Discovery and Isolation of Bioactive Natural Products from

Discovery and Isolation of Bioactive Natural Products from Actinomycete Bacteria

With an Examination of

Infectious Disease Drug Development and Application: A Comparison of the Developing

and Developed Worlds

By

Gregory Petrossian

A THESIS

submitted to

Oregon State University

in partial fulfillment of

the requirements for the

degree of

Bachelors of Science in BioResource Research with an Option in

Biotechnology and Bioproducts/Bioenergy

And

Bachelors of Arts in International Studies

Presented June 8, 2012

Commencement June 17, 2012

AN ABSTRACT OF THE THESIS OF:

Gregory Petrossian

Bachelor of Sciences in Bioresource Research, Microbiology, and International Studies

6/8/2012

Discovery and Isolation of Bioactive Natural Products from Actinomycete Bacteria

With an Examination of

Infectious Disease Drug Development and Application: A Comparison of the Developing

and Developed Worlds

Abstract Approved:

___________________________________

Dr. Mark Zabriskie

___________________________________

Dr. Kerry McPhail

The rise of resistant strains of bacteria (e.g., methicillin resistant Staphylococcus

aureus) has motivated the pursuit of new antibiotic compounds with novel mechanisms

of action. Rare non-Streptomyces actinomycetes are prime targets for the discovery of

novel natural products due to their tremendous metabolic diversity. Non-Streptomyces

actinomycetes have been isolated from soil samples collected from a unique Black Water

Ecosystem in Indonesia. As shown by screening in antimicrobial microtiter plate assays,

different culture media used to grow the bacteria elicited the production of diverse

biologically active natural products. In biological activity assays against Staphylococcus

aureus (S. aureus) extracts of test strains ICBB8352 (Amycolatopsis halotolerans) from

GOT medium culture and ICBB8340 (Nocardia seriolae) from M2 medium culture

inhibited bacterial growth, which was measured by comparing the optical density

(absorbance of light) of treated and unstreated S. aureus culture. The metabolites

produced by ICBB8352 and ICBB8340 were not effective at low concentrations (≤ 10

mg/mL), and thus were not good candidate antibacterials against S. aureus. The screening

process was successful in filtering out biologically inactive strain isolates. However, no

novel antibiotic natural products were found. Nevertheless, focusing on improvement of

natural product-based screening rather than high-throughput screening of synthetic

compound libraries, which possess limited structural diversity, has been the aim of

current pharmaceutical research and development.

Enduracidin was semi-purified from the commercial poultry feed additive

Enradin® for future semi-synthetic modification. On route to isolating enduracidin from

crude fermentation biomass, a bioactive fraction posed an opportunity for antibacterial

discovery. Eight compounds that inhibit the growth of S. aureus were detected in the

Enradin® extract. These compounds were determined to be fatty acid metabolites. This

class of compounds is a nuisance when screening natural products because fatty acids

often inhibit bacterial growth, but do not make good pharmaceutical drug candidates.

Discovery of novel natural products for antibiotic development by pharmaceutical

companies is confounded by competing interests with regard to the diseases targeted. The

developing and developed worlds have disparate challenges characterizing their fights

against infectious diseases. Furthermore, the developed world has experienced an

epidemiological transition of disease characterized by a considerable decline in the

burden of infectious diseases while chronic degenerative diseases have become more

prevalent. In contrast, the developing world has not had the infrastructure to implement

the same strategies that were successful in controlling infectious disease in the developed

world. A subset of infectious diseases defined as neglected tropical diseases (NTDs)

plague the developing world. Although NTDs are not currently a burden for the

developed world, the necessity for new drug development targets and new strategies for

equitable pharmaceutical distribution on a global scale is imminent.

Discovery and Isolation of Bioactive Natural Products from Actinomycete Bacteria

With an Examination of

Infectious Disease Drug Development and Application: A Comparison of the

Developing and Developed Worlds

By

Gregory Petrossian

A THESIS

submitted to

Oregon State University

in partial fulfillment of

the requirements for the

degree of

Bachelors of Science in BioResource Research with an Option in

Biotechnology and Bioproducts/Bioenergy

And

Bachelors of Arts in International Studies

Presented June 8, 2012

Commencement June 17, 2012

APPROVED:

_____________________________________________________________________

Dr. Mark Zabriskie, Department of Pharmaceutical Sciences

_____________________________________________________________________

Dr. Kerry McPhail, Department of Pharmaceutical Sciences

_____________________________________________________________________

Nick Fleury, Head Advisor, International Degree Program

_____________________________________________________________________

Dr. Katharine G. Field, BioResource Research Director

I understand that my thesis will become part of the

permanent collection of Oregon State University

Libraries. My signature below authorizes release

of my thesis to any reader upon request.

__________________________________________________________

Gregory Petrossian, Author

ACKNOWLEDGEMENTS

The author expresses sincere appreciation for Subsurface Biosphere Initiative (SBI)

summer undergraduate internship program, NIH research grant R01-AI073784,

Intervet/Schering Plough Animal Health, and Oregon State University.

CONTRIBUTION OF AUTHORS

Neal Goebel assisted with data collection and writing of Chapter 2. Dr. Dahai Zhang

assisted with data collection of Chapter 3. Dr. Mark Zabriskie assisted in the

interpretation of the data and writing of Chapter 2 and 3. Dr. Kerry McPhail assisted in

the writing of Chapter 1 and 4.

TABLE OF CONTENTS

Chapter 1

Section Page

Summary .............................................................................................................................1

Chapter 2

Section Page

Introduction ........................................................................................................................3

General Experimental Methods........................................................................................6

Results ...............................................................................................................................10

Discussion..........................................................................................................................13

Work Cited .......................................................................................................................14

Chapter 3

Section Page

Introduction ......................................................................................................................15

General Experimental Methods......................................................................................19

Results ...............................................................................................................................22

Discussion..........................................................................................................................32

Work Cited .......................................................................................................................34

Chapter 4

Section Page

Examination......................................................................................................................35

Work Cited .......................................................................................................................47

LIST OF FIGURES

Chapter 2

Figure Page

1 Simple media culture of strain ICBB8352,

Amycolatopsis halotolerans ..................................................................................11

2 Simple media culture of strain ICBB8340,

Nocardia seriolas...................................................................................................11

3 Microtiter bioassay results of strains 8340 and

8352 including growth inhibition (%) of S.aureus ............................................12

Chapter 3

Figure Page

1 Structure of enduracidin A .................................................................................24

2 Semi-pure enduracidin HPLC analysis of the

70% aq IPA extract fraction ...............................................................................26

3 Semi-pure enduracidin HPLC Area analysis ....................................................26

4 Disk diffusion assay of Enradin® bioactive

fractions with kanamyacin control .....................................................................27

Figure Page

5 Microtiter plate bioassay results of bioactive

fractions 37-41 from Enradin® against S. aureus .............................................28

6 HPLC chromatogram of bioactive fraction

with identified active peaks .................................................................................29

7 Mass spectra results of compound D ..................................................................30

8 Mass spectra results of compound E ..................................................................30

9 Compound E structure ........................................................................................31

LIST OF SCHEMES

Chapter 3

Scheme Page

1 Quantitative enduracidin isolation scheme ............................................................ 25

LIST OF TABLES

Chapter 4

Table Page

1 New antibiotics approved by the FDA ...............................................................46

2 Prevalence of resistance in hospital-acquired infections ..................................46

Chapter 1

Summary

New antibiotic compounds with novel mechanisms of action have become

essential in response to the recent paradigm of increasingly resistant strains of bacteria.

