1. Answering the Call to Arms: Introduction and Overview

Alita A. Miller and Paul F. Miller

A global public health crisis due to antibiotic resistance may be imminent. Several organizations are working to mitigate the lack of new, effective drugs either in development or in the clinic by proposing strategies for re-investment in antibacterial research. Although it is imperative that regulatory issues be resolved and strategic policies be put in place, it is equally important to define the scientific path required to address this crisis. The goal of this textbook, therefore, is to offer new ways of thinking about antibiotics and technical solutions for the resistance problems we face. By summarizing innovative new concepts and approaches from leading experts around the world, we hope to enable the implementation of the re-investment strategies that are so urgently needed.

2. Renewing Investment in Antibacterial Research

L. Silvia Munoz-Price, and John P. Quinn

We summarize the epidemiology, clinical presentation, and current treatment options for the most clinically relevant multidrug resistant Gram-positive and Gram-negative organisms. Additionally, we describe the challenges faced by pharmaceutical companies within the antimicrobial research and development field, especially the disproportion between the degree of investment (both monetary and time) required and the relatively small profit antimicrobial agents bring. Finally, some potential solutions for the lack of antimicrobial agents are discussed. These include more widespread use of the Orphan Drug Act, patent extensions, and the Biomedical Advanced Research and Development Authority (BARDA).

3. Mining Bacterial Cell Division for New Antibacterial Drugs

Leigh G. Monahan, Michael A. D'Elia and Elizabeth J. Harry

The alarming rise of antibiotic resistant bacteria in hospitals and the community has exposed a critical need for new drugs that are not merely variants of older antibiotics, but target previously unexploited proteins and pathways. The wealth of available knowledge on the process of bacterial cell division implicates the division pathway as an excellent potential target, and has aided target-driven approaches to identify novel inhibitors. In this chapter we discuss the therapeutic potential of inhibiting bacterial divison based on a strong foundation of basic research into the division mechanism and its regulation in model bacteria, and more recently, clinically relevant pathogens. In addition, we review the progress made towards identifying division inhibitors, describe new approaches for antibacterial drug development targeting division and discuss the potential challenges for the future of this exciting new area of antibacterial discovery.

4. Efflux Pumps from Gram-negative Bacteria: From Structure and Function to Inhibition

Olga Lomovskaya and Helen I. Zgurskaya

Multidrug efflux pumps adversely affect both the clinical effectiveness of existing antibiotics as well as the discovery process to find new ones. In this chapter, we summarize recent advances in structural and functional analyses of multi-component efflux pumps from Gram-negative bacteria with the focus on transporters belonging to the Resistance-Nodulation-cell Division superfamily. The unquestionably significant impact of these pumps on the effectiveness of antibiotics in clinical settings and their emerging role in bacterial pathogenesis makes them attractive targets for inhibition. We discuss modes of inhibition and current efforts to develop effective inhibitors of multidrug efflux pumps.

5. Metabolite-sensing Riboswitches as Antibacterial Drug Targets

Elaine R. Lee, Kenneth F. Blount and Ronald R. Breaker

The need for new antibacterial drug targets increases as antibiotic resistant pathogens continue to arise. Researchers have recently begun to investigate whether structured noncoding RNAs such as riboswitches can be exploited as targets for new classes of antimicrobial compounds. Riboswitches are gene control elements made entirely of RNA, and in bacteria they are usually located in the 5' untranslated regions (UTRs) of messenger RNAs. These elements are capable of forming complex structures that selectively bind to specific fundamental metabolites and often control the expression of proteins critical for bacterial metabolism and survival. In principle, novel ligands could be designed that target specific riboswitches and alter the expression of the critical genes they regulate. Several riboswitch classes have begun to be examined as potential targets for new classes of antibacterial compounds. Herein we present some of the data generated by efforts to validate riboswitches as drug targets and discuss some of the key unanswered questions that will determine the ultimate success of antibacterial compounds that interact with these RNAs.

