Antimicrobial natural products II - Terrestrial and marine ... · PDF filecompounds having pharmacological value ... and marine biodiversity as a source ... property of the plant derived

Antimicrobial natural products II - Terrestrial and marine organisms

Ashba Hassan and Imran Sajid

Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore, Pakistan

Key words: Terrestrial and marine natural products

1. Introduction

According to the written records, ancient Sumerians used the herbs and described the medicinal use of plants approximately 5000 years ago. The ancient Egyptian medicinal compilation, by Ebers papyrus (1552 BC) recorded a list of folk remedies and magical medical practices. The significance of the medicinal plants is also mentioned in different religious books as well. A number of writers of the ancient era (Greeks, Chinese, Indians, Roman and Egyptians etc.) have documented the usage of medicinal plants for treating different diseases. The writers of those compilations travelled the world and explored the flora of different regions for medicinal applications. The Greek physician, Pedanius Dioscorides known as “father of pharmacognosy” gave an encyclopedia which included a number of medicinal plants and their therapeutic compounds. Similarly, the Roman author Pliny the Elder, wrote the book of “Historia naturalis” enlisted an extensive list of the medicinal plants.Theophrastus who is known as ‘Father of Botany’ also wrote a couple of books on the plant’s medicinal usage. With the passage of time, different cultures added different plants to the literature, like a significant contribution came from Arabians, Iranians and from central Asian regions. The scientific pharmacy began in 19th century and then the detailed chemistry of medicinal compounds started. In the late 19th and early 20thcentury, a turn comes in folk medicine and efforts have been established to search for more resources including microorganisms, animals and marine life. In 1940, the accidental discovery of the antibiotic, Penicillin by British scientist, Alexander Fleming led the scientists to search the antimicrobial natural compounds from microorganisms and from various other life forms. As a result various pharmaceutical industries started projects under the name of “Natural Product Discovery”. The pipeline of isolation and commercialization of antimicrobial compounds from environment started in 1950s including all the kingdoms of life and from all the ecological niches. Now a days, the growing chart of the resistance among the infectious entities insists on the discovery of new and useful compounds to provide assistance and relief to humans for the management of their healthy life style. According to the geological time scale, the first organism which appears in the proterozoic era was the ancient father from which different kingdoms of life diverge. These all kingdoms starting from the primitive, unicellular archaea to the complex multicellular mammals produces the natural antimicrobials as their defense weapons contributing to their successful survival as well as their healthy being. Here’s different groups of life on the planet earth are shown in the Fig. 1which would be discussed in the later sections of this chapter with reference to the antimicrobial products obtained from them.

Fig.1 Classification scheme showing various life forms which are the major sources of antimicrobial natural products [1]

1.1. Significance of Natural Product Discovery

Nature is more appropriate and valuable house for achieving the discovery of new drugs as compared to the chemically synthesized compounds. This makes the natural bioactive or antimicrobial compounds, the lead compounds which

Antimicrobial research: Novel bioknowledge and educational programs (A. Méndez-Vilas, Ed.)

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could be manipulated and modified according to the manufacturer demands. The situation in the present days is quite unfortunate for pharmaceutical companies due to the replication and re-discovery of the same compounds with no activity or with the same activity or narrow pattern of new activity. Drug discovery by targeting the natural products is advantageous due to following reasons: native unexplored resource high biological potency and broad range of biological activity increased chance of chemically diverse structured compounds nature selected them and they became dormant which depicted their better interaction with the biological systems would be valuable for the designing of new synthetic compounds their discovery would lead the scientist in the finding of new biosynthetic pathways as well as new mode of action Both terrestrial and marine environments constituting the prokaryotes and eukaryotes would be precious sources to get a number of the natural bioactive compounds (drugs) (Fig. 2).

2. Terrestrial environment

Terrestrial environment or ecosystem constitutes that part of the earth which includes forests, grasslands, deserts, snowy areas and rainforests. Their successful inhabitant includes following groups of life; the prokaryotic microbes and the eukaryotic vascular plants, animals, and mammals which have been targeted for obtaining antimicrobial drugs as well as industrially important compounds. The description of these few resources will be discussed in this section.

