Discovery of Novel Natural Products with Therapeutic Potential

Japan

PREFACE

Although effective drugs for many of the diseases that afflict humankind have been discovered, many health problems remain untreatable. These problems include various types of cancer; viral infections such as HIV; severe fungal infections, particularly in immunocompromised patients; cardiovascular diseases; and inflammatory and allergic disorders. Even when significant progress is made, as in the treatment of bacterial infections, resistant, highly pathogenic organisms can appear. However, as knowledge of various disease processes expand, new targets for intervention are discovered. Therefore, the search for novel therapeutic agents continues.

From where are drugs derived? There are only two sources of drugs, natural products and synthetic chemicals. The trend in recent years has been to focus attention on rational drug design, highlighting the synthetic chemical approach. However, rational drug design has proven to be effective in refining structural leads that interact with a particular target, for example, an enzyme or receptor. The need still remains to uncover the initial structural lead that interacts with the therapeutic target. The myriad of structurally diverse compounds found in nature offers a unique source for drug discovery. It is in this scenario that natural products play an important role.

The authors of each chapter are a diverse group of experts, who, together, present the knowledge base required to discover potential therapeutic agents from natural sources. The subject matter included in each chapter is intended to be useful for scientists currently working in this broad field, as well as to enlighten those interested in the current status and complexity of natural product research. The authors have highlighted successes, but, for the most part, they have concentrated on the strategies employed. I am deeply indebted to them for their efforts.

The purpose of this book is to capture what we perceive as the rational process of natural product drug discovery. The book has been divided into three major sections that represent this process. Chapters 1-5 describe some of the diverse sources of natural products. Both terrestrial and marine environments represent ecological niches where therapeutically active, complex molecules can be discovered. Chapters 6-12 describe how molecular biological and biochemical research have increased knowledge of biological systems and human disease. This knowledge has led to the design of targeted assays, amenable to high-volume screening. Chapter 13 and 14 describe the current methods that natural product chemists employ to isolate and identify compounds with biological activity from natural product extracts.

Vincent P. Gullo

PART I

THE SOURCE OF CHEMICAL DIVERSITY

Outline

Chapter 1: Aerobic Actinomycetes: A Continuing Source of Novel Natural Products

Chapter 2: Bacteria As a Source of Novel Therapeutics

Chapter 3: Potential of Fungi in the Discovery of Novel, Low-Molecular Weight Pharmaceuticals

Chapter 4: The Discovery of Drugs from Higher Plants

Chapter 5: The Discovery of Marine Natural Products with Therapeutic Potential

CHAPTER 1

Aerobic Actinomycetes: A Continuing Source of Novel Natural Products

Ann C. Horan

Publisher Summary

This chapter describes the importance of aerobic actinomycetes as a continuing source of novel natural products. The micromonosporae have been crucial to the success of the Natural Products Program at Schering-Plough. The exploitation of these bacteria proved that nonstreptomycetes were valuable sources of novel natural products. Micromonosporae are the second largest group of actinomycetes in soil, surpassed only by the streptomycetes. They have been isolated from a variety of habitats. Jensen isolated micromonosporae from dry, neutral, and alkaline soils. They are found to be widely distributed, occurring preferentially in moist Russian soils. Shearer reported that two or three micromonosporae can be isolated from most soil types. Cross places the number at 10 × 106 cfu per gram of soil. Micromonosporae have been isolated from freshwater lakes and lake mud, where they are the most frequently isolated mesophilic actinomycetes. The surface of lake mud was reported to contain 2.4 × 106 micromonosporae cfu per gram, which represented 50% of the total population and deep mud contained 4 × 105 cfu per gram, and in which the micromonosporae predominated. Micromonosporae have also been isolated from the marine environment, from a salt marsh, from sea water and sand, and antibiotic producers have been isolated from marine sediments.

A Natural Products Program begins at the source, whether it be extracts of plants and/or marine organisms or fermentation products of actinomycetes, fungi, and bacteria. All have yielded abundant diverse compounds, many with therapeutic utility. Ten of the top selling 30 branded prescription products for 1990 (Scripts Review Issue 1990, p. 21) were either natural products or their derivatives and accounted for U.S. $8.2 billion in sales.

