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Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
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Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
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Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
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Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
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Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
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Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
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Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
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Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
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Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
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Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
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Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
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Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
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Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
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Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
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Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
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Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
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Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
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Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
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Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
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Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 20
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 21
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 22
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 23
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 24
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 25
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 26
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 27
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 28
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 29
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 30
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 31
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 32
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 33
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 34
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 35
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 36
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 37
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 38
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 39
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 40
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 41
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 42
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 43
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 44
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 45
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 46
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 47
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 48
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 49
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 50
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 51
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 52
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 53
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 54
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 55
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 56
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 57
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 58
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 59
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 60
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 61
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 62
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 63
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 64
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 65
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 66
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 67
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 68
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 69
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 70
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 71
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
Page 72
Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
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Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
×
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Suggested Citation:"Workshop Overview." Institute of Medicine. 2009. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/12586.
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Below is the uncorrected machine-read text of this chapter, intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text of each book. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.

Workshop Overview1 MICROBIAL EVOLUTION AND CO-ADAPTATION: A WORKSHOP IN HONOR OF JOSHUA LEDERBERG Prologue To a great extent, the Forum on Microbial Threats (hereinafter, the Forum) owes its very existence to the life and legacies of the late Dr. Joshua Lederberg. Dr. Lederberg’s death on February 2, 2008, marked the departure of a central figure of modern science. It is in his honor that the Forum hosted this public workshop on “microbial evolution and co-adaptation” on May 20 and 21, 2008. Along with the late Robert Shope and Stanley C. Oaks, Jr., Lederberg orga- nized and co-chaired the 1992 Institute of Medicine (IOM) study, Emerging Infections: Microbial Threats to Health in the United States (IOM, 1992). The Emerging Infections report helped to define the factors and dynamic relation- ships that lead to the emergence of infectious diseases. The recommendations of this report (IOM, 1992) addressed both the recognition of and interventions against emerging infections. This IOM report identified major unmet challenges in responding to infectious disease outbreaks and monitoring the prevalence of endemic diseases, and ultimately led to the Forum’s creation in 1996 (Morse, 2008). As the first chair of the Forum, 1996-2001, Dr. Lederberg was instrumen- tal in establishing it as a venue for the discussion and scrutiny of critical and sometimes contentiousscientific and policy issues of shared concern related to 1The Forum’s role was limited to planning the workshop, and this workshop summary has been prepared by the workshop rapporteurs as a factual summary of what occurred at the workshop. 

 MICROBIAL EVOLUTION AND CO-ADAPTATION research on and the prevention, detection, and management of infectious diseases and dangerous pathogens. Lederberg’s influence may readily be appreciated in the 2005 Forum work- shop Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship (IOM, 2006a). Its central theme was derived from a comprehensive essay entitled “Infectious History” that he published several years earlier in Science (Lederberg, 2000; reprinted as Appendix WO-1). Under the heading, “Evolving Metaphors of Infection: Teach War No More,” Lederberg argued that “[w]e should think of each host and its parasites as a superorganism with the respective genomes yoked into a chimera of sorts.” Thus began a dis- cussion that developed the concept of the microbiome—a term Lederberg coined to denote the collective genome of an indigenous microbial community—as a forefront of scientific inquiry (Hooper and Gordon, 2001; Relman and Falkow, 2001). Having reviewed the shortcomings and consequences of the war metaphor of infection, Lederberg suggested, in the same essay, a “paradigm shift” in the way we collectively identify and think about the microbial world around us, replacing notions of aggression and conflict with a more ecologically—and evolutionarily—informed view of the dynamic relationships among and between microbes, hosts, and their environments (Lederberg, 2000). This perspective recognizes the participation of every eukaryotic organism—moreover, every eukaryotic cell—in partnerships with microbes and microbial communities, and acknowledges that microbes and their hosts are ultimately interdependent upon one another for survival. It also encourages the exploration and exploitation of these ecological relationships in order to increase agricultural productivity and to improve animal, human, and environmental health. The agenda of the present workshop demonstrates the extent to which con- ceptual and technological developments have, within a few short years, advanced our collective understanding of microbial genetics, microbial communities, and microbe-host-environment relationships. Through invited presentations and dis- cussions, participants explored a range of topics related to microbial evolution and co-adaptation, including: methods for characterizing microbial diversity; model systems for investigating the ecology of host-microbe interactions and microbial communities at the molecular level; microbial evolution and the emer- gence of virulence; the phenomenon of antibiotic resistance and opportunities for mitigating its public health impact; and an exploration of current trends in infectious disease emergence as a means to anticipate the appearance of future novel pathogens.

 WORKSHOP OVERVIEW Organization of the Workshop Summary This workshop summary was prepared for the Forum membership by the rapporteurs and includes a collection of individually authored papers 2 and com- mentary. Sections of the workshop summary not specifically attributed to an individual reflect the views of the rapporteurs and not those of the Forum on Microbial Threats, its sponsors, or the IOM. The contents of the unattributed sections are based on presentations and discussions at the workshop. The workshop summary is organized into chapters as a topic-by-topic sum- mation of the presentations and discussions that took place at the workshop. Its purpose is to present lessons from relevant experience, to delineate a range of pivotal issues and their respective problems, and to offer potential responses as discussed and described by workshop participants. Although this workshop summary provides an account of the individual presentations, it also reflects an important aspect of the Forum philosophy. The workshop functions as a dialogue among representatives from different sectors and allows them to present their beliefs about which areas may merit further attention. The reader should be aware, however, that the material presented here expresses the views and opinions of the individuals participating in the workshop and not the deliberations and conclusions of a formally constituted IOM study committee. These proceedings summarize only the statements of participants in the workshop and are not intended to be an exhaustive exploration of the subject matter or a representation of consensus evaluation. THE LIFE AND LEGACIES OF JOSHUA LEDERBERG This workshop continued the tradition established by the late Joshua Lederberg, this Forum’s first chairman, of wide-ranging discussion among experts from many disciplines and sectors, honoring him by focusing on fields of inquiry to which he had made important contributions. At the same time, this gathering was unique in the history of the Forum, for it also offered participants a chance to reflect upon Lederberg’s life (see Box WO-1) and his extraordinary contribu- tions to science, academia, public health, and government. Formal remarks by David Hamburg of Cornell University’s Weill Medical College, Stephen Morse of Columbia University, and Adel Mahmoud of Princeton University (collected in Chapter 1) inspired open discussion of Lederberg’s life and legacy, as well as personal reminiscences about his role as mentor, advisor, advocate, and friend. Recalling the words of Ralph Waldo Emerson, who likened institutions to the lengthened shadows of their founders (Emerson, 1841), Morse observed that Lederberg’s influential shadow reaches into many places, but is most imposing in 2 Some of the individually authored manuscripts may contain figures that have appeared in prior peer-reviewed publications. They are reprinted as originally published.

 MICROBIAL EVOLUTION AND CO-ADAPTATION BOX WO-1 Joshua Lederberg: An Extraordinary Life • orn on May 23, 1925, in Montclair, New Jersey, to Zvi Lederberg, an orthodox B rabbi, and Esther Schulman, a homemaker and descendant of a long line of rabbinical scholars; Lederberg’s family moved to the Washington Heights area of upper Manhattan when he was six months old. • rom 1938-1940, attended Stuyvesant High School in New York City (a public, F highly competitive school of science and technology). • In 1941, enrolled at Columbia University, majoring in zoology. • n 1943, enrolled in the United States Navy’s V-12 training program, which I combined an accelerated premedical and medical curriculum to fulfill the armed services’ projected need for medical officers. • n 1944, received his bachelor’s degree in zoology at Columbia and began I medical training at the university’s College of Physicians and Surgeons. the area of infectious diseases, as epitomized by the Forum. Indeed, Forum mem- ber Stanley Lemon,3 of the University of Texas Medical Branch in Galveston, observed that the Forum’s mission—“tackling tough problems and addressing them with the best of science from the academic perspective and the active involvement of government”—is now borne by scores of people who can only hope to carry out what Lederberg once undertook single-handedly. As stated previously, it was largely due to Lederberg’s efforts, and particularly his co-chairmanship of the IOM Committee on Emerging Microbial Threats to Health, that the idea for a Forum became a reality. In recognition of the profound 3Vice-Chair from July 2001 to June 2004; Chair from August 2004 to July 2007.

 WORKSHOP OVERVIEW • n 1946, during a year-long leave of absence from medical school, carried I out experiments on Escherichia coli in the laboratory of Edward Tatum at Yale University. Lederberg’s findings demonstrated that certain strains of bacteria can undergo a sexual stage, and that they mate and exchange genes. • n 1947, having extended his collaboration with Tatum for another year in order I to begin mapping the E. coli chromosome, received his Ph.D. degree from Yale. He then received an offer of an assistant professorship in genetics at the University of Wisconsin, which caused him to abandon his plans to return to medical school in order to pursue basic research in genetics. He was accom- panied by his new wife, Esther Zimmer Lederberg, who received her doctorate in microbiology at Wisconsin and who also rose to prominence in that field. • n 1957, founded and became chairman of the Department of Medical Genet- I ics at Wisconsin and was elected to the National Academy of Sciences. • n 1958, became the first chairman of the newly established Department I of Genetics at Stanford University’s School of Medicine, days before being awarded the Nobel Prize in Physiology or Medicine, along with Tatum and George Beadle, for “discoveries concerning genetic recombination and the organization of the genetic material of bacteria.” • n 1966, his marriage to Esther Lederberg ended in divorce; in 1968 he mar- I ried Marguerite Stein Kirsch, a clinical psychologist, with whom he had two children. • rom 1966-1971, published “Science and Man,” a weekly column on science, F society, and public policy in The Washington Post. • In 1978, accepted the presidency of Rockefeller University. • In 1989, awarded the National Medal of Science. • n 1990, retired from the presidency and continued at Rockefeller as Raymond I and Beverly Sackler Foundation Scholar. • In 2006, awarded the Presidential Medal of Freedom. • On February 2, 2008, died of pneumonia at New York-Presbyterian Hospital. SOURCE: NLM (2008); photo courtesy of The Rockefeller University. impact of Emerging Infections: Microbial Threats to Health in the United States (IOM, 1992)—which provided the U.S. government with a basis for developing a national strategy on emerging infections and informed the pursuit of international negotiations to address this threat—the Centers for Disease Control and Preven- tion (CDC) and the National Institute of Allergy and Infectious Diseases (NIAID) asked the IOM to create a forum to serve as a follow-on activity to the national disease strategy developed by these agencies. In 1996, the IOM launched the Forum on Emerging Infections (now the Forum on Microbial Threats). Lederberg chaired the Forum for its first five years and remained an avid participant in its workshops and discussions until his failing health precluded travel.

 MICROBIAL EVOLUTION AND CO-ADAPTATION Even in his physical absence, the Forum has continued—and undoubtedly will continue—to be inspired by Lederberg’s expansive vision: a command of science that forged connections between microbiology and a broad range of disciplines, that was profoundly informed by history and literature, and that embraced the fullness of human imagination and possibility. Scientist “Joshua Lederberg has been the dominant force that shaped our thinking, responses, and intellectual understanding of microbes for much of the last half of the twentieth century,” Mahmoud remarked. From his early, Nobel Prize–winning work on bacterial recombination, accomplished while he was barely 20, through the last years of his life, when he continued to provide much sought-after advice to global policy makers on emerging infectious diseases and biological warfare, Lederberg extended his command of microbiology to profoundly influence a host of related fields, including biotechnology, artificial intelligence, bioinformatics, and exobiology. Exobiology, the study of extraterrestrial life, was one among many widely used terms coined by Lederberg, according to Stephen Morse. He also noted along with several other participants that the hero of the classic sci- ence fiction novel The Andromeda Strain4 (Crichton, 1969), Dr. Jeremy Stone, may well have been based on Lederberg. Ultimately, Lederberg viewed his wide- ranging scientific interests through the lens of evolution. According to Morse, the unifying theme of Lederberg’s scientific studies was to characterize sources of genetic diversity and natural selection. Nowhere is Lederberg’s comprehensive view of microbial evolution and its consequences more evident than in his essay, “Infectious History” (Lederberg, 2000), which informed the workshop’s agenda and serves as a framework for this workshop overview. Referring to that landmark publication as “the Bible of infectious diseases,” Mahmoud observed that it laid out “fundamental concepts that we are still debating about [including] the evolutionary biology and the ecol- ogy of microbes.” From his earliest years, Lederberg embodied scientific curiosity and innova- tion, David Hamburg noted. He recalled Lederberg’s knack for “turning an issue on its head, and thereby illuminating it,” and added that he “took deep, deep sat- isfaction in discovery, his own and others,” which was apparent in his relentless questioning. Lederberg “was a great challenger of the scientific community to pursue many ramifications of questions that appeared to be, at least for the time being, answered but were never answered for him,” Hamburg said. “This inter- 4 TheAndromeda Strain (1969), by Michael Crichton, is a techno-thriller novel documenting the efforts of a team of scientists investigating a deadly extraterrestrial microorganism that rapidly and fatally clots human blood. The infected show Ebola-like symptoms and die within two minutes (see http://en.wikipedia.org/wiki/The_Andromeda_Strain; accessed December 15, 2008).

 WORKSHOP OVERVIEW related set of attributes characterized Josh all his life and had much to do with his great accomplishments.” Hamburg recounted that Lederberg entered medical school at Columbia University with this intense curiosity and sense of discovery, as well as a desire to improve the lot of humanity and to relieve human suffering. Fascinated with bacterial genetics, however, Lederberg took a one-year leave from medical school to work on Escherichia coli with Edward Tatum, at Yale University, in 1946. “This was groundbreaking, highly imaginative work on the nature of microorgan- isms, especially their mechanisms of inheritance,” Hamburg said. “It opened up bacterial genetics, including the momentous discovery of genetic recombination,” a line of inquiry that paved the way for Lederberg’s being awarded the Nobel Prize in Physiology or Medicine in 1958, along with Tatum and George Beadle for “discoveries concerning genetic recombination and the organization of the genetic material of bacteria.” Following an extremely successful first year of research in Tatum’s labo- ratory, Lederberg decided to take another year away from medical school and continue to explore bacterial genetics. “We lost the budding physician in Joshua Lederberg by the end of the second year, because he was offered a faculty posi- tion at the University of Wisconsin,” Mahmoud explained, “but that did not stop Joshua Lederberg from being at the forefront of those concerned about human health and well-being.” According to Forum member Jo Handelsman, professor of bacteriology at the University of Wisconsin, Lederberg’s influence reverberates to this day. “He left behind the great legacy of his research and the spirit of a truly great mind in science,” she said, as well as stories that have attained the status of “urban legends.” At Wisconsin, Lederberg also established the legendary habit of appearing to sleep during seminars, after which he would ask difficult and prob- ing questions. This habit was still in evidence in the early 1990s during his co- chairmanship of the first IOM study on emerging infections, according to Forum member Enriqueta (Queta) Bond, president of the Burroughs Wellcome Fund. “I was the executive officer at the Institute of Medicine when the first Emerging Infections report was done,” she recalled. “I remember coming to one of the first meetings of the committee, and . . . Josh would sit there and you would think, ‘Is he awake? He’s supposed to be chairing this committee.’ . . . Then you would get the zingers from Josh: just the perfect question to move the agenda, develop the next topic, and so forth.” Indeed, Morse said, Lederberg “was never happier than when he was absorb- ing knowledge and questioning it. I like to think of this, with all of us here, as being an important part of Josh’s legacy,” he added. Hamburg recalled Lederberg’s “rare capacity to range widely with open eyes and open mind, and also dig deeply at times into specialized topics; to combine these capacities in research, educa- tion, and intellectual synthesis led to so much fruitful stimulation in a variety of fields.”

 MICROBIAL EVOLUTION AND CO-ADAPTATION “He believed that there are no limits to what the human mind can accom - plish, especially when its power is hitched to a willingness to think boldly and unconventionally, and to hard work,” Mahmoud said. “Until almost the day he died, Joshua could be found in his office, in his apartment, working. His mind was always thinking, always probing, always questioning.” Indeed, during his last days, Lederberg offered insightful advice to his longtime friend Hamburg, who was editing the final draft of his recently published book, Preventing Genocide: Practical Steps Toward Early Detection and Effective Action (Hamburg, 2008). “We had a couple of very intensive hours in which he asked his usual penetrat- ing questions and clarified key issues, and then was obviously quite exhausted,” Hamburg recalled. “We were prepared to take him back home. He said, ‘No. I’d like to rest for an hour or so and come back. I have one more chapter I want to discuss.’” “We did that,” Hamburg continued. “It was vintage Josh. He mobilized him- self to address an important problem with a friend that he valued and made an important contribution. The final changes in the book—all improvements—were due to that conversation.” Academic Another tribute to Lederberg’s remarkable capacities was institutional inno- vation, Hamburg observed. When Lederberg created departments of genetics in the medical schools at the University of Wisconsin and Stanford University, Hamburg recalled, “[the field of] genetics had been marginal or nonexistent in medical schools. There was a widely shared assumption, in the middle of the twentieth century, that genetics might be intrinsically interesting, but it would never have much practical significance for medicine.” “In teaching and in institution building, Lederberg emphasized the mutu- ally beneficial interplay of basic and clinical research,” Hamburg continued. Lederberg, he said, helped clinical departments at Stanford University’s School of Medicine build interdisciplinary groups and identify research opportunities and promising lines of innovation. He fostered many lines of inquiry within his own Department of Genetics at Stanford—including molecular genetics, cellu- lar genetics, clinical genetics, population genetics, immunology, neurobiology, and exobiology (particularly in relation to the National Aeronautics and Space Admininstration’s [NASA’s] Mariner and Viking missions to Mars)—and hired a superb group of internationally-known researchers, including Walter Bodmer and Eric Shooter from the United Kingdom, Luca Cavalli-Sforza from Italy, and Gus Nossal from Australia, Hamburg recalled. He also recruited from within the uni- versity, including speaker Stanley Cohen, who eventually succeeded Lederberg as chairman of the genetics department at Stanford. By taking this action, Hamburg said, Lederberg “was not robbing another department, but rather opening up an

 WORKSHOP OVERVIEW opportunity that Stan [Cohen] wanted and needed, and, of course, in which he made tremendous contributions.” While at Stanford, Lederberg also made a major contribution to undergradu- ate education, establishing a cross-disciplinary program in human biology that remains one of the university’s most sought-after majors. Hamburg—who as chairman of Stanford’s psychiatry and behavioral science department, assisted in this effort along with Donald Kennedy, then the chairman of Stanford’s biol- ogy department—remarked that the program might not have had such a long and illustrious history if Lederberg had not insisted that it include endowed chairs. Following his years at Stanford, Lederberg’s “rich experience, knowledge, skill, and wisdom were brought to bear on Rockefeller University under his presidency, broadening the scope of its great faculty, opening new opportunities for young people, and greatly improving the facilities,” Hamburg said. Although admitting that he did not at first think university administration was the best use of his friend’s talents, Hamburg recognized that Lederberg adapted well to his new responsibilities and proved adept both as a financial and a human resources manager who was deeply concerned about the personal well-being of his faculty. While it seems that nothing was too big for Lederberg to tackle, Forum mem- ber Gerald Keusch of Boston University described how he had benefited from Lederberg’s willingness to address what might have seemed a small issue. Dur- ing the mid-1990s, National Institutes of Health (NIH) director Harold Varmus was thinking about the impact on NIH of shrinking the number of institutes and centers, beginning with the Fogarty International Center. “Harold is a very smart person and knew there were going to be problems in trying to change the status quo. How to proceed? You form a committee to give you the recommendation that allows you to go ahead and act,” Keusch recalled. “So he asked Josh and Barry Bloom5 to do a review of the Fogarty and all international programs at the NIH.” Lederberg and Bloom proceeded to conduct an exhaustive study, which ultimately recommended that the Fogarty be strengthened, not disbanded. As a result, a new position was created—for which Keusch was hired—to direct the Fogarty International Center and serve as the NIH’s associate director for inter- national research. After five years in this position, Keusch asked Lederberg and Bloom to return and review the Fogarty’s progress. Although unwell and not traveling as he once had, Lederberg did not hesitate “to come back to do an honest, objective review and [once again] come out strongly in favor of the Fogarty’s international mission,” Keusch said. “You might have thought, in 1996, that Fogarty and the 5 In the mid-1990s, Barry Bloom was a Howard Hughes Medical Institute investigator and served on the National Advisory Board of the Fogarty International Center at the National Institutes of Health; see http://www.hsph.harvard.edu/administrative-offices/deans-office/dean-barry-r-bloom/.

