Chapter 4

Discussion

Chapter 4: Discussion

Soil is the major repository of microorganisms that produce antibiotics. Actinomycetes

are well known for production of antimicrobial compounds. These are ubiquitious in
nature and major component of microbial population found in natural and man-made
habitats. Actinomycetes were isolated from diverse ecological habitats including

agricultural, desert, dump site, dung, forest, garbage, garden, industrial areas, pesticide
treated, pond, radiation treated, ridge, sanitary landfill and valley soils which represented

a range of different habitats. The logic behind this approach was that actinomycetes
present in different ecological niches may exhibit variable metabolic activity and may be

the potential source of new bioactive metabolites. It has been reported in earlier studies

that actinomycetes isolated from diverse ecological habitats are producers of novel and

diverse chemical entities (Baur et al., 2006; Ji et al., 2007; Solanki et al., 2008; Nachtigall

et al., 2011; Poulsen et al., 2011; Hamedi et al., 2012; Schulz et al., 2012). Soil samples
were taken from a depth of 0-5cm of top layer. It has been known that top soil is rich in

organic matter and microbes where most of the biological activities occur. The population
of actinomycetes decreased significantly with soil depth as there is decrease in organic

substrate as well as aeration is poor in deeper layers of soils (Corke & Chase, 1964;
Essien & Udosen, 2000). Krishna et al., 2012 have analysed different soils and found that
there was remarkable decrease in bacterial, fungi and actinomycetes population with soil

depth. Takahashi & Omura, 2003 studied the vertical distribution of actinomycetes in
soils taken at 10cm intervals between 0 and 1 m and demonstrated that actinomycete
population was maximum in surface layer of soils and decreased with soil depth.

Soil samples were subjected to different physical and chemical pretreatment methods for

selective isolation of actinomycetes. These have been developed and used by researchers

for selective isolation of actinomycetes and some of these are genus specific (Hayakawa,

2008; Khanna et al., 2011). Air drying of samples was done for a variable duration
depending on the moisture content. Drying of the soil samples kills contaminant Gram-

negative bacteria where as desiccation resistant actinomycetes survive (Tamura et al.,

1997; Seong et al., 2001; Baskaran et al., 2011). Therefore, number of actinomycete

colonies on isolation plates increased. Incubation of soil samples with calcium carbonate

also effectively decreased the number of contaminant microbes, but actinomycetes

selected were morphologically alike as reported earlier (El Nakeeb & Lechevalier; 1963;

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Chapter 4: Discussion

Otoguro; 2001a; Qin et al., 2009a). Differential centrifugation was also tried, but no
actinomycete colonies could be isolated on yeast extract malt extract agar media. Other
researchers have also used this method but in conjunction with some specific media like

humic acid vitamin agar for selective isolation of actinomycetes (Hayakawa et al., 2000;
Otoguro et al., 2001 a,b). A range of selective media were tested for facilitating isolation

of actinomycetes including Yeast Extract-Malt Extract (YM agar) (Shirling & Gottlieb,
1966), Selective Media (SM-1, SM-2 and SM-3) (Tan et al., 2006), Starch Casein (SC

agar) (Kuester & Williams, 1964), Water Agar (WA agar) (Berd, 1973), Arginine

Glycerol (AG agar) (EL-Nakeeb & Lechevalier, 1963), Organic Agar Gause 2 (Gause et
al., 1983), Glycerol Asparagine agar (GA agar) (Shirling & Gottlieb, 1966) and MC agar

(Nonomura & Ohara, 1971). Among those, arginine glycerol agar, glycerol asparagine
agar, organic agar Gause 2 and starch casein agar were found to be most effective in this

study. These media have also been used by other research groups for isolation of
actinomycetes (Kuester & Williams, 1964; Shirling & Gottlieb, 1966; Gause et al., 1983;

Kutzner, 1986). Barcina et al. 1987, found that addition of glycerol and asparagine in the

isolation media favored the growth of actinomycetes. Zhang & Zhang, 2011 used glycerol

asparagine as the medium for selective isolation of actinomycetes. Seong et al., 2001 and

Kokare et al., 2004 found starch casein agar to be effective whereas Qiu et al., 2008 and
George et al., 2011 found glycerol arginine an efficient medium for isolation of
actinomycetes. Bredholdt et al., 2007 and Solanki et al., 2011 found effective isolation of

actinomycetes on organic agar Gause 2. Antibiotics cycloheximide and streptomycin

sulphate were added to isolation media for decreasing occurrence of contaminant fungi

and bacteria without affecting the growth of actinomycetes. This has proved to be a

general strategy for promoting growth of slow growing actinomycetes in the absence of
contaminant fast growing colonies (Seong et al., 2001; Saintpierre-Bonaccio et al., 2004,
2005; Bredholdt et al., 2007; Sajid et al., 2009; Baskaran et al., 2011).