2.1. Drug producing microbes from soil

Microbes are always an important source of bioactive secondary metabolites since the discovery of penicillin and then streptomycin in the beginning of 20thcentury. The fierce living style of the microbes and their adaptations according to the environmental influence makes them significant leaders for the discovery of new classes of antibiotics. The low molecular weight of the microbial secondary metabolitesmakes them the superior candidates in medicines for the treatment of human diseases due to constructive acquiescence and easy bioavailability. Also, thousands of microbial samples urge on the usage of highly sophisticated and elaborated techniques with low sensitivity and good reliability [2] andscientific efforts are always required for increasing the chance of getting new microbes and their metabolites by employing cultural and molecular techniques. Some of the significant microbial sources which produce a chunk of valuable metabolites and commercial drugs include the following bacterial group; general bacteria (Bacillus, Pseudomonas), filamentous bacteria (Actinomycetes), endophytic microbes (bacteria and fungi). • Bacteria Bacillus spp., the aerobic spore forming rods, is the common soil bacteria and a chief candidate for obtaining industrial products including, enzymes, insecticides, vitamins and antibiotics etc. Some of the antibiotics reported from this group of bacteria are; iturin and bacillomycin(Bacillus subtilis), fusaricidin (Paenibacillus polymyxa), polymyxins (P. polymyxa), mattacin and polymyxin M (Paenibacillus kobensis M), sattabacins and sattazolins (Bacillus sp.) [3] and fusaricidin (Paenibacillus polymyxa SQR-21) [4]. Pseudomonas; The gram negative aerobic, spore forming and rod shaped bacteria, Pseudomonas, is another producer of antibiotics. Some of the derived antibiotics from this group are; viscosinamide, tensin, 2-acetamidophenol, pyrrolnitrin, pyocyanin, 2,4-diacetylphloroglucinol [5]. • Actinomycetes (Filamentous bacteria) This class of bacteria includes the members which are the most exploited group for the industrial applications [6]. Among actinomycetes, the most important genus is Streptomyces which is known to be the producer of almost 7600 compounds having pharmacological value [7]. In 1952, Stanley Waksman‘s got the nobel prize owing to the discovery of antibiotic, streptomycin from Streptomyces griseus which was effective in treating the infections caused by, Mycobacterium tuberculosis. This discovery diverts the attention of the scientists to see the different sights of this

Fig. 2 Terrestrial and marine biodiversity as a source of natural antimicrobials


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group of filamentous bacteria. Some of its other famous genera apart of Streptomyces includes; Micromonospora, Nocardia and Actinomyces etc. Presently, almost two third of the antimicrobials in the market which are used for treating different ailments in humans and animals are actually the metabolic products (secondary metabolites) produced by the members of the genus Streptomyces. The major classes of antibiotics which are produced by actinomycetes are;

Fig. 3 Structural classes of antibiotics which have been isolated from actinomycetes

• Fungi Penicillium notatum, the fungi having mycelium body belongs to microscopic eukaryotic group, was the first reported fungi as the producer of the beta-lactam antibiotic, penicillin. The group of the fungi, Aspergillales is the most important one from the kingdom fungi as antibiotics producer. Until now, three types of penicillins; penicillin G, penicillin F, and penicillin V have been obtained from the genus Penicillium which includes the following members; Penicllium notatum and P. chrysogenum [8]. The other members of kingdom fungi are also known to be the significant producers of bioactive metabolites including, basidiomycetes, Penicillium, Trichoderma, yeast and slime molds. Some of the antibiotics have been reported from the different groups of kingdom fungi (Fig. 4).