Secondary metabolites (natural products) from bacteria such as Actinobacteria serve as

lead compounds for the development of pharmaceutical drugs widely used to fight

bacterial, viral and fungal infections, as well as cancer and immune system disorders.

Pursuing the cultivation, isolation, and identification of rare non-Streptomyces

actinomycetes is necessary for antibiotic drug discovery (Chapter 2). The improvement of

natural products-based screening, rather than relying on synthetic sources, has been the

aim of current pharmaceutical research and development. Alternative strategies to

consider include the identification of potential new antibiotics from commercial crude

bacterial fermentations. For example, Enradin® by Intervet/Schering-Plough could

contain metabolites other than the major active ingredient presumed to be responsible for

its antibacterial action (Chapter 3). In examining the driving forces for novel drug

development one cannot ignore the fact that developing and developed worlds have

disparate challenges that characterize their fights against infectious diseases.

Pharmaceutical companies typically invest in research for diseases that will boost

financial returns rather than those diseases that impose the greatest global burden,

attributed largely to the developing world. A subset of infectious diseases defined as

neglected tropical diseases (NTDs) plague the poorest populations in the world and has

not been met with the same attention as infectious diseases directly impacting the

developed world. Analyzing the similarities and differences between the developing and

2

developed worlds in their fight against infectious diseases reveals the necessity for new

drug development targets and new strategies for equitable pharmaceutical distribution

catering to NTD disparities (Chapter 4).

3

Chapter 2

Discovery and Isolation of Bioactive Natural Products from Rare Non-Streptomyces Actinomycete Bacteria

Introduction

The cultivation, isolation, and identification of natural product-producing bacteria

are necessary tasks to pursue the identification of novel compounds for drug discovery.

One way to search for novel natural products begins with finding unique strains of

microorganisms. Multitudes of actinobacterial strains are noteworthy both as human

pathogens and as essential producers of antibiotics. Natural products produced by

actinomycetes most importantly include those with antibacterial activity against resistant

strains of pathogens, which have been on the rise since the introduction of modern

antibiotic chemotherapy in the 1940s. Actinomycetes are widely distributed in the

natural environment, and synthesize numerous natural products. These natural products,

or derivatives thereof are widely used in medicine to fight bacterial, viral and fungal

infections, as well as cancer and immune system disorders.1

The discovery of the antituberculosis agent streptomycin from the culture broth of

Streptomyces griseus by Waksman in 1944 provided the foundation for targeting the

genus Streptomyces and related Actinomycetales.2 Streptomyces have provided

antibacterials (e.g., erythromycin, and tetracycline), antifungals (e.g., amphotericin B),

anticancer (e.g., mitomycin C, lipstatin, thienamycin), antiparisitic (e.g., ivermectin), and

immunosuppressive (e.g., rapamycin) agents. The order Actinomycetales consists mostly

of Gram-positive, aerobic, and chemoorganotrophic bacteria. They grow in branching

filaments resembling mycelia, which sometimes break off into rod-shaped structures.

4

This fungus-like characteristic once caused wrong classification of these bacteria as

fungi, but proves to be a very beneficial trait. Their metabolic diversity and particular

growth characteristics, forming fungus-like mycelia and relatively rapid colonization of

selective substrates, signify well-suited agents for bioremediation of metal and organic

compounds.3 Searching for genes encoding for secondary metabolite synthesis reveals

gene clusters from just a couple to well over 30 pathways. Some families of

Actinomycetes regularly possess more than twenty gene clusters for secondary

metabolism, dedicating over 5% of their coding capacity to secondary metabolic

processes.4 With such metabolic diversity, there remains a vast potential for finding more

drug leads from actinomycetes. Only about 1-3% of the total number of natural products

produced by Streptomyces has been characterized.5

Despite the genetically-encoded metabolic diversity, the chance of finding

genuinely new biologically active molecules from the common Streptomyces sp.

actinomycetes is greatly reduced because many produce similar compounds.1 In

particular, repeatedly cultured “cosmopolitan” strains of Streptomyces result in the

reisolation of known natural products. The pursuit of rare non-Streptomyces

actinomycetes has been growing to investigate ways to eliminate the antibiotic discovery

bottleneck from Streptomyces. Traditional techniques focusing on Streptomyces

cultivation overlooked the development of rare non-Streptomyces screening. These rare

strains have been considered difficult to grow. However, altering media, heat treatment,

or adding antibiotics reduces competition from cosmopolitan species to promote the

growth of rare actinomycetes.6-9

Developing methods such as bacteriophage markers,10,11

ultrasonic treatments,12

and extremely high frequency irradiation,13

have promoted

5

selectivity for rare actinomyctes, allowing for more successful screening. Exploring

ecological niches and unique environmental conditions also play a crucial role in finding

rare strains.

Highly diverse microbial collections isolated from different environments should

be considered for development of novel natural product sourcing.14,15

Focusing on

specific sources and systematic approaches in the exploitation of ecosystems is required

to improve screening and success rate of novel natural product discovery.14,16

Marine

ecosystems are additionally investigated, and have proven to be a promising resource for

rare actinomycetes.17

Therefore rare, non-Streptomyces actinomycetes from soil samples

collected from an exclusive Black Water Ecosystem in Indonesia were screened for the

production of novel bioactive natural products. A number of distinct culture media were

utilized to provoke the formation of biologically active natural products.

6

General Experimental Methods

Turbidity of 96-well plate cultures was measured with a SpectraMAX 190

spectrometer reading 600 nm (Molecular Devices, Sunnyvale, California). Centrifugation

was performed on a GS-6R Beckman with CH3.7 rotor. Complex media cultures were

grown in sterile 6-well Falcon plates (BD Biosciences, New Jersey). Antimicrobial

assays were performed with sterile 96-well plates (Greiner Bio-One, North Carolina).

Brain and Heart Infusion Agar (BHIA) used for S.aureus culture medium (EMD

Chemicals, Darmstadt, Germany). Culture medium reagents obtained from BD

Biosciences and EMD Chemicals were used as received.

Simple Media - YPG medium: yeast extract (2 g), soy peptone (2 g), glucose (4

g), and agar (16 g) were dissolved in 1 L deionized (DI) H2O. TSB medium: tryptic soy

broth (30 g) and agar (16 g) were dissolved in 1 L DI H2O. NB medium: nutrient broth (8

g) and agar (16 g) were dissolved in 1 L DI H2O. ISP2 medium: yeast extract (4 g), malt

extract (10 g), mannitol (4 g), and agar (16 g) were dissolved in 1 L DI H2O. SWS

medium: soy peptone (1 g), soluble starch (10 g), and agar (16 g) were dissolved in 1 L

DI H2O. A-1 medium: soluble starch (10 g), yeast extract (4 g), soy peptone (2 g), and

agar (16 g) were dissolved in 1 L DI H2O.

Complex Media - M2 medium: glycerol (24 g), mannitol (25 g), soluble starch (25

g), glutamine (5.84 g), arginine (1.46 g), sodium chloride (1 g), potassium phosphate

monobasic (1 g), and magnesium sulfate (0.5 g) were dissolved in 1 L of DI H2O, then

added 2 mL of trace solution [0.1 g FeSO4, 0.01 g MnSO4, 0.01 g CuSO4, 0.01 g ZnSO4,

dissolved in 100 mL DI H2O]. M6 medium: casamino acids (5 g), calcium carbonate (4

g), molasses (10 g), peptone (5 g), and glycerol (10 g) were dissolved in 1 L DI H2O.