Pure actinomycete cultures from different ecological habitats were subjected to

preliminary antimicrobial screening against five standard 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). These strains are used routinely for assessing

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Chapter 4: Discussion

antimicrobial activity of actinomycetes (Kokare et al., 2004; Augustine et al., 2005;

Anansiriwattana et al., 2006; Selvin et al., 2009). Cross streak and agar plug methods

were used for primary screening. These methods were adopted as these were easy to

perform and a large number of strains could be tested simultaneously in short time.
Various modifications of these methods have been adopted for antimicrobial studies

(Kokare et al., 2004; Xie et al., 2005; Selvin et al., 2009). Each method has its own

advantage, by cross streak method efficiency of a single actinomyete culture for

production of antimicrobial compounds against different sensitive strains can be

checked whereas agar plug method helps in the comparative analyses of different

sensitive strains simultaneously. During primary screening, 128 isolates from

different ecological habitats were randomly chosen and subjected to antimicrobial

analyses. 61 isolates were found to show activities against sensitive strains. 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. The results of primary screening revealed that most of the isolates were

active against gram positive bacteria (Bacillus cereus and Staphylococcus aureus) as

compared to gram negative bacteria, yeast and fungus (Escherichia coli, Fusarium

oxysporum and Candida albicans). The reason for differential activity against Gram
positive and negative bacteria might be due to the morphological differences between

them. Gram negative bacteria have an outer polysaccharide membrane carrying the

structural lipopolysaccharide components which makes the cell wall of bacteria

impermeable to lipophilic solutes. On the other hand, gram positive bacteria possess

an outer peptidoglycan layer which is not an effective permeability barrier making

them more susceptible to antimicrobial compounds (Scherrer & Gerhardt, 1971). This
kind of difference in the activities of actinomycetes against pathogenic strains have

been reported earlier by Basilio et al., 2003; Oskay et al., 2004; Anansiriwattana et
al., 2006; Charoensopharat et al., 2008.

Depending 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. Nearly all

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Chapter 4: Discussion

of them had substantial activities against more than one sensitive strain and had been

collected from various ecological habitats (Table 3.2). Bioactive compounds from

these isolates were extracted both from culture broth and solid media plates.

Appropriate solvent systems had to be selected for extraction of compounds

depending on their polarity as per literature survey (Augustine et al., 2005; Igarashi et
al., 2006; Arasu et al., 2008; Selvin et al., 2009). It has been found in the present

studies that compounds active against Bacillus cereus could be extracted in ethyl

acetate whereas compounds active against Candida albicans, Escherichia coli,

Staphylococcus aureus in methanol. Ethyl acetate extracts either showed very low

activity or no activity against Candida albicans, Escherichia coli and Staphylococcus

aureus. Activity against Fusarium oxysporum was observed by use of both ethyl
acetate and methanolic culture extracts.

Extraction method has definite effect on the isolation of bioactive compounds. This
could be due to difference in their polarity. Compounds extracted in ethyl acetate are

less polar as compared to those extracted in methanol. Polar antibiotics were found to

be effective against Gram negative bacterium Escherichia coli. As per literature

survey, the possible explanation for this could be that the outer membrane of Gram

negative bacteria is coated by a lipopolysaccharide layer (O-antigen, a core

polysaccharide and a lipid A) and excludes large hydrophobic antibiotics from
entering the cell. Porins (beta barrel proteins) are present which transverse outer

membrane and facilitate the entry of polar antibiotics (Nikaido, 2003; Davin-Regli et

al., 2008). Both polar and nonpolar extracts worked against Fuarium oxysporum and

Candida albicans. It has been known that both these type of antibiotics act on fungi

and yeast by destroying selective permeability of their membrane, allowing cations to

leak out and facilitating entry of protons to neutralize the charge. This results in

internal acidification and death of the cell (Hammond & Kliger, 1976). Gram positive
bacterium Bacillus cereus was inhibited by ethyl acetate extracts that entered the cell
through its less selective peptidoglycan layer. Actinomycin D, soluble in ethyl acetate

enters the bacterial cell and intercalates in DNA to inhibit bacterial growth (Guy &

Taylor, 1978). Polar extracts inhibited Staphylococcus aureus in this study. This is in
agreement to observations that polar aminoglycosides like kanamycin disrupt the

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Chapter 4: Discussion

integrity of bacterial cell membrane and inhibit protein production (Gourevitch et al.,

1958-59). Effect of extraction methods on activity of extracts has been mentioned

earlier by many other authors. Selvin et al., 2009 reported that extraction with ethyl

acetate and other organic solvents did not show activity against Candida albicans
whereas anticandida compound could be extracted with ammonium sulphate
precipitation method. Boudjella et al., 2006 used five different solvents n-hexane,

ethyl acetate, dichloromethane, benzene and n-butanol for extraction of bioactive

compounds and found n-butanol as most appropriate solvent for extraction of

antibiotic. Augustine et al., 2005 tested different solvent systems including n-butanol,
n-hexane, ethyl acetate, petroleum ether, chloroform, benzene and xylene to find the

ideal solvent for extraction of the non-polyene antifungal antibiotic from culture
supernatant of Streptomyces albidoflavus PU 23 and hexane was found to be most

effective for extraction.

