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The right David O. Carter to be identified as the author of this work has been asserted in accordance with law.

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ISBN: 9781119062554

Cover Design: WileyCover Image: Courtesy of David O. Carter

This work is dedicated to all donors (human and nonhuman) that allowed for much of this research to be completed. You are not forgotten and your contribution to science is timeless.

David O. Carter would like to thank his wife, Charlotte, for her never‐ending support and encouragement.

Jeffery K. Tomberlin would like to thank his family (Laura, Celeste, and Jonah) for putting up with his late nights with his computer, constant texting about work, and simply allowing him to enjoy his pursuit of knowledge. He would like to extend a special thanks to Tawni L. Crippen for being patient with him as he explored the world of microbiology under her tutelage and his constant leaning on her sterile bench tops. She has truly opened his eyes to the wonderful world of bacteria and their importance in nature. You are a great friend and colleague.

M. Eric Benbow would like to thank Melissa, Arielle, and Alia for their tremendous support, encouragement, and understanding while he travels to continue his passion that leads to efforts like this project. He would also like to thank Jen Pechal for her extra attention to reviewing several of the chapters of this book and for “maintaining the ship” of writing and productivity while he explores new avenues of science like that related to many topics of the book.

Jessica L. Metcalf would like to thank her forensic collaborators for being excellent and patient colleagues, coauthors, and coeditors. She would like to thank Rob Knight for his support of pursuing microbial tools for forensic science. She would also like to thank her family for their support and contributions to her scientific endeavors.

About the editors

Dr. David O. Carter is Director and Associate Professor of Forensic Sciences at Chaminade University of Honolulu. He also serves as Principal Investigator of the Laboratory of Forensic Taphonomy. His primary research interest is the decomposition of human remains, particularly in tropical environments. Current research projects focus on the structure and function of antemortem and postmortem microbial communities: using microbiomes as spatial and temporal evidence. He is interested in understanding the relationships between decomposing remains, microbial communities, and the environment. Dr. Carter’s ultimate goal is to get quality science and technology in the hands of first responders and investigators.

Dr. Carter is an active member of the forensic science community with a significant interest in undergraduate education. He is a Fellow in the Pathology/Biology Section of the American Academy of Forensic Sciences and has recently served as Program Chair for the Pathology/Biology Section. Dr. Carter also serves on the Medicolegal Death Investigation Subcommittee in the Organization of Scientific Area Committees, a joint endeavor between the US Department of Justice and US Department of Commerce. He incorporates this experience into undergraduate education where he plays an active role in curriculum development, assessment, academic advising, and recruiting.

Dr. Jeffery K. Tomberlin is an Associate Professor and Co‐Director of the Forensic & Investigative Sciences Program and Principle Investigator of the Forensic Laboratory for Investigative Entomological Sciences (FLIES) Facility (forensicentomology.tamu.edu) in the Department of Entomology at Texas A&M University. Research in the FLIES Facility examines species interactions on ephemeral resources such as vertebrate carrion, decomposing plant material and animal wastes in order to better understand the mechanisms regulating arthropod behavior as related to arrival, colonization and succession patterns. The goals of his program are to refine current methods used by entomologists in forensic investigations. His research also is focused on waste management in confined animal facilities and the production of alternate protein sources for use as livestock, poultry, and aquaculture feed. Since arriving on campus at Texas A&M University in 2007, eight Ph.D. and fourteen M.S. students have completed their degrees under his supervision.

Dr. Tomberlin has been very active within the forensic science community. He, along with a colleague, initiated the first forensic entomology conference in North America as well as the formation of the North America Forensic Entomology Association of which he served as its first president. He is also a Fellow in the American Academy of Forensic Sciences and has served as the Chair of the Pathology/Biology Section. Dr. Tomberlin is also one of 18 entomologists board certified by the American Board of Forensic Entomology (ABFE). He has served a number of roles with the ABFE including Secretary and Chair.