Fig. 4 Some of the antibiotics which have been reported from the kingdom fungi

2.1.1 Endophytic microbes in terrestrial plants

The richest biological diversity with reference to the endophytes is found in the plants of the terrestrial territory. Endophytes are the microbes that spend at least one part of their life in the intercellular spaces of the plant tissue, without causing any apparent damage to them. The scientific study also supports the friendly aspect of these endophytes to the plant but if they are the causative agent for any disease in them then that damage may not be remarkable or immediately non-obvious. The host plant helps the endophytic microbes in the production of their unique secondary products (chemical moieties). The benefit with respect to the endophytes is that the yield of the drug from the rare yew plant could be increased by isolating their residential microbes because these microbes would have adopt some genetic pairment with the plant metabolism and produces the same compounds produced by the host plant. One of the anticancer as well as antibiotic compound, torreyanic acid, was found to be a metabolic product of endophyte, P. microspora which was an isolated fungus from Torreya taxifolia (Florida torreya) an endangered tree [10]. So the isolation of the microbes from endangered species of the medicinal plants can minimize the usage of that plant and thus we can save our endangered valuable flora. The genus, Streptomyces, is the most targeted endophytic bacterial group due to its diverse genome encoding a number of secondary metabolites. The antibiotic, munumbicins was the first antibiotic which was reported from an endophytic Streptomyces, isolated from snakevine plant. Accordingly, an unknown reservoir of the endophytic microbe does exist inside the plant which is yet to be explored [11]. Some of the reported antibiotics from endophytic Streptomyces sp. are mention in table 1[12].


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Table 1: Plant sources, their endophytic Streptomyces, and reported antibiotics from them

Plants Endophytic Actinomycetes Antibiotics Allium fistulosum Streptomyces. sp. Antifungal Aucuba japonica Streptomyces. sp. Novobiocin analogs Cryptomeria japonica Streptomyces. sp. Cedarmycins Alnus glutinosa Streptomyces sp. Alnumycin Streptomyces galbus Actinomycin X2 and

fungichromin Perennial ryegrass Streptomyces 1-N-methylalbonoursin

(antifungal) Snakevine plant Streptomyces sp. NRRL30562 Munumbicins A-D Grevillea pteridifolia Streptomyces sp. NRRL30566 Kakadumycins Monstera sp. Streptomyces sp. MSU-2110 r Coronamycin,

3. Terrestrial plants as a source of antimicrobials compounds According to the ancient category of the medicine, the “Ethnomedicine” or “Ethnobotanic medicine” “the usage of the plants as medicine by the population of the world was the main concept since Paleolithic era. The plants have always been chief source for getting medicinal compounds to cure different infections. The history’s famous essential compound, Quinine was finally extracted from the plant; cinchona in the earlier 19th century whose tree extracts has been used since 16th century against Plasmodium falciparum for treating malaria. Today, in the under developed countries and in some rural areas of developed countries, still plants are considered as the cheapest and safest source for curing illness. The most interesting property of the plant derived compounds is their potential of having both antioxidant and antimicrobial activities. Some of these plant components includes; phenols, saponin, flavonoids, thiosulfinates, glucosinolates, phenolics and organic acids (Fig. 5).

Fig. 5 Chemical components of the plants having antimicrobial and anti-oxidant activities

The central attention is always grabbed by phenolic compounds because of their strong anti-oxidant properties as well as antimicrobial potential. Here some of the medicinal plants with their efficacy of treating different infections in mention in table 2. Table 2: Some of the commonly used plants and their efficacy for treating various ailments

Plants Treatment Plants Treatment Spinach Anti-inflammatory Blueberry UTIs and anti-diabetic Grape Anti-cancer, kidney disorders, low

cholesterol, migraine, constipation, Alzheimer’sdisease

Blackberry Cognitive functions

Red Onion Yeast infection Beet Anti-inflammatory Garlic Anti-bacterial Bell pepper Anti-cancerous Craneberry UTIs Oak Antibacterial, antifungal, antiviral and anti-

parasitic Cucumber Alzheimer’s disease Ginger Anti-inflammatory Cinchona Anti-malarial Caraway remedy for bronchitis and irritable bowel

syndrome Brussel sprouts

Anti-inflammatory Guava Diarrhea & Dysentery Cough & cold

Parsely Skin infection (acne and pimples), UTIs

Eucalyptus Antiseptic

Rosemary Anti-fungal Sage Antiviral and antibacterial (throat infections, dental abscesses)

Coriander UTIs Aloe vera Anti-cancerous, skin acne causing Fenugreek Sore

throat, constipation and diarrhea, Tea plant Antiseptic and treating of nasal infections

Cinnamon UTIs Olives anti-inflammatory and anti-cancerous Oregano Antibacterial, antifungal, antiviral

and anti-parasitic

Abbreviation; UTIs: Urinary tract infections


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4. Antimicrobials from terrestrial animals