0 MICROBIAL EVOLUTION AND CO-ADAPTATION international programs at NIH would not have attracted [Lederberg’s] attention. But they did, and I think the Fogarty is certainly the better for it, [as] is NIH.” Global Citizen Achieving the Nobel Prize at the age of 33 gave Lederberg a global per- spective that he fully embraced in the subsequent half-century, according to Mahmoud. In so doing, Lederberg undertook multiple roles, including advisor to governments, institutions, and industry, as well as educator of the general public. “Every president from John F. Kennedy to the current administration sought Joshua’s advice and consultation,” Hamburg said. “He chaired and studied issues from space science to human and artificial intelligence, to human-microbe inter- play.” Lederberg advised many agencies in the United States, most notably the NIH, the Centers for Disease Control and Prevention (CDC), the National Sci- ence Foundations (NSF), NASA, the Office of Science and Technology Policy (OSTP), and the Department of the Navy. He also served as an advisor to the World Health Organization (WHO) and was particularly influential as that orga- nization attempted to establish regional surveillance centers for emerging infec- tious diseases. Forum member James Hughes, of Emory University, remarked that Lederberg was “very engaged in Geneva, to the point that he took it upon himself to meet with the director-general of the WHO at the time, Dr. Hiroshi Nakajima. I am sure this is one of the reasons that WHO went on to develop its emerging infections focus.” “Josh used to go to Washington sometimes three times a week, back and forth, to give scientific advice,” Morse recalled. “He was the model of the sci- entific adviser. His advice was honest and dispassionate and in no way self- interested. His interest was furthering the cause of science and humanity.” Morse observed Lederberg had been concerned that samples obtained from space or spaceships might contain extraterrestrial life forms. NASA asked Lederberg how to decontaminate such samples and what precautions should be taken with them. “He gave very freely of his advice,” Morse said. “This led, I think, to one of the most interesting job descriptions I have ever seen. NASA created a position called ‘planetary quarantine officer.’6 Those of us who talk about emerging infections on this world have to realize that Josh’s purview extended far beyond that.” Emerging infections on Earth did, however, feature prominently in Lederberg’s advisory efforts, as many participants readily acknowledged. According to Mah- moud, “It was Josh, and Josh alone, who articulated and brought to the forefront of the scientific agenda the subject of emerging and reemerging infections.” Concern about emerging infections has grown following the appearance of new diseases, such as HIV/AIDS, and the reemergence of others, such as dengue, 6This position was later renamed by NASA as “Planetary Protection Officer, Earth.”

 WORKSHOP OVERVIEW and from appreciation of the complex determinants of their emergence—including microbial adaptation to new hosts (HIV infection, severe acute respiratory syn- drome [SARS]), population immunity pressures (influenza A), travel (acute hemorrhagic conjunctivitis), animal migration and movement (West Nile virus infection, H5N1 avian influenza), microbial escape from antibiotic pressures (multidrug-resistant and extensively drug-resistant tuberculosis), mechanical dis- persal (Legionnaires’ disease), and others (panel, Figure WO-1; Morens et al., 2008).7 Lederberg was also “a pioneer in biological warfare and bioterrorism defense, applying his farsighted vision to efforts to understand the danger and find ways to cope with it,” Morse said, long before that threat was widely acknowledged. “He strongly influenced the negotiation of the biological weapons disarmament treaty.”8 When Lederberg first voiced his concerns regarding emerging microbial threats in the late 1980s, Mahmoud recalled, “half of the scientific community was just smiling [as if to say], ‘the old man is just babbling about the subject.’” Instead, the advent of “a fundamental platform,” the 1992 IOM report, “really opened the way for a new way of thinking about microbes . . . [and also] forced the whole community to come back, in 2003, for the second report on the sub- ject.” Lederberg also co-chaired the committee that produced this second report, Microbial Threats to Health (IOM, 2003), along with current Forum co-chair, Margaret (“Peggy”) A. Hamburg of the Nuclear Threat Initiative/Global Health and Security Initiative (and daughter of David Hamburg). At an early conference on emerging viruses, in 1989, “somebody asked Josh, when should we declare that a virus is a new species or a new unknown virus?” Morse recalled, to which Lederberg gave the Solomonic answer, “When it matters.” “That was very much Josh’s way, to cut through all of the red tape and all of the inconsistencies and see straight to the heart of the matter,” Morse concluded. Lederberg strongly believed in educating the public about science and encour- aging public discussion of complex and politically and emotionally charged top- ics, Peggy Hamburg said. The tangible evidence of this belief can be found in the columns on science and society that Lederberg wrote for The Washington Post between 1966 and 1971, and that have been collected by the National Library of Medicine at its website, “Profiles in Science” (NLM, 2008). As David Hamburg remembered, “many in the scientific community thought, why would a person of his gifts devote that kind of time to the public?” Lederberg believed, however, 7 For more information, see also IOM (1992, 2003); Morens et al. (2004); Parrish et al. (2008); and Stephens et al. (1998). 8The Convention on the Prohibition of the Development, Production and Stockpiling of Bacterio- logical (Biological) and Toxin Weapons and on their Destruction; signed on April 10, 1972; effective March 26, 1975. As of July 2008, there were 162 states party to this international treaty to prohibit an entire class of weapons.

 A Human African trypanosomiasis Cholera Marburg haemorrhagic fever MDR/XDR tuberculosis Plague Human monkeypox Chikungunya fever Enterovirus 71 Hendra virus Nipah virus Vancomycin-resistant Staphylococcus aureus H5N1 influenza Escherichia coli O157:H7 SARS Anthrax bioterrorism† Hantavirus pulmonary syndrome Typhoid fever Dengue Rift Valley fever Yellow fever Diphtheria HIV Drug-resistant malaria Lassa fever Ebola haemorrhagic fever Lyme disease Cryptosporidiosis Hepatitis C West Nile virus vCJD Cyclosporiasis B The French pox (syphilis), 1494 The American plague (yellow fever), 1793 Hueyzahuatl (smallpox), 1520† Anthrax, 1770 † Cholera, 1832 HIV/AIDS, circa 1930

HIV Drug-resistant malaria Lassa fever Ebola haemorrhagic fever Lyme disease Cryptosporidiosis Hepatitis C West Nile virus vCJD Cyclosporiasis B The French pox (syphilis), 1494 The American plague (yellow fever), 1793 Hueyzahuatl (smallpox), 1520† Anthrax, 1770 † Cholera, 1832 HIV/AIDS, circa 1930 Spanish influenza, 1918 Measles, 1875 The Black Death (plague), 1347–50 The Plague of Athens (unidentified disease), 430 BC Newly emerging Re-emerging Deliberately emerging† FIGURE WO-1 Newly emerging, reemerging or resurging, and deliberately emerging diseases. (A) Selected emerging diseases of public-health importance in the past 30 years (1977-2007), with representative examples of where epidemics occurred. (B) Selected emerging diseases of public-health importance in previous centuries (430 B.C. to 1981 A.D.). MDR = multidrug-resistant; SARS = severe acute respiratory syndrome; Figure WO-1A COLOR.eps vCJD = variant Creutzfeldt-Jakob disease; XDR = extensively drug-resistant. SOURCE: Reprinted from Morens et al. (2008) with permission from Elsevier. landscape  could be larger if presented on 2-page spread

 MICROBIAL EVOLUTION AND CO-ADAPTATION that an informed public was essential not only for good science policy, but ulti - mately for human survival, Hamburg said. It should be noted that many of his columns in The Washington Post addressed the health implications of environ- mental conditions. Forum member Terence Taylor, director of the International Council for Life Sciences, asked Dr. David Hamburg to speculate on what Lederberg would say to the next U.S. president if he had been asked to name priorities for enhancing scientific advice to the nation’s leadership. Hamburg observed that Lederberg “had a fundamental concern about the relationship between scientific expertise and political leadership. On the one hand, he thought it was enormously impor- tant for our political leaders to have access to and appreciation of the scientific community. . . . However, he was equally concerned that we as scientists might inadvertently mislead political leaders, with even the best of intentions.” Hamburg believed that Lederberg would stress the importance of a diversity of expert advice, through processes that invite experts from different arenas to challenge each other. “He never, I think, to the end of his life, was satisfied that we had found the right formula for that,” Hamburg added, “but I’m sure he would tell the new president, ‘Make much more use of the scientific community than your predecessor has done and do it with much less ideology or political slant. . . . Don’t just pick people you know, but reach out to get people that you don’t know and have never heard of that you have some reason to believe are excellent.’” Moreover, Hamburg said, Lederberg would encourage further efforts toward improving the yet-unresolved and vital relationship between scientific expertise and political leadership. “I simply know of no eminent scientist of such immense stature, who gave so much serious analysis of public policy and social problems,” Hamburg concluded. “Our country and the world are in his debt. Those of us here today profoundly appreciate what he did, not just for science, but for humanity. His life exempli- fied the finest attributes of the great institution in which we meet today to honor his memory.” Mentor, Colleague, and Friend In the course of remembering Lederberg’s prodigious accomplishments, workshop participants also reflected upon the ways in which he had touched their lives and careers, further revealing his extraordinary character. Forum co-chair Peggy Hamburg—whose experiences with Lederberg evolved from those of a young daughter of a colleague (exploring tidal pools) to that of a professional peer (co-chairing the IOM Committee on Microbial Threats to Health in the 21st Century)—recalled a man who “loved to go out and walk and talk and study the life on the beach and in the tide pools and see what you could discover in there and how it changed.” He was also the first person she knew who owned a computer: “I remember being brought over and sort of ushered into the room, as

 WORKSHOP OVERVIEW though it was almost a temple.” As a child, Lederberg seemed to her “the epitome of a mad scientist.” However, “as I got older and learned more, I realized that he was, in fact, this extraordinary presence in the field of science.” Peggy Hamburg went on to observe, “I would say that one of the things that I actually appreciated most about Josh was that even though he had gotten to know me when I was just a kid, he was able to make the transition—and I don’t know exactly when it happened—to really treating me as a peer and a colleague. That is, I think, quite extraordinary, particularly in someone of his generation.” As one might expect, Lederberg’s leisure interests were largely intellectual, including technology in all its forms and reading widely and voraciously (he had a particular fondness for the Times Literary Supplement), according to Morse. “A kind of recreation for him was to meet someone about whom he had heard good things, in a completely, even wildly different field . . . and through conversation with that person, to get some idea of what was going on in many different fields,” David Hamburg recalled. He was also a phenomenal correspondent, as attested by many workshop participants who received handwritten notes, telephone calls, and e-mails from him over the years. Lederberg had special memo pads upon which he would write notes that were challenged and challenging to many people, Hamburg included. “At first I thought he was just picking on me,” he said. “He explained to me that he didn’t really expect that the person receiving it would respond or necessarily act on it, but he thought from what he knew of the person’s interest that this was something that he or she ought to know about. It was kind of a way of needling us all to broaden our horizons.” When he became director of what was then the Hospital Infections Program at the Centers for Disease Control and Prevention, Hughes was at first amazed to be receiving notes from Lederberg, who had been a figure of awe to Hughes as a medical student at Stanford. Then, Hughes said, “I began to get notes from him asking very interesting and challenging questions that I had never been asked before and that, of course, I never knew the answers to and had great difficulty finding anyone else who knew the answers to his questions.” Speaker Bruce Levin, of Emory University, was equally challenged by com- munications he received from Lederberg. “It was always a delight for me to receive those e-mails,” he said. “The questions Josh asked would sometimes keep me busy for a day, making me think about things I thought I knew, but really didn’t. While I don’t know whether he got much out of my answers, I know I learned a great deal by thinking about his questions.” “Josh’s notes have always been insightful,” added Cohen. “I can’t imagine how he found the time to write all of the notes he has written to all of us over so many years, and keep track of our interests, and pick out exactly the relevant things to say at particular times. . . . I really miss them.” Speakers Mark Woolhouse, of the University of Edinburgh, and Margaret

 MICROBIAL EVOLUTION AND CO-ADAPTATION McFall-Ngai, of the University of Wisconsin, were both surprised to hear from Lederberg when their work caught his attention. In Woolhouse’s case, it was a catalog of human pathogen species (see Woolhouse and Gaunt in Chapter 5), which caused him to reflect that while his group employs various forms of sophis- ticated mathematics and modeling in many of their studies, “Josh Lederberg liked our work because we can count. So when somebody of that eminence says he likes your work because you can count, you count some more.” McFall-Ngai was a young associate professor, in 1998, when Lederberg e-mailed her after reading a piece she wrote for the American Zoologist (McFall- Ngai, 1998). “At first I thought it was spam,” she admitted. “Why would Joshua Lederberg write to me? I was getting ready to trash it and I thought, okay, I’ll open this up. It started an e-mail volley between him and me, several back-and- forths, about the role of beneficial microbes.” In Memoriam Recalling his own childhood in Egypt, Mahmoud observed that imposing monuments, such as the Great Sphinx, were a testament to the enormous egos of the rulers who ordered their construction. “They wanted to be sure that long after they were gone, people would be able to gaze upon their mighty works and remember that a great man once ruled here,” he said. “Joshua Lederberg, of course, needs no [such] monuments to ensure that his life and work are long remembered. In a very real sense, his accomplishments are embedded in the DNA of many whose lives have been shaped because of his work. That work and those concepts will be passed on to every generation yet to come, long after the Great Sphinx has crumbled into dust.” “Joshua believed very strongly in the work of this Forum,” Mahmoud con- tinued. “He had great confidence in the ability of scientists and researchers to continue to solve some of the riddles that still confront science in the fight against infectious diseases. By remembering him with this tribute, we are also remem- bering the many things that his life and career can teach all of us. I hope that every time we meet at this Forum, Joshua Lederberg will be an inspiration and a reminder that our work can truly change the world, just as his life and career certainly did.” MICROBIAL ECOLOGY AND ECOSYSTEMS Perhaps one of the most important changes we can make is to supercede the 0th-century metaphor of war for describing the relationship between people and infectious agents. A more ecologically informed metaphor, which includes the germs’-eye view of infection, might be more fruitful. Consider that microbes

 WORKSHOP OVERVIEW occupy all of our body surfaces. Besides the disease-engendering colonizers of our skin, gut, and mucous membranes, we are host to a poorly cataloged en- semble of symbionts to which we pay scant attention. Yet they are equally part of the superorganism genome with which we engage the rest of the biosphere. Joshua Lederberg, “Infectious History” (2000) More than a century of research, sparked by the germ theory of disease and rooted in historic notions of contagion that long precede Pasteur and Koch’s nineteenth-century research and intellectual synthesis, underlies current knowl - edge of microbe-host interactions. This pathogen-centered understanding attrib- uted disease entirely to the actions of “invading” microorganisms, thereby drawing the lines of battle between “them” and “us,” the injured hosts (IOM, 2006a). The paradigm of the systematized search for the microbial basis of disease, followed by the development of antimicrobial and other therapies to eradicate these disease-causing “agents,” is now firmly established in human and veterinary clinical practice. The considerable impact of this approach, assisted by improvements in sanitation, diet, and living conditions in the industrialized world, once led us to believe that we humans were engaged in a war against pathogenic microbes, and that we were winning (IOM, 2006a; Lederberg, 2000). By the mid-1960s, experts opined that, since infectious disease was all but controlled, researchers should focus their attention on other chronic disease challenges, such as heart disease, cancer, and psychiatric disorders. This optimism coupled with several decades of complacency was profoundly shaken by the appearance in the early 1980s of HIV/AIDS, and was dealt a fur- ther blow with the emergence and spread of multidrug-resistant bacteria (IOM, 2006a). As these experiences began to lead researchers to reexamine the host- microbe relationship, additional reasons to do so began to accumulate: pandemic threats from newly emergent (e.g., SARS) and reemergent (e.g., influenza) infec- tious diseases; lethal outbreaks of Ebola, hantavirus, and other exotic viruses of animal origin; and a new appreciation for the infectious etiology of a variety of chronic diseases, including the association of peptic ulcer with Helicobacter pylori infection, liver cancer with hepatitis B and C viruses, and Lyme arthritis with Borrelia burgdorferi, to name a few. In certain lines of inquiry the advantages of adopting an ecological frame- work for understanding the dynamic equilibria of host-microbe-environment interactions have become evident. Studies of the microbiota of the human gas- trointestinal tract—a complex, dynamic, and spatially diversified community comprising at least 1013 organisms of more than 1,000 species, most of which are anaerobic bacteria—reveal that these microbes comprise an exquisitely tuned metabolic “organ” that mediates both energy harvest and storage (Bäckhed et al., 2004). Research on the biocontrol agent Bacillus cereus suggests that it reduces

 MICROBIAL EVOLUTION AND CO-ADAPTATION disease in alfalfa and soybeans by modifying the composition of the microbial community associated with the plants’ roots to make it resemble that of the sur- rounding soil (Gilbert et al., 1994). Such promising discoveries were anticipated by Lederberg, who also noted that superinfections associated with antibiotic therapy attested to the protection naturally conferred by microbial communities in dynamic equilibria. “Understanding these phenomena affords openings for our advantage, akin to the ultimate exploitation by Dubos and Selman Waksman of intermicrobial competition in the soil for seeking early antibiotics,” he wrote (Lederberg, 2000). “Research into the microbial ecology of our own bodies will undoubtedly yield similar fruit.” This challenge has been taken up, and elaborated upon, by several workshop presenters, including Forum chair David Relman of Stanford University, who noted that the scientific community has known for hundreds of years—beginning with van Leeuwenhoek’s observations of the morphological diversity of microbes in his own dental plaque—that a complex microbiota exists within the human body. Equally complex “host-less” microbial communities exist in the form of biofilms—complex aggregations of microorganisms that grow on solid sub- strates—as described by speaker Jill Banfield of the University of California, Berkeley. The diversity of mutually beneficial host-microbe interactions was reflected in a pair of presentations by Margaret McFall-Ngai and Jean-Michel Ané, both of the University of Wisconsin, Madison, who described the symbiotic relationships between bacteria and eukaryotes that either allow squids to cam- ouflage themselves from aquatic predators, or enable plants to acquire nutrients through their roots. Communities of Microbes and Genes Exploring the Human Microbiome9 Microbes colonize the human body during its first weeks to years of life and establish themselves in relatively stable communities in its various microhabitats (Dethlefsen et al., 2007). The human microbiome is far from being fully appreci- ated or definitively described. Research to date suggests that while site-specific communities (such as skin, mouth, intestinal lumen, small intestine, and large bowel, to name a few) of most individual humans contain characteristic microbial families and genera, the exact mix of species and strains of microbes present in any given individual may be as unique as a fingerprint. The microbiomes of other 9The microorganisms that live inside and on humans are known as the microbiota; together, their genomes are collectively defined as the microbiome, a term coined by Lederberg (Hooper and Gordon, 2001). However, since most of the organisms that make up the microbiome have resisted cultivation in the laboratory, and thus are known only by their genomic sequences, the microbiota and the mi- crobiome are largely one and the same.