Activity of extracts was quantified and compared with each other as well as with standard
antibiotics by two different methods including agar well method and MIC determination

(Solanki et al., 2011). L3.41 ethyl acetate extract showed maximium activity against

Bacillus cereus followed by extracts of L3.46, B.69, RI.24, S.43 and SL.4. The control

antibiotic kanamycin acid sulphate produced inhibition zones bigger in size as compared
to all extracts. Among all extracts used against Fusarium oxysporum, L3.46 methanolic

extract showed maximum activity followed by L3.46 and L3.41 ethyl acetate extracts by

using the agar well diffusion method. Among controls used against Fusarium oxysporum,

sizes of inhibition zones were higher for cycloheximide as compared to amphotericin B.

On the other hand, L3.46 methanolic extract produced bigger inhibition zones as
compared not just to RI.30 methanolic extract but also produced by amphotericin B.

Activity of RI.24 methanolic extract was lower against Staphylococcus aureus and

Escherichia coli as compared to that produced by the control kanamycin acid sulphate. It
has been known that size of inhibition zones depends both on diffusion parameters of

antibiotic in the agar medium as well as on activity of the compound being tested (Linton,
1983; Antal et al., 2005; Augustine et al., 2005; Bonev et al., 2008).

MICs of these compounds were thereafter determined by microdilution method. MICs

of extracts gave an insight into their effectiveness against sensitive strains. MICs of
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Chapter 4: Discussion

L3.41 and L3.46 ethyl extracts were comparable to MIC of the control antibiotic

kanamycin acid sulphate against Bacillus cereus, while MICs of B.69, RI.24, S.43 and

SL.4 ethyl acetate extracts were higher than that of the control antibiotic. MIC of
amphotericin B was lowest as compared to MIC of cycloheximide and of all extracts
tested against Fusarium oxysporum. L3.46 methanolic extract had lowest MIC against

Fusarium oxysporum followed by L3.41 and L3.46 ethyl acetate extracts. MIC of

RI.30 extract was lower as compared to L3.46 methanolic extract against Candida

albicans. MIC of amphotericin B was very low against Candida albicans in

comparison to any of the extracts. MICs of RI.24 methanolic extract against
Staphylococcus aureus and Escherichia coli were higher as compared to kanamycin

acid sulphate. It has been found that in most cases activities of control antibiotics were

higher as compared to the extracts being tested both by agar well and MIC methods. A
possible explanation could be that extracts being tested were crude containing
proportionately lesser amount of bioactive moiety in comparison to highly purified
commercial antibiotics being used as controls. There was also some discrepancy in

activity of extracts as well as of control antibiotics when compared by agar well and
MIC methods. In agar well method size of inhibition zones produced by amphotericin
B against Fusarium oxysporum were smaller as compared to those generated by

extracts and cycloheximide whereas MIC of amphotericin B against Fusarium

oxysporum was the lowest. L3.46 ethyl acetate extract produced bigger inhibition
zones as compared to L3.41 ethyl acetate extract whereas MIC of former was higher
than off latter. Similarly, sizes of inhibition zones produced by amphotericin B against

Candida albicans were smaller as compared to extract whereas MIC was low. The
possibility of smaller inhibition zones in agar well diffusion method might be due to

less diffusion of respective compound in adjacent medium. Similar observations have

been reported by other authors. Del Poeta et al., 1994 determined MIC of

amphotericin B and flucytosine by both agar and broth dilution methods and observed

significant differences in the results obtained by these two methods. Vander Heijden et

al., 2007 determined MICs of carbapenem-resistant Pseudomonas aeruginosa to

polymyxins and observed discrepancies in results of agar diffusion and broth dilution
methods. Sakoulas et al., 2012 found that agar dilution consistently gave higher MIC

values than broth microdilution method indicating differences in MIC determination

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Chapter 4: Discussion

by both methods. Suthindhiran & Kannabiran, 2010 used combination of above

methods for analyses of antimicrobial compounds from Streptomyces sp.,

Actinopolyspora sp., Sachharopolyspora sp. and Micromonospora sp. and came to the
same conclusion. Arasu et al., 2008 extracted bioactive compounds from culture plates

of Streptomyces using methanol, MIC of extracts was higher as compared to the

control antibiotic streptomycin. Researchers have adopted different methods for MIC

determination. Matsuzawa et al., 1980 and Schimana et al., 2000 determined MIC of

newly discovered antibiotics from Streptomyces galilaeus and Streptomyces

antibioticus respectively by broth dilution method whereas Lu & Shen, 2007 used disk
diffusion assay for Naphthomycin K obtained from Streptomyces sp.

