Applied Microbiology and Biotechnology Springer-Verlag 2008 10.

1007/s00253-008-1731-8

Mini-Review
Prospects of using marine actinobacteria as probiotics in aquaculture Surajit Das1, 2 , Louise R. Ward1 and Chris Burke1

(1) National Centre for Marine Conservation and Resource Sustainability, Australian Maritime College, University of Tasmania, Locked Bag 1370, Launceston, Tasmania, 7250, Australia (2) Amity Institute of Biotechnology, Amity University, Noida, 201 303, Uttar Pradesh, India Surajit Das Email: sdas@amc.edu.au Email: surajit@myself.com Received: 18 August 2008 Revised: 21 September 2008 Accepted: 23 September 2008 Published online: 8 October 2008 Abstract Chemotherapeutic agents have been banned for disease management in aquaculture systems due to the emergence of antibiotic resistance gene and enduring residual effects in the environments. Instead, microbial interventions in sustainable aquaculture have been proposed, and among them, the most popular and practical approach is the use of probiotics. A range of microorganisms have been used so far as probiotics, which include Gram-negative and Gram-positive bacteria, yeast, bacteriophages, and unicellular algae. The results are satisfactory and promising; however, to combat the latest infectious diseases, the search for a new strain for probiotics is essential. Marine actinobacteria were designated as the chemical factory a long time ago, and quite a large number of chemical substances have been isolated to date. The potent actinobacterial genera are Streptomyces; Micromonospora; and a novel, recently described genus, Salinispora. Despite the existence of all the significant features of a good probiont, actinobacteria have been hardly used as probiotics in aquaculture. However, this group of bacteria promises to supply the most potential probiotic strains in the future. Keywords Aquaculture - Actinobacteria - Streptomyces - Probiotics - Growth - Survival An erratum to this article can be found at http://dx.doi.org.pbidi.unam.mx:8080/10.1007/s00253008-1747-0 Introduction Aquaculture is a fast-growing and rapidly expanding multibillion dollar industry. Marine capture fisheries and aquaculture supplied the world with about 104 million tons of fish in 2004 (FAO

2007). Of this total, marine aquaculture accounted for about 18%, where shrimp from aquaculture continues to be the most important commodity traded in terms of value (2.4 million tons). Worldwide, the aquaculture sector has been expanding at an average compounded rate of 9.2% per year since 1970, compared with only 1.4% for capture fisheries and 2.8% for terrestrial-farmed meat production systems. However, the serious concern about this industry is disease. Aquatic animals are constantly and intimately related with the composition and changes in the surrounding environment. The aquatic environment supports their pathogens as well, which can reach densities sufficient to cause disease or to render the animals immunocompromised (Moriarty 1998). In addition, overstocking or poor seed conditions contribute significantly to the destruction of hostpathogen environment equilibrium and, ultimately, to disease outbreak. The use of antibiotics to control diseases was widely practiced and the indiscriminate use of chemotherapeutic agents led to the emergence of numerous antibiotic-resistant bacteria; thereby, the production crashed in many Asian countries (Karunasagar et al. 1994). As a result, currently, antibiotics are no longer effective in treating luminescent vibriosis (Defoirdt et al. 2007). The consequences of emerging antibiotic-resistant bacteria on aquaculture pose the risk of transferring the antibiotic-resistance plasmid to human pathogenic bacteria too. Considering these factors, as well as the fatal effect of residual antibiotics of aquaculture products on human health, the European Union and USA implemented bans on, or restricted the use of, antibiotics (Kesarcodi-Watson et al. 2008). The norms are stringent and there are many events of returning consignments to the exporting countries for not maintaining the prescribed standards. In this context, microbial intervention can play a vital role in aquaculture production, and effective probiotic treatments may provide broad spectrum and greater nonspecific disease protection (Rengpipat et al. 2000; Panigrahi and Azad 2007). The range of probiotic microorganisms examined for use in aquaculture includes both Gram-negative and Grampositive bacteria, bacteriophages, yeasts, and unicellular algae (Irianto and Austin 2002). The selection for probiotic candidate organisms was based on in vitro antagonism (Verschuere et al. 2000), as well as on the results of adhesion, colonization, and growth in intestinal mucus (Irianto and Austin 2002; Vine et al. 2004). Several other methods of reducing pathogenic microbes in aquaculture, viz., filtration of water, addition of sodium chloride, ozonation, use of ultraviolet light, etc., are also useful but not as much as probiotics. The use of probiotics to augment production has been reported to be the most successful method. Therefore, a constant search for new and potent strains as probionts is necessary to combat recently emerged diseases. Probiotics: definition and principles Probiotics that compete with bacterial pathogens for nutrients and/or inhibit the growth of pathogens could be a valid alternative to the prophylactic application of chemicals, antibiotics, and biocides. The term probiotic means for life, originating from the Greek words pro and bios (Gismondo et al. 1999). The word probiotic was introduced by Parker (1974), who defined it as organisms and substances which contribute to intestinal microbial balance.