Dr. M. Eric Benbow is an Associate Professor in the Department of Entomology and the Department of Osteopathic Medical Specialties at Michigan State University. The research in his lab focuses on microbial‐invertebrate community interactions in aquatic ecosystems, disease systems and carrion decomposition. These research foci use basic science to inform applications in areas such as human health, natural resources management and forensics. Dr. Benbow has authored or co‐authored a collection of over 120 peer‐reviewed papers, book chapters, and proceedings, many of which relate to carrion decomposition ecology, forensic entomology and forensic microbiology. He has served on two National Research Council committees related to aquatic ecology, and is regularly invited as a speaker at international and national academic meetings related to decomposition ecology and forensics. Dr. Benbow has led workshops at the international level discussing experimental design, statistical analyses and the importance of novel basic ecological concepts in advancing the field of carrion ecology and applications in forensics. Dr. Benbow was part of the inaugural executive committee for the North American Forensic Entomology Association (NAFEA) where he served as the Editor‐in‐ Chief of the annual NAFEA Newsletter and NAFEA Webmaster (www.nafea.net) for eight years. He was the president of NAFEA from 2012–2013 and has served as an expert witness and worked on several cases that involved insects as evidence during investigations or water resource litigation. He continues a recognized and collaborative research program in microbe‐insect interactions that supports undergraduate and graduate students and postdoctoral associates. Dr. Benbow continues to mentor and co‐mentor students and postdoctoral associates through research and teaching. He sees the future of ecology, evolution and applications in forensics to be fundamentally in the hands of students and early career scientists worldwide.

Dr. Jessica L. Metcalf is an Assistant Professor in the Department of Animal Sciences at Colorado State University. Her research uses high‐throughput sequencing and bioinformatic tools in an ecological and evolutionary framework to understand changes of the human microbiome during life and after death. Her microbiome research projects span the fields of forensic science, medicine, agriculture, and vertebrate ecology. A main focus of her research is developing a microbial stopwatch for estimating the postmortem interval. Dr. Metcalf strives to provide foundational microbiome science that can be developed into tools for the justice system.

Dr. Metcalf has a background in evolutionary biology, ancient DNA, experimental microbial ecology, and microbiome science. Her training includes expertise in generating genomic data from a broad range of sample types, including fossil remains and other low‐biomass samples, and analyzing large genomic data sets. Dr. Metcalf is currently building her microbiome science program as part of the microbiome cluster at Colorado State University.

List of contributors

Kate M. BarnesDepartment of Natural SciencesUniversity of DerbyDerby, UK

M. Eric BenbowDepartment of Entomology and Department of Osteopathic Medical SpecialtiesMichigan State UniversityEast Lansing, MI, USA

Sibyl R. BucheliDepartment of Biological SciencesSam Houston State UniversityHuntsville, TX, USA

Michael BunceTrace and Environmental DNA (TrEnD) Laboratory, Department of Environment and AgricultureCurtin UniversityPerth, Western Australia, Australia

Zachary M. BurchamDepartment of Biological SciencesMississippi State UniversityStarkville, MS, USA

Danielle M. ButzbachToxicology SectionForensic Science SAAdelaide, South Australia, Australia

David O. CarterLaboratory of Forensic Taphonomy, Forensic Sciences Unit, Division of Natural Sciences and MathematicsChaminade University of HonoluluHonolulu, HI, USA

Jared W. CastleSchool of Chemical and Physical SciencesFlinders UniversityAdelaide, South Australia, Australia

Tawni L. CrippenSouthern Plains Agricultural Research Center Agricultural Research ServiceUnited States Department of AgricultureCollege Station, TX, USA

Franklin E. DamannCentral Identification Laboratory—Offutt Air Force BaseDefense POW/MIA Accounting AgencyBellevue, NE, USA

David R. ForanForensic Science Program, School of Criminal JusticeandDepartment of Integrative BiologyMichigan State UniversityEast Lansing, MI, USA

Jack A. GilbertDepartment of Ecology and Evolution and Department of SurgeryUniversity of ChicagoChicago, ILandInstitute for Genomics and Systems BiologyArgonne National LaboratoryLemont, ILandMarine Biological LaboratoryWoods Hole, MA, USA

Valery HedouinLille University School of Medicine, CHU LilleForensic Taphonomy Unit, UTML, Lille, France