 WORKSHOP OVERVIEW terrestrial vertebrates are dominated by organisms related to, but distinct from, those found in humans. This suggests that host species have co-evolved with their microbial flora and fauna. Through their explorations of the human microbiome, Relman and cowork- ers seek to understand the role of indigenous microbial communities associated with human health, disease, and the various transition states in between. By understanding essential features of symbiotic relationships between microbial communities and their human hosts, they hope eventually to be able to predict host phenotypes—such as health status—that are associated with particular fea- tures of indigenous communities, and potentially manipulate these communities to restore or preserve health. This effort is at an early stage of development, with research focused on identifying elements of microbial communities that can be monitored and measured to assess physical and metabolic interactions within and among microbial communities and between human and microbial cells. One such important, and measurable, characteristic of microbial communi- ties is their diversity, as reflected in the number of different ribosomal RNA sequences present in a given location in the human body. These highly-conserved sequences also reveal microbial ancestry and phylogenetic relatedness, permit- ting the construction of phylogenetic trees (see Relman in Chapter 2, especially Figure 2-1). The organisms represented by these sequences remain largely uncul- tivated. Sequences derived by Relman and coworkers in 2005, from the microbial inhabitants of human colonic tissue, suggested that approximately 80 percent had not yet been cultured, and about 60 percent had not been previously described (Eckburg et al., 2005). This analysis also revealed a striking diversity of microbes at the genus and species level, but affiliated with relatively few phyla, a pattern apparently com- mon among indigenous microbial communities of vertebrates, but not among microbial communities found in external environments. The dearth of microbial phyla on or within the human body probably results from multiple influences, including selection, environmental factors, and even early opportunistic environ- mental exposures to particular microorganisms. Further, samples collected from various locations in the gut of several subjects revealed greater variation in the diversity of microbial communities between these hosts than was present within an individual host (Eckburg et al., 2005). Similarly distinct gut communities were found by Relman and coworkers in each of 14 babies, whose feces were sampled periodically throughout the first year of life (Palmer et al., 2007). The composi- tion and temporal patterns of the microbial communities varied widely from baby to baby, especially early in the first year of life, but the patterns converged by the end of the first year towards a distinct signature for each baby, as well as towards a generic adult signature (Figure WO-2). Clinical problems associated with the human microbiota include chronic peridontitis, Crohn’s disease and other forms of inflammatory bowel disease, tropical sprue, antibiotic-associated diarrhea, bacterial vaginosis, and premature

20 MICROBIAL EVOLUTION AND CO-ADAPTATION Relative Abundance (% Total Bacteria) FIGURE WO-2  Temporal  profiles  of  the COLOR.eps Figure WO-2 most  abundant  level  3  taxonomic  groups.  bitmap image Level 3 taxonomic groups were selected for display if their mean (normalized) relative  abundance across all baby samples was greater than 1 percent. The x-axis indicates days  since birth and is shown on a log scale, and the y-axis shows estimated (normalized) rela- tive abundance. For some babies, no values are plotted for the first few days because the  total amount of bacteria in the stool samples collected on those days was insufficient for  microarray-based analysis.  SOURCE: Palmer et al. (2007). labor  and  delivery  (see  Relman  in  Chapter  2).  Considerable  evidence  suggests  that  the  indigenous  microbiota  is  altered  during  states  of  infectious  disease,  especially  those  diseases  that  involve  the  mucosal  or  skin  surfaces  that  serve  as a contact boundary. In some cases, it appears that the indigenous microbiota  propagates  the  disease  process. Treatment  of  such  clinical  problems  with  anti-

 WORKSHOP OVERVIEW biotics, or diversion of the luminal flow away from a segment of bowel, reduces inflammation and other symptoms. These features suggest a system in which the collective microbial community acts as a pathogen, and in which disease results from community disturbance, rather than from infection by a specific organism or group of organisms. It also invites an ecological view of infectious disease control that seeks to restore community equilibrium following disturbance. In order to study the effects of such disturbances, Relman and coworkers have examined patterns of microbial diversity in the human gut before, during, and after deliberate, periodic, exposure of healthy human subjects to the antibiotic ciprofloxacin. They identified approximately 5,800 different species or strains of bacteria from these samples, of which only 6 percent had been seen before. All three subjects studied so far showed significant reduction in the number of bacterial species present following antibiotic treatment, the result of which was a partial elimination of the differences in community structure that distinguished the three host individuals. “What makes the human microbiome so intrinsically interesting, at least to me, is the degree to which it may reflect who we are as individuals and as a host species,” Relman said, and this individuality has implications for health and dis- ease. While the human microbiome remains largely uncharacterized, Relman held out hope that, thanks to the progress of microbiology since van Leeuwenhoek, we now possess sufficient experimental technology and clinical opportunities to explore the microscopic terra incognita10 within and upon us all. Biofilms and the Processes That Shape Them Microbe-microbe relationships include nutritional interactions (e.g., the step- wise processing of plant polysaccharides in the human gut by members of the microbiota) and genetic exchanges that occur through transformation, phage transduction, and conjugation (IOM, 2006a). The last of these processes, bacte- rial conjugation, first described by Lederberg and coworkers, earned him the Nobel Prize in 1958. Indeed, horizontal gene transfer—also known as lateral gene transfer (Eisen, 2000)—among members of some microbial communities appears to be an extremely pervasive process, but perhaps not to the extent as to call into question whether the concept of speciation applies to communal microbes (Eppley et al., 2007). Investigations of microbial biofilm communities—which grow on substrates such as rocks in freshwater streams, drains, and teeth11—are providing insights into the ecological and evolutionary processes that shape microbial communi- ties. The microbial constituents of the biofilm known as dental plaque include 10 Latin term for “unknown land.” 11 Biofilms are not restricted to streams, drains, and teeth. They are also found on natural and man- made objects, including catheters and other indwelling devices.

 MICROBIAL EVOLUTION AND CO-ADAPTATION hundreds of species and strains of bacteria,12 as well as various methanogens (Archaea) whose collective metabolic activities are associated with gum dis- ease and tooth decay (Lepp et al., 2004). Biofilms containing iron- and sulfur- oxidizing microbes also thrive in mines and in watersheds where mine wastes drain, resulting in the release of acids and toxic metals into creeks and streams (Banfield, 2008a). This process, called acid mine drainage (AMD), impairs bio- diversity and ecological productivity in aquatic ecosystems and, in some cases, precludes inhabitation by macroorganisms altogether (Klemow, 2008). Horizontal gene exchange appears to help microbes in these biofilms adapt to this extreme environment (Lo et al., 2007). Banfield and coworkers use mine-derived biofilms as a model system to examine how relatively simple microbial communities (that is, communities dominated by a few types of organisms) organize themselves and how their mem- bers interact with each other and their physical surroundings (Banfield, 2008b). Biofilms “grow”—that is, they add or accumulate increasingly large populations of microbes—in stages. In this setting, a biofilm nucleus begins at a stream’s margins and extends across the water’s surface toward its center, while simultane- ously increasing in thickness. In her workshop presentation, Banfield described her group’s efforts to characterize this process by comparing genomic and protein profiles of biofilms at early and late stages of development. Using metagenomic13 methods, Banfield and coworkers have constructed near-complete collective genomes from several different mine-derived biofilm communities (see Chapter 2 Overview). These proved to be dominated by mem- bers of bacterial Leptospirillum groups II and III, but the biofilm communities also contained several uncultivated Archaea species, as well as some novel organ- isms. In comparisons of 27 early- and late-stage biofilms, the researchers found that early biofilms, which were dominated—in some cases, almost exclusively— by Leptospirillum group II, later developed into more complex communities with more diverse members, including greater numbers of Leptospirillum group III bacteria and more species of Archaea. To examine how this changing cast of organisms functions in the commu- nity, and how their functions change as the community develops, the researchers used proteomic14 methods to determine whether, much like the community’s 12Anton van Leeuwenhoek was the first to see and describe plaque bacteria through a microscope in 1674. For more information about this inventor, see http://inventors.about.com/library/inventors/ blleeuwenhoek.htm (accessed December 15, 2008). 13 Metagenomics involves obtaining DNA from communities of microorganisms, sequencing it in a “shotgun” fashion, and characterizing genes and genomes comparisons with known gene sequences. With this information, researchers can gain insights into how members of the microbial community may interact, evolve, and perform complex functions in their habitats (Jurkowski et al., 2007; NRC, 2007). 14Analogous to genomic methods, proteomics permits the identification of expressed proteins from an individual or community.

 WORKSHOP OVERVIEW taxonomic composition, the genes being expressed by its members changed over the course of development (see Chapter 2 Overview). Significant shifts in protein expression correlated with the sequential domination of the community by two different but closely-related strains from Leptospirillum group II. This result suggests that different suites of proteins, as well as genotypes, perform different functions at different times in these communities, Banfield concluded. Further characterization by Banfield and coworkers of 27 biofilms of various stages of development, sampled from eight different microenvironments at the same iron mine, revealed the presence of six distinct genotypes (Lo et al., 2007); each contained blocks of sequence from the two closely-related Leptospirillum group II strains. Many of the biofilm samples were found to contain only one genotype; others had several (Denef et al., 2009). The researchers also examined the distribution of genotypes across the eight sampling sites (Figure WO-3). Over the course of more than two years, they consistently found the same genotype at one site—despite the fact that biofilms at this site would have had constant expo- sure to other genotypes. Thus, Banfield concluded, there appeared to be strong local selection for this particular genotype, which has “achieved a fine level of adaptation to environmental opportunity” (Figure WO-3). Banfield’s group has also examined the role of viruses in biofilms, and particularly the viral “predators” of the dominant bacterial species in these com- munities. Their investigations were inspired by recent reports (Makarova et al., 2006; Mojica et al., 2005) that the genomes of most Bacteria and Archaea con- tain repeat regions, known as clustered regularly interspaced short palindromic repeats (CRISPRs). Derived from coexisting viruses, CRISPRs appear to provide immunity (perhaps via RNA interference) to their possessors for the virus of its derivation. Thus, Banfield said, “a microbe has a level of immunity to a virus, so long as it has the spacers that match it or silence it. It has been shown experi- mentally by the Danisco Group that should a mutation occur such that the spacer is no longer effective, the virus may proliferate and the microbe will suffer” (Barrangou et al., 2007). However, she added, another component of the bacte- rial system, CRISPR-associated proteins, rapidly sample the local viral DNA and incorporate new spacers, conferring the population with a range of immunity levels to different mutant viruses as they arise (Tyson and Banfield, 2008). Taking advantage of the correspondence in CRISPR sequences between viruses and their host microbes (see Chapter 2 Overview), Banfield and cowork- ers identified the sequences of viruses that target Bacteria and Archaea present in acid mine drainage biofilms (Andersson and Banfield, 2008; Figure WO-4). Their investigation revealed a picture of microbial interaction within the biofilm, where a “cloud of viruses” maintains high levels of sequence diversity by various means in order to defeat host microbes, while the hosts counter by rapidly acquir- ing viral spacers, and, thereby, immunity. Overall, Banfield said, this dynamic system is probably in stasis; nevertheless, she added, “it’s clearly an example of co-evolution in a virus and host community.”

 MICROBIAL EVOLUTION AND CO-ADAPTATION FIGURE WO-3 Six genotypes of Leptospirillum group II bacteria were detected in the Figure WO-3 COLOR.eps Richmond Mine (Iron Mountain, CA) by proteomic-inferred genome typing and inferred bitmap image to have arisen via homologous recombination between parental genotypes. The types (shown schematically as mixtures of red and blue genome segments) were observed in biofilms at locations shown on the map. Each pie chart displays the genome type com- position in each sample (samples 34 and 35 were typed via community genomics). The selection for specific genotypes, despite system-wide dispersal of all types, indicates that recombination serves as a mechanism for fine-scale adaptation. SOURCE: Adapted with permission from Denef et al. (2009).

 WORKSHOP OVERVIEW FIGURE WO-4 Virus-host associations in AMD biofilms. Putative viral (SNC) contigs Figure WO-4 COLOR.eps were clustered based on tetranucleotide frequencies (left panel), and CRISPRs were clus- bitmap image tered based on patterns of SNC contig matching (right panel). Columns in the left panel represent tetranucleotides (reverse complementary pairs are combined); colors indicate frequencies (gray indicates absence). Columns in the right panel represent CRISPRs; colors indicate number of distinct spacer sequences matching SNC contigs. The majority of Cluster 1 (C1) contigs belong to the AMDV1 population, Cluster 2 (C2) to AMDV2, and Cluster 3 (C3) to AMDV3, AMDV4, and AMDV5. SOURCE: Andersson and Banfield (2008). Models of Coexistence and Cooperation Quoting Heinrich Anton de Bary15 in his 1879 monograph Die Erscheinung der Symbios, Ané defined symbiosis as “a prolonged living-together of different 15 Heinrich Anton de Bary (January 26, 1831-January 19, 1888) was a German botanist whose researches into the roles of fungi and other agents in causing plant diseases earned him distinction as a founder of modern mycology and plant pathology. De Bary determined the life cycles of many fungi, for which he developed a classification that has been retained in large part by modern my- cologists. Among the first to study host-parasite interactions, he demonstrated ways in which fungi penetrate host tissues (see www.britannica.com/EBchecked/topic/54513/heinrich-anton-de-bary, ac- cessed March 10, 2009).

 MICROBIAL EVOLUTION AND CO-ADAPTATION organisms that is beneficial for at least one of them.” He noted that this general description applies to a continuum of interactions ranging from the extreme of strict mutualism, which benefits both partners, to the opposite extreme of parasit- ism, which benefits one partner and is detrimental to the other (Figure WO-5). Individual symbioses evolve over time, and under the influence of a variety of environmental, physiological, and developmental factors. In order to capture certain aspects of this complexity and gain insights into the bases of symbioses, biologists develop models of colonization. These fall into two main categories, according to McFall-Ngai: constructed models, based on germ-free hosts such as mice and zebrafish, which allow investigators to study colonization as microbes are introduced in a controlled manner (see, for example, IOM, 2006a); and natural models, which permit researchers to observe the process of colonization, typically by only a few microbial phylotypes, as it occurs naturally in a variety of hosts and sites. Existing models of the latter type include the guts of certain insects (such as the gypsy moth, described below, and by Handelsman in Chapter 4), as well as the two systems described in workshop presentations and discussed below: plant roots and the light organ of the Hawai- ian squid. Plant Root Symbionts In relationships somewhat analogous to those that exist between mammals and their gastrointestinal microbiota, plants establish mutualistic associations CHEATER + Parasitism Parasitism Commensalism Mutualism Species 0 Competition Neutralism Commensalism “A” – Competition Competition Parasitism – 0 + Species “B” FIGURE WO-5 The symbiotic continuum. SOURCE: Figure courtesy of John Meyer, North Carolina State University.

 WORKSHOP OVERVIEW with several microorganisms (see Chapter 2 Overview). The roots of most higher plant species form arbuscular mycorrhiza, associations with specific fungal spe- cies that significantly improves the plant’s ability to acquire phosphorus, nitrogen, and water from the soil (Brelles-Mariño and Ané, 2008). This type of interaction dates back approximately 460 million years and has played a central role in the evolution of land plants, according to Ané. A more recent association, over the past 60 million years, involves legumes and nitrogen-fixing bacteria named rhizobia. The bacteria induce and colonize new organs on the plant’s roots, called nodules; there, they receive energy in the form of carbon from the plant and convert atmospheric nitrogen to ammonia for the plant’s use. This partnership furnishes much of Earth’s biologically available nitrogen and boosts productivity in non-leguminous crops that are grown in rota- tion with legumes. Symbiotic relationships between plants and bacteria or fungi are established through chemical and genetic “cross-talk.” As shown in Figure WO-6, legume roots release compounds that trigger nitrogen-fixing rhizobia to express modified chitin oligomers called Nod factors, which in turn facilitate infection of the root by the bacteria, as well as nodule development (Brelles-Mariño and Ané, 2008; Riely et al., 2006). Plants also produce chemical signals called strigolactones that increase branching of fungal hyphae, and thereby increase their contact with arbuscular mycorrhizal fungi. The fungi release diffusible compounds known as ARBUSCULAR HOST LEGUME PLANT RHIZOBIA MYCORRHIZAL FUNGI FIGURE WO-6 Symbiotic relationship between plants and bacteria. Legume roots re- lease compounds that trigger nitrogen-fixing rhizobia to express modified chitin oligomers called Nod factors, which in turn facilitate infection of the root by the bacteria, as well as New WO-6 nodule development. SOURCE: Figure courtesy of Jean-Michel Ané.

 MICROBIAL EVOLUTION AND CO-ADAPTATION Myc factors, which, when recognized by the plant, activate symbiosis-related genes. The discovery that a largely shared signaling pathway makes possible both arbuscular mycorrhization and legume nodulation—despite their apparent differences—has led to the conclusion that plants have a single, highly-conserved genetic program for recognizing beneficial microbes, according to Ané. Both microbial Nod and Myc factors also appear to have common features, including the ability to promote plant growth, which may benefit microbes by increasing the availability of infection sites, he said. Plant-microbe symbioses do not exist in a vacuum, but are challenged by “cheaters” and parasites (see Chapter 2 Overview). The cheaters include indi- vidual rhizobial colonists of legume nodules that do not fix nitrogen efficiently, and thereby act as parasites, receiving carbohydrates without offering anything in return (and without expending the considerable energy involved in fixing nitrogen), Ané explained. However, their hosts appear to have ways of detect- ing these microbial freeloaders and “sanctioning” them. Some researchers have hypothesized that the plant decreases oxygen supplies to under-performing nod- ules (Kiers et al., 2003). While the actual mechanism remains unknown, Ané said, he suspects that the plant may starve the cheaters by reducing their access to carbohydrates. Parasites on plant roots include root-knot nematodes, nearly ubiquitous pathogens that account for up to 10 percent of global crop loss, according to Ané. Evidence suggests that these nematodes infect legume roots by using genetic pathways adapted for rhizobial colonization, perhaps by producing molecular mimics of Nod factors (Weerasinghe et al., 2005). Human pathogens, including Salmonella and E. coli O157:H7, also take advantage of the symbiotic signaling pathway to colonize legume roots, such as alfalfa sprouts, that have been linked to several outbreaks of foodborne illness (Taormina et al., 1999). Characterizing the plant and microbe genes involved in these infections, and understanding how these pathogens override or constrain the plant’s defenses against invading microbes, may reveal ways to prevent such outbreaks. The Squid and the Bacterium The Hawaiian squid Euprymna scolopes forms a persistent association with the gram-negative luminous bacterium Vibrio fischeri (Nyholm and McFall-Ngai, 2004). Incorporated in the squid’s light organ, the bacterium emits luminescence that resembles moonlight and starlight filtering through ocean waters, camouflag- ing the squid—a nocturnal animal—from predators. In her presentation, McFall- Ngai described the process by which the bacterium colonizes the squid’s light organ, which begins within an hour after hatching and appears to occur in stages, each enabling greater specificity between host and symbiont, as shown in Figure WO-7. McFall-Ngai referred to this progression as “a fairly well-orchestrated

 WORKSHOP OVERVIEW Non-flagellated V. fischeri Flagellated V. fischeri Gram-negative non-symbiotic bacteria Gram-positive bacteria FIGURE WO-7 The “winnowing.” This model depicts the progression of light-organ colonization as a series of steps, each more specific for symbiosis-competent Vibrio fischeri. (a) In response to gram-positive and gram-negative bacteria (alive or dead) the Figure WO-7 COLOR.eps bacterial peptidoglycan signal causes the cells of the ciliated surface epithelium to secrete mucus. (b) Only viable gram-negative vector type elements bitmap image w/bacteria form dense aggregations. (c) Motile or non-motile V. fischeri out-compete other gram-negative bacteria for space and become dominant in the aggregations. (d) Viable and motile V. fischeri are the only bacteria that are able to migrate through the pores and into the ducts to colonize host tissue. (e) Follow- ing successful colonization, symbiotic bacterial cells become non-motile and induce host epithelial cell swelling. Only bioluminescent V. fischeri will sustain long-term colonization of the crypt epithelium. SOURCE: Reprinted from Nyholm and McFall-Ngai (2004) with permission from Macmillan Publishers Ltd. Copyright 2004. minuet between the host and the symbiont” that induces the maturation of the squid’s light organ, as well as developmental changes that appear to exclude colo- nization of the organ by other bacterial cells. She noted that much of this process is signaled by microbe-associated molecular patterns (MAMPs). 16 16 Investigations of the innate immune system, which enables both plants and animals to detect pathogens and mount defensive responses, have identified a series of receptor proteins that recognize

0 MICROBIAL EVOLUTION AND CO-ADAPTATION Using a range of molecular approaches, McFall-Ngai and coworkers are engaged in characterizing the colonization of the squid light organ and the main- tenance of its symbionts in exacting detail, “to get an hour-by-hour view of the conversation that the host has with its bacterial partner,” McFall-Ngai explained. Among the many squid genes that are transcriptionally upregulated in response to colonization by V. fisheri, the researchers identified 18 genes that are also upregu- lated during the colonization of both mouse and zebrafish guts, as determined in constructed models (Chun et al., 2008; see McFall-Ngai in Chapter 2). These shared genes encode proteins that are components of cellular pathways involved in transcriptional regulation, oxidative stress, and apoptosis—responses that typi- cally have been associated with pathogenesis, McFall-Ngai noted. Instead, she said, these pathways constitute a “language of symbiosis,” by which a host “talks” to a bacterial colonist, which in most cases is not a pathogen (see the following section for an extended discussion of microbial pathogenesis and the host response). “What I think this demands of biologists . . . is to go back and question our basic premises about how bacteria and animals work together, what virulence fac- tors really are, and such host behaviors as inflammation, tolerance, and carriage,” she concluded. “The horizon, then, is how these characters of host and symbiont are controlled to result in a mutualistic, commensal, or beneficial association.” MICROBIAL EVOLUTION, ADAPTIVE MECHANISMS, AND THE EMERGENCE OF VIRULENCE AND RESISTANCE Most successful parasites travel a middle path. It helps for them to have aggres- sive means of entering the body surfaces and radiating some local toxicity to counter the hosts’ defenses, but once established they also do themselves (and their hosts) well by moderating their virulence. Joshua Lederberg, “Infectious History” (2000) As they explored the effects of adaptation, virulence, and antimicrobial resis- tance on the host-microbe equilibrium, workshop participants were reminded of Lederberg’s important contributions to research on these topics. Presenter Stanley Falkow, of Stanford University, described how Lederberg’s discovery of bacterial conjugation and characterization of plasmids—the machinery of horizontal gene conserved molecular patterns specific to bacteria, viruses, and fungi. These signaling elements, which are displayed on the surfaces of pathogenic, commensal, and mutualistic microbes, are known as microbe-associated molecular patterns, or MAMPs. The binding of MAMPs by host receptor proteins elicits a transcriptional response that in some cases triggers host defenses against pathogens, but in others—such as the squid light organ—is associated with host colonization (Didierlaurent et al., 2001; Nyholm and McFall-Ngai, 2004; Yokoyama and Colonna, 2008).

