Genomic and Metagenomic Approaches for Predicting Pathogen Evolution VERONICA CASAS and STANLEY MALOY Center for Microbial Sciences, San Diego State University, San Diego, CA 92182
ABSTRACT Global climate change can alter the distribution of microbial pathogens and vectors that transmit infectious diseases, exposing humans to newly emerging or reemerging diseases. Early detection of potential pathogens and vectors in the environment can facilitate upstream interventions that limit the spread of infectious disease. Metagenomics is the analysis of DNA sequences from a population of microorganisms in a particular environment, followed by the computational reconstruction of the data to determine what organisms are present and predict their role in the environment. Defining the microbial populations associated with humans, animals, and their environment provides insight into the structure of microbial communities in any particular niche, including the abundance, diversity, and composition of the microbes and viruses present. It can also reveal the distribution of virulence genes within that niche. These data can be used to identify reservoirs of pathogens in an environment and predict environments with a high probability for evolution of new pathogens or outbreaks caused by known pathogens, thereby facilitating approaches to prevent infections of animals or humans before serious outbreaks of infectious disease.
OVERVIEW Emerging infectious diseases have been defined as microbial infections “whose incidence in humans has increased in the past 2 decades or threatens to increase in the near future” (1). Microbial causes of emerging infectious diseases generally fall into two groups: (i) new pathogens that arise following environmental disturbance or acquisition of novel virulence traits and (ii) existing and known pathogens that have spread to new geographic areas or populations. In both cases, human activities play a key role in the evolution and transmission of these diseases. Human disruption of the
natural environment alters not only the physical landscape but also the invisible microbial landscape. This process can facilitate transfer of virulence genes to new hosts or exposure of naïve hosts to previously unidentified pathogens. Due to the tremendous impact of emerging diseases on human and animal populations, we need a better understanding of how new diseases evolve and how to detect emerging diseases in order to develop more effective ways to predict outbreaks before they happen.
GLOBAL CLIMATE CHANGE One example of the broad impact of human activities is global climate change induced by the extensive use of fossil fuels. Global warming promotes climate change that results in more frequent and damaging extreme weather conditions such as hurricanes and other tropical storms and also affects the ecology and physiology of marine and terrestrial plants and animals (2, 3). In addition to larger organisms, climate change also affects the microbes that determine the health and disease of animals, humans, and the environment (4). Climate change Received: 3 April 2013, Accepted: 8 April 2013, Published: 31 January 2014 Editors: Ronald M. Atlas, University of Louisville, Louisville, KY; and Stanley Maloy, San Diego State University, San Diego, CA Citation: Casas V, Maloy S. 2014. Genomic and metagenomic approaches for predicting pathogen evolution. Microbiol Spectrum 2(1):OH-0019-2013. doi:10.1128/microbiolspec.OH-0019-2013. Correspondence: Veronica Casas, [email protected] © 2014 American Society for Microbiology. All rights reserved.