Thereafter, several modifications were proposed to trim the original definition (Gram et al. 1999; Salminen et al. 1999; Irianto and Austin 2002). However, the widely accepted definition was made by Fuller (1989) as a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance. A modified definition was also proposed by Verschuere et al. (2000)a live microbial adjunct which has a beneficial effect on the host by modifying the host-associated or ambient microbial community, by ensuring improved use of the feed or enhancing its nutritional value, by enhancing the host response towards disease, or by improving the quality of its ambient environment. By definition, a probiotic should benefit the host either nutritionally or by changing its immediate environment (Kesarcodi-Watson et al. 2008). Commonly studied probiotics include the spore-forming Bacillus spp. and the yeast Saccharomyces cerevisiae. Bacillus spp. have been shown to possess adhesion abilities and produce bacteriocins, and the yeast was reported to show immunostimulatory activity and the production of inhibitory substances. Mechanisms of action of probiotics Probiotic bacteria can inhibit pathogens by the production of antagonistic compounds and/or by competitive exclusion (competition for nutrients and attachment sites). Probiotic bacteria directly take up or decompose the organic matter and improve the water quality of an aquatic ecosystem. Beneficial microbial cultures produce a variety of exoenzymes like amylase, protease, lipase, etc., which help to degrade the unconsumed feed and feces in the pond, in addition to the possible role of these enzymes in the nutrition of the animals by improving feed digestibility and feed utilization. Among all the microbial interventions to augment the production, use of probiotics is in the central dogma (Fig. 1). Fig. 1 Microbial intervention in aquaculture where use of probiotics has given greater importance (after Panigrahi and Azad 2007) The modes of action of probiotics include the inhibition of a pathogen through the production of bacteriocin-like compounds, competition for attachment sites, competition for nutrients (particularly iron in marine microbes), alteration of enzymatic activity of pathogens, immunostimulatory functions, and nutritional benefits such as improving feed digestibility and feed utilization (Fuller 1989; Kesarcodi-Watson et al. 2008). Marine Actinobacteria: an overview Actinobacterial taxonomy and distribution Actinobacteria is a new class with five subclasses (Fig. 2) proposed by Stackebrandt et al. (1997) to group the highly diverse so-called actinomycetes based on the chemotaxonomy that detects differences in the chemical composition, DNADNA reassociation experiments, and 16S rRNA gene sequence similarities. Bacteria belonging to the order Actinomycetales, commonly referred to actinomycetes cover the culturable group of actinobacteria from diverse ecological niches. Fig. 2 Hierarchic classification system of the class Actinobacteria based on molecular sequence data (according to Stackebrandt et al. 1997)

