Introduction

Introduction

Emergence of multidrug resistant pathogens has triggered the need for discovery and

development of new antibiotics with unique modes of action. Disease causing

microbes including Staphylococcus aureus, Mycobacterium tuberculosis, Neisseria

gonorrhoeae, Streptococcus pneumoniae, Pseudomonas aeruginosa, Enterococcus

faceium, Enterococcus faecalis, Clostridium difficile, Salmonella enterica have

acquired resistance against antibiotics being used for their treatment. Acquisition of
resistance has been by production of enzymes which either degrade or inactivate the

target drug, efflux of antibiotics resulting in reduced intracellular accumulation,

reduced permeability to drugs and by an alteration of target sites (French, 1998;

Charpentier & Tuomanen, 2000; Tapsall, 2001; Lambert, 2002; Quinn et al., 2006;

O'Connor et al., 2008). Consequently, there is an urgent requirement to discover new

antimicrobial compounds to combat resistant pathogens.

Natural products from microbes are a good source of antibiotics. Among all bacteria,

actinomycetes are best known for their ability to produce antimicrobial compounds.
Actinomycetes are Gram positive bacteria characterized by formation of branching
filaments in the form of substrate and aerial mycelium. They are commercially

important as they have potential for synthesis of valuable secondary metabolites

including antibiotics and extracellular enzymes. Among different genera of
actinomycetes, genus Streptomyces alone is responsible for production of 60% of

antibiotics with little contribution by other genera including Actinomadura,

Actinoplanes, Amycolatopsis, Kibdelosporangium, Micromonospora, Nocardia,

Saccharopolyspora, Streptosporangium, Streptoverticillium and Thermoactinomyces

(Okudoh, 2001; Watve et al., 2001; Demain, 2002; Berdy, 2005). These antibiotics
are categorized on the basis of their chemical structures and possess a broad range of

biological activities. There is further for discovery of novel chemical entities from
these commercially important bacteria.

In the light of above information, the present investigation was undertaken with the
following objectives i) selective isolation of actinomycetes from diverse ecological

habitats, ii) qualitative and quantitative antimicrobial analyses of bioactive

compounds, iii) structure elucidation of purified compounds and iv) taxonomic

studies of selected strains.

Introduction

Actinomycetes are ubiquitous in nature and make up the major component of soil
microbial fauna. Samples were collected from agricultural, desert, dump site, dung, forest,
garbage, garden, industrial areas, pesticide treated, pond, radiation treated, ridge, sanitary

landfill and valley sites representing natural and man-made habitats. Soil samples were
subjected to physical and chemical pretreatment methods including air drying, calcium
carbonate treatment, use of antibacterial and antifungal agents in the isolation media and

were subsequently plated on a range of selective media including Yeast extract malt

extract agar (Shirling & Gottlieb; 1966), Selective medium 1,2 3 (Tan et al., 2006), Starch

casein agar (Kuester & Williams; 1964), Water agar (Berd, 1973), Arginine glycerol agar
(EL-Nakeeb & Lechevalier; 1963), Organic agar Gause 2 (Gause et al., 1983), Glycerol
asparagine agar (Shirling & Gottlieb; 1966) and MC agar (Nonomura & Ohara, 1971).

Among the media tested, arginine glycerol agar, glycerol asparagine agar, organic agar

Gause 2 and starch casein agar promoted prolific growth of actinomycetes. Combination
of appropriate physical and chemical pretreatment methods facilitated selective isolation
of slow growing actinomycetes with a concomittant reduction in the occurrence of other
bacterial and fungal contaminants.

Isolates were subjected to primary antimicrobial screening against five sensitive

strains including Escherichia coli (Gram negative bacterium), Bacillus cereus (Gram

positive bacterium), Staphylococcus aureus (Gram positive bacterium), Fusarium

oxysporum (Fungus) and Candida albicans (Yeast). This was done to ascertain

antimicrobial potential of the strains. Out of 128 colonies tested, 61 were found to
possess antimicrobial activities. Most of the active isolates were from pond, radiation

treated, desert, dump site, pesticide treated, garden, agricultural and industrial soils.

Among 61 active isolates, 31%, 17%, 12%, 15% and 8% isolates had shown activities

against Bacillus cereus, Staphylococcus aureus, Escherichia coli, Fusarium

oxysporum and Candida albicans, respectively. Primary screening revealed that

isolates not only possessed noticeable activity against Gram positive bacteria but also
against Gram negative bacteria, yeast and fungi.

Based on the results of primary screening, eight isolates namely B.69, L3.41, L3.46,
RI.24, RI.30, S.4A, S.43 and SL.4 were selected for further studies since they

represented various ecological habitats and had substantial activities against more
2

Introduction

than one sensitive strain. Bioactive compounds were extracted both from culture broth

and media plates. Appropriate solvent systems had to be selected for extraction of
compounds depending on their polarity (Augustine et al., 2005; Igarashi et al., 2006;
Arasu et al., 2008; Selvin et al., 2009). Ethyl acetate was used for extraction of

antimicrobial compounds that displayed activities against Bacillus cereus and

Fusarium oxysporum. On the other hand, bioactive compounds extracted in methanol

were active against Candida albicans, Escherichia coli and Staphylococcus aureus.
Activity of extracts was quantified and compared with each other as well as with
standard antibiotics by agar well method and MIC determination. Some of the extracts
showed comparable activities to those of control antibiotics whereas others possessed

lower activities, perhaps due to presence of impurities that hampered complete

diffusion of the compounds in the testing medium.

