Natural Products Analysis: Instrumentation, Methods, and Applications

This book highlights analytical chemistry instrumentation and practices applied to the analysis of natural products and their complex mixtures, describing techniques for isolating and characterizing natural products.

• Applies analytical techniques to natural products research – an area of critical importance to drug discovery
• Offers a one-stop shop for most analytical methods: x-ray diffraction, NMR analysis, mass spectrometry, and chemical genetics
• Includes coverage of natural products basics and highlights antibacterial research, particularly important as efforts to combat drug resistance gain prominence
• Covers instrumental techniques with enough detail for both current practitioners and beginning researchers

• Language: English
• Publisher: Wiley
• Release date: Sep 17, 2014
• ISBN: 9781118876022

Book preview

Chapter 1

Natural Products Analysis: Instrumentation, Methods, and Applications

Vladimír Havlíček and Jaroslav Spížek

Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic

This book aims at highlighting the newest trends in analytical chemistry that have recently been, or soon will be, employed in the analysis of natural products and their complex mixtures. All contributing authors were motivated to stress the innovative aspects in emerging natural product chemistries and were asked to formulate their own personal visions clearly indicating which milestones can be achieved in their fields of expertise in a five-year frame. The book is structured according to analytical instrumental approaches used either routinely or experimentally for structure characterization and/or determination of both low- and high-molecular-weight natural products.

1.1 BOOK MOTIVATION

This book enumerates the most recent and cutting-edge analytical approaches including those that have not yet been commercialized into the rejuvenated natural products field. For example, less-traditional applications of synchrotron irradiation to small molecules are reported when referring to standard X-ray diffraction. Likewise, examples of the newest hyphenation techniques with impact on screening and secondary metabolism studies are described in cases in which well-known multidimensional NMR spectroscopy is discussed.

The revitalization of the natural product field is documented by an increase in the number of peer-reviewed articles illustrated by a Web of Science search (Figure 1.1). The number of hits is seen to have increased threefold if the term natural product activity is evaluated. Antibacterial, antifungal, antineoplastic, anti-inflammatory, and other activities are also reported in patent literature. SciFinder returned constant data for the 2007–2013 period oscillating between 60 and 80 patent applications published annually. Diverse applications of natural products are also subjects of many review articles and book chapters. Interestingly, no monograph focused on instrumentation used for identification of natural products has been published in the past decade. This market gap was identified by Wiley senior editor Jonathan T. Rose: In my opinion, given that plants and natural products are major sources for current and potential drugs, there is need for a book geared to researchers and professionals to facilitate natural product analysis, synthesis, and drug discovery. This type of book could explain the basics of natural products as pharmaceuticals, analytical tools and techniques, methods for isolation and elucidation, and applications for library design and in drug discovery. Such a book will find a welcome audience in organic and medicinal chemists, biochemists, analytical and medicinal chemists, microbiologists, and biomedical researchers.

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Figure 1.1 Report of published items accessed from the Web of Knowledge (Thomson Reuters) on December 31, 2013 illustrates the number of papers published annually in the field of Natural Product Structure.

In this book the instrumentation represents the common denominator. The contributors were motivated to make a very brief introduction to physicochemical principles of their methods and give an up-to-date overview of the most important applications relevant to natural products. In a limited number of chapters the tutorial part was extended, giving the reader the opportunity to get acquainted with both the fundamentals and future trends in one place. Personal views and mutual instrumental evaluations will help the newcomers to find a suitable technique. For instance, whereas nuclear magnetic resonance spectroscopy is nonselective and less sensitive (always tells the truth), mass spectrometry is selectively sensitive (tells you what you want to hear).

1.2 THE BROAD FIELD OF NATURAL PRODUCTS

Chapters 2–4 represent medically oriented introductory chapters. Chapter 2 focuses specifically on fungi and malaria and defines the current microbiology challenges in the field of natural product discovery. These two application areas were deliberately selected because they are rather underestimated in the review literature. The importance of tackling antimicrobial resistance and the application of standardized combination therapies is stressed. Drug degradation products arising from enzyme-specific reactions, drug target reprogramming, or ejecting the drug out of the bacterial or fungal cells belong to known mechanisms by which microbes fight against antimicrobial drugs. In the field of drug resistance, cultivation of microorganisms in drug-containing stable isotope-labeled media are particularly promising. Mass spectrometry (MS) is then used for the determination of natural isotope shift reflecting the viability of the microorganism and its ability to consume and metabolize the labeled nutrients. The potential and limitations of NextGen or NextNext sequencing methods are briefly described in the perspective section in Chapter 3. The importance of peptidogenomic methods for the determination of virulence mechanisms of pathogens is accentuated by means of imaging mass spectrometry in Chapter 10.

The introductory segment of this book is terminated by Chapter 4, in which the major fractionation and isolation procedures of natural products are briefly outlined. Major attention is dedicated to the respective biological activities of natural products. The chapter is subdivided according to plant and marine origin of most important metabolites that have found significant medical applications. The authors faced a difficult task to select the clinically most important active principles, of both marine and plant origin, and align their pharmacokinetic and biological properties with medical applications. Attention was paid to organic compounds in different phases of biological trials. Most important applications of natural compounds in cardiovascular, infectious, cancer and other areas are summarized.

1.3 DISCOVERY PHASES

Recent applications of metabolomics, proteomics, mutagenomics, and genomics in exploiting bacterial natural products are summarized in Chapter 5. In mass spectrometry-based metabolomics, the problem of silent or cryptic NP biosynthesis pathways (the silent parvome) is discussed in the context of the quest for novel chemistries. Mass spectral alignment strategies are outlined (XCMS, MZMine, commercial products) and supported by principal component analysis program packs (SIMCA, MATLAB, etc.), the importance of which is documented (for example) on strain prioritization. Two proteomic approaches in natural product discoveries are reviewed. The first is the Kelleher group proteomic investigation of secondary metabolism (PrISM) utilizing the phosphopantetheinyl ejection assay [1]. The second proteomic technique is represented by an Orthogonal Active Site Identification System (OASIS) [2]. In the (meta)genomic part, the amplicon sequencing, shotgun libraries/metagenomics, and single-cell genomics methods are outlined and supported by success stories. Genome annotation pipelines are provided (CloVR-microbe, AntiSMASH, NCBI, SMART). The importance of concerted application of density functional theory and 2D NMR spectroscopy for absolute structure determination in natural products is stressed in the final part of the chapter. The applications of residual dipolar couplings in nuclear magnetic resonance (NMR), circular dichroism, and classical chemistry are also emphasized and create the bridge to molecular tools.

Some of them are further structured in tutorial Chapter 6 referring to the applications of electronic and vibrational spectroscopies. Advances and challenges in optical molecular spectroscopy of biomolecules and natural products are supported by chiroptic methods and placed in the context with surface-enhanced techniques and surface plasmon resonance (SPR) sensing. NMR users and fans will appreciate Chapter 7, a substantial part of which is dedicated to sample preparation and handling. Attention is also paid to LC-NMR setup, with most common instrumental variants and practical recipes (on-flow, stop-flow, and the combination of solid-phase extraction and MS). Their properties in terms of sensitivity, sample concentration, and sample nature are discussed in detail. Similarly, both supervised and unsupervised methods of statistical data evaluation are reported. Differential analysis is addressed in specialized subchapters dedicated to statistical heterospectroscopy, statistical total correlation spectroscopy, and other methods. The reader can benefit from public databases of NMR spectra and web servers dedicated to NMR metabolomics. Covariance NMR data processing of TOCSY and NOESY spectra is described. Virtual NMR chromatography (including its 3D variant) is used for distinguishing signals coming from small or large molecules. Food adulteration, plant extract analysis, and tens of other NMR applications in metabolomics are presented.