7

M23 medium: glucose (1 g), soluble starch (24 g), peptone (3 g), meat extract (3 g), yeast

extract (5 g), and calcium carbonate (4 g) were dissolved in 1 L DI H2O. M33 medium:

glucose (10 g), glycerol (5 g), corn steep liquor (3 g), beef extract (3 g), malt extract (3

g), yeast extract (3 g), calcium carbonate (2 g), glutamine (5.84 g), and arginine (1.46 g)

were dissolved in 1 L DI H2O. M50 medium: peptone (10 g), beef extract (8 g), yeast

extract (3 g), glucose (5 g), lactose (5 g), potassium phosphate dibasic (2.5 g), potassium

phosphate monobasic (2.5 g), magnesium sulfate (0.2 g), and manganous sulfate (0.05 g)

were dissolved in 1 L DI H2O. GOT medium: glycerol (60 g), oatmeal (15 g), tomato

paste (5 g), and calcium carbonate (3 g) were dissolved in 1 L DI H2O. All medium’s pH

adjusted within range of 7.3-7.6 with 2 N NaOH and 2 N HCl and sterilized at 121°C for

35 min.

Organism Collection. Bacteria were cultured from soil samples collected in Black Water

Ecosystems located at: Pangkoh Limalibu Village, Gandang Dubdistrict, Pulang Pisau

Regency, and Central Kalimantan Province in Indonesia. The river is identified as a

unique Black Water ecosystem, about 150 km from the coast of South Kalimantan. The

Actinomycete spp. ICBB8340, 8352, 8400, 8316, 8368, 8313, 8346, 8360, 8329, and

8314 were deposited from ICBB-CC (Indonesian Center for Biodiversity and

Biotechnology Culture Collection of Microorganisms) as 0.5 mL of a 20% glycerol stock

stored at -80 °C.

8

Simple Media Culture. Organisms were transferred from ICBB-CC stock and streaked

on petri-dishes with each medium: YPG, TSB, NB, ISP2, SWS, and A-1. Organisms

were incubated for 3-5 days at 30 °C.

Small-scale Complex Media Fermentation and Isolation. Organisms were transferred

from simple media culture and cultivated in 6-well plates with each M2, M6, M23, M33,

M50, and GOT mediums (7.5 mL) at 29° C for 6-9 days on a rotary shaker (125 rpm).

Mycelia from each culture were harvested and extracted with 1:1 acetone:MeOH (7.5

mL). Insoluble materials were filtered and concentrated. Metabolites were then

redissolved in 1:1:1 EtoAc:MeOH:acetone, transferred, and reconcentrated to remove

excess media. Metabolites were tested for activity against S.aureus by antimicrobial

assay in concentrations of: 50 and 5 mg/mL.

96-Well Plate Bioassay. Antimicrobial assays were performed using Greiner bio-one 96-

well plate, following the method described by Smith et al.15

Kanamycin was employed in

serial dilutions (256, 128, 64, 32, 16, 8, 4, 2, 1 ug/mL) as a susceptibility control.

Samples dissolved in 70% aq MeOH were transferred to a sterile 96-well plate and

concentrated. Samples were redissolved in 10% aq DMSO (20 µL) and then media (80

µL) was added. The wells were then inoculated with media (100 µL) containing S. aureus

(~5×10^5 CFU/mL). Initial absorbance for each well was measured and the plate was

incubated for 17 hrs at 37 ºC. The absorbency for each well was then measured and used

to identify active metabolites.

9

Large-scale Complex Media Fermentation and Isolation. Organisms were transferred

from simple media cultures and cultivated in respective complex media (250 mL) at 30

°C for 6-9 days on a rotary shaker (125 rpm). Mycelia and culture broth were separated

via centrifugation (20 min at 3000 rpm). Culture broth was extracted with ethyl acetate

(250 mL) in a separation funnel, decanted, and concentrated. The mycelia pellet was

extracted with 1:1:1 MeOH:DCM:acetone (120 mL), filtered, and concentrated. The

mycelium and culture broth extracts were tested for activity against S.aureus by

antimicrobial assay in serial dilution concentrations (10, 5, 2.5, 1, 0.5, 0.25 mg/mL).

10

Results

Small-scale complex media cultures of strains ICBB8316, 8340, 8352, 8368, 8400

were screened for bioactivity by 96-well plate bioassay. Significant growth inhibition was

considered to be a greater than 50% decrease in absorbance when measured by

spectrophotometry. Strains ICBB8316, 8352, 8368, and 8400 from all complex medium

cultures did not produce significant growth inhibition of S.aureus. Strain ICBB 8352

(Figure 1) from the GOT medium culture (50 mg/mL) resulted in growth that was 16%,

15%, and 15% of controls (Figure 3); indicating bioactivity. Strain ICBB8340 (Figure 2)

from M2 medium culture (30 mg/mL) resulted growth that was 31%, 45%, and 53% of

controls (Figure 3); indicating bioactivity. A large-scale culture of ICBB8340 in M2

medium produced a pastel pink culture broth. Crude extracts were prepared from the

mycelia and culture broth as indicated in the experimental section. The mycelia extract

was a pinkish orange and the culture broth extract was bright orange. Both extracts were

tested by 96-well plate bioassay and resulted in no bioactivity at any concentration.

Strains 8340 and 8352 were given to Dr. Dahai Zhang for verification of

bioactivity by disk diffusion bioassay. Results returned with no significant activity

against S.aureus. The metabolites produced by ICBB8352 in GOT medium and

ICBB8340 from M2 medium were not effective at low concentrations (≤ 10 mg/mL), thus

not powerful agents against S.aureus. Dr. Zhang carried out the screening of remaining

rare non-Streptomyces actinomycete soil bacteria isolated strains.

11

Figure 1 – Simple media culture of strain ICBB8352, Amycolatopsis halotolerans

Figure 2 – Simple media culture of strain ICBB8340, Nocardia seriolae

12

Figure 3: Microtiter bioassay results for strains 8340 and 8352 including growth

inhibition (%) of S.aureus. Labels for experimental wells: 1C is M2 media, 2C is M23

media, 3C is M33 media, 4C is M50 media, 5C is M6 media, and 6C is GOT media.

Positive control was kanamycin.

13

Discussion

The extracts exhibiting bioactivity were effective at 30 and 50 mg/mL, where as

the positive control was effective at 8 µg/mL. The bioactive strains cultured in their

specific medium were screened at ≤10 mg by Dr. Dahai Zhang, and did not inhibit

S.aureus growth. Although these were crude metabolite extracts, ideal crude metabolites

produce inhibitions below 1 mg. Different fermentation mediums were successful in

triggering alternative metabolite production. Each bioactive strain in their respective

medium did not produce the same S.aureus growth results when fermented in other

mediums. Strain ICBB8340 from M2 medium and strain ICBB8352 from GOT medium

share a glycerol ingredient, but differ in sources of proteins and sugars. The M2 medium

relies on mannitol and soluble starch for carbohydrate sources, and arginine and

glutamine for nitrogen sources. The GOT medium relies on commercially available

oatmeal and tomato paste for both protein and carbohydrate sources.

Use of these unique mediums for future fermentation endeavors will be beneficial

in provoking alternative metabolite pathways. This is an important technique for

screening actinomycete strains due to their propensity for secondary metabolic synthesis.

These findings support the fact that actinomycete gene clusters4 house a vast array of

secondary metabolic pathways. The unique mediums activate these genes from the stress

of different nutrient sources. Dr. Zhang carried out the screening of the remaining rare

non-Streptomyces actinomycete soil bacteria isolated strains using the same six unique

mediums.

14

Work Cited

(1) Bredholdt, Harald, et. al. Environ. Microbiol. 2007, 9, 2756-2764.