The major goal which will be set in the coming years of this century by medical professionals and pharmaceuticals would be the search of the novel strategies for getting new antimicrobial peptides for fighting the deadly pathogens. Until now multicellular organisms (higher eukaryotes) has been the source of 2500 antimicrobials [13].The group of terrestrial animals is one of the leading candidates for obtaining antimicrobial peptides which are the components of their non-specific immune system. Thedefensive properties of these peptides makes them attractive contestant [14] due to their broad spectrum antimicrobial activity. The most notable antimicrobials of animal origin are; lactoferrin, pleurocidin, protamine, defensins, lactoperoxidase, maiginin along with lipids of animal origin. Various compounds from animals are under various phasesof clinical trials. One of the broad spectrum antimicrobial compounds having animal origin is magainans whose analogue, pexiganan is under phaseIII clinical trial [15].

5. Antimicrobial peptides and insects

In insects, the antimicrobial peptides and lysozymes,naturally occurs in their body protects them from different infections. The mosquitoes, produces defensins and cecropinsas protective components. The malarial parasite, Plasmodium cannot use/infect the mosquito, Anopheles gambiae as an intermediate host due to the production of antimicrobial peptide, gambicin by the mosquito which acts as a protective shield against malarial parasite [16].

6. Antimicrobial peptides from mammals

The two major natural antimicrobial peptides produced by the human defensive system includes, defensins and cathelicidins that have potent antimicrobial activity against a number of microbial pathogens [17]. Lactoferrin is another antimicrobial peptide found in the mammalian secretions which provide protection against infections [18]. The skin of the vertebrates which is at higher assault risk protects them by producing antimicrobial peptides. In an experiment, the peptide Cramp which is expressed in the wild mouse as skin secretion, when was mutated, that mouse with this mutant protein developed the infection [19]. This experiment indicated the importance of these peptides secreted in mammalian skin for their defensive action against pathogens. Similarly, other sites in the body of mammals also involves in providing the protective shield against invading microbes. Likewise, the paneth cells at the base of the intestinal tract in humans secretes antimicrobial peptides and protects the individual from infections but these peptides are insusceptible towards the commensal of the gut flora [20]. Some of the antimicrobial peptides from mammals which are under clinical trials are; hLF1-11 derived from lactoferricin (human) is in Phase I/II, novexatin (NP-213) derived bioactive compounds from human defensins, PXL01 from lactoferricin (human) and AC-113 derived from histatin 3 (human saliva) are in Phase II clinical trials [15]. The Fig. 6 depicts the picture of some of the antimicrobial peptides produced by eukaryotes.

Fig. 6 Antimicrobial peptides produced by some eukaryotic groups

7. Marine life and natural antimicrobial products

The marine ecosystem includes the deep sea, sea floor as well as salty marshy areas of the earth covering about 70% of the earth crust. This ecosystem is being the richest one with reference to the biodiversity. The call for newly diversified natural bioactive compounds is the key driver to start a hunt of bioactive metabolites from this environment. The possibility of the presence of novel chemical moieties in the marine environment fascinates the scientist to apply the field research in marine ecosystem. The scale of the annually reported bioactive compounds from the marine environment is increasing with time [21]. The marine life includes invertebrates and microbial flora present in the sediment, floating free in marine water or establishing the symbiotic associations with animals or with other microbes. One of the history’s program for the discovery of natural products from oceanswhich reported a number of natural bioactive compounds was started by the Roche research institute in Australia, from 1974-1981, [22]. The isolation of the pyrrole rich antibiotic from Pseudomonas bromoutilis became the pulling force for isolating the marine derived bioactive compounds in the 20th century [23]. Then some of the other compounds which came into focus includes; thiomarinol and the siderophores (alterobactins A and B), y-Indomycinone bioxalomycins, and salinosporamide A


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(marizomib) etc. Extensive systematic approaches should be developed for treasuring the world’s oceans for gettingnew potential drugs. Different groups of marine life which are the main source of obtaining bioactive compounds are mention in Fig. 7a. Hu, Chen [21] published a meta-analysis on the discovery of marine natural products on the basis of the available data of the almost last three decades and provided a picture of the eight groups of the compounds from marine environment (Fig. 7b).

