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1. INTRODUCTION
[“The role of the infinitely small in nature is infinitely large”- Louis Pasteur.]
1.1 Marine environment
Nearly three quarters of the earth’s surface is comprised of the marine
environment and it can be considered as a storehouse of basically all conceivable types
of microbes (Konig & Wright, 1999). They may present in suspended form on
inanimate or animate surfaces as epibionts or as symbionts. Microorganisms are
intimately involved in ecological phenomena, e.g. settlement, biofouling and
metamorphosis as they play important roles in all the foremost elemental cycles which
occur in the oceans (Hawksworth, 1991). The marine environment is radically
distinctive in terms of its unique composition for both organic and inorganic
substances, as well as pressure conditions and temperature ranges. Ecological niches
like mangrove forests, deep-sea hydrothermal vents, sponge, algae, and fish supply
habitats for the assessment of specific microorganisms (Kohlmeyer, 1979). Many
research groups were motivated by the difficulties coupled with the collection of
marine macroorganisms and the insufficient amount of the isolated bioactive substance
(Edrada et al., 2000) to investigate the microbes linked with them, or those constituted
in the marine sediments or water columns (Konig & Wright, 1996). There are some
noticeable advantages while looking into microbes comparing with macroorganisms.
These comprise isolation of the compounds by large scale cultivation of the
microorganisms biotechnological fermentations with different parameters without
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ecological exploitation and microorganisms being easily genetically manipulated.
Based on this, marine microbes have emerged as a central topic for many groups
investigating natural products intending to find pharmaceutical drugs or compounds
valuable for agriculture (Osterhage, 2001).
1.2 Mangrove environment
Mangrove ecosystem is one of the world’s most productive ecosystem that
yields commercial forest products, enriches coastal waters, support coastal fisheries and
protect coastlines. Nevertheless, mangroves survive under extreme tides, condition of
high salinity, high temperature, strong winds, and muddy and anaerobic soils. No other
group of plants has been reported with such highly evolved ecological, morphological,
physiological and biological adaptations to extreme conditions.
Mangroves are the coastal wet land forests mainly found in the intertidal zone
of creeks, estuaries, back waters, marshes, deltas, lagoons and also mud flats of the
tropical and subtropical latitudes (Sahoo et al., 2009). The ecosystems where the
mangrove plants grow are termed as “Mangrove Ecosystem” which occupies millions
of hectors across the world coastal areas (Spalding et al., 1997; Alongi, 2002).
Mangrove marine ecosystems are largely unwrap source for screening and isolation of
new microbes with rich potential to produce the important active secondary bioactive
metabolites. The environment of the mangrove ecosystem is saline and highly rich in an
organic matter because of its various microbial enzymatic and metabolic activities
(Kizhekkedathu and Parukuttyamma, 2005). The products of natural origin remain to
be the most important source of antibiotics (Bull and Starch, 2007). Marine derived
compounds are more efficient in action against the pathogens that are resistant to the
existing antibiotics (Donia and Hamman, 2003). The risk undermining the health care
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system is because of the relentless and rapid spread of the multiple antibiotic resistant
pathogens causing life threatening infection (Talbot et al., 2006) and therefore the
demand for new antibiotics grows continually. Though considerable progress has been
made within the fields of chemical synthesis and engineered biosynthesis of
antibacterial compounds, nature remains the richest and the most versatile source for
new antibiotics (Baskaran et al., 2011). Since actinomycetes have an important proven
capacity to produce novel antibiotics (Bentley et al., 2002), the practice in screening
such organisms for the new bioactive compounds is continued (Berdy, 2005). However,
difficulty to discover the commercially potent secondary metabolites from well-known
Actinomycetes is becoming increasingly difficult due to the practice of wasteful
screening that is leading to rediscovery of the known bioactive compounds (Kui et al.,
2009). This stringent condition emphasizes the need to screen and isolate the
undiscovered representatives of the unexplored actinomycetes taxa. It is a clear object
that the mangrove ecosystem is a rich source of novel actinomycetes that have the
capacity to produce interesting new bioactive compounds including antibiotics.
Screening for the microbial species is an important aspect as there is a remarkable
source for the production of structurally diverse secondary metabolites that possess
pharmaceutically relevant biological activities (Berdy, 2005). It has long been an
observed fact that the search for the new secondary metabolites from microorganisms
in general has been confounded because different strains belonging to the same species
produce different types of secondary metabolites (Waksman and Bugie, 1943) but
identical secondary metabolites are produced by taxonomically diverse strains (Larsen
et al., 2005).
To differentiate among closely related actinomycetes contribute to the former
research. Sequence based approaches can now tackle the challenges that determine the
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taxonomic arrangements and provides opportunities to extract the relationship between
the groups of related strains and the secondary metabolites they produce. In addition,
this method is the tool to probe the evolutionary history of the metabolic pathways and
thus deduce the root and action of the LGT (lateral gene transfer) responsible for the
unrelated organisms to produce similar compounds (Paul et al.,) In view of the above
discussion, the present study is taken up to isolate, screen and characterize the
biologically diverse strains of actinomycetes from the mangrove sediment samples for
bioactive secondary metabolites. Taxonomic characterization was carried out based on
16S rRNA sequence analysis in combination with morphological, biochemical and
physiological data. The ability to produce antibacterial and antifungal compounds was
also investigated.
1.3 The Actinomycetes
Actinomycetes are Gram-positive bacteria with DNA rich in guanine and cytosine
(Urakawa, et al., 1999). They are unicellular filamentous microorganisms that branch
monopodially, more rarely dichotomously. These filaments can be either of a single
type called substrate or vegetative, or of two types, substrate and aerial. Some
Actinomycetes, like Mycobacterium, do not form mycelia and grow as pleomorphic or
coccoid elements. Due to their filamentous aspect, actinomycetes were thought to be
fungi, explaining the origin of the name actinomycetes which in Greek means “radiant
fungi”. Actinomycetes used to form a group on their own between the bacteria and the
fungi but in the 1950s, after investigation of their chemical composition and fine
structure, they were confirmed as prokaryotes and joined the bacterial domain.
Actinomycetes belong to the class Actinobacteria (Stackebrandt et al., 1997), order
Actinomycetales which includes 10 suborders and 30 families. This relatively recent
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Actinobacteria class was proposed based on the 16S rRNA analysis of hundreds of
actinomycete sequences.
1.3.1 Some of the general characters of Actinomycetes
1. Most of the Actinomycetes are chemoorganotrophic although some of them
grow on simple mineral media.
2. Cell wall has some peptidoglycan as in gram negative bacteria, but there is
variety in the peptidoglycan composition more than in gram negative bacteria.
3. Majority of them have a branched mycelia body.
4. The filaments is made up of several cells but in some, septa are absent and the
members of coenocytes.
5. Reproduction is by conidia borne on conidiophores. Endospore formation is
generally not seen.
6. Except for some (Actinomycetaceae), majority are aerobic.
7. They are not sheathed, stalked or photosynthetic.
8. They are prokaryotic and gram positive.
9. In certain families, filaments tend to break and fragmentation leads to coccoid
or elongate cells which develop into new individuals.
10. Some species are motile but most of them are not.
11. Cells have a diameter 0.5µ to 5µ. In some branched members the filaments may
be as long as several millimetres.
12. Most of them are saprophytic, widely distributed in organic matter in soil, dung
and marine and fresh waters. Some are pathogenic parasites (e.g.
Mycobacterium).
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1.3.2 Actinomycetes differ from true bacteria and fungi in the following aspects
1.3.2.1 Difference between Bacteria and Actinomycetes
1. Actinomycetes are not sheathed or photosynthetic while bacteria (at least some)
are.
2. Actinomycetes do not accumulate irons, sulphur or other free elements in or on
the cells while bacteria do.
3. Actinomycetes do not form endospores.
4. Actinomycetes have true branching, bacteria do not.
1.3.2.2 Differences between Actinomycetes and Fungi
1. Cell walls of Actinomycetes contain mucopolysaccharides and both muramic
and diaminopimelic acid as in bacteria while fungal cell walls are chitinous.
2. Actinomycetes are prokaryotic.
3. Actinomycetes are much smaller (1-5µ in diameter and not more than few µm
in length) than fungi (10-20 µ in diameter; mycelial length varies).
4. Sexual reproduction seen in fungi is absent in Actinomycetes.
1.3.3 Habitat of Actinomycetes
Actinomycetes are found in a wide range of habitats. They are present in the
frozen soils of polar regions and in the dry soils of deserts. They can be found in crude
oil, heavily metal contaminated soil and sediments and fresh and salt water
environments. They are not extremophiles and seem to be absent in highly acidic
(pH<1) and extremely hot (hot spring) environments. Actinomycetes are mostly
saprophytes though some can form parasitic or symbiotic associations with animals and
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plants. Selman Waksman in the early part of the 20th century contributed greatly to the
understanding of actinomycete ecology by publishing more than two hundred papers
and many books on the subject and established the predominance of actinomycetes in
soil (Waksman & Curtis, 1916, 1918). The techniques described in these studies along
with those from Stanley Williams (Williams et al., 1983, 1984) another major
contributor to the field of actinomycete ecology, are still in use in today’s laboratories.
In the last quarter of the 20th century, investigation of the marine environment such as
near-shore and deep-sea sediments, have revealed the presence of actinomycetes
(Jensen et al., 1991; Weyland, 1969, 1981). It is worth mentioning that despite the fact
that oceans cover 70% of the Earth surface and contain the most diverse ecosystems on
the planet, they have not been widely recognized as an important source for novel
actinomycetes. The distributions of actinomycetes in the marine environment and their
ecological roles remain largely undescribed. For a long time, the existence of
indigenous populations of marine actinomycetes was challenged. Actinomycetes
produce resistant spores that can remain viable but dormant for many years and it was
argued that the actinomycetes recovered from the marine environment were in fact the
result of spores from soil actinomycetes that had washed into the oceans.
This theory persisted despite evidence that actinomycetes can be recovered from
deep-sea sediments (Weyland, 1969) and that marine actinomycetes can be
metabolically active (Moran et al., 1995) and physiologically adapted to the salt
concentration encountered in the sea (Jensen et al., 1991; Mincer et al., 2002).
Rhodococcus marinonascens was the first actinomycete species that was described and
accepted as an autochthonous marine species. Mincer et al., (2002) studied 212
actinomycete isolates from a group called Mar 1. These bacteria were isolated from
geographically distant sediments collected from tropical or subtropical locations. The
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strains were differentiated by morphological characteristics like small-subunit rRNA
gene signature nucleotides and by an obligate requirement for sea water for growth.
Phylogenetic analysis of 16S rRNA gene sequences of seven strains showed that they
formed a monophyletic clade within the family Micromonosporaceae suggesting
novelty at the genus level. The Mar 1 strains were provisionally called ‘Salinospora’.
Later the taxon was formally named Salinispora, belonging to the family
Micromonosporaceae (Maldonado et al., 2005).
Actinomycetes are capable of producing several types of secondary metabolites and
being gram-positive bacteria they grow extensively in soils having profuse organic
matter (Henis, 1986 and Demain, 1999). The dispersion and presence of Actinomycetes
have been exhibited to be related with their different ecological habitats, including
seawater (Takizawa et al., 1993) and beach sand (Suzuki et al., 1994). Actinomycetes
capable of yielding antimicrobial compounds have been isolated from terrestrial
habitats as well as marine environments (Grein and Meyers, 1958.; Zobell and Upham,
1944) which suggests that Actinomycetes from marine sediments are a rich source of
natural bioactive compounds. Lately, new species and genera of marine Actinomycetes
species have been reported (Fenical and Jensen, 2006; Maldonado et al., 2005). The
present study was designed to evaluate various types of samples from different marine
environments as sources of Actinomycetes in order to screen for bioactive compounds.