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influences the emergence and spread of many infectious diseases by providing new environments for pathogens and expanding the range of disease vectors. Acquisition or enhancement of virulence traits is facilitated by the presence of environmental reservoirs of virulence genes. For example, metagenomics has revealed that a reservoir of exotoxin-encoding genes exists in the environment (5–7). As the temperature increases, there is increased stress on ecosystems such as coral reefs, and this stress makes organisms more susceptible to infectious disease. A change in the average temperature by as little as 1 to 2°C can have a profound effect on the population of some organisms, reducing the survival of some beneficial microbes while increasing the survival of insect vectors that spread disease and microbes that cause disease (8, 9). Flooding and drought can increase the spread of waterborne pathogens like Vibrio cholerae and Salmonella, and an increase in temperature allows mosquitoes to thrive at higher elevations, promoting the spread of diseases transmitted by mosquitoes, including malaria, dengue, and yellow fever. Several examples demonstrate these concepts. The emergence of hantavirus was precipitated by a particularly wet season in the southwestern United States in 1993. The elevated rainfall led to increased plant growth in this typically dry desert region. The resulting verdant vegetation provided an abundant food supply for rodents as well as protection from predators; thus the rodent population surged. Hantaviruses are shed in the urine and feces of infected rodents, but they do not make the animal carrier sick. The deer mouse (Peromyscus maniculatus) is the host for Sin Nombre virus, the causative agent of hantavirus pulmonary syndrome in the Four Corners outbreak. The deer mouse is common and widespread in this region, and approximately 10% of deer mice tested show evidence of infection with Sin Nombre virus. When the population of deer mice burgeoned in response to the unusually wet season, the mice moved into new niches that included the living quarters of the local human population, exposing humans to a previously unrecognized disease from wild animals (10). Humans become infected from contaminated dust containing virus in the urine or feces of mice, resulting in high mortality for the infected people. Thus, the Four Corners hantavirus outbreak serves as an example of a disease whose etiologic agent is widespread in the environment but whose transmission to humans was precipitated by environmental change (11). In many areas of the developing world, fresh water is a scarce resource, and flooding destroys access to uncontaminated water supplies. This is clear from the recent
epidemic of cholera in Haiti (12). Since 2005, the reemergence of cholera has paralleled the growing population of vulnerable humans living in regions subject to frequent, extensive flooding. The increased flooding in Southeast Asia has expanded the distribution of V. cholerae in its natural estuarine habitats and led to an increase in the incidence of cholera in human water supplies. The pattern of disease suggests that cholera outbreaks begin in coastal regions prior to spreading to inland water supplies. The El Niño-Southern Oscillation, a major source of climate variability from year to year, influences periodic outbreaks of cholera. The incidence of cholera increases after warm cycles and decreases after cold cycles, while in the intervening years the climatecholera connection breaks down. The outbreaks of cholera are closely correlated with an increase in the abundance of plankton that occurs when the ocean water temperature increases, events that can be tracked by satellite imaging of the marine chlorophyll (13). There is also a strong correlation between extreme weather events and diarrheal diseases caused by Salmonella and other pathogens that are spread by food and water contaminated with animal or human waste. This correlation between certain climate conditions and disease can be used to develop computational models that predict when and where major epidemics are likely to occur, project the severity of disease outbreaks, and provide insight into how we can develop upstream interventions (14, 15). An outbreak of plague that began in Central Africa in 2004 and abruptly ended in 2009 also shows a strong correlation with El Niño cycles. From 2004 to 2009, eight countries were impacted with more than 12,000 cases and more than 800 deaths attributable to plague. In 2009, there was a sudden decrease in the incidence in plague. The decrease coincided with the worst El Niño cycle since 1998. The wet season in Africa started late, and this might have had a significant impact on the fecundity of the reservoir and movement of wild animals and the associated abundance of fleas that transmit the pathogens (16). Outbreaks of plague are not restricted to developing countries: changes in precipitation also lead to an increase in plague in rodent populations in the southwestern United States. These examples focus on global climate change. However, the important point is that in each of these examples the emergence of infectious diseases followed an environmental disruption that provided a new niche for microbes, exposing animals to pathogens they had previously not experienced, with subsequent transmission of disease from animals to humans.
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DETECTION OF VIRULENCE FACTORS IN THE ENVIRONMENT Rapid detection of pathogens in the environment will allow the development of approaches to predict disease outbreaks before they happen, thereby facilitating public health approaches to prevent the spread of these emerging infectious diseases instead of treating diseases after they occur. Society often experiences considerable fear and hysteria when there is an outbreak of a new disease, while scientists are trying to identify the cause, find an appropriate drug or vaccine, and distribute the therapy to the exposed population. A One Health approach will allow development of upstream approaches to predict outbreaks and build upstream barriers to transmission. This approach will demand close collaboration among environmental scientists, veterinarians, and physicians coupled with computational scientists and mathematicians who can build powerful predictive models of disease transmission. Metagenomics has provided insights into the impact of environmental change on the distribution and transmission of pathogens in the environment, in domestic and wild animal populations, and in humans. Given these insights, it is possible to design molecular assays that provide simple, exquisitely sensitive methods for sampling any environment using DNA signatures that probe the biology of that environment and the impact of environmental disturbances. For example, our work has demonstrated that bacterial exotoxin genes are widespread in the environment (5–7). These gene pools of virulence factors exist in diverse habitats and can undergo genetic exchange between microbes, leading to the emergence of new animal and human pathogens. Detection of these genes in the environment provides a sensitive test for new emerging infectious diseases.