1.4 ABSOLUTE STRUCTURE

Chapter 8 describes the general technique of X-ray diffraction including the single-crystal and powder methods, and it covers advances in the instrumentation currently in use. The central argument that X-ray diffraction has a great potential and plays an increasingly important role in the structure determination of natural products is well documented and supported by the possibility to provide absolute structure determination, packing of molecules in the crystal, and structure determination in the presence of solvents in the crystal unit. Public academic software programs Sir2011, SuperFlip, CRYSTALS, and checkCIF are referred to and Cambridge Structural Database is stressed. The chapter discusses in a reader-friendly manner the common myths of X-ray diffraction (excessive time and high amount of samples needed for analyses, samples do not crystallize). It also provides a set of examples showing cases of natural product whose stereochemistry or absolute configuration originally suggested by other tools was completely revised or reassigned by X-ray. Practical comparison of what can be achieved with both laboratory or synchrotron sources and what can also be achieved with given crystal size and quality is reported as well. The chapter is concluded with a belief that the number of research groups producing a mere amorphous white solid will steadily decrease. Neutron diffraction and electron diffraction are briefly outlined. A short notice on the analysis of NRPS/PKS domains by crystallography is also presented in Chapter 12.

1.5 MASS SPECTRAL APPLICATIONS IN CONCERT

Although mass spectrometry is a mature technique celebrating its 100-year anniversary, some of its newer applications have revolutionized the emerging fields of peptidogenomics and metabolomics and also significantly contributed to revitalization of the natural product field. Chapters 9 to 15 are thus dedicated to both the instrumentation (inductively coupled plasma, imaging, ion mobility, affinity, ultrahigh resolution) and applications of mass spectrometry (ribosomal and nonribosomal natural products). In Chapter 9, solid or semisolid samples are probed by inductively coupled plasma (ICP) mass spectrometry with special attention to heteroelements—that are metals, metalloids, and nonmetals. For beginners in the field, the instrumental setup is briefly outlined with numerous applications to inorganic and organic matter analysis, including proteins separated by native polyacrylamide gel electrophoresis. Particular attention is paid to laser ablation, also when combined with 2D or 3D bioimaging approaches. The importance of elemental fractionation phenomena in quantitative determination is described and key variables defined (e.g., sample planarity, aerosol transport, vaporization, or ionization efficiency). Suppression of spectral interferences by collisional or dynamic reactive interactions in the gas phase is placed in context with the resolving power of a mass analyzer. The analytical limitations of ICP-MS are defined in a fair manner. The part on laser ablation ICP-MS comparison to other techniques of surface analysis will also be of interest to the reader. EMPA, XRD, XFA, XRA, XRF, PIXE, NAA, and some other instrumental tools are mutually compared and appropriate applications defined (including imaging). The chapter concludes with a critical personal view of quantitation in selected peer-reviewed papers reporting misleading results.

Imaging mass spectrometry is addressed in Chapter 10. In the introductory part, ionization techniques (SIMS, MALDI, and DESI) used for mass spectrometry imaging are reviewed in a tutorial manner while their practical limits and prospects for future technical development are described in the final, visionary part of the chapter. nanoDESI is defined as a central technique for bacterial imaging mass spectrometry. Ionization enrichment by derivatization and labeling strategies are outlined with a special attention to analysis of carbohydrates, oligonucleotides, and other less common molecules. Experimental considerations are defined with respect to applications in microbiology. Particular emphasis is devoted to ecology and elemental analysis with submicron spatial resolution. Biosynthesis, secretion, exchange, symbiotic interaction, or competitions are described. Many topics make this chapter interesting not only for analytical chemists and natural product fans but also for (micro)biologists and biochemists in general; for instance, the role of siderophores in iron piracy is outlined. The importance of peptidogenomic approaches is documented, for example, by characterization of cannibalistic phenomena in bacteria and other bacterial or interkingdom interactions.

Chapter 11 is devoted to the specific exploration of primary and secondary metabolites by ion mobility mass spectrometry (IM-MS). In addition to a historical overview, fundamentals and instrumentation approaches introduce the reader into the field of natural product prioritization and dereplication without chromatographic separation. IM-MS offers a 10–4 lower peak capacity but is much faster (10 ms) compared to LC-FTMS separations. Different classes of biomolecules are separated in the order of increasing gas-phase packing efficiencies or densities: lipids < peptides/proteins < carbohydrates < oligonucleotides. IM structural separation can readily resolve isobaric species resulting from conformational isomers. Various arrangements for performing tandem mass spectrometry on IM instruments as well as the computational approaches for collision cross sections are provided. Contemporary efforts are underway to construct an atlas of conformation space to direct the rapid molecule identification. Future trends are targeted to peak resolution improvement and development of ion mobility imaging area.

Dereplication—that is, elimination of already known compounds from further investigation—is also depicted in Chapter 12. High-resolution tandem mass spectrometry combined with collisionally activated dissociation and/or electron-induced dissociation was used for the purpose. In its introductory part, the chapter gives basic information on the biosynthesis of nonribosomal peptides and polyketides providing the reader with the initial orientation in the enzyme systems and corresponding natural products subsequently targeted by other analytical tools (mainly NMR and X-ray) and strategies (Ppant ejection, OASIS, PrISM) [3]. The second part of the chapter shows representative success stories, where tandem mass spectrometry was used as the main tool.

The direct and indirect affinity mass spectrometry assays for drug discovery are addressed in Chapter 10. Techniques such as fragment-based lead assembly, nanoelectrospray ionization, multitarget affinity specificity screening, detection of oligonucleotide-ligand complexes by electrospray ionization (ESI), and ESI-electron capture dissociation are included in the section of direct affinity mass spectrometry assays (MASS and DOLCE involving nucleic acid binding). Frontal affinity chromatography, affinity capillary electrophoresis, ultrafiltration, gel permeation chromatography, size exclusion chromatography, and automated ligand identification system are also reported.

1.6 COMPLEX STRUCTURES AND COMPLEX MIXTURES

The last part of the book contains three application chapters that show the complexity of natural product structures and indicate that state-of-the-art equipment is a prerequisite, but not a guarantee, for successful structure elucidation or even characterization. Chapter 13 is dedicated to ribosomally synthesized peptide toxins. Because these peptide natural products have been overviewed elsewhere [4], G. Norris and M. Patchett focused on characterizing glycosylated ribosomal products and began with Richard Phillips Feynman's principle of Science: The first principle is that you must not fool yourself, and you are the easiest person to fool (Caltech's commencement address, 1974). This principle was then applied to the exciting story of glycocin reflecting its long-lasting development and final contributions of 2D NMR, circular dichroism spectroscopy, Edman sequencing, and Fourier transform ion cyclotron resonance mass spectrometry. The chapter also refers to useful specific or broader antibacterial peptide and prokaryotic glycoprotein repositories, including manually curated ones. Non-ribosomally synthesized and glycosylated antimicrobial peptides, as well as potential biological functions and roles of glycosylation in the discussed organisms, are also reviewed. The authors conclude with applications of venom glycopeptides, bacteriocins, and glycocins in biotechnology and the food industry.

Chapter 14 is dedicated to the description of the organic chemical diversity within complex biological and geochemical systems studied by ultrahigh-resolution mass spectrometry. Basic principles of ion cyclotron resonance are reported and various metabolome databases useful for searches based on elemental composition are described. The chapter also reports on basic statistics and mathematical tools for data visualization. The utility of Van Krevelen diagrams or Kendrick mass defect plots for selective displaying of compounds of interest, including endogenous small molecules or drugs and their metabolites, is demonstrated by the success story of Chlamydia-infected human cells.