Fig. 7a: Groups of marine life which produces a number of bioactive compounds b: Group of compounds which can be obtained from marine source So, on the basis of the groups mentioned in Fig 7a the following marine inhabitants will be discussed in this section. • Antimicrobials from mangroves and intertidal zones Mangroves are the forest wetlands which grow best in the intertidal zones of the tropical and sub-tropical regions. They are marshy plants, loves to grow in the condition of high saline conditions, anaerobic soil and changes sea levels. They have pneumatophoric roots, their leaves eject the salts and seeds have water diffusing quality and makes productive ecosystems along the coastal areas[24]. Mangroves and their associated microbes are the reservoir of the biologically significant compounds which includes; saponins, triterpenes, steroids, alkaloids, flavonoids and tannins. So it can be worthy to screen mangrove plants for obtaining antibacterial compounds. The mangrove plant, Avicenna marina showed different level of antimicrobial potential against the infectious eye pathogens [25]. The mangrove territory considered as the most dynamic ecotone between habitat of the terrestrial and marine environment. Mangrove fungi are the second largest group of marine fungi, one of themarine fungi isolated from brazilian mangrove plant, Laguncularia racemosa revealed their potential as producer of broad spectrum antimicrobial compounds [24]. • Sponges The medicinal history of sponges dated back to the ancient time when sponges were used for the treatment of heartaches, poisonous, stomach aches, dropsy, testicular tumors, wounds, bone fractures and infectious disease due to which they always considered as gold mines of future drugs [26]. Badiaga and Stodal syrup is one of the homeopathic versions of extracts from fresh water sponge which have been used in Poland, Russia and western world. But microbes are always being the suspect of the actual producers of bioactive compounds in the sponges, due to the favorable environment to carry out secondary metabolism. Bacterial genera which have been reported from the inside of the marine sponges are; Proteobacteria, Acidobacteria, Cyanobacteria, Archaea, Bacteriodetes, Planctomycetes, Actinobacteria and Acidobacteria [27]. The antimicrobial peptides diketopiperazines obtained from the extracts of the sponge Tedania ignis was actually produced by the symbiotic bacteria Micrococcus and hence proved the bacterial association with the sponges[28]. The antibiotics, urauchimycins A and B, were identified from a symbiotic actinomycetes sp. living in association with a sponge [29].These microbial communities resides in the mesohyl tissues of the sponges [30]. But still the extracts of the sponge have the potential pharmaceutical properties. The two of the clinically approved sponge derived antiviral compounds reported are;

Fig. 8 Clinically approved compounds derived from sponges • Antimicrobial natural products from marine algae and sea weeds Marine algae (green, brown and red algae), which are the major biomass producers, reportedly contributes about 9% of the active metabolites of the total marine natural products [31]. The antimicrobial substances which can be obtained from algal groups are; phlorotannins, lectins, fatty acids, polysaccharides (alginates, carrageenan, fucoidans, laminarans, ulvans and derivatives), carotenoids, pigments, terpenoids, alkaloids and halogenated compounds. Alginates and carrageenan have been used for decades in traditional pharmaceutics worldwide [32]. The antimicrobial metabolite, phlorotannins produced by brown algae can be used to control the infective agents especially the food spoiling microbes [33].

b. a.


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Sea weeds (marine macro-algae) the edible one, produces antimicrobial and especially antioxidant compounds. Researches symbolizes the sea weeds as prebiotic for humans and animals and can be used in various medical applications for improving health[34]. Some of the important bioactive compounds from sea weeds that have been reported to possess the antimicrobial, antiviral, anti-oxidant and anti-cancerous activities are;