6. Targeting Bacterial Secretion Systems for the Development of Novel Therapeutic Agents

Heather B. Felise, Toni Kline and Samuel I. Miller

Antibiotic resistance is threatening our ability to treat bacterial diseases. Scientific development to define new antibacterial targets, including those that inhibit microbial virulence rather than target essential cellular functions, is required to develop the therapeutics of the future. In this chapter we will discuss the feasibility of Gram-negative secretion systems as therapeutic targets, provide a synopsis of current research on the identification and development of secretion inhibitors, and discuss their possible future utility as antimicrobial agents.

7. Recent Developments in Natural Products: Potential Impact on Antibacterial Drug Discovery

Ronald J. Quinn and Jeffrey E. Janso

Natural products and derivatized natural products, produced mainly by actinomycetes, have been one of the most successful sources of drugs used to treat and cure infectious diseases. However, many bacteria have quickly become resistant to the majority of antibiotics in use today prompting an urgent need to discover new classes of antibacterial compounds. The goal of this chapter is to summarize some of the recent advances that favorably position natural products drug discovery in the quest to discover new antibacterial agents. This includes new sources of biodiversity such as plants and the oceans as well as the overlooked potential within common soil-derived actinomycetes. Other encouraging advancements include: (1) the development of new culturing techniques, which have enabled the isolation of microbes that were once thought to be uncultivable, (2) the impact of sequencing technology and bioinformatics that have made strain dereplication more reliable and revealed that actinomycete genomes encode far more secondary metabolite gene clusters than originally thought and (3) the use of innovative methods to express and exploit these orphan biosynthetic pathways. Finally, the ability to dereplicate, isolate and elucidate the structure of natural products from less and less sample quantity will also be discussed.

8. Biosynthetic Engineering of Antibacterial Natural Products

Jay Fitzgerald, Younjoo Lee and Chaitan Khosla

Since the discovery of penicillin, the development of anti-infective drugs has been a central theme in the pharmaceutical industry through much of the 20^th century. However, the pace of developing new anti-infective agents has precipitously declined in the past two decades. The main reason for this change is an economic one - whereas the technical and regulatory risks associated with the development of a new broad-spectrum antibiotic are deemed unacceptably high, the financial returns derived from a targeted (narrow-spectrum) antibiotic are unattractive to the pharmaceutical industry. Meanwhile, the need for new anti-infective agents continues to be as urgent as ever. New business models are called for, ones that are grounded in the possibilities and realities of 21st century technologies for antibiotic discovery and development. This chapter discusses, using four selected examples, the opportunities for harnessing modern biosynthetic insights and engineering methods to discover new antibiotics.

9. Bacterial Phenotypes Refractory to Antibiotic-Mediated Killing: Mechanisms and Mitigation

Alex J. O'Neill

Antibiotic resistance is conferred by heritable genetic determinants that enable a bacterium to grow and cause disease in the presence of therapeutically-achievable concentrations of the corresponding antibiotic. However, bacteria may also become refractory to the killing action of antibacterial agents in ways that do not fit this definition, and which are collectively referred to here as 'antibiotic survival'. These phenomena, which include drug indifference, tolerance, persistence, and the recalcitrance of biofilms to antibacterial agents, are believed to play a central role in antibacterial treatment failure. In addition, they can extend the duration of treatment required to resolve bacterial infections, and facilitate the emergence of acquired antibiotic resistance. This chapter will provide an overview of the different types of antibiotic survival, and will discuss chemotherapeutic approaches to minimising or overcoming the problems that they present to effective antibacterial treatment.