Methods for detecting biological activity with therapeutic potential have shifted from simple determinations of antibacterial and antifungal activity to assays using genetically engineered eukaryotic cells that can detect regulators of specific gene expression, inhibitors of signal transduction, small molecular weight agonists/antagonists of cytokines, and/or specific receptors and receptor subtypes. The shift in emphasis away from antibacterials is reflected in the reported activities of novel secondary metabolites found in The Journal of Antibiotics. During the years from 1986 to 1991 (Table 1-1), the total number of novel compounds increased 20%; anti-infectives were reduced by 24%, and pharmacologically active compounds increased 87%. When new targets for drug intervention are defined and new assays developed to exploit the target, chemical leads are not available as starting points for potent new drugs. Random screening will be initiated and should include diverse natural products as well as synthetic compounds.

TABLE 1-1

Novel Secondary Metabolites Reported in The Journal of Antibiotics by Therapeutic Area, 1986 versus 1991

Aerobic, saprophytic actinomycetes remain the major source of novel, fermentation derived, secondary metabolites (Table 1-2) and are well suited to the production of secondary metabolites. Their minimum nutritional requirements seem to leave large segments of the genome available for functions other than growth. This may not be true for organisms whose Survival requires complex growth conditions, such as anaerobes and nitrogen fixers. The natural reservoir of the aerobic actinomycetes is soil, where their primary ecological role appears to be decomposing organic matter (Lechevalier and Lechevalier 1981). Labeda and Shearer (1990) reported that the number of recoverable actinomycetes per gram of agricultural soil is in excess of 10⁶ colony forming units (cfu) per gram.

TABLE 1-2

Novel Secondary Metabolites Reported in The Journal of Antibiotics by Producing Organism, 1986 versus 1991

Our Natural Products Program has focused on isolating aerobic actinomycetes that contain meso-diaminopimelic acid in their cell walls (Lechevalier and Lechevalier 1981, 1985). We have developed a testing paradigm that evaluates the effect of soil type, media constituents, and selective pressure. Physiological data used for taxonomic analysis of the various genera of actinomycetes formed the basis for our initial choices of selective pressures (antibiotics) and media constituents. The purpose of these investigations has been to discover appropriate conditions for isolating various groups of actinomycetes, thereby enhancing the ability to discover novel natural products. Numerous reviews (Cross 1981a, 1982; Goodfellow and Williams 1983; Williams and Wellington 1981, 1982; Williams and Vickers 1988; Williams et al. 1984) have detailed procedures for the isolation of a wide variety of actinomycetes from soil. A recent review by Labeda and Shearer (1990) is highly recommended. This chapter will detail experiments performed in our laboratory directed toward the isolation of micromonosporae, actinomadurae, and nocardioform actinomycetes.

1.1 MICROMONOSPORAE

The micromonosporae (Kawamoto 1989) have been crucial to the success of the Natural Products Program at Schering-Plough. The exploitation of these bacteria proved that non-streptomycetes were valuable sources of novel natural products. For a detailed history of the micromonosporae’s role in Schering-Plough’s Natural Products Program, the reader is referred to Dr. George Luedemann’s recent publication, Free Spirit of Inquiry, The Uncommon Common Man in Research and Discovery, The Gentamicin Story (Luedemann 1991).

Micromonosporae are the second largest group of actinomycetes in soil, surpassed only by the streptomycetes. They have been isolated from a variety of habitats. Jensen (1932) isolated micromonosporae from dry, neutral, and alkaline soils. Solovieva and Singal (1972) found them to be widely distributed, occurring preferentially in moist Russian soils. Shearer (1987) reported that two or three micromonosporae can be isolated from most soil types. Cross (1981b) places the number at 10 × 10⁶ cfu per gram of soil. Micromonosporae have been isolated from freshwater lakes and lake mud, where they are the most frequently isolated mesophilic actinomycetes (Willoughby 1969; Johnston and Cross 1976). The surface of lake mud was reported to contain 2.4 × 10⁶ micromonosporae cfu per gram, which represented 50% of the total population; deep mud contained 4 × 10⁵ cfu per gram, and in which the micromonosporae predominated. Micromonosporae have also been isolated from the marine environment, from a salt marsh by Hunter et al. (1981), from sea water and sand by Watson and Williams (1974), and antibiotic producers have been isolated from marine sediments (Okazaki and Okami 1976).

