Microbes: Concepts and Applications

Copyright © 2012 by Wiley-Blackwell. All rights reserved

Published by John Wiley & Sons, Inc., Hoboken, New Jersey

Published simultaneously in Canada

Wiley-Blackwell is an imprint of John Wiley & Sons, formed by the merger of Wiley's global Scientific, Technical, and Medical business with Blackwell Publishing.

No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, or online at http://www.wiley.com/go/permission.

Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages.

For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) 762-2974, outside the United States at (317) 572-3993 or fax (317) 572-4002.

Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic formats. For more information about Wiley products, visit our web site at www.wiley.com.

Library of Congress Cataloging-in-Publication Data:

Bisen, Prakash S.

Microbes : concepts and applications/Prakash S. Bisen, Mousumi Debnath, Godavarthi B.K.S. Prasad.

p. cm.

Includes bibliographical references and index.

ISBN 978-0-470-90594-4

1. Microbiology. 2. Microbial diversity. 3. Microbial ecology. 4. Microbial biotechnology. 5. Microorganisms. I. Debnath, Mousumi. II. Prasad, Godavarthi B. K. S. III. Title.

QR41.2.B576 2012

616.9′041–dc23

2011044258

Preface

Microbiology embraces functional disciplines at all levels, from the behavior of population of atoms to the behavior of population of microorganisms. Microorganisms undergo profound changes during their transition from planktonic organisms to cells that are part of a complex, surface-attached community. These changes are reflected in the phenotypic characteristics developed in response to a variety of environmental conditions. This book gives an insight into the microbial world. It features a comprehensive account of various topics including systematics; diversity of microbes in the environment; microbes in agriculture, food, and industry; microbial interactions; microbial metabolism; microbial diseases; and the diagnostic approaches.

In the coming few decades, it is the Archaea and the extremophilic microorganisms found in harsh and unusual environment that will occupy the center stage of microbiology in view of their tremendous importance in industrial applications. However, the excitement of modern microbiology extends far beyond Archaea and extremophiles. Microbiologists are debating the novel, philosophical, and controversial but fascinating concept of bacteria as global superogranisms, and focus on bacterial biofilms is likely to have a profound influence on environmental and medical microbiology in the twenty-first century. We have attempted to cover these and all other similar aspects of modern microbiology that are not commonly seen in existing books. We have covered the older traditional microbiology rather briefly and concisely but the major focus has been on recent development.

The authors have designed a chapter on Gene Technology, covering latest technologies relevant to the understanding of molecular aspects of microbes. This chapter offers a straightforward approach to learning the core principles associated with Omics technologies and their applications to the world of microbes. Omics technology if studied in isolation provides us certain specific snapshots of complex biological systems. Genomics, transcriptomics, proteomics, and metabolomics have enhanced our understanding of biological system by several folds and helped in providing insights into the biomedical research. Each of these omics technologies is limited in scope, as it can explain only one aspect of biological system. It is desirable to integrate snapshots provided by different omics technologies to get a holistic picture of complex biological system. Such challenging task of integration of omics technology is undertaken by the new discipline Systems Biology.

Microbes are of major economic, environmental, and social importance and are being exploited for production of a wide range of products of commercial significance. The chapter on industrial applications of microbes deals with their role in production of enzymes, foods, probiotics, chemical feedstock, fuels and pharmaceuticals, biofertilizers, traditional fermentation-based beverages, and development of clean technologies used in waste treatment and pollution control. Developments in bacterial genomics of lactobacilli and bifidobacteria would dramatically alter the scope and impact of the probiotic field, offering tremendous new opportunities with accompanying challenges for research and industrial application. The genetic and molecular approaches used to study bacterial and fungal biofilms, host immune evasion, and drug resistance have been discussed. Current evidence indicates that several microbes, especially bacteria and viruses, encode miRNAs, and over 200 viral miRNAs involved in manipulation of both cellular and viral gene expression have now been identified. Small noncoding RNAs have been found in all organisms, primarily as regulators of translation bioterrorism. This book discusses our current knowledge of these regulatory genetic elements and their relationship to infection.

Each chapter starts with a prologue to the concept and finally ends with a vision for future approach and challenges. The authors have tried to foresight some of the innovative areas of microbes and their involvement in the near future. Some of the topics of interest are metagenomics, pharmacogenomics, biochip technology, aptamers, microorganisms on space, functional genomics for improvement of plants, and next generation diagnostic industry.

This book responds to the requirement of students of undergraduates and graduates in microbiology and medicine. Each topic has been approached as a separate topic, and large illustrations in the chapters can help students and researchers to understand better. An effort has been done to reach at the individual level. Students can master key concepts and develop insights into the latest technologies of microbial relevance. This book is self-explanatory and written in a lucid language. A large compilation of references have been added at the end of each chapter. This book can be a source reference to all innovative people interested in microbes. We welcome suggestions and comments for improvement of this book.

Prakash S. Bisen

Gwalior, India

Mousumi Debnath

Kishangarh, Ajmer, India

Godavarthi B. K. S. Prasad

Gwalior, India

Acknowledgments

We wish to thank Dr. R. Vijayaraghavan, Outstanding Scientist and Director, Defence Research Development Establishment, Defence Research Development Organization, Ministry of Defence, Government of India, Gwalior, India, and Professor G. P. Agarwal, former Dean and Senior Microbiologist, Faculty of Life Sciences, R. D. University, Jabalpur, India, for their valuable guidance and encouragement and for extending all necessary facilities to complete the task smoothly.

Mr. Rakesh Singh Rathore, CEO, Vikrant Group of Institutions, Gwalior and Dr. Gurudev S. Davar and Mr. Puneet Davar, Tropilite Foods Pvt. Limited, Gwalior, are gratefully acknowledged for extending all secretarial assistance and for their help in various ways for completing this work without any hindrances.

We thank Mr. Devendra Singh and Mr. Avinash Dubey for all the computational help in preparation of figures and tables and our research students Ruchika Singh Raghuvanshi, Bhagwan S. Sanodiya, Gulab S. Thakur, Rakesh Baghel, and Rohit Sharma for their valuable assistance in the preparation of this book. Thanks are also due to Karen E. Chambers and Anna Ehler, Wiley-Blackwell Publishing, John Wiley & Sons, for their full support in publishing this book in time with patience and interest. Finally, we thank our families for their constant support, cooperation, and understanding. We are thankful to the Council of Scientific and Industrial Research (CSIR), New Delhi, for the award of Emeritus Scientist to Professor Prakash S. Bisen.