Thin layer chromatography was used for fractionation of extracts. An appropriate

solvent system needs to be used for proper fractionation. Various solvent systems

were tested for ethyl acetate and methanolic extracts. Among all the solvent systems

tested for ethyl acetate extracts, Dichloromethane: methanol (9:1) was found most
effective whereas in case of methanolic extracts Butanol:acetic acid:water (3:1:1)

gave the best results for separation of fractions. Taddei et al., 2006 also found best

separation of ethyl acetate extracts in Dichloromethane: methanol (9:1). Boudjella et

al. 2006 used Butanol:acetic acid:water (3:1:1) for separation of culture extract. On

the other hand, Selvameenal et al, 2009 found good separation of extracts in

chloroform: methanol (30:70) as compared to their fractionation using n-

hexane:chloroform (40:60, 50:50, 60:40), n-butanol:acetic acid:water (70:20:10 and

60:30:10) and chloroform:methanol (60:40, 50:50, 70:30, 30:70, 80:20 and 20:80).

Extracts were subjected to chemical screening for visualizing metabolic profile of

strains so as to establish similarities or differences between extracts of multiple isolates

and had a tentative idea about the possible chemical moieties present in our extracts.
Most of the bands showed UV absorption at 254 and 365nm in both ethyl acetate and

methanolic extracts. Secondary metabolic pattern was visualized with a variety of

staining reagents but best results were obtained with vanillin/H2SO4 in case of ethyl
acetate extract and with anisaldehyde/ H2SO4 for methanolic extracts. Brown and grey

color fractions obtained by staining with these two chemicals indicated the presence of

sugar moieties in our extracts whereas yellow, orange, pink, and slate color fractions
99

Chapter 4: Discussion

were those which did not have any sugar moiety (Taddei et al., 2006). Aminoglycoside,
nucleoside, glycopeptide, macrolide and anthracycline antibiotics have sugar moieties

in their chemical structures on the other hand polypeptides, xanthone antibiotics do not

have. In the initial stage, we could predict that our compounds having sugar moieties
may belong to macrolide and anthracycline antibiotics based on physical properties

whereas others may belong to polypeptides and xanthones. Taddei et al., 2006 showed
usefulness of chemical screening for avoiding strain duplication on the basis of their

metabolic fingerprint and found best results with anisaldehyde/ H2SO4 staining. Sajid et

al., 2009 used chemical screening for selection of best actinomycetes on the basis of

their secondary metabolite pattern. Therefore, chemical screening is one of the

important components in the natural product discovery. Selvameenal et al., 2009
identified active compound to be a sugar moiety tentatively after staining TLC
chromatogram with naphthoresorcin-sulphuric acid.

Subsequent to fractionation of extracts by chromatography, the actual bioactive

fraction(s) need to be recognized. This was done by bioautography which is

considered as an efficient technique for determination of bioactive fractions in crude

extracts. Immersion biautography was used, bioactive zones were visualized and
identified after staining with 3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium
bromide (MTT). L3.41 had one bioactive fraction with Rf value of 0.67. Three

fractions having Rf values of 0.60, 0.56 and 0.45 from L3.46 ethyl acetate extract

were found bioactive whereas in L3.46 methanolic extract one fraction with Rf value

0.64 was found bioactive. In RI.30 methanolic extract the fraction with Rf value of

0.55 was bioactive whereas in S.43 ethyl acetate extract two fractions with Rf values

of 0.86 and 0.49 were found bioactive. In SL.4 ethyl acetate extract two fractions

having Rf values of 0.78 and 0.66 were found bioactive. Selvameenal et al., 2009
identified bioactive fraction having Rf value of 0.768 from extract of Streptomyces
hygroscopicus subsp. ossamyceticus corresponding to a bioactive sugar compound by

using bioautography. Boudjella et al., 2006 could detect four active spots having Rf

values 0.74, 0.68, 0.61 and 0.36 belonging to the group of glycosylated aromatics,

from culture extract of Streptosporangium strain whereas Boudjelal et al., 2011

identified a bioactive fraction at a Rf value of 0.44 belonging to aminoglycoside or

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Chapter 4: Discussion

dioctyl phthalate. Different authors have used various bioautography methods for
identification of bioactive fraction. Sajid et al., 2009 used contact bioautography,

Selvameenal et al., 2009 used immersion bioautography and Rakotoniriana et al.,

2012 used direct bioautography for identification of bioactive fractions.