Viable micromonosporae spores have been found from 100-year-old sediments (Cross and Attwell 1974), and there has been much discussion over the ecological role these organisms play in mud and marine sediments. We have isolated numerous strains of micromonosporae from lake mud. Macroscopic and microscopic evaluation of the isolates did not reveal a wide range of diversity nor were these organisms prolific producers of secondary metabolites.

Lake mud isolates and type species were tested for their ability to utilize glucose fermentatively using the Hugh and Leifson test as described by Gordon and Horan (1968). None of the strains tested gave a positive fermentative response, nor were they able to grow along the stab line in the aerobic tube. Growth was limited to the surface of the agar, indicating that all the strains were strict aerobes. The results suggest that micromonosporae are not physiologically active in lake mud or sediments. Their presence may be the result of the spores’ physical properties. Micromonosporae spores are hydrophilic, wettable, and easily removed from soil by water (Ruddick and Williams 1972). They withstand ultrasonication, moist heat treatment for 20 minutes at 60°C, dry heat up to 75°C, and are resistant to various chemical solutions (Vobis 1992). Their physical nature suggests that micromonosporae spores that occur in soil along rivers, streams, and lakes are easily washed into the water, eventually falling to the bottom and accumulating in the mud and sediments.

In addition to being more resistant than most actinomycete spores, micromonosporae spores may also have a different density. Karwowski (1986) was able to separate micromonosporae spores from streptomycete and fungal spores using CsCl2 density gradient centrifugation of saline extracted soil. W. Reiblein (unpublished observations), from our laboratory, experimented with the use of renographin-76 density gradients in order to isolate micromonosporae from soils that were heavily contaminated with bacteria. He was able to isolate 3 × 10³ cfu per gram of soil in specific regions of the gradient, which was free from bacteria, fungi, and streptomycetes.

Micromonosporae hydrolyze complex carbons (Luedemann 1971), degrade biopolymers (Erikson 1941), attack lignin complexes (McCarthy and Broda 1984), and are proteolytic (Umbreit and McCoy 1941), preferring hydrolyzed vegetable protein over meat protein. Jensen (1932) emended soil

with cellulose and lignic acid to enhance the isolation of micromonosporae. Soil samples were plated on dextrose-casein medium (Jensen 1930) and incubated at 30 to 32°C. Cross (1981a) used colloidal chitin agar (Lingappa and Lockwood 1962) for the selective isolation of micromonosporae while Sandrak (1977) isolated strains from the rhizosphere of winter wheat and corn using Kadoto’s medium emended with 2% sodium benzoate. Species of Micromonospora are sensitive to acid conditions (Luedemann 1971) and NaCl, rarely growing at saline levels greater than 3% (Luedemann 1971; A.C. Horan, unpublished observations). Kawamoto et al. (1983) found inorganic nitrogen to be a poor source of nitrogen; one-half of the strains tested were unable to grow in the presence of sodium nitrate or potassium nitrate, but all the strains tested grew to varying degrees in the presence of ammonium nitrate. We have used soil isolation media that exploit the physiological properties of the micromonosporae. Sodium nitrate has been used successfully in isolation media and results in a significant reduction of the bacterial population. The media constituents are presented in Table 1-3.

TABLE 1-3

Soil Isolation Media for Micromonosporae

¹All media contain CaCO3, 1.0 g; Difco agar, 15.0 g; tap water, 1000 ml; nystatin, 75 μg/ml.

²Lingappa and Lockwood 1962.