Prakash S. Bisen

Gwalior, India

Mousumi Debnath

Kishangarh, Ajmer, India

Godavarthi B. K. S. Prasad

Gwalior, India

Chapter 1

Human and Microbial World

1.1. Prologue

The microbial world is vast, diverse, and dynamic. The Earth hosts over 10³⁰ microorganisms, representing the largest component of the planet's biomass. Microbes include bacteria, archaea, mollicutes, fungi, microalgae, viruses, and protozoa, and many more organisms with a wide range of morphologies and lifestyles. All other life-forms depend on microbial metabolic activity. Microorganisms have colonized virtually every environment on earth ranging from deep sea thermal vents, polar sea ice, desert rocks, guts of termites, roots of plants, to the human body. Much as we might like to ignore them, microbes are present everywhere in our bodies, living in our mouth, skin, lungs, and gut. Indeed, the human body has 10 times as many microbial cells as human cells. They are a vital part of our health, breaking down otherwise indigestible foods, making essential vitamins, and even shaping our immune system. While microbes are often feared for the diseases they may cause, other microorganisms mediate the essential biogeochemical cycles of key elements that make our planet habitable. Ancient lineages of microorganisms may hold the key to understanding the earliest history of life on earth.

1.2. Innovations in Microbiology for Human Welfare

The human society is overburdened with infectious diseases. Despite worldwide efforts toward prevention and cure of these deadly infections, they remain major causes of human morbidity and mortality. Microbes play a role in diseases such as ulcers, heart disease, and obesity. Over the past century, microbiologists have searched for more rapid and efficient means of microbial identification. The identification and differentiation of microorganisms has principally relied on microbial morphology and growth variables. Advances in molecular biology over the past few years have opened new avenues for microbial identification, characterization, and molecular approaches for studying various aspects of infectious diseases. Perhaps, the most important development has been the concerted efforts to determine the genome sequence of important human pathogens. The genome sequence of the pathogen provides us with the complete list of genes, and, through functional genomics, a potential list of novel drug targets and vaccine candidates can be identified (Hasnain, 2001). The genetic variation inherent in the human population can modulate success of any vaccine or chemotherapeutic agent. This is why, the sequencing of the human genome has attracted not only the interest of all those working on human genetic disorders but also the interest of scientists working in the field of infectious diseases.

1.2.1. Impact of Microbes on the Human Genome Project

Technology and resources generated by the Human Genome Project and other genomic research are already having a major impact on research across life sciences. The elucidation of the human genome sequence will have a tremendous impact on our understanding of the prevention and cure of infectious diseases. The human genome sequence will further advance our understanding of microbial pathogens and commensals and vice versa (Relman and Falkow, 2001). This will be possible through efforts in areas such as structural genomics, pharmacogenomics, comparative genomics, proteomics, and, most importantly, functional genomics. Functional genomics includes not only understanding the function of genes and other parts of the genome but also the organization and control of genetic pathway(s). There is an urgent need to apply high throughput methodologies such as microarrays, proteomics (the complete protein profile of a cell as a function of time and space), and study of single nucleotide polymorphisms, transgenes and gene knockouts. Microarrays have a tremendous potential in

1. Determining new gene loci in diseases

2. Understanding global cellular response to a particular mode of therapy

3. Elucidating changes in global gene expression profiles during disease conditions; and so on.

Increasingly detailed genome maps have aided researchers seeking genes associated with dozens of genetic conditions, including myotonic dystrophy, fragile X syndrome, neurofibromatosis types 1 and 2, inherited colon cancer, Alzheimer's disease, and familial breast cancer.

The Human Microbiome Project (HMP) has published an analysis of 178 genomes from microbes that live in or on the human body. The core human microbiome is the set of genes present in a given habitat in all or the vast majority of humans (Fig. 1.1). The variable human microbiome is the set of genes present in a given habitat in a smaller subset of humans. This variation could result from a combination of factors such as host genotype, host physiological status (including the properties of the innate and adaptive immune systems), host pathobiology (disease status), host lifestyle (including diet), host environment (at home and/or work), and the presence of transient populations of microorganisms that cannot persistently colonize a habitat. The gradation in color of the core indicates the possibility that, during human microevolution, new genes might be included in the core microbiome, whereas other genes might be excluded (Turnbaugh et al., 2007). The researchers discovered novel genes and proteins that serve functions in human health and disease, adding a new level of understanding to what is known about the complexity and diversity of these organisms (http://www.nih.gov). Currently, only some of the bacteria, fungi, and viruses can grow in a laboratory setting. However, new genomic techniques can identify minute amounts of microbial DNA in an individual and determine its identity by comparing the genetic signature with known sequences in the project's database. Launched in 2008 as part of the NIH Common Fund's Roadmap for Medical Research, the HMP is a $157 million, five-year effort that will implement a series of increasingly complicated studies that reveal the interactive role of the microbiome in human health (http://www.eurekalert.org). The generated data will then be used to characterize the microbial communities found in samples taken from healthy human volunteers and, later, those with specific illnesses.

Figure 1.1 The concept of human microbiome.

Studies were also conducted to evaluate the microbial diversity present in the HMP reference collection. For example, they found 29,693 previously undiscovered, unique proteins in the reference collection; more proteins than there are estimated genes in the human genome. The results were compared to the same number of previously sequenced microbial genomes randomly selected from public databases and reported 14,064 novel proteins.

These data suggest that the HMP reference collection has nearly twice the amount of microbial diversity than is represented by microbial genomes already in public databases (http://www.ncbi.nlm.nih.gov/genomeprj). One of the primary goals of the HMP reference collection is to expand researchers' ability to interpret data from metagenomic studies. Metagenomics is the study of a collection of genetic material (genomes) from a mixed community of organisms. Comparing metagenomic sequence data with genomes in the reference collection can help determine the novel or already existing sequences (Hsiao and Fraser-Liggett, 2009). A total of 16.8 million microbial sequences found in public databases have been compared to the genome sequences in the HMP reference collection and it was found that 62 genomes in the reference collection showed similarity with 11.3 million microbial sequences in public databases and 6.9 million of these (41%) correspond with genome sequences in the reference collection (http://www.ncbi.nlm.nih.gov/genomeprj).