10 . Quorum Sensing Inhibitors Disable Bacterial Biofilms

Thomas Bjarnsholt, Tim Tolker-Nielsen and Michael Givskov

It is now evident that bacteria assume the biofilm mode of growth during chronic infections. The important hallmarks of biofilm infections are development of local inflammations, extreme tolerance to the action of conventional antimicrobial agents and an almost infinite capacity to evade the host defense systems in particular innate immunity. In the biofilm mode, bacteria use cell to cell communication termed quorum-sensing (QS) to coordinate expression of virulence, tolerance towards a number of antimicrobial agents and shielding against the host defense system. Chemical biology approaches may allow for the development of new treatment strategies focusing on interference with cell to cell communication with the aim of primarily disabling expression of virulence, immune shielding and antibiotic tolerance. Here we present our experience with screening and testing small molecule chemistry for N-acyl homoserine lactone dependent QS inhibition. In addition we present our thoughts with respect to advantages and potential limitations of the intervention strategies described.

11. The Indigenous Human Microbiota

Adam M. Nelson and Vincent B. Young

Recent technological advances have expanded the tools available for study of the indigenous human microbiota. One of the early limitations in this field was the difficulty in recovering most residents of the community via standard culture-based methods. Many residents of the flora are anaerobic or microoxic, require specific nutrients, or are dependant on microbe-microbe/microbe-host interactions that are difficult to replicate in vitro, thus making their cultivation difficult. Naturally, the easiest species to grow in the laboratory have been the best studied. However, these cultivatable species are only a fraction of the total population of the microbiota. This chapter will introduce both the culture and non-culture based techniques being used to look deeper into the population structure both on a temporal and spatial scale. It will also discuss how disruptions (including those mediated by the administration of antibiotics) of the microbiota can produce changes in human health, and outline ongoing efforts by the National Institutes of Health and international investigators to study the indigenous microbiota.

12. Prospects for the Development of New Anti-TB Drugs Based on Novel Targets Related to the Host-Parasite Relationship in Tuberculosis

Haruaki Tomioka

Worldwide, tuberculosis (TB) remains the most frequent and important infectious disease to cause morbidity and death. However, the development of new drugs for the treatment and prophylaxis of TB has been slow. Therefore, novel types of antituberculous drugs, which act on the unique drug targets in MTB pathogens, particularly the drug targts related to the establishment of mycobacterial dormancy in host's macrophages, are urgently needed. In this context, it should be noted that current anti-TB drugs mostly target the metabolic reactions and proteins which are essential for the growth of MTB in extracellular milieus. It may also be promising to develop another type of drug that exerts an inhibitory action against bacterial virulence factors which cross talk and interfer with signaling pathways of MTB-infected host immunocompetent cells such as lymphocytes, macrophages and NK cells, thereby changing the intracelluar milieus favorable to intramacrophage survival and growth of infected bacilli. In this chapter, I will describe recent approaches to identify and establish novel potential drug targets in MTB, especially those related to mycobacterial dormancy and cross-talk with cellular signaling pathways.

13. Current Strategies for Antibacterial Vaccine Development

Robert G.K. Donald and Annaliesa S. Anderson

Prophylactic anti-bacterial vaccines have been responsible for a drastic reduction in global bacterial diseases. Older vaccines made from attenuated whole cells or lysates have been largely replaced by less reactogenic acellular vaccines made with purified components, including capsular polysaccharides and their conjugates to protein carriers, inactivated toxins (toxoids) and proteins. Examples of vaccines in each category are reviewed to illustrate underlying strategies and associated technological advances such as polysaccharide conjugation and recombinant protein expression. In addition, progress and the current status in the development of new vaccines to prevent diseases caused by N. meningitidis serogroup B, S. aureus and C. difficle is summarized. Future progress will likely bring to the clinic passive immunotherapies based on monoclonal antibodies and new adjuvants, especially for use in vaccines against intracellular pathogens.