Bioactive constituents from ethyl acetate extracts of L3.41, L3.46, B.69 and

methanolic extract of L3.46 were isolated and purified by repeated rounds of TLC and

subjected to 1H NMR and mass spectrometry for structure elucidation. Igarashi et al.,

2005; Maskey et al., 2006 and Rakotoniriana et al., 2012 have used these techniques
for structure elucidation of their compounds.

Molecular weight of compound L3.41 (1) isolated and purified from L3.41 culture
was found to be 1255g/mol which is similar to that of the antibiotic Actinomycin D.

Mass fragmentation pattern of both compounds was similar indicating structural

resemblance between them (Barber et al., 1988). 1H NMR data of L3.41 (1) showed

similarity to that of Actinomycin D. Actinomycin D and L3.41 (1) compound also

possessed similarity in their physical and biological activities. On the basis of these
studies, it was confirmed that compound isolated from L3.41 is Actinomycin D. This

antibiotic is a polypeptide and has been reported from various Streptomyces species

including Streptomyces antibioticus, Streptomyces chrysomallus, Streptomyces

parvulus, Streptomyces plicatus, Streptomyces griseoruber (Katz & Pugh, 1961; Lam

et al., 2002; Praveen & Tripathi, 2009). Actinomycin D has also been isolated from

marine derived Streptomyces sp. (Chen et al., 2012). It is reported to possess

antibacterial, antifungal and antitumor activities. Actinomycin D has clinical

applications owing to its anti-tumour properties and works by intercalating into
duplex DNA resulting in inhibition of DNA-dependent RNA polymerase and thus of

protein synthesis (Wadkins et al., 1998). It is used in the treatment of several types of

cancers including sarcomas, Wilms tumour, germ cell cancers, testicular cancer,

melanoma, choriocarcinoma and childhood rhabdomyosarcoma (Green, 1997; Womer

1997). Structure activity relationship studies revealed that chromophoric group
phenoxazine (Figure 4.1) present in actinomycin D is responsible for its antibacterial
and antifungal activities. This group binds to DNA thereby interfering with functions

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Chapter 4: Discussion

such as RNA transcription. Strong binding occurs by intercalation of the phenoxazine

group into the base pairs of double helical DNA (Guy & Taylor, 1978).
H

H
C

NH2

O
CH3

CH3

Figure 4.1: Bioactive moiety of Actinomycin D and compound L3.41 (1) (Phenoxazine)

Similarly, the compound L3.46 (1) isolated from L3.46 ethyl acetate had a molecular

weight of 681 g/mol which is similar to mycinamycin III, belonging to the macrolide
antibiotic class. Although, mass fragmentation pattern indicated structural similarity

with mycinamycin III but NMR data showed peaks which correspond to additional

presence of an aromatic moiety. Consequently, L3.46 (1) compound may be an

isomer of mycinamycin or may be an antibiotic having structural similarity to
mycinamycin. Mycinamicins I, II, III, IV and V are macrolide antibiotics produced by

Micromonospora griseorubida sp. nov (Satoi et al., 1980) but not yet reported from

Streptomyces sp. However, a non mycinamycin producing but tylosin producing strain

was mutated that resulted in production of desosaminyl-5-O-mycaminosyl-10,11-

dihydromycinamicin IV (Lotvin et al., 1982). There are similar reports from literature

where same or related compounds are known to be produced by actinomycetes

belonging to different genera. Kosinostatin, a quinocycline antibiotic produced by

Micromonspora (Igarashi et al., 2002; EI-Naggar; 2007) has structure resemblance to

quinocycline B isolated from Streptomyces aureofaciens (Celmer et al., 1958). It has

generally been found that strains belonging to different species of same genus may

produce dissimilar chemical moieties whereas taxonomically unrelated genera may

produce identical compounds (Waksman & Bugie, 1943; Larsen et al., 2005). There is

molecular and phylogenetic evidence that genes involved in secondary metabolism

are subjected to lateral or horizontal gene transfer which leads to this ambiguity (Egan
et al., 2001; Kinashi et al., 1987; Koonin et al., 2001; Jensen et al., 2007).