On the horizon is a new era of molecular medicine characterized less by treating symptoms and more by looking at the most fundamental causes of diseases. Rapid and more specific diagnostic tests will make possible earlier treatment of countless maladies. Medical researchers will also be able to devise novel therapeutic regimens on the basis of new classes of drugs, immunotherapy techniques, avoidance of environmental conditions that may trigger disease, and possible augmentation or even replacement of defective genes through gene therapy.

Despite our reliance on the inhabitants of the microbial world, we know little of their number or their nature. Less than 0.01% of the estimated all microbes have been cultivated and characterized. Microbial genome sequencing will help to lay the foundation for knowledge that will ultimately benefit human health and the environment. The economy will benefit from further industrial applications of microbial capabilities.

Information gleaned from the characterization of complete microbial genomes will lead to insights into the development of such new energy-related biotechnologies as photosynthetic systems and microbial systems that function in extreme environments and organisms that can metabolize readily available renewable resources and waste material with equal facility. Expected benefits also include development of diverse new products, processes, and test methods that will open the door to a cleaner environment. Biomanufacturing will use nontoxic chemicals and enzymes to reduce the cost and improve the efficiency of industrial processes. Microbial enzymes have been used to bleach paper pulp, stone wash denim, remove lipstick from glassware, break down starch in brewing, and coagulate milk protein for cheese production. In the health arena, microbial sequences may help researchers to find new human genes and shed light on the disease-producing properties of pathogens.

Microbial genomics will also help pharmaceutical researchers to gain a better understanding of how pathogenic microbes cause disease. Sequencing these microbes will help to reveal vulnerabilities and identify new drug targets. Gaining a deeper understanding of the microbial world will also provide insights into the strategies and limits of life on this planet, and the human genome sequence will further strengthen our understanding of microbial pathogens and commensals, and vice versa.

Data generated in HMP have helped scientists to identify the minimum number of genes necessary for life and confirm the existence of a third major kingdom of life. Additionally, the new genetic techniques now allow us to establish, more precisely, the diversity of microorganisms and identify those critical to maintaining or restoring the function and integrity of large and small ecosystems; this knowledge can also be useful in monitoring and predicting environmental changes. Finally, studies on microbial communities provide models for understanding biological interactions and evolutionary history.

1.2.2. Microbial Biosensors

Biosensors are defined as analytical devices combining biospecific recognition systems with physical or electrochemical signaling. They have been used for many years to provide process control data in the pharmaceutical, fermentation, and food-processing industries. The generic system comprises three components: the biospecific interaction, the signal emitted when the target is bound, and the platform that transduces the binding reaction into a machine-readable output signal. Significant progress has been made in developing platforms that exploit recent technological advances in microfabrication, optoelectronics, and electromechanical nanotechnology. Dramatic improvements in device designs facilitated by new tools and instrumentation (Fig. 1.2) have increased biosensor sensitivities by several magnitudes (Tepper and Shlomi, 2010). The designed biosensors facilitate high throughput detection and quantification of chemicals of interest, enabling combinatorial metabolic engineering experiments aiming to overproduce them (http://www.cs.technion.ac.il). New platforms can be arrayed in panels to reduce costs and simplified methods can be employed to detect and validate against numerous hazards.

Figure 1.2 Concept of microbial biosensor (Tepper and Shlomi, 2010). (See insert for color representation of the figure).

Majority of the described piezoelectric (PZ) devices are based on immunosensors. Targeting intact bacteria as antibody is relatively simple to immobilize, and entrapped bacterial cells can accumulate a significantly large, detectable mass. Many PZ devices are used in clinical and food pathogen identifications, for example, quartz crystal microbalance (QCM) devices coated with antibody, protein A, or other specific receptor molecules have been applied for a wide range of antibodies, pathogenic organisms (including Vibrio, Salmonella, Campylobacter, Escherichia coli, Shigella, Yersinia, viruses, and protozoa) and PCR amplicons (Hall, 2002). The simplicity, flexibility, and utility of the PZ systems in a range of foodborne and clinical applications make it well accepted for QCM format, although the sensitivity of these sensors is suitable only for very dense bacterial cultures. The QCM device coated with specific antibody and integrated into the culture enrichment tube resolves several problems in food safety testing, albeit at the expense of rapidity and cost. There are numerous biological components that can be coupled to mass aggregation and deposition chemistries, largely because the signal amplification required in most ELISA, western hybridization, and dot blot detection systems entails accumulation of precipitated mass in the process of generating a detectable signal. Among the ELISA systems that have been modified for mass sensors is Ag/Abs binding and DNA–DNA hybridization, and indirect, amplified systems such as double anti-angiogenic protein (DAAP) and avidin/streptavidin enzyme-conjugated secondary antibodies.

Microbial biosensors for environmental applications range in their development stages from proof of concept to full commercial availability, and the target detection specificity may fall in one of the following two groups (Bogue, 2003; Rodriguez-Mozaz et al., 2004):

Biosensors that measure general biological effects/parameters.

Biosensors for specific detection of target compounds.

The first group of biosensors is aimed to measure integral toxicity, genotoxicity, estrogenicity, or other general parameters of the sample, which affect living organisms. They essentially include whole microorganisms as biorecognition elements. The most-often reported cell-based biosensors include genetically modified bacteria with artificially constructed fusions of particular regulatory system (native promoter) with reporter genes. The presence of effectors (nonspecific stressor such as DNA damaging agents, heat shock, oxidative stress, toxic metals, and organic environmental pollutants) results in transcription and translation of fused target genes, generating recombinant proteins that produce some measurable response. Frequently used reporter genes are lux (coding for luciferase) and gfp (coding for green fluorescence protein), expression of which correlates with luminescence- or fluorescence-based light emission (Kohlmeier et al., 2006). Colorimetric determination of target gene expression is possible by fusing it to reporter genes coding for β-galalactosidase (lacZ) or alkaline phosphatase (phoA).