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1.4 Rare Actinomycetes
Actinomycetes are widely distributed in natural and manmade environments
where they play an important role in the degradation of organic matter. Being well
known as a rich source of antibiotics and bioactive molecules they are of considerable
importance in industries. While applying conventional isolation techniques, most of the
isolates recovered on agar plates have been identified as genus Streptomyces, the
dominant actinomycetes found in soil. Different factors must be considered for the
function of screening novel bioactive molecules: choice of source of screening,
pretreatment procedure, selective media, the culture condition and identification of
candidate colonies on a primary isolation plate. Re-isolation of previously known
antibiotics strains is a major problem in new drug discovery. Less well studied
organisms such as non-streptomycete species (rare actinomycetes) provide attractive
opportunities for developing new antibiotics (Nareeluk N et al., 2009). The successful
discovery of novel rare actinomycetes needs ecological study of their distribution. New
methods for isolating them from diverse habitats and culturing them in the laboratory
are needed for such studies because complicated procedures for isolation and
cultivation are currently required (Lazzarini et al., 2000).
The role of rare actinomycetes as bioactive molecule sources became apparent
as these organisms provided about 25% of the antibiotics of actinomycete origin
reported during 1975 to 1980. Usually rare actinomycetes have been considered as
strains of actinomycetes whose isolation frequency by conventional methods is much
lower than that of streptomycete strains. Subsequently, employing pretreatments of soil
by drying and heating stimulated the isolation of rare actinomycetes. An alternative
approach was to make the isolation procedure more selective by adding chemicals such
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as phenol to the soil suspension. Many actinomycetes have shown multiple resistances
to wide ranges of antibiotics. Different antibiotic molecules were used in selective
medium to inhibit the competing bacteria including fast-growing actinomycetes.
Macromolecules such as hair hydrolysate, casein, humic acid and chitin and were
chosen as carbon and nitrogen sources for isolation of rare actinomycetes.
After isolating an actinomycete, it was initially identified on the basis of
morphological characters so as to have a preliminary determination of the genus.
Actinomycetes can be observed under the light microscope using coverslip culture
(Arifuzzaman et al., 2010, Khan et al., 2008), and slide culture techniques (Kavitha &
Vijayalakshmi, 2007). Strains are observed for several characters such as presence or
absence of aerial mycelium, fragmentation or non fragmentation of substrate and aerial
mycelium, presence of sclerotia, spore chain morphology and color of spore mass
(Kavitha &Vijayalakshmi, 2007). Genera of purified isolates can be identified based on
morphological comparisons to the existing description of known genera as given in
Bergey's Manual of Determinative Bacteriology. It is important to avoid strain
duplication by an accurate identification of isolates. However taxonomic
characterization based only on morphological and biochemical characteristics, is
tedious (Singh et al., 2009). There is a need to develop molecular methods that are used
in conjunction with the earlier techniques would help in differentiating between the rare
and common genera of Actinomycetes (Valenzuela-Tovar et al., 2005).
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Table 1. Summary of methods developed for the selective isolation of rare -actinomycetes from soil (1987-2007), (Masayuki Hayakawa 2008).
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1.5 Antimicrobial property of Actinomycetes
1.5.1 Antibacterial property of Actinomycetes
Infectious diseases are the leading cause of death worldwide which accounts for
13.3 billion deaths constituting about 25% of all deaths. Presently, resistance capacity
to the drugs used in the treatment and cure of many infectious diseases is increasing,
however microbial infections are being found to be responsible for more number of life
threatening diseases than previously thought. The reasons for the addition in incidence
of infectious disease are not properly understood. One of the reasons is the emergence
of multidrug resistant pathogens (Cassell and Mekalanos, 2001). Among the different
drug resistant pathogens, methicillin-resistant Staphylococcus aureus (MRSA),
vancomycin-resistant Staphylococcus aureus (VRSA), extended spectrum β-lactamases
(ESBL) producing bacteria such as E. coli, Klebsiella sp. and Pseudomonas aeruginosa
and multi drug resistant Mycobacterium tuberculosis (MDR-MTB) are of major
concern.
The demand for new antibiotics continues to grow due to the rapid emergence
of antibiotic resistant pathogens causing life threatening infections in spite of
considerable progress in the fields of chemical synthesis and engineered biosynthesis of
antimicrobial compounds. The changing pattern of diseases as well as the emergence of
resistant bacterial strains to currently used antibiotics continuously put demand on the
drug discovery scientists to search for novel antibiotics (Baltz, 2007).
Actinomycetes make important biogeochemical functions in terrestrial soils and
are highly assessed for their unique ability to produce biologically dynamic secondary
metabolites. Altogether 22,500 bioactive secondary metabolites have been reported and
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out of which 16,500 compounds show antibiotic activities. Out of the total 22,500
bioactive secondary metabolites, 45% (10,100) are reported to be produced by
actinomycetes in which 7630 from Streptomycetes and 2470 from rare actinomycetes.
A search for recent literature brought out that at least 4607 patents have been issued on
actinomycetes related products and processes (Berdy J. 2005).
1.5.2 Antifungal properties of Actinomycetes
Plant pathogens are estimated to cause yield reductions in crops of almost 20%
worldwide. Fungal diseases are among the main causes of low yields, viz. the effect of
a disease caused by Pyricularia grisea Sacc, reaches up to 80% due to its destructive
capacity (Fabregat, 1984; Cárdenas, 1999) under favorable conditions. Each year it is
estimated to destroy enough rice to feed more than 60 million people. The fungus is
known to occur in approximately 85 countries worldwide. The extensive use of
chemicals viz. fungicides can not be considered as an optimum solution because it
enhances the risk of the chemical pollution of the environment and agricultural
production.
An important study published by the US environment protection agency
indicates that in the US alone 3000-6000 cancer cases are induced annually by pesticide
residues on foods and another 50-150 by exposure to pesticides during application
(Goud, 2004).This type of findings increasingly put emphasis on drawbacks of many
chemical fungicides, pesticides in terms of their effect on the environment as well as on
the grower and consumer of agriculture products (Cool, 1993).Large demands for
fungicides exist in agriculture, food protection and medicine. In order to cope with the
needs of the fast-growing world population, yields must be improved by optimizing
inputs, including fungicides (Knight et al., 1997).
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Actinomycetes are known to have the capacity to synthesize bioactive
secondary metabolites which include enzymes, herbicides, pesticides, and antibiotics.
Almost 80% of the world’s antibiotics are known to come from actinomycetes, mostly
from the genera Micromonospora and Streptomyces (Pandey et al., 2004).
Actinomycetes are also used to control plant diseases. For example, Streptomyces strain
5406 has been used in China for more than 30 years now to protect cotton crops against
soilborne pathogens (Yin et al., 1965). One decade ago, Kemira Oy has developed a
biofungicide that contains living cells of S. griseoviridis Anderson, Erlich, Sun and
Burkholder to protect crops against Fusarium and Alternaria infections (Lahdenpera et
al., 1991).
Soil inoculation with specific streptomycete strains could significantly reduce
damages caused by Pythium or Phytophthora species in ornamental (Bolton 1980;
Malajczuk, 1983), legume (Filnow and Lockwood, 1985) and horticultural productions
(Crawford et al., 1993; Turhan and Turhan, 1989). Moreover, food quality has to be
guaranteed by controlling fungi that produce mycotoxins. Filamentous fungi can also
cause opportunistic systemic mycoses, associated primarily with patients with AIDS or
those receiving treatment with immunosuppressive agents.
Antifungal chemotherapy relies heavily on fungicides and many efforts have
been made to standardize test procedures in order to increase reproducibility between
laboratories (Cormican and Pfaller, 1996).
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Table 2. Some of the metabolites of Actinomycetes
Sl. No
Name References
1 Abamectin T. W. Miller et al.: Antimicrob. Agents Chemother. 15: 368 (1979) 2 G. Alber-Schoenberg et al.: J. Am. Chem. Soc. 103: 4216 (1981) 3 Aclarubicin T. Oki et al.: J. Antibiot. 28: 830 (1975) 4 Actaplanin M. Debono et al.: J. Antibiot. 37: 85 (1984) A. H. Hunt et al.: J. Org. Chem. 49: 635, 641 (1984) 5 Actinobolin T. H. Haskell et al.: Antibiot. Ann. 1958-1959: 505 6 Actithiazic acid E. Tejera et al.: Antibiot. Chemother. 2: 233 (1952) 7 Albomycin G. F. Gauze et al.: Novosti Med. Akad. Med. Nauk. (USSR) 23: 3
(1951) 8 Amdinocillin D. S. Reeves et al.: J. Antimicrob. Chemother. 3 (Suppl. B): 5 (1977) J. W. Krajewski et al.: J. Antibiot. 34: 282 (1981) 9 Amicetin A C. DeBoer et al.: J. Am. Chem. Soc. 75: 499, 5864 (1953) M. H. McKormich et al.: Antibiot. Chemother. 3: 718 (1953)
10 Amidinomycin S. Nakamura et al.: J. Antibiot. 14A: 103, 193 (1961) S. Nakamura et al.: Chem. Pharm. Bull. 9: 641 (1961)
11 6-Aminopenicillanic acid F. R. Batchelor et al.: Nature 183: 257 (1959) 12 Amoxicillin Beecham, series: Antimicrob. Agents Chemother. 1970: 407-430
Long et al.: J. Chem. Soc. (C), 1020 (1971) R. Sutherland et al.: Antimicrob. Agents Chemother. 1971: 411
13 Amphomycin B. Heinemann et al.: Antibiot. Chemother. 3: 1239 (1953) Bodanszky et al.: J. Am. Chem. Soc. 95: 2352 (1973) (Glumamycin)
14 Ampicillin G. N. Rolinson, S. Stevens: Brit. Med. J. 2: 191 (1961) Beecham, series: Brit. Med. J. 2: 193 (1961) Doyle et al.: J. Chem.Soc. 1440 (1962)
15 Ansamitocin E. Higashide et al.: Nature 270: 721 (1977) 16 Anthramycin M. D. Tendler et al.: Nature 199: 501 (1963)
W. Leimgruber et al.: J. Am. Chem. Soc. 87: 5791, 5793 (1965) N. Komatsu et al.: J. Antibiot. 33: 54 (1980)
17 Aplasmomycin Y. Okami et al.: J. Antibiot. 29: 1019 (1976) H. Nakamura et al.: J. Antibiot. 30: 714 (1977)
18 Augmentin C. P. Robinson et al.: Med. Actual. 18: 213 (1982) D. J. Weber et al.: Pharmacotherapy (Carlisle, Mass.) 4: 122 (1984)
19 Aureothricin H. Umezawa et al.: Japan Med. J. 1: 512 (1948) 20 Avoparcin M. P. Kunstmann et al.: Antimicrob. Agents Chemother. 8: 242 (1968) 21 Azalomycin F M. Arai et al.: J. Antibiot. 13A: 46 (1960)
M. Meguro et al.: Antibiot. Chemother. 12: 554 (1962) 22 Azithromycin S. C. Aronoff et al.: Antimicrob. Chemother. 19: 275 (1987) 23 Benzylpenicillin A. Fleming: Br. J. Exp. Pathol. 10: 226 (1929)
E. B. Chain et al.: Lancet II: 226 (1940) 24 Bicozamycin T. Miyoshi et al.: J. Antibiot. 25: 569 (1972)
T. Kamiya et al.: J. Antibiot. 25: 576 (1972) M. Nishida et al.: J. Antibiot. 25: 582, 594 (1972)
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25 Butirosin Woo et al.: Tetrahedron Lett. 2617, 2621, 2625 (1971) Dion et al.: Antimicrob. Agents Chemother. 2: 84 (1972)
26 Cactinomycin H. Brockmann, Grubhofer: Naturwiss. 36: 376 (1949) 27 Candicidin D Lechevalier et al.: Mycologia 45: 155 (1953) 28 Carbenicillin E. T. Kundsen et al.: Br. Med. J. 3: 75 (1967) 29 Carbomycin F. W. Tanner et al.: Antibiot. Chemother. 2: 441 (1952)
F. A. Hochstein, K. Murai: J. Am. Chem. Soc. 76: 5080 (1954) 30 Carumonam R. L. Then et al.: Chemotherapy 30: 398 (1984) 31 Cefetamet Pivoxil Takeda: Ger. Pat. (1977)
US Pat. (1987) 32 Cefotaxime R. Heymes et al.: Infection (Munich) 5: 529 (1977)
R. Weise et al.: Antimicrob. Agents Chemother. 14: 807 (1978) 33 Ceftezole T. Noto et al.: J. Antibiot. 29: 1058 (1976)
Fujisawa, series: Chemotherapy (Tokyo) 24: 619, 655, 671, 722, 1006 (1976)
34 Ceftriaxone R. Reiner et al.: J. Antibiot. 33: 783 (1980) M. Seddon et al.: Antimicrob. Agents Chemother. 18: 240 (1980) P. Angehrn et al.: Antimicrob. Agents Chemother. 18: 913 (1980)
35 Cephalexin Muggeleton et al.: Antimicrob. Agents Chemother. 353 (1968) Kind et al.: Antimicrob. Agents Chemother. 361 (1968)
36 Cephaloglycin Kurita et al.: J. Antibiot. 19A: 243 (1966) J. L. Spencer et al.: J. Med. Chem. 9: 746 (1966)
37 Cephalosporin C G. Brotzu: Lay. Ist. Igiene Cagliari (1948) G. G. F. Newton, E. Abraham: Nature 175: 548 (1955)
38 Chloramphenicol J. Ehrlich et al.: Science 106: 417 (1947) G. Keiser: Dtsch. Med. Wochensch. 96: 1544 (1971)
39 Coumermycin H. Kawaguchi et al.: J. Antibiot. 18A: 1, 11, 220 (1965) 40 Cycloheximide A. J. Whiffen et al.: J. Bacteriol. 52: 610 (1946)
B. E. Leach et al.: J. Am. Chem. Soc. 69: 474 (1947) 41 Cyclosporin A A. Ruegger et al.: Helv. Chim. Acta 59: 1075 (1976) 42 Dactinomycin S. A. Waksman et al.: Proc. Soc. Exp. Biol. Med. 45: 609 (1940)
Manaker et al.: Antibiot. Ann. 1954-55: 853 43 Dermostatin M. J. Thirumalachar, S. K. Menon: Hindustan Antibiot. Bull. 4: 106
(1962) D. S. Bhate et al.: Hindustan Antibiot. Bull. 4: 159 (1962) R. C. Pandey et al.: J. Antibiot. 26: 475 (1973) revised str.