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Chapter 4: Discussion

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. Mass fragmentation

pattern resembled that of mycinamicin III and L3.46 (1) compound and could be
interpreted as a glycoside of mycinamicin or L3.46 (1) compound. Sugars like

desosamine, rhamnose and mycinose are known to be part of antibiotic structure

(Hayashi et al., 1980). Rhamnose could be the likely sugar in B.69 (1) compound as it

has molecular weight of 164 g/mol which is approximately equivalent to the

difference in molecular weight of compound isolated from B.69 and that of

mycinamicin III. Structure activity relationship revealed that the aglycone functional

group (Figure 4.2) of L3.46 (1) and B.69 (1) compounds imparted antibacterial
activity against Bacillus cereus (Kaysser et al., 2010).
Et
O

OH

CH3

Me
Me
O

Figure 4.2: Bioactive moiety of compounds L3.46 (1) and B.69 (1) (Aglycone)

Compound L3.46 (2) isolated from methanolic extract of L3.46 possessed a molecular
weight of 771 g/mol. On the basis of NMR data, it showed similarity to xantholipin in its
core structure but differs from it in possessing extra side chains and the structure could be

interpreted on the basis of mass fragmentation pattern. Addition of these extra

hydrocarbon and methoxy side chains attributed to difference in molecular weight,

physical properties as well as biological activities of compound L3.46 (2) compared to
xantholipin. Xantholipin belongs to xanthone group of compounds and isolated from a

Streptomyces sp (Terui et al., 2003). Actinoplanones A and B, Sch 56036, Sch 42137,
albofungin, cervinomycins, simaomicins are other xanthone antibiotics that have been
reported from Streptomyces, Actinoplanes and Actinomadura (Gurevich et al., 1972;

Omura et al., 1982; Kobayashi et al., 1988; Nakagawa et al., 1987; Maiese et al., 1990;
Cooper et al., 1992; Chu et al., 1998). Structure activity relationship of the compound
was studied and suggested that presence of 1,4 dioxygenated xanthone moiety (Figure

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Chapter 4: Discussion

4.3) was responsible for bioactivity against Candida albicans and Fusarium oxysporum
(Nicholas et al; 2011). In addition, presence of ketonic functional group attached with

ring also imparted antifungal activity to the L3.46 (2) compound.

O
C

CH3

O
O

HO

Figure 4.3: Bioactive moiety of compound L3.46 (2) and (Xanthone group and ketonic ring moiety)

In this study, isolates named as B.69, L3.41, L3.46, RI.24, RI.30, S.4A, S.43 and SL.4

showing antimicrobial activities were subjected to taxonomic characterization.

Morphological, microscopic and biochemical studies were performed according to

methods of Shirling & Gottlieb; 1966. Morphological studies on different ISP media

(ISP 1-7) were performed to study growth, sporulation, color of substrate and aerial

mycelium and production of any diffusible and melanin pigment. All strains showed
good growth and sporulation on ISP media no. 2,3,4,5 and 7. Color of aerial
mycelium of different strains belonged to white, grey, pink, black and brown series

and substrate mycelium showed different colors including beige, brown, cream,

yellow, white, grey, black, red, orange. These colors of substrate and aerial mycelium

are commonly observed in the Streptomyces species (Jiang et al., 2007; Le Roes-Hill

et al., 2009; le Roes-Hill & Meyers, 2009; Pimentel-Elardo et al. 2009; Reddy et al.
2011). Strain B.69, L3.41 and S.43 produced brick-red, yellow and orange colored
diffusible pigment on some of the ISP media while other strains lack this property.
Comparable situation occur in Streptomyces where some species produce pigment

while others do not (Miyajima et al., 1998; Meyers et al., 2004; Zhu et al., 2007;

Labeda et al., 2009; Pimentel-Elardo et al. 2009; Luo et al., 2011; Reddy et al. 2011;
Sazak et al., 2011). Spore chain configuration is an important taxanomic marker in

actinomycetes taxonomy helping in species description of genus Streptomyces. The

three categories recognized and adopted for the International Streptomyces Project
were straight to flexuous (Rectiflexibles); hooks, loops or spirals with one to two

turns (Retinaculiaperti) and spirals (Spirales) (ISP, Shirling & Gottlieb, 1966). Strains
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Chapter 4: Discussion

in the present study also possessed spore chains which belonged to one of the three

described categories. Spore chains of strains L3.41 and L3.46 were of spirales type,
strains RI.30 and B.69 possessed rectiflexibles type of spore chains and spore chains

of RI.24, S.4A, S.43 and SL.4 belonged to retinaculiaperti type (Li et al., 2002;

Meyers et al., 2004; Jiang et al., 2007; Le Roes-Hill et al., 2009; Le Roes-Hill &

Meyers, 2009; Loqman et al., 2009; Pimentel-Elardo et al., 2009).