MICROBIAL METAGENOMICS While microbial genomics is the study of the genomes of individual microbes, microbial metagenomics is the culture-independent functional and sequence-based analysis of the heterogeneous microbial genomes from a particular environment (17–20). Metagenomics has revolutionized the way researchers investigate the community structure, composition, and distribution of populations of bacteria and bacteriophages (phages) from a multitude of environments, from the mundane to the extreme— including the microbiomes normally associated with animals or humans (21–24). The improvements in the technology for sequencing microbes directly from their habitats have made metagenomics research less expensive
and more accessible and also generated an abundance of data that has expanded and transformed our understanding of the microbes that surround us (25). Sequencing of phages from some of the same environments from which bacterial metagenomes have been constructed has provided invaluable insight into the dynamic interactions and genetic exchange occurring between bacteria and their viruses (26–32). Phages play an important role in determining the types and numbers of bacteria present in a particular population and in the transfer of genetic material between bacteria (19, 33). Many of the novel genes present in bacteria are of phage origin. When the phage genes transferred to the bacterial genome encode virulence factors, such as exotoxin genes, they can serve to confer or enhance the pathogenesis of that bacterial host. Moreover, if these genes are found in alternative, noncognate environmental hosts, they serve as the genetic “raw material” for evolution of novel animal and human pathogens. As described below, the phage-encoded genes for Shiga toxin and staphylococcal enterotoxin A have been found in alternative hosts cultivated from environmental samples (5, 6). Exotoxins are proteins secreted by certain bacteria that can quickly and effectively cause damage to eukaryotic cells by targeting essential functions (34). Very minute amounts of some exotoxins are sufficient to kill a cell and ultimately cause death. For example, 1 mg of botulinum or tetanus exotoxin can be fatal to an adult human (35), and as little as 100 to 150 ng/kg of body weight of diphtheria toxin is lethal (36). In many cases, exotoxins are responsible for most or all aspects of their associated diseases. As the bacteria grow within the infected individual, the secreted exotoxins exert their deleterious effects at the cellular level, causing damage that leads to symptoms associated with the disease (34). Due to their tremendous potency and the ability of the genes that code for these exotoxins to be horizontally transferred between bacteria and phages, exotoxins are critical virulence factors in a number of emerging infectious diseases of significance to public health. Recently food-borne illnesses caused by exotoxinproducing pathogens such as Escherichia coli O157:H7 have been on the rise worldwide (37–44). The origin of many of these outbreaks has been linked to contaminated food originating from our complex agricultural system; however, the specific source of a pathogen is often difficult to identify definitively. The inability to quickly and precisely identify pathogens in our food sources poses a clear threat to public health. This is exacerbated when the pathogens we encounter contain a novel combination of virulence traits, such as the E. coli O104:H4 strain
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responsible for the 2011 outbreak in Germany (45–47). In the case of E. coli O104:H4, genome sequencing provided the necessary clues for the rapid development of diagnostic tests that led to the source of this pathogen. In addition, the genome sequence led to insight into the evolution of E. coli O104:H4 by the sequential acquisition of chromosomal mutations, antibiotic resistance plasmids, and Shiga toxin-producing phages. Survival in the environment is an essential step for the transmission of many pathogens. Recent evidence suggests that the exotoxin genes may enhance the ecological fitness of the bacteria (5, 6). In addition, the phages themselves survive harsh environmental conditions much better than bacteria, allowing transfer of virulence genes to a new bacterial host when the appropriate conditions prevail. Thus, it has become clear that phages are common elements of our ecosystem and have a major impact on the ecosystem—influencing the population dynamics of bacteria and playing a direct role in bacterial growth and physiology. Phage-encoded genes may increase a bacterium’s fitness by providing new metabolic capabilities, allowing evasion of predators, or increasing virulence. Phages may also facilitate the movement of exotoxin genes between microbial hosts, thereby converting avirulent bacteria into toxigenic pathogens. The phage-mediated transfer of virulence traits between bacteria occurs in a variety of environments and facilitates evolution of bacterial pathogens (48–54). The properties of phages that make them particularly effective vehicles for gene transfer are their abundance and prevalence in natural environments, in animals, and in humans. There are roughly 10-fold more phages than bacteria in each of these environments. Phages are also hardier than their bacterial hosts and can persist without bacteria in harsh environments (55, 56). Phages can survive chlorination and heat treatments, are resistant to inactivation by sunlight, and have been shown to escape sewage/water treatment processes (56–62). Since phages are more impervious to extreme environmental conditions, they can maintain a pool of virulence genes in natural environments, thereby increasing the potential for exchange. Furthermore, the ability of many phages to fluctuate between lysogenic and lytic lifestyles also increases their fitness and the pool of associated virulence genes.