44 Detoxin H. Yonehara et al.: J. Antibiot. 21: 369 (1968) 45 Dicloxacillin C. Gloxhuger et al.: Arzneimittel-Forsch. 15: 322 (1965)
H. Yoshioka et al.: J. Antibiot. B 20: 34 (1967) 46 Dirithromycin P. Luger, R. Maier: J. Cryst. Mol. Struct. 9: 329 (1979)
F. T. Counter et al.: Antimicrob. Agents Chemother. 15: 1116 (1991) 47 Doxorubicin F. Arcamone et al.: Tetrahedron Lett. 1007 (1969) 48 Erythromycin
Estolate V. C. Stephens et al.: J. Am. Pharm. Assoc. Sci. Ed. 48: 620 (1959)
49 Erythromycin J. M. McGuire et al.: Antibiot. Chemother. 2: 281 (1952) 50 Floxacillin R. Sutherland et al.: Br. Med. J. 4: 455 (1970) 51 Fungichromin M. C. McCowen et al.: Science 113: 292 (1951)
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52 Fusidic acid W.O. Godtfredsen et al.: Nature 193: 987 (1962) 53 Gentamicin M. J. Weinstein et al.: Antimicrob. Agents Chemother. 1 (1963)
J. Black et al.: Antimicrob. Agents Chemother. 138 (1964) D. J. Cooper et al.: J. Chem. Soc. (C), 960, 2876, 3126 (1971)
54 Gibberelic acid P. J. Curtis, B. E. Cross: Chem. & Ind. 1066 (1954) B. E. Cross: J. Chem. Soc. 4670 (1954)
55 Gusperimus H. Iwasawa et al.: J. Antibiot. 35: 1665 (1982) Y. Umeda et al.: J. Antibiot. 38: 886 (1985)
56 Griseofulvin A. E. Oxford et al.: Biochem. J. 33: 240 (1939) J. F. Grove et al.: J. Chem. Soc. 3977 (1952)
57 Herbimycin S. Omura et al.: J. Antibiot. 32: 255 (1979) S. Omura et al.: Tetrahedron Lett. 4323 (1979) A. Furusaki et al.: J. Antibiot. 33: 781 (1980)
58 Hetacillin Hardcastle et al.: J. Org. Chem. 31: 897 (1966) Ueda et al.: J. Antibiot. 20B: 206 (1967)
59 Idarubicin F. Arcamone et al.: Cancer Treat. Rep. 60: 829 (1976) (Carminomycin I)
M. J. Broadhurst et al.: J. Chem. Soc, Chem. Commun. 158 (1982) 60 Imipenem-Cirastatin F. M. Kahan et al.: J. Antimicrob. Chemother. 12: Suppl. D, 1-35
(1983) Merck, series: J. Antimicrob. Chemother. 12: Suppl. D, 1-155 (1983) J. Birnbaum et al.: Am. J. Med. 78: Suppl. 6A, 3-21 (1985)
61 Isepamicin T. L. Nagabhushan et al.: J. Antibiot. 31: 681 (1978) 62 Josamycin T. Osono et al.: J. Antibiot. 20: 174 (1967) 63 Kanamycin H. Umezawaet al.: J. Antibiot. A10: 181 (1957) 64 Kasugamycin H. Umezawa et al.: J. Antibiot. 18A: 101 (1965) 65 Kitasamycin T. Hata et al.: J. Antibiot. 6A: 87 (1953)
Y. Sano et al.: J. Antibiot. 7A: 93 (1954) 66 Lactacystin S. Omura et al.: J. Antibiot. 44: 113, 117 (1991) 67 Laidlomycin F. Kitame et al.: J. Antibiot. 27: 884 (1974)
F. Kitame et al.: J. Antibiot. 29: 759 (1976) R. D. Clark et al.: J. Antibiot. 35: 1527 (1982) R. D. Clark et al.: J. Antibiot. 39: 1765 (1986)
68 Lenampicillin F. Sakamoto et al.: Chem. Pharm. Bull. 32: 2241 (1984) 69 Leptomycin T. Hamamoto et al.: J. Antibiot. 36: 639, 646 (1983) 70 Lincomycin D. J. Mason et al.: Antimicrob. Agents Chemother. 555 (1962) 71 Lividomycin T. Mori et al.: J. A antibiot. 24: 339 (1971) 72 Macbecin S. Tanida et al.: J. Antibiot. 33: 199 (1980) 73 Maduramicin C. M. Liu et al.: J. Aantibiot. 36: 343 (1983) 74 Maridomycin H. Ono et al.: J. Antibiot. 26: 191 (1973) 75 Micronomicin R. Okachi et al.: J. Antibiot. 27: 793 (1974)
R. S. Egan et al.: J. Antibiot. 28: 29 (1975) 76 Mycobactin P Francis et al.: Nature 163: 365 (1949)
Francis et al.: Biochem. J. 55: 596 (1953) 77 Nanaomycin S. Omura et al.: J. Antibiot. 27: 363 (1974)
H.Tanaka et al.: J. Antibiot. 28: 860, 868, 925 (1975)
18
78 Nocardicin H. Aoki et al.: J. Antibiot. 29: 492 (1976) M. Hashimoto et al.: J. Antibiot. 29: 890 (1976)
79 Nogalamycin B. K. Bhuyan et al.: Antimicrob. Agents Chemother. 836 (1965) 80 Paromomycin Parke Davis: US Pat. 2,916,485 (1959)
Pfizer: US Pat. 2.895,876 (1959) 81 Pecilocin S. Takeuchi et al.: J. Antibiot. 12A: 109, 195 (1959)
S. Takeuchi et al.: J. Antibiot. 17A: 267 (1964) 82 Piperacillin T. Saito et al.: Jap. J. Antibiot. 30: 835 (1977)
K. Ueo et al.: Antimicrob. Agents Chemother. 12: 455 (1977) 83 Pivampicillin von Daehne et al.: J. Med. Chem. 13: 607 (1970)
von Daehne et al.: Antimicrob. Agents Chemother. 1970: 430 84 Quatrimycin McCormick et al.: J. Am. Chem. Soc. 79: 2849 (1957)
Kaplan et al.: Antibiot. Chemother. (Basel) 7: 569 (1957) 85 Quinacillin Richards et al.: Nature 199: 354 (1963) 86 Rifamycin Maggi et al.: Chemotherapia 11: 285 (1966)
P. Sensi et al.: Antimicrob. Agents Chemother. 699 (1967) 87 Roxithromycin R. N. Jones et al.: Antimicrob. Agents Chemother. 24: 209 (1983) 88 Salinomycin H. Kinashi et al.: Tetrahedron Lett. 4955 (1973)
Y. Miyazaki et al.: J. Antibiot. 27: 814 (1974) 89 Streptolydigin T. E. Eble et al.: Antibiot. Ann. 1955-1956: 893
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(1944) S. A. Waksman: Streptomycin. The Williams and Wikins Co.
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L. H. Conover et al.: J. Am. Chem. Soc. 75: 4622 (1953) 94 Tetracycline,
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95 Thienamycin J. S. Kahan et al.: Abstracts Papers of 16th Intersci. Conf. Antimicrob. Agents
96 Tobramycin W. M. Stark et al.: Antimicrob. Agents Chemother. 314-348 (1967) 97 Tunicamycin A. Takatsuki et al.: J. Antibiot. 24: 215 (1971) 98 Ubenimex H. Umezawa et al.: J. Antibiot. 29: 97 (1976)
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19
2. AIMS AND OBJECTIVES
Isolation of Actinomycetes from Marine environment.
Isolation of Marine rare Actinomycetes by Pre treatment of sediment samples.
Morphological and Biochemical studies of isolated Actinomycetes.
To study Antimicrobial activity of Marine Actinomycetes.
Isolation and partial screening of bioactive molecules from selected
Actinomycetes.
Molecular studies and Identification of selected Marine Actinomycetes by
16S rRNA sequencing.
20
3. REVIEW OF LITERATURE
3.1 Natural habitat and Isolation of Actinomycetes
Weyland. H. (1969) opined that Bacteria belonging to the family
Actinomycetaceae are well known for their ability to produce secondary metabolites,
some of which are active against pathogenic microorganisms. Traditionally, these
bacteria have been isolated from terrestrial sources although the first report of
mycelium-forming actinomycetes being recovered from marine sediments.