14. Recent Advances in Vaccine Adjuvants

Risini D. Weeratna and Michael J. McCluskie

Infectious disease remains one of the main causes of mortality and morbidity worldwide. Vaccination has had the greatest impact of any medical intervention technique in controlling infectious diseases. Most notably, eradication of smallpox was achieved through concerted and rigorous mass vaccination programs, and the incidence of diphtheria, pertussis, polio and other childhood diseases have been significantly reduced through routine infant immunization. However, with a move away from whole-killed vaccines for safety reasons, a key challenge in realizing the full potential of vaccination has been the lack of immunogenicity of many novel vaccines especially in certain populations such as the elderly and the immunocompromised. Adjuvants are a key component in enhancing immunogenicity of vaccines. Furthermore, adjuvants can play a vital role in facilitating the induction of the appropriate type of immunity that is required to either prevent, such as in prophylactic vaccines, or to treat, such as in therapeutic vaccines. Therefore, careful consideration of the choice of adjuvants becomes quintessential for developing an effective vaccine. This chapter focuses on the importance of choosing the correct adjuvant or adjuvant combination to induce the appropriate immune responses to control the target pathogen.

15. Host Defense Peptides

Olga M. Pena, John D. F. Hale and Robert E.W. Hancock

The increasing problem of resistance to antimicrobial agents, combined with the limited development of novel agents to treat infectious diseases is a serious threat to human morbidity and mortality around the world. Among the available strategies available to create new therapeutic agents is the enhancement of the multifunctional properties of the natural anti-infectives, cationic host defense (antimicrobial) peptides (HDPs). This chapter will provide a summary of our current understanding of the different types of HDPs including natural and synthetic peptides and their antimicrobial and immunomodulatory modes of action. Additionally, we will describe new approaches to peptide design and discuss both the therapeutic potential and prospective challenges in the utilization of peptides for antibacterial

16. . Antibodies for Antibacterials

Bret R. Sellman and C. Ken Stover

Prior to the use of antibiotics, antibody (or serum) therapy was used with some success to treat bacterial infections. Antibiotics almost completely replaced the use of antibody therapies for bacterial disease with few exceptions. Based upon the information available at the time, this was an obvious progression given the broader spectrum activity of antibiotics. Antibiotics revolutionized medicine and the approach to treating infectious disease. In addition to their broad spectrum, they exhibited few side-effects relative to the potential for serum sickness (following the administration of equine immune serum) and they were inexpensive. But bacterial resistance to antibiotics became evident in the decades to follow, and we are now faced with a shortage of effective antibiotics and a need for alternative approaches to stand-alone antibiotic therapy. One such approach which could supplement antibiotic use, thereby removing some of the selective pressure from antibiotics, is monoclonal antibody therapy or prophylaxis. Recent advances in monoclonal antibody technology and discovery strategies and the ability to make a fully human antibody have led to the marketing of ~30 recombinant antibodies and Fc fusion proteins to treat a variety of human diseases. Although this technology has yet to yield an antibacterial product, many clinical and preclinical programs are underway to explore varied and novel approaches to monoclonal antibody-based anti-infectives.