(2) Waksman, S. A. Science. 1953, 118, 259–266.

(3) Polti, A. Marta, Amoroso, Maria, and Abate, M. Carlos. Chemosphere. 2006, 67, 660-

667.

(4) Nett, Markus, Ikeda, Harou, and Moore, S. Bradley. Nat. Prod. Rep. 2009, 26, 1362-

1384.

(5) Baltz, R. H. J. Ind. Microbiol. Biotechnol. 2006, 33, 507-513.

(6) Bredholdt, H., et. al. Environ Microbiol. 2007, 9, 2756-2764.

(7) Qio, D., Ruan, J, and Huang, Y. Appl. Environ. Microbiol. 2008, 74, 5593-5597.

(8) Hamaki, T., et. al. J. Biosci. Bioeng. 2005, 99, 485-492.

(9) Lazzarini, A., Cavaletti, L., Toppo, G., and Marinelli, F. A. Van. Leeuw. J. Microb.

2001, 79, 399-405.

(10) Gathogo, W.N. Esther, et al. Biotech. Letters. 2004, 26, 897-900.

(11) Kurtboke, D.I. Appl. Microbiol. Biot. 2010, 89, 931-936.

(12) Jiang, Y., et al. Microbiol. 2010, 50, 1094-1097.

(13) Li, Y. V., Terekhova, P., and Gapochka M.G. Microbiol. 2003 71, 105-108.

(14) Lam, K. S. Trends Microbiol. 2007, 15, 279-289.

(15) Knight, V. Sanglier, et. al. Appl. Microbiol. Biotech. 2003, 62, 446-458.

(16) Czaran, T. L., Hoekstra R. F., and Pagie L. Proc. Natl. Acad. Sci. 2002, 99, 786-790.

(17) Fiedler, Hans-Peter. A. Van Leeuw. J. Microb. 2005, 87, 37-42.

15

Chapter 3

Isolation of a New Antibiotic from a Commercial Feed Additive

Introduction

The loosely defined word “antibiotic” has become a descriptor for any molecule

with the ability to inhibit growth of microbes. Therapeutic agents may be derived from

animals, plants or microbes, or produced synthetically. These compounds have improved

life expectancy ever since the commercialization of the first antibiotic, penicillin, in the

1940s. Natural products have had a central role in the development of new therapeutics

such as: cholesterol lowering statin drugs (e.g., lovastin), antibacterial agents (e.g., β-

lactams, macrolides, aminoglycosides, tetracyclines), antifungals (e.g., echinocandins,

griseofulvin), antimalarials (e.g., artemisinin, quinine), anticancer agents (e.g,. paclitaxel,

vinblastine, doxorubicin), and immunosuppressants (e.g. cyclosporine).1 These

compounds are sought after through means which must boast bioactivity as well as

financial returns. Financial burdens strain the attractiveness of natural products due to

lengthy isolation protocols and screening. Commercially available compound collections

provide rapid entry into screening endeavors without the tedious fermentation/collection,

isolation, and structural determination intrinsic to natural products research. The

pharmaceutical industry’s growing reliance on curated archives and combinatorial

libraries of “drug-like” compounds was seen as a hope to speed up drug discovery. These

methods have profoundly affected the quantity of new drug leads in many areas, but not

antibiotics.2

Interest in natural product-based drug discovery declined in the 1980s and 1990s

due to advances in combinatorial chemistry coupled with high-throughput screening

16

methods. The accompanying decline in the raw number of novel drug candidates,

however, has sparked an interest in re-expanding natural product-based efforts.3

Pharmaceutical companies and academic labs working with compound libraries based

closely upon the core scaffold of individual complex natural products mimicking

structural properties have better screening results.2 Focusing on improvement of natural

product-based screening rather than relying on synthetic alternatives has thus been the

aim of current pharmaceutical research and development.

Natural products have been more widely sought after in tandem with the creation

of archives to push the envelope for novel antibiotics. Compared to synthetic compounds,

natural products inherently have higher molecular weights; more stereogenic centers;

fewer rotatable bonds; larger, more complex ring systems; lower nitrogen, sulfur, and

halogen content; higher oxygen content; and more hydrogen bond donors and acceptors.4

These characteristics allow for a multitude of modifications to potentially create analogue

bioactive compounds. The complex structures of natural products inspire creative

advancements in synthetic organic chemistry as the scaffolds that medicinal chemistry

has exploited in the development of numerous new synthetic pharmaceuticals.1

Importantly, natural products have evolved specifically to interact with biological

macromolecules, namely proteins and nucleic acids.5 Consequently natural products

yield higher bioactivity hit rates and greater potency rather than synthetically produced

small molecule libraries.6 Pursuing these natural products as the foundation for antibiotic

discovery must be emphasized before relying on synthetic and combinatorial libraries.

Opponents to compound libraries claim that specific structures with track records of

biological activity must be pursued instead of randomly synthesized compounds.7

17

As part of a project to identify new antibiotics from bacterial fermentations, we

sought to determine if we could find new bioactive compounds from commercially

produced fermentation biomass products. Crude, unpurified fermentation products such

as Enradin® by Intervet/Schering-Plough, contain metabolites other than the target

bioactive compounds. In the case of Enradin, this is enduracidin, which composes only 4-

8% dried weight. Enduracidin is a potent peptide antibiotic used as a commercial poultry

feed additive first marketed in the 1970’s. Enduracidin demonstrates strong in vitro and

in vivo antibacterial activity against a wide range of Gram-positive bacteria, including

methicillin-resistant Staphylococcus aureus (MRSA).8,9

Tests in 1971 against bacteria

with resistance to numerous different antibiotics revealed no cross-resistance or acquired

mutational resistance to enduracidin.8 Since then there still has been no known

mechanism of resistance to enduracidin. This is attributed to its unique mechanism of

inhibiting peptidoglycan biosynthesis.10

However, controversy over the use of antibiotics

in animal feeds inducing resistant-strain bacteria, remains prevalent.

Animals such as the cow, pig, and chicken have become staples in the human diet.

These animals have overlapping reservoirs of drug resistant bacteria that thus impact

human health. Public health concerns, exemplified by VanA-type glycopeptide-resistant

enterococci (GRE) being transferred from an agricultural reservoir to humans, have

caused the ban of avoparcin. Avoparcin, an antibacterial commercial chicken feed

additive introduced in 1986, showed a direct association between the use of avoparcin

and the occurrence of GRE.11,12

The banning of avoparcin began in Denmark and Norway

in 1995, in Germany in 1996, and finally in all the European Union in 1997. It was

assumed that removal of the selective antimicrobial pressure would reverse glycopeptides

18

resistance by causing GRE to be less competitive than non-GRE counterparts, due to the

biological cost of harboring the resistance plasmid. However, Norwegian poultry farms

retain a high prevalence of GRE-positive poultry flocks 3 to 8 years after the avoparcin

ban.13

This persistence is disturbing because the resistance determinant may be

transferable to other bacterial species pathogenic to humans. A pathogenic strain,

glycopeptide-resistant Enterococcus faecium, has already been isolated from human

samples. Agricultural exposure to antibiotics has additionally become an environmental

concern. Environmental releases of antibiotics from the previously described animal

feeding operations have caused regulatory action.

Environmental concerns are important when considering antibiotic residues

reaching surface water, groundwater collection systems, and surrounding soil.