E. coli biosensor capable of detecting both genotoxic and oxidative damage has been developed. This was achieved by introducing two plasmids: fusion of katG (gene encoding for an important antioxidative enzyme) promoter to the lux reporter genes and recA (gene encoding a crucial enzyme for DNA repair) promoter with the gfp reporter gene (Mitchell and Gu, 2004). Besides genetically modified microorganisms (also named bioreporters), some other types of cellular biosensors have also been constructed. An example is the algal biosensor, which functions based on amperometric monitoring of photosynthetic O2 evolution—the process affected by toxic compounds—was developed by coupling Clark electrode to the cyanobacterium Spirulina subsalsa (Campanella et al., 2000). Biosensors for specific determination of chemical compounds frequently contain molecules such as enzymes, receptors, and metal-binding proteins as recognition elements. A number of enzymes have been shown to be inhibited by toxic metals, pesticides, and some other important contaminants such as endocrine disrupting compounds. Limitations for the potential applications of many enzyme biosensors include limited sensitivity and selectivity, as well as interference by environmental matrices Marinšek Logar and Vodovnik, 2007. One recently introduced strategy to overcome the first two of these limitations uses inhibition ratio of two enzymes for the detection of specific compounds. Acetylcholinesterase and urease, coentrapped in the sol–gel matrix with the sensing probe (FITC-dextran), have successfully been used for the detection of Cu, Cd, and Hg (Tsai and Doong, 2005). Besides molecular biosensors, bioreporter cells may also be used for the detection of specific target compounds. A biosensor for nitrate monitoring has been constructed by transformation of plasmid containing nitrate reductase operon fused to gfp reporter gene to E. coli cells (Taylor et al., 2004).

1.2.3. Molecular Diagnostics

Traditionally, the clinical medical microbiology laboratory has functioned to identify the etiologic agents of infectious diseases through the direct examination and culture of clinical specimens. Direct examination is limited by the number of organisms present and by the ability of the laboratories to successfully recognize the pathogen. Similarly, the culture of the etiologic agent depends on the ability of the microbe to propagate on artificial media and the choice of appropriate media for the culture. When a sample of limited volume is submitted, it is often not possible to culture for all pathogens. In such instances, close clinical correlation is essential for the judicious use of the specimen available. Commercial kits for the molecular detection and identification of infectious pathogens have provided a degree of standardization and ease of use that has facilitated the introduction of molecular diagnostics into the clinical microbiology laboratory. The use of nucleic acid probes for identifying cultured organisms and for direct detection of organisms in clinical material was the first exposure that most laboratories had to explore commercially available molecular tests. Although these probe tests are still widely used, amplification-based methods are increasingly employed for diagnosis, identification, quantization of pathogens, and characterization of antimicrobial-drug-resistant genes.

The tools of molecular biology have proven readily adaptable for use in the clinical diagnostic laboratory and promise to be extremely useful in diagnosis, therapy, and epidemiologic investigations and infection control (Cormican and Pfaller, 1996; Pfaller, 2000, 2001). Although technical issues such as ease of performance, reproducibility, sensitivity, and specificity of molecular tests are important, cost and potential contribution to patient care are also of concern (Kant, 1995). Molecular methods may be an improvement over conventional microbiologic testing in many ways. Currently, their most practical and useful application is in detecting and identifying infectious agents for which routine growth-based culture and microscopy methods may not be adequate (Fredricks and Relman, 1996; Fredricks and Relman, 1999; Tang and Persing, 1999; Woods, 2001).

Nucleic-acid-based tests used in diagnosing infectious diseases use standard methods for isolating nucleic acids from organisms and clinical material, and restriction endonuclease enzymes, gel electrophoresis, and nucleic acid hybridization techniques to analyze DNA or RNA (Tang and Persing, 1999). Because the target DNA or RNA may be present in very small amounts in clinical specimens, various signal amplification and target amplification techniques have been used to detect infectious agents in clinical diagnostic laboratories (Fredricks and Relman, 1999; Tang and Persing, 1999). Nucleic acid sequence analysis coupled with target amplification is clinically useful to detect and identify previously uncultivable organisms and characterize antimicrobial resistant gene mutations, thus aiding both diagnosis and treatment of infectious diseases (Fredricks and Relman, 1999). Automation and high-density oligonucleotide probe arrays (DNA chips) also hold great promise for characterizing microbial pathogens (Tang and Persing, 1999).

Although most clinicians and microbiologists enthusiastically welcome the new molecular tests for diagnosing infectious diseases, the high cost of these tests is of concern. Molecular methods will be increasingly used for pathogen identification, microbial quantification, and resistance testing.

The use of these detection methods in microbiology laboratories has resolved many problems and has initiated a revolution in the diagnosis and monitoring of infectious diseases. Some microorganisms are uncultivable at present, extremely fastidious, or hazardous to laboratory personnel. In these instances, the diagnosis often depends on the serologic detection of a humoral response or culture in an expensive biosafety level II–IV facility. In community medical microbiology laboratories, these facilities may not be available, or it may not be economically feasible to maintain the special media required for the culture of all of the rarely encountered pathogens. Thus, cultures are often sent to referral laboratories. During transit, fragile microbes may lose viability or become overgrown by contaminating organisms or competing normal flora.

Although direct detection of organisms in clinical specimens by nucleic acid probes is rapid and simple, it suffers from lack of sensitivity. Most direct probe detection assays require at least 10⁴ copies of nucleic acid per microliter for reliable detection, a requirement rarely met in clinical samples without some form of amplification. Amplification of the detection signal after probe hybridization improves sensitivity to as low as 500 gene copies per microliter and provides quantitative capabilities. This approach has been extensively used for quantitative assays of viral load HIV, hepatitis B virus (HBV), and hepatitis C virus (HCV) but does not match the analytical sensitivity of target-amplification-based methods such as polymerase chain reaction (PCR) for detecting organisms.

Probe hybridization is useful in identifying organisms that grow slow after isolation from culture using either liquid or solid media. Identification of mycobacteria and other slow-growing organisms such as the dimorphic fungi (Histoplasma capsulatum, Coccidioides immitis, and Blastomyces dermatitidis), has certainly been facilitated by commercially available probes. All commercial probes for the identification of organisms are produced by Gen-Probe, which uses acridinium-ester-labeled probes directed at species-specific rRNA sequences. Gen-Probe products are available for the culture identification of Mycobacterium tuberculosis, Mycobacterium avium-intracellulare complex, Mycobacterium gordonae, Mycobacterium kansasii, Cryptococcus neoformans, the dimorphic fungi (listed above), Neisseria gonorrhoeae, Staphylococcus aureus, Streptococcus pneumoniae, E. coli, Haemophilus influenzae, Enterococcus spp., Streptococcus agalactiae, and Listeria monocytogenes. The sensitivity and specificity of these probes are excellent, and they provide species identification within one working day. Because most of the bacteria listed and C. neoformans can be easily and efficiently identified by conventional methods within 1–2 days, many of these probes are not widely used. The mycobacterial probes, on the other hand, are accepted as mainstays for the identification of M. tuberculosis and related species.