 WORKSHOP OVERVIEW transfer, and, thereby, the means to virulence17—built upon prior discoveries of bacterial transformation and mutagenesis and helped to set the stage for present- day research on bacterial pathogenicity18 (see Falkow in Chapter 3). Stanley Cohen, Lederberg’s colleague in the Department of Genetics at Stanford Univer- sity, pointed out that Lederberg had invented the term “plasmid” for extrachromo- somal genetic elements. He noted Lederberg’s long-standing concerns about the challenge posed by disease-producing microbes and discussed Lederberg’s early work demonstrating the genetic basis for antimicrobial drug resistance. Many in attendance at this workshop, and certainly the scientists whose pre- sentations are summarized herein, would echo the following remark by Falkow: “I consider all of the scientists whose discoveries expanded Lederberg’s initial work on bacterial conjugation to be giants standing on his shoulders, and they made possible my own experimental work.” The Nature of Bacterial Pathogenicity As Lederberg’s observation above suggests, and studies of indigenous micro- bial communities attest, coexistence between host and microbe is a dynamic equilibrium (Blaser, 1997; Lederberg, 2000). In the case of microbes that cause persistent, asymptomatic infections, physiological or genetic changes in either host or microbe may shift the relationship toward microbial invasion of host tissue, which typically results in an immune response that destroys the invading microbes, but which may also injure or kill the host (Dethlefsen et al., 2007; Merrell and Falkow, 2004). Research in the decades following Lederberg’s ground-breaking work on bacterial conjugation has revealed the following fundamental characteristics of bacterial pathogens, as noted by Falkow (see also Chapter 3): • acteria manipulate the normal functions of host cells in ways that benefit B the bacteria (see Falkow’s Figure 3-1 in Chapter 3). • orizontal gene transfer via mobile genetic elements has shaped the evo- H lution of bacterial specialization. • athogenicity is generally conferred through the inheritance of blocks of P genes, called pathogenicity islands. In order to establish themselves within their hosts, reproduce, and find a new suitable host, pathogenic and commensal bacteria alike must overcome many similar challenges posed by the host’s immune system and by competition with 17Virulence is the degree of pathogenicity of an organism as evidenced by the severity of resulting disease and the organism’s ability to invade the host tissues. 18Pathogenicity reflects the ongoing evolution between a parasite and host, and disease is the prod- uct of a microbial adaptive strategy for survival.

 MICROBIAL EVOLUTION AND CO-ADAPTATION other microbes. Pathogens have an inherent ability—largely conferred by the products of pathogenicity islands, known as virulence factors—to breach host barriers and defenses that commensals cannot penetrate, Falkow explained. When pathogenic bacteria cross the intestinal epithelium of their mammalian hosts, usu- ally through areas known as Peyer’s patches, they are engulfed by phagocytes: immune cells that destroy invaders by digesting them. Successful pathogens are able to avoid this fate and survive and sometimes replicate within phagocytes, however, and thereafter are distributed to the liver and spleen. Some pathogens establish persistent, systemic—and sometimes asymptomatic—infections in their hosts and may be shed for the remainder of the host’s life. “In my view,” Falkow said, “pathogens choose to live in a dangerous place [exposed to the host’s immune system] to avoid competition and to get nutrients.” However, Falkow also observed, several members of the human bacterial microbiota that typically live uneventfully in the nasopharynx—including Strep- tococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae type b, and Streptococcus pyogenes—sometimes cause disease. These microbes have vir- ulence factors, suggesting that they interact with the host’s immune system, and they persistently infect a significant proportion of the human population, the vast majority of whom are asymptomatic carriers. The existence of such “commensal pathogens” suggests that virulence factors represent one form of a larger class of adaptive factors that allow microbes to colonize and survive in particular niches, and that these factors have been selected on this basis, rather than for their ability to produce disease in host organisms. Indeed, Falkow remarked, it may also be the case that the continual interaction of persistent, asymptomatic bacterial infections with the host immune system keeps it “primed for defensive action.” The conceptualization of virulence factors as colonization factors underlies the larger notion of a distinction between pathogenicity and disease, Falkow observed. “I submit that medicine’s focus on disease really distracts us from understanding the biology of pathogenicity,” he said. “Disease does not encom- pass the biological aspects of pathogenicity and the evolution of the host-parasite relationship.” Thus, he continued, “If the nature of microbial pathogenicity is schizophrenic—characterized by inconsistent or contradictory elements—then it is important to study every aspect of its biology, and not be distracted by its role in causing disease.” Microbial Virulence and the Host Response Just as there is more to microbial pathogenicity than disease, there is more to infectious disease than the actions of virulence factors on host cells and sys- tems. Rather, as workshop presenter Bruce Levin, of Emory University, bluntly asserted, virulence almost always results from “screw-ups” by the host’s immune system. These immunological failings include responding more vigorously than needed, as occurs in bacterial sepsis; responding incorrectly to a pathogen, as

 WORKSHOP OVERVIEW occurs in lepromatous leprosy; or responding to the wrong signals, as occurs in toxic shock syndrome (see Margolis and Levin, 2008, reprinted in Chapter 3). “Sometime in the future, we will look at antimicrobial chemotherapy as a primi- tive approach to treating infections; we will treat diseases such as sepsis and meningitis by controlling the host response,” Levin predicted. “It’s going to be a hard job, and we can’t do it by episodic dosing. Effective treatment will require real-time monitoring and response to changes in the immune response. I believe it will be possible to treat infections in this way, but to do so we have to know a lot more about the immune response and its control than we do now.” Tying this “it’s the host’s fault” perspective into existing hypotheses for the evolution of virulence, which focus primarily on the parasite, raises some inter- esting issues (see Margolis and Levin in Chapter 3). According to “conventional wisdom,” as described by May and Anderson (1983), virulence is an early stage in the association between a parasite and its host after which, over the course of evolution, a successful parasite “learns” not to bite the hand that feeds it. Levin suggested that the host, too, could evolve such that its immune system “learns” not to overreact to the parasite, and that eventually, on “equilibrium day,” all such host-parasite relationships would achieve mutualism (Levin et al., 2000). He also considered the trade-off hypothesis, which postulates that a too-virulent parasite will kill its host too rapidly to permit efficient transmission. Natural selection in the parasite population, therefore, favors some—but not too much—virulence. Levin further wondered whether this trade-off could be achieved by the parasite evolving restraint in its production of agents that inflame the host’s immune system. By contrast, in his prepared remarks Levin presented experimental findings suggesting that the host effects of certain bacterial products (e.g., Shiga toxin produced by Escherichia coli O157:H7; Steinberg and Levin, 2007) appear to have evolved coincidentally as virulence determinants, having been selected for different functions and the advantages that they confer upon a microbe. Other vir- ulent microbes (e.g., Falkow’s “commensal pathogens”) may have been selected within the host, under local circumstances that favor more pathogenic members of a colonizing population, even if they are at a disadvantage in the community of hosts, Levin said. But how to account, in evolutionary terms, for the disadvantages of host “immunoperversity”: the tendency to overreact to pathogens, resulting in host morbidity and mortality? This phenomenon may be an artifact of the relative slowness of human evolution, Levin explained, coupled with the low efficiency of infectious disease-mediated selection in our species. It may also result from selection pressures associated with maintaining a large microbiota, McFall-Ngai suggested. “By and large, the invertebrates (with the exception of the termites and the cockroaches, which do have large consortia) generally have very limited persistent coevolved interactions with microbes,” she said. Thus, it is possible that the adaptive immune system of jawed vertebrates evolved as a mechanism

 MICROBIAL EVOLUTION AND CO-ADAPTATION by which to control the large populations of microbes—a task that may require extreme responses that occasionally result in disease. Pathogen Evolution, as Illustrated by Salmonella Setting aside the inconsistencies and contradictions inherent to pathogenicity, Falkow and fellow workshop speakers Gordon Dougan and Julian Parkhill, of the Wellcome Trust Sanger Institute in Cambridge, United Kingdom, described , approaches to discovering how certain microbes have evolved to cause disease in their hosts (see Chapter 3). In particular, each presentation discussed bacterial pathogens of the genus Salmonella. Serovars19 of Salmonella enterica include S. typhimurium, which infects a wide range of hosts and is a major cause of gastroenteritis in humans, and S. typhi, the human-specific agent of the systemic infection typhoid fever (Lawley et al., 2006; Monack et al., 2004b). In humans, S. typhimurium infections are generally (but not always) con- tained within the intestinal epithelium, while S. typhi evades destruction by the immune system and is transported, via the liver and spleen, to the gall bladder and bone marrow, in which the bacteria can persist (Figure WO-8; Monack et al., 2004b). Thus, significant numbers of people infected with typhoid—including those asymptomatically infected with S. typhi—become chronic carriers of the pathogen and reservoirs of a disease that poses a considerable threat to public health. From the perspective of S. typhi, however, this “stealth” strategy is essen- tial to its survival. Workshop presentations described how evolution—both ancient and recent—has shaped pathogenicity in Salmonella, from its initial acquisition of genes that confer invasiveness to the loss of gene function in some serovars, leading to a reduction in host range and increasing virulence, to the recent chal- lenge of antibiotics, which the bacterium has quickly met with resistance. Genes Make a Pathogen In order to identify genes and gene products that enable Salmonella to establish systemic infection, Falkow and coworkers employed a mouse model of persistent, systemic infection by S. typhimurium, which resembles that of S. typhi in humans (Monack et al., 2004a). Using a microarray-based strategy, they screened the entire Salmonella genome for genes associated with different stages of persistent Salmonella infection (see Falkow in Chapter 3). Some of the genes they identified enable Salmonella to excrete proteins that kill macrophages during initial infection, while others allow the bacterium to replicate and persist within the vacuoles of macrophages, invisible to the host’s immune system. Using existing technology, “we can now identify all such genes quite readily,” Falkow said, “but we may not be able to determine their exact function.” 19 Strains distinguished serologically, based on the antigens displayed on their surfaces.

 WORKSHOP OVERVIEW FIGURE WO-8 Comparison of pathogenesis of infection associated with Salmonella Figure WO-8 COLOR.eps typhimurium versus S. typhi (human restricted). SOURCE: Reprinted from Young bitmap image et al. (2002) with permission from Macmillan Publishers Ltd. Copyright 2002. Loss of Function Leads to Specialization While bacterial pathogenicity is associated with the acquisition of novel virulence genes (typically through horizontal transfer), research by Parkhill and colleagues indicates that host-restricted, virulent pathogens such as S. typhi have evolved those characteristics following a loss of function in genes that control interactions with host cells (thereby limiting their host range) and that modulate the expression of virulence factors (see Box WO-2 and Chapter 3). The genomes of S. typhi and another systemic, host-restricted pathogen, S. paratyphi A—each independently descended from S. enterica—contain approximately 200 inac- tivated genes, of which about 30 are common to both serovars. Many of these encode functions involved in determining virulence or host range. Most of these shared pseudogenes, however, do not bear the same inactivating mutations, sug- gesting that their loss conferred a selective advantage.

 MICROBIAL EVOLUTION AND CO-ADAPTATION BOX WO-2 Host-Restriction Versus Virulence in Bordetella spp. Genomic comparisons of three Bordetella species by workshop presenter Julian Parkhill and colleagues provide clues to the evolution of virulence in bacte- rial pathogens (Parkhill et al., 2003). B. pertussis, the primary causative agent of whooping cough in humans, can survive only within its single host species. B. parapertussis also causes whooping cough; some strains are restricted to humans, others to sheep. Both of these species have apparently evolved from the less virulent B. bronchiseptica, which causes chronic and often asymptomatic disease in a wide range of animals. These three species are genetically identical at the 16S RNA level. By most measures (except phenotype), they constitute a single species. There is little sequence variation among strains of either B. pertussis or B. parapertussis worldwide, indicating that these species are very recently evolved; by contrast, B. bronchiseptica strains vary considerably. Their genome structures also differ significantly: compared with B. bronchiseptica, the B. pertussis genome is approximately 25 percent smaller and contains a large number of genes inacti- vated by mutation (pseudogenes). A large majority of these pseudogenes resulted from single mutations, indicating that they were recently inactivated (since further mutations have not accumulated in the inactive gene). Approximately one-third of the pseudogenes in B. pertussis were inactivated by an insertion sequence (IS) element, a mobile genetic sequence that, once introduced into a bacterial ge- nome, can reproduce and reinsert in multiple locations. The B. pertussis genome contains 240 copies of a single IS element, which have undoubtedly produced high levels of recombination and deletion. B. parapertussis contains different IS elements that have also proliferated in its genome. In evolving from B. bronchiseptica toward host restriction and greater virulence, B. pertussis and B. parapertussis did not acquire novel virulence factors, but in- stead lost function in genes associated with host interaction (thereby narrowing their host ranges) and in regulation of the expression of virulence factors, such as the pertussis toxin. Parkhill and coworkers hypothesize that these changes occurred when humans began living in close proximity to each other, increasing opportunities for pathogen transmission, and easing selection against virulence (which formerly might have killed a host before it could transmit the pathogen). These circumstances could have created an evolutionary bottleneck, causing the increased fixation of advantageous (and potentially disadvantageous) point mu- tations and IS element insertions in the population, giving rise to the new, more virulent, and host-restricted species. Similar events appear to have influenced the evolution of a variety of human pathogens, including S. typhi and S. paratyphi, independent derivatives of the ancestral S. enterica, and Yersinia pestis, the causative agent of plague, from its ancestor Y. pseudotuberculosis. An evolutionary bottleneck—perhaps the result of domestication—may also have enabled the descent of Burkholderia mallei, a pathogen restricted to horses, which causes glanders, from B. pseudomallei, a broad host-range pathogen. Likewise, the planting of crops as monocultures may have separated Clavibacter michiganensis subspecies sepedonicus, an en- dophytic pathogen of potato with a narrow host range, from C. michiganensis subspecies michiganensis, an epiphytic pathogen with a broad host range.

 WORKSHOP OVERVIEW Emergence of Resistance As is typical of human-restricted (and therefore recently evolved) pathogens, S. typhi strains exhibit scant genetic variation (see Chapter 3). A sequencing study conducted by Dougan and colleagues that compared 200 gene fragments of approximately 500 base pairs each from 105 globally representative S. typhi isolates identified only 88 single nucleotide polymorphisms (SNPs; Roumagnac et al., 2006). Considerable numbers of these SNPs—at least 15 independent mutations to the same crucial gene encoding a DNA gyrase subunit—arose fol- lowing the introduction of fluoroquinolone antibiotics in the late 1980s (see the following section for a general discussion of antimicrobial resistance). Another route to antibiotic resistance appears recently to have been taken by non-typhoidal serovars of Salmonella, including S. typhimurium, Dougan noted. These strains cause invasive infections—instead of the usual gastroenteritis— and have become a major cause of morbidity and mortality in African children (Gordon et al., 2008; Graham, 2002). Sequences of strains causing non-typhoidal salmonellosis (NTS) proved genetically distinct from Salmonella strains (of the same serovars) that cause gastroenteritis in Western populations: they bore plasmids containing two distinct genetic elements (integrons20) that conferred resistance to multiple antibiotics, as well as to quaternary ammonium disinfec- tants. Dougan warned that these resistance genes could spread rapidly through horizontal transfer to other Salmonella strains following the planned introduction of large-scale antibiotic prophylaxis for HIV-infected African children. Antibiotic Resistance: Origins and Countermeasures Reports of antibiotic-resistant bacterial infections followed within a few years of the first widespread use of penicillin at the close of World War II. By the mid-1950s, multidrug-resistant bacterial strains began to emerge (Figure WO-9) and have since become ubiquitous. Indeed, mortality rates due to bacterial infec- tions threaten to return to the levels of the pre-antibiotic era, according to speaker Julian Davies of the University of British Columbia. Perhaps these developments could have been anticipated based on Lederberg’s work on bacterial conjugation, a key route by which plasmids carrying drug- resistance genes are horizontally transferred between bacteria. Certainly, the ongoing impact of antibiotic resistance has confirmed the importance of under- standing its evolutionary, genetic, and ecological origins, as several workshop presentations attested. 20 Integrons are gene elements that facilitate horizontal gene transfer by allowing bacteria to inte- grate and express DNA in the form of “gene cassettes”: mobile genes bearing attC recombination sites. Integrons catalyze the integration of foreign genes into a DNA molecule that is already recog- nized by the native replication machinery of the chromosome or plasmid, and under the control of a promoter that allows gene expression in the host (Nemergut et al., 2008).

 MICROBIAL EVOLUTION AND CO-ADAPTATION 120 100 Frequency of isolation of multi-resistant 1960 Production of antibiotics in Japan 1955 Identification 100 First report of of transfer of 75 MDR strain resistance 80 Shigella (%) (tonnes) 60 50 Streptomycin Multidrug 40 resistance 25 Tetracycline 20 Chloramphenicol 0 0 1950 1955 1960 1965 Year FIGURE WO-9 The relationship between antibiotic resistance development in Shigella Figure WO-9.eps dysentery isolates in Japan and the introduction of antimicrobial therapy between 1950 and 1965. In 1955, the first case of plasmid determined type elements bitmap image w/ vector resistance was characterized. MDR = multidrug resistance. Transferable, multi-antibiotic, resistance was discovered five years later in 1960. SOURCE: Reprinted from Davies (2007) with permission from Macmillan Publishers Ltd. Copyright 2007. The biochemical mechanisms by which bacteria achieve resistance are many and varied, and the genes to accomplish each of them can be acquired by horizontal transfer, Davies said. Mechanisms conferring resistance include increased efflux of antibiotic, enzymatic inactivation, target modification, target overexpression, sequestration, and intracellular localization. Yet although we have gained consider- able understanding of the biochemical and genetic bases of antibiotic resistance, we have failed dismally to control the development of antibiotic resistance, or to stop its transfer among bacterial strains, Davies observed. Novel antibiotics are unlikely to be developed without significant financial incentives for the pharmaceutical industry, which has largely abandoned infectious disease therapeutic discovery for more profitable targets, such as chronic conditions (Spellberg et al., 2008). Work- shop participants considered a variety of means to address these considerable chal- lenges, including investigating the environmental origins of antibiotic resistance, identifying sources of novel antibiotics, and developing alternatives to conventional antibiotic therapies.

 WORKSHOP OVERVIEW Environmental Sources of Resistance Genes and Antibiotics As Lederberg and others have shown, genes that confer resistance to clini- cal antibiotics exist in bacterial populations that have never encountered these compounds. Many such naturally-occurring resistant bacterial strains have been isolated (or activities recognized through metagenomic methods, as will be sub- sequently described) from the soil—as were the bacterial strains from which antibiotics were initially derived (Dantas et al., 2008; D’Costa et al., 2006; Riesenfeld et al., 2004). In naïve bacterial populations, “resistance” genes are likely to encode other functions (e.g., metabolism, regulation) that nevertheless offer a selective advantage, Davies explained. “Resistance genes in the environ- ment, in general, are not resistant,” he said. “They become resistant when picked up and overexpressed in a foreign cytoplasm.” Opportunities for such acquisitions are presented by the flow of water among the various environments in which bacterial resistance genes exist, Davies observed. In particular, wastewater treatment plants—which he described as “an incredible mixing pot of genes and plasmids”—provide an ideal opportunity for pathogenic bacteria to acquire new resistance genes, and new virulence genes as well (see Davies in Chapter 4). He noted recent studies by Szczepanowski and coworkers, who isolated and sequenced antibiotic-multiresistant plasmids from bacteria present in sludge in wastewater treatment plants, and found that they also contained several virulence-associated genes and integrons (Szczepanowski et al., 2004, 2005). Such plasmids, moreover, were detectable in effluents released from the treatment plant into the environment (Szczepanowski et al., 2004). Research- ers from the same laboratory have also performed a metagenomic analysis of such bacteria and determined that their collective plasmid DNA encoded resistances to all major classes of antimicrobial drugs (Szczepanowski et al., 2008). The pervasiveness of antibiotic resistance in the environment suggests that antibiotics—that is, molecules with antibiotic activity—are equally abundant in nature, produced by bacteria (and also by plants) to serve a variety of purposes, Davies said. Thus, to find novel antibiotics, his laboratory is pursuing a strat- egy of identifying organisms that produce bioactive compounds, then analyz- ing these compounds for their antibiotic properties. Similarly, Handelsman (see Chapter 4) described a process by which she and coworkers are searching the soil metagenome—DNA derived from soil, mainly of bacterial and archaeal origins, digested and ligated into a vector used to transform Escherichia coli—for both antibiotic and antibiotic resistance activities. One compound they have discov- ered is a single enzyme possessing two antibiotic resistance domains: one that disables penicillin-like compounds; the other, cephalosporin-like compounds. Although never before seen, such an enzyme may someday find its way into the human microbiome (or microbial community), Handelsman said, and if so, its potential to confer broad-spectrum antibiotic resistance might pose a serious threat to public health.