James M. HopkinsForensic Science Program, School of Criminal JusticeMichigan State UniversityEast Lansing, MI, USACurrent address:St. Jude Children’s Research HospitalMemphis, TN, USA

Embriette R. HydeKnight Laboratory, Department of PediatricsUniversity of California, San DiegoSan Diego, CA, USA

Miranda M.E. JansCentral Identification Laboratory—Hickam Air Force BaseDefense POW/MIA Accounting AgencyHonolulu, HI, USA

Ellen M. JesmokForensic Science Program, School of Criminal JusticeMichigan State UniversityEast Lansing, MI, USACurrent address:Minnesota Bureau of Criminal Apprehension LaboratorySt. Paul, MN, USA

Heather R. JordanDepartment of Biological SciencesMississippi State UniversityStarkville, MS, USA

Emily N. JunkinsLaboratory of Forensic Taphonomy, Forensic Sciences Unit, Division of Natural Sciences and MathematicsChaminade University of HonoluluHonolulu, HI, USAandDepartment of Microbiology and Plant Biology, University of OklahomaNorman, OK, USA

K. Paul KirkbrideSchool of Chemical and Physical SciencesFlinders UniversityAdelaide, South Australia, Australia

Rob KnightDepartment of PediatricsandDepartment of Computer Science and EngineeringUniversity of California, San DiegoSan Diego, CA, USA

Whitney A. KodamaLaboratory of Forensic Taphonomy, Forensic Sciences Unit, Division of Natural Sciences and MathematicsChaminade University of HonoluluHonolulu, HI, USA

Simon LaxDepartment of Ecology and EvolutionUniversity of Chicago, Chicago, ILandInstitute for Genomics and Systems BiologyArgonne National LaboratoryLemont, IL, USA

Claire E. LenehanSchool of Chemical and Physical SciencesFlinders UniversityAdelaide, South Australia, Australia

Aaron M. LynneDepartment of Biological SciencesSam Houston State UniversityHuntsville, TX, USA

Vadim MesliLille University School of Medicine, CHU LilleForensic Taphonomy Unit, UTMLLille, France

Jessica L. MetcalfDepartment of Animal SciencesColorado State UniversityFort Collins, CO, USA

Dáithí C. MurrayTrace and Environmental DNA (TrEnD) Laboratory, Department of Environment and AgricultureCurtin UniversityPerth, Western Australia, Australia

Christel NeutClinical Bacteriology LaboratoryINSERM U995 LIRIC, University of LilleLille, France

Jennifer L. PechalDepartment of EntomologyMichigan State UniversityEast Lansing, MI, USA

Mohammad RazaDepartment of Medical Microbiology and VirologySheffield Teaching Hospitals NHS Foundation TrustSheffield, UK

Frank ReithSchool of Biological Sciences Sprigg Geobiology CentreUniversity of AdelaideandCSIRO Land and WaterEnvironmental Contaminant Mitigation and Technologies, PMB2Adelaide, South Australia, Australia

Elisabeth J. RidgwayDepartment of Medical Microbiology and VirologySheffield Teaching Hospitals NHS Foundation TrustSheffield, UK

Baneshwar SinghDepartment of Forensic ScienceVirginia Commonwealth UniversityRichmond, VA, USA

Bala M. SubramanianDepartment of Medical Microbiology and VirologySheffield Teaching Hospitals NHS Foundation TrustSheffield, UK

Jeffery K. TomberlinDepartment of EntomologyTexas A&M UniversityCollege Station, TX, USA

Silvana R. TridicoForensic Science & Wildlife MattersAdelaide, South Australia, Australia

G. Stewart WalkerSchool of Chemical and Physical SciencesFlinders UniversityAdelaide, South Australia, Australia

Foreword

In recent years, technology improvements have enabled forensic scientists to analyze smaller and smaller samples. However, with new and expanded capabilities often comes the challenge of providing appropriate meaning to that evidence. Use of information from microbial communities in forensic investigations may open new avenues for answering important questions in crime‐solving efforts. This book provides a look into current applications, and future possibilities, with microbial analysis aiding forensic investigations.