Agricultural-use antibiotics and their metabolites are excreted in feces and urine,

affecting terrestrial and aquatic organisms, which can lead to development of antibiotic-

resistant strains of microorganisms. Current studies show that antibiotics are omnipresent

at the surface and in the waste-stream of dairy farms, but do not accumulate extensively

in the soil.14

Further research must be done to evaluate whether the low but continual

occurrence of antibiotics at farm surfaces affects the ecosystem and microbial community

in developing antibiotic resistance.

19

General Experimental Procedures

HPLC was performed on a Shimadzu Prominence instrument equipped with SIL-

20A autosampler and PDA detector reading 210, 254, 278, 310 nm (Shimadzu

Corporation, Kyoto, Japan). A Phenomenex Synergi 4u Hydro-RP 80A (250×10.0 mm

4µm) and a Phenomenex Synergi 4u Fusion-RP 80A (250×10.0 mm 4µm) were used for

HPLC purification (Torrance, California). A Phenomenex Synergi 4u Fusion-RP 80A

(250×4.60 mm 4µm) and a Phenomenex Gemini 5u C18 110A (150×4.6 mm 5µm) were

used for HPLC analysis. Mass spectra were obtained using a Thermo Finnigan LCQ

Advantage (Thermo Fisher Scientific, Massachusetts) instrument in ESI negative ion

mode. Turbidity of 96-well plate cultures were measured via SpectraMAX 190

spectrometer reading at 600 nm (Molecular Devices, Sunnyvale, California).

Antimicrobial assays were performed with sterile 96-well plates (Greiner Bio-One, North

Carolina). Thin-Layer Chromatography (TLC) was performed on aluminum-backed silica

gel 60 F254 (EMD Chemicals Inc., Darmstadt, Germany). Centrifugation was performed

on a GS-6R Beckman with CH3.7 rotor. Brain and Heart Infusion (BHI) broth (EMD

Chemicals Darmstadt, Germany) was used for S.aureus culture medium. Solvents were

obtained from VWR International LLC (Sacramento, California) and used as received.

Enradin® was a gift from Intervet/Schering-Plough Animal Health (Whitehouse Station,

New Jersey).

Enduracidin Isolation. Enradin® (20 g) was extracted with 2:1 75 mM phosphoric

acid:acetone (200 mL) for 24 hours by stirring with magnetic stir bar. The insoluble

material was removed via centrifugation (20 min at 3000 rpm). The crude extract was

20

decanted, mixed with silica gel (220 g) and concentrated under reduced pressure. The

dried silica gel mixture was packed in a column with fritted glass filter. The column was

eluted with the following solvents, in order: THF (1 L), DCM (1 L), 90% aq MeCN (1

L), and 80% aq MeCN (2 L). The eluted fractions were concentrated and analyzed by

TLC and compared to an enduracidin standard. The dried material from the 80% aq

MeCN elution (1.0 g) and XAD-16 resin (25 g) was mixed in 75% aq MeOH (100 mL)

then concentrated. The dried resin and elution mixture was packed in a column with a

fritted glass filter. The column was eluted with solvents, in order: deionized H2O (0.5 L),

20% aq IPA (1.5 L), 25% aq IPA (250 mL), 30% aq IPA (250 mL), 70% aq IPA (250

mL), and MeOH (250 mL). HPLC was employed to analyze the purity of enduracidin

dispersed among the various fractions.

Isolation of Bioactive Compounds from Enradin. Enradin® (60 g) was extracted in 2:1

75 mM phosphoric acid:acetone (900 mL) for 24 hours by stirring with magnetic stir bar.

The insoluble material was removed via centrifugation (20 min at 3000 rpm). The crude

extract was decanted, mixed with silica gel (150 g) and concentrated under reduced

pressure. The dried silica gel mixture was packed in a fritted funnel and extracted with

THF (2 L). The THF fraction was loaded on to a silica gel column (5 cm ×13 cm) and

eluted with 1% MeOH in DCM (250 mL) and 5% MeOH in DCM (500 mL). The

fractions were pooled according to TLC analysis and tested for bioactivity via disk

diffusion assay against S.aureus. Indicator dye Thiazolyl Blue Tetrazolium was utilized

for the disk diffusion assay.

21

Purification of Bioactive Compounds from Enradin. The bioactive fraction was

analyzed by HPLC using a Synergi 4u Fusion column. Individual peaks were collected in

a 96 well plate and tested for activity against S.aureus by antimicrobial assay. Bioactive

peaks were labeled A-H and isolated via a HPLC method for larger scale isolation.

96-Well Plate Bioassay. Antimicrobial assays were performed using a 96-well plate,

following the method described by Smith et al.15

Kanamyacin was employed in serial

dilutions (256, 128, 64, 32, 16, 8, 4, 2, 1 ug/mL) as a susceptibility control. Samples

dissolved in 70% aq MeOH were transferred to a sterile 96-well plate and concentrated.

Samples were redissolved in 10% aq DMSO (20 µL) and then BHI media (80 µL) was

added. The wells were then inoculated with media (100 µL) containing S. aureus

(~5×10^5 CFU/mL). Initial absorbance for each well was measured and the plate was

incubated for 17 hrs at 37 ºC. The absorbency for each well was then measured and used

to identify active compounds.

22

Results

The isolation and purification of enduracidin (Figure 1) from Enradin® is

outlined by Scheme 1. This isolation described yielded a total of 16.1 mg of semi-pure

enduracidin from 20 g of the commercial product. HPLC analysis of the semi-pure

enduricidin (Figure 2) revealed a 77.77% total composition of pure enduracidin by area

analysis (Figure 3).

On route to isolating enduracidin other bioactive compounds were found,

highlighting the effectiveness to screen all fractions for bioactivity. The bioactive THF

fractionation of Enradin® was further separated by silica gel column as explained in the

experimental procedures. Fractions 37 – 41 identified by TLC resulted in bioactivity

against S.aureus by disk diffusion assay (Figure 4).

Individual peaks from fractions 37 – 41 were collected by HPLC and tested for

bioactivity by 96-well plate bioassay (Figure 5). The HPLC utilized Synergi Fusion 4u

column (250 x 4.6 mm) with a gradient from 20% B to 100% B (A: water + 0.1% acetic

acid, B: MeCN) over 45 minutes. Well 17 (peak A) inhibited growth to 0.07%. Well 20

(peak B) inhibited growth to -0.23%. Wells 23 and 24 (peak C) inhibited growth to -

0.27% and -0.29% respectively. Wells 29, 30, and 31 (peak D) inhibited growth to

21.4%, -6.37%, and -0.21% respectively. Wells 32 and 33 (peak E) inhibited growth to -

11.84% and 8.72% respectively. Wells 52 and 53 (peak F) inhibited growth to 20.96%

and 13.57% respectively. Well 54 (peak G) inhibited growth to 14.54%. Wells 62 and 63

(peak H) inhibited growth to 1.56% and 3.91% respectively. These peaks were presented

and labeled by HPLC chromatogram (Figure 6).

23

Compounds A-C & F-H: These compounds were determined to be fatty acid analogues

of the characterized compound E. Compound D and E were pursued due to unique UV

absorbencies (Figure 6).

Compound D: White powder; retention time was 23 minutes. Mass spectrum results

provided (Figure 7).

Compound E: White powder; retention time was 24.5 minutes. Mass spectrum results

provided (Figure 8). Structure determined to be unsaturated fatty acid Figure 9.

24

Figure 1 – Structure of enduracidin A.