HOW ENVIRONMENTAL DISRUPTION SELECTS FOR NEW PATHOGENS What happens to the structure, composition, and dynamics of microbial communities upon deforestation, irrigation, coastal zone degradation, wetland modifica-
tion, expansion of urban areas, global climate change, or other human interventions that disrupt the environment (63)? Reduction of the relative biodiversity of an environment provides an opportunity for invasive plant and animal species that can wreak havoc with the ecosystem. The microbial ecosystem responds similarly, facilitating the evolution and transmission of infectious diseases that affect plants, animals, and humans. For example, the reduced biodiversity associated with the reduction in natural forests and their inhabitants was an important factor in the spread of Lyme disease in the northeastern United States (64). Global expansion of the human population and the growing proximity of humans to wild and domesticated animals have been linked to emergence of Nipah virus in Malaysia (65, 66), as well as the global increase in food-borne illnesses (67). Use of metagenomics to survey the microbial landscape before and after human activities provides an approach to examine the impact of such changes. The microbial communities associated with humans, animals, and their environment must be investigated concurrently in order to be able to draw a clearer picture of the microbial landscape and the interplay between the microbiomes from these interdependent niches. Prior to the metagenomics era, identification and modeling of the uncultivable microorganisms from a given environment were nearly impossible. Metagenomics has provided novel insights into the structure of the microbial communities in the environment, including the abundance, diversity, and composition of the microbes and viruses. Metagenomics also reveals the distribution of particular genes, including virulence genes, within an environment. Once we know the microbes and potential virulence genes in these environments, it becomes important to better understand what they are doing in these environments, the interactions between organisms and the physical environment, how they interact, and how these dynamics could lead to selection or transmission of organisms that can cause disease. Thus, with some vision and creativity, metagenomics can be used to survey the changing environmental microbial landscape to generate data for models that can predict, and in turn help prevent, impending outbreaks.
ONE HEALTH Human disruption of the environment promotes alterations in the patterns of infectious disease. For example, global climate change increases food- and waterborne diseases by contamination of fresh water supplies, increases vector-borne diseases by changing the rate and
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distribution of insect vectors, and alters the vulnerability of organisms to infectious disease by changing the dynamics of the ecosystem. Understanding these factors will demand the integration of a variety of types of data that probe the health of the environment, animals, and humans. The approaches needed extend from the molecular approaches of genomics and metagenomics to geographic information systems, satellite imaging, and mathematical models that can model and predict future outbreaks (68). Bringing together scientists with a broad understanding of computer sciences, mathematics, and the ecology of microbes in humans, animals, and diverse environments can generate predictions that will allow us to anticipate when and where a disease outbreak may occur and provide upstream interventions that protect the health of humans, animals, and the environment. ACKNOWLEDGMENTS The authors of this manuscript do not have any commercial affiliations, consultancies, stock or equity interests, or patentlicensing arrangements that could be considered to pose a conflict of interest regarding the manuscript.