0 MICROBIAL EVOLUTION AND CO-ADAPTATION Alternatives to Antimicrobials In his workshop presentation, Stanley Cohen noted that the public health crisis of antimicrobial drug resistance in bacteria and viruses has resulted largely from the practice of treating infectious diseases with therapeutics designed to attack pathogens, resulting in the spread of mutant microbes that are insensitive to drug therapy. He described an approach that recognizes that to be successful, many pathogens require the cooperation of host cells, which furnish the invader with genes and gene products necessary for pathogen propagation and transmis- sion. Interfering with host functions that are recruited by pathogens provides an alternative to drugs that target pathogens (see Chapter 4). Possible toxicity from the targeting of such host genes, which Cohen termed host-oriented therapeutics, must be addressed, he acknowledged—just as they must for infectious disease therapies aimed at microbial targets. However, he reminded workshop partici- pants that drugs targeting normal host cell functions are used routinely in the treatment of other types of disease. To find genes in eukaryotic host organisms that enable pathogens to propa- gate, Cohen and coworkers developed antisense RNA-based methods for identify- ing mammalian cells that show altered biological properties when mutagenized with randomly-integrating retroviruses (Li and Cohen, 1996). This random homo- zygous knockout (RHKO) strategy and other global gene inactivation strategies, as described in Cohen’s contribution to this volume (see Chapter 4), have enabled investigation of host-cell genes and genetic pathways required for viral and bacterial pathogenicity. Tsg101, the first gene isolated using RHKO, has been implicated in the egress of a broad range of viruses (including HIV and Ebola) from infected cells and is currently being pursued as a therapeutic target (Lu et al., 2003). Using an assay that identifies mammalian cells resistant to anthrax tox- icity, Cohen and coworkers have also used host-gene-inactivation approaches to discover the previously unsuspected role of the host cell surface protein ARAP3 and LRP6 in anthrax toxin internalization. Cells deficient in the function of the genes encoding these proteins demonstrate increased survival in the presence of anthrax toxin (Lu et al., 2004; Wei et al., 2006). Research is underway to determine the potential efficacy of therapies directed against these proteins (see Cohen in Chapter 4). A further departure from conventional antimicrobial therapeutics was intro- duced by Handelsman, who is investigating the possible manipulation of indig- enous microbial communities to defend their hosts against pathogens. “To figure out how to use communities in order to protect us, we need to understand the process of invasion,” she said. Her laboratory is pursuing this line of inquiry as part of ongoing efforts to characterize the composition, dynamics, and functions of model indigenous communities. Their studies of the microbial community of the gypsy moth gut have yielded intriguing evidence that commensal bacteria

 WORKSHOP OVERVIEW interact in ways that can influence host health—and in surprising ways, including collaborating with invaders to kill their hosts (see Handelsman in Chapter 4). Handelsman and coworkers have also demonstrated various ways by which they can alter the response of their model microbial community to pathogens. “We find that perturbation by a number of means, including antibiotic addition, can increase the susceptibility of the community to invasion,” she explained. Exposure to antibiotics also increased microbial diversity in their community model. Based on these results, the researchers plan to undertake experiments to measure community robustness—a composite of resistance to change, stability, and resilience—and to identify organisms and genes that create robust commu- nities. They also hope to reveal the genetic attributes of invading microbes that permit them to overcome a robust community. INFECTIOUS DISEASE EMERGENCE: ANALYZING THE PAST, UNDERSTANDING THE PRESENT, ANTICIPATING THE FUTURE The future of humanity and microbes likely will unfold as episodes of a suspense thriller that could be titled Our Wits Versus Their Genes. Joshua Lederberg, “Infectious History” (2000) Having considered a wide spectrum of examples of microbial evolution and co-adaptation, and the diverse outcomes for microbes and hosts as indi- viduals, species, and communities, workshop participants considered the global effects of host-microbe relationships that manifest as emerging infectious dis- eases. As defined by presenter Stephen S. Morse of Columbia University, such diseases have shown a rapid increase in the number of new cases (incidence) or in their geographic range; often, they are caused by novel—that is, previously undiscovered—pathogens, and their emergence is often driven by anthropogenic factors (e.g., land use, travel and trade, food handling; see, for example, IOM, 1992, 2004a,b, 2006b,c, 2007, 2008a,b). Workshop presentations and discussions examined knowledge gained from recent experiences of disease emergence, charted progress toward predicting how and where new diseases might emerge, and explored key challenges and opportunities for understanding and addressing future infectious threats. “We have had years of complacency about infectious diseases,” Morse noted, thanks to the advent of antibiotics, immunizations, and improved public health measures, all of which have led chronic diseases to displace infections as the major cause of death in wealthy countries (Figure WO-10). However, this has not been the case in much of the world, where acute infectious diseases have remained the primary cause of morbidity and mortality. Figure WO-11 depicts the global distribution of a number of recently emerged diseases, as well as “reemerg-

 MICROBIAL EVOLUTION AND CO-ADAPTATION FIGURE WO-10 Deaths resulting from infectious diseases decreased markedly in the Figure WO-10.eps United States during most of the twentieth century. However, between 1980 and 1992, the death rate from infectious diseases increasedimage bitmap 58 percent. The sharp increase in infectious disease deaths in 1918 and 1919 was caused by an influenza pandemic, which killed more than 20 million people. SOURCE: Hughes (2001). ing” diseases21 and the “deliberate” emergence of Bacillus anthracis, an agent of bioterrorism (see also Morse in Chapter 5). A somewhat different perspective on disease emergence was provided by Mark Woolhouse of the University of Edinburgh, whose group has characterized 87 “novel” pathogens—organisms that have been discovered since 1980, and, thus, do not include antimicrobial-resistant strains of previously-known pathogens—among the approximately 1,400 human pathogens recognized in 2007 (see Woolhouse and Gaunt, 2007, reprinted in Chapter 5). Are emerging diseases really on the rise? Yes, according to analyses by presenter Peter Daszak of the Consortium for Conservation Medicine. “This is a trend that has gone up, and it should continue to go up,” he observed. While more than half of emergent diseases can be attributed to antibiotic resistance, Daszak said, zoonoses—infectious diseases that can be transmitted from verte- brate animals to humans—have also increased significantly, particularly those 21 Familiardiseases that have recently expanded their geographic range and/or demonstrated intensi- fied virulence due to such factors as reduced public health measures or antibiotic resistance.

FIGURE WO-11 Global examples of emerging and reemerging infectious diseases, some of which are discussed in the main text. Red rep- Figure WO-11 COLOR.eps resents newly emerging diseases; blue, reemerging or resurging diseases; black, a “deliberately emerging” disease. bitmap image SOURCE: Reprinted from Morens et al. (2004) with permission from Macmillan Publishers Ltd. Copyright 2004.  landscape

 MICROBIAL EVOLUTION AND CO-ADAPTATION emerging from wildlife, such as SARS, Ebola hemorrhagic fever, and Nipah viral encephalitis (see Daszak in Chapter 5). The IOM reports Emerging Infections: Microbial Threats to Health in the United States (IOM, 1992) and Microbial Threats to Health (IOM, 2003), pro- duced by ad hoc committees co-chaired by Lederberg, provided a crucial frame- work for understanding the drivers of infectious disease emergence. The list of six “factors in emergence” in the first report was expanded to 13 in the second, as shown in Box WO-3, which also conceptualized interrelationships among these factors in the model shown in Figure WO-12. As the following summary of workshop presentations and discussions illustrates, this framework has guided, and continues to guide, research to elucidate the origins of emerging infectious threats. These concepts also inform the analysis of recent patterns of disease emergence in order to identify risks for future disease emergence events and, thereby, target surveillance to enable early detection and response in the event of an outbreak. BOX WO-3 Factors in Emergence 1992 • Microbial adaptation and change • Economic development and land use • Human demographics and behavior • International travel and commerce • Technology and industry • Breakdown of public health measures 2003 • Microbial adaptation and change • Human susceptibility to infection • Climate and weather • Changing ecosystems • Human demographics and behavior • Economic development and land use • International travel and commerce • Technology and industry • Breakdown of public health measures • Poverty and social inequality • War and famine • Lack of political will • Intent to harm SOURCE: IOM (2003).

 WORKSHOP OVERVIEW FIGURE WO-12 The convergence model. At the center of the model is a box represent- ing the convergence of factorsFigureto the emergence of an infectious disease. The in- leading WO-12.eps bitmap image terior of the box is a gradient flowing from white to black; the white outer edges represent what is known about the factors in emergence, and the black center represents the unknown (similar to the theoretical construct of the “black box” with its unknown constituents and means of operation). Interlocking with the center box are the two focal players in a microbial threat to health—the human and the microbe. The microbe-host interaction is influenced by the interlocking domains of the determinants of the emergence of infection: genetic and biological factors; physical environmental factors; ecological factors; and social, political, and economic factors. SOURCE: IOM (2003).

 MICROBIAL EVOLUTION AND CO-ADAPTATION Patterns of Emergence The Process As several workshop speakers observed, infectious disease emergence occurs incrementally, and can be accelerated or hindered at various stages in the process (see Chapter 5). The introduction of a pathogen to a new host population drives many emergent events, particularly those involving zoonoses (for example, see the case studies of hantavirus pulmonary syndrome and influenza described by Morse in his contribution to Chapter 5). The “zoonotic pool” provides a rich source of extremely diverse pathogens, Morse noted. Woolhouse added that humans share nearly 60 percent of their pathogen species—and nearly 80 percent of the 87 “novel” pathogen species—with nonhuman vertebrates (see Woolhouse and Gaunt in Chapter 5). Opportunities for such introductions are provided by many of the “factors in emergence” described in Microbial Threats to Health (IOM, 2003) and Box WO-3. Woolhouse and Gaunt found that, among their group of “novel” pathogen species, “the most commonly cited drivers [of emer- gence] fall within the following IOM categories: • economic development and land use; • human demographics and behavior; • international travel and commerce; • changing ecosystems; • human susceptibility; and • hospitals.” Although humans are exposed to many potential novel pathogens, a relatively small number succeed in causing severe infectious disease, which also requires pathogen establishment and transmission among humans. Woolhouse described a “pathogen pyramid” (depicted in Figure 5-8 of Woolhouse and Gaunt’s contribu- tion to Chapter 5), in which about 500 out of the total 1,400 pathogens capable of infecting humans are also able to be transmitted to another human. Of these, fewer than 150 have the potential to cause epidemic or endemic disease. The potential for novel pathogens to become established in, and transmitted among, humans is also influenced by factors of emergence, particularly human migration and travel (which disseminate localized diseases), the use of hospitals (which intensify exposure to pathogens), and medical technologies such as injection equipment (which, if contaminated, can also serve to transmit disease). Envi- ronmental changes may also expand the geographic range occupied by species that serve as hosts or vectors for infectious diseases. In the case of SARS and HIV/AIDS, it is unknown how (or how many times) these diseases entered human populations, but it is clear that human migration and health care practices served to amplify their emergence (see Morse in Chapter 5).

 WORKSHOP OVERVIEW The Pathogens Woolhouse and coworkers used a rigorous, formal methodology to produce and refine their catalog of the nearly 1,400 recognized human pathogen species, of which the 87 “novel” species constitute a subset (see Woolhouse and Gaunt in Chapter 5). As shown in Figure WO-13, while known human pathogens are dominated by bacterial species, the vast majority of novel pathogen species are viruses. Indeed, the researchers identified four attributes of these novel pathogens that they expect will describe most future emergent microbes: a preponderance of RNA viruses; pathogens with nonhuman animal reservoirs; pathogens with a broad host range; and pathogens with some (perhaps initially limited) potential for human-to-human transmission (Woolhouse and Gaunt, 2007). “These new pathogens that we are reporting are coming from the same sorts of places, the same sorts of animal populations, we have always shared our viruses and other pathogens with,” Woolhouse explained, adding that host proximity, not taxonomy, seems to be the main driver for pathogens to jump between host species. The Origin of Novelty While anthropogenic factors provide plenty of fuel for infectious disease emergence, pathogens clearly can and do evolve, and at a far faster rate than do humans, as Lederberg (2000) observed. Viruses evolve fastest of all, which may contribute to their lead position among novel pathogen species, Woolhouse said. He noted that the evolution of viral pathogens could occur either subsequent to FIGURE WO-13 While known igure pathogens are dominated by bacterial species, the F human WO-13.eps bitmap image vast majority of novel pathogen species are viruses. SOURCE: Adapted from data in Woolhouse and Gaunt (2007).

 MICROBIAL EVOLUTION AND CO-ADAPTATION the infection of humans (adaptation) or in reservoir hosts prior to the first infec - tion of humans (resulting in human-infecting variants; Woolhouse and Antia, 2007). Microbes evolve new functions through various mechanisms, according to speaker Jonathan Eisen, of the University of California, Davis. These include de novo invention of new genes, small sequence changes that lead to changes in function of existing genes, duplication and divergence of one or both of the duplicates, and shuffling and swapping of large domains of different genes. In addition to generating novelty within their own genomes, microbes engage, through symbiosis, in sharing functions among species. Perhaps the best known example of this sharing is horizontal gene trans- fer, which allows one organism to acquire genes encoding new functions from another. In addition, microbes greatly facilitate the acquisition of functions by “macrobes” such as plants and animals by engaging in symbiotic interactions. For example, from the gut of the glassy-winged sharpshooter—the insect vector of Pierce’s disease of grapes—Eisen and coworkers characterized a pair of mutually- dependent symbionts that enable their host to survive on nutrient-poor sap from the water-conducting (xylem) tissues of plants (Wu et al., 2006). Eisen also discussed how the rate of the generation of novelty can be further accelerated in many microbes through the loss of mutation-damping mismatch repair genes. These comparisons between endosymbiotic and free-living bacterial species were undertaken using phylogenomic analysis, a method for integrating infor- mation about the evolution of an organism (expressed through its relationships with other organisms, or phylogeny) and the contents of its genome (see Eisen in Chapter 5). Eisen then described some of the phylogenomic methods he and colleagues developed and used in the analysis of various microbial genomes. For example, using one such method, Eisen and coworkers compared the genomic sequence of Vibrio cholerae to that of closely related bacteria, searching for gene families that had recently expanded in copy number, suggesting a recent diver- sification of function. They found that a family of proteins involved in chemi- cal gradient sensing—the methyl-accepting chemotaxis proteins (MCPs)—had undergone such an expansion (Heidelberg et al., 2000). “If a gene is not under strong selection within a bacterial genome, it usually disappears relatively rapidly over evolutionary time,” Eisen said. Moreover, the presence of large numbers of closely related genes suggests that some might have evolved new functions recently. Indeed, Eisen continued, the fact that genes usu- ally disappear rapidly if they are not under some strong selective pressure also enables researchers to make useful inferences about a class of genes that encode “conserved hypothetical proteins”—proteins that are conserved across species for which a function has yet to be identified. The way this works, he explained, is as follows: if a pathway is not being used, the genes required for the pathway are usually lost as a unit. Furthermore, if a microbe acquires a process by lateral gene transfer, it will have to acquire

 WORKSHOP OVERVIEW all the genes required for that process. It follows that one can identify sets of genes that likely work together in a pathway by looking for the correlated gain and loss of genes over evolutionary time. In his contribution to Chapter 5, Eisen describes the use of a method based on these principles, called phylogenetic pro- filing, to identify genes for sporulation in Carboxydothermus hydrogenoformans, a hydrogen-producing bacterium (Wu et al., 2005). Potential for Prediction The considerable knowledge gained over recent years regarding ecologi- cal and evolutionary processes that drive disease emergence makes possible the measurement and prediction of future patterns of disease emergence, Daszak asserted. “I’m not going to try to say that we can predict in great detail where, when, and what the next new emerging disease is going to be,” he said, “but I think we can use ecological approaches to make some predictions about future trends.” Bringing together information from genetic sequencing, phylogenetic analyses, and ecological studies, Daszak’s group has produced predictions on disease emergence on scales ranging from the local to the global (see Daszak in Chapter 5). At the global level, Daszak and coworkers developed a predictive model identifying regions (“hotspots”) where new infectious diseases are likely to emerge (Jones et al., 2008). From a database of 335 emerging infectious disease (EID) “events”22 that occurred between 1940 and 2004, the researchers found that zoonoses were responsible for a majority (60 percent) of these events, of which 72 percent originated in wildlife—including SARS and Ebola hemorrhagic fever. Bacteria caused more than 50 percent of these disease events, and many were associated with the development of antibiotic resistance. The hotspots shown in Figure WO-14 were identified based on correlations between the disease events and five socio-economic, environmental, and ecological variables. Concentrated in lower-latitude developing countries, these areas largely lack infectious disease surveillance and control efforts, which are disproportionately focused on the world’s healthiest, wealthiest citizens. Thus, Daszak concluded, “we are misal- locating our global efforts to deal with emerging infections.” Our Wits Versus Their Genes Discussion at the workshop’s conclusion (and immediately following the session on emerging infectious diseases) brought together many of Lederberg’s passions, in the tradition of Infectious History (Lederberg, 2000). As Daszak remarked of the potential for predicting the next emerging zoonosis, “I think this 22 Defined as the original case or cluster of cases representing an infectious disease in human populations for the first time.

0 FIGURE WO-14 Global distribution of relative risk of an EID event. Maps are derived for EID events caused by (a) zoonotic pathogens from wildlife; (b) zoonotic pathogens from nonwildlife;Figure WO-14 COLOR.eps (c) drug-resistant pathogens; and (d) vector-borne pathogens. SOURCE: Reprinted from Jones et al. (2008) with permission from Macmillan Publishers Ltd. Copyright 2008. bitmap image landscape

 WORKSHOP OVERVIEW is the next ‘Holy Grail’ for emerging disease, a way to fuse different disciplines: evolution, ecology, virology, microbiology.” Workshop participants explored how best to pursue this prize, as well as the larger objective of anticipating, detect- ing, and responding to emerging infectious threats, and the even greater goal of developing a truly comprehensive understanding of the relationships between and among microbes and host species. Addressing Immediate Infectious Threats The paramount importance of surveillance,23 recognized in Microbial Threats to Health as the “foundation for infectious disease prevention and control” (IOM, 2003), was evident in workshop discussions as well. Woolhouse cited a recent U.K. government study (Foresight, 2006) that deemed disease detection, identifi- cation, and monitoring essential for timely, effective, and cost-efficient response to infectious disease outbreaks, and which recommended support for a range of new technologies (e.g., genomic and post-genomic analyses, hand-held diagnos- tic devices, high-throughput screening) to enable the detection and diagnosis of multiple infections. “These are clearly the tools that we are going to need for effective global surveillance,” he concluded. Despite strong scientific support for strengthening infectious disease sur- veillance, from local to global levels, several participants noted that mustering the political will to expand such activities will be a daunting challenge. Indeed, support for public health—which Forum member Gerald Keusch of Boston Uni- versity described as “the active art of making sure nothing happens”—is increas- ingly difficult to obtain. Additional efforts beyond those directed at improving surveillance strategies and technologies will be needed to alter a political climate in which descriptive surveillance programs have been dismissed as “stamp col- lecting” and a dearth of outbreaks and epidemics is interpreted as a reason to curtail “unnecessary” public health initiatives. Participants offered various strategies for educating policy makers and the public on such matters as the accelerating emergence of infectious diseases, the true cost of infectious disease in comparison to that of surveillance, and the cost- effectiveness of broad-based surveillance for multiple infectious threats (e.g., zoonoses). Daszak is currently working with economists to develop a cost-benefit analysis for a global infectious disease surveillance program, which he views as a form of insurance—and, as he noted, begs the question: Who pays? “If you look at what causes emerging disease, quite often it is livestock production or trade,” he observed. “Does the group doing the trade or the livestock production pay for [infectious disease surveillance] as part of their global insurance package?” Similarly thorny questions are plaguing the establishment of surveillance 23 Defined as “the continuing scrutiny of all aspects of occurrence and spread of a disease that are pertinent to effective control” (IOM, 2003).

 MICROBIAL EVOLUTION AND CO-ADAPTATION systems mandated for signatories to the recently revised International Health Regulations (IHRs), the legal framework for international cooperation on infec- tious disease surveillance (WHO, 2008a,b). It is far from clear how many coun- tries will muster the resources to develop such systems. Although the possibility of incentives for resource-poor countries has been raised, such a program has yet to be established. Even if the goal of global infectious disease surveillance is realized, however, another problem looms: how to interpret the ensuing flood of information. There will be a critical need for technology capable of processing enormous amounts of data, quickly enough that it can be acted upon to prevent infectious outbreaks. Surveillance programs that target species and ecosystems at the highest risk for infectious disease outbreaks can conserve time and resources, including com- puting capacity. While current predictive efforts focus on identifying pathogen species and families likely to emerge as disease threats, “smart surveillance” will become even smarter when conducted in ecosystems determined to be ripe for disease emergence, Daszak noted. “With the technology we have now to discover new viruses, I think it makes sense to target [emergence-prone] ecological niches a bit better,” he said. Expanding Knowledge of Microbial Evolution and Co-Adaptation Metagenomic studies suggest that the vast majority of microbial species have yet to be identified (DOE, 2007; see also NRC, 2007). Finding and characterizing new microbes will undoubtedly enrich and expand our understanding of micro- bial biology, ecology, and evolution. Parkhill noted that his group and others are attempting to use high-throughput sequencing techniques to search for unknown viruses; however, he added, “the jury is still out [on this approach]. You can find things you know about, but it’s very difficult to recognize things you don’t know about.” As technology advances to enable the reading of ever-longer sequences, the genomes of unknown microbes should become easier to identify; however, Parkhill noted, such efforts are expected to produce so much data, so quickly, that it would overwhelm current analytical systems. Knowledge of microbial genomes, and the functions they encode, is also severely limited. Among 40 phyla of Bacteria, for example, most of the avail- able genomic sequences were from only three phyla as of 2002, Eisen said. He added that sequencing of Archaea and Eukaryote genomes has proceeded in a similarly sporadic manner. “This is not a very good sampling of the diversity of life in terms of genome sequence space,” Eisen remarked. The difficulty of culturing most microbes presents a major obstacle to accomplishing this goal. His group has embarked on an effort to sequence representative cultured bacte- rial and archaeal species from the missing phyla, thereby filling out a genomic “tree of life” that can be used to study the biology of organisms (see Eisen in Chapter 5).