Fig. 9 The major active components produced by the sea weeds • Echinoderms Echinoderms is another ancient group of invertebrate composed of a number of species, including; sea cucumber, sea lilies and sea urchin, living near the coastal areas and hence are in continual exposure to a number of pathogens, so they are required to maintain their self-integrity. They, like other invertebrates produce humoral compounds to resist infectious particles. Some of the reported antimicrobials from echinoderms are, strongylocins and centrocins isolated and characterized from Strongylocentrotus droebachiensis, paracentrin from Paracentrotus lividus and holothuroidin from Holothuria tubulosa [35]. • Bioactive compounds from cnidarians The phylum cnidarians composed of ecological friendly invertebrates with 11000 extant species which includes jellyfish, hydra, corals and sea anemone, from which 3000 significant natural compounds have been determined. Mainly, the reporting of the compounds from this phylum started in 21st century [36] and almost 2000 compounds being reported in the first decade of this century (2000-2010). Cnidarians got attention due to their capability of producing toxins and venom which play a major role in their survival strategy. Antimicrobials which were reported from this phylum are; pseudopteroxazole, pseudopterosin P, pseudopterosin Q and homopseudopteroxazole from Pseudopterogorgia elisabethae, litosterol and nephalsterol C from Nephthea sp., sarcophytolide from Sarcophyton glaucum, cembranolide from Sarcophyton trocheliophorum and aurelin (peptide) from Aurelia aurita etc.[37]. • Bioactive compounds from marine mollusks Mollusks including; whelks, slugs, mussels, octopods, oysters, squids, scallops etc. Gastropods which is a significant group of mollusks have been recognized as the potential producer of a number of bioactive compounds. The antimicrobial factors produces by the mollusks are their indole and glycerol derives compounds, terpens, chlorinated acetylenes, polysaccharides, macrolides, proteins, lysozyme and glycoproteins. The crude extracts of various mollusks species, depicted in-vitro activity against bacteria in cuttlefish [38]. Anti-HIV activity was studied in 1997 from the mussel Perna viridis which was patented later on, kelletinin from marine mollusk, diemenesin A and B from the gastropod mollusk Siphonaria diemenesis [39]. • Antimicrobials from teleost From the fish group, teleost, has become a dominant group of fish in the aquatic environment and different antimicrobials have been identified from them which includes; pleurocidin, pardaxin, misgurnin, HFA-1, piscidins, parasin, steroidal antibiotic, squalamine, (shark), parasin and hepcidin [40]. • Antimicrobials from the crustaceans Crustaceans being the most primitive group which have survived evolutionary events of millions of years depicting their successful survival strategies. Antimicrobial peptide (AMPs), armadillidin, was characterized from the crustacean Armadillidium vulgare, homarin from the lobster Homarus americanus in 2008. Carcinus maenas is the source of the AMPs crustin, but latter on its various types were obtained from different crustaceans species [41] . • Marine tunicates Marine tunicates are famous for the nitrogen rich bioactive compounds coded by their genome. Yondelis (ecteinascidin 743) and aplidine (dehydrodidemnin B) are the marketed tunicate metabolites [42]. Rubrolides is a potent antibiotic which was obtained from the colonial tunicate Ritterella rubra [43]. • Naturally occurring AMPs of miscellaneous origin or Amphibians The glandular tissues present in the amphibian skin produced the active compounds i.e. dermaseptins (frog) magainins obtained from clawed frog, phylloseptins the novel antibacterial and anti-protozoan compound was isolated from amazonian frog Phyllomedusa hypochondrialis [44]. One of the antimicrobial compound, temporins was obtained from the skin secretions of the frog, Rana temporaria in1996. Some of the antimicrobials which have been found in different marine groups are;


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Fig 10. Antimicrobial peptides derived from some marine groups