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ABSTRACT Global climate change can alter the distribution of microbial pathogens and vectors that transmit infectious diseases, exposing humans to newly emerging or reemerging diseases. Early detection of potential pathogens and vectors in the environment can facilitate upstream interventions that limit the spread of infectious disease. Metagenomics is the analysis of DNA sequences from a population of microorganisms in a particular environment, followed by the computational reconstruction of the data to determine what organisms are present and predict their role in the environment. Defining the microbial populations associated with humans, animals, and their environment provides insight into the structure of microbial communities in any particular niche, including the abundance, diversity, and composition of the microbes and viruses present. It can also reveal the distribution of virulence genes within that niche. These data can be used to identify reservoirs of pathogens in an environment and predict environments with a high probability for evolution of new pathogens or outbreaks caused by known pathogens, thereby facilitating approaches to prevent infections of animals or humans before serious outbreaks of infectious disease.
OVERVIEW Emerging infectious diseases have been defined as microbial infections “whose incidence in humans has increased in the past 2 decades or threatens to increase in the near future” (1). Microbial causes of emerging infectious diseases generally fall into two groups: (i) new pathogens that arise following environmental disturbance or acquisition of novel virulence traits and (ii) existing and known pathogens that have spread to new geographic areas or populations. In both cases, human activities play a key role in the evolution and transmission of these diseases. Human disruption of the
natural environment alters not only the physical landscape but also the invisible microbial landscape. This process can facilitate transfer of virulence genes to new hosts or exposure of naïve hosts to previously unidentified pathogens. Due to the tremendous impact of emerging diseases on human and animal populations, we need a better understanding of how new diseases evolve and how to detect emerging diseases in order to develop more effective ways to predict outbreaks before they happen.
GLOBAL CLIMATE CHANGE One example of the broad impact of human activities is global climate change induced by the extensive use of fossil fuels. Global warming promotes climate change that results in more frequent and damaging extreme weather conditions such as hurricanes and other tropical storms and also affects the ecology and physiology of marine and terrestrial plants and animals (2, 3). In addition to larger organisms, climate change also affects the microbes that determine the health and disease of animals, humans, and the environment (4). Climate change Received: 3 April 2013, Accepted: 8 April 2013, Published: 31 January 2014 Editors: Ronald M. Atlas, University of Louisville, Louisville, KY; and Stanley Maloy, San Diego State University, San Diego, CA Citation: Casas V, Maloy S. 2014. Genomic and metagenomic approaches for predicting pathogen evolution. Microbiol Spectrum 2(1):OH-0019-2013. doi:10.1128/microbiolspec.OH-0019-2013. Correspondence: Veronica Casas, [email protected] © 2014 American Society for Microbiology. All rights reserved.
