1 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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1
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
2
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
10
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).
11
Table 1. Summary of methods developed for the selective isolation of rare -actinomycetes from soil (1987-2007), (Masayuki Hayakawa 2008).
12
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),
(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).
14
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).
15
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)
16
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)
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)
17
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)
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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.
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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