Biochemical studies were performed using sugars D-fructose, D-mannitol, D-xylose, L-

arabinose, L-rhamnose, meso-inositol, raffinose and sucrose. Among all the sugars tested

D-fructose, D-mannitol, D-xylose, L-arabinose, L-rhamnose, meso-inositol were

preferably utilized by most of the strains as compared to raffinose and sucrose as carbon
sources. Biochemical studies were also done to observe metabolization or degradation of

casein, hypoxanthine, starch, tween and urea. Tween and urea were metabolized by all
strains. Hypoxanthine was preferably metabolized as compared to starch and casein

(Labeda et al., 2009; Luo et al., 2011; Reddy et al., 2011; Sazak et al., 2011).
Morphological, microscopic and biochemical characteristics of strains were compared

with descriptions given in Bergeys Manual of Systematic Bacteriology (Lechevalier et

al., 1989) and it was suggested that all the strains belonged to the genus Streptomyces.

Homology studies of 16S rRNA gene sequences with those of validly described

species of genus Streptomyces followed by phylogenetic tree construction for strains

B.69, L3.41, L3.46, RI.24, RI.30, S.4A and SL.4 confirmed the hypothesis that
isolates were species of Streptomyces. 16S rRNA gene is the most commonly used

marker for phylogenetic studies because of its ubiquitous and highly conserved nature

and the fundamental role of ribosomes in protein synthesis. Furthermore, 16SrRNA is

not subjected to horizontal gene transfer and its evolutionary rate represents an

appropriate level of variation between different bacteria. 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. High 16S rRNA gene sequence

similarities are often found between members of Streptomyces (Guo et al., 2008). Zhu
et al., 2007 found 99.4% 16S rRNA gene sequence similarity of their strain with

Streptomyces bikiniensis ATCC 11062T, Liu et al., 2012 found 99.61 % similarity of

their Streptomyces strain to Streptomyces neyagawaensis strain ATCC 27449T, Zhao

et al., 2010 found 99.90% gene sequence similarity to Streptomyces armeniacus

105

Chapter 4: Discussion

NBRC 12555T. However, novelty in all cases could still be confirmed by DNA-DNA
hybridization (Zhu et al., 2007; Zhao et al., 2010; Liu et al., 2012).

The closest relatives of B.69, L3.41, L3.46 and SL.4 were Streptomyces atriruber NRRL

B-24165T (EU812169) (Labeda et al., 2009), Streptomyces parvulus NBRC 13193T

(AB184326) (Reddy et al., 2011), Streptomyces samsunensis M1463T (EU077190)

(Sazak et al., 2011) and Streptomyces atrovirens NRRL B-16357T (DQ026672)

(Preobrazhenskaya & Terekhova, 1986), respectively. Streptomyces rochei NBRC

12908T (AB184237) (Williams et al., 1989) and Streptomyces enissocaesilis NRRL B-

16365T (DQ026641) (Williams et al., 1989 & Ningthoujam et al., 2011) was closest

relatives of RI.24 and S.4A respectively whereas Streptomyces globosus LMG 19896T
(AJ781330) (Luo et al., 2011) was relative of RI.30. Strain RI.24 and S.4A shared 100%
16S rRNA gene sequence similarities and exhibited similarity in their morphological,
microscopic and biochemical features. S.43 16S rRNA gene could not be amplified with

standard universal primers. Gradient PCR was also tried with different annealing
temperatures but no amplification of 16S rRNA gene could be observed. The strain is
known to produce a copious amount of pigment that possibly hindered binding of primers
to the DNA not allowing amplification of gene to occur (Cook & Meyers, 2003).

Comparison of strains with their nearest relatives in terms of phenotypic and

biochemical features indicated similarities and dissimilarities on the basis of literature
review (Williams et al., 1989; Labeda et al., 2009; Luo et al., 2011; Ningthoujam et

al., 2011; Reddy et al., 2011; Sazak et al., 2011; Khanna et al., 2012). Antibiotic

production profile of strains B.69, L3.41, L3.46, RI.24, RI.30, S.4A, S.43 and SL.4
were also compared with those of their clade members (Tables 4.1-4.7). Though

considerable dissimilarities were found between structures and antimicrobial

activities, but biosynthetic gene clusters of strains and relatives were all part of either

the Polyketide synthases (PKSs) or Nonribosomal peptide synthetases (NRPS)

systems.

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Chapter 4: Discussion
Table 4.1: Antibiotics produced by clade members of B.69
Strains

Streptomyces
atriruber

Streptomyces
durhamensis

Streptomyces
filipinensis

Streptomyces
coastaricanus

16S rRNA
similarity

99.71%

98.16%

97.93%

97.94%

Compounds

Not known

Durhamycin
(Gordon & Lapa,
1966)
Chemistry

Filipin (Bergy &

Eble, 1968)
Chemistry

Antinematodal and
antifungal compound
(Esnard et al., 1995)

Polyene

Pentane

Activity

Activity

Antifungal

Antifungal

Table 4.2: Antibiotics produced by clade members of L3.41

Strains
16S rRNA
similarity

Compounds

Streptomyces parvulus

Streptomyces olivaceus

Streptomyces pactum
var. pactum

100%

99.29%

99.29%

Actinomycin D (Williams
& Katz; 1977)
Chemistry
Polypeptide
Activity
Antibacterial, Antifungal,
Antitumor