Nucleic acid techniques such as plasmid profiling, various methods for generating restriction fragment length polymorphisms, and the PCR are making increasing inroads into clinical laboratories. PCR-based systems to detect the etiologic agents of diseases directly from clinical samples, without the need for culture, have been useful in rapid detection of uncultivable or fastidious microorganisms. Additionally, sequence analysis of amplified microbial DNA allows for the identification and better characterization of the pathogen. Subspecies variation, identified by various techniques, has been shown to be important in the prognosis of certain diseases. New advances in real-time PCR promise results that will come fast enough to revolutionize the practice of medicine.

Commercial-amplification-based molecular diagnostic systems for infectious diseases have focused largely on systems for detecting N. gonorrhoeae, Chlamydia trachomatis, M. tuberculosis, and specific viral infections [HBV, HCV, HIV, CMV (cytomegalovirus), and enterovirus]. Given the adaptability of PCR, numerous additional infectious pathogens have been detected by investigator-developed PCR assays. This novel, fully integrated device, coupled with appropriate databases, will insure better management of patients, should reduce health costs, and could have an impact on the spread of antibiotic resistance (Boissinot and Bergeron, 2002). Another exciting technology that has demonstrated clinical diagnostic utility is DNA microarray science. DNA microarray enables simultaneous analyses of global patterns of gene expression in microorganisms or host cells. In addition, genotyping and sequencing by microarray-based hybridization have been successfully used for organism identification and molecular resistance testing.

Microbial phenotypic characteristics, such as protein, bacteriophage, and chromatographic profiles, as well as biotyping and susceptibility testing, are used in most routine laboratories for identification and differentiation. Other important advances include the determination of viral load and the direct detection of genes or gene mutations responsible for drug resistance. Increased use of automation and user-friendly software makes these technologies more widely available. In all, the detection of infectious agents at the nucleic acid level represents a true synthesis of clinical chemistry and clinical microbiology techniques.

Ou et al. (2007) have beautifully illustrated an example of how to use whole genome sequence analyses of Salmonella enterica paratyphi A and existing comparative genomic hybridization data to design a highly discriminatory multiplex PCR assay that can be developed in any molecular diagnostic laboratory. In a time of overwhelmingly rapid expansion of genomic information, various navigation tools and a recipe for mining the genomic databases to design species, serovar, or pathotype specific PCR assays for accurate identification have been developed (Wenyong et al., 2007).

Molecular methods can rapidly detect antimicrobial drug resistance in clinical settings and have substantially contributed to our understanding of the spread and genetics of resistance. Conventional broth and agar-based antimicrobial susceptibility testing methods provide a phenotypic profile of the response of a given microbe to an array of agents. Conventional methods are slow and fraught with problems, although useful for selecting potential therapeutic agents. The rapid evolution in microbial genomics will transform the process of accurate identification of novel, difficult to culture, or phenotypically indistinguishable pathogens and research and development in diagnostics, or diagnomics.

Molecular typing methods have allowed investigators to study the relationship between colonizing and infecting isolates in individual patients. Most available DNA-based typing methods may be used in studying infections when applied in the context of a careful epidemiologic investigation. Molecular testing for infectious diseases includes testing for the host's predisposition to diseases, screening for infected or colonized persons, diagnosis of clinically important infections, and monitoring the course of infection or the spread of a specific pathogen in a given population.

There are many areas of molecular diagnostics and many tools related to this field (Fig. 1.3). Not all molecular diagnostic tests are extremely expensive. Direct costs vary widely, depending on the complexity and sophistication of the test performed. Inexpensive molecular tests are generally kit based and use methods that require little instrumentation or technologist experience. DNA probe methods that detect C. trachomatis or N. gonorrhoeae are examples of low-cost molecular tests. The more complex molecular tests such as resistance genotyping often have high labor costs because they require experienced, well-trained technologists. Although the more sophisticated tests may require expensive equipment (e.g., DNA sequencer) and reagents, advances in automation and the production of less expensive reagents promise to decrease these costs as well as technician time.

Figure 1.3 Applications and areas of interest related to molecular diagnostics.

In general, molecular tests for infectious diseases have been more readily accepted for reimbursement; however, reimbursement is often on a case-by-case basis and may be slow and cumbersome. FDA approval of a test improves the likelihood that it will be reimbursed but does not ensure that the amount reimbursed will equal the cost of performing the test.

Molecular screening programs for infectious diseases are developed to detect symptomatic and asymptomatic diseases in individuals and groups. Persons at high risk, such as immuno-compromised patients or those attending family planning or obstetrical clinics, are screened for CMV and Chlamydia, respectively. Likewise, all blood donors are screened for blood borne pathogens. The financial outcome of such testing is unknown. The cost must be balanced against the benefits of earlier diagnosis and treatment and societal issues such as disease epidemiology and population management.

One of the most highly touted benefits of molecular testing for infectious diseases is the promise of earlier detection of certain pathogens. The rapid detection of M. tuberculosis directly in clinical specimens by PCR or other amplification-based methods is quite likely to be cost-effective in the management of tuberculosis. Other examples of infectious diseases that are amenable to molecular diagnosis and for which management can be improved by this technology include HSV (herpes simplex virus) encephalitis, Helicobacter pylori infection, and neuroborreliosis caused by Borrelia burgdorferi. For HSV encephalitis, detection of HSV in cerebrospinal fluid (CSF) can direct specific therapy and eliminate other tests including brain biopsy. Likewise, detection of H. pylori in gastric fluid can direct therapy and obviate the need for endoscopy and biopsy. PCR detection of B. burgdorferi in CSF is helpful in differentiating neuroborreliosis from other chronic neurologic conditions and chronic fatigue syndrome.

Molecular tests may be used to predict disease response to specific antimicrobial therapy (Fig. 1.4, Chinnery et al., 1999). Detection of specific resistance genes (mec A, van A) or point mutations resulting in resistance has been proved to be efficacious in managing diseases. Molecular based viral load testing has become a standard practice for patients with chronic hepatitis and AIDS. Viral load testing and genotyping of HCV are useful in determining the use of expensive therapy such as interferon therapy, and can be used to justify decisions on the extent and duration of therapy. With AIDS, viral load determinations and resistance genotyping have been used to select among the various protease inhibitor drugs available for treatment, improving patient's response and decreasing incidence of opportunistic infections.

Figure 1.4 Molecular tests may be used to predict disease response to specific antimicrobial therapy.