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influences the emergence and spread of many infectious diseases by providing new environments for pathogens and expanding the range of disease vectors. Acquisition or enhancement of virulence traits is facilitated by the presence of environmental reservoirs of virulence genes. For example, metagenomics has revealed that a reservoir of exotoxin-encoding genes exists in the environment (5–7). As the temperature increases, there is increased stress on ecosystems such as coral reefs, and this stress makes organisms more susceptible to infectious disease. A change in the average temperature by as little as 1 to 2°C can have a profound effect on the population of some organisms, reducing the survival of some beneficial microbes while increasing the survival of insect vectors that spread disease and microbes that cause disease (8, 9). Flooding and drought can increase the spread of waterborne pathogens like Vibrio cholerae and Salmonella, and an increase in temperature allows mosquitoes to thrive at higher elevations, promoting the spread of diseases transmitted by mosquitoes, including malaria, dengue, and yellow fever. Several examples demonstrate these concepts. The emergence of hantavirus was precipitated by a particularly wet season in the southwestern United States in 1993. The elevated rainfall led to increased plant growth in this typically dry desert region. The resulting verdant vegetation provided an abundant food supply for rodents as well as protection from predators; thus the rodent population surged. Hantaviruses are shed in the urine and feces of infected rodents, but they do not make the animal carrier sick. The deer mouse (Peromyscus maniculatus) is the host for Sin Nombre virus, the causative agent of hantavirus pulmonary syndrome in the Four Corners outbreak. The deer mouse is common and widespread in this region, and approximately 10% of deer mice tested show evidence of infection with Sin Nombre virus. When the population of deer mice burgeoned in response to the unusually wet season, the mice moved into new niches that included the living quarters of the local human population, exposing humans to a previously unrecognized disease from wild animals (10). Humans become infected from contaminated dust containing virus in the urine or feces of mice, resulting in high mortality for the infected people. Thus, the Four Corners hantavirus outbreak serves as an example of a disease whose etiologic agent is widespread in the environment but whose transmission to humans was precipitated by environmental change (11). In many areas of the developing world, fresh water is a scarce resource, and flooding destroys access to uncontaminated water supplies. This is clear from the recent
epidemic of cholera in Haiti (12). Since 2005, the reemergence of cholera has paralleled the growing population of vulnerable humans living in regions subject to frequent, extensive flooding. The increased flooding in Southeast Asia has expanded the distribution of V. cholerae in its natural estuarine habitats and led to an increase in the incidence of cholera in human water supplies. The pattern of disease suggests that cholera outbreaks begin in coastal regions prior to spreading to inland water supplies. The El Niño-Southern Oscillation, a major source of climate variability from year to year, influences periodic outbreaks of cholera. The incidence of cholera increases after warm cycles and decreases after cold cycles, while in the intervening years the climatecholera connection breaks down. The outbreaks of cholera are closely correlated with an increase in the abundance of plankton that occurs when the ocean water temperature increases, events that can be tracked by satellite imaging of the marine chlorophyll (13). There is also a strong correlation between extreme weather events and diarrheal diseases caused by Salmonella and other pathogens that are spread by food and water contaminated with animal or human waste. This correlation between certain climate conditions and disease can be used to develop computational models that predict when and where major epidemics are likely to occur, project the severity of disease outbreaks, and provide insight into how we can develop upstream interventions (14, 15). An outbreak of plague that began in Central Africa in 2004 and abruptly ended in 2009 also shows a strong correlation with El Niño cycles. From 2004 to 2009, eight countries were impacted with more than 12,000 cases and more than 800 deaths attributable to plague. In 2009, there was a sudden decrease in the incidence in plague. The decrease coincided with the worst El Niño cycle since 1998. The wet season in Africa started late, and this might have had a significant impact on the fecundity of the reservoir and movement of wild animals and the associated abundance of fleas that transmit the pathogens (16). Outbreaks of plague are not restricted to developing countries: changes in precipitation also lead to an increase in plague in rodent populations in the southwestern United States. These examples focus on global climate change. However, the important point is that in each of these examples the emergence of infectious diseases followed an environmental disruption that provided a new niche for microbes, exposing animals to pathogens they had previously not experienced, with subsequent transmission of disease from animals to humans.
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DETECTION OF VIRULENCE FACTORS IN THE ENVIRONMENT Rapid detection of pathogens in the environment will allow the development of approaches to predict disease outbreaks before they happen, thereby facilitating public health approaches to prevent the spread of these emerging infectious diseases instead of treating diseases after they occur. Society often experiences considerable fear and hysteria when there is an outbreak of a new disease, while scientists are trying to identify the cause, find an appropriate drug or vaccine, and distribute the therapy to the exposed population. A One Health approach will allow development of upstream approaches to predict outbreaks and build upstream barriers to transmission. This approach will demand close collaboration among environmental scientists, veterinarians, and physicians coupled with computational scientists and mathematicians who can build powerful predictive models of disease transmission. Metagenomics has provided insights into the impact of environmental change on the distribution and transmission of pathogens in the environment, in domestic and wild animal populations, and in humans. Given these insights, it is possible to design molecular assays that provide simple, exquisitely sensitive methods for sampling any environment using DNA signatures that probe the biology of that environment and the impact of environmental disturbances. For example, our work has demonstrated that bacterial exotoxin genes are widespread in the environment (5–7). These gene pools of virulence factors exist in diverse habitats and can undergo genetic exchange between microbes, leading to the emergence of new animal and human pathogens. Detection of these genes in the environment provides a sensitive test for new emerging infectious diseases.