These Gram-positive, filamentous actinobacteria are well adapted to the soil environment and are able to break down complex biological polymers. In particular, Streptomycetes are well known as important antibiotic producers, which help them to defend their nutrient sources (Davelos et al. 2004). The genus Streptomyces was classified under the family Streptomycetaceae, which includes Gram-positive aerobic members of the order Actinomycetales and suborder Streptomycineae within the new class Actinobacteria (Stackebrandt et al. 1997; Anderson and Wellington 2001), with a DNA G+C content of 6978 mol%. Actinomycetes are a prolific source of secondary metabolites, and the vast majority of these compounds are derived from the single genus Streptomyces (Fig. 3). Streptomyces species are distributed widely in aquatic and terrestrial habitats (Pathom-aree et al. 2006) and are of commercial interest due to their unique capacity to produce novel bioactive compounds. It was also expected that Streptomyces species will have a cosmopolitan distribution, as they produce abundant spores that are readily dispersed (Antony-Babu et al. 2008). Fig. 3 A typical grown-out culture of marine Streptomyces on agar medium However, the existence of marine actinomycetes in the marine environment has been questioned frequently in the recent past, and the land run-off hypothesis was postulated. However, based on culture-independent and culture-dependent studies (Moran et al. 1995; Urakawa et al. 1999; Mincer et al. 2002; Das et al. 2008a), it was confirmed that marine actinomycetes are the autochthonous flora in the marine environment. A series of papers describing the distribution of actinomycetes in the marine environment, published in the dedicated volume of the Antonie van Leeuwenhoek journal (Bull and Goodfellow 2005), confirmed the indigenous nature of marine actinobacteria. This view was best supported by the discovery of the first obligate new marine actinomycete genus, Salinispora (formerly known as Salinospora) (Mincer et al. 2005). While early research estimated low numbers (Jensen et al. 1991) and patchy distribution (Mincer et al. 2002) of actinomycetes in the marine environment, more recent studies suggested higher abundance and diversity of actinobacteria with numerous novel taxa (Gontang et al. 2007). Thus, bona fide actinomycetes not only exist in the oceans, but they are also widely distributed in different marine ecosystems (Lam 2006). In the marine environment, the representatives from six families have been listed based on molecular studies (Fig. 4). The representative families are Micromonosporaceae, Nocardiaceae, Nocardiopsaceae, Pseudonocardiaceae, Streptomycetaceae, and Thermonosporaceae. However, greater sequence coverage or improved DNA extraction efficiencies may be required to detect the rare phylotypes. Besides, new strategies need to be developed for the cultivation of frequently observed but yet-to-be-cultured marine actinobacteria (Jensen and Lauro 2008). Fig. 4 Radial tree depicting the phylogenetic relationships of 13 groups of marine-derived actinomycetes (after Fenical and Jensen 2006) Actinobacterial genera isolated from the marine environment include Aeromicrobium, Dietzia, Marinispora, Marinophilus, Salinispora, Solwaraspora, Salinibacterium, Kocuria, Williamsia, and Verrucosispora, in addition to Actinomyces, Actinopolyspora, Micromonospora, Micropolyspora, Nocardia, Rhodococcus, Streptomyces, Streptosporangium, and

Streptoverticillium (reviewed by Das et al. 2006a). More actinobacterial genera are expected to be discovered and reported with culture-independent studies in the years to come. However, regardless of the geographical origin, marine actinomycetes were shown to follow a well documented pattern in secondary metabolite production (Jensen et al. 2007). Marine Actionbacteria in secondary metabolite production The marine environment is a source of novel actinobacteria, many of which produce bioactive compounds that supply over half of the bioactive compounds in the present day. Although they promise a persistent population in the oceans (Mincer et al. 2002), studies on the diversity, distribution, and ecology of actinobacteria in the oceans are scarce (Stach et al. 2003). Though more than 50% of the microbial antibiotics discovered so far originate from actinomycete bacteria, only a few selected soil-derived genera (Streptomyces and Micromonospora) account for most of these compounds (Berdy 2005). Of late, obligate marine actinobacteria Salinispora strains have been reported to be a prolific source of structurally diverse secondary metabolites. Salinispora spp. have proven to be a rich source of new chemical structures, including the potent proteasome inhibitor salinosporamide A, new classes of terpenoids, amino acid-derived metabolites, and polyene macrolides (Feling et al. 2003; Jensen et al. 2007). It was later remarked that, instead of the old paradigm secondary metabolites are strain specific, the phylotype can predict the chemotype in Salinispora spp. (Newman and Hill 2006). The genus Streptomyces is the source of the vast majority of actinomycete secondary metabolites that have been discovered. From the genome sequence of Streptomyces coelicolor, it has been found that this genus possesses a single linear chromosome flanked by two nonconserved arms. These arms contain large DNA and are the location for the most contingency genes that code for secondary metabolite production (Bentley et al. 2002; Jensen et al. 2007). Furthermore, to understand the importance of marine-derived actinomycetes in ecological terms, perceptions on the extent to which they are capable of growing in the ocean, the degree to which they display specific marine adaptations, and the extent to which these adaptations have affected secondary metabolite production are required (Jensen et al. 2005). Potential mode of action of marine Actinobacteria Search for the rationale Biological control or biocontrol is a naturally occurring phenomenon in the aquaculture environment through which pathogens can be killed or reduced in number by antagonism (Maeda et al. 1997). By definition, and of course, logically, probiotics must not be harmful to the host, and they need to be effective over a range of temperature extremes and variations in salinity (Fuller 1989). They should provide actual benefits to the host, be able to survive in the digestive tract, be capable of commercialization, and be stable and viable for prolonged storage conditions and in the field. Application could be either via feed or by immersion or injection. Considering the above facts, we now look into the positive traits of the actinobacteria. Neither pathogenic organisms in aquaculture nor potentially hazardous microorganisms for human health