A study was done by Paul R. Jensen et al., (2005) on natural product discovery
to characterize marine actinomycete diversity and how adaptations to the marine
environment affect secondary metabolite production. It would create a better
understanding of the possible utility of these bacteria as a source of useful products for
biotechnology. In Marinophilus and Salinospora strains, there appears to be a
correlation between phylogeny and biosynthetic capacity.
Abou-elela et al., (2005) worked on phenotypic characterization and numerical
taxonomy of some actinomycetes strains isolated from Burullos Lake. Twenty nine
actinomycetes isolates randomly selected of 130 from Burullos Lake were investigated.
These isolates were characterized taxonomically for 63 phenotypic characters including
morphological, biochemical, nutritional, substrate utilization and anti-microbial
analyses. A representative strain from sample site was chosen, they were identified as
21
Streptoverticillum morookaense, Nocardiabrasiliensis, Streptomyces alanosinicus,
Streptomyces globosus and Streptomyces gancidicus.
Jensen et al., (2005) reported that major populations of marine Actinomycetes
reside in ocean sediments and that these bacteria display highly evolved marine
adaptations including the requirement of seawater for growth. These findings will
hopefully encourage additional studies addressing the ecological roles of
Actinomycetes in the marine environment, their diversity, distributions, culture
requirements, and evolutionary responses to life in the sea.
A study was conducted by Crawford et al., (1993) on the use of selective media
where they included 267 actinomycete strains that were isolated from rhizosphere-
associated and non-rhizosphere-associated British soils. For isolating diverse group of
actinomycetes the organic media which has low nutrient concentrations were found to
be very good by avoiding contamination and overgrowth of isolation media by
eubacteria and fungi. All isolates grew well at the pH range of 6.5 to 8.0 and only some
strains were unable to grow at pH 6.0. A significant number of isolated strains failed to
grow at the pH 5.5.
Luis A. Maldonado and Fragoso-Yanez (2009) Reported Seventeen different
media known to support the growth and isolation of members of the class
Actinobacteria were evaluated as selective isolation media for the recovery of this
microbial group from marine sediments samples collected in the Gulf of California and
the Gulf of Mexico. Complete 16S rRNA gene sequencing revealed that the isolates
belonged to several actinobacterial taxa, notably to the genera Actinomadura, Dietzia,
Gordonia, Micromonospora, Nonomuraea, Rhodococcus, Saccharomonospora,
Saccharopolyspora, Salinispora, Streptomyces, ‘‘Solwaraspora’’ and Verrucosispora.
22
Previous works on marine sediments have been restricted to the isolation of members
of the genera Micromonospora, Rhodococcus and Streptomyces. This study provides
further evidence that Actinobacteria present in marine habitats are not restricted to the
Micromonospora- Rhodococcus-Streptomyces grouping. Indeed, this first systematic
study shows the extent of actinobacterial diversity that can be found in marine
sediments collected in Mexico and probably worldwide.
Streptomycete growth in nonsaline media was reduced by 39% compared with
that in seawater. The actinoplanetes had a near obligate requirement of seawater for
growth, and this is presented as evidence that actinomycetes can be physiologically
active in the marine environment Jensen, (1991).
A study was conducted by Vijayakumar et al., (2007) by the collection of 192
actinomycetes. Colonies were isolated from 18 marine sediment samples of Palk Strait
region of Bay of Bengal, India. Among those colonies, 68 isolates were
morphologically distinct on the basis of colour of spore mass, aerial and substrate
mycelia formation, production of diffusible pigment, sporophore morphology and
reverse side colour. Thirty-nine isolates were assigned to the genus Streptomyces (1),
Actinopolyspora (10), Saccharopolyspora (7), Actinomadura (4), Nocardiopsis (3),
Micromonospora (2), Actinomyces (1), Actinoplanes (1) and Microbispora (1). From
these, 64 isolates with aerial mycelia, 65 isolates with substrate mycelia and 61 isolates
had both aerial and substrate mycelia.
Actinomycetes population from continental slope sediment of the Bay of Bengal
was studied by Das et al., (2008). The diverged range of actinomycete population is
from 5.17 to 51.94 CFU/g and 9.38 to 45.22 CFU/g dry sediment weight. From stations
in 1000 m depth no actinomycete colony was isolated. Populations in stations in 500 m
23
depth in were higher than that of 200 m depth stations. Three actinomycetes genera
were identified. The found Streptomyces was the dominating one which was followed
by Micromonospora and Actinomyces. Spiral spore chain showed the maximum
abundance and the spore surface was smooth.
Actinomycetes population has been identified as one of the major groups of the
soil population Kuster, (1968). They are gram-positive organisms and assumed to be
the transition group between fungi and bacteria. They belong to the order
Actinomycetales (Super kingdom: Bacteria, Phylum: Firmicutes, Class:
Actinobacteria, Subclass: Actinobacteridae). According to Bergey's Manual
actinomycetes are divided into eight diverse families: Actinomycetaceae,
Mycobacteriaceae, Actinoplanaceae, Frankiaceae, Dermatophilaceae, Nocardiaceae,
Streptomycetaceae, Micromonosporaceae Holt, (1989) and they comprise 63 genera
Nisbet and Fox, (1991). Based on 16s rRNA classification system they have recently
been grouped in ten suborders: Actinomycineae, Corynebacterineae, Frankineae,
Glycomycineae, Micrococineae, Micromonosporineae, Propionibacterineae,
Pseudonocardineae, Streptomycineae and a large members of actinomycetes are still
remained to be grouped (www.ncbi.nlm.nih.gov).
Vijayakumar and Remya. (2008) studied 173 actinomycetes colonies which
were isolated from near shore marine environment and mangrove ecosystem at 8
different locations of Kerala, West Coast of India. Among them, 64 isolates were
morphologically distinct on the basis of spore mass colour, reverse side colour, aerial
and substrate mycelia formation and production of diffusible pigment. The majority
(47%; n=30) of these isolates were assigned to the genus Streptomyces.
24
The concept of biocontrol of plant diseases includes disease reduction or
decrease in inoculums potential of a pathogen brought about directly or indirectly by
other biological agencies Johnson and Carl, (1972). Outside the host, the biocontrol
agent may be antagonistic and thereby reduce the activity, efficiency and inoculums
density of the pathogen through antibiosis, competition and predation/hyper parasitism.
This leads to a reduction in inoculum potential of the pathogens Baker, (1977). The
biocontrol agent may operate primarily in the host tissue, there by indicating a
resistance response in the host, by transmitting factors that render the pathogens
avirulent Cook and Baker, (1983). These interactions are mediated by environment and
may have an overriding impact in determining whether biocontrol operates in a system
or not.
3.2 Antimicrobial properties and Identification of Actinomycetes
Kathiresan et al., (2005) Reported 160 isolates of marine Actinomycetes were
isolated from the sediment sample drawn from mangroves, estuary, sand dune, and
industrially polluted coast. Of these, mangrove sediments were rich sources of marine
Actinomycetes. Each isolate was tested against four phytopathogenic fungi, viz.
Pyriculariaoryzae, Rhizoctoniasolani, Helminthosporiumoryzae (causing blast, sheath
blight and leaf spot diseases of rice) and Colletotrichum falcatum (causing red rot
disease of sugar cane). The isolates appeared to produce high antifungal compounds at
120 hrs of incubation period of production medium culture. Glucose and soybean meal
were the best carbon and nitrogen sources, respectively and 17.5 ppt was the best
salinity level for maximum antibiotic production. Cylinder plate method was better for
antifungal assay than the disc diffusion method. Based on the morphological and
culture characteristics, the potent strains were identified as the species belonged to the
25
genus Strepomyces. These strains may prove to be the potent source for isolation of
agro based fungicides.
Lam (2006) reported that marine actinomycetes produce different types of new
secondary metabolites. Many of these metabolites possess biological activities and have
the potential to be developed as therapeutic agents. Marine actinomycetes are a prolific
but underexploited source for the discovery of novel secondary metabolites.
Hong et al. (2009) conclude that actinomycetes isolated from mangrove habitats
are a potentially rich source for the discovery of anti-infection and anti-tumor
compounds, and of agents for treating neurodegenerative diseases and diabetes.
A study was done by Debananda S. Ningthoujam et al. (2009) on screening of
Actinomycete isolates. From niche habitats in Manipur for Antibiotic Activity from 172
lake sediment (SCNA, LS1series), 35 lake sediment (CA, LSCH series), 120 river
(NRP, NRB and….Series), 39 forest (AML series), 35 cave (KC1 series), 101 salt
spring (NH, N3S and …..Series), 46 Shirui jungle (SJ series) and 66 Shirui hill (SH
series) actinomycetes isolates were obtained. About 18 potent antibacterial, 1 anti
pseudomonas, 1exclusively antifungal and 3 broad-spectrum antimicrobial
actinomycetes were chosen for further studies.
A study was done by Jeffrey et al., (2008) on actinomycetes and screening
results indicate that 48, 46 and 41 numbered isolates showed the ability to secrete the
enzyme cellulase, lipase and protease respectively. By the selection of phytopathogens
as test strains, antimicrobial test was done and it was observed that four isolates 3, 25,
35 and 37 showed antagonistic reaction with Fusarium palmivora, Pantoae dipersa,
26
Bacillus subtilis and Ralstonia solanacearum respectively. The most promising six
isolates were selected and identified by their 16SrRNA sequence.
Vellar Estuary was investigated by Dhanasekaran et al., (2009) as a source of
actinomycetes to screen for the production of novel bioactive compounds. Insignificant
variation was shown by the physiochemical characteristics of soils samples in
temperature, pH and dissolved phosphate, and total variation was shown by nitrogen
compounds and organic matter. Among the 20 actinomycetes isolate, some of the very
putative antibiotics producing actinomycetes were isolated which were strongly
inhibiting the growth of both Gram positive and Gram-negative bacteria including yeast
like fungi. Only 4 isolates exhibited the antimicrobial activity and only the strain
DPTD-5 showed broad-spectrum activity and was further characterized and identified.
Earlier studies done by Joe D' Souza and Nelson De Souza (2000) proved that estuarine
soils are rich in actinomycetes and they can produce antibiotics. The samples from
estuary were reported as very rich habitat for the microbial diversity. After this, the
morphological, physiological characterization and its DNA homology suggested that
the strain DPTD-5 was very similar to Streptomyces bikiniensis.
A study was done by Nathan et al., (2004) which recommended a unique
selective enrichment procedure resulted in the identification and isolation of two new
genera which were marine-derived actinobacteria. By this study it was revealed that
approximately 90% of the microorganisms were cultured by using the presented
method which was from the prospective new genera, it indicated as a result of its high
selectivity. From the Bismarck Sea and the Solomon Sea off coast of Papua New
Guinea, 102 actinomycetes were isolated from the subtidal marine sediment. Among
the isolated actinomycetes two new genera (represented by strains of the PNG1 clade
27
and strain UMM518) within the family Micromonosporaceae, showed activities against
multidrug-resistant gram-positive pathogens, vaccinia virus replication and malignant
cells.
A study conducted by Arora et al., (2005) on actinomycete strain Streptomyces
griseus B1, isolated from soil showed that when it was grown on cellulose powder as
submerged culture it produced high levels of all the three components i.e. filter paper
lyase (FPase), CM-Cellulase and β-glucosidase of the cellulolytic enzyme system.
Extracellular activity was shown by FP activity and CMCellulase whereas
β-glucosidase was both intra- and extra-cellular. When grown on hardwood powder
under submerged culture it showed highest FPase activity. It was not able to use lignin
monomers (ferulic acid, syringic acid and vanillic acid) as carbon source. When it
grows on hardwood and softwood powders under solid-state conditions, it depleted the
cellulose (36.3% in the case of softwood and 14.4% in the case of hardwood). And it
also caused partial loss of lignin content in both the substrates by solubilizing them.