Pharmacogenomics is the use of molecular based tests to predict the response to specific therapies and to monitor the response of the disease to the agents administered (Pfaller, 2001). The best examples of pharmacogenomics in infectious diseases are the use of viral load and resistance genotyping to select and monitor antiviral therapy of AIDS and chronic hepatitis. This application improves disease outcome, shortens the period of stay at hospital, reduces adverse events and toxicity, and facilitates cost-effective therapy by avoiding unnecessary expensive drugs, optimizing doses and timing, and eliminating ineffective drugs.

Molecular strain typing of microorganisms is now well recognized as an essential component of a comprehensive infection control program that also involves the infection control department, the infectious disease division, and pharmacy. The sequences of 16S ribosomal RNA sequences can be used to study the evolutionary relationship between bacteria. This region is highly conserved. The sequence in these variable regions is species specific. In this approach PCR primers, complementary to flanking conserved sequences are used to amplify the variable regions. The product is then sequenced and sequence compared against the database of the 16S sequence to identify the bacteria it is derived from. This approach to identification of the bacteria has particular advantage with organisms that cannot be easily cultured in the laboratory, as the DNA is amplified by PCR rather than the organisms being amplified by growing in a culture.

Amplification of 16S genes by PCR can be very effective when combined with oligonucleotide hybridization probes or molecular beacon technology to identify bacteria in the mixture. A PCR reaction with a single set of primers complementary to conserved sequences will amplify species-specific sequence from a range of different bacteria in a mixture. These can then be probed with molecular beacons complementary to the individual species-specific sequences and labeled with different fluorophores. This makes it possible to identify more than one type of pathogenic bacterium in a mixture. By measuring the fluorescence levels in real time, it is also possible to make quantitative measurements and to detect the presence of a rare pathogen in the more abundant one.

One topical application of this technology is in devising tests to detect and identify bioterrorism agents. In the case of suspected bioterorrist attack, there is an urgent need for a robust and rapid assay for the selection of possible bioterrorist agents. A real-time PCR assay has been devised to simultaneously detect four bacteria having the potential to be used as bioterrorism agents by using a single set of PCR primers and four species-specific molecular beacons. In the case of bioterrorism accident, it would be vital to be able to identify the bacteria concerned. Technology has obvious applications in routine clinical laboratories where patient care could be improved by reducing the time taken.

1.2.4. Nanomedicine

The early genesis of the concept of nanomedicine sprang from the visionary idea that tiny nanorobots and related machines could be designed, manufactured, and introduced into the human body to perform cellular repairs at the molecular level. Today, it has branched out in hundreds of different directions, each of them embodying the key insight that the ability to structure materials and devices at the molecular scale can bring enormous immediate benefits in the research and practice of medicine. Nanomedicine is defined as the application of nanotechnologies including nanobiotechnologies in medicine. Evidently, dimensional parameters alone are insufficient to refer someone or other work to the field of nanotechnology (e.g., nanomedicine). Fundamental novelty of nanomedicine as a branch of knowledge and technology is exemplified by the developments in pharmacology and design of medicinal products that brought about new drugs (nanomedical/nanopharmaceutical). These products are multicomponent supramolecular compounds designed for a specific purpose whose intricate structure is intended not so much to impart new properties as to properly deliver the active ingredient to the biological target. Accordingly, nanomedicine should be regarded as the use of supramolecular complexes with a well-differentiated surface, manufactured by purposeful assembly of selected components for diagnostic and/or therapeutic application (Piotrovsky, 2010). Nanopharmaceuticals are defined as a big part of what nanomedicine is today. Nanopharmaceuticals can be developed either as drug delivery systems or as biologically active drug products (Table 1.1). Various types of nanomedicine are already in clinical use these days (Fig. 1.5).

Figure 1.5 Various types of nanomedicines. (See insert for color representation of the figure).

Table 1.1 Various Types of Nanomedicinea

DermaVir vaccine is a novel pathogen-like nanomedicine containing a plasmid DNA complexed with a polyethylenimine (pDNA/PEIm) that is mannobiosylated to target antigen presenting cells and to induce immune responses. A commercially viable vaccine product was developed and the variability of raw materials and their relationship with the product's biological activity was investigated and found that the cGMP quality requirements are not sufficient to formulate the nanomedicine with optimal biological activity. The high cationic concentration of the pDNA favored the biological activity, but did not support the stability of the nanomedicine (Toke et al., 2010).

Nanomedicine also offers the prospect of powerful new tools for the treatment of human diseases and the augmentation of human biological systems (Table 1.2). They have been used as liposomes (3–100 nm) and nanoparticles (iron oxide, 5–50 nm) in clinical laboratories. To cure cancer, targeted drug delivery system is used using liposomes (Park, 2002). For hepatic diseases, nanoparticles are used as the contrast agent for generating resonance imaging (Thorek et al., 2006). Nanomedicine using dendrimers (Bharali et al., 2009), fullerenes (Partha and Conyers, 2009), and gold-coated nanoshells (Hirsch et al., 2003) is also under the developmental stage. In the cardiovascular phase III clinical trials, dendrimers are used as the contrast agent for magnetic resonance (Saha, 2009). Diamodoid-based medical nanorobotics may offer substantial improvements in capabilities over natural biological systems, exceeding even the improvements possible via tissue engineering and biotechnology. For example, the respirocytes, the artificial red blood cells comprise microscopic diamondoid pressure tanks that are operated at high atmospheric pressure and could carry >200 times respiratory gases than an equal volume of natural red blood cells.

Table 1.2 Application of Nanomedicine for Health

Nanomedicine has been used to cure many diseases (Table 1.2). The clottocytes are artificial platelets that can stop human bleeding within ∼ 1 s of physical injury, but using only 0.01% the bloodstream concentration of natural platelets in other words, nanorobotic clottocytes would be ∼ 10, 000 times more effective as clotting agents than an equal volume of natural platelets. In neurodegenerative diseases, carbon buckyballs (2–20 nm) are used as antioxidants. In preclinical cancer therapy during hyperthermia, gold-coated silica (60 nm) nanoshells are used.

Microbiovores constitute a potentially large class of medical nonorobots intended to be deployed in human patients for a wide variety of antimicrobial therapeutic purposes. They can also be useful in treating infections of the meninges or the CSF and respiratory diseases involving the presence of bacteria in the lungs or sputum, and can also digest bacterial biofilms. These handy nanorobots can quickly rid the blood of nonbacterial pathogens such as viruses (viremia), fungus cells (fungemia), or parasites (parasitemia).