17. Therapeutic Applications of Phage Biology: History, Practice and Recommendations

Jason Gill and Ryland F. Young III

18. Role of Within-Host Models in Target Identification, Compound Optimization and Dose Selection

Juilee Thakar and Eric T. Harvill

Integrated pharmacokinetic-pharmacodynamic models are commonly used to study the in vivo dynamics of antimicrobial agents and bacterial pathogens. These models are extremely useful for understanding the properties of antimicrobial agents such as absorption, transport, rate of binding, etc. However, they fail to consider within-host aspects of the infectious process that are likely to affect the bacterial-host interactions. For example, immune-mediated mechanisms to contain bacteria or limit their access to nutrients can also affect the access of a drug to its bacterial target. Alternatively, pathogens have various strategies to sequester themselves from host immune mechanisms that can also affect the access of therapeutic agents. The search for new antibacterial agents that will be effective in vivo can be substantially informed by an understanding of the within-host dynamics of bacterial pathogens. Mathematical modeling of immune responses can assist in this process by providing new predictions, by offering mechanistic understanding and by revealing the gaps in our current understanding. Such models are based on experiments that reveal the components of the immune system that play important roles during infections. But knowing the components alone usually provides only a static picture of bacterium-host interactions. Mathematical models aim to use the information obtained from experiments to construct the interactions and dependencies between various components. Thus mathematical models offer a mechanistic understanding of the interplay between various immunological processes and simulations of these models give a dynamic view of the entire process. In this chapter we will first provide an overview of pharmacokinetic and pharmacodynamic models followed by a review of some of the immunological processes involved in bacterial infections which are generally ignored in pharmacodynamic models but are likely to affect access or activity of treatments. We will then discuss the development of mathematical models by different approaches. We will end the chapter by exploring implications of these models in the discovery of new antibacterial agents.

19. Advances and Applications of Diagnostic Microbiology in Changing Antibacterial Discovery

Audrey N. Schuetz and Yi-Wei Tang

Despite the rising numbers of multidrug resistant pathogens, and their continuously emerging resistance patterns, few novel antibacterial agents have been approved or released recently. In order to combat this problem, efforts are being made to extend the utility of existing antibiotics as long as possible, while attempting to develop new drugs. The clinical practice of evidence-based therapy, based on diagnosing early and narrowing antimicrobial coverage, with timely administration of an antibiotic, may help alleviate the problem. Diagnostic procedures optimized for accuracy and turn-around time further improve patient therapy. We review techniques currently in use in diagnostic microbiology, such as direct microscopic examination, rapid biochemical and antigen testing, microorganism culture, serologic diagnosis, and a variety of molecular diagnostic techniques. In addition, we introduce various emerging diagnostic techniques, which show promise in their application towards a more exact antibacterial practice. Such emerging technologies include ultra high-throughput sequencing, microarray science, quantum dots, PCR electrospray ionization mass spectrometry, atomic force microscopy, and carbon nanotubes. Point-of-care testing devices are also reviewed. As diagnostic methods have changed over the years, the novel applications of these technologies hold promise in their rapidity and accuracy, while showing potential application in drug target testing and drug discovery.

20. Changing the Therapeutic Paradigm in Antibacterial Drug Therapy and Discovery

Arturo Casadevall

At the beginning of the 21^st century the therapeutic paradigm for the treatment of infectious diseases can be summarized by three words: kill the bug. In other words, the overwhelming majority of therapeutic interventions against microbial diseases are designed to help the host by damaging the microbe directly and/or interfering with its ability to replicate in tissue (Casadevall, 2006). This strategy has been termed the second age of antimicrobial therapy and was preceded by the era of serum therapy, which differed in the fundamental manner that serum was primarily an immunotherapeutic agent than enhanced host defenses (Casadevall, 2006). First and second age therapeutics differed in other ways including the chemistry of the therapeutic agent, their specificity and the form of manufacturing (Table 1). Second age therapeutics have been were tremendously successful and brought numerous drugs to the market that have saved countless lives. However, there are major trends at work that have significantly reduced the overall efficacy of second age therapeutics including widespread antimicrobial resistance, the emergence of new pathogenic microbes for which there are few drugs available and an epidemic of immunocompromised hosts where antimicrobial therapy is often less effective. Microbe-targeting strategies are limited in that they neglect the host; consequently, there are very few treatment strategies that aim to achieve a therapeutic outcome by enhancing host defenses. Microbe-targeting strategies include both microbe-specific and -non-specific drugs, each of which can put tremendous selection pressure on microbes that often result in the emergence of resistance. Non-specific microbe-targeting strategies have the additional problem that they can select for resistance in non-targeted microbes and their effects on host flora can have a variety of unintended deleterious consequences on host homeostasis. This chapter will consider these strategies in light of their historical development and analyze the advantages and disadvantages of specific and non-specific antimicrobial strategies.