Mangamuri et al. (2012) reported that Pseudonocardia species VUK-10 strain checked
against the pathogens of Staphylococcus aureus MTCC 3160, Streptococcus mutans
MTCC 497, Bacillus subtilis ATCC 6633, E.coli ATCC 35218, Enterococcus faecalis
MTCC 439, Pseudomonas aeruginosa ATCC 9027, Candida albicans ATCC 10231,
Fusarium oxysporum MTCC 3075, Aspergillus niger and reported that this strain
inhibited growth of Gram positive and Gram negative bacteria, yeast and fungi
suggesting a broad spectrum nature of the active compound.
28
Oskay et al., (2004) worked on antibacterial activity of some Actinomycetes
isolated from farming soils of Turkey worked on 50 different actinomycete strains were
recovered from farming soil samples collected from Manisa Province and its
surrounding. These were then assessed for their antibacterial activity against four
phytopathogenic and six pathogenic bacteria. Results indicated that 34% of all isolates
are active against, at least, one of the test organisms; Agrobacterium tumefaciens,
Erwiniaamylovora, Pseudomonas viridiflova, Clavibactermichiganensis subsp.
michiganensis, Bacillus subtilis ATTC 6633, Klebsiellapneumoniae ATTC 10031,
Enterococcus feacalis ATCC 10541, Staphylococcus aureus ATCC 6538, Esherichia
coli ATCC 29998 and Sarcinalutea ATCC 9341. According to antibacterial activity
and spectrum broadness, seven of the isolates were selected and characterized by
conventional methods. The unusual antibiotic profile of these isolates underlined their
potential as a source of novel antibiotics.
Nakashima et al., (2005) worked on the Actinomycetes as host cells for
production of recombinant proteins and reported that the host-vector systems of
Actinomycetes are suitable for expressing proteins of Actinomycetes and proteins from
closely related organisms as well as from higher eukaryotes. However, further
development of host-vector system in Actinomycetes is required, particularly with
respect to the modification of host cells.
In a study performed by Baskaran et al., (2011), various pre-treatment methods
and three different media were employed for the isolation of bioactive actinomycetes
from mangrove sediments of Andaman and Nicobar Islands, India. Collection of
sediment sample was done from four different sites of mangrove forest and pre-treated
by dry heat method, after that the media were supplemented with nalidixic acid 75
29
µg/mL and cycloheximide 80 µg/mL. In sediment samples, the mean actinomycetes
population density were recorded as 22 CFU-10-6/gm in KUA medium followed by 12
CFU-10-6/gm in AIA medium and 8 CFU-10-6/gm in SCA medium. Total 42
actinomycetes strains were isolated. All the isolates were evaluated for their
antibacterial activity against pathogenic bacteria on two different media. Among all the
tested isolates, antibacterial metabolite production was shown by 22 species. They were
tested against test bacteria namely, Bacillus subtilis, Staphylococcus aureus, Klebsiella
pneumoniae and Salmonella typhi. The strain A107 was identified as Streptomyces spp.
which had shown the maximum activity against all used pathogens.
Several antibiotics were tested by Williams and Davies, (1964) against a range
of actinomycetes, fungi and bacteria representing types found in soil. From these tests
four antibiotics polymyxin-B sulphate (5.0 pg/ml), actidione (50 pg/ml), nystatin (50
pg/ml.) and sodium penicillin (1.0 µg/ml) were selected for incorporation into a starch
casein medium to achieve selective growth of actinomycetes on soil dilution plates. The
antifungal ones (nystatin, actidione) did not inhibit any strains not even at the highest
concentration of 100µg/ml among all the seven antibiotics tested against a range of
actinomycetes. Same types of results were obtained by Porter et al. (1960). Polymyxin-
B sulphate has shown the least inhibition by antibacterial antibiotics. Most appropriate
mixture for the enumeration of soil actinomycete colonies was starch casein medium
with the two antifungal antibiotics (nystatin, actidione). And for isolation of
actinomycete colonies the use of same medium with all four antibiotics was most
satisfactory.
Actinobacteria producing bioactive compounds were isolated by Kumar et al.,
(2011) by the serial dilution method from marine sediments collected from Bay of
30
Bengal at a depth of 10-40m near pudimadaka coast of Andhra pradesh. During the
study total 78 isolates were obtained and among all the isolates Streptomycetes is
predominant. Among all the 78 isolates antibacterial and antifungal activity exhibited
by 22 isolates exhibited antibacterial and antifungal activity, respectively. Promising
activities were shown by the active isolates viz. BTS-112, BTS-314 and BTS-401.
After this the strains were further characterized and identified to be belonging to the
genus Rhodococcus and Streptomyces.
A total of 55 actinomycetes isolated from soil sample of Karanjal region in
Sundarbans were characterized by Arifuzzaman et al., (2010) for morphological
recognition as well as antimicrobial activity. The total numbers of isolates were 14, 11,
27, and 3 which belong to Nocardia, Streptomyces, Actinomyces and Micromonospora
respectively, as they were identified from the sample. Against one or more gram
negative pathogenic bacteria such as Shigella dysenterriae type-1, Shigella boydii,
Shigella sonnei, Pseudomonas, Vibrio cholerae-0139, Salmonella typhi-Ao-12014,
Shigella flexneri-AN-31153,Plesiomonas, Hafnia spp., Escherichia coli- 186LT and
Vibrio cholerae-OGET, twenty actinomycetes isolates were found which could produce
antibiotics. A varied group of actinomycetes were found in sundarbans soil and among
them three of the tested isolates had a broader spectrum antibacterial activity that
showed there potential as a source of antibiotics for pharmaceutical interest.
A study was performed by the Santhi et al., 2010 by the assortment of total of
two marine actinomycetes isolated from different locations of the Manakudi Estuary in
Arabian Sea of Tamilnadu, India. All the isolated strains exhibits higher antagonistic
activity against the Gram positive bacteria; methicillin resistant Staphylococcus aureus,
Salmonella typhi, Enterobacter sp, Bacillus subtilis, Proteus vulgaris and Klebsiella
31
pneumoniae. Intermediate activity was shown by them against Gram negative organism
Pseudomas auregionosa and it shows no antagonistic effect towards yeast like Candida
albicans. Pink colored actinomycetes (PJS) with white aerial mycelium and pink
substrate mycelium and black colonies (BJS) of white aerial mycelium and yellowish
white substrate mycelium shows potent inhibiting effect of other microorganisms. And
after that 16S rDNA phylogenetic typing gave ~1500 bp amplified product and it was
cloned in pGEMT easy vector. The sequencing of amplified product will give the
phylogeny of isolated actinomycetes and the further study on this organism may
provide a new antibiotic for the welfare of human being.
A total of 94 actinomycete strains were isolated by the You et al., 2005 from the
marine sediments of a shrimp farm, 87.2% belonged to the genus Streptomyces, others
were Micromonospora spp. Fifty-one percent of the actinomycete strains among them
showed activity against the pathogenic Vibrio spp. strains. Almost thirty-eight percent
of marine Streptomyces strains produced siderophores on chrome azurol S (CAS) agar
plates. From the total strains seven strains of Streptomyces were found to produce
siderophores and they inhibit the growth of Vibrio spp. in vitro. Two strains belonged
to the Cinerogriseus group, which was the most frequently isolated group of
Streptomyces. From the obtained results it could be assumed that in aquaculture it
could be use as biocontrol agent.
A study was performed by Imeda (2005) on several enzyme-inhibitor-producing
actinomycetes isolated from various samples collected from the marine environment
and were characterized. Among them it was found that they could produce novel
compounds which were useful in medicine and agriculture. From neurotic sea water a
strain of actinomycetes was isolated and characterized which produced antibiotics
32
against gram positive bacteria only in the presence of sea water. And the production of
antibiotics was observed at seawater concentrations ranging from 60 to 110% (v/v).
Hence, the production was seawater-dependent. They showed the production of
tetrodotoxin (TTX), it was known otherwise as puffer fish toxin, and it was investigated
in various actinomycetes collected from the marine environment. Among the 10
isolates from various regions of sea, 9 produced TTX as judged by their retention times
on high performance liquid chromatography (HPLC).
A study was done by Thenmozhi and Kannabiran (2010) to screen the
antifungal activity of the crude extract prepared from the 8 strain of Streptomyces spp.
isolated from the Puducherry coast of India. Primarily, eight strains were screened
against three species of Aspergillus namely A. fumigatus, A. flavus and A. niger for
antifungal activity. This search resulted in the isolation of a potential strain VITSTK7.
The optimization was done by the production media for the maximum yield of
secondary metabolites. And the metabolites were extracted using ethyl acetate; it is
than lyophilized and screened for antifungal activity against the three Aspergillus
species by well diffusion method. Maximum zone of inhibition observed was 21mm for
A. fumigatus in comparison with the standard antifungal antibiotic Nystatin which
shows 20 mm. The molecular taxonomy and phylogeny revealed that the strain
belonged to the genus Streptomyces. The sequence of 16s rRNA of the strain
Streptomyces spp.VITSTK7 was submitted to the database of GenBank under the
accession number GQ 499369.
Seventy-nine Actinomycetes were isolated from soils of Kalapatthar (5545m),
Mount Everest region by Gurung et al., 2009. Among all the isolates twenty
seven(34.18%) of the isolates showed an antibacterial activity against at least one test
33
bacteria among two Gram positive and nine Gram negative bacteria in primary
screening by the technique of perpendicular streak method. In secondary screening
thirteen (48.15%) showed antibacterial activity. After that the MIC test was done and
the minimum inhibitory concentration (MIC) of antibacterial metabolites of the isolate
K.6.3 was 1mg/ml whereas that of isolates K.14.2 and K.58.5 was 2mg/ml. Each of the
metabolites showed two spots on thin layer chromatography plate which was
completely different from the spot produced by vancomycin. And the active isolates
from primary screening were heterogeneous in their overall biochemical, macroscopic
and physiological characteristics.
A study was carried by Harald Bredholt et al., (2007) on Actinomycetes from
Norway for Diversity and Biological Activity. Approximately 3,200 actinomycete
bacteria were isolated using four different agar media from the sediment samples
collected at different locations and depths (4.5 to 450 m). Grouping of the isolates first
according to the morphology followed by characterization of isolates chosen as group
representatives by molecular taxonomy revealed that Micromonospora was the
dominating actinomycete genus isolated from the sediments. Micromonospora was
found at higher relative amount in the deep water sediments compared to the shallow
water samples. The nine percent of the isolates clearly required sea water for normal
growth which suggests that these strains represent obligate marine organisms.
Zhonghui Z. et al., (2000) screened the actinomycetes for antimicrobial or
antitumor activity, which were isolated sea plants and animals collected from the
Taiwan Strait, China. MTT assay was used to study the antitumor activity and DNA
target activity was studied by the biochemical induction assay while antimicrobial
activity was determined by observing bacterial and fungal growth inhibition. 20.6% of
34
marine actinomycete cultures displayed cytotoxic activity on P388 cells at dilutions at
and below 1:320 and 18.6% on KB cells. 2.96% of marine actinomycete cultures
showed inducing activity. Among isolated all marine actinomycetes, the genus
Micromonospora had the highest positive rate of inducing activity. Even though, most
antimicrobial activity was found in the genus Streptomyces. These results indicate that
actinomycetes from marine organism could be used as source for antitumor and
antimicrobial bioactive agents.