 WORKSHOP OVERVIEW In addition to understanding and surveying microbial biodiversity at the taxo- nomic and genomic level, Eisen also aspires to the more challenging goal of sam- pling functional diversity across a broad range of microbes. “What we need are more experiments across the tree of life . . . for all processes that we are interested in,” he said, “not just in a couple of model organisms. [Microbial] diversity is immense.” He proposed developing a “field guide to the microbes”—akin to those for birds—that not only describes their taxonomy, but their behavior, ecology, and distribution patterns. With such comprehensive knowledge, he concluded, “we can really integrate all this information and maybe predict the future.” Participants also discussed the feasibility of obtaining and applying detailed knowledge of intraspecies diversity, as evidenced by phylogeny. The method used by Dougan and coworkers (see previous discussion and Chapter 3) to discern the complete phylogeny of 200 S. typhi strains, known as DNA-based signature typ- ing, could potentially be used to conduct genetic studies of bacteria in the field or to type bacteria for diagnostic or surveillance purposes, Dougan said; moreover, it could be used in clinical settings to investigate associations between specific mutations in a pathogen strain and its phenotype, manifested in the characteristics (e.g., transmissibility, virulence) of disease. It also illustrates Lederberg’s hopeful description of the present era of infectious history: “Together with wiser insight into the ground rules of pathogenic evolution, we are developing a versatile plat- form for developing new responses to infectious disease” (Lederberg, 2000). APPENDIx WO-1 INFECTIOUS HISTORY24 Joshua Lederberg, Ph.D.25 In 1530, to express his ideas on the origin of syphilis, the Italian physician Girolamo Fracastoro penned Syphilis, sive morbus Gallicus (Syphilis, or the French disease) in verse. In it he taught that this sexually transmitted disease was spread by “seeds” distributed by intimate contact. In later writings, he expanded this early “contagionist” theory. Besides contagion by personal contact, he described contagion by indirect contact, such as the handling or wearing of clothes, and even contagion at a distance, that is, the spread of disease by some- thing in the air. Fracastoro was anticipating, by nearly 350 years, one of the most important 24This article was originally published in Science 288(5464):287-293. Reprinted with permission from AAAS. Copyright 2000. 25 Joshua Lederberg (1925-2008) was a Sackler Foundation Scholar heading the Laboratory of Molecular Genetics and Informatics at The Rockefeller University in New York City, and a Nobel laureate (1958) for his research on genetic mechanisms in bacteria. He worked closely with the Institute of Medicine and the Centers for Disease Control and Prevention on analytical and policy studies on emerging infections.

 MICROBIAL EVOLUTION AND CO-ADAPTATION turning points in biological and medical history—the consolidation of the germ theory of disease by Louis Pasteur and Robert Koch in the late 1870s. As we enter the 21st century, infectious disease is fated to remain a crucial research challenge, one of conceptual intricacy and of global consequence. The Incubation of a Scientific Discipline Many people laid the groundwork for the germ theory. Even the terrified masses touched by the Black Death (bubonic plague) in Europe after 1346 had some intimation of a contagion at work. But they lived within a cognitive frame- work in which scapegoating, say, of witches and Jews, could more “naturally” account for their woes. Breaking that mindset would take many innovations, including microscopy in the hands of Anton van Leeuwenhoek. In 1683, with one of his new microscopes in hand, he visualized bacteria among the animal- cules harvested from his own teeth. That opened the way to visualize some of the dreaded microbial agents eliciting contagious diseases. There were pre-germ-theory advances in therapy, too. Jesuit missionaries in malaria-ridden Peru had noted the native Indians’ use of Cinchona bark. In 1627, the Jesuits imported the bark (harboring quinine, its anti-infective ingredient) to Europe for treating malaria. Quinine thereby joined the rarified pharmacopoeia— including opium, digitalis, willow (Salix) bark with its analgesic salicylates, and little else—that prior to the modern era afforded patients any benefit beyond placebo. Beginning in 1796, Edward Jenner took another major therapeutic step—the development of vaccination—after observing that milkmaids exposed to cowpox didn’t contract smallpox. He had no theoretical insight into the biological mecha- nism of resistance to the disease, but vaccination became a lasting prophylactic technique on purely empirical grounds. Jenner’s discovery had precursors. “Hair of the dog” is an ancient trope for countering injury and may go back to legends of the emperor Mithridates, who habituated himself to lethal doses of poisons by gradually increasing the dose. We now understand more about a host’s immuno- logical response to a cross-reacting virus variant. Sanitary reforms also helped. Arising out of revulsion over the squalor and stink of urban slums in England and the United States, a hygienic movement tried to scrub up dirt and put an end to sewer stenches. The effort had some health impact in the mid-19th century, but it failed to counter diseases spread by fleas and mosquitoes or by personal contact, and it often even failed to keep sewage and drinking water supplies separated. It was the germ theory—which is credited to Pasteur (a chemist by training) and Koch (ultimately a German professor of public health)—that set a new course for studying and contending with infectious disease. Over the second half of the 19th century, these scientists independently synthesized historical evidence with their own research into the germ theory of disease.

 WORKSHOP OVERVIEW Pasteur helped reveal the vastness of the microbial world and its many practi - cal applications. He found microbes to be behind the fermentation of sugar into alcohol and the souring of milk. He developed a heat treatment (pasteurization, that is) that killed microorganisms in milk, which then no longer transmitted tuberculosis or typhoid. And he too developed new vaccines. One was a veteri- nary vaccine against anthrax. Another was against rabies and was first used in humans in 1885 to treat a young boy who had been bitten by a rabid dog. One of Koch’s most important advances was procedural. He articulated a set of logical and experimental criteria, later restated as “Koch’s Postulates,” as a standard of proof for researchers’ assertions that a particular bacterium caused a particular malady. In 1882, he identified the bacterium that causes tuberculosis; a year later he did the same for cholera. Koch also left a legacy of students (and rivals) who began the systematic search for disease-causing microbes: The golden age of microbiology had begun. Just as the 19th century was ending, the growing world of microbes mush- roomed beyond bacteria. In 1892, the Russian microbiologist Dmitri Ivanowski, and in 1898, the Dutch botanist Martinus Beijerinck, discovered exquisitely tiny infectious agents that could pass through bacteria-stopping filters. Too small to be seen with the conventional microscope, these agents were described as “filtrable [sic] viruses.” With a foundation of germ theory in place even before the 20th century, the study of infectious disease was ready to enter a new phase. Microbe hunting became institutionalized, and armies of researchers systematically applied scien- tific analyses to understanding disease processes and developing therapies. During the early acme of microbe hunting, from about 1880 to 1940, how- ever, microbes were all but ignored by mainstream biologists. Medical micro- biology had a life of its own, but it was almost totally divorced from general biological studies. Pasteur and Koch were scarcely mentioned by the founders of cell biology and genetics. Instead, bacteriology was taught as a specialty in medi- cine, outside the schools of basic zoology and botany. Conversely, bacteriologists scarcely heard of the conceptual revolutions in genetic and evolutionary theory. Bacteriology’s slow acceptance was partly due to the minuscule dimen- sions of microbes. The microscopes of the 19th and early 20th centuries could not resolve internal microbial anatomy with any detail. Only with the advent of electron microscopy in the 1930s did these structures (nucleoids, ribosomes, cell walls and membranes, flagella) become discernible. Prior to that instrumental breakthrough, most biologists had little, if anything, to do with bacteria and viruses. When they did, they viewed such organisms as mysteriously precellular. It was still an audacious leap for René Dubos to entitle his famous 1945 mono- graph “The Bacterial Cell.” The early segregation of bacteriology and biology per se hampered the scien- tific community in recognizing the prospects of conducting genetic investigation with bacteria. So it is ironic that the pivotal discovery of molecular genetics—that

 MICROBIAL EVOLUTION AND CO-ADAPTATION genetic information resides in the nucleotide sequence of DNA—arose from stud- ies on serological types of pneumococcus, studies needed to monitor the epidemic spread of pneumonia. This key discovery was initiated in 1928 by the British physician Frederick Griffith. He found that extracts of a pathogenic strain of pneumococcus could transform a harmless strain into a pathogenic one. The hunt was then on to identify the “transforming factor” in the extracts. In 1944, Oswald Avery, Colin MacLeod, and Maclyn McCarty reported in the Journal of Experimental Medi- cine that DNA was the transforming factor. Within a few years, they and others ruled out skeptics’ objections that protein coextracted with the DNA might actu- ally be the transforming factor. Those findings rekindled interest in what was really going on in the life cycle of bacteria. In particular, they led to my own work in 1946 on sexual conjugation in Escherichia coli and to the construction of chromosome maps emulating what had been going on in the study of the genetics of fruit flies, maize, and mice for the prior 45 years. Bacteria and bacterial viruses quickly supplanted fruit flies as the test-bed for many of the subsequent developments of molecular genetics and the biotechnology that followed. Ironically, during this time, we were becoming nonchalant about microbes as etiological agents of disease. Despite its slow emergence, bacteriology was already having a large impact. Its success is most obviously evidenced by the graying of the population. That public health has been improving—due to many factors, especially our better understanding of infectious agents—is graphically shown by the vital statistics. These began to be diligently recorded in the United States after 1900 in order to guide research and apply it to improving public health. The U.S. experience stands out in charts (Figure WO-15) depicting life expectancy at birth through the century. The average life-span lengthened dramatically: from 47 years in 1900 to today’s expectation of 77 years (74 years for males and 80 for females).26 Similar trends are seen in most other industrialized countries, but the gains have been smaller in economically and socially depressed countries. Other statistics reveal that the decline in mortality ascribable to infectious disease accounted for almost all of the improvement in longevity up to 1950, when life expectancy had reached 68. The additional decade of life expectancy for babies born today took the rest of the century to gain. Further improvements now appear to be on an asymptotic trajectory: Each new gain is ever harder to come by, at least pending unpredictable breakthroughs in the biology of aging. The mortality statistics fluctuated considerably during the first half of the last 26This sex difference in life expectancy is partly explained by the ability of two X chromosomes to buffer against accumulated recessive mutations and is illustrated by the prevalence in males of color blindness and hemophilia. Another factor is the gender-related difference in self-destructive behaviors.

 WORKSHOP OVERVIEW 90 80 70 Age 60 50 40 1900 1920 1940 1960 1980 2000 Year FIGURE WO-15 Longer life. Anti-infection medicine and other factors have helped Figure WO-15 COLOR.eps dramatically lengthen the average life expectancy in the United States. SOURCE: Social Security Officew/ the Chief Actuary. bitmap image of vector type elements on axes century. Much of this instability was due to sporadic outbreaks of infections such as typhoid fever, tuberculosis, and scarlet fever, which no longer have much sta- tistical impact. Most outstanding is the spike due to the great influenza pandemic of 1918-19 that killed 25 million people worldwide—comparable to the number of deaths in the Great War. Childhood immunization and other science-based medical interventions have played a significant role in the statistical trends also. So have public health measures, among them protection of food and water sup- plies, segregation of coughing patients, and personal hygiene. Overall economic growth has also helped by contributing to less crowded housing, improved work- ing conditions (including sick leave), and better nutrition. As infectious diseases have assumed lower rankings in mortality statis- tics, other killers—mostly diseases of old age, affluence, and civilization—have moved up the ladder. Heart disease and cancer, for example, have loomed as larger threats over the past few decades. Healthier lifestyles, including less smok- ing, sparer diets, more exercise, and better hygiene, have been important coun- termeasures. Prophylactic medications such as aspirin, as well as medical and surgical interventions, have also kept people alive longer.

 MICROBIAL EVOLUTION AND CO-ADAPTATION The 1950s were notable for the “wonder drugs”—the new antibiotics peni- cillin, streptomycin, chloramphenicol, and a growing list of others that at times promised an end to bacteria-based disease. Viral pathogens have offered fewer routes to remedies, except for vaccines, such as Jonas Salk’s and Albert Sabin’s polio vaccines. These worked by priming immune systems for later challenges by the infectious agents. Old vaccines, including Jenner’s smallpox vaccine, also were mobilized in massive public health campaigns, sometimes with fantastic results. By the end of the 1970s, smallpox became the first disease to be eradi- cated from the human experience. Confidence about medicine’s ability to fight infectious disease had grown so high by the mid-1960s that some optimists were portraying infectious microbes as largely conquered. They suggested that researchers shift their attention to consti- tutional scourges of heart disease, cancer, and psychiatric disorders. These views were reflected in the priorities for research funding and pharmaceutical develop- ment. President Nixon’s 1971 launch of a national crusade against cancer, which tacitly implied that cancer could be conquered by the bicentennial celebrations of 1976, was an example. Few people now sustain the illusion that audacious medical goals like conquering cancer or infectious disease can be achieved by short-term campaigns. TABLE WO-1 An Infectious Disease Timeline 1300s 1346 Black Death begins spreading in Europe. 1400s 1492 Christopher Columbus initiates European-American contact, which leads to transmission of European diseases to the Americas and vice versa. 1500s 1530 Girolamo Fracastoro puts forward an early version of the germ theory of disease. 1600s 1627 Cinchona bark (quinine) is brought to Europe to treat malaria. 1683 Anton van Leeuwenhoek uses his microscopes to observe tiny animalcules (later known as bacteria) in tooth plaque. 1700s 1796 Edward Jenner develops technique of vaccination, at first against smallpox.

 WORKSHOP OVERVIEW TABLE WO-1 Continued 1800s 1848 Ignaz Semmelweis introduces antiseptic methods. 1854 John Snow recognizes link between the spread of cholera and drinking water supplies. 1860s Louis Pasteur concludes that infectious diseases are caused by living organisms called “germs.” An early practical consequence was Joseph Lister’s development of antisepsis by using carbolic acid to disinfect wounds. 1876 Robert Koch validates germ theory of disease and helps initiate the science of bacteriology with a paper pinpointing a bacterium as the cause of anthrax. 1880 Louis Pasteur develops method of attenuating a virulent pathogen (for chicken cholera) so that it immunizes but does not infect; in 1881 he devises an anthrax vaccine and in 1885, a rabies vaccine. Charles Laveran finds malarial parasites in erythrocytes of infected people and shows that the parasite replicates in the host. 1890 Emil von Behring and Shibasaburo Kitasato discover diphtheria antitoxin serum, the first rational approach to therapy for infectious disease. 1891 Paul Ehrlich proposes that antibodies are responsible for immunity. 1892 The field of virology begins when Dmitri Ivanowski discovers exquisitely small pathogenic agents, later known as viruses, while searching for the cause of tobacco mosaic disease. 1899 Organizing meeting of the Society of American Bacteriologists—later to be known as the American Society for Microbiology—is held at Yale University. 1900s 1900 Based on work by Walter Reed, a commission of researchers shows that yellow fever is caused by a virus from mosquitoes; mosquito-eradication programs are begun. 1905 Fritz Schaudinn and Erich Hoffmann discover bacterial cause of syphilis— Treponema pallidum. 1911 Francis Rous reports on a viral etiology of a cancer (Rous sarcoma virus). 1918-1919 Epidemic of “Spanish” flu causes at least 25 million deaths. 1928 Frederick Griffith discovers genetic transformation phenomenon in pneumococci, thereby establishing a foundation of molecular genetics. Continued

0 MICROBIAL EVOLUTION AND CO-ADAPTATION TABLE WO-1 Continued 1929 Alexander Fleming reports discovering penicillin in mold. 1935 Gerhard Domagk synthesizes the antimetabolite Prontosil, which kills Streptococcus in mice. 1937 Ernst Ruska uses an electron microscope to obtain first pictures of a virus. 1941 Selman Waksman suggests the word “antibiotic” for compounds and preparations that have antimicrobial properties; 2 years later, he and colleagues discover streptomycin, the first antibiotic effective against tuberculosis, in a soil fungus. 1944 Oswald Avery, Colin MacLeod, and Maclyn McCarty identify DNA as the genetically active material in the pneumococcus transformation. 1946 Edward Tatum and Joshua Lederberg discover “sexual” conjugation in bacteria. 1948 The World Health Organization (WHO) is formed within the United Nations. 1952 Renato Dulbecco shows that a single virus particle can produce plaques. 1953 James Watson and Francis Crick reveal the double helical structure of DNA. Late 1950s Frank Burnet enunciates clonal selection theory of the immune response. 1960 Arthur Kornberg demonstrates DNA synthesis in cell-free bacterial extract. François Jacob and Jacques Monod report work on genetic control of enzyme and virus synthesis. 1970 Howard Temin and David Baltimore independently discover that certain RNA viruses use reverse transcription (RNA to reconstitute DNA) as part of their replication cycle. 1975 Asilomar conference sets standards for the containment of possible biohazards from recombinant DNA experiments with microbes. 1979 Smallpox eradication program of WHO is completed; the world is declared free of smallpox. 1981 AIDS first identified as a new infectious disease by U.S. Centers for Disease Control and Prevention. 1982 Stanley Prusiner finds evidence that a class of infectious proteins, which he calls prions, cause scrapie in sheep. 1983 Luc Montagnier and Robert Gallo announce their discovery of the human immunodeficiency virus that is believed to cause AIDS.