MICROBIAL METAGENOMICS While microbial genomics is the study of the genomes of individual microbes, microbial metagenomics is the culture-independent functional and sequence-based analysis of the heterogeneous microbial genomes from a particular environment (17–20). Metagenomics has revolutionized the way researchers investigate the community structure, composition, and distribution of populations of bacteria and bacteriophages (phages) from a multitude of environments, from the mundane to the extreme— including the microbiomes normally associated with animals or humans (21–24). The improvements in the technology for sequencing microbes directly from their habitats have made metagenomics research less expensive
and more accessible and also generated an abundance of data that has expanded and transformed our understanding of the microbes that surround us (25). Sequencing of phages from some of the same environments from which bacterial metagenomes have been constructed has provided invaluable insight into the dynamic interactions and genetic exchange occurring between bacteria and their viruses (26–32). Phages play an important role in determining the types and numbers of bacteria present in a particular population and in the transfer of genetic material between bacteria (19, 33). Many of the novel genes present in bacteria are of phage origin. When the phage genes transferred to the bacterial genome encode virulence factors, such as exotoxin genes, they can serve to confer or enhance the pathogenesis of that bacterial host. Moreover, if these genes are found in alternative, noncognate environmental hosts, they serve as the genetic “raw material” for evolution of novel animal and human pathogens. As described below, the phage-encoded genes for Shiga toxin and staphylococcal enterotoxin A have been found in alternative hosts cultivated from environmental samples (5, 6). Exotoxins are proteins secreted by certain bacteria that can quickly and effectively cause damage to eukaryotic cells by targeting essential functions (34). Very minute amounts of some exotoxins are sufficient to kill a cell and ultimately cause death. For example, 1 mg of botulinum or tetanus exotoxin can be fatal to an adult human (35), and as little as 100 to 150 ng/kg of body weight of diphtheria toxin is lethal (36). In many cases, exotoxins are responsible for most or all aspects of their associated diseases. As the bacteria grow within the infected individual, the secreted exotoxins exert their deleterious effects at the cellular level, causing damage that leads to symptoms associated with the disease (34). Due to their tremendous potency and the ability of the genes that code for these exotoxins to be horizontally transferred between bacteria and phages, exotoxins are critical virulence factors in a number of emerging infectious diseases of significance to public health. Recently food-borne illnesses caused by exotoxinproducing pathogens such as Escherichia coli O157:H7 have been on the rise worldwide (37–44). The origin of many of these outbreaks has been linked to contaminated food originating from our complex agricultural system; however, the specific source of a pathogen is often difficult to identify definitively. The inability to quickly and precisely identify pathogens in our food sources poses a clear threat to public health. This is exacerbated when the pathogens we encounter contain a novel combination of virulence traits, such as the E. coli O104:H4 strain
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responsible for the 2011 outbreak in Germany (45–47). In the case of E. coli O104:H4, genome sequencing provided the necessary clues for the rapid development of diagnostic tests that led to the source of this pathogen. In addition, the genome sequence led to insight into the evolution of E. coli O104:H4 by the sequential acquisition of chromosomal mutations, antibiotic resistance plasmids, and Shiga toxin-producing phages. Survival in the environment is an essential step for the transmission of many pathogens. Recent evidence suggests that the exotoxin genes may enhance the ecological fitness of the bacteria (5, 6). In addition, the phages themselves survive harsh environmental conditions much better than bacteria, allowing transfer of virulence genes to a new bacterial host when the appropriate conditions prevail. Thus, it has become clear that phages are common elements of our ecosystem and have a major impact on the ecosystem—influencing the population dynamics of bacteria and playing a direct role in bacterial growth and physiology. Phage-encoded genes may increase a bacterium’s fitness by providing new metabolic capabilities, allowing evasion of predators, or increasing virulence. Phages may also facilitate the movement of exotoxin genes between microbial hosts, thereby converting avirulent bacteria into toxigenic pathogens. The phage-mediated transfer of virulence traits between bacteria occurs in a variety of environments and facilitates evolution of bacterial pathogens (48–54). The properties of phages that make them particularly effective vehicles for gene transfer are their abundance and prevalence in natural environments, in animals, and in humans. There are roughly 10-fold more phages than bacteria in each of these environments. Phages are also hardier than their bacterial hosts and can persist without bacteria in harsh environments (55, 56). Phages can survive chlorination and heat treatments, are resistant to inactivation by sunlight, and have been shown to escape sewage/water treatment processes (56–62). Since phages are more impervious to extreme environmental conditions, they can maintain a pool of virulence genes in natural environments, thereby increasing the potential for exchange. Furthermore, the ability of many phages to fluctuate between lysogenic and lytic lifestyles also increases their fitness and the pool of associated virulence genes.