O OHN

NH

HN

NH

HN

NH

NH

O

O

O

HN

O

HN

OH

O

OHO

OH

O

HO

NH

HN

NH

HN

O

O

Cl

OH

Cl

O

OHO

O

OH

HN NH

HN

OH

O NHOH

HN

NH

O

O

HN

NH

NH

NH

H2N

O

O

NHHO2C

Enduracidin A

L-Asp1 D-Hpg3

L-allo-Thr8

L-Thr2

L-Hpg17

D-Orn4

D-Ala16

D-End10L-End15

Gly14

L-Dpg13

D-Ser12

L-Hpg11

L-Cit9

D-Hpg7

L-Hpg6

D-allo-Thr5

NH2

O

25

Scheme 1 - Quantitative enduracidin isolation scheme. Enduracidin was purified from

30% aq IPA and 70% aq IPA (total yield: 16.1 mg) fractions.

26

Figure 2 – Semi-pure enduracidin HPLC analysis of the 70% aq IPA extract fraction.

Figure 3 – Semi-pure enduracidin HPLC Area analysis (70% aq IPA).

27

Figure 4 – Disk diffusion assay of Enradin® bioactive fractions with kanamyacin (256

µg/mL) control.

28

Figure 5: Microtiter plate bioassay results of bioactive fraction 37-41 from Enradin®

against S. aureus.

29

Figure 6: HPLC chromatogram of bioactive fraction with identified active peaks: A = 17,

B = 20, C = 23/24, D = 29/30/31, E = 32/33, F = 52/53, G = 54, H = 62/63 (compound ID

= Figure 1.5 well numbers).

30

Figure 7 – Mass spectrum for compound D.

Figure 8 – Mass spectrum of compound E.

31

Figure 9 – Compound E structure.

32

Discussion

The HPLC separation method (Figure 1.6) allowed the immediate pursuit of

individual peak purification due to good separation between compounds. This effective

HPLC method bypassed further fractionation and method development. Purified

compound E was determined by NMR to be a fatty acid, as was the related compound D

analogue. The structure of compound E was pursued because it exhibited a broad range

of UV absorbencies by HPLC (Figure 1.9). All of the isolated compounds were reasoned

to have similar fatty acid properties. This finding is a common nuisance for natural

product discovery because fatty acids often inhibit bacterial growth, but do not make

ideal antibacterials. Fatty acids can incorporate themselves into the cell’s membranes and

disrupt continuity. These fatty acids act like a surfactant by lysing cells and solubilizing

molecules. Thus Enradin® does not contain other significant bioactive compounds

besides enduracidin.

Although manual screening detected alternative compounds or secondary

metabolites, they were not necessarily good antibacterial candidates. The process of 96-

well plate bioassay succeeded at detecting µg amounts of metabolite affecting growth of

S.aureus. The bioassay method proved to be an accurate method for bioactivity screening

with consistent controls. This holds potential for screening other fermentation products

from novel bacterial strains. The same method was used in Part II to screen multiple

strains of fermented cultures. The enduracidin isolation scheme outlined by Scheme 1.1

yielded ample semi-pure enduracidin (16.1 mg) for future work of semi-synthetic

modification. Neal Goebel, a graduate student in the Zabriskie lab, will be using with this

33

enduracidin to make derivatives with improved solubility, while retaining antimicrobial

activity.

34

Work Cited

(1) Berdy, J. J. Antibiot. 2005, 58, 1-26.

(2) Tan, Derek S. Comb. Chem. High T. Scr. 2004, 7, 631-643. (3) Koehn, F. E., and Carter, G. T. Nat. Rev. Drug Discovery 2005 4, 206–220.

(4) Feher, M., Schmidt, J.M. J. Chem. Inf. Comput. Sci. 2003, 43, 218-227.

(5) Piggott, A. M., and Karuso, Comb. Chem. High T. Scr. 2004 7, 607–630.

(6) Koch, M. A., et. al. H. Proc. Natl. Acad. Sci. U.S.A. 2005 102, 17272–17277.

(7) Rouhi, A.M. Chem. Eng. News 2003, 81, 104-107.

(8) Goto, S.; Kuwahara, S.; Okubo, N.; Zenyoji, H. J. Antibiot. 1968, 21, 119–125.

(9) Tsuchiya, K.; Kondo, M.; Oishi, T.; Yamazaki, I. J. Antibiot. 1968, 21, 147–153.

(10) Fang, Xiao, et. al. Mol. Biosyst. 2005, 2, 69-76.

(11) Aerestrup, F. M. Microb. Drug Resist. 1995, 1, 255-257.

(12) Kruse, H., B. K. Johansen, L. M. Rorvik, and G. Schaller. Microb. Drug Resist.

1999, 5, 135-139.

(13) Sorum, M., et. al. Environ. Microbiol. 2005, 72, 516-521.

(14) Watanabe, Naoko. Environ. Sci. Technol 2010, 44, 6591-6600

(15) Smith et al. J. Microbiol. Methods. 2006, 72, 103-106

35

Chapter 4

Infectious Disease Drug Development and Application: A Comparison of the Developing and Developed Worlds

Infectious diseases have been the leading cause of the global health burden for

most of recorded history. The developed and developing worlds have diverged in their

approaches fighting against infectious diseases. In the developed world there has been an

epidemiological transition of disease as tremendous progress was made in healthcare over

the 19th

and 20th

centuries. The epidemiological transition is characterized by a

considerable decline in the burden of infectious diseases while chronic degenerative

diseases have become more prevalent. In contrast, the developing world has not had the

infrastructure to implement the same strategies that were successful in controlling

infectious disease in the developed world. Immunization programs have been successful

and cost-effective public health programs worldwide accounting for the prevention of 2

million child deaths in 2003 alone. However, there were still an estimated 2.5 million

childhood deaths from vaccine-preventable diseases, most of which occur in the

developing world.1 The World Health Organization describes establishing and

maintaining high immunization coverage rates in many of the poorest developing

countries as “challenging due to high population growth rates, limited infrastructure and

resources, and fluctuating demand for services”.2 It is estimated that mortality due to

infectious diseases in developing countries accounts for >25% of deaths worldwide and

over 40% of deaths in developing countries, significantly more than in developed

countries.3 Other important measures of disease burden (or morbidity), such as cognitive

36

and developmental delays or economic impact, reveal an even greater difference between

the epidemiological transitions of the developed versus developing world.1 There are

characteristic differences between the developed and developing worlds attributed to

infectious diseases.

The developed world may have better access to care and a stronger infrastructure

than the developing world, but infectious diseases are still a significant challenge. The

epidemiological transition began in the 19th

century with the development of “germ

theory” and significant advances in three main areas that lead to dramatic declines in

death rates due to infectious diseases: sanitation and hygiene, immunizations, and

antibiotics.4 Immunizations that prevent the spread of infectious diseases had the greatest

impact on reducing the burden of infectious diseases in the 20th

century.5 The growing

number of vaccines has led to the near elimination of diseases that had once been

common causes of childhood morbidity and mortality. The greatest success in the use of

vaccines was in 1977 after a decade-long campaign involving 13 countries which lead to

the eradication of smallpox from the world.6 In addition, over the last 60 years new

classes of antibiotics against a range of disease-causing pathogens have been developed

and have contributed significantly to reducing mortality from many infectious diseases.

However, with the rise of resistant strains these antibiotics are becoming obsolete.