7.1. Marine microbes

Almost 90% of the marine ecosystem is occupied by the microbes. Here, like in terrestrial environment, the endophytic one surpass the others groups for getting secondary active metabolites. Zhang et al.[45]isolated epi- and endophytic fungus from sponges and algae and found that the endophytic microbes were more active against pathogens. Some of the microbial classes whether they belong to terrestrial and marine ecosystem possess the antimicrobial producing potential in their metabolic machinery. The fungal genera, Penicillium and Fusarium spp. present in the both terrestrial and marine environments are attractive source of drug discovery [45, 46]. In 1979, the gram negative, marine bacterium Pseudoalteromonas phenolica sp. which produces anti-MRSA compounds against the superbug S. aureus was isolated [47]. This genus is considered as the foremost and highest number of antibiotics producing marine bacteria from the marine microbiota. Some of the reported antibiotics from marine microorganisms includes loloatins (Bacillus), agrochelin and sesbanimides (Agrobacterium), pelagiomicins (Pelagiobacter variabilis), indomycinone and dihydrophencomycin methyl ester (Streptomyces) etc.[48]. • Marine Fungi In the past fifty years, the unique compounds from the marine bio-resources have been characterized and reported, like other microbes, a vital role with respect to the discovery of the drugs is played by marine fungi. The β-lactam antibiotic, cephalosporin C, was first time discovered from marine fungal specie, Cephalosporium acremonium, isolated from Sardinian coast [49]. Pestalotia sp. a marine fungus which lives in symbiosis with the brown algae produced anti-MRSA pestalone. Reports suggest that the production of this antibiotic (pestalone) by the fungus is due to the competition faced by them in microbial community. Similarly, the epiphytic and endophytic fungi living with invertebrates as their chemical guards are also promising to find new drugs. In 2002, according to a statistical analysis, out of 272 marine origin compounds, 85% were obtained from the epi or endophytic fungi [45]. These figures suggests the importance of this microbial group as reservoir of diversified therapeutic compounds. • Marine Actinomycetes potential Due to the presence of the competitive biosynthetic capabilities, marine actinomycetes targeted by the researchers as the prime candidate for producing natural products. The hit rate would be higher from marine actinomycetes due to its vast and diverse genome containing biosynthetic gene clusters.Some of the marine actinomycetes strains and their reported antimicrobials are given in the Fig. 11a. • Marine Cyanobacteria Cyanobacteria, the most primitive organism (microbes), on the earth is primitive photosynthetic microbial group. Cyanobacterial species produces a rich diversity of metabolites that possess different antimicrobial activity. From decades, the pharmaceutically active and important members for clinically significant compounds were obtained from the actinomycetes and hyphomycetes (fungi) but the recent trend is shifting towards the cyanobacteria. They may become a classical source of some interesting compounds of clinical importance in the future due to the variety of compounds reported from this group.

7.1.1 Marine product has terrestrial origin

Marine products can have terrestrial origin because the current of water flow carries the soil from sea shore to the deep of the sea. In this way horizontal transfer of the genes may occurs which would result in the origin of isoform of the bioactive compounds present in both environments. Like, Harman, the compound which was extracted from the terrestrial plant was also reported from marine dinoflagellates, bryozoans as well as from marine bacteria (Fig 11b) [50].


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Fig 11.a: Some of the marine actinomycetes and their bioactive secondary metabolites b:Harman, the bioactive compound found in both terrestrial and marine environments • Overall number of natural products and relative percentage of the bioactive compounds derived from

different life forms According to the statistical overview given by Berdy, 2012 [9], the total number of natural products derived from plants, animals and microbes, is about 500,000. As per this report,of the 70,000 microbial derived products 47% are bioactive compounds, while among 100,000 animal derived compounds, only 3% are bioactive and from 350,000 plant derived compounds only 7% are bioactive compounds. Thus, microbes living in the diversified environments with their complex genome and adaptive living strategies are found to be more significant with reference to the bioactive secondary metabolites. So, although alarge number of compounds can be derived from animals and plantssources as compared to the microbes but the probability of having the bioactive compounds is higher in microbial derived products as compared to all other life forms (Fig 12a). Berdy, 2012 [9] also compared the relative number of bioactive metabolites from different microbial groups from 1940s-2010 in three different time periods (Fig 12b).

Fig. 12 a: Approximate number of the total natural compounds and their origin from various life forms [9]

Fig. 12 b:Approximate percentages of microbial metabolites in three time periods; 1940-1974, 1975-2000 and 2001-2010

The application of different molecular (PCR based screening and metagenomics etc.) and strategies of high throughput screening [HTS] could increase the number of drugs from these natural resources in the future.

8. Molecular approaches to target the drugs from microbes

• Biosynthetic genes encoding antimicrobials The biosynthetic gene clusters, PKS (Polyketide synthase), NRPS (Non Ribosomal Peptide Synthase), glycopeptides and polyenes etc. are the basis for encoding many pharmacological active compounds including; antibiotics, immunosuppressant, anti-cancer and anti-cholesterolic drugs. Some of the PKS encoded reported compounds are; tetracycline, macrolides, sirolimus, erythromycin, lovastatin and epothilone B [51]. Polyenes gene clusters have been known to encode the following bioactive compounds; pimaricin, candicidin, nystatin, and amphotericins [52]. NRPS encodes beta-lactams, cephalosporins, daptomycins, cyclosporins and glycopeptides [53]. PCR based gene screening


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can be used to trace these genes and their expression can be further analyzed through RT-PCR and detailed analysis of the encoded compounds can be traced by using different available databases.