Kanchanamycins (Fiedler
et al., 1996)
Chemistry
36 membered polyol
macrolide
Activity
Antibacterial, Antifungal

Pactamycin (Weller et
al., 1978)
Chemistry
Aminocyclitol
Activity
Antitumor

Table 4.3: Antibiotics produced by clade members of L3.46

Strains
16S rRNA similarity
Compounds

Streptomyces samsunensis

99.85%

Not known

99.43%

Streptomyces malaysiensis

i) Mitomalcin (McBride et al., 1969)

Chemistry
Proteinaceous
Activity
Antileukemic
ii) Azalomycin F complex (Cheng et al., 2010)
Chemistry
Macrocyclic lactone
Activity
Antifungal

107

Chapter 4: Discussion
Table 4.4: Antibiotics produced by clade members of RI.24
Strains
16S rRNA
similarity

Compounds

Streptomyces
vinaceusdrappus

Streptomyces
plicatus

Streptomyces
rochei

99.85%

100%

Amicetin
(Zhang et al., 2012)
Chemistry
Nucleoside
Activity
Antibacterial
Antiviral

i) Amicetin
(Haskell et al., 1958)
Chemistry
Nucleoside
Activity
Antibacterial
Antiviral
ii) Plicacetin
(Haskell et al., 1958)
Chemistry
Nucleoside
Activity
Antibacterial
iii) Norplicacetin
(Evans & Weare, 1977)
Chemistry
Nucleoside
Activity
Antibacterial

Streptomyces
enissocaesilis

100%

100%

Borrelidin
(Berger et al.,
1949)
Chemistry
18
membered
macrolide
Activity
Antibacterial,
Antimalarial

Not known

Table 4.5: Antibiotics produced by clade members of RI.30

Strains
16S
similarity

Compounds

rRNA

99.36%

Streptomyces globosus

2-deoxystreptamine (El-khatib
2008)
Chemistry
Aminocyclitol aminoglycoside
Activity
Antibacterial, Antifungal

Streptomyces toxytricini

99.36%
et

al.,

Lipstatin (Weibel et al., 1987)

Chemistry
lactone with hydrocarbon
Activity
Inhibitor of pancreatic lipase

Table 4.6: Antibiotics produced by clade members of S.4A

Strains
16S rRNA
similarity

Compounds

Streptomyces
vinaceusdrappus

Streptomyces
plicatus

99.85%

100%

Amicetin
(Zhang et al., 2012)

i) Amicetin
(Haskell et al., 1958)

108

Streptomyces
rochei

Streptomyces
enissocaesilis

100%

100%

Borrelidin
(Berger et al.,

Not known

Chapter 4: Discussion
Chemistry
Nucleoside
Activity
Antibacterial
Antiviral

Chemistry
Nucleoside
Activity
Antibacterial
Antiviral
ii) Plicacetin
(Haskell et al., 1958)
Chemistry
Nucleoside
Activity
Antibacterial
iii) Norplicacetin
(Evans & Weare, 1977)
Chemistry
Nucleoside
Activity
Antibacterial

1949)
Chemistry
18 membered
macrolide
Activity
Antibacterial,
Antimalarial

Table 4.7: Antibiotics produced by clade members of SL.4

Strains
16S rRNA
similarity

Compounds

Sreptomyces
atrovirens

Streptomyces albogriseolus

Streptomyces
viridodiastaticus

99.65%

99.65%

99.64%

Antibiotic
(Cho & Kim; 2012)
Chemistry
Benzaldehyde
Activity
Antibacterial

Antibiotic complex
(Gauze et al., 1980)
Chemistry
Amphomycin group
Activity
Antibacterial

Not known

In conclusion, actinomycetes were isolated from different ecological habitats using

combination of physical and chemical pretreatment methods. Strains from some

habitats had better antimicrobial activities as compared to others. Isolates showed

noticeable activity 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. Bioactive compounds were extracted using

different 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

109

Chapter 4: Discussion

identification of actual bioactive fractions using bioautography. Structure elucidation

of selected compounds revealed structures which either showed partial or complete
resemblance with known antibiotics indicating the presence of novel chemical
moieties in most of these extracts. These antimicrobial compounds have been

identified as Actinomycin D, Mycinamicin III derivative, glycoside isomer of

Mycinamicin III derivative and xantholipin derivative. Furthermore, these compounds

belonged to different chemical groups including polypeptides, macrolides and

xanthones. 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. Antibiotic producing strains

can further be subjected to strain improvement and combinatorial biosynthesis for

production of more effective analogues or hybrid bioactive molecules.

110