The whole book is concluded with an application chapter (Chapter 15), in which the analytical armory is represented by electron and atomic force field microscopies. Basic principles and experimental setups in both techniques are briefly discussed in the introductory part. Amyloid fibrils represent the central subject of structural studies supported by recent literature including patents. Although these protein aggregates have been associated with more than 30 serious illnesses including Alzheimer's, Parkinson's, Huntington's, prion diseases, or atherosclerosis, the chapter also highlights the important nontoxic biological functions of amyloids. Potential and proven properties of most important polypeptides (e.g., chaplins, rodlins, or hydrophobins), are reported in the context of recent structural studies shedding light on processes like biofilm formation, microbial adhesion, initiation of aerial growth, and so on. Important applications include coating of catheters, improving biocompatibility of implants, detergent-resistant glass coatings, stainless steel lubrication to reduce friction, drug delivery systems, and many others. The fibrils may also be important in infection: They interfere with blood clotting and activate the immune system. Conversely, fibril disruption and detachment by d-amino acids can define new emerging applications in this fascinating field.

References

1. Dorrestein, P. C., Bumpus, S. B., Calderone, C. T., Garneau-Tsodikova, S., Aron, Z. D., Straight, P. D., Kolter, R., Walsh, C. T., Kelleher, N. L. (2006). Facile detection of acyl and peptidyl intermediates on thiotemplate carrier domains via phosphopantetheinyl elimination reactions during tandem mass spectrometry. Biochemistry45, 12756–12766.

2. Meier, J. L., Niessen, S., Hoover, H. S., Foley, T. L., Cravatt, B. F., Burkart, M. D. (2009). An orthogonal active site identification system (OASIS) for proteomic profiling of natural product. ACS Chemical Biology4, 948–957.

3. Chen, Y. Q., Ntai, I., Ju, K. S., Unger, M., Zamdborg, L., Robinson, S. J., Doroghazi, J. R., Labeda, D. P., Metcalf, W. W., Kelleher, N. L. (2012). A proteomic survey of nonribosomal peptide and polyketide biosynthesis in actinobacteria. J. Proteome Res.11, 85–94.

4. Arnison, P. G., Bibb, M. J., Bierbaum, G., Bowers, A. A., Bugni, T. S., Bulaj, G., Camarero, J. A., Campopiano, D. J., Challis, G. L., Clardy, J., Cotter, P. D., Craik, D. J., Dawson, M., Dittmann, E., Donadio, S., Dorrestein, P. C., Entian, K.-D., Fischbach, M. A., Garavelli, J. S., Goeransson, U., Gruber, C. W., Haft, D. H., Hemscheidt, T. K., Hertweck, C., Hill, C., Horswill, A. R., Jaspars, M., Kelly, W. L., Klinman, J. P., Kuipers, O. P., Link, A. J., Liu, W., Marahiel, M. A., Mitchell, D. A., Moll, G. N., Moore, B. S., Mueller, R., Nair, S. K., Nes, I. F., Norris, G. E., Olivera, B. M., Onaka, H., Patchett, M. L., Piel, J., Reaney, M. J. T., Rebuffat, S., Ross, R. P., Sahl, H.-G., Schmidt, E. W., Selsted, M. E., Severinov, K., Shen, B., Sivonen, K., Smith, L., Stein, T., Suessmuth, R. D., Tagg, J. R., Tang, G.-L., Truman, A. W., Vederas, J. C., Walsh, C. T., Walton, J. D., Wenzel, S. C., Willey, J. M., van der Donk, W. A. (2013). Ribosomally synthesized and post-translationally modified peptide natural products: Overview and recommendations for a universal nomenclature. Natural Product Reports30, 108–160.

Chapter 2

The Need for New Antifungal and Antimalarial Compounds

Jaroslav Spížek

Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic

Arnold L. Demain

Charles A. Dana Research Institute for Scientists Emeriti (RISE), Drew University, Madison, NJ, USA

2.1 INTRODUCTION

Infectious diseases were the leading cause of death in 1900s, and today they are the second most important killer in the world, number three in developed nations, and fourth in the United States [1]. Although not all statistics may be absolutely correct and some may even be misleading, it has been clearly demonstrated that infectious diseases are the leading causes of mortality and morbidity [2]. It is particularly drastic in elderly and debilitated populations. Americans are infected with bacteria at a rate of 2.5 million people per year, resulting in 100,000 deaths. In our previous reviews [3, 4], we concentrated mainly on bacterial infections. In the present review, we focus on fungal diseases and malaria.

Antibiotic resistance has rapidly developed, and this fact was originally interpreted as a relatively modern phenomenon. This assumption has long been supported by the fact that collections of microorganisms from the pre-antibiotic period are highly sensitive to antibiotics. Relatively recent molecular-biological studies, however, indicated that the occurrence of resistance genes in the environment is much more frequent than originally thought [5]. In a recent paper, D'Costa et al. [6] showed that antibiotic resistance precedes the use of antibiotics and propose that it is an ancient phenomenon commonly detected in the environment. This is in agreement with the facts that antibiotic resistance is so often observed and occurs at a high frequency. This indicates that new antibiotics select preexisting resistance determinants that had circulated inside the microbial pangenome for millennia. This should be kept in mind in management of the presently used and new antibiotics. In addition, the pipeline for new antibiotics is running dry and new antibiotics are desperately needed. Many excellent reviews on the uses of antibiotics and antibiotic resistance have recently been published [7, 8]. We previously concentrated on bacterial infections [3, 4]. In this review, resistant microorganisms are classified as those that are not inhibited by antibiotics or antimicrobials or antimicrobial compounds (these words will be used interchangeably). Resistance has steadily increased since systemic antibiotics were introduced in the 1930s and 1940s. In the present review, we focus on fungal infections and malaria since these diseases appear to be among the most serious threats to the human population. We will primarily, but not always, discuss compounds described as antibiotics, according to the definition of Selman Waksman, the Nobel Prize laureate in 1949. Waksman suggested the use of the word antibiotics, classifying these compounds as chemical substances that are produced by microorganisms and that have the capacity, in dilute solution, to selectively inhibit the growth and even to destroy other microorganisms. However, this definition has been modified many times. In this review, we discuss natural compounds produced by microorganisms and plants, as well as semisynthetic and synthetic compounds, that have antimicrobial activity.

2.2 FUNGAL INFECTIONS

2.2.1 Fungal Diseases

Fungal infections have continued to increase due to population aging, increases in immunocompromized individuals, and use of central venous catheters and broad-spectrum antibacterials. In an excellent review, Calderone et al. [9] classified fungal diseases as cutaneous and subcutaneous infections, mucosal invasions, and bloodstream infections. They stated that of all fungal diseases, dermatophytosis is probably the most prevalent, but also the least studied in regard to host–fungus interactions. Also, according to the authors, fungal diseases are endemic (histoplasmosis, blastomycosis, coccidiomycosis, penicilliosis, paracoccidiomycosis) or pandemic (invasive candidiasis, aspergillosis including invasive aspergillosis, cryptococcosis, fusariosis, mucormycoses, dermatophytosis). It is probable that the greatest threats to life today include those pathogens that cause common bloodstream infections.

Treatment of patients with fungal diseases has become a serious burden. Candidaemia is estimated to involve about 300,000 cases per year worldwide, and mortality can be as high as 30–55%. In the United States, Candida albicans kills between 3000 and 11,000 people per year due to nosocomial candidemia. Invasive aspergillosis can occur in different patient groups. It is estimated that around 10% of new leukemia cases develop invasive aspergillosis—that is, 30,000 per year—of stem cell transplants. Fifty-four thousand patients in the United States, the United Kingdom, and Europe will need treatment for Aspergillus infections. In chronic destructive pulmonary disease, roughly 1.2% will need antifungals for aspergillosis—that is, roughly 216,000 per year. Over 50% of invasive aspergillosis patients will die from their infection, even if treated. In AIDS patients, 1 million contract cryptococcal meningitis resulting in 600,000 deaths, 70% of which are in sub-Saharan Africa. Less fatal infections affecting large numbers of people worldwide include cutaneous fungal infections—namely, nail infections and athlete's foot—affecting some 1.5 billion people or 25% of the world population. Hair infection, which is common in young children, is predicted to affect some 200 million people worldwide. In addition, according to Vandenputte et al. [10], resistance to antifungals rapidly increases. The authors propose how to cope with this problem. According to them, it is estimated that 1728 billion people contract a fungal disease, with 1 million deaths occurring.