8.1 In-situ cultivation of unculturable microbes

About, 99% of all microbial species in external environments are uncultured under standard laboratory conditions but they are capable of coding a reservoir of new antibiotics. Researchers develops various methods of cultivations for these fastidious microbes. Recently, an uncultured symbiotic microbe, Entotheonella sp. which produces proteases (lassomycin) against mycobacterium was isolated from the marine sponge, Theonella swinhoei by applying in-situ cultivation technique [54]. The NRPS encoded antibiotic teixobactin was derived by the culturing of uncultivable soil community bacteria, Eleftheria terrae which was reported to be the member of the genus Aquabacteria. This bacteria was isolated by using iChip, which is a multivalent chip and is significant in isolating and growing the unculturable microbes. This method approaches almost 50% of the uncultivable microbial community in comparison to their 1% recovery on a nutrient petri dish [55].

8.1.1 Metagenomics

Metagenomic profiling, the biotechnological culture independent technique, has been emerged and rapidly spread in alleviating the biases which were caused by traditional inappropriate laboratory culturing methods. It constructs the metagenomic libraries of the environmental DNA. Gillespie, Brady [56] isolated the broad spectrum antibiotics tobramycin A and B through metagenomic approach from the soil bacterial community. The molecular approaches of 16S rDNA and DNA/DNA hybridization in silico can be applied on the metagenomic DNA from which the biosynthetic genes can be traced and hence new bioactive compounds can be identified. Here’s the schematic diagram showing thedifferent steps in the technique of metagenomics;

Fig. 13 Schematic diagram showing different steps in accessing metagenomic DNA So by applying those molecular and technical approaches, the unculturable microbes which are the reservoir of a pool of antibiotics like texiobactin and tobramycin etc. can be screened and we would be able to have new compounds with better therapeutic options.

9. High Throughput screening (HTS)

The development of drug discovery or the probability of getting successful bioactive compounds depends on the applied biological test system as well as the structurally diverse test compounds [57]. The hurdles during this process of screening of a new bioactive compound from the compound libraries can be overcome by using the platform of high throughput screening (HTS). The future dilemma related to the new compounds in the pharmaceutical companies needs to be overcome by the combination of the following complementary technologies;

• Natural-product discovery, HTS, genomics, proteomics, combinatorial biosynthesis, and combinatorial chemistry. [58].

In the past few decades, the incorporation of the compounds in pharmaceuticals through high-throughput screening (HTS) is due to its elaborated screening strategies and applied techniques. These techniques involved; X-ray crystallography for the virtual screening in order to have docking models, physiological screening in which the effect of the drug is tested on the tissues, and finally the NMR (nuclear magnetic resonance spectroscopy) which identifies the compound (Fig 14)[59].

Fig. 14a Different screening approaches used in HTS

The process of HTS from the target identification to the clinical trials includes the following steps [59]:


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Fig. 14b The process of HTS from Hit discovery phase to lead discovery phase The better exploitation of the natural antipeptides requires continual improvement in screening strategies to make it possible to screen large number of samples in shorter time period. Presently the techniques of computational biology would be friendly in exploring and recognizing the gene clusters, activating them through heterologous expression and getting the desired products. • Conclusion

Antimicrobial natural products are the major source for new pharmaceutical therapeutic compounds against the emerging deadly pathogens to control the endemics and epidemics caused by these pathogenic entities worldwide. The hit rate, for getting the valuable therapeutics from the environmental samples is always high. The recent year’s trend points towards the importance of the marine environment in context of getting unique/new compounds with broad antimicrobial activity but still the terrestrial source for obtaining therapeutic compounds can never be ignored as still a reservoir of effective bioactive compounds is locked in terrestrial biomass. We just need to have competitive strategies to explore these environments deeply and their residential prokaryotes and eukaryotes. Thus, we need to continue our investigation in terrestrial and marine ecosystems by applying the successful strategies to get full benefit from the produced metabolites of their inhabitants.

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