Culture extracts were fractionated by thin layer chromatography. Various solvent

systems were tested and it was found that Dicholomethane:methanol (9:1) and
Butanol:acetic acid: water (3:1:1) gave better separation of ethyl acetate and
methanolic extracts, respectively. Fractionated extracts were thereafter subjected to

chemical screening. This technique involved separation of metabolites from a microbe

on thin layer chromatography (TLC) plate and visualization of bands under UV at

365nm/254nm followed by staining with chemical reagents like anisaldehyde/H2SO4,

vanillin/H2SO4 and methanolic H2SO4. Chemical screening enabled metabolic

profiling of each strain and provided a preliminary estimate of possible chemical
moieties present in extracts.

Once the antimicrobial compound has been fractionated on TLC plate, it is important
to identify the actual bioactive fraction(s) and their Rf values. This was accomplished
by bioautography. It included fractionation of crude culture broths by TLC followed
by spraying with respective pathogen and staining with appropriate dye. White

inhibition zone against dark background indicates the presence of bioactive

fraction(s). The single bioactive fraction from L3.41 ethyl acetate extract showed

broad range activity against both Bacillus cereus and Fusarium oxysporum. Similarly,

L3.46 methanolic extract displayed a single bioactive fraction active against Candida

Introduction

albicans and Fusarium oxysporum. Ethyl acetate extracts from L3.46, Sl.4 and S.43

showed multiple bioactive fractions but active against a single pathogen Bacillus
cereus. Ethyl acetate extract from B.69 and methanolic extract from RI.30 showed

single bioactive fraction displaying activity only against single pathogen Bacillus

cereus and Candida albicans, respectively.

Bioactive extracts were purified by repeated rounds of TLC and subjected to structure

elucidation by mass spectrometry and nuclear magnetic resonance (NMR). The yellow
colored bioactive compound L3.41 (1) purified from L3.41 ethyl acetate extract had
molecular mass of 1255 g/mol which is similar to that of the antibiotic Actinomycin D.

Mass fragmentation pattern of both compounds were compared and found similar. 1H
NMR data of compound L3.41(1) also showed similarity to that of Actinomycin D. It was
concluded that compound from L3.41 is Actinomycin D which belongs to polypeptide

class of antibiotics. The L3.46 (1) compound from L3.46 ethyl acetate extract had a
molecular weight of 681 g/mol comparable to mycinamycin III which belongs to the
macrolide antibiotic class. Although, mass fragmentation pattern indicated structure

similarity with mycinamycin III but NMR data showed extra peaks corresponding to
presence of an aromatic moiety which is missing in mycinamycin III. Consequently,

L3.46 (1) compound may be an isomer of mycinamycin or may be an altogether different

antibiotic having structure similarity to mycinamycin. Furthermore, bioactive compound
B.69 (1) from B.69 had a molecular weight of 840 g/mol, which could be due to an

additional sugar moiety in mycinamycin III or L3.46 compound. Mass fragmentation

pattern resembled that of mycinamicin III and of antibacterial compound from L3.46.
From preliminary studies, the B.69 (1) compound could be interpreted as a mycinamicin

or L3.46 compound glycoside. Compound L3.46 (2) isolated from L3.46 methanolic
extract had a molecular weight of 771 g/mol. Core structure of L3.46 (2) was similar to

that of xantholipin which belongs to xanthone group of antibiotics but differed from
xantholipin in possessing extra side chains. Therefore, it could be interpreted as a
xantholipin derivative.

Introduction

Isolates B.69, L3.41, L3.46, RI.24, RI.30, S.4A, S.43 and SL.4 subjected to
exhaustive antimicrobial analyses were taxonomically characterized on the basis of
microscopic, morphological, biochemical and phylogenetic studies. Spore chain

morphology of these strains were either straight to flexuous (Rectiflexibles) or as

hooks, loops or spirals with one to two turns (Retinaculiaperti) or in the form of
spirals (Spirales). This was suggestive of the isolates belonging to the actinomycete

genus Streptomyces. Morphological and growth studies on different ISP media as well
as biochemical studies consisting of utilization of various sugars as carbon sources

and degradation of substrates including hypoxanthine, starch, tween and urea also
indicated that the strains belonged to genus Streptomyces. This was further confirmed

by phylogenetic studies which included amplification and sequencing of 16S rRNA

gene. Related sequences were retrieved from Eztaxon database and phylogenetic trees

were constructed. Homology studies based on 16S rRNA gene sequences confirmed

the hypothesis that isolates were species of genus Streptomyces. 16S rRNA gene

sequences of our strains exhibited sequence similarity in the range of 99-100% with
those of validly described species of genus Streptomyces.

In conclusion, actinomycetes were isolated from several ecological habitats using a

combination of physical and chemical pretreatment methods. Actinomycetes from some

habitats possessed appreciable antimicrobial activities as compared to others. Isolates

showed noticeable antimicrobial activities not only against Gram positive bacteria but
also against Gram negative bacteria, yeast and fungi. Primary antimicrobial analyses
helped in selection of potent strains for further studies. Eight strains were selected which

represented varied ecological habitats and with substantial activities against sensitive

strains. Bioactive compounds were extracted with a combination of solvent systems.

Secondary screening and MIC determination helped in quantifying activity of various

culture extracts. Some of the bioactive compounds were as effective as well known

antibiotics and showed broad spectrum activities. Thin layer chromatography helped in
fractionation of extracts followed by identification of actual bioactive fractions using

bioautography. Structure elucidation of selected compounds revealed structures which

Introduction

either showed partial or complete resemblance with known antibiotics indicating the
presence of novel chemical moieties in most of these compounds. Selected strains were
taxonomically characterized on the basis of microscopic, morphological, biochemical and
phylogenetic studies and it was confirmed that all strains belong to genus Streptomyces.