2.2.2 Antifungal Therapy

In the paper by Waksman et al. [11], the authors stated that:

The great majority of antibiotics that have been tested in the numerous screening programmes concerned with the search for new chemotherapeutic agents have been tested primarily for their activity against different bacteria. Only limited consideration has been given to those antibiotics which possess mainly antifungal properties. With the growing importance of various antibiotics in clinical medicine, however, especially in the treatment of diseases caused by bacteria and some larger viruses, the need for substances with antifungal properties, especially substances that are not too toxic and which offer promise in human and animal therapy, has become of great importance.

We will use the above statement as a motto and will now concentrate our attention on antifungal compounds.

The 2011 antifungal market amounted to $6.1 billion. Antifungal compounds have different mechanisms of action in fungal cells, yeast cells in particular [12]. Flucytosine affects pyrimidine metabolism, as well as RNA and DNA biosynthesis. Azoles inhibit ergosterol biosynthesis pathway at different steps. Echinocandins inhibit cell wall β-glucan synthesis in fungal cells, and polyene antifungals target fungal membrane sterols.

However, there are similarities in the mode of action of some antifungal compounds, due mainly to the fact that many fungi have their own form of cholesterol, that is, ergosterol. The enzymes involved in biosynthesis of ergosterol differ from those of biosynthesis of cholesterol to such an extent that antifungal compounds that target the fungal enzymes practically do not affect those involved in human cell cholesterol biosynthesis. Such compounds thus have a relatively broad spectrum.

Treating and caring for patients with fungal diseases is becoming a serious burden. It is commonly accepted that fungal infections are caused by two types of fungi: (a) primary pathogens and (b) opportunistic pathogens. Whereas primary pathogens can cause infections in healthy populations, opportunistic pathogens could be, in general, commensal microorganisms in healthy humans that do not cause any problems and become pathogenic only under specific conditions, namely in immunocompromised individuals. Most primary pathogens are filamentous fungi, whereas most of the opportunistic pathogens are yeasts.

There are not too many classes of antifungal drugs. In general, we can recognize four main types of compounds that are used in the clinical practice: fluoropyrimidine analogs, polyenes, azoles, and echinocandins [10]. As with any other drugs, antifungal drugs should be nontoxic and preferably highly water-soluble, their effect should be selective, and resistance should not develop easily. It is usually the case that not all of the above requirements are met simultaneously. It should also be noted that a number of antifungal drugs are also used, or have even been primarily used, for the treatment of other diseases—for example, different types of cancer. Examples are the fluoropyrimidine drugs that are primarily used for the treatment of colorectal cancer.

2.2.3 Fluoropyrimidines

Among fluoropyrimidines, flucytosine is probably the most important antifungal compound. Its mode of action is based on interference with pyrimidine metabolism and the synthesis of RNA/DNA and proteins. Flucytosine is indicated for the treatment of infections caused by strains of Candida and Cryptococcus neoformans and some filamentous fungi [13]. Some in vitro studies showed emerging resistance to flucytosine. Papon et al. [14] described the molecular mechanisms of flucytosine resistance. Due to increased flucytosine resistance, it has been suggested that flucytosine should always be used in combination with other antifungal agents as the standard of care for the treatment of fungal infections.

2.2.4 Azoles

Azoles are cyclic organic molecules that can be divided into two groups according to the number of nitrogen atoms in the azole ring. Imidazoles contain two nitrogen atoms and triazoles contain three nitrogen atoms. Azole drugs intervene with ergosterol biosynthesis, and their effect on fungal infections is thus selective. Triazoles continue to be a critical component of therapy for most forms of invasive mycoses, as well as the key to the successful management of patients at all levels of invasive mycoses [15]. Of the azole compounds, fluconazole, itraconazole, variconazole, and posaconazole are the most important [16]. As with other antimicrobial compounds, their widespread use has resulted in increasing resistance of fungal pathogens and, even worse, in cross-resistance to commonly used triazoles and to a new generation of triazoles [10]. In addition, the relative toxicity of azoles complicates their use.

Fungi are an important component of nosocomial bloodstream infections that kill 40% of the infected patients. Since the azole compound, ketoconazole (KTC) inhibits lanosterol 14-methylase, blocking sterol synthesis in fungi and mammals, it has been used against infections by Candida and molds. However, it is toxic, causing hepatitis. Studies were carried out by Zhang et al. [17] to find compounds that synergize the effect of a low dose of KTC (i.e., 0.01μg/mL), which produces about 20% of maximal antifungal activity. Screening of 20,000 microbial extracts resulted in 12 compounds with broad spectra of activity. Of these, seven showed only minor cytotoxicity against human hepatoma cells. The most efficient was beauvericin (BEA). In mice infected with Candida parapsilosis, the combination of BEA at 0.5 mg/kg and KTC at 0.5 mg/kg prolonged survival and reduced fungal counts in their kidneys, lungs, and brains. Such effects were not observed even at a very high dose of KTC alone (50 mg/kg). Candida parapsilosis, used in this study, is the second most commonly isolated fungus in clinical laboratories, following C. albicans. The antifungal effect of the combination was much better than for one of these alone in vitro or in an immunocompromised mouse model. BEA in the combination broadened the KTC spectrum on drug-resistant strains and reduced its side effects. Although BEA has little antifungal activity, the combination was fungicidal, compared to the fungistatic activity of KTC. The combination showed no negative effect on human liver HerpG2 cells.

2.2.5 Echinocandins

Echinocandins are antifungals introduced into practice within the last 15 years. They were the first anifungals found to inhibit cell wall biosynthesis. This group of compounds includes natural, semisynthetic, and synthetic antibiotics derived from lipopeptides. They are cyclic lipohexapeptides synthesized on nonribosomal peptide synthase complexes by different ascomycotic fungi. Although weakly soluble and relatively narrow spectrum in activity, echinocandins have been used against Candida and Aspergillus infections due to their unique mode of action. Their mechanism of action is noncompetitive inhibition of the beta-1,3-glucan synthase complex, targeting the cell wall. It is worth noting that human cells do not contain 1,3-β-glucan, thus avoiding direct human cell toxicity. The first echinocandin-type antimycotic, echinocandin B, was discovered in the 1970s. It was discovered independently at the Ciba-Geigy, Sandoz, and Eli Lilly companies from Asperillus nidulans var echinolatus, A. nidulans var roseus, and Aspergillus rugulosus. This was followed by the isolation of more than 20 natural echinocandins, including aculeacins, mulundocandins, pneumocandins, sporiofungins, catechol-sulfate echinocandins and, cryptocandin. Of the echinocandins, the semisynthetic cyclic lipohexapeptides caspofungin, micafungin, and anidulafungin are currently approved for use. Since they show good fungicidal (vs. Candida spp.) or fungistatic (vs. Aspergillus spp.), activity against the most important human pathogenic fungi, including azole-resistant strains, they are important additions to the list of antifungal drugs [18]. Natural echinocandins are produced by several fungal species—for example, Aspergillus spp. [19]. Echinocandin antifungals were compared by Eschenauer et al. [20]. Of the clinically used echinocandins, the authors compared the pharmacokinetic parameters of caspofungin, micafungin, and anidulafungin in adults and in pediatrics. What are still needed are new derivatives for oral administration or with better activity against Pneumocystis carnii, a fungus of great importance. Other desirable targets are C. neoformans and Histoplasma capsulatum.

2.2.6 Other Peptides

With the increase in resistance to commercial antibiotics, antimicrobial peptides are being considered for medical use [21]. They contain 15 to nearly 50 amino acids, are generally positively charged, are synthesized by the ribosome, and are modified post-translationally. More than 2000 are known, some of which are toxic.