A nanorobotic device that can safely provide quick and complete eradication of blood borne pathogens using relatively low doses of devices would be a welcome addition to the physician's therapeutic armamentarium Freitas, 2005. The ultimate tool of nanomedicine is the medical nanorobot—a robot to the size of a bacterium, composed of thousands of molecule-size mechanical parts perhaps resembling macroscale gears, bearings, and ratchets, possibly composed of a strong diamond-like material.

A nanorobot will need motors to make things move, and manipulator arms or mechanical legs for dexterity and mobility. It will have a power supply for energy, sensors to guide its actions, and an onboard computer to control its behavior. But, unlike a regular robot, a nanorobot will be very small. A nanorobot that would travel through the bloodstream must be smaller than the red cells—tiny enough to squeeze through even the narrowest capillaries in the human body.

Medical nanorobots could also be used to perform surgery on individual cells. In one proposed procedure, a cell repair nanorobot called a chromallocyte, controlled by a physician, would extract all existing chromosomes from a diseased cell and insert fresh new ones in their place. This process is called chromosome replacement therapy. The replaced chromosomes are manufactured outside the patient's body using a desktop nanofactory optimized for organic molecules.

The patient's own individual genome serves as the blueprint to fabricate the new genetic material. Each chromallocyte is loaded with a single copy of a digitally corrected chromosome set. After injection, each device travels to its target tissue cell, enters the nucleus, replaces old worn out genes with new chromosome copies, then exits the cell and is removed from the body. If the patient chooses, inherited defective genes could be replaced with nondefective base pair sequences, permanently curing any genetic disease and even permitting cancerous cells to be reprogrammed to a healthy state. Perhaps, most importantly, chromosome replacement therapy could correct the accumulating genetic damage and mutations that lead to aging in every one of our cells. At present, medical nanorobots are just theory. Nanorobots will have several advantages. Firstly, they can physically enter cells and scan the chemicals present inside. Secondly, they can have onboard computers that allow them to do calculations not available to immune cells. Thirdly, nanorobots can be programmed and deployed after a cancer is diagnosed, whereas the immune system is always guessing about whether a cancer exists. Given such molecular tools, a small device can be designed to identify and kill cancer cells (Saha, 2009).

The potential impact of medical nanorobotics is enormous. Rather than using drugs that act statistically and have unwanted side effects, we can deploy therapeutic nanomachines that act with digital precision, have no side effects, and can report exactly what they did back to the physician. Continuous medical monitoring by embedded nanorobotic systems will provide automatic collection of long-baseline physiologic data permitting detection of slowly developing chronic conditions that may take years or decades to develop, such as obesity, diabetes, calcium loss, or Alzheimer's. Nanorobot life cycle costs can be very low because nanorobots, unlike drugs and other consumable pharmaceutical agents are intended to be removed intact from the body after every use, then refurbished and recycled many times, possibly indefinitely. Even if the delivery of nanomedicine does not reduce total health-care expenditures—which it should—it will likely free up billions of dollars that are now spent on premiums for private and public health-insurance programs.

1.2.5. Personalized Medicine

The war against infectious agents has produced a powerful arsenal of therapeutics, but treatment with drugs can sometimes exacerbate the problem. The drug-resistant strains and the infectious agents that are least susceptible to drugs survive to infect again. They become the dominant variety in the microbe population, a present day example of natural selection in action. This leads to an ever-present concern that drugs can be rendered useless when the microbial world employs the survival of the fittest strategy of evolution. And, frequently used drugs contribute to their own demise by strengthening the resistance of many enemies.

Some engineering challenges are to develop better systems to rapidly assess a patient's genetic profile; another is collecting and managing massive amounts of data on individual patients; and yet another is the need to create inexpensive and rapid diagnostic devices such as gene chips and sensors able to detect minute amounts of chemicals in the blood. In addition, improved systems are necessary to find effective and safe drugs that can exploit the new knowledge of differences in individuals. The current gold standard for testing a drug's worth and safety is the randomized controlled clinical trial—a study that randomly assigns people to a new drug or to nothing at all, a placebo, to assess how the drug performs, but this approach essentially decides a drug's usefulness based on average results for the group of patients as a whole, not for the individual (Bottinger, 2007).

New methods are also needed for delivering personalized drugs quickly and efficiently to the site in the body where the disease is localized (Kalow, 2006). For instance, researchers are exploring ways to engineer nanoparticles that are capable of delivering a drug to its target in the body while evading the body's natural immune response (Fig. 1.6). For example, when the drug-packed liposome is injected into the bloodstream, the amino acids on the nanoparticles attache to the proteins. The heat is pushed to the surface of the tumor and more of the drug is delivered to the tumor.

Figure 1.6 Drug delivery by nanoparticles.

Such nanoparticles could be designed to be sensitive to the body's internal conditions, and, therefore, could, for example, release insulin only when the blood's glucose concentration is high. In a new field called synthetic biology, novel biomaterials are being engineered to replace or aid in the repair of damaged body tissues. Some are scaffolds that contain biological signals that attract stem cells and guide their growth into specific tissue types. Mastery of synthetic tissue engineering could make it possible to regenerate tissues and organs (Lutolf and Hubbell, 2005).

Ultimately, the personalization of medicine should have enormous benefits. It ought to make disease (and even the risk of disease) evident much earlier, when it can be treated more successfully or prevented altogether. It could reduce medical costs by identifying cases where expensive treatments are unnecessary or futile. It will reduce trial-and-error treatments and ensure that optimum doses of medicine are applied sooner.

More optimistically, personalized medicine could provide the path for curing cancer, by showing why some people contract cancer and others do not, or how some cancer patients survive when others do not. Thus, personalized medicine involves the use of laboratory-based molecular diagnostics and medical imaging to select suitable candidates for treatment with a particular drug(s), to rule out those patients who would suffer unacceptable side effects from the proposed drug treatment, and to monitor the health status of the patient postinitiation of therapy to assess therapeutic drug levels and the continuing efficacy of the agent in suppressing or curing the disease. Hence, personalized medicine is a new trend in drug development based on tailoring drugs to patients based on their individual genetic profiles.

Personalized-medicine-based pharmaceuticals avoid these issues by marketing to patients with specific genetic profiles that maximize both the safety and efficacy of the drug on each patient. Promising personalized-medicine-based pharmaceuticals in the pipeline have shown close to 100% efficacy in patients. Major pharmaceutical firms have responded to the growing emphasis on individualized therapy to improve drug efficacy and safety with large investments in pharmacogenomics research (Mancinelli et al., 2000); Table 1.3). Examples of drugs in the market that have used genetic markers to achieve improved safety and efficacy in patients include Gleevec [Novartis AG (NVS)] and Herceptin [Genentech (DNA)] (http://www.wikinvest.com). Of course, a transition to personalized medicine is not without its social and ethical problems.