Li and Liu (2006) worked on the Marine sponge Craniella austrialiensis-
associated bacterial diversity revelation based on 16S rDNA library and biologically
active Actinomycetes screening, phylogenetic analysis and reported that bacterial
diversity associated with South China Sea sponge C. austrialiensis was assessed using a
16S rDNA clone library alongside restriction fragment length polymorphism and
phylogenetic analysis. It was found that the C. austrialiensis-associated bacterial
community consisted of alpha, beta and gamma-Proteobacteria, Firmicutes,
Bacteroidetes as well as Actinobacterium. Actinomycetes were isolated successfully
using seawater medium with sponge extracts. According to the BLAST and
phylogenetic analysis based on about 600-bp 16S rDNA sequences, 11 of the
representative 23 isolates closely matched the Streptomyces sp. while the remaining 12
matched the Actinomycetales. Twenty Actinomycetes have antimicrobial potentials, of
which 15 are found to possess broad-spectrum antimicrobial potentials and finally
conclude the sponge C. austrialiensis-associated bacterial community is very abundant
including Proteobacteria, Firmicutes, Bacteroidetes and Actinobacterium while
Actinomycetes is not predominant. Artificial seawater medium with sponge extracts is
suitable for Actinomycetes isolation. Most of the isolated C. austrialiensis-associated
Actinomycetes have a broad spectrum of antimicrobial activity.
35
4. MATERIALS AND METHODS
In the present work it has been envisaged to investigate the marine
Actinomycetes and their biological properties i.e. Isolation, Characterization,
Antimicrobial properties, partial isolation of bioactive compounds and Molecular
studies for identification.
4.1 Area of Study
Marine soil samples were collected from four different stations, seashore soils
covering Karwar (Uttar Kannada District), Karnataka, Central West Coast of India.
Name and Geographical position of the study cites are shown in the Table 1. Locations
of the study sites were fixed during the time of collection with the help of Global
Position System (GPS) and Google map with four study stations.
Table 3. Locations of Study area
Station Name Latitude Longitude
Station 1-Kinner. 140 51` 28.12``N. 740 10` 38.72``E.
Station 2-Sunkeri. 140 50` 32.33``N. 740 10` 06.16``E.
Station 3-Devbhag. 140 51` 22.49``N. 740 06` 40.52``E.
Station 4-Majaali. 140 51` 21.29``N. 740 06` 38.51``E.
36
Figure 1. Location of Study stations
4.1.1 Samples Collection
Samples were collected during the low tide from above four stations. Marine
sediment samples were collected with hand core and sterile polythene bags from 0-6
inch depth (15.24 cm) as upper surface of sediments consist more microbial load
Nevine B. G. et al., (2000) and transported to the laboratory for process as described by
Jensen et al., (1991). The samples were stored in refrigerator for further study.
4.2 Isolation and enumeration of Actinomycetes
Along with the collection of sediment samples, some Hydrological parameters
like Temperature, Salinity, Water pH (Standard Methods, APHA 20th edition) and
dissolved oxygen (winklers method) were also recorded on the site itself (Table 2).
In the present study, Starch Casein Agar, Starch Nitrite Agar and
Actinomycetes Isolation Agar (Hi Media) were used for the isolation of Actinomycetes
37
by spread plate technique. The samples were subjected to serial dilution (up to 10-6
dilution) by adding 1g of sediment sample in 10 mL of distilled water and an aliquot of
0.1 ml from these samples were spread on the agar media (Singh and Agrawal, 2002 &
2003). After incubation of 11 – 21days at 280C-300C, Actinomycetes were recognized
by their characteristic tough, leathery colonies, branched vegetative mycelia, aerial
mycelia and spore formation. On the bases of these criteria, only actinomycetes with
well-developed and branched hyphae were included in this study. Streak plate method
was used to purify cultures of actinomycetes (Williams and Cross, 1971, Singh and
Agrawal 2002; Agrawal 2003). The actinomycete colonies those developed on the
plates were enumerated and expressed as colony forming units (CFU), (Williams and
Cross, 1971, Singh and Agrawal 2002; Agrawal 2003) (Table 9).
4.2.1 Rare actinomycetes
The actinomycetes, especially Streptomyces, are remarkable producers of
antibiotics arising from their unlimited capacity to produce secondary metabolites with
diverse chemical structures and biological activities. Tens of thousands of such
compounds have been isolated and characterized, and many have been developed into
drugs for treatment of a wide range of human diseases (Bull et al., 1992; Franco &
Coutinho, 1991). Re-isolation of previously known antibiotics strains is a major
problem in new drug discovery. Less well studied organisms such as non-streptomycete
species (rare actinomycetes) provide attractive opportunities for developing new
antibiotics (Nareeluk et al., 2009). The successful discovery of novel rare
actinomycetes needs ecological study of their distribution. New methods for isolating
them from diverse habitats and culturing them in the laboratory are needed for such
studies because complicated procedures for isolation and cultivation are currently
required (Lazzarini et al., 2000).
38
4.2.2 Sampling and pretreatment of soil
Collected sediment samples were subjected to five different pretreatment
methods namely control (without any Treatment), antibiotics, Dry heat, Air dry, wet
heat and Phenol (Hayakawa et al., 1995) as described in Table 3, were carried out in
the first 24h after sampling.
4.2.3 Selective isolation of rare actinomycetes
Serially diluted soil suspensions were spread onto selective isolation medium,
and incubated for upto 4 weeks at 28°C. Starch casein agar (SCA), humic acid vitamin
agar (HVA), hair hydrolysate vitamin agar (HHVA), and Bennet's agar (BA) were used
for the selective isolation of rare actinomycetes agar (Hayakawa & Nonomura, 1987).
Preliminary designation of rare actinomycete colonies were done by microscopic
observation with a long working distance microscope.
4.3 Morphological and Biochemical characterization of actinomycetes
Morphological studies of isolates include microscopic and macroscopic
observations. Microscopic observations were made with a light microscope by using
the method of Shirling and Gottlieb (1966). Active purified isolates of actinomycetes
were identified by comparing their morphology of spore bearing hyphae with entire
spore chain and structure of spore chain in 1000x (Oil immersion) (Figure 3) and Gram
nature with the actinomycetes colony morphologies such as configuration, margin and
elevation (Figure 2) as described in Bergey’s manual of Determinative Bacteriology,
Ninth edition (2000) and the organism was identified.(Cross, 1989; Lechevalier, 1989;
Locci, 1989; Wendisch and Kutzner, 1991; Williams et al., 1989). Various biochemical
tests were performed for the identification of the potent isolates.
39
In the present study with respect to sampling station, Out of selected 55 isolates
first A1-A10 actinomycetes were from Kinner station, A11-A20 actinomycete isolates
from Sunkeri, A21-A30 isolates from Devbhag and A31-A42 actinomycetes from
Majaali station. Whereas in case of rare actinomycetes were obtained from four
sampling stations in following manner RA43-RA47 from Kinner, RA48-RA52 isolates
from Sunkeri, RA53 and RA54 isolates from Devbhag and RA55 isolate from Majaali.
Figure 2. Different Colony characteristics of Actinomycetes on Agar medium (Madigan et al., 1997).
40
Figure 3. Different types of spore arrangement on actinomycetes mycelia
4.3.1 Gram staining
Gram staining was performed for pure strain cultures to determine whether Gram
negative or Gram positive. Gram staining detects a fundamental difference in the wall
composition of bacteria. All the chemicals required for Gram staining were procured
from Hi media, Mumbai, India.
A. Bacterial smears were prepared by using following steps
1. A drop of distilled water was taken on a clean glass slide.
2. Isolated colony was transferred to the slide with a sterile loop or needle
touch and mixed in the water drop.
41
3. Mixed until just slightly turbid and made smear on glass slide.
4. Smears were air dried and heat fixed.
5. Slides preparation allowed cooling.
B. Slides were flooded with crystal violet, and allowed the slide for 60 seconds.
C. Washed off the crystal violet with running tap water.
D. Slides were flooded with Gram’s iodine, and allow the slide for 60 seconds.
E. Washed off with running tap water.
F. Decolorized with 95% alcohol and 5% water solution until the solvent flows
colorless from the slide (approximate 5-10 seconds).
G. Washed off the crystal violet and Gram’s iodine complex with running tap
water.
H. Slides were flooded with counter stain, safranine for 60 seconds.
I. Rinsed with water and allow to air dry.
J. Gram negative: Bacterial cells are decolorized by the 95% alcohol solution and
take on a red to pink color when counterstained with safranine. They appeared
pink in colour under microscope.
Gram positive: Bacterial cells retain the crystal violet and remained purple to
dark blue in colour under microscope.
4.3.2 Biochemical tests for actinomycetes
All isolates were tested using the Rapid Biochemical kit (Himedia) according to
the manufacturer’s instructions and were tested for esculin hydrolysis and for acid
production from arabinose, cellobiose, fructose, glycogen, inositol, lactose, mannitol,
ribose and trehalose (at 1% w/v). Isolates were also tested for the presence of
preformed glycosidic enzyme activities such as (N-acetyl-β-glucosaminidase, N-acetyl-
42
bgalactosaminidase, α-L-fucosidase, β-glucosidase, α-glucosidase, α-arabinosidase, α-
galactosidase and β-galactosidase).
Catalase test
The catalase activity and ability of each isolate to grow in air was also determined.
2-3 ml of the hydrogen peroxide solution was poured in to a small test tube. Using a
sterile inoculation loop, test organisms were removed from Starch casein agar plate and
immersed in the hydrogen peroxide solution. The test tube was observed for Bubbles
formation. This test is used to detect the soil bacteria.
Lactose and mannitol fermentation test
This test is used to differentiate the microorganisms fermenting carbohydrate (such as
lactose and mannitol).
Voges-Proskauer test
To detect the production of acetylmethylcarbinolacetoin, a natural product formed from
pyruvic acid in the course of glucose fermentation. The glucose broth together with the
organism was inoculated and incubated at 28°C for 3 days. Approximately 3 ml of
alpha naphthol was added followed by 1 ml of 40% KOH and mixed well for 30
minutes. Pink coloration of solution indicate VP (+) and no coloration indicate VP (-).
ONPG Test
This test determines the presence of β-galactosidase. A fermenter may produce slow
results if it lacks the enzyme is permease. If this enzyme is missing, but the
β-galactosidase is present within the cell, fermentation can take place. The ONPG test
uses the reagent, orthonitrophenyl- β -D-galactopyranoside. When added to a bacterial
suspension, this reagent will produce a yellow color if β-galactosidase is present.
43
Urease test
This is determination test for the hydrolysis of urea to ammonia and water. In this test a
dense suspension of the test organism was prepared 2 ml saline in a small tube. A
urease tablet was added into the tube and incubated at 28- 30oC for up to 4 h or
overnight. When the medium becomes alkaline and an indicator produces a pink colour.
Decarboxylation of Arginine
Many species of bacteria possess enzyme capable of de-carboxylating specific
aminoacids in the test medium. The decarboxylase enzymes remove a molecule CO2
from an aminoacid to form alkaline-reacting amines. The following are the amino acids
most commonly tested and their amine degradation products. Arginine-Citruline, the
amines resulting from the decarboxylation reaction can be detected with Ninhydrin
reagent after extraction from the broth culture with chloroform.
Esculin hydrolysis
The purpose was to see if the microbe can hydrolyze the compound esculin as a carbon
source, Bile escullin agar medium was used. This is a nutrient agar-based medium
containing 0.1% esculin and 10% bile salts, and allowed to solidify at a slant. The bile
salts inhibit some bacteria, and so the ability to grow in the presence of bile salts
represents a second test use for the medium.