The use of antibiotics as selective pressures that enhance the isolation of actinomycetes from soil has been extensively reported and reviewed (Cross 1982; Labeda and Shearer 1990). Early studies in our laboratories, confirmed by Sveshnikova et al. (1976), identified novobiocin as an effective selective pressure in isolation media, enhancing the growth of micromonosporae while suppressing streptomycetes. Prior to the reports of antibiotic gene clustering and the presence of resistance genes within the cluster, we had devised a specific screen to search for novel aminoglycoside antibiotics by plating soil samples on M1 and M2 agars containing 50 to 100 μg/ml gentamicin. Interestingly, as high as 90% of the micromonosporae, the predominating organisms under these conditions, were aminoglycoside producers. Ivanitskaya et al. (1978), using 1, 5, and 10 μg/ml of gentamicin, showed a marked increase in the frequency of micromonosporae detected, 1 μg/ml being optimal. Antibiotic producers were three times higher than on media without gentamicin. Tunicamycin at 25 to 50 μg/ml resulted in the isolation of four different micromonosporae from one soil sample, on average (Wakisaka et al. 1982). Data, generated in our laboratory, on the ability of secondary metabolite producing species of Micromonospora to grow in the presence of antibiotics, are presented in Table 1-4.

TABLE 1-4

Ability of Micromonospora Species to Grow in the Presence of Antibiotics

¹The left-hand column under each type of antibiotic represents a 10 μg/ml concentration, the right represents a 50 μg/ml concentration.

Micromonospora have proven to be prolific producers of a wide variety of antibacterial antibiotics. However, certain chemical classes produced by other actinomycetes, including polyenes, β-lactams, and tetracycline, have never been detected or isolated. The micromonosporae-produced compounds isolated at Schering-Plough are presented in Table 1-5.

TABLE 1-5

Micromonosporae Isolated at Schering-Plough and Associated Products

1.2 NON-MICROMONOSPORAE-NON-STREPTOMYCETES

The micromonosporae’s inability to produce certain chemical classes of secondary metabolites has led to a systematic examination of isolation conditions that favor other actinomycetes. Although no specific group was targeted, we began by incorporating antibiotics into soil isolation agar that did not inhibit actinomadurae (Table 1-6). The resulting actinomycetes were isolated, examined microscopically, and identified as either micromonosporae or non-micromonosporae. Beneficial results were those antibiotics (rifamycin, mefoxitin, lincomycin, rosaramicin, gentamicin, everninomicin, neomycin, streptomycin) resulting in greater than 50% non-micromonosporae. The data are presented in Figure 1-1. Although some treatments such as neomycin, streptomycin, and lincomycin, resulted in a high percentage of non-micromonosporae, the yield of isolates was extremely low (Figure 1-2). Rifamycin resulted in the greatest percentage of non-micromonosporae having a moderate yield, confirming the early results of Gordon and Barnett (1977). Novobiocin and spectinomycin selected for and enhanced the micromonosporae; 54% of the total isolates were from these treatments, while the other 12 antibiotics accounted for the remaining isolates. The large numbers of micromonosporae isolated in the presence of these antibiotics obscured the effects of soil type and media constituents.

TABLE 1-6

Ability of Actinomadura Species to Grow in the Presence of Antibiotics¹

¹See Horan and Brodsky (1982) for methods.

²The left-hand column under each type of antibiotic represents a 10 μg/ml concentration, the right represents a 50 μg/ml concentration.

FIGURE 1-1 The effect of antibiotics on the isolation of non-micromonosporae and micromonosporae.

FIGURE 1-2 Effect of antibiotics on the yield of actinomycetes.