The Human Microbiome effort over the past decade or so has built on information gained from the earlier Human Genome Project. Likewise, forensic microbiology will in many cases build on previous work with human forensic DNA testing. Forensic DNA analysis with human DNA began in 1984 with the pioneering work of Alec Jeffreys at Leicester University in the United Kingdom and a Nature publication the following year (Gill et al., 1985). Forensic microbiology is poised to have a similar trajectory, with several research groups having their work disseminated in several high impact publications. It remains to be seen whether recent advances in forensic microbiology will impact forensic science as human DNA testing, but it nevertheless has the potential to be equally revolutionary in some cases (Fierer et al., 2010). Bioterrorism and forensic pathology are two important areas where microbial analysis plays an important role, but these applications are being expanded as we understand the temporal and spatial value of microorganisms.

Perhaps in a future day the microbial environment around crime scenes and persons of interest will be rapidly assessed to answer important investigative questions. Massively parallel (next‐generation) sequencing can provide microbial and human DNA sequences on an unprecedented scale.

This book is part of a larger effort by the American Academy of Forensic Sciences and Wiley to bring forensic science issues and capabilities into focus. It is hoped that the completed product is satisfying to both the authors and their future readers.

John M. Butler, Ph.D.NIST Fellow and Special Assistant to the Director for Forensic ScienceNational Institute of Standards and TechnologyGaithersburg, MD, USA

Series preface

The forensic sciences represent diverse, dynamic fields that seek to utilize the very best techniques available to address legal issues. Fueled by advances in technology, research and methodology, as well as new case applications, the forensic sciences continue to evolve. Forensic scientists strive to improve their analyses and interpretations of evidence and to remain cognizant of the latest advancements. This series results from a collaborative effort between the American Academy of Forensic Sciences (AAFS) and Wiley to publish a select number of books that relate closely to the activities and objectives of the AAFS. The book series reflects the goals of the AAFS to encourage quality scholarship and publication in the forensic sciences. Proposals for publication in the series are reviewed by a committee established for that purpose by the AAFS and also reviewed by Wiley.

The AAFS was founded in 1948 and represents a multidisciplinary professional organization that provides leadership to advance science and its application to the legal system. The 11 sections of the AAFS consist of Criminalistics, Digital and Multimedia Sciences, Engineering Sciences, General, Pathology/Biology, Questioned Documents, Jurisprudence, Anthropology, Toxicology, Odontology, and Psychiatry and Behavioral Science. There are over 7000 members of the AAFS, originating from all 50 States of the United States and many countries beyond. This series reflects global AAFS membership interest in new research, scholarship, and publication in the forensic sciences.

Douglas H. UbelakerSenior ScientistSmithsonian InstitutionWashington, DC, USASeries Editor

Preface

Microorganisms have been used as physical evidence in “forensic” investigations for nearly as long as their existence has been known. These organisms have been used to reconstruct events as diverse as cause of death, food spoilage, and bioterrorism. Such diverse applications represent our definition of modern forensic microbiology. Ultimately, this text will serve as a resource for those interested in the relationship between microbiology and forensic science, particularly criminal and medicolegal death investigations.

We have approached the text with the perspective that forensic microbiology primarily draws from three fundamental disciplines: medical microbiology, environmental microbiology, and food microbiology (Figure P.1). The current text was inspired by a workshop presented at the 2014 American Academy of Forensic Sciences Conference in Seattle, Washington. From this workshop germinated the topics covered in the text. Topics reviewed include primarily newly emerging applications of microbiology to criminal and medicolegal death investigations. To do this, the contents focus on the characterization and interpretation of microbial communities through culturing and sequencing techniques. Our goal is to position forensic microbiology in a modern context of improved microbiome sequencing and analytical techniques beyond those that have been traditionally covered in microbial forensics that include bioterrorism and pathology (Caplan and Koontz, 2001; Budowle et al., 2011; Ray and Bhunia, 2013). Recent developments in sequencing technology and corresponding discoveries have allowed scientists to examine microbial communities at unprecedented resolution and in multidisciplinary contexts. The current text will highlight the potential new applications that these technologies can provide.