An inoculum from a pure culture is transferred aseptically to a sterile tube of
bile esculin agar and streaked along the slant. After incubation at 28-30oC for
appropriate time abundant growth on the slant indicates a positive test for growth in the
presence of bile. If growth is present, esculin hydrolysis can be observed if the medium
has taken on an intense, chocolate brown coloration due to the production of dark
brown compound esculetin.
44
4.4 Screening of actinomycetes and rare actinomycetes for antimicrobial activity
The synthesis level of secondary metabolites of microorganisms has been found
to be at its peak at the late stationary phase of their life cycle. The arrows head indicates
direction and increase in different parameter like time, number of viable microbes or
cell and number of metabolites synthesis (Figure 4). The antimicrobial screening
method consists of two steps, primary screening and secondary screening.
Figure 4. Primary and secondary metabolites levels in viable cell growth phase
4.4.1 Primary screening
In primary screening the antimicrobial activity of pure isolates were determined
by perpendicular streak method on Nutrient agar (NA). In vitro screening of isolates for
antagonism Nutrient agar plates were inoculated with Actinomycetes isolate by a single
streak of inoculum in the petri plate. After 4 days of incubation at 28°C the plates were
seeded with perpendicular test organism’s single streak. The microbial interaction were
analyzed by the determination of the size of the of the clear/inhibition zone (Madigan et
al., 1997).The test organisms used were; Bacillus subtilis (MTCC 441), Staphylococcus
aureus (MTCC 737), Enterobacter aerogens (MTCC 111), Escherichia coli (MTCC
1668), Klebsiella pneumonia (MTCC 109), Proteus vulgaris (MTCC 1771),
Pseudomonas aeruginosa (MTCC 2453), Salmonella typhi (MTCC 733) and Shigella
sp (MTCC 1457).
45
4.4.1.2 Actinomycetes as biocontrol against fungal pathogens
Actinomycetes isolates were tested for their inhibitory activity against five
fungal plant pathogens, viz., Alternariasp, Rhizoctonia bataticola, Sclerotium rolfsii,
Fusarium udum and Pyricularia species by dual culture technique (Ganesan and
Gnanamanickam, 1987) as described below. These plant pathogens were obtained from
Department of Agricultural microbiology, University of Agricultural Sciences (U.A.S),
Dharwad.
In primary screening each fungal pathogen was grown on a PDA plate till it
covered the whole surface of the agar. With the help of a sterile 5 mm cork borer, a disc
of fungal growth from this plate was taken and placed at the center of a fresh PDA
plate, which consist 4 day old culture of actinomycetes isolates were streaked parallel
on either side of the fungal disc 3 cm away from the disc. The plates were kept for
incubation at 30oC for 96 hours. Visual observations on the inhibition of growth of
fungal pathogen were recorded after 96 hours of incubation in comparison with the
control PDA plate which was simultaneously inoculated and incubated with respective
fungal pathogen.
Figure 5. Different steps of antimicrobial assay
Isolation of Actinomycetes and Rare actinomycetes from sediments
Actinomycetes Isolates on agar slants
Growth on agar plates Cultivation in different growth media
Liquid fermentation Ethyl acetate, Methanol, Petroleum ether
Crude extract
Assay for antimicrobial activity
46
4.4.2 Secondary screening
Secondary screening was performed by disk diffusion (Kirby-Bauer et al.,
1966) method against the above mentioned standard test organisms.
4.4.3 Fermentation and Isolation of antimicrobial metabolites
Fermentation and Isolation of antimicrobial metabolites consists different steps
as shown in (figure 5). Fermentation was carried out using Starch Casein Broth in an
Erlenmeyer flask. Antimicrobial compound was recovered from the filtrate by solvent
extraction method. Ethyl acetate was added to the filtrate in the ratio of 1:1(v/v) and
shaken vigorously for 1 hour for complete extraction. The ethyl acetate phase that
contains bioactive compounds was separated from the aqueous phase. It was evaporated
to dryness in water bath at 80°-90°C and the residue obtained was weighed. Thus
obtained extract was used to determine antimicrobial activity and to perform
Bioautography (Pandey et al., 2004). In the present study diffusion methods were used
for Antimicrobial susceptibility test.
4.4.4 Disc Diffusion method
1. Turbidity standard for inoculum preparation:
To standardize the inoculum density for a susceptibility test, a BaSO4 turbidity
standard, equivalent to a 0.5 McFarland standard or its optical equivalent (e.g. latex
particle suspension), was used. A BaSO4 0.5 McFarland standard prepared by 0.5 ml
aliquot of 0.048 mol/L BaCl2 (1.175% w/v BaCl2. 2H2O) was added to 99.5 ml of 0.18
mol/L H2SO4 (1% v/v) with constant stirring to maintain a suspension. The correct
density of the turbidity standard was verified by using a spectrophotometer. The
absorbance at 625 nm should be 0.008 to 0.10 for the 0.5 McFarland standard.
47
2. Inoculums Preparation, with the help of sterile loop pure colony culture was
transferred to a tube containing 5 ml of Starch casein broth medium.
3. The broth cultures were incubated at 28±2C until they achieves the turbidity of the
0.5 McFarland standard.
4. The turbidity of the actively growing broth culture was adjusted with sterile saline or
broth to obtain turbidity optically comparable to that of the 0.5 McFarland standard.
This results in a suspension containing approximately 1-2 x 108 CFU/ml.
5. Test Plates were inoculated, within 15 minutes after adjusting the turbidity of the
inoculum suspension, a sterile cotton swab was dipped into the adjusted suspension.
The swab should be pressed gently on inner wall of the tube to remove excess
inoculum from the swab.
6. The dried surface of a Mueller-Hinton agar (Himedia) for bacteria and Potato
Dextrose Agar (Himedia) for fungi, plates were inoculated with respective inoculums
by streaking the swab over the entire sterile agar surface and ensure for an even
distribution of inoculum.
7. The petriplate lid partially kept open for 5 to 7 minutes, in Laminar air flow hood, to
allow for any excess surface moisture to be absorbed before applying the antimicrobial
impregnated disks.
8. The predetermined series of crude extract contained discs from the secondary screening
were dispensed onto the surface of the inoculated agar plate. Each disc pressed gently
to ensure complete contact with the agar surface.
48
9. The petriplates with discs were incubated at 37C for 24 to 48 hours for bacteria and
ambient temperature for 48 to 72 hours for fungi. Solvent Ethyl Acetate and broth
media were used as negative control (10µl/disc). Each plate was examined for the
uniform growth and uniformly circular resulting inhibition zones. The diameters of the
complete inhibition zones were measured, including the diameter of the disc. Zones
are measured to the nearest whole millimeter, using a scale, which is held on the back
of the inverted Petri plate.
4.5 Thin Layer Chromatography
Riguera (1997) described various strategies used for isolation and structural
elucidation of pharmacologically active metabolites from marine organisms.
Procedures adapted to the physical and chemical characteristics of the compounds
isolated, particularly to their polarity and lipo- or hydrophilic character have been
mentioned. After biomass extraction with an adequate solvent system (usually
methanol or acetone), the first step in the isolation of a natural compound from the
main extract or broth usually consists of a sequential gradient partition with solvents
(petroleum ether, hexane, CCl4, CHCl3, CH2Cl2, Ethyl acetate, n-butanol and water).
The fractions so obtained contain compounds distributed according to their polarity. In
the case of a bioactive extract, this process can be guided by the appropriate assay to
localize the active component. In this way, water-soluble organic material represented
mainly by alkaloid salts, amino acids, polyhydroxysteroids, and saponins is found in
the n-Butanol fraction. The CH2Cl2, EA, fraction affords compounds of medium
polarity such as peptides and depsipeptides, while in the hexanes, PE and CCl4, only
low polarity metabolites (hydrocarbons, fatty acids, acetogenins, terpenes, etc.) are
found.
49
According to their increasing polarity different solvent systems and ratios were
checked, among in the Petroleum ether: ethyl acetate (1:1) solvent system
actinomycetes crude extracts showed distinctive and clear separation on chromatogram.
Thin Layer Chromatography of the crude extracts and control (SCB medium)
was done using Petroleum ether: ethyl acetate (1:1) solvent systems. The procedure was
as follows.
Procedure:
1. Commercial Thin Layer Chromatography plates using alumina, backed sheets
(Silica gel 60 F254, 0.25mm thick Merck, Darmstadt, Germany) were used.
2. Using a pencil and ruler, a line was drawn 1 cm from the bottom of the plate. Points
were marked and labeled at a distance of approximately 1 cm from each other
corresponding to the samples to be loaded.
3. The 20µl of extracts and control were spotted on their respective labeled spots.
Followed by dry and spotting method.
4. The spots were allowed to dry.
5. The chromatography chamber was rinsed clean and dried. 100 ml of the solvent
system was added into it.
6. The TLC plate loaded with sample was placed in it with the bottom edge just
touching the solvent system.
7. The chamber was closed with the lid to prevent solvent from evaporating and the
solvent was allowed to move till it reached approximate 2cm below the top edge.
8. Once the solvent had reached the solvent front, it is removed from the developing
chamber and air-dried. The separated components were visualized by the following
methods
50
i. Placing the plate in the UV chamber and observed at 254nm and 366nm.
ii. Placing the plate in the Iodine chamber till the bands were observed.
iii. By spraying 5% methanolic sulphuric acid and placing it in the oven.
4.5.1 Bioautography of Thin Layer Chromatogram
The Ten microliters of the ethyl acetate fractions and reference antibiotic
(Tetracycline and Nystatin in the concentration 10µg/ml, as antibacterial and antifungal
respectively) were applied on the plates and the chromatogram was developed using
Petroleum ether: ethyl acetate (1:1) as solvent system. The plates are run in triplicate;
one set was used as the reference chromatogram and the other two were used for
Bioautography (Pandey et al., 2004). The spots in the chromatogram are visualized in
the iodine vapor chamber and UV chamber.
In case of fungi, the organic solvent of chromatogram was evaporated by a
stream of air. TLC plates were homogeneously sprayed with about 10 mL potato
dextrose broth containing conidia. Plates were incubated for 3 days in a moist chamber
at 25°C in the dark and the appearance of clear zones in the mycelium layer indicated
antifungal activity. Exposure (15–30 min) of fungi with unpigmented mycelia and
spores to iodine vapors significantly enhanced contrast in order to detect inhibition
zones (Hadacek & Greger 2000).
4.6 Isolation of Genomic DNA from Actinomycetes and 16S rRNA sequencing
4.6.1 Isolation of Genomic DNA from Actinomycetes by HiPurATM Miniprep
Purification Spin kit
(All the chemicals required for molecular characterization were procured from Hi
media, Mumbai, India.)
51
Introduction
The DNA purification procedure using the miniprep spin columns comprises of three
steps viz,
Adsorption of DNA to the membrane,
Removal of residual contaminants and
Elution of pure genomic DNA.
4.6.1.2 Concentration, yield and purity of DNA
In the present study, Nano drop instrument (Quawell) UV Spectrophotometer
and agarose gel electrophoresis techniques were used to reveal the concentration and
the purity of the genomic DNA.
The calibration was done by the spectrophotometer and the absorbance was
measured at 260 nm and 320 nm. Absorbance reading at 260 nm falls between 1.0
corresponds to approximately 50 µg/ml of DNA. The A260-A320/A280-A320 ratio should
be 1.5-2.0. Purity is determined by calculating the ratio of absorbance at 260 nm to
absorbance at 280 nm (Table 15).