Experiments to further evaluate selective pressures and to determine whether media constituents and soil type affected the isolation of actinomycetes were initiated. Eight soil types, four media constituents, and six selective pressures were included in the analysis (Table 1-7). Selective pressures (antibiotic additions) were chosen from previous studies that resulted in greater than 50% non-micromonosporae-non-streptomycetes (NMNS). Soils were air-dried for 24 to 48 hours in a fume hood, 1.0 g samples were suspended in 10 ml of sterile distilled water, serially diluted (1:10, 1:100, 1:1000), and 0.1 ml of all dilutions streaked onto the agar surface using glass rods or sterile cotton swabs presoaked in sterile water. All media contained 75 μg/ml nystatin. Plates were incubated for 3 to 4 weeks at 30°C. Resulting actinomycete colonies were removed to ATCC medium 172 agar (Gherna et al. 1989), incubated 7 to 14 days at 30°C, and transferred to ATCC medium 172 broth, 10 ml/25 mm tubes. The broth was incubated, shaken at 30°C, and shaken at 250 rpm for 3 to 5 days, and the resulting biomass restreaked onto ATCC medium 172 and water agars (Sigma agar, 15 g; tap water 1000 ml). Plates were incubated for 14 to 21 days at 30°C and examined microscopically. The organisms were classified as NMNS, streptomycetes (STMY), or micromonosporae (MOSO). Approximately 2000 actinomycetes were isolated and identified using this technique.

TABLE 1-7

An Examination of Environmental Variables

¹All ingredients at 1.0 g. All media contained Difco agar, 15.0 g; CaCO3, 1.0 g; tap water, 1000/ml, pH 7.0.

The elimination of novobiocin and spectinomycin permitted the effect of soil type to be seen (Figure 1-3). Cultivated soils and mud resulted in greater than 50% NMNS. Sandy soils were the richest in streptomycetes; wet and organic soils were the richest in micromonosporae. The largest number of isolates per soil sample (Figure 1-4) was found in dry soils; wet, waterlogged soils were the least productive. The addition of the antibiotics gentamicin, neomycin, rosaramicin, everninomicin, and rifamycin to the isolation agar resulted in greater than 50% NMNS (Figure 1-5). Rifamycin resulted in the most isolates, neomycin the fewest (Figure 1-6). A simple carbon source in combination with organic nitrogen favored NMNS (Figure 1-7). This effect was negated in the presence of antibiotics, as illustrated in Figure 1-8. Without antibiotic additions, none of the media resulted in greater than 50% NMNS. Soil type, media constituents, and selective pressures all affected the isolation of NMNS. Antibiotic additions to the agar exerted the greatest effect.

FIGURE 1-3 Effect of soil type on the isolation of actinomycetes.

FIGURE 1-4 Number of actinomycete colonies isolated per soil type.

FIGURE 1-5 Effect of selective pressures on the isolation of actinomycetes.

FIGURE 1-6 Effect of selective pressures on the yield of actinomycetes.

FIGURE 1-7 Effect of media constituents without selective pressures on the isolation of actinomycetes.

FIGURE 1-8 The effect of selective pressures in various media on the isolation of NMNS.

The procedures outlined have led to the isolation of a wide variety of secondary metabolite-producing maduramycetes (Williams and Wellington 1981; Goodfellow and Cross 1984). Their high level of resistance to rifamycin (Gordon and Barnett 1977; Goodfellow and Orchard 1974; Athalye et al. 1981; see Table 1-6) and their relative numbers in soil (Goodfellow 1992; Kroppenstedt and Goodfellow 1992) contributed to the ease with which maduramycetes were isolated. The cultures and their products are presented in Table 1-8.

TABLE 1-8

Maduramycetes Isolated at Schering-Plough and Their Products

1.3 NOCARDIOFORM ACTINOMYCETES

After establishing a protocol capable of evaluating some of the variables involved in isolating non-streptomycete actinomycetes from natural habitats, we applied the same methods to isolating nocardioform actinomycetes. Nocardioform is a general term used to describe actinomycetes that reproduce by fragmentation of the vegetative and aerial mycelium into irregular to rodlike to coccoid elements that give rise to new mycelia (Figures 1-9 1-10) (Prauser 1976). We were interested in the aerobic, saprophytic nocardioforms that have a cell wall composition of Type III or IV (Lechevalier and Lechevalier 1985).

FIGURE 1-9 Typical fragmenting zig-zag vegetative mycelium of a nocardioform actinomycetes isolated from soil. Culture was grown on water agar for 21 days at 28°C.

FIGURE 1-10 Fragmenting aerial mycelium of a nocardioform actinomycete isolated from soil. Culture was grown on water agar for 21 days at 28°C.