Figure P.1 Forensic microbiology and microbial ecology is a broad field that contributes to investigations ranging from pathology and bioterrorism to the quality of food and the environment. This text will focus on newly emerging areas of forensic microbiology related to postmortem microbiology and trace evidence (indicated in gray). The text will also serve to direct readers to established texts on other aspects of forensic microbiology, particularly bioterrorism and food microbiology

February 2017

David O. CarterJeffery K. TomberlinM. Eric BenbowJessica L. Metcalf

References

Budowle, B., Schutzer, S.E., Breeze, R.G.

et al.

(eds) (2011)

Microbial Forensics

, 2nd edn, Academic Press, Burlington.

Caplan, M.J. and Koontz, F.P. (2001)

Cumitech 35 Postmortem Microbiology

, American Society for Microbiology Press, Washington, DC.

Ray, B. and Bhunia, A. (2013)

Fundamental Food Microbiology

, 5th edn, CRC Press, Boca Raton.

CHAPTER 1A primer on microbiology

David O. Carter1, Emily N. Junkins1,2 and Whitney A. Kodama1

1Laboratory of Forensic Taphonomy, Forensic Sciences Unit, Division of Natural Sciences and Mathematics, Chaminade University of Honolulu, Honolulu, HI, USA

2Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, USA

1.1 Introduction

In many ways microorganisms are an ideal form of physical evidence. They can be found virtually everywhere and are certainly present in every habitat occupied by humans. Thus, microbes could be collected from every scene under a forensic investigation, yet not all microorganisms are everywhere; like many forms of trace evidence, some microbes are found only in certain locations due to having a preferred habitat, much like how insects, birds, and reptiles have a preferred habitat range. Another valuable characteristic of microorganisms is that many of them can transform themselves into a highly durable structure that is designed to survive harsh conditions, which increases the likelihood of their survival and discovery. Considering all of these attributes, it is probably not surprising that microorganisms have been used as physical evidence since the early days of forensic science, particularly to establish the cause of death (e.g., MacCallum and Hastings, 1899). Forensic microbiology has since grown into an exciting discipline relevant to several areas of forensic science including medicolegal death investigation (Caplan and Koontz, 2001; Forbes et al., 2016), bioterrorism (Budowle et al., 2011), and product authenticity (Brzezinski and Craft, 2012). It will be absolutely fascinating to learn of the new discoveries in forensic microbiology over the next few decades.

Historically microbes have been used almost exclusively as spatial evidence—physical evidence that is used to associate people with diseases, objects, and/or locations (Locard, 1930a, b, c; Caplan and Koontz, 2001; Tridico et al., 2014; Wiltshire et al., 2014; Young et al., 2015). This application is similar to the use of any other form of trace evidence, such as soil (Bisbing, 2016), paint (Kirkbride, 2016), glass (Almirall and Trejos, 2016), and fibers (Houck, 2016). However, recent research has shown that microorganisms represent a relatively unique form of physical evidence that can also serve as temporal evidence, evidence that is used to establish a timeline. This application uses the ability of microorganisms to respond rapidly to changes in their environment (e.g., Carter and Tibbett, 2006), and these changes are temporally predictable (Metcalf et al., 2013; Pechal et al., 2014; Guo et al., 2016; Metcalf et al., 2016), with an apparent ability to serve as an estimate of the postmortem interval (Chapter 2) and human habitation (Chapter 13) interval.

We are currently in an exciting time when multiple research groups around the world are leading advances in postmortem microbiology and trace microbiology (Fierer et al., 2010; Benbow et al., 2015; Lax et al., 2015; Metcalf et al., 2016). These advances are occurring rapidly and have great potential to significantly change how microorganisms are used as physical evidence. Microorganisms will likely play a greater role as physical evidence in the future, so the purpose of the current chapter is to provide an introduction to some fundamental aspects of microbiology and microbial ecology to help the reader develop an appreciation for the vast diversity of microorganisms and how they can be used to identify a location or time period of investigative interest. It is not possible for this chapter to review all known microorganisms, so the contents hereinafter will place an emphasis on bacteria that are of interest to the most recent research relevant to the scope of this book, postmortem microbiology and trace microbiology (e.g., Benbow et al., 2015; Iancu et al., 2015; Finley et al., 2016; Metcalf et al., 2016). However, domains Archaea and Eukarya are also highly relevant, and the current chapter will reference relevant work, when possible, that focuses on these very important taxa within a forensic context.