Developed countries have strict regulations governing pharmaceuticals to try and

reduce resistant strains of bacteria and viruses. Diagnostic tools aid doctors to pin point

causes of disease and reduce the use of broad spectrum antibiotics, further combating

pharmaceutical drug resistance. Disease awareness and information is also more

efficiently disseminated in developed countries. The emergence of HIV and other

37

infectious diseases such as Ebola, bovine spongiform encephalopathy (“mad cow

disease”), severe acute respiratory syndrome (SARS), avian influenza, and West Nile

virus resulted in increased awareness of infectious diseases in the developed world.7

There has also been growing recognition in developed countries of the global

vulnerability to diseases emerging from developing countries, as well as a genuine

humanitarian concern for those in need. This has led to greater interest in diseases

endemic to the developing world.8 Fortunately numerous philanthropic governmental and

non-governmental organizations are making efforts to provide financial and technical

support to aid the developing world in reaching their epidemiological transition. Several

of the more advanced “developing” countries around the world appear to be on their way

to achieving the same transition the developed world experienced, with declining

mortality rates from infectious diseases. However, not all countries have been able to

achieve such progress. There has been ongoing debate and concern regarding the delayed

transition, or even regression, of some of the developing world.9

The developing world has experienced slower progress in increasing life

expectancy and control of infectious diseases due to multiple compounding factors. The

first obvious disadvantage is the socioeconomic discrepancy between population growth

and limited resources. It has been estimated that if the “under the age of five” mortality

rate in the poorest 80% of the population of the developing world could be reduced to

that of the richest 20%, global mortality would be reduced by 40% theoretically.10

The

WHO describes three key factors that contribute to the burden of infectious diseases in

developing countries: failure to use existing tools effectively, inadequate or non-existent

diagnostic tools, and insufficient knowledge of diseases.11

The lack of diagnostic tools

38

results in use of broad spectrum antibiotics and repeated trial-and-error prescriptions,

further exacerbating antibiotic resistance. Additionally, a subset of infectious diseases

defined as neglected tropical diseases (NTDs) plague the poorest populations in the

world. This subset includes 13 parasitic and bacterial infections: helminth infections

(ascariasis, hookworm infection, and trichuriasis), lymphatic filariasis, onchocerciasis,

dracunculiasis, schistosomiasis, Chagas disease, human African trypanosomiasis,

leishmaniasis, Buruli ulcer, leprosy, and trachoma.12

The term “neglected” refers to the

lack of attention to these diseases of the developing world due to the focus on tackling

HIV/AIDS, tuberculosis, and malaria. Without exact figures on the extent of neglect it is

difficult to catalyze change and demand further support from governmental and non-

governmental bodies.13

The top ten leading causes of healthy life lost to long-term

disability and premature deaths worldwide are due to NTDs.14

Sub-Saharan Africa bears

the largest burden of many NTDs with over 90% of the world’s burden for these diseases.

Such NTDs have severe socioeconomic consequences on a developing nation’s

population as they cause long-term illness, disfigurement, social stigma, and decreased

productivity. Since NTDs affect worker productivity, households and employment firms

must adapt their productive activities in response. These coping strategies can lead to low

savings and investment, lost capital and purchasing power, and inefficient labor

substitution.15

This macroeconomic impact further hinders the progress of developing

countries. Treatments have been lacking for these diseases mainly because they do not

offer sufficient financial returns for the pharmaceutical industry to engage in research and

development. Pharmaceutical drug development discrepancies emphasize the

39

disadvantages of developing countries fighting against infectious diseases, specifically

NTDs.

The pharmaceutical industry must combat the double-edged sword facing drug

development. The dilemma of pharmaceutical development is fighting against antibiotic

resistance in the face of an “antibiotic drought”. The innovation gap for novel antibiotics

is evident in the dramatically reduced number of new antibiotic approvals by the United

States Federal Drug Administration (FDA) over the past two decades (Table 1). Market

saturation of antibiotics and over prescribing in the 1970’s when resistance was not an

issue caused rapid resistance to first generation antibiotics. Pharmaceutical company

profits for new products have fallen remarkably, affecting investment in research and

development. The growing trend of doctors prescribing older antibiotics as first-line and

new antibiotics only as second-line may reduce the rate of development of resistance but

also makes new antibiotics much less profitable. The decline in the raw number of novel

drug candidates has sparked interest in revisiting natural product-based efforts even

though the screening of biologically diverse organisms, such as rare actinomycete

bacteria, for production of metabolites with antibiotic properties is an expensive and time

consuming process. The cosmopolitan actinomycete genus Streptomyces is estimated to

be capable of producing antimicrobials to the order of 100,000 – only a tiny fraction of

which has been discovered so far.16

Initial investment in natural products drug discovery

from actinomycete bacteria involves searching for and isolating unique strains of

actinomycetes from samples harvested from equally unique ecosystems. These

ecosystems are often found in the biologically diverse Tropics, particularly in

inaccessible, mostly uninhabited, and thus under-developed regions of the world.

40

Consequently it is ironic that the brunt of the innovation gap in drug development is

borne by the infectious diseases characteristic of the developing world, which includes

most of the Tropics. Recent advances in microsale, high throughput technologies have

made the natural product screening process more efficient. Similarly, advances in

laboratory cultivation techniques, arising from a better understanding of microbial

metabolism, have permitted the induction of a broader array of diverse secondary

metabolite (natural product) pathways, many of which are only expressed in response to

certain environmental stimuli.

New antibiotic developments against NTDs are necessary to replace inadequate

current treatments that require particular repeated injectable doses. Current treatments are

difficult for those living in the developing world due to the lack of access to treatment

centers and poor implementation of proper antibiotic repeat dose regimens. The NTDs

represent a substantial global burden at 11.4%. Only 1% of the 1393 new chemical

entities marked between 1975 and 1999 were registered for these diseases: 13 for tropical

disease indications and three for tuberculosis.11

The imbalance is also evident in the

overall level of pharmaceutical industry-investments in research and development: of the

$35.3 billion invested in 1999, 10.1% was spent on infectious diseases.11

The costly and

high-risk activity of pharmaceutical research and development is the argument put forth

by the pharmaceutical industry to justify the exorbitant cost of new chemical entities.

Maintaining a low cost for NTD control is imperative and thus must be incentivized to

encourage private investment in the development of new cost-effective medications or

public-private partnerships. Low costs for NTD control are determined by four factors:

the commitment of pharmaceutical companies to provide subsidized medicines; the scale

41

of the program; the potential for synergizing delivery methods; and the volunteer

contribution of communities (including educators) in the distribution of medicines.12

Provision of cost-effective medicines is often referred to as push and pull mechanisms.

Push mechanisms are incentives that operate upstream during the research and

development process, involving costs to the public sector without a guarantee that a

viable medicine will be delivered. Incentives, for example, can be tax credits to lower

costs. Pull mechanisms operate downstream to offer public incentives for development of

a product. In exchange for the increased market attractiveness (subsidy), private

pharmaceutical companies are expected to increase their research and development

efforts in the area of infectious diseases.11

Public-private partnerships (PPPs) attempt to

meet the needs of developing countries through establishing public-private

collaborations, networks, and partnerships. This new paradigm for drug development

activities has had a large impact on the international health market, particularly the

pharmaceutical sector. Initiatives focus mainly on converting therapeutic candidates into

registered entities using a social venture capital model funded by the public and

philanthropic sectors. These PPPs must be intricately structured to shift the industrial

strategy from maximizing profits to establishing an equitable pricing policy worldwide.

Economic analysis can forecast financial and economic implications of any change in

implementation strategy, medicines, and technological developments. Threshold analysis

can help decision makers and PPPs decide when it is cost effective to switch

interventions, delivery strategies, or first-line medicines due to resistance.12

Comparing

the difference between infectious disease control strategies of the developed and

developing world highlights the difficulties faced by the developing world.