HOW ENVIRONMENTAL DISRUPTION SELECTS FOR NEW PATHOGENS What happens to the structure, composition, and dynamics of microbial communities upon deforestation, irrigation, coastal zone degradation, wetland modifica-
tion, expansion of urban areas, global climate change, or other human interventions that disrupt the environment (63)? Reduction of the relative biodiversity of an environment provides an opportunity for invasive plant and animal species that can wreak havoc with the ecosystem. The microbial ecosystem responds similarly, facilitating the evolution and transmission of infectious diseases that affect plants, animals, and humans. For example, the reduced biodiversity associated with the reduction in natural forests and their inhabitants was an important factor in the spread of Lyme disease in the northeastern United States (64). Global expansion of the human population and the growing proximity of humans to wild and domesticated animals have been linked to emergence of Nipah virus in Malaysia (65, 66), as well as the global increase in food-borne illnesses (67). Use of metagenomics to survey the microbial landscape before and after human activities provides an approach to examine the impact of such changes. The microbial communities associated with humans, animals, and their environment must be investigated concurrently in order to be able to draw a clearer picture of the microbial landscape and the interplay between the microbiomes from these interdependent niches. Prior to the metagenomics era, identification and modeling of the uncultivable microorganisms from a given environment were nearly impossible. Metagenomics has provided novel insights into the structure of the microbial communities in the environment, including the abundance, diversity, and composition of the microbes and viruses. Metagenomics also reveals the distribution of particular genes, including virulence genes, within an environment. Once we know the microbes and potential virulence genes in these environments, it becomes important to better understand what they are doing in these environments, the interactions between organisms and the physical environment, how they interact, and how these dynamics could lead to selection or transmission of organisms that can cause disease. Thus, with some vision and creativity, metagenomics can be used to survey the changing environmental microbial landscape to generate data for models that can predict, and in turn help prevent, impending outbreaks.
ONE HEALTH Human disruption of the environment promotes alterations in the patterns of infectious disease. For example, global climate change increases food- and waterborne diseases by contamination of fresh water supplies, increases vector-borne diseases by changing the rate and
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distribution of insect vectors, and alters the vulnerability of organisms to infectious disease by changing the dynamics of the ecosystem. Understanding these factors will demand the integration of a variety of types of data that probe the health of the environment, animals, and humans. The approaches needed extend from the molecular approaches of genomics and metagenomics to geographic information systems, satellite imaging, and mathematical models that can model and predict future outbreaks (68). Bringing together scientists with a broad understanding of computer sciences, mathematics, and the ecology of microbes in humans, animals, and diverse environments can generate predictions that will allow us to anticipate when and where a disease outbreak may occur and provide upstream interventions that protect the health of humans, animals, and the environment. ACKNOWLEDGMENTS The authors of this manuscript do not have any commercial affiliations, consultancies, stock or equity interests, or patentlicensing arrangements that could be considered to pose a conflict of interest regarding the manuscript.
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