Table 1.3 Selected Pharmaceutical Companies Focusing on Genomics and Pharmacogenomics

Even if the technical challenges can be met, there are issues of privacy when unveiling a person's unique biological profile, and it is likely that there will still be masses of people throughout the world unable to access its benefits deep into the century.

The drug resistance problem is not limited to bacteria and antibiotics. Antiviral drugs for fighting diseases such as AIDS and influenza face similar problems from emerging strains of resistant viruses (Wright and Sutherland, 2007). In fact, understanding the development of resistance in viruses is especially critical for designing strategies to prevent pandemics. The use of any antimicrobial drug must be weighed against its contribution to speeding up the appearance of resistant strains.

The engineering challenges for enabling drug discovery mirror those needed to enable personalized medicine development: more effective tools and techniques. This helps in rapid analysis and diagnosis so that a variety of drugs can be quickly screened and proper treatments can be promptly applied (West et al., 2006). Current drugs are often prescribed incorrectly or unnecessarily, promoting the development of resistance without real medical benefit. Quicker, more precise diagnosis may lead to more targeted and effective therapies. Antibiotics that attack a wide range of bacteria have typically been sought, because doctors could not always be sure of the precise bacterium causing an infection. Instruments that can determine the real culprit right away could lead to the use of more narrowly targeted drugs, reducing the risk of promoting resistance. Developing organism-specific antibiotics could become one of the century's most important biomedical engineering challenges. Personalized medicine will reshape pharmaceutical research and development and the calculation of cost-effectiveness by health services. The previous business model was based on so-called blockbuster drugs, intended for general use in the population and generating annual global profits in excess of $1 billion. Profits from blockbuster drugs offset the expenses of regulatory approval and investment in research and development (http://www.parliament.uk/documents/post/postpn329.pdf).

This could be especially challenging in the case of biological agents specifically designed to be weapons. A system must be in place to rapidly analyze their methods of attacking the body and quickly produce an appropriate medicine. In the case of a virus, small molecules might be engineered to turn off the microbe's reproductive machinery. Instructions for making proteins are stored by genes in DNA. Another biochemical molecule, called messenger RNA, copies those instructions and carries them to the cell's protein factories (Dietel and Sers, 2006). Sometimes other small RNA molecules can attach to the messenger RNA and deactivate it, thereby preventing protein production by blocking the messenger, a process known as RNA interference. Viruses can be blocked by small RNAs in the same manner, if the proper small RNAs can be produced to attach to and deactivate the molecules that reproduce the virus. The key is to decipher rapidly the sequence of chemicals comprising the virus so that effective small RNA molecules can be designed and deployed.

Traditional vaccines have demonstrated the ability to prevent diseases, and even eradicate some such as smallpox. It may be possible to design vaccines to treat diseases as well. Personalized vaccines might be envisioned for either use. But, more effective and reliable manufacturing methods are needed for vaccines, especially when responding to a need for mass immunization in the face of a pandemic (Heymann, 2006). A healthy future for the world's population will depend on engineering new strategies to overcome multiple drug resistances (Gerard and Sutherland, 2007). One major challenge in this endeavor will be to understand more fully how drug resistance comes about, how it evolves, and how it spreads. Furthermore, the system for finding and developing new drugs must itself evolve, and entirely novel approaches to fighting pathogens may also be needed (Kalow, 2006).

Drug resistance is nothing new. The traditional approach to this problem is still potentially useful in expanding the search for new antibiotics. Historically, many drugs to fight disease-producing microbes have been found as naturally occurring chemicals in soil bacteria, which is still a source of promising candidate (Lesko, 2007). Even more drug candidates, though, may be available from microbes in more specialized ecological niches or from plants or from bacteria living in remote or harsh environments, namely, deep lakes and oceans.

1.2.6. Biowarfare

Biological warfare agents are a group of pathogens and toxins of biological origin that can be potentially misused for military or criminal purposes (Pohanka and Kuca, 2010). In early December 2002, the National Security Council learned of a smallpox outbreak in Oklahoma. Twenty cases were confirmed by the Centres for Disease Control and Prevention (CDC), with 14 more suspected. There were 16 more reported cases in Georgia and Pennsylvania. Federal and State authorities quickly informed the public and implemented a vaccine distribution policy to those people most at risk of being exposed to the smallpox virus (http://learn.genetics.utah.edu). Three days before the Christmas holiday and 13 days after the initial outbreak, a total of 16,000 smallpox cases were reported in 25 states, and 1000 people were dead. Ten other countries reported cases of smallpox, likely to be caused by visitors from the United States. Canada and Mexico closed their borders to the United States. Vaccine supplies were depleted, and health officials predicted that by February, there will be three million cases of smallpox, leading to as many as one million deaths. The above scenario was, in fact, a game.

In their natural state, bacteria, viruses, and fungi can make pretty good biological weapons. If genetic engineering is used, more harmful agents can emerge. During the Cold War, several offensive biowarfare programs were run to develop the so-called Super Bugs. One such program, Project Bonfire, worked to create bacteria that were resistant to about 10 varieties of antibiotics. This was done by identifying and cutting out genes that conferred antibiotic resistance in many different strains of bacteria. By pasting these genes into the DNA of the anthrax bacterium, the Project Bonfire created a strain of anthrax that resisted any existing cure, making it impossible to treat (Fig. 1.7). The Hunter Program was another biological warfare research program that focused on combining whole genomes of different viruses to produce completely new hybrid viruses (Fig. 1.8). These artificial viruses could cause unpredictable symptoms that have no known treatment. In an innovative twist, the Hunter Program also created bacterial strains that carried pathogenic viruses inside them. These strains would be double trouble: a person who contracted the bacterial disease would likely be treated with an antibiotic, which would stop the infection by disrupting the bacterial cells. This would release the virus, resulting in an outbreak of viral disease. Such a scenario would confuse medical personnel, making treatment very difficult.

Figure 1.7 Project Bonfire: creation of antibiotic-resistant bacteria.

Figure 1.8 Hunter Program: creation of hybrid viruses.

It is not known whether the biological agents were ever actually used to infect people. At the same time, there was mounting fear over the offensive biological warfare agenda, focusing on the difficult