 WORKSHOP OVERVIEW TABLE WO-1 Continued 1984 Barry Marshall shows that isolates from ulcer patients contain the bacterium later known as Helicobacter pylori. The discovery ultimately leads to a new pathogen-based etiology of ulcers. 1985 Robert Gallo, Dani Bolognesi, Sam Broder, and others show that AZT inhibits HIV action in vitro. 1988 Kary Mullis reports basis of polymerase chain reaction (PCR) for detection of even single DNA molecules. 1995 J. Craig Venter, Hamilton Smith, Claire Fraser, and colleagues at The Institute for Genomic Research elucidate the first complete genome sequence of a microorganism: Haemophilus influenzae. 1996 Implied link between bovine spongiform encephalopathy (“mad cow disease”) and human disease syndrome leads to large-scale controls on British cattle. 1999 New York experiences outbreak of West Nile encephalopathy transmitted by birds and mosquitoes. 2000s c. 2000 Antibiotic-resistant pathogens are spreading in many environments. NOTE: For more extensive chronological listings, see “Microbiology’s fifty most significant events during the past 125 years,” poster supplement to ASM News 65(5), 1999. Wake-Up Calls The overoptimism and complacency of the 1960s and 1970s was shattered in 1981 with the recognition of AIDS. Since then, the spreading pandemic has overtaken one continent after another with terrible costs. Its spread has been coin- cident with another wake-up call—the looming problem of antibiotic-resistant microbes. This was a predictable consequence of the evolutionary process operat- ing on microbes challenged by the new selection pressure of antibiotics, arising in part from medical prescriptions and in part from unregulated sales and use in feed for crop animals. AIDS’s causative agent, the human immunodeficiency virus (HIV), is a member of the retrovirus family. These viruses had been laboratory curiosities since 1911, when Francis Peyton Rous discovered the Rous sarcoma virus (RSV) in chickens. Early basic research on retroviruses later helped speed advances in HIV research. By the time AIDS began to spread, RSV had been studied for years as a model for cancer biology, because it could serve as a vector for transferring

 MICROBIAL EVOLUTION AND CO-ADAPTATION oncogenes into cells. That work accelerated the characterization of HIV as a retrovirus, and it also helped guide our first steps toward medications that slow HIV infection. AIDS and HIV have spurred the most concentrated program of biomedi- cal research in history, yet they still defy our counterattacks. And our focus on extirpating the virus may have deflected less ambitious, though more pragmatic, aims, including learning to live with the virus by nurturing in equal measure the immune system that HIV erodes. After all, natural history points to analogous infections in simians that have long since achieved a mutually tolerable state of equilibrium. Costly experiences with AIDS and other infectious agents have led to wide- spread reexamination of our cohabitation with microbes. Increased monitoring and surveillance by organizations such as the U.S. Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) have revealed a stream of outbreaks of exotic diseases. Some have been due to the new impor- tation of microbes (such as cholera in the Southern Hemisphere); some to older parasites (such as Legionella) that have been newly recognized as pathogenic; and some to newly evolved antibiotic-resistant pneumonia strains. Even maladies that had never before been associated with infectious agents recently have been revealed as having microbial bases. Prominent among these are gastric ulcers, which previously had been attributed almost entirely to stress and other psychosomatic causes. Closer study, however, has shown a Helico- bacter to be the major culprit. Researchers are now directing their speculations away from stress and toward Chlamydia infection as a cause of atherosclerosis and coronary disease. The litany of wake-up calls goes on. Four million Americans are estimated to be infected with hepatitis C, mainly by transfusion of contaminated blood prod- ucts. This population now is at significant risk for developing liver cancer. Those harboring hepatitis C must be warned to avoid alcohol and other hepatotoxins, and they must not donate blood. Smaller but lethal outbreaks of dramatic, hypervirulent viruses have been raising public fear. Among these are the Ebola virus outbreak in Africa in 1976 and again in 1995 and the hantavirus outbreak in the U.S. Southwest in 1993. In hindsight, these posed less of a public health risk than the publicity they received might have suggested. Still, studying them and uncovering ecological factors that favor or thwart their proliferation is imperative because of their potential to mutate into more diffusible forms. Our vigilance is mandated also by the facts of life: The processes of gene reassortment in flu viruses, which are poorly confined to their canonical hosts (birds, swine, and people), goes on relentlessly and is sure to regenerate human- lethal variants. Those thoughts were central in 1997 when the avian flu H5N1 transferred into a score of Hong Kong citizens, a third of whom died. It is likely that the resolute actions of the Hong Kong health authorities, which destroyed 2

 WORKSHOP OVERVIEW million chickens, stemmed that outbreak and averted the possibility of a world- wide spread of H5N1. Complacency is not an option in these cases, as other vectors, including wildfowl, could become carriers. In Malaysia, a new infectious entity, the Nipah virus, killed up to 100 people last year; authorities there killed a million livestock to help contain the outbreak. New York had a smaller scale scare last summer with the unprecedented appearance of bird- and mosquito-borne West Nile encephali- tis, although the mortality rate was only a few percent of those infected. We need not wonder whether we will see outbreaks like these again. The only questions are when and where? These multiple wake-up calls to the infectious disease problem have left marks in vital statistics. From mid-century to 1982, the U.S. mortality index (annual deaths per 100,000) attributable to infection had been steady at about 30. But from 1982 to 1994, the rate doubled to 60. (Keep in mind that the index was 500 in 1900 and up to 850 in 1918-19 due to the Spanish flu epidemic.) About half of the recent rise in deaths is attributable to AIDS; much of the rest is due to respiratory disease, antibiotic resistance, and hospital-acquired infection. Our Wits Versus Their Genes As our awareness of the microbial environment has intensified, important questions have emerged. What puts us at risk? What precautions can and should we be taking? Are we more or less vulnerable to infectious agents today than in the past? What are the origins of pathogenesis? And how can we use deeper knowledge to develop better medical and public health strategies? Conversely, how much more can the natural history of disease teach us about fundamental biological and evolutionary mechanisms? An axiomatic starting point for further progress is the simple recognition that humans, animals, plants, and microbes are cohabitants of the planet. That leads to refined questions that focus on the origin and dynamics of instabilities within this context of cohabitation. These instabilities arise from two main sources loosely definable as ecological and evolutionary. Ecological instabilities arise from the ways we alter the physical and bio- logical environment, the microbial and animal tenants (humans included) of these environments, and our interactions (including hygienic and therapeutic interventions) with the parasites. The future of humanity and microbes likely will unfold as episodes of a suspense thriller that could be titled Our Wits Versus Their Genes. We already have used our wits to increase longevity and lessen mortality. That simultaneously has introduced irrevocable changes in our demographics and our own human ecology. Increased longevity, economic productivity, and other factors have abetted a global population explosion from about 1.6 billion in 1900 to its present level above 6 billion. That same population increase has

 MICROBIAL EVOLUTION AND CO-ADAPTATION fostered new vulnerabilities: crowding of humans, with slums cheek by jowl with jet setters’ villas; the destruction of forests for agriculture and suburbanization, which has led to closer human contact with disease-carrying rodents and ticks; and routine long-distance travel. Travel around the world can be completed in less than 80 hours (compared to the 80 days of Jules Verne’s 19th-century fantasy), constituting a historic new experience. This long-distance travel has become quotidian: Well over a million passengers, each one a potential carrier of pathogens, travel daily by aircraft to international destinations. International commerce, especially in foodstuffs, only adds to the global traffic of potential pathogens and vectors. Because the transit times of people and goods now are so short compared to the incubation times of disease, carriers of disease can arrive at their destination before the danger they harbor is detectable, reducing health quarantine to a near absurdity. Our systems for monitoring and diagnosing exotic diseases have hardly kept pace with this qualitative transformation of global human and material exchange. This new era of global travel will redistribute and mix people, their cultures, their prior immunities, and their inherited predispositions, along with pathogens that may have been quiescent at other locales for centuries. This is not completely novel, of course. The most evident precedent unfolded during the European conquest of America, which was tragically abetted by pan- demics of smallpox and measles imported into native populations by the invad- ing armies. In exchange, Europeans picked up syphilis’s Treponema, in which Fracastoro discerned contagion at work. Medical defense against the interchange of infectious disease did not exist in the 16th century. In the 21st century, however, new medical technologies will be key parts of an armamentarium that reinforces our own immunological defenses. This dependence on technology is beginning to be recognized at high levels of national and international policy-making. With the portent of nearly instant global transmission of pathogenic agents, it is ever more important to work with international organizations like WHO for global health improvement. After all, the spread of AIDS in America and Europe in the 1980s and 1990s was due, in part, to an earlier phase of near obliviousness to the frightful health conditions in Africa. One harbinger of the kind of high-tech wit we will need for defending against outbreaks of infectious disease is the use of cutting-edge communications technology and the Internet, which already have been harnessed to post prompt global alerts of emerging diseases (see osi.oracle.com:8080/promed/promed. home). Moving Targets “Germs” have long been recognized as living entities, but the realization that they must inexorably be evolving and changing has been slow to sink in to the ideology and practice of the public health sector. This lag has early roots. In

 WORKSHOP OVERVIEW the 19th century, Koch was convinced that rigorous experiments would support the doctrine of monomorphism: that each disease was caused by a single invari- ant microbial species rather than by the many that often showed up in culture. He argued that most purported “variants” were probably alien bacteria that had floated into the petri dishes from the atmosphere. Koch’s rigor was an essential riposte to careless claims of interconvertibility— for example, that yeasts could be converted into bacteria. It also helped untangle confusing claims of complex morphogenesis and life cycles among common bac- teria. But strict monomorphism was too rigid, and even Koch eventually relented, admitting the possibility of some intrinsic variation rather than contamination. Still, for him and his contemporaries, variation remained a phenomenological and experimental nuisance rather than the essence of microbes’ competence as pathogens. The multitude of isolable species was confusing enough to the epi- demic tracker; it would have been almost too much to bear to have to cope with constantly emerging variants with altered serological specificity, host affinity, or virulence. Even today it would be near heresy to balk at the identification of the great plague of the 14th century with today’s Yersinia pestis; but we cannot readily account for its pneumonic transmission without guessing at some intrinsic adap- tation at the time to aerosol conveyance. Exhumations of ancient remains might still furnish DNA evidence to test such ideas. We now know and accept that evolutionary processes elicit changes in the genotypes of germs and of their hosts. The idea that infection might play an important role in natural selection sank in after 1949 when John B. S. Haldane conjectured that the prevalence of hemoglobin disorders in Mediterranean peoples might be a defense against malaria. That idea developed into the first concrete example of a hereditary adaptation to infectious disease. Haldane’s theory preceded Anthony C. Allison’s report of the protective effect of heterozygous hemoglobinopathy against falciparum malaria in Africa. The side effects of this bit of natural genetic engineering are well known: When this beneficial polymorphism is driven to higher gene frequencies, the homozy- gous variant becomes more prevalent and with it the heavy human and societal burden of sickle cell disease. We now have a handful of illustrations of the connection between infection and evolution. Most are connected to malaria and tuberculosis, which are so prevalent that genetic adaptations capable of checking them have been strongly selected. The same prevalence also makes their associated adaptations more obvious to researchers. A newly reported link between infection and evolution is the effect of a ccr (chemokine receptor) deletion, a genetic alteration that affords some protection against AIDS. It would be interesting to know what factors—another pathogen perhaps—may have driven that polymorphism in ear- lier human history. One lesson to be gleaned from this coevolutionary dynamic is how fitful and

 MICROBIAL EVOLUTION AND CO-ADAPTATION sporadic human evolution is when our slow and plodding genetic change is pitted against the far more rapidly changing genomes of microbial pathogens. We have inherited a robust immune system, but little has changed since its early vertebrate origins 200 million years ago. In its inner workings, immunity is a Darwinian struggle: a randomly generated diversification of leukocytes that collectively are prepared to duel with a lifetime of unpredictable invaders. But these duels take place in the host soma; successful immunological encounters do not become genetically inscribed and passed on to future generations of the host. By contrast, the germs that win the battles quickly proliferate their successful genes, and they can use those enhancements to go on to new hosts, at least in the short run. The human race evidently has withstood the pathogenic challenges encoun- tered so far, albeit with episodes of incalculable tragedy. But the rules of encoun- ter and engagement have been changing; the same record of survival may not necessarily hold for the future. If our collective immune systems fail to keep pace with microbial innovations in the altered contexts we have created, we will have to rely still more on our wits. Evolving Metaphors of Infection: Teach War No More New strategies and tactics for countering pathogens will be uncovered by finding and exploiting innovations that evolved within other species in defense against infection. But our most sophisticated leap would be to drop the man- ichaean view of microbes—“We good; they evil.” Microbes indeed have a knack for making us ill, killing us, and even recycling our remains to the geosphere. But in the long run microbes have a shared interest in their hosts’ survival: A dead host is a dead end for most invaders too. Domesticating the host is the better long-term strategy for pathogens. We should think of each host and its parasites as a superorganism with the respective genomes yoked into a chimera of sorts. The power of this sociological development could not be more persuasively illustrated than by the case of mito- chondria, the most successful of all microbes. They reside inside every eukaryote cell (from yeast to protozoa to multicellular organisms), in which they provide the machinery of oxidative metabolism. Other bacteria have taken similar routes into plant cells and evolved there into chloroplasts—the primary harvesters of solar energy, which drive the production of oxygen and the fixed carbon that nourishes the rest of the biosphere. These cases reveal how far collaboration between hosts and infecting microbes can go. In the short run, however, the infected host is in fact at metastable equi- librium: The balance could tip toward favorable or catastrophic outcomes. On the bad side, the host’s immune response may be excessive, with autoim- mune injuries as side effects. Microbial zeal also can be self-defeating. As with rogue cancer cells, deviant microbial cells (such as aggressive variants from a

 WORKSHOP OVERVIEW gentler parent population) may overtake and kill the host, thereby fomenting their own demise and that of the parent population. Most successful parasites travel a middle path. It helps for them to have aggressive means of entering the body surfaces and radiating some local toxicity to counter the hosts’ defenses, but once established they also do themselves (and their hosts) well by moderating their virulence. Better understanding of this balancing act awaits further research. And that may take a shift in priorities. For one, research has focused on hypervirulence. Studies into the physiology of homeostatic balance in the infected host qua superorganism have lagged. Yet the latter studies may be even more revealing, as the burden of mutualistic adaptation falls largely on the shoulders of the para- site, not the host. This lopsided responsibility follows from the vastly different evolutionary paces of the two. But then we have our wits, it is to be hoped, for drafting the last word. To that end, we also need more sophisticated experimental models of infec- tion, which today are largely based on contrived zoonoses (the migration of a parasite from its traditional host into another species). The test organism is usu- ally a mouse, and the procedure is intended to mimic the human disease process. Instead, it is often a caricature. Injected with a few bugs, the mouse goes belly up the next day. This is superb for in vivo testing of an antibiotic, but it bears little relation to the dynamics of everyday human disease. Natural zoonoses also can have many different outcomes. In most cases, there will be no infection at all or only mild ones such as the gut ache caused by many Salmonella enteritidis species. Those relatively few infectious agents that cause serious sickness or death are actually maladapted to their hosts, to which they may have only recently gained access through some genetic, environmental, or sociological change. These devastatingly virulent zoonoses include psittacosis, Q fever, rickettsiosis, and hantavirus. Partly through lack of prior coevolutionary development with the new host, normal restraints fail. I suggest that a successful parasite (one that will be able to remain infec- tious for a long time) tends to display just those epitopes (antigen fragments that stimulate the immune system) as will provoke host responses that (a) moderate but do not extinguish the primary infection, and (b) inhibit other infections by competing strains of the same species or of other species. According to this speculative framework, the symptoms of influenza evolved as they have in part to ward off other viral infections. Research into infectious diseases, including tuberculosis, schistosomiasis, and even AIDS, is providing evidence for this view. So are studies of Helico- bacter, which has been found to secrete antibacterial peptides that inhibit other enteric infections. We need also to look more closely at earlier stages of chronic infection and search for cross-protective factors by which microbes engage one another. HIV, for one, ultimately fails from the microbial perspective when

 MICROBIAL EVOLUTION AND CO-ADAPTATION opportunistic infections supervene to kill its host. That result, which is tragic from the human point of view, is a byproduct of the virus’s protracted duel with the host’s cellular immune system. The HIV envelope and those of related viruses also produce antimicrobials, although their significance for the natural history of disease remains unknown. Now genomics is entering the picture. Within the past decade, the genomes of many microbes have been completely sequenced. New evidence for the web of genetic interchange is permeating the evolutionary charts. The functional analyses of innumerable genes now emerging are an unexplored mine of new therapeutic targets. It has already shown many intricate intertwinings of hosts’ and parasites’ physiological pathways. Together with wiser insight into the ground rules of pathogenic evolution, we are developing a versatile platform for developing new responses to infectious disease. Many new vaccines, antibiotics, and immune modulators will emerge from the growing wealth of genomic data. The lessons of HIV and other emerging infections also have begun taking hold in government and in commercial circles, where the market opportunities these threats offer have invigorated the biotechnology industry. If we do the hard work and never take success for granted (as we did for a while during the last century), we may be able to preempt infectious disasters such as the influenza outbreak of 1918-1919 and the more recent and ongoing HIV pandemic. Perhaps one of the most important changes we can make is to supercede the 20th-century metaphor of war for describing the relationship between people and infectious agents. A more ecologically informed metaphor, which includes the germs’-eye view of infection, might be more fruitful. Consider that microbes occupy all of our body surfaces. Besides the disease-engendering colonizers of our skin, gut, and mucous membranes, we are host to a poorly cataloged ensem- ble of symbionts to which we pay scant attention. Yet they are equally part of the superorganism genome with which we engage the rest of the biosphere. The protective role of our own microbial flora is attested to by the superinfec- tions that often attend specific antibiotic therapy: The temporary decimation of our home-team microbes provides entrée for competitors. Understanding these phenomena affords openings for our advantage, akin to the ultimate exploitation by Dubos and Selman Waksman of intermicrobial competition in the soil for seeking early antibiotics. Research into the microbial ecology of our own bodies will undoubtedly yield similar fruit. Replacing the war metaphor with an ecological one may bear on other impor- tant issues, including debates about eradicating pathogens such as smallpox and polio. Without a clear strategy for sustaining some level of immunity, it makes sense to maintain lab stocks of these and related agents to guard against possible recrudescence. An ecological perspective also suggests other ways of achieving lasting security. For example, domestication of commensal microbes that bear relevant cross-reacting epitopes could afford the same protection as vaccines based on the virulent forms. There might even be a nutraceutical angle: These

 WORKSHOP OVERVIEW commensal epitopes could be offered as optional genetically engineered food additives, clearly labeled and meticulously studied. Another relevant issue that can be recast in an ecological model is the rise in popularity of antibacterial products. This is driven by the popular idea that a superhygienic environment is better than one with germs—the “enemy” in the war metaphor. But too much antibacterial zeal could wipe out the very immuno- genic stimulation that has enabled us to cohabit with microbes in the first place. BOX WO-4 The Microbial World Wide Web The field of molecular genetics, which began in 1944 when DNA was proven to be the molecule of heredity in bacteria-based experiments, ushered microbes into the center of many biological investigations. Microbial systems now provide our most convenient models for experimental evolution. Diverse mechanisms for genetic variation and recombination uncovered in such systems are spelled out in ponderous monographs. Assays for chemical mutagenesis (e.g., the Ames test using Salmonella) are now routinely carried out on bacteria, because microbial DNA is so accessible to environmental insult. Mutators (genes that enhance variability) abound and may be switched on and off by different environmental factors. The germs’ ability to transfer their own genetic scripts, via processes such as plasmid transfer, means they can exchange biological innovations including resistance to antibiotics. Indeed, the microbial biosphere can be thought of as a World Wide Web of informational exchange, with DNA serving as the packets of data going every which way. The analogy isn’t entirely superficial. Many viruses can integrate (download) own DNA into host genomes, which subsequently can be copied and passed on: Hundreds of segments of human DNA originated from historical encounters with retroviruses whose genetic information became integrated into our own genomes. What makes microbial evolution particularly intriguing, and worrisome, is a combination of vast populations and intense fluctuations in those populations. It’s a formula for top-speed evolution. Microbial populations may fluctuate by factors of 10 billion on a daily cycle as they move between hosts, or as they encounter antibiotics, antibodies, or other natural hazards. A simple comparison of the pace of evolution between microbes and their multicellular hosts suggests a million fold or billion fold advantage to the microbe. A year in the life of bacteria would easily match the span of mammalian evolution! By that metric, we would seem to be playing out of our evolutionary league. Indeed, there’s evidence of sporadic species extinctions in natural history, and our own human history has been punctuated by catastrophic plagues. Yet we are still here! Maintaining that status within new contexts in which germs and hosts inter- act in new ways almost certainly will require us to bring ever more sophisticated technical wit and social intelligence to the contest. -J. L.

0 MICROBIAL EVOLUTION AND CO-ADAPTATION Ironically, even as I advocate this shift from a war metaphor to an ecology metaphor, war in its historic sense is making that more difficult. The darker corner of microbiological research is the abyss of maliciously designed biologi- cal warfare (BW) agents and systems to deliver them. What a nightmare for the next millennium! What’s worse, for the near future, technology is likely to favor offensive BW weaponry, because defenses will have to cope with a broad range of microbial threats that can be collected today or designed tomorrow. As a measure of social intelligence and policy, we should push for enforce- ment of the 1975 BW disarmament convention. The treaty forbids the devel- opment, production, stockpiling, and use of biological weapons under any circumstances. One of its articles also provides for the international sharing of biotechnology for peaceful purposes. The scientific and humanistic rationale is self-evident: to enhance and apply scientific knowledge to manage infectious disease, naturally occurring or otherwise. Further Readings W. Bulloch, History of Bacteriology (Oxford University Press, London, 1938). R. Dubos, Mirage of Health: Utopias, Progress, and Biological Change (Rutgers University Press, New Brunswick, New Jersey, 1987). J. Lederberg, R. E. Shope, S. C. Oaks Jr., Eds., Emerging Infections: Microbial Threats to Health in the United States (National Academy Press, Washington, D.C., 1992) (see www.nap.edu/books/0309047412/html/index.html). G. Rosen, A History of Public Health (Johns Hopkins University Press, Balti- more, 1993). T. D. Brock, The Emergence of Bacterial Genetics (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1990). S. S. Morse, Ed., Emerging Viruses (Oxford University Press, New York, 1993). Journal of the American Medical Association theme issue on emerging infections, August 1996. W. K. Joklik et al., Eds., Microbiology, a Centenary Perspective (ASM Press, Herndon, Virginia, 1999). S. C. Stearns, Ed., Evolution in Health and Disease (Oxford University Press, Oxford, New York, 1999). P. Ewald, “Evolution of Infectious Disease,” in Encyclopedia of Microbiology, J. Lederberg, Ed. (Academic Press, Orlando, Florida, 2000).