Concentration of DNA sample (µg/ml) = 50 X A260 X dilution factor.
Materials and Reagents required
1. 370C water bath or heating block.
2. 550C water bath or heating block.
3. Tabletop Microcentrifuge (with rotor for 2.0 ml tubes).
4. Ethanol (96-100%).
5. Molecular Biology Grade water.
52
Procedure:
1. Actinomycetes were grown in a Luria medium till they reach log phase.
2. Lysozyme solution was prepared using lysozyme from chicken egg white,
which is provided in the kit. A 45 mg/ml stock solution of lysozyme was
prepared as described under general preparation (200 µl of lysozyme solution
was required per isolation procedure).
3. Cell Harvestment: 1.5 ml of actinomycetes broth culture was pelleted by
centrifuging for 2 minutes at 12,000-16,000Xg (=13,000-16,000 rpm), the
culture medium (Luria broth) was removed completely and discarded.
4. Cell Resuspension: the pellet was resuspended thoroughly in 200 µl of
lysozyme solution (prepared in step 1) and incubated for 30 minutes at 37°C.
5. Cell Lysate: 20 µl of the proteinase K solution (20 mg/ml) was added to the
sample. Residual RNA was removed by following methods:
Optional RNase A treatment: The RNA-free genomic DNA was obtained, by
adding 20 µl of RNaseA solution; it was mixed and incubated for 5 minutes at
room temperature (15-25°C) then step 6 was continued.
RNaseA enzyme treatment: RNase A is a type of RNase that is commonly
used in research. RNase A (e.g., bovine pancreatic ribonuclease A) is one of the
sturdiest enzymes in common laboratory usage. It cleaves 3' end of unpaired C
and U residues.
6. 200 µl of Lysis solution was added. It was thoroughly vortexed for few seconds
and incubated at 550C for 10 minutes. A homogeneous mixture is essential for
efficient cell lysis.
7. Preparation for binding:
200 µl of ethanol (95-100%) was added to the lysate and was mixed thoroughly
by vortexing for few seconds.
53
8. Lysate loading onto Spin Column :
The lysate obtained was transfered from step 6 onto spin column. It was
centrifuged at >6,500Xg (=10000 rpm) for 1 minute. The flow-through liquid
was discarded and the spin column was placed in same 2.0 ml collection tube.
9. Prewashing:
500 µl of Prewash Solution was added to the spin column and centrifuge at
>6500Xg (=10,000rpm) for 1 minute. The flow through liquid was discarded
and the same collection tube was re-used with the column.
10. Washing:
500 µl of wash solution (WS) was added to the spin column and was
centrifuged for 3 minutes at maximum speed (12,000-16,000 rpm). The spin
column was transfered to new collection tube; it was centrifuged again at same
speed for the additional 1 minute to dry, the column must be free of ethanol
before the elution of the DNA.
11. DNA Elution:
The spin column was transfered to new collection tube. 200 µl of the Elution
Buffer (ET) was directly pipetted into the column without spilling to the sides.
It was incubated for 1 minute at room temperature. It was centrifuged at
>6500Xg (=10,000rpm) for 1 minute to elute the DNA.
12. Storage of the elute with Purified DNA:
Elute contained pure genomic DNA. For short-term storage (24-48 hrs) of the
DNA, 2-8°C is recommended for long–term storage, 20°C or lower temperature
(-80°C) is recommended. The elution Buffer would help to stabilize the DNA at
these temperatures.
54
4.6.2 DNA quantitation and electrophoresis
Electrophoretic analysis of DNA using agarose gels can confirm DNA integrity.
Typically, intact genomic DNA would be up to 40KB in size, depending upon the
species. 0.8% agarose gel was prepared by adding required quantity of agarose to 1X
Tris-Acetate-EDTA (TAE) buffer and mixed well. The mixture was heated in
microwave oven until it became clear and care was taken to avoid over boiling and
evaporation. The mixture was cooled to ~500 C and ethidium bromide was added to
make a final concentration of 0.001µg/ml. The entire mixture was poured in to a tray in
which combs were fixed to make wells in the gel. It was cooled to form uniform gel.
After gel formation, the tray was placed in buffer tank containing 1X TAE buffer for
submerged gel electrophoresis and combs were removed with care to avoid rupture of
wells. 3µl of each DNA sample was mixed with 1µl of loading dye and the mixture was
loaded into the wells. Gel was subjected to electrophoresis at 100V for 30 minutes and
visualized using gel documentation system (Vilber Lourmet. Germany), (Plate).
Chemicals and buffers used for gel electrophoresis
1. Tris-Acetate-EDTA (TAE) buffer 20X
2. Agarose (Himedia)
3. Ethidium Bromide
4. Loading Dye (Stock)
Preparation of DNA working Dilutions
100µl of DNA working dilutions were prepared at a concentration of 50ng/µl by
dissolving required amount of stock DNA sample in MB grade water. After preparation
of working dilutions the uniformity of the samples were checked by performing
electrophoresis on a 1% agarose gel. Samples were stored at -200C.
55
Polymerase Chain Reaction
The target sequences amplified on a ABI Veriti thermal cycler (Applied
Biosystems, USA) with an initial denaturation at 960C for 5 minutes and later on for 35
cycles at 950C for 60 seconds, at estimated annealing temperature of the primer for 45
seconds, extension at 720 C for 2 minutes and a final extension at the end of 35th cycle
at 680C for 7 minutes in a final volume of 20 µl containing 1.5mM MgCl2, 5pm of each
primer(Sigma Aldrich), 200 µM deoxy-NTP and 1U Taq polymerase (NEB, UK) .
Primer Details:
Table 4. Oligonucleotides used in the present study
Sl.
No
Primers
name Sequence (5’-3’)
No.of
bp
Temperat
ure in ºC Reference Specificity
1 27f AGA GTT TGA
TCA TGG CTC AG
20 56 Lane 1991;
Magarvey A.
et al.,2004.
Bacterial 16S
rRNA gene
(Universal)
2 1492r TAC GGC TAC
CTT GTT ACG
ACT T
22 56 Lane 1991;
Magarvey A.
et al.,2004.
Bacterial 16S
rRNA gene
(Universal)
Amplicon Check by Agarose Gel Electrophoresis
After the completion of 35 cycles of polymerase chain reaction 5µl of the
amplicon was electrophorased on a 1.2% agarose gel containing ethidium bromide.
DNA bands were visualized using gel documentation system (Vilber Lourmet,
Germany). The samples which were amplified successfully were used for the
sequencing after post PCR cleanup (Plate).
56
Post PCR cleanup
DNA fragments should be purified prior to sequencing to remove proteins, salts,
left out primers and dNTPs which may have detrimental effects on the sequencing
reaction. A combination of exonuclease I and shrimp alkaline phosphatase enzymes
(ExoI/SAP) were used to clean the PCR product. PCR cleanup master mix was prepared
by adding 5U of ExoI, 0.5U of SAP in final volume of 8.5 µl MilliQ water (Millipor
lifesciences, USA) and this master mix was added to 8µl of PCR product.
ExoI/SAP enzymatic reaction was allowed to proceed by heating the samples up
to 37°C for 30 minutes using a thermal cycler and then denatured the enzymes by
heating to 80°C for 15 minutes. Further 0.1 volumes of 3M sodium acetate (1.65µl) and
2.0 volumes of chilled 100% ethanol (33µl) were added and mixed well. Samples were
centrifuged at 4,000rpm for 30 minutes at 4°C. The ethanol was decanted and folded
paper towels were used to remove the excess ethanol by blotting the plate. 100µl of
cold 70% ethanol was added and centrifuged at 4,000rpm for 10 minutes at 4°C. After
decanting the ethanol PCR plate was blotted on folded paper towels to remove the
excess ethanol and the plate was centrifuged inversely for 30 seconds at 180rpm to
remove all ethanol. The pellet was resuspended in 8µl of water and stored at 40C.
4.6.3 DNA Sequencing
BigDye-terminator sequencing is a modification of the Sanger’s dideoxy chain
termination method. It utilizes labeling of the chain terminator dNTPs, which permits
sequencing in a single reaction. In BigDye-terminator sequencing, each of the four
dideoxynucleotide chain terminators is labeled with fluorescent dyes, each of which
with different wavelengths of fluorescence and emission. The dye labeled DNA
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fragments will be capillary electrophoresed and a detection system would identify the
labelled bases when they pass through a laser that activated the dye.
Cycle sequencing
BigDye labeling and chain termination was carried out by cycle sequencing
method. To label each base PCR amplicon was subjected to cycle sequencing reaction
with single primer was done using ABI Prism® BigDyeTM terminator v3.1 cycle
sequencing ready reaction kits (Applied Biosystems, USA) following the manufacturer’s
guidelines.
Sequencing Cleanup
To remove the remnants of the above reaction, to the each sample 2µl of 3M
sodium acetate, 50µl of 100% ethyl alcohol were added and incubated at room
temperature for 15 minutes to precipitate the DNA. The samples were centrifuged at
4000rpm for 30 minutes at 40C. The supernatant was discarded and the reaction plate
was centrifuged inversely at 300 rpm for 20 seconds. 100µl of 75% alcohol was added
to each sample and centrifuged at 4000rpm for 15 minutes at 250C. The supernatant was
discarded and plate was inversely centrifuged at 300 rpm for 20 seconds to remove
alcohol completely. The plate was dried at room temperature until left out alcohol was
dripped off.
Denaturation and Snap chilling of labeled amplicon
10µl of Hi-Di formamide was added to each well of the sample plate. The
samples were heated to 960C for 5 minutes and immediately cooled to 40C to denature
and linearise the cycle sequencing products.
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Sequencing run
Sample information sheets which contain analysis protocol along with the
sample details were prepared and imported into the data collection software. Prepared
samples were analysed on ABI 3500 genetic analyzer (Applied Biosystems, USA) to
generate DNA sequences.
Sequence quality check
After completion of sequencing reaction, the quality of generated sequence was
checked by using Sequencing Analysis v1.0 software (Applied Biosystems, USA). The
Applied Biosystems Sequencing Analysis Software v1.0 is designed to analyse, display,
edit, save, and print sample files generated ABI genetic analyzers. The program has a
basecaller algorithm that performs basecalling for pure and mixed base calls also. It
provides quality values (QV) for every single base and sample scores for the assessment
of average quality value of the bases in the clear range sequence for the sample. The QV
is a per-base estimate of the basecaller accuracy. The QVs are calibrated on a scale
corresponding to:
QV= –10 log10 (Pe)
Where, Pe is the probability of error.
For this study, typical high-quality pure bases QVs were set to 20 to 50 and typical high-
quality mixed bases QVs were set to 10 to 50. The samples which didn’t follow the
above conditions were re-sequenced after fresh PCR amplification.
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Sequence Alignment
The generated sequences were aligned to their respective reference sequences
with the use of SeqScape v2.5 software (Applied Biosystems, USA). SeqScape is one of
the suits of Applied Biosystems designed for automated sequence data analysis. It
performs sequence comparisons for variant identifications, SNP discovery and
validation. It allows analysis of the re-sequenced data, comparing the consensus
sequences to a known reference sequence. The reference sequences for the gene studied
were obtained from NCBI Gen bank data base.
To set clear range of the sequence, a method that considers quality values of the bases
was used which removes bases from the ends of sequences until fewer than 4 bases out
of 20 have QVs <20. Filter setting values to filter the inappropriate sequences were set
as maximum mixed bases to 20 and minimum sample score to 25. Depending on the
sequence quality and the criteria specified for filtering the samples with low quality
were not assembled by the program. These unassembled samples were re-sequenced
until it satisfied the quality.