A lipopeptide-producing Bacillus amyloliquefaciens strain inhibits clinical isolates of C. albicans by producing a cyclic lipopeptide containing a hexapeptide and a 3-hydroxy fatty acid with 15 carbon atoms [22]. The purified lipopeptide kills drug-resistant C. albicans and also inhibits other yeasts. Candida albicans is the most virulent agent infecting immunocompromised patients with HIV infections. Most of the available drugs against C. albicans have toxicity problems. Previous cyclic lipopeptides included surfactin, iturin, and fengymycin. Of the yeasts inhibited by the new compound, Candida tropicalis is also a pathogen.

Novexatin of Novabiotics (Aberdeen, UK) is a cyclic and highly cationic (arginine-rich) peptide targeting fungal infections of toenails. It is in Phase II clinical trials. Another group under study are the peptoids, which have a natural amino acid backbone with synthetic side-chain residues conferring protease resistance and increased hydrophobicity, thus enhancing membrane permeability.

2.2.7 Polyenes

Polyenes are broad-spectrum antifungal antibiotics produced by soil actinomycetes, especially the bacterial genus Streptomyces. They are active against fungi, parasites, enveloped viruses, and prion diseases. This large family of polyketides includes nystatin, amphotericins, candicidin, pimaricin, and rimocidin. Polyene antibiotics are formed as a macrolide ring with polyunsaturations, closed by an ester or lactone [12]. They act by interacting with ergosterol to form transmembrane channels that cause leakage of cellular K+ and Mg²+, leading to fungal death.

Nystatin was isolated from the fermentation broth of Streptomyces noursei and is still used as a topical antifungal agent. Amphotericin B and its analogs are produced by Streptomyces nodosus. After the discovery of amphotericin B, roughly 90 polyenes were discovered but most of them were not used in clinical practice due to their poor solubility, stability, oral bioavailability, and/or toxicity [16]. The effect of amphotericin B is based on the complex formation between the antifungal molecule and the ergosterol-containing membrane that results in altered permeability and leakage of important cell components, thus killing the cell. Nystatin binds to ergosterol in the fungal membrane, producing membrane permeability changes leading to release of K+, sugars, and metabolites. Disruption of the membrane is apparently rersponsible for death of the fungal cells; however, the modes of action of amphotericin B and nystatin are apparently different [12]. As compared with the above-mentioned polyene antibiotics that form pores in the membrane, the relatively recently described polyene antibiotic natamycin inhibits growth of yeasts and molds via inhibition of amino acid and glucose transport across the plasma membrane. This is attributable to ergosterol-specific and reversible inhibition of membrane transport proteins [23].

Although polyene macrolide antibiotics, including nystatin and amphotericin B, possess fungicidal activity, they show toxicity and poor distribution in tissues. Thus, their clinical applications are relatively limited. With the aim of generating new, less toxic nystatin derivatives, Brautaset et al. [24], using genetic engineering, designed, purified, and tested four new nystatin derivatives with promising therapeutic effects.

Van Minnebruggen et al. [25] concluded that there is still an unmet medical need to further improve antimycotic therapy and that interdisciplinary research bringing together chemical, molecular and clinical expertise can help make the antimycotic drugs of tomorrow to fulfill the promise of today. One of the approaches could be genome mining of different organisms—actinomycetes and fungi in particular.

2.2.8 Miscellaneous Antifungals

An interesting fact is that ants cultivate fungi as a food source. The ants contain a fungal garden which, unfortunately for the ants, can be parasitized by other fungi that can destroy the garden. The fungus-growing Allonerus ant has been recently shown to harbor antifungal-producing actinobacteria of the genera Streptomyces and Amycolatopsis [26]. Perhaps, these actinomycetes will some day be a source of new and useful antifungal agents.

2.3 MALARIA

2.3.1 The Disease

Several Plasmodium parasites cause malaria (the most serious being Plasmodium falciparum) and are transmitted to people through bites by infected night-flying mosquitoes. Infection with malaria parasites may result in a wide variety of symptoms, ranging from absent or very mild symptoms to severe disease and even death. Malaria can be categorized as uncomplicated or severe (i.e., complicated). In general, malaria is a curable disease if diagnosed and treated promptly and correctly. Forty percent of the world's population are exposed to malaria, and there is a constant need for new antimalarials in the face of the ever-present and ever-emerging resistance of parasites to currently available drugs, whether used in monotherapy or in combination. According to Ginsburg and Deharo [27],

Research on natural compounds has already contributed to the discovery of new antimalarial drugs. Atovaquone, artemisinin and its semi-synthetic derivatives, as well as clindamycin, azithromycin, chlortetracycline, tetracycline, oxotetracycline and doxycycline, are noteworthy examples of the varied contribution of natural products for the development of effective antimalarial drugs, particularly valuable for the treatment of chloroquine-resistant parasites.

Combination of doxycycline, clindamycin, fosfidomycin, or azithromycin with a synthetic antimalarial agent has shown some success [28].

Malaria kills twice as many people as previously thought, that is., 1–2 million people each year; 300–500 million cases of malaria are reported annually. Malaria is a particularly devastating disease in sub-Saharan Africa, where about 90% of the cases and deaths occur. In addition, interactions between HIV and malaria remain a major public health concern in areas affected by both diseases. Very few studies have evaluated the role of quinine in the management of malaria in HIV-infected populations. Malaria is also a serious public health problem in certain regions of Southeast Asia and South America. As with many other antimicrobial drugs, the development of resistance to mainstay drugs like chloroquine, along with controlled use of new artemisinin analogs, has created an urgent need to discover new antimalarial agents. In their excellent review, Kaur et al. [29] described a number of natural compounds including alkaloids, terpenes, quassinoids, flavonoids, limenoids, chalcones, peptides, xanthones, quinones, coumarins, and miscellaneous antimalarials from nature. In the present review, we discuss only a limited number of antimalarial drugs, namely, quinine, artemisinine, tetracycline, and clindamycin.

2.3.2 Quinine

Quinine was the first natural product used for malaria. Quinine remains an important antimalarial drug, almost 400 years after Jesuit priests first documented its effectiveness. It was isolated from the bark of Cinchona spp. The 2010 World Health Organization (WHO) guidelines recommended a combination of quinine plus doxycycline, tetracycline, or clindamycin as second-line treatment for uncomplicated malaria (to be used when the first-line drug fails or is not available) and quinine plus clindamycin for treatment of malaria in the first trimester of pregnancy. It has been proposed that intravenous artesunate, rather than quinine, should be used for the treatment of severe falciparum malaria in adults and children [30]. However, as compared with other drugs, quinine is cheap and is often the only available option. Of the quinine compounds, the 4-aminoquinoline derivatives, like chloroquine and amodiaquine, are particularly important. Although drugs other than quinine are available at present, they are more expensive and thus not readily available. According to Achan et al. [30], in the near future, quinine will continue to play a significant role in the management of malaria, particularly in resource-limited settings. At present, quinine is usually only used as an alternative when artesunate is not available. The drug continues to be used in pregnant women and will probably still be used until safer alternatives become available.