The following experiments employed 1 g soil samples that were air-dried for 48 hours, suspended in 10 ml of sterile distilled water, and sonicated for 1 minute in an ice bath to break up clumps. Soil suspensions were serially diluted (1:10, 1:100, 1:1000), plated on agar media containing 75 μg/ml nystatin, incubated 14 to 21 days at 28°C, and the resulting colonies removed to ATCC medium 172 agar. The cultures were evaluated morphologically after growth on water agar for 14 to 21 days following the procedures outlined previously. The data on number of nocardioforms isolated were calculated as the mean number per variable tested.

The effect of soil type is illustrated in Figure 1-11. One hundred and forty-four soils, plated on all media and under all conditions, were evaluated. Rain forest and conifer forest soils were the most productive, yielding between three and four isolates per soil type. Like the NMNS, wet, water-logged soils yielded the fewest nocardioforms as well as the fewest total isolates. Five random soils were chosen to evaluate the effect of air-drying and heating soils to 120°C for 1 hour (Table 1-9). Heating reduced the numbers of nocardioforms whereas air-drying reduced the growth of spreading bacteria.

TABLE 1-9

Effect of Pretreatments on the Isolation of Nocardioform Actinomycetes¹

¹Five soils/all media/all additions.

²Plates overgrown with bacteria.

FIGURE 1-11 Effect of soil type on the isolation of nocardioform actinomycetes.

Antibiotic additions were based on the taxonomic data generated by Goodfellow and Orchard (1974) and on our previous results in reducing streptomycetes with minimum effect on other actinomycetes. Of the selective pressures evaluated, rifamycin was the most effective (Figure 1-12). Three media frequently used to isolate actinomycetes (Shearer 1987), AV-agar (Nonomura and Ohara 1969), starch-casein-nitrate agar (Kuster and Williams 1964), and glycerol-asparagine agar (Waksman 1961) were used. The three media were equally effective in supporting the growth of isolated nocardioforms (see Table 1-10). However, adjusting the media to pH 6.0 (Figure 1-13) increased nocardioform isolations.

TABLE 1-10

Effect of Media on the Isolation of Nocardioform Actinomycetes¹

¹All soil types/soil air-dried/all additions.

²AV, arginine B vitamin (Nonomura and Ohara 1969); SCN, starch casein nitrate (Küster and Williams 1964); and GA, glycerol asparagine (Waksman 1961).

FIGURE 1-12 Effect of selective pressures on the isolation of nocardioform actinomycetes.

FIGURE 1-13 Effect of pH on the isolation of nocardioform actinomycetes.

An experiment to optimize isolation conditions consisted of examining 12 soils, six conifer forest and six dry tropical soils, plated on glycerol-asparagine and starch-casein-nitrate agars at pH 6.0 and 7.0, comparing no selective pressure to rifamycin at 5 μg/ml. These data are presented in Table 1-11. Conifer forest soils plated on starch-casein-nitrate agar at pH 6.0 in the presence of rifamycin resulted in 5.8 nocardioform isolates per soil plated.

TABLE 1-11

Optimization of Variables for the Isolation of Nocardioform Actinomycetes¹

¹Twelve soils/all soils air-dried.

We have isolated two novel secondary metabolite-producing nocardioform actinomycetes using these techniques, and both have been identified as members of the genus Saccharothrix (Labeda 1992). One strain produces a series of 14 novel indolocarbazole metabolites, which are potent inhibitors of protein kinase C (Brodsky et al. 1991). The second series of novel metabolites are currently being evaluated.

1.4 CONCLUSIONS

Procedures can be defined that will successfully enhance the isolation of specific groups of actinomycetes from natural habitats. The most effective method appears to be the incorporation of antibiotics into isolation media comprised of nutrients that favor the group being pursued. However, the soil harbors a myriad of organisms that have eluded isolation. Scanning electron micrographs of soil samples clearly show that only a fraction of the organisms present have been cultured.