1.2 Microbial characteristics

Microorganisms can differ in many ways including their morphology, method of movement (motility), metabolic strategy, environmental requirements, and several other characteristics (Brown, 2015). The current chapter will address this issue with relative simplicity by describing how microorganisms survive with a brief description of some relevant taxa.

1.2.1 Microbial taxonomy and function

Classification of life has proven to be a challenge. Presently, there are a number of opinions on how we should organize organisms in terms of their relationship to one another. Although not the focus of this chapter, this topic is of great importance as it impacts our ability to assess microbial communities in general. Thus, we suggest individuals with an interest in forensic microbiology remain cognizant of the ever‐shifting landscape of microbial taxonomy.

For this text, we focus our discussion on three major groups of microorganisms organized as domains: Archaea, Bacteria, and Eukarya (Woese et al., 1990) although a new perspective on this classification was presented recently (Hug et al., 2016). The List of Prokaryotic Names with Standing in Nomenclature (http://www.bacterio.net) currently divides Bacteria into 30 phyla (Table 1.1) and Archaea into five phyla (Crenarchaeota, Euryarchaeota, Korarchaeota, Nanoarchaeota, Thaumarchaeota). These microbes can vary morphologically, with spherical (cocci; Figure 1.1a) and rod‐shaped (historically termed bacilli; Figure 1.1b) being the most common. Variations on these general morphologies exist (Figure 1.1c), as do other morphologies such as club‐shaped cells (coryneform; Figure 1.1d) and curved rods (e.g., vibrio). Thus, referring to a bacterium as a rod or a coccus is a helpful way to begin the identification process.

Table 1.1 Phyla of domain Bacteria included in the list of prokaryotic names with standing in nomenclature, http://www.bacterio.net

Acidobacteria

Cyanobacteria

Nitrospira

Actinobacteria

Deferribacteres

Planctomycetes

Aquificae

Deinococcus–Thermus

Proteobacteria

Armatimonadetes

Dictyoglomi

Spirochaetes

Bacteroidetes

Elusimicrobia

Synergistetes

Caldiserica

Fibrobacteres

Tenericutes

Chlamydiae

Firmicutes

Thermodesulfobacteria

Chlorobi

Fusobacteria

Thermomicrobia

Chloroflexi

Gemmatimonadetes

Thermotogae

Chrysiogenetes

Lentisphaerae

Verrucomicrobia

Figure 1.1 Scanning electron micrograph showing some of the contrasting morphologies observed in microbial cells including the widely observed coccus (a: Staphylococcus cohnii) and rod (b: Bacillus subtilis) shapes, which can vary and occur in association with other morphologies (c). Microbial cells can be observed as several other shapes including spiral (spirilla), curved rods (vibrio), and club shaped (coryneform), the latter of which is observed in Sporosarcina contaminans (d)

As the term microbiology indicates, microbes are small. A bacterial cell will likely have a diameter of 1–5 µm (see Figure 1.1), which means that microscopy is necessary to view individual microbial cells. Thus, the shape and size of microbial cells can be used for a general identification, usually to exclude possible identities. Other commonly used characteristics to identify microbes include the reaction to the Gram stain and the ratio of nucleotides in a cell, which is presented as guanine–cytosine (GC) content.

Stains play a significant role in the identification of microorganisms. For example, the Gram stain was developed in the nineteenth century to help visualize microbial cells. Without staining, many microbial cells are transparent and difficult to see. The Gram‐positive and Gram‐negative designation also provides some insight into the structure of the microbial cell wall. The cell wall of a Gram‐positive bacterium is approximately 90% peptidoglycan, whereas the cell wall of a Gram‐negative bacterium is approximately 10% peptidoglycan (Madigan et al., 2012). Interestingly, the Gram designation also provides information about the taxonomy of bacteria. Gram‐positive bacteria are generally found in phyla Actinobacteria and Firmicutes. Some relatively well‐known Gram‐positive bacteria include genera Bacillus (rod), Clostridium (rod), and Streptococcus (coccus), all of which are in phylum Firmicutes. The GC content of a cell can be used to generally distinguish between Actinobacteria and Firmicutes. The GC content represents the proportion of the bacterial genome that comprises GC base pairs, rather than adenine–thymine base pairs, and is presented as high GC (>50% GC content) or low GC (<50% GC content). Phylum Actinobacteria includes high GC bacteria, while phylum Firmicutes includes low GC bacteria.