 WORKSHOP OVERVIEW G. L. Mandell, J. E. Bennett, R. Dolin, Principles and Practice of Infectious Diseases (Churchill Livingstone, 5th ed., Philadelphia, 2000). Notable Web Sites www.lib.uiowa.edu/hardin/md/micro.html www.idsociety.org www.asmusa.org osi.oracle.com:8080/promed www.cdc.gov/ncidod/EID WORKSHOP OVERVIEW REFERENCES Andersson, A. F., and J. F. Banfield. 2008. Virus population dynamics and acquired virus resistance in natural microbial communities. Science 320(5879):1047-1050. Bäckhed, F., H. Ding, T. Wang, L. V. Hooper, G. Y. Koh, A. Nagy, C. F. Semenkovich, and J. I. Gordon. 2004. The gut microbiota as an environmental factor that regulates fat storage. Proceed- ings of the National Academy of Sciences 101(44):15718-15723. Banfield, J. F. 2008a. Geomicrobiology program UC Berkeley, http://eps.berkeley.edu/~jill/banres. html (accessed July 31, 2008). ———. 2008b. Subproject : integrating genomics, proteomics, and functional analyses into ecosystem-level studies of natural microbial communities, http://eps.berkeley.edu/~jill/gtl/ subproject_1.htm (accessed July 31, 2008). Barrangou, R., C. Fremaux, H. Deveau, M. Richards, P. Boyaval, S. Moineau, D. A. Romero, and P. Horvath. 2007. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315(5819):1709-1712. Blaser, M. J. 1997. Ecology of Helicobacter pylori in the human stomach. Journal of Clinical Inves- tigation 100(4):759-762. Brelles-Mariño, G., and J. M. Ané. 2008. Nod factors and the molecular dialogue in the rhizobia- legume interaction. In Nitrogen fixation research progress, edited by G. N. Couto. Hauppauge, NY: Nova Science Publishers, Inc. Chun, C. K., J. V. Troll, I. Koroleva, B. Brown, L. Manzella, E. Snir, H. Almabrazi, T. E. Sheetz, M. de Fatima Bonaldo, T. L. Casavant, M. B. Soares, E. G. Ruby, and M. J. McFall-Ngai. 2008. Effects of colonization, luminescence, and autoinducer on host transcription during development of the squid-vibrio association. Proceedings of the National Academy of Sciences 105(32):11323-11328. Crichton, M. 1969. The andromeda strain. 1st ed. New York: Knopf. Dantas, G., M. O. Sommer, R. D. Oluwasegun, and G. M. Church. 2008. Bacteria subsisting on antibiotics. Science 320(5872):100-103. Davies, J. 2007. Microbes have the last word: A drastic re-evaluation of antimicrobial treatment is needed to overcome the threat of antibiotic-resistant bacteria. EMBO Reports 8(7):616-621. D’Costa, V. M., K. M. McGrann, D. W. Hughes, and G. D. Wright. 2006. Sampling the antibiotic resistome. Science 311(5759):374-377. Denef, V. J., N. C. VerBerkmoes, M. B. Shah, P. Abraham, M. Lefsrud, R. L. Hettich, and J. F. Banfield. 2009. Proteomics-inferred genome typing (PIGT) demonstrates inter-population recombination as a strategy for environmental adaptation. Environmental Microbiology 11(2):313-325. Dethlefsen, L., M. McFall-Ngai, and D. A. Relman. 2007. An ecological and evolutionary perspective on human-microbe mutualism and disease. Nature 449(7164):811-818.

 MICROBIAL EVOLUTION AND CO-ADAPTATION Didierlaurent, A., J.-C. Sirard, J.-P. Kraehenbuhl, and M. R. Neutra. 2001. How the gut senses its content. Cellular Microbiology 4(2):61-72. DOE (Department of Energy). 2007. Understanding how a cell works: background, http://microbial genomics.energy.gov/MicrobialCellProject/background.shtml (accessed December 10, 2008). Eckburg, P. B., E. M. Bik, C. N. Bernstein, E. Purdom, L. Dethlefsen, M. Sargent, S. R. Gill, K. E. Nelson, and D. A. Relman. 2005. Diversity of the human intestinal microbial flora. Science 308(5728):1635-1638. Eisen, J. A. 2000. Horizontal gene transfer among microbial genomes: new insights from complete genome analysis. Current Opinion in Genetics and Development 10(6):606-611. Emerson, R. W. 1841. Self-reliance. In Essays, first series, http://www.emersoncentral.com/ selfreliance.htm (accessed August 22, 2008). Eppley, J. M., G. W. Tyson, W. M. Getz, and J. F. Banfield. 2007. Genetic exchange across a species boundary in the Archaeal genus Ferroplasma. Genetics 177(1):407-416. Foresight. 2006. Infectious diseases: preparing for future threats. London, UK: Office of Science and Innovation. Gilbert, G. S., J. Handelsman, and J. L. Parke. 1994. Root camouflage and disease control. Phyto- pathology 8(3):222-225. Gordon, M. A., S. M. Graham, A. L. Walsh, L. Wilson, A. Phiri, E. Molyneux, E. E. Zijlstra, R. S. Heyderman, C. A. Hart, and M. E. Molyneux. 2008. Epidemics of invasive Salmonella enterica serovar enteritidis and S. enterica serovar typhimurium infection associated with multidrug resistance among adults and children in Malawi. Clinical Infectious Diseases 46(7):963-969. Graham, S. M. 2002. Salmonellosis in children in developing and developed countries and popula- tions. Current Opinion in Infectious Diseases 15(5):507-512. Hamburg, D. A. 2008. Preventing genocide: practical steps toward early detection and effective ac- tion. Boulder, CO: Paradigm Publishers. Heidelberg, J. F., J. A. Eisen, W. C. Nelson, R. A. Clayton, M. L. Gwinn, R. J. Dodson, D. H. Haft, E. K. Hickey, J. D. Peterson, L. Umayam, S. R. Gill, K. E. Nelson, T. D. Read, H. Tettelin, D. Richardson, M. D. Ermolaeva, J. Vamathevan, S. Bass, H. Qin, I. Dragoi, P. Sellers, L. McDonald, T. Utterback, R. D. Fleishmann, W. C. Nierman, O. White, S. L. Salzberg, H. O. Smith, R. R. Colwell, J. J. Mekalanos, J. C. Venter, and C. M. Fraser. 2000. DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature 406(6795):477-483. Hooper, L. V., and J. I. Gordon. 2001. Commensal host–bacterial relationships in the gut. Science 292(5519):1115-1118. Hughes, J. M. 2001. Emerging infectious diseases: a CDC perspective. Emerging Infectious Diseases 7(3):494-496. IOM (Institute of Medicine). 1992. Emerging infections: microbial threats to health in the United States. Washington, DC: National Academy Press. ———. 2003. Microbial threats to health: emergence, detection, and response. Washington, DC: The National Academies Press. ———. 2004a. Learning from SARS: preparing for the next disease outbreak. Washington, DC: The National Academies Press. ———. 2004b. The threat of pandemic influenza: are we ready? Washington, DC: The National Academies Press. ———. 2006a. Ending the war metaphor: the changing agenda for unraveling the host-microbe relationship. Washington, DC: The National Academies Press. ———. 2006b. The impact of globalization on infectious disease emergence and control: exploring the consequences and opportunities. Washington, DC: The National Academies Press. ———. 2006c. Addressing foodborne threats to health: policies, practices, and global coordination. Washington, DC: The National Academies Press. ———. 2007. Global infectious disease surveillance and detection: assessing the challenges. Wash- ington, DC: The National Academies Press.

 WORKSHOP OVERVIEW ———. 2008a. Vector-borne diseases: understanding the environmental, human health, and ecologi- cal connections. Washington, DC: The National Academies Press. ———. 2008b. Global climate change and extreme weather events: understanding the contributions to infectious disease emergence. Washington, DC: The National Academies Press. Jones, K. E., N. G. Patel, M. A. Levy, A. Storeygard, D. Balk, J. L. Gittleman, and P. Daszak. 2008. Global trends in emerging infectious diseases. Nature 451(7181):990-993. Jurkowski, A., A. H. Reid, and J. B. Labov. 2007. Metagenomics: a call for bringing a new science into the classroom (while it’s still new). CBE Life Sciences Education 6(4):260-265. Kiers, E. T., R. A. Rousseau, S. A. West, and R. F. Denison. 2003. Host sanctions and the legume- rhizobium mutualism. Nature 425(6953):78-81. Klemow, K. M. 2008. Environmental effects of mining in the anthracite region: problems and possible solutions, http://www.wilkes.edu/pages/2280.asp (accessed July 31, 2008). Lawley, T. D., K. Chan, L. J. Thompson, C. C. Kim, G. R. Govoni, and D. M. Monack. 2006. Genome-wide screen for salmonella genes required for long-term systemic infection of the mouse. PLoS Pathogens 2(2):e11. Lederberg, J. 2000. Infectious history. Science 288(5464):287-293. Lepp, P. W., M. M. Brinig, C. C. Ouverney, K. Palm, G. C. Armitage, and D. A. Relman. 2004. Methanogenic archaea and human periodontal disease. Proceedings of the National Academy of Sciences 101(16):6176-6181. Levin, B. R., V. Perrot, and N. Walker. 2000. Compensatory mutations, antibiotic resistance and the population genetics of adaptive evolution in bacteria. Genetics 154(3):985-997. Li, L., and S. N. Cohen. 1996. Tsg101: a novel tumor susceptibility gene isolated by controlled ho- mozygous functional knockout of allelic loci in mammalian cells. Cell 85(3):319-329. Lo, I., V. J. Denef, N. C. Verberkmoes, M. B. Shah, D. Goltsman, G. DiBartolo, G. W. Tyson, E. E. Allen, R. J. Ram, J. C. Detter, P. Richardson, M. P. Thelen, R. L. Hettich, and J. F. Banfield. 2007. Strain-resolved community proteomics reveals recombining genomes of acidophilic bacteria. Nature 446(7135):537-541. Lu, Q., L. W. Hope, M. Brasch, C. Reinhard, and S. N. Cohen. 2003. TSG101 interaction with HRS mediates endosomal trafficking and receptor down-regulation. Proceedings of the National Academy of Sciences 100(13):7626-7631. Lu, Q., W. Wei, P. E. Kowalski, A. C. Chang, and S. N. Cohen. 2004. EST-based genome-wide gene inactivation identifies ARAP3 as a host protein affecting cellular susceptibility to anthrax toxin. Proceedings of the National Academy of Sciences 101(49):17246-17251. Makarova, K. S., N. V. Grishin, S. A. Shabalina, Y. I. Wolf, and E. V. Koonin. 2006. A putative RNA- interference-based immune system in prokaryotes: Computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biology Direct 1(7): http://www.biology-direct.com/content/1/1/7 (accessed August 28, 2008). Margolis, E., and B. R. Levin. 2008. Evolution of bacterial-host interactions: virulence and the im- mune overresponse. In Evolutionary biology of bacterial and fungal pathogens, edited by F. Bequero, C. Nombela, G. H. Cassel, and J. A. Gutierrez. Washington, DC: ASM Press. Pp. 3-12. May, R. M., and R. M. Anderson. 1983. Parasite-host coevolution. In Coevolution, edited by D. J. Futuyama and M. Slatkin. Sunderland, MA: Sinauer Associates. . McFall-Ngai, M. J. 1998. The development of cooperative associations between animals and bacteria: establishing détente among domains. American Zoologist 38(4):593-608. Merrell, D. S., and S. Falkow. 2004. Frontal and stealth attack strategies in microbial pathogenesis. Nature 430(6996):250-256. Mojica, F. J., C. Diez-Villasenor, J. Garcia-Martinez, and E. Soria. 2005. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. Journal of Molecular Evolution 60(2):174-182.

 MICROBIAL EVOLUTION AND CO-ADAPTATION Monack, D. M., D. M. Bouley, and S. Falkow. 2004a. Salmonella typhimurium persists within mac- rophages in the mesenteric lymph nodes of chronically infected Nramp+/+ mice and can be reactivated by IFNγ neutralization. Journal of Experimental Medicine 199(2):231-241. Monack, D. M., A. Mueller, and S. Falkow. 2004b. Persistent bacterial infections: the interface of the pathogen and the host immune system. Nature Reviews Microbiology 2(9):747-765. Morens, D., G. K. Folkers, and A. S. Fauci. 2004. The challenge of emerging and re-emerging infec- tious diseases. Nature 430(6996):242-249. Morens, D., G. K. Folkers, and A. S. Fauci. 2008. Emerging infections: a perpetual challenge. Lancet Infectious Diseases 8(11):710-719. Morse, S. S. 2008. Retrospective: Joshua Lederberg (1925-2008). Science 319(5868):1351. Nemergut, D. R., M. S. Robeson, R. F. Kysela, A. P. Martin, S. K. Schmidt, and R. Knight. 2008. Insights and inferences about integron evolution from genomic data. BMC Genomics 9(261): http://www.biomedcentral.com/1471-2164/9/261 (accessed August 26, 2008). NLM (National Library of Medicine). 2008. The Joshua Lederberg papers: biographical information, http://profiles.nlm.nih.gov/BB/ (accessed August 4, 2008). NRC (National Research Council). 2007. The new science of metagenomics: revealing the secrets of our microbial planet. Washington, DC: The National Academies Press. Nyholm, S. V., and M. J. McFall-Ngai. 2004. The winnowing: establishing the squid-vibrio symbiosis. Nature Reviews Microbiology 2(8):632-642. Palmer, C., E. M. Bik, D. B. Digiulio, D. A. Relman, and P. O. Brown. 2007. Development of the human infant intestinal microbiota. PLoS Biology 5(7):e177. Parkhill, J., M. Sebaihia, A. Preston, L. D. Murphy, N. Thomson, D. E. Harris, M. T. Holden, C. M. Churcher, S. D. Bentley, K. L. Mungall, A. M. Cerdeno-Tarraga, L. Temple, K. James, B. Harris, M. A. Quail, M. Achtman, R. Atkin, S. Baker, D. Basham, N. Bason, I. Cherevach, T. Chillingworth, M. Collins, A. Cronin, P. Davis, J. Doggett, T. Feltwell, A. Goble, N. Hamlin, H. Hauser, S. Holroyd, K. Jagels, S. Leather, S. Moule, H. Norberczak, S. O’Neil, D. Ormond, C. Price, E. Rabbinowitsch, S. Rutter, M. Sanders, D. Saunders, K. Seeger, S. Sharp, M. Simmonds, J. Skelton, R. Squares, S. Squares, K. Stevens, L. Unwin, S. Whitehead, B. G. Barrell, and D. J. Maskell. 2003. Comparative analysis of the genome sequences of Bordetella pertussis, Borde- tella parapertussis and Bordetella bronchiseptica. Nature Genetics 35(1):32-40. Parrish, C. R., E. C. Holmes, D. M. Morens, E. C. Park, D. S. Burke, C. H. Calisher, C. A. Laughlin, L. J. Saif, and P. Daszak. 2008. Cross-species virus transmission and the emergence of new epidemic diseases. Microbiology and Molecular Biology Reviews 72(3):457-470. Relman, D. A., and S. Falkow. 2001. The meaning and impact of the human genome sequence for microbiology. Trends in Microbiology 9(5):206-208. Riely, B. K., J.-H. Mun, and J.-M. Ané. 2006. Unravelling the molecular basis for symbiotic signal transduction in legumes. Molecular Plant Pathology 7(3):197-207. Riesenfeld, C. S., R. M. Goodman, and J. Handelsman. 2004. Uncultured soil bacteria are a reservoir of new antibiotic resistance genes. Environmental Microbiology 6(9):981-989. Roumagnac, P., F. X. Weill, C. Dolecek, S. Baker, S. Brisse, N. T. Chinh, T. A. Le, C. J. Acosta, J. Farrar, G. Dougan, and M. Achtman. 2006. Evolutionary history of Salmonella typhi. Science 314(5803):1301-1304. Spellberg, B., R. Guidos, D. Gilbert, J. Bradley, H. W. Boucher, W. M. Scheld, J. G. Bartlett, J. Edwards Jr., and Infectious Diseases Society of America. 2008. The epidemic of antibiotic- resistant infections: a call to action for the medical community from the Infectious Diseases Society of America. Clinical Infectious Diseases 46(2):155-164. Steinberg, L. M., and B. R. Levin. 2007. Grazing protozoa and the evolution of the Escherichia coli O157:H7 Shiga toxin-encoding prophage. Proceedings of the Royal Society: Biological Sciences 274(1621):1921-1929.

 WORKSHOP OVERVIEW Stephens, D. S., E. R. Moxon, J. Adams, S. Altizer, J. Antonovics, S. Aral, R. Berkelman, E. Bond, J. Bull, G. Cauthen, M. M. Farley, A. Glasgow, J. W. Glasser, H. P. Katner, S. Kelley, J. Mittler, A. J. Nahmias, S. Nichol, V. Perrot, R. W. Pinner, S. Schrag, P. Small, and P. H. Thrall. 1998. Emerging and reemerging infectious diseases: a multidisciplinary perspective. American Journal of the Medical Sciences 315(2):64-75. Szczepanowski, R., I. Krahn, B. Linke, A. Goesmann, A. Puhler, and A. Schluter. 2004. Antibiotic multiresistance plasmid pRSB101 isolated from a wastewater treatment plant is related to plas- mids residing in phytopathogenic bacteria and carries eight different resistance determinants including a multidrug transport system. Microbiology 150(Pt 11):3613-3630. Szczepanowski, R., S. Braun, V. Riedel, S. Schneiker, I. Krahn, A. Puhler, and A. Schluter. 2005. The 120 592 bp IncF plasmid pRSB107 isolated from a sewage-treatment plant encodes nine different antibiotic-resistance determinants, two iron-acquisition systems and other putative virulence-associated functions. Microbiology 151(Pt 4):1095-1111. Szczepanowski, R., T. Bekel, A. Goesmann, L. Krause, H. Kromeke, O. Kaiser, W. Eichler, A. Puhler, and A. Schluter. 2008. Insight into the plasmid metagenome of wastewater treatment plant bac- teria showing reduced susceptibility to antimicrobial drugs analysed by the 454-pyrosequencing technology. Journal of Biotechnology 136(1-2):54-64. Taormina, P. J., L. R. Beuchat, and L. Slutsker. 1999. Infections associated with eating seed sprouts: an international concern. Emerging Infectious Diseases 5(5):626-634. Tyson, G. W., and J. F. Banfield. 2008. Rapidly evolving CRISPRs implicated in acquired resistance of microorganisms to viruses. Environmental Microbiology 10(1):200-207. Weerasinghe, R. R., D. M. Bird, and N. S. Allen. 2005. Root-knot nematodes and bacterial nod fac- tors elicit common signal transduction events in Lotus japonicus. Proceedings of the National Academy of Sciences 102(8):3147-3152. Wei, W., Q. Lu, G. J. Chaudry, S. H. Leppla, and S. N. Cohen. 2006. The LDL receptor-related protein LRP6 mediates internalization and lethality of anthrax toxin. Cell 124(6):1141-1154. WHO (World Health Organization). 2008a. About the IHR, http://www.who.int/csr/ihr/prepare/en/ index.html (accessed July 27, 2008). ———. 2008b. Core capacity requirements for surveillance and response, http://www.who.int/csr/ ihr/capacity/en/index.html (accessed July 27, 2008). Woolhouse, M., and R. Antia. 2007. Emergence of new infectious diseases. In Evolution in health and disease, 2nd ed., edited by S. C. Stearns and J. K. Koella. Oxford, UK: Oxford University Press. Pp. 215-228. Woolhouse, M., and E. Gaunt. 2007. Ecological origins of novel human pathogens. Critical Reviews in Microbiology 33(4):231-242. Wu, D., S. C. Daugherty, S. E. Van Aken, G. H. Pai, K. L. Watkins, H. Khouri, L. J. Tallon, J. M. Zaborsky, H. E. Dunbar, P. L. Tran, N. A. Moran, and J. A. Eisen. 2006. Metabolic complemen- tarity and genomics of the dual bacterial symbiosis of sharpshooters. PLoS Biology 4(6):e188. Wu, M., Q. Ren, A. S. Durkin, S. C. Daugherty, L. M. Brinkac, R. J. Dodson, R. Madupu, S. A. Sullivan, J. F. Kolonay, D. H. Haft, W. C. Nelson, L. J. Tallon, K. M. Jones, L. E. Ulrich, J. M. Gonzalez, I. B. Zhulin, F. T. Robb, and J. A. Eisen. 2005. Life in hot carbon monoxide: the complete genome sequence of Carboxydothermus hydrogenoformans z-2901. PLoS Genetics 1(5):e65. Yokoyama, W. M., and M. Colonna. 2008. Innate immunity to pathogens. Current Opinion in Im- munology 20(1):1-2. Young, D., T. Hussell, and G. Dougan. 2002. Chronic bacterial infections: living with unwanted guests. Nature Immunology 3(11):1026-1032.

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Dr. Joshua Lederberg - scientist, Nobel laureate, visionary thinker, and friend of the Forum on Microbial Threats - died on February 2, 2008. It was in his honor that the Institute of Medicine's Forum on Microbial Threats convened a public workshop on May 20-21, 2008, to examine Dr. Lederberg's scientific and policy contributions to the marketplace of ideas in the life sciences, medicine, and public policy. The resulting workshop summary, Microbial Evolution and Co-Adaptation, demonstrates the extent to which conceptual and technological developments have, within a few short years, advanced our collective understanding of the microbiome, microbial genetics, microbial communities, and microbe-host-environment interactions.

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