42

Globally South-East Asia has the highest burden of three major NTDs:

leishmaniasis, leprosy, and lymphatic filariasis. Three countries – Bangladesh, India, and

Nepal – together account for 60% of all leishmaniasis cases in the world.17

Although the

prevalence of leprosy in the region has declined, 67% of the leprosy cases detected

globally in 2008 occurred in this region.18

Lymphatic filariasis is endemic throughout

South-East Asia except Bhutan and the Democratic People’s Republic of Korea.19

Several factors make eliminating these NTDs an attainable goal by the WHO. Safe and

effective diagnostic tools and interventions are available for each of these diseases and

should be scaled up accordingly. For leishmaniasis, the use of a rapid dipstick diagnostic

screening test known as rK39 followed by treatment with an effective drug and vector

control by indoor residual spraying is presently underway. Pilot exercises in 11 districts

of India were expanded to all 52 endemic districts by the end of 2010. For leprosy,

multidrug therapy to treat and cure patients and reduce the reservoir of infection has

already cured 12.8 million cases of the 15 million reported in 2010.17

For lymphatic

filariasis, mass drug administration to reduce microfilaraemia levels and transmission

rates is a goal to eliminate the disease for all countries in the region by 2020. In 2008,

425 million people in the region, equivalent to 86% of those treated worldwide, were

reached by mass drug administration exercises. A comparison of these methods of

disease eradication with those practiced in the developed world emphasizes the

challenges met by sustaining political commitment and providing adequate resources,

along with ensuring uninterrupted drug supplies and wider health service coverage to

underserved population groups.

43

Although NTDs are not a significant burden in the developed world, a growing

recognition of vulnerability to such diseases that might emerge from developing

countries, as well as genuine humanitarian concern, has led to greater interest. In the late

1960’s public health leaders of the developed world were confident that they were

witnessing the end of infectious diseases; even to the extent that the US Surgeon General

William H. Stewart was frequently quoted as saying “it is time to close the book on

infectious diseases and pay more attention to chronic ailments”. This success was short-

lived because due to emerging and re-emerging diseases, death rates from infections in

the United States increased 58% between 1980 and 1992.20

The infrastructure of the

developed world allows for quick and effective control of infectious disease outbreaks.

The recent pandemic of influenza A (H1N1), which first emerged in Mexico in April

2009, was met with a swift reaction by the United States. Although social distancing and

use of antiviral agents are partially effective at slowing the spread of infection, mass

vaccination remains the most effectively utilized means of pandemic control. Currently,

more than 20 manufacturers are in various stages of production for pandemic H1N1

vaccines in the United States and could have been delivered starting in September 2009.21

In the United States, monitoring remains localized at the state level allowing for swift

response. This quick mobilization of vaccine production by pharmaceutical companies

and effective distribution is the hallmark of infectious disease control by the developed

world. Developing new therapeutic agents to fight antibiotic resistance must also focus

on combating other infectious diseases aside from NTDs.

The widespread and rapid development of antibiotic resistance can be credited to

the sheer amount of antibiotic usage (about 50 million pounds per year). The prevalence

44

of resistance in hospital-acquired infections in the United States (US) averages about

30% whereas methicillin-resistant strains in particular account for 60% of hospital-

acquired resistant infections (Table 2). Antibiotics are not distributed solely through

physician prescriptions. Indiscriminate use of antibiotics, particularly in the developing

world, aids increased development of resistance. Antibiotics and antiparasitics are often

sprayed on crops that are consumed by livestock and humans. Ingestion of these trace

amounts of antibiotics exposes gut microbiota in both livestock and humans to sustained

low doses of these chemicals, which promotes the development of resistance as further

discussed below. In the developed world, antimicrobial chemicals are ubiquitous in

household products, and are largely unnecessary for maintaining a safe home

environment. The use of antibiotics in animal feed makes up 40% of the antibiotic use in

the US.22

The amounts of antibiotic administered are often too low to combat infections

but in theory promote animal health, especially in the crowded conditions typical of

livestock rearing. It is difficult to determine the impact of antibiotics as feed additives for

food-animals because they are simultaneously being used for treatment and prevention of

infections. The direct transmission of resistant enterococci between animals and farm

workers has been demonstrated in several studies.22

Transmission of resistant bacteria

from food-animals to humans results in healthy humans in society carrying resistant

bacteria. This has been well documented for glycopeptides-resistant enterococci (GRE).

Several studies from Europe have found relatively high carrier rates of GRE in healthy

humans in the community during hospital admissions.23

Growth promoter use creates a

major food animal reservoir of resistant bacteria that have the potential to spread to

humans. Recent studies conclude that the use of antimicrobials to promote growth of

45

food-animals provides insignificant benefits to agriculture and could be terminated

(antimicrobials omitted from animal feed). The only solution to reducing antibiotic

resistance is to take careful measures when implementing antibiotic uses. Reducing or

terminating the use of antibiotics in animal feed is predicted to decrease widely the cross-

species distribution of resistant bacteria. However, it is remarkable that some feed

additives, such as enduracidin (the subject of the following chapter), have not resulted in

detectable resistant strains since their introduction. Ending the indiscriminate prescription

and use of antibiotics would require increased public awareness through widespread

education. Limiting the use of antimicrobial agents in household products would also be

advantageous. Implementing such regulations is an enormous hurdle for the developing

world but is necessary to attenuate the surge of resistant bacteria.

Analyzing the differences surrounding infectious disease in the developed and

developing worlds may reveal many disparate challenges, but also highlights the

conserved consequences of the development of untreatable infectious diseases. The

shifted focus from infectious diseases in the developed world may allow vulnerabilities

for emerging infectious diseases due to globalization promoted by ease of travel and

climate change (warming). Although NTDs are not currently a burden for the developed

world, the necessity for new drug development targets and new strategies for equitable

pharmaceutical distribution on a world-wide scale is imminent. Fighting antibiotic

resistance is a challenge for both the developed and developing world that must be

addressed promptly before widespread prevalence of drug and multi-drug resistant strains

is manifested. Fostering the same progress previously experienced by the developed

46

world in the developing world could relegate the overwhelming burden of infectious

diseases to something of the past.

Table 1- New antibiotics approved by the FDA

Year Novel Antibiotics (#)

1983-1987 16

1988-1989 14

1993-1997 10

1998-2002 7

2002-2005 3

According to published FDA data online24

Table 2 – Prevalence of resistance in hospital-acquired infections in the US, 2004

Reproduced from Wilson, B., A.

25

47

Work Cited

(1) Sanders, W., John, et al. Sci. Prog. 2008, 91, 1-38

(2) Brenzel, Logan, et. al. Disease Control Priorities in Developing Countries. Oxford

University Press: New York 2006; Chapter 20, pp 389-412.

(3) Morens, D.M., Folkers, G.K., and Fauci, A.S. Nature. 2004, 242-249.

(4) Cutler, D. and Miller, G. Demo. 2005, 48, 1-22.

(5) Pollard, A.J. Arch. Dis. Child. 2007, 92, 426-433.

(6) Control of infectious diseases. Morb. Mortal. Wkly. Rep. 1999, 48, 621-629.

(7) Fauci, A., S. Acad. Med. 2005, 80, 1079-1085.

(8) Fauci, A., S. Cell. 2006, 124, 665-670.

(9) Frenk, J., Bobadilla, et. al. Health Transit Rev. 1995, 1, 21-38.

(10) Victora, C., G. Lancet. 2003, 362, 233-241.

(11) Trouiller, Patrice, et. al. Public Health. 2002, 359, 2188-2194.

(12) Conteh, Lesong, Engels, Thomas, and Molyneux, H., David. Lancet. 2010, 375, 239-

247.

(13) Payne, Lara, and Fitchett, R., Joseph. Cell. 2010, 9, 421-423.

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