2.3.3 Artemisinin

The best therapeutic agent for malaria is artemisinin, which is produced from extracts of the Wormwood tree, Artemisia annua. Long used in Chinese traditional medicine, it exhibits a significant antimalarial activity against both drug-resistant and cerebral malaria-causing strains. Chemically, it is a sesquiterpene lactone containing an unusual peroxide bridge. It is assumed that this peroxide is responsible for the mechanism of action of this drug. Of the artemisinin derivatives, artemether, artesunate, dihydroartemisinin, and arteether are the most frequently used drugs in clinical practice. Dihydroartemisinin is more stable and 10 times more active than artemisinin [31]. Artemisinin and artesunate also exhibit activities against viruses, hepatitis B, shistosomiasis, and cancer [32]. Artemisinin and its derivatives have become essential components of antimalarial treatment. These agents kill young malaria parasites inside the erythrocytes and thus prevent their further development. Artemisinin derivatives are now used in combination with other drugs for the treatment of uncomplicated malaria caused by P. falciparum. However, the access to artemisinin combination treatments is limited in most countries in which malaria is endemic. As with any other antimicrobial drugs, tolerance to artemisinin derivatives may emerge in some countries [33, 34]. The history of artemisinin discovery and its application have been reviewed by Miller and Su [35]. As with any other antimicrobial drug, artemisinin resistance develops rapidly. Artemisin drugs are quite often used in combination with other antimalarials, viz. lumefantrine, piperaquine, and pyronaridine. Recently, partial artemisinin-resistant P. falciparum malaria has emerged on the Cambodia–Thailand border. According to Dondorp et al. [36], exposure of the parasite population to artemisinin monotherapies in subtherapeutic doses for over 30 years, along with the availability of substandard artemisinins, has probably been the main driving force in the selection of the resistant phenotype in the region. In spite of this, artemisinins are still established antimalarial agents with an excellent safety profile, However, there are concerns that artemisinin resistance would be harmful for global malaria control. The high cost of preparing artemisinin from plants has been a problem. To combat this problem, genetic engineering has been used to produce the intermediate artemisinic acid with an engineered version of the yeast Saccharomyces cerevisiae. A leader in this field is Jay Keasling [37] of the University of California at Berkeley. His group [38] first used metabolic engineering of Escherichia coli to produce artemisinic acid but the titer was low (100 mg/L). The genes originated from a truncated mevalonic acid pathway in S. cerevisiae. Then, a genetically engineered strain of S. cerevisiae was used to produce 2.5 g/L of artemisinic acid in a fed-batch fermentation [39, 40]. Seebeager and Lexesque devised a chemical method to convert artemisinic acid to artemisinin [41]. An improved process for making artemisinic acid plus semisynthesis to artemisinin has recently been devised [42]. The new procedure achieved a titer of 25 g/L of artemisinic acid. The process was improved by applying synthetic biology technology to the S. cerevisiae process.

2.3.4 Tetracyclines

Tetracyclines are broad-spectrum drugs exhibiting activity against a wide range of microorganisms including Gram-positive and Gram-negative bacteria, chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites including P. falciparum. Due to their low cost, they have been used extensively in the prophylaxis and therapy of human and animal infections and, unfortunately, at low concentrations also as animal growth promoters, which later resulted in a considerably increased microbial resistance to these antibiotics. Chlortetracycline and oxytetracycline were the first members of the tetracycline group discovered. They are produced by Streptomyces aureofaciens and Streptomyces rimosus, respectively. Other tetracyclines were discovered later, either as natural molecules––for example, tetracycline from S. aureofaciens, S. rimosus, and Streptomyces viridofaciens and demethyltetracycline from S. aureofaciens––or as semisynthetic products such as methacycline, doxycycline, and minocycline [43]. The emergence and rapid extension of P. falciparum resistance to various antimalarial compounds has gradually limited malarial therapeutic possibilities. Doxycycline is apparently a useful drug in this respect, in spite of its limited use in children less than eight years old and in pregnant women. Doxycycline has already been successfully used in areas where resistance to other antimalarial drugs has occurred. It has also been shown to be effective and well-tolerated in the prevention of malaria. It is extremely important that resistance to doxycycline has not yet been observed [44].

With increasing resistance to existing antimalarials, there is an urgent need to discover new drugs at affordable prices for countries in which malaria is endemic. With this aim in mind, Draper et al. [45] synthesized 18 novel tetracycline derivatives and tested their activity against malarial parasites. Compounds with potent in vitro activity and other favorable drug properties were further tested in a rodent malaria model. Ten compounds inhibited the development of cultured P. falciparum, demonstrating activity much higher than that of doxycycline. In a murine Plasmodium berghei model, 13 compounds demonstrated improved activity in comparison with that of doxycycline. The authors proposed that optimized compounds may allow lower doses for treatment and chemoprophylaxis. If safety margins are adequate, dosing in children, the group at greatest risk for malaria in countries in which it is endemic, may be feasible.

2.3.5 Clindamycin

Clindamycin is a semisynthetic chlorinated derivative of natural lincomycin. The group of lincosamides, to which lincomycin and clindamycin belong, also includes celesticetin and several semisynthetic derivatives of lincomycin of which only clindamycin is usually used. Pirlimicin, a clindamycin analog in which the subsituted pyrrolidine moiety is replaced by ethylpiperic acid, is generally less active than clindamycin. Clindamycin, which is usually used to treat infections with anaerobic bacteria, can be used against methicillin-resistant Staphylococcus aureus infections and also to treat some protozoal diseases, including malaria. As reviewed by Lell and Kremsner [46], clindamycin monotherapy is an effective treatment but the drug must be given for at least five days and at least twice a day; under these conditions, it has a mean efficacy of 98%. However, it is a slowly acting drug.

Correspondingly, with clindamycin treatment the rate of clinical cure is slow, with fever clearance times in the range of three to five days. According to the above authors, clindamycin monotherapy for falciparum malaria cannot be recommended. The combination of clindamycin with a fast-acting drug is necessary to take advantage of its full antimalarial potential. The adoption of combination therapy, the simultaneous administration of two or more drugs with independent modes of action and different biochemical targets in the parasite, is thought to improve treatment efficacy and to delay the emergence of drug resistance to the individual components of the combination. Clindamycin plus quinine is a potential combination that can be applied. In general, clindamycin is a well-tolerated drug with mild and transient side effects [47].

2.3.6 Combinations of Drugs

As mentioned several times in the preceding paragraphs, combination treatment of malaria with several drugs has been recommended. In an excellent review article, Noedl [48] proposed that there are good reasons to routinely combine antimalarials, such as artemisinins or quinine, with broad-spectrum antibiotics with antimalarial activity in standardized combination therapies for the treatment of severe falciparum malaria. According to the author,

a prudently selected antibiotics-based combination (ABC) therapy for severe malaria cases could address many of the most common conditions that can be misdiagnosed as severe malaria, while at the same time treating malaria. In addition, such a treatment should neutralize possible concomitant bacterial infections.

Combination therapy has become the gold standard for treating uncomplicated falciparum malaria. According to Noedl [48],

There is a limited armory of drugs in widespread use for falciparum malaria with relatively few new developments in the past three decades. Antibiotics, particularly azithromycin, a relatively new macrolide antibiotic, clindamycin, a lincosamide antibiotic, and members of the tetracycline group, have shown to be highly active and generally very well tolerated antibiotics.

In such combinations, the glycylcycline tigecycline was found to be six times more active than the previously used doxycycline. Although treatment with azithromycin alone was found to be inefficient [49], its combination with quinine or artesunate has had success [50]. Fosfidomycin–clindamycin was proposed for the combination treatment of P. falciparum malaria [51], but it has been recently demonstrated [52] that it is not as efficient as previously thought.

2.4 CONCLUSIONS

In the present review, we concentrated on fungal diseases and malaria. It appears to us that although numerous drugs including antibiotics are currently available, none of them is absolutely efficient, which is not surprising. In addition, resistance to most of these drugs regularly increases. This situation leads to the use of combinations of two or more drugs. It is evident that new antibiotics against pathogenic fungi and plasmodia should be looked for as we previously proposed for pathogenic bacteria [3]. As far as the treatment of malaria is concerned, the use of nanotechnology reviewed by Santos-Magalhães and Mosqueira [53] seems to be interesting. Another approach could be genome mining of different organisms, actinomycetes and fungi in particular. Due to the increased need for new antibiotics, genome mining has become a common method for finding novel antibiotics by investigating cryptic gene clusters [54]. Genome mining and combinations of genes specifying production of antibiotics in a single producer could lead to new antibiotics against pathogenic bacteria, fungi, plasmodia, and other pathogenic microorganisms.

References

1. Kraus, G. N. (2008). Low hanging fruit in infectious disease drug development. Current Opinion in Microbiology11, 434–438.

2. Vicente, M., Hodgson, J., Massida, O., Tonjum, T., Henriques-Normark, B., Ron, E. (2006). The fallacies of hope: Will we discover new antibiotics to combat pathogenic bacteri in time? FEMS Microbiology Reviews30, 841–852.