Microbes can also be identified based on their function. In fact, one of the reasons that microbes are so important to life on Earth is the vast diversity of functions that they carry out. Microbes decompose organic material, utilize carbon dioxide, fix nitrogen, and help plants and animals to acquire nutrients, and they contribute many other vital functions that keep habitats stable. These functions are the result of microorganisms competing for that which is essential to microbial life, water, energy, and nutrients, where the energy source is often organic (contains carbon) and the nutrients include several essential elements such as nitrogen, phosphorus, potassium, sulfur, and calcium. As seen with other organisms, the ability of microbes to function is greatly influenced by their environment: oxygen availability, temperature, chemistry (particularly pH and Eh), and light (Ball, 1997). Thus, humans live in habitats that are the result of several processes, many of which are carried out by microorganisms. To effectively use microorganisms as physical evidence, we must therefore understand where microbes live, what their function is within that habitat, and how their environment affects their function and survival.

Microbes use several strategies to acquire energy and nutrients, referred to here as metabolic strategy. Some microbes even have the ability to change their metabolic strategy depending on environmental conditions. A microbe that is restricted to a single metabolic strategy is known as obligate. An obligate aerobe is a microbe that requires oxygen, for example. A microbe that can change metabolic strategies is termed facultative. A facultative anaerobe is a microbe that can maintain metabolic function regardless of oxygen availability. Understanding these metabolic strategies is important for forensic microbiology because it forms the foundation for interpreting microbial evidence or identifying a particular microorganism. For example, one would expect to see decomposing remains associated with chemoorganotrophic microbes, like the Enterobacteriaceae (e.g., Benbow et al., 2015), because a dead body is a high quality source of water, energy, and nutrients (Carter et al., 2007b).

The metabolic terms defined in Table 1.2 are presented to help understand the diversity of metabolic strategies used by microbes and show that they are regularly described by their metabolic strategy. Phototrophs, such as those in the phylum Cyanobacteria, use light to conduct photosynthesis. Chemotrophs, such as those in class Gammaproteobacteria, consume organic compounds (e.g., human remains, plant detritus) to generate energy. Because of this they are often referred to as decomposers. These decomposers represent the bulk of microorganisms associated with decomposing remains and trace evidence. It is important to accept that some taxa have physiologies that are not easily described using the terminology in Table 1.2 because of the vast array of metabolic strategies employed by microorganisms (e.g., Slonczewski and Foster, 2011; Madigan et al., 2012)

Table 1.2 Glossary of terms commonly used to describe the habitat preferences and metabolic strategies of microorganisms

Aerobe

An organism that lives in the presence of oxygen

Aerotolerant

An organism that can live in aerobic and anaerobic conditions

Anaerobic

An organism that lives in the absence of oxygen

Autotrophy

Using carbon dioxide as the only source of carbon

Chemoautotrophy

Oxidizing chemical compounds to obtain energy while acquiring carbon only from carbon dioxide

Chemolithotrophy

Oxidizing inorganic compounds to obtain energy

Chemoorganotrophy

Oxidizing organic compounds to obtain energy

Chemotrophy

Oxidizing chemical compounds to obtain energy

Extremotroph, ‐phile

An organism that lives in extreme environmental conditions such as temperature and pH

Facultative

Not required, optional

Fermentation

Obtaining energy by using an organic compound as both an electron donor and an electron acceptor

Halotroph, ‐phile

An organism that lives in high salt concentrations

Heterotrophy

Using organic compounds as sources of carbon

Hyperthermotroph, ‐phile

An organism with an optimal growth temperature of ≥80°C

Mesotroph, ‐phile

An organism that lives at moderate temperature with an optimal growth temperature from 15 to 40°C