If an organism has not been isolated, its nutritional requirements cannot be defined; therefore determining appropriate conditions for its isolation is impossible. Yet, these are exactly the organisms we want to evaluate for the production of novel secondary metabolites. When faced with the dilemma of isolating organisms that have not been characterized, it will be the microbiologist skilled in the isolating, identifying, and classifying the actinomycetes who will be able to recognize rare and unusual strains. We tried to measure the effect of a microbiologist’s training for successfully isolating NMNS. Two groups of microbiologists were given the same soil samples, media, and selective pressures, and were asked to isolate non-streptomycete actinomycetes. All isolates were identified to their major group by an actinomycete taxonomist (A.C. Horan) not directly involved in culture isolation. The results are presented in Figure 1-14. Group 1 consisted of microbiologists who were familiar with some genera of actinomycetes but were not trained in the macroscopic and microscopic identification of a broad range of soil isolates. They isolated predominantly streptomycetes. Group 2, however, had extensive experience in isolating and identifying actinomycetes. The results clearly show that they were better able to distinguish NMNS. The extent of the participating microbiologist’s experience and training in actinomycete isolation and identification is a key element in a successful Natural Products Program.

FIGURE 1-14 Effect of microbiologist training on the isolation of NMNS.

Actinomycetes remain a major source of novel, therapeutically relevant natural products. To best exploit this group, a continuing source of geographically and ecologically diverse soil samples along with experienced microbiologists trained in the identification, classification, and preservation of actinomycetes are required. Taxonomic data on newly described genera should be evaluated in order to define media constituents and selective pressures that favor their growth over other actinomycetes, particularly streptomycetes. New materials and methods should then be incorporated into an isolation protocol and monitored for effectiveness in enhancing the isolation of novel, secondary metabolite-producing actinomycetes.

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CHAPTER 2

Bacteria As a Source of Novel Therapeutics

J.C. Hunter–Cevera and Angela Belt

Publisher Summary

This chapter discusses the bacteria as a source of novel therapeutics. In a study described in the chapter, Gluconobacter and Acetobacter species isolated from 41 samples were the two genera most frequently found to produce monobactams and were, therefore, positive in the Colworth agar droplet/B-lactamase screen for related beta lactams. The isolation methods that incorporated natural extract and low pH isolation media did favor development of these colony types. These strains produced acid and still maintained metabolic activity at low pH. The fermentation media was not buffered and thus, it can be hypothesized that low pH may trigger the formation of monobactams. It is also interesting that Takedas monobactam-producing strains were probably isolated from a low pH sample, as the strains were identified as Pseudomonas acidophila and P. mesoacidophila, both of which grow better at low pH compared with other members of this genus. Pseudomonas strains, which produced the beta-lactones, are another example where microbial ecology and metabolite production are correlated. Most isolates originated from a variety of old or decaying mushrooms. Considerable detail and attention should be given to sampling when trying to isolate the natural biota from a given ecosystem.

A naturalist once stated that the most important discoveries of the laws, methods and progress of nature have nearly always sprung from the examination of the smallest objects which she contains (Slater et al. 1983). Bacteria, being small objects in nature, come in a variety of sizes and shapes, and so do their secondary metabolites, some of which have proven to be useful as therapeutic agents for humans and animals.

Babes, a biochemist, in 1885 was the first to realize that microbial antibiosis was due to the production of an inhibitory chemical substance by the antagonistic organism (Vandamme 1984). Years later, microbial physiologists, Bouchard, Emmerich, and Loew made the extract pyocyanase from Pseudomonas aeruginosa, which was active against Corynebacterium diphteriae, Salmonella typhi, Pasteurella pestis, and other pathogenic cocci (Vandamme 1984). Pyocyanase was used for two decades as a treatment for a variety of infectious diseases. However, due to its toxicity, further research on this compound as an antimicrobial agent was not continued.

Interest in examining bacteria for antimicrobial activity was rekindled in 1939 when Dubos discovered tyrothricin, produced by Bacillus brevis  (Vandamme 1984). This work marked the beginning of the Golden Era of Antibiotics, during which in 1945, bacitracin, produced by Bacillus subtilis and B. licheniformis, was discovered along with polymyxins (B. polymyxa) and the gramicidins A, C, D, and S (B. brevis) (Berdy 1974,