3. Spížek, J., Novotná, J., Řezanka, T., Demain, A. L. (2010). Do we need new antibiotics? The search for new targets and new compounds. Journal of Industrial Microbiology and Biotechnology37, 1241–1248.

4. Demain, A. L., Spizek, J. (2012). The antibiotic crisis, in Antimicrobial Drug Discovery: Emerging Strategies, G. Tegos and E. Mylonakis, eds., CAB International, Wallingford, UK, pp. 26–43.

5. D'Costa, V. M., McGrann, K. M., Hughes, D. W., Wright, G. W. (2006). Sampling the antibiotic Resistome. Science311, 374–377.

6. D'Costa, V. M. D., King, C. E., Kalan, L., Morar, M., Sung, W. W. L., Schwarz, C., Froese, D., Zazula, G., Calmels, F., Debruyne, R., Golding, C. B., Poinar, H. N., Wright, G. D. (2011). Antibiotic resistance is ancient. Nature477, 457–461.

7. Shapiro, S. (2013). Speculative strategies for new antibacterials: All roads should not lead to Rome. Journal of Antibiotics66, 371–400.

8. Bérdy, J. (2012). Thoughts and facts about antibiotics: Where we are now and where we are heading? Journal of Antibiotics65, 385–395.

9. Calderone, R., Fonzi, W., Gay-Andrieu, F., Sun, N., Li, D., Deepu, A. (2012). Antifungals and antifungal drug discovery, in Antimicrobial Drug Discovery, Emerging Strategies, G. Tagos and E. Mylonakis, eds., CAB International, Wallingford, UK, pp. 247–264.

10. Vandenputte, P., Ferrari, S., Coste, A. T. (2012). Antifungal resistance and new strategies to control fungal infections, International Journal of Microbiology2012 Article ID 713687, 26 pages. doi.10.1155/2012/7113687.

11. Waksman, S. A., Romano, A. H., Lechevalier, H., Raubitschek, F. (1952). Antifungal antibiotics. Bulletin of the World Health Organization6, 163–172.

12. Carillo-Muñoz, A. J., Giusano, G., Ezkurra, P. A., Quindós, G. (2006). Antifungal agents: Mode of action in yeast cells. Revista Espanola Quimioterapia19, 130–139.

13. Enoch, D. A., Ludlam, H. A., Brown, N. M. (2006). Invasive fungal infections: A review of epidemiology and management options. Journal of Medical Microbiology88, 809–818.

14. Papon, N., Noël, T., Florent, M., Gibot-Leclerc, S., Jean, D., Chastin, C., Villard, J., Chapeland-Leclerc, F. (2007). Molecular mechanism of flucytosine resistance in Candida lusitaniae: Contribution of the FCY2, FCY1, and FUR1 genes to 5-fluorouracil and fluconazole cross-resistance. Antimicrobial Agents and Chemotherapy51, 369–371.

15. Perfect, J. R. (2011). Azoles: Back to the future. Current Opinion in Infectious Disease24, S41–S58.

16. Denning, D. W., Hope, W. W. (2010). Therapy for fungal diseases: Opportunities and priorities. Trends Microbiol.18, 195–204.

17. Zhang, L., Yan, K., Zhang, Y., Huang, R., Bian, J., Zheng, C., Sun, H., Chen, Z., Sun, N., An, R., Min, F., Zhao, W., Zhuo, Y., et al. (2007). High-throughput synergy screening identifies microbial metabolites as combination agents for the treatment of fungal infections. Proceedings of the National Academy of Sciences of the USA104, 4606–4611.

18. Emri, T., Majoros, L., Tóth, V., Pócsi, I. (2013). Echinocandins: Production and applications. Applied Microbiology and Biotechnology97, 3267–3284.

19. Goswama, S., Runi, A., Priydarshini, R., Bhunia, B., Mandal, T. (2012). A review on production of echinocandins by Aspergillus sp. Journal Biochemical Technology4, 568–575.

20. Eschenauer, G., DePestel, D. D., Carver, P. G. (2007). Comparison of echinocandin antifungals. Therapeutics and Clinical Risk Management 3, 71–97.

21. Fox, J. L. (2013). Antimicrobial peptides stage a comeback. Nature Biotechnology31, 379–382.

22. Song, B., Rong, Y.-J., Zhao, M.-X., Chi, Z.-M. (2013). Antifungal activity of the lipopeptides produced by Bacillus amyloliquefaciens anti-CA against Candida albicans isolated from clinic. Applied Microbiology and Biotechnology87, 7141–7150.

23. Welscher, Y. M., van Leeuwen, M. R., de Kruijff, B., Dijksterhuis, J., Breukink E. (2012). Polyene antibiotic that inhibits membrane transport proteins. Proceedings of the National Academy of Sciences of the USA109, 11156–11159.

24. Brautaset, T., Sletta, H., Degnes, K. F., Sekurova, O. N., Bakke, I., Volokhan, O., Andreassen, T., Ellingsen, T. E., Zotchev, S. B. (2011). New nystatin-related antifungal macrolides with altered polyol region generated via biosynthetic engineering of Streptomyces noursei. Applied and Environmentel Microbiology77, 6636–6643.

25. Van Minnebruggen, G., François, I. E. J. A., Cammue, B. P. A., Thevissen, K., Vroome, V., Borgers, M., Shroot, B. (2010). General overview on past, present and future antimycotics. The Open Mycology Journal4, 22–32.

26. Seipke, R. F., Barke, J., Mario, X., Ruiz-Gonzalez, M. X., Yu, D. W., Hutchings, M. L. (2012). Fungus-growing Allomerus ants are associated with antibiotic-producing actinobacteria. Anton van Leeuwenhoek101, 443–447.

27. Ginsburg, H., Deharo, E. (2011). A call for using natural compounds in the development of new antimalarial treatments—An introduction. Malaria Journal10, 51.

28. Schlitzer, M. (2008). Antimalarial drugs—What is in use and what is in the pipeline. Archives of Pharmaceutical, Chemical, and Life Sciences341, 149–163.

29. Kaur, K., Jain, M., Kaur, M., Jain, R. (2009). Antimalarials from nature. Bioorganic and Medicinal Chemistry17, 3229–3256.

30. Achan, J., Talisuna, A. O., Erhart, A., Yeka, A., Tibenderana, J. K, Baliraine, F. N., Rosenthal, P. J., D'Alessandro, U. (2011). Quinine, an old anti-malarial drug in a modern world: Role in the treatment of malaria. Malaria Journal10, 144. doi:0.1186/1475-2845-10-144doi:0.1186/1475-2845-10-144.

31. Tu, Y. (2011). The discovery of artemisinin (qinghaosu) and gifts from Chinese medicine. Nature Medicine17, 1217–1220.

32. Efferth, T., Romero, M. R., Wolf, D. C., Stamminger, T., Marin, J. J. G., Marschall, M. (2008). The antiviral activities of artemisinin and artesumate. Clinical Infectious Diseases47, 804–811.

33. White, N. J. (2008). Qinghaosu (Artemisinin): The price of success. Science320, 330–334.

34. Maude, R. J., Pontavornpinyo, W., Saralamba, S., Aguas, R., Dondorp, A. M., Day, N. P. J., White, N. J., White, L. J. (2009). The last man standing is the most resistant: Eliminating artemisinin-resistant malaria in Cambodia. Malaria J. 8, 31. 8:31 doi:10.1186/1475-2875-8-31doi:10.1186/1475-2875-8-31.

35. Miller, L. H, Su, X. (2011). Artemisinin: Discovery from the Chinese Herbal Garden. Cell146, 855–858.

36. Dondorp, A. M., Yeung, S., White, L., Nguon, C., Nicholas, P. J., Day, N. P. J., Socheat, D., von Seidlein, L. (2010). Artemisinin resistance: Current status and scenarios for containment. Nature Reviews Microbiology8, 272–280.