Metabolism

All reactions, anabolic and catabolic, in a cell

Microaerotroph, ‐phile

An organism that requires low levels of oxygen

Obligate

Required

Photoautotrophy

Using light as an energy source while acquiring carbon only from carbon dioxide

Photoheterotrophy

Using light as an energy source while using organic compounds as sources of carbon

Phototroph

An organism that obtains energy from light

Psychrotroph, ‐phile

An organism that lives at low temperature with an optimal growth temperature of <15°C

Respiration

Obtaining energy by oxidizing chemical compounds through a series of reactions to a terminal electron acceptor

Thermotroph, ‐phile

An organism that lives at high temperature with an optimal growth temperature from 45 to 80°C

See also Madigan et al. (2012) and Slonczewski and Foster (2011).

1.2.2 Enzyme activity

Enzymes are important in microbial ecology. Microbial activity and metabolism can be measured in many different ways (e.g., carbon mineralization, calorimetry), but these metrics are often the result of enzyme‐substrate reactions, if not a direct measure of potential enzyme activity (e.g., Carter et al., 2008). Microbial enzymes can be classified in many ways, for example, as hydrolase, lyase, oxidoreductase, and transferase enzymes (Table 1.3). They can also be classified as intracellular and extracellular, which are important distinctions to consider; intracellular enzymes react with substrates within the microbial cell, while extracellular enzymes are released from microbial cells so that the enzyme–substrate product can then be transported across the cell membrane into the cell. Microorganisms commonly release extracellular enzymes into their habitat so that they can use the products to acquire the resources necessary for survival. This degradative and overall enzyme profile can be used to determine the range of substrates that can be used by a microbe (e.g., Pechal et al., 2013), known as community level physiological profiling (Degens and Harris, 1997). This potential substrate use is then modified by resource availability/quality, decomposer community, and physicochemical environment (Swift et al., 1979; Killham and Prosser, 2007).

Table 1.3 General classification of enzymes commonly associated with microorganisms

Enzyme

Examples

Function

Hydrolase

Amylase, chitinase, lipase, peptidase, phosphatase, phosphodiesterase, protease, sulfatase, urease

Catalyzes the hydrolysis of chemical bonds

Lyase

Aldehyde lyase, amino acid decarboxylase, cyclase, dehydratase

Catalyzes an elimination reaction and oxidation to break chemical bonds

Oxidoreductase

Alcohol oxidoreductase, amino acid oxidoreductase, ammonia monooxygenase, glucose oxidase, nitrite oxidoreductase, methane monooxygenase, monoamine oxidase, peroxidase

Catalyzes the transfer of electrons from the electron donor (reductant) to the electron acceptor (oxidant)

Transferase

Amino transferase, CoA transferase, methyltransferase, polymerase, kinase

Catalyzes the transfer of a functional group from one molecule (donor) to another (acceptor)

The release of extracellular enzymes into a habitat does introduce some complexity into interpreting the ecology of a microbe. Once extracellular enzymes are released from a microbial cell, they can function independently of the microbial cell. This ability means that microbial habitats can contain free enzymes capable of reacting with substrates. As a result, a microbial cell might take up the product of a reaction that used an enzyme it did not release. To further complicate matters, these free enzymes do not necessarily react with substrates soon after release from the microbial cell. They can be bound to organic material and inorganic surfaces, such as soil minerals, and even accumulate in habitats so that the measurement of enzyme activity might provide a misleading metric of microbial activity (Carter et al., 2007a). This phenomenon must be researched in greater detail before we truly understand postmortem microbial ecology.

1.3 Microorganisms and their habitats

Microorganisms and their activity are greatly influenced by their environment. Temperature, relative humidity, pH, oxidation–reduction potential (Eh), and oxygen availability are all environmental parameters that play a role in defining the microbial community. For example, the Psychrobacter sp. observed by Carter et al. (2015) in gravesoil during the winter in Nebraska, United States, is a taxon that prefers cold temperature. Many of these environmental parameters, such as ambient temperature and precipitation, can be measured with relative ease (see Chun et al.