37. Anonymous: IB Interview. (2013). A conversation with Jay Keasling, PhD. Industrial Biotechnology9, 152–155.

38. Martin, V. J., Pitera, D. J., Withers, S. T., Newman, J. D., Keasling, J. D. (2003). Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature Biotechnology21, 796–802.

39. Ro, D. K., Paradise, E. M., Ouellet, M., Fisher, K. J., Newman, K. L., Ndungu, J. M., Ho, K. A., Eachus, R. A., Ham, T. S., Kirby, J., Chang, M. C. Y., Withers, S. T., Shiba, Y., Sarpong, R., Keasling, J. D. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature440, 940–943.

40. Lenihan, J. R., Tsuruta, H., Diola, D., Renninger, N. S., Regentin, R. (2008). Developing an industrial artesimic acid fermentation process to support the cost- effective production of anti-malarial artemisinin-based combination therapies (ACTs). Biotechnology Progress24, 1026–1032.

41. Halford, B. (2012). Easier path to leading antimalarial drug. Chemical and Engineering News90, 21.

42. Paddon, C. J., Westfall, P. J., Pitera, D. J., Benjamin, K., Fisher, K., McPhee, D., Leavell, M. D., Tai, A., Main, A., Eng, D., Polichuk, D. R., Teoh, K. H., Reed, D. W., et al. (2013) High-level semi-synthetic production of the potent antimalarial artemisin. Nature496, 528–532.

43. Chopra, I., Roberts, M. (2001). Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology and bacterial resistance. Microbiology and Molecular Biology Reviews65, 232–260.

44. Briolant, S., Fusai, T., Rogier, C., Pradines, B. (2008). Tetracycline antibiotics in malaria. The Open Tropical Medicine Journal1, 31–46.

45. Draper, M. P., Hatia, B., Asssefa, H., Honeyman, L., Garity-Ryan, L. K., Verma, A. K, Gut, J., Larson, K., Donatelli, J., Macone, A., Klausner, K., Leahy, R. G., Odinecs, A., Ohemeng, K., Rosenthal, P. J., Nelson, M. L. (2013). In vitro and in vivo antimalarial efficacies of optimized tetracyclines. Antimicrobial Agents and Chemotherapy57, 3131–3146.

46. Lell, B., Kremsner, P. G. (2002). Clindamycin as an antimalarial drug: Review of clinical trials. Antimicrobial Agents and Chemotherapy46, 2315–2320.

47. Obonyo, C. O., Juma, E. A. (2012). Clindamycin plus quinine for treating uncomplicated falciparum malaria: A systematic review and meta-analysis. Malaria Journal11: 2. doi:10.1186/1475-2875-11-1210.1186/1475-2875-11-12.

48. Noedl, H. (2009). ABC–antibiotics-based combinations for the treatment of severe malaria? Opinion. Trends in Parasitology25, 540–544.

49. van Eijk, A. M., Terluw, D. J. (2011). Azithromycin for treating uncomplicated malaria. Cochrane Database of Systematic Reviews16. CD006688.doi 10.1002/14561858.CD 006688.pub2CD006688.doi 10.1002/14561858.CD 006688.pub2.

50. Starzengurba, P., Thriesen, K., Haque, R., Khan, A., Fuehrer, H. P., Siedl, A., Hofecker, V., Ley, B., Wernsdorfer, W. H., Noedl, H. (2009). Antimalarial activity of tigecycline, a novel glycylcycline antibiotic. Antimicrobial Agents and Chemotherapy53, 4040–4042.

51. Borrmann, S., Issifou, S., Esset, G., Adegnika, A. A., Rambarter, M., Matsiegui, P., Oyakhirome, S., Mawili-Mbhoumba, D. P., Mssinou, M. A., Kun, J. F. K, Jomaa, H., Kremsner, P. G. (2004). Fosfidomycin–clindamycin for the treatment of Plasmodium falcifarum malaria. Journal Infectious Diseases190, 1534–1540.

52. Lanaspa, M., Moraleda, C., Machevo, S., Gonzáles, R., Serrano, B., Macete, E., Cisteró, P., Mayor, A., Hutchinson, D., Kremsner, P. G., Alonso, P., Menéndez, C., Bassat, Q. (2012). Inadequate efficacy of a new formulation of fosfidomycin–clindamycin combination in Mozambican children less than three years old with uncomplicated Plasmodium falcifarum malaria. Antimicrobial Agents and Chemotherapy56, 2923–2928.

53. Santos-Magalhães, N. S., Mosqueira, V. C. F. (2010). Nanotechnology applied to the treatment of malaria. Advanced Drug Delivery Reviews82, 562–575.

54. Scheffler, R. J., Colmer, S., Tynan, H., Demain, A. L., Gullo, V. P. (2013). Antimicrobials, drug discovery, and genome mining. Applied Microbiology and Biotechnology97, 969–978.

Chapter 3

Emerging Instrumental Methods for Antimicrobial Resistance and Virulence Testing

Plamen A. Demirev

Johns Hopkins University, Applied Physics Laboratory, MD, USA

3.1 INTRODUCTION

Drug-resistant strains of pathogenic microorganisms—from viruses to single-cellular eukaryotic parasites—are rapidly emerging and exacerbating public health problems in both the developed and the developing world [1]. Present-day health scourges inflicting humanity, such as HIV, tuberculosis, and malaria, are becoming harder, more expensive, and more time-consuming to treat as a result of the emergence of novel drug-resistant strains. New more potent drugs and vaccines, as well as new more rapid and accurate diagnostic tools, are sorely needed if we want to be successful in the fight against both old and emerging pathogens. Thus, rapid and accurate determination of drug resistance is very important, with applications in a number of fields—from clinical microbiology to timely response of a bioterrorism attack. Rapid detection of drug-resistant strains will benefit patient care by optimizing antibiotic therapies and reducing the threat of uncontrollable outbreaks/epidemics. Comprehensive large-scale epidemiological monitoring of emergence of drug resistance provides valuable information for the spread of pathogens and the available opportunities for treatment and eradication. A number of classical microbiology techniques—for example, broth dilution or disk diffusion—have been developed to determine drug resistance [2]. These techniques include organism proliferation monitoring in the presence of the drug and the resulting biosynthesis of organism-specific molecules (DNA, proteins, etc.). For example, a change in optical density (turbidity) of culture suspensions is an indication of growth. However, these classical techniques for determining drug resistance are not rapid, typically taking between 24 and 48 h. This is problematic in, for example, infectious disease spread or bioterrorism scenarios, where time is of the essence in identifying, treating, or eradicating particularly virulent and unknown pathogens. In addition, existing classical tests with higher false-positive or false-negative rates can lead to complications, due to, for example, lack of containment and/or proper treatment.

Cell wall damage and/or blocking major biosynthetic pathways are among the main mechanisms by which drugs affect microorganisms. Respectively, bacteria fight antibiotics and acquire resistance by either destroying the drug directly in enzyme-specific reaction, by reprogramming the structure of the drug target and thus reducing binding, or by ejecting the drug outside of the bacterial cell [3]. Examples of the first major pathway include hydrolysis of beta-lactam antibiotics. Examples of the second line of defense are protein sequence variants that lower the susceptibility to vancomycin in, for example, vancomycin-resistant Enterococci. The third pathway of acquiring drug resistance is illustrated by the action of transmembrane efflux pumps that lower the intracellular drug concentration in, for example, Pseudomonas. Biophysical technologies, based on recent advances in molecular biology, are revolutionizing the practice of infectious disease diagnostics [4]. Among such technologies are mass spectrometry (MS), nucleic acid amplification (polymerase chain reaction, PCR) and sequencing, and a diverse array of micro- and nano-sensors and microfluidics devices. Several such technologies have been successfully applied for screening, monitoring, and detection of drug resistance and susceptibility in microorganisms. Selected examples of these molecular level technologies and methods, namely MS and nucleic acid amplification, will be discussed here.

3.2 MASS SPECTROMETRY