have yet to be reported so far. Virulence factors of pathogenic bacteria (adhesins, toxins, invasins, protein secretion systems, iron uptake systems, and others) may be encoded by particular regions of the prokaryotic genome termed pathogenicity islands (PAIs) (Hacker and Kaper 2000). PAIs carry genes encoding one or more virulence factors that are present in the genomes of pathogenic microorganisms but absent from the genomes of nonpathogenic forms. PAIs occupy relatively large genomic regions and are often associated with transfer RNA genes. The virulence factor database (Yang et al. 2007) (http://www.mgc.ac.cn/VFs/) has not yet included Streptomyces (accessed on 14 September 2008), and the Pathogenicity Island Database (Yoon et al. 2006) (http://www.gem.re.kr/paidb) detected PAIs only in a plant pathogen Streptomyces turgidiscabies. In this context, we contend that Streptomyces will not cause harm to the target animals in aquaculture. Actinobacteria are primarily saprophytic, soil-dwelling organisms and contribute significantly to the turnover of complex biopolymers, such as lignocellulose, hemicellulose, pectin, keratin, and chitin (Williams et al. 1984), which shows the potential to involve in mineralization and nutrient cycles in the aquaculture ponds. Once it gets colonized into the host intestine, the exoenzymes produced by actinomycetes may be helpful in facilitating feed utilization and digestion. In addition, the colonized microflora play an important role in the resistance to infectious diseases by producing antibacterial substances. Moreover, as suggested by Wang et al. (2008), adhesion and colonization of the probiotic strain on the mucosal surfaces are possible protective mechanisms against pathogens. The protection occurs through competition for binding sites and nutrients (Westerdahl et al. 1991) or immune modulation (Salminen et al. 1999) (Fig. 5), and these characteristics are also expected to be fulfilled by actinobacteria through the production of secondary metabolites. Actinomycetes are well studied for secondary metabolites; however, well studied taxa have the potential to yield new metabolites because of unanticipated biosynthetic gene clusters (Udwary et al. 2007). The genes encoding the enzymes of secondary metabolism are usually chromosomal, but a few have been shown to be plasmid-borne. Fig. 5 Complex microbial ecology of the intestinal tract provides protection against pathogens. Probiotics provide protection by the production of antimicrobial compounds, competing for essential nutrients and adhesion sites, or by modulating the immune response (following Balcazar et al. 2006) Retention of viability during preparation and storage represents a particular challenge and can be regarded as a major setback in commercial probiotic production. In most cases, bacterial probiotics are stored at low temperatures to keep them viable during the shelf life. The longer the shelf life, the better the acceptability in the aquaculture industry is. The streptomycetes are distinguished by the formation of substrate mycelium that grows in and onto the culture medium and an aerial mycelium, which develops in the air and forms a chain of spores. Therefore, actinomycetes are more resistant to soil desiccation than other bacteria and retain viability under exposure to harsh conditions, e.g., a w as less as 0.50 (Doroshenko et al. 2005). The tolerance of actinomycetes to hydration is determined by the resistance of the spores. Due to these features, the actinomycetes strains are supposed to have a longer shelf life. Likely mode of action of actinobacterial probionts

It is realized that the biogeochemical turnover in the aquatic environment is mainly due to the metabolism of the microbial population, and this is performed through the processes of aerobic and anaerobic decomposition by which the microbial cells get their energy. In addition, they also play an important role in the decomposition of organic matter, dissolution of inorganic insoluble salts, and regeneration of nutrients. The distributions and ecological roles of actinomycetes in the marine environment and the extent to which obligate marine species occur have remained unresolved issues in marine microbiology (Jensen et al. 2005). The degradation and turnover of various materials are critical in the recycling of carbon and nitrogen compounds in the aquatic environment, and it is a continuous process mediated by the action of a variety of microorganisms. The increase or decrease of a particular enzyme-producing microorganism may indicate the concentration of the natural substrate and conditions of the environment. Actinomycetes are also considered to contribute to the breakdown and recycling of organic compounds (Goodfellow and Haynes 1984). Marine actinomycetes are reported to be the producers of many hydrolytic enzymes involved in productivity (Table 1). Actinobacteria are recognized as a ubiquitous yet small component of the marine bacterioplankton, where they perform yet-undefined ecological roles (Jensen and Lauro 2008) Therefore, the ecological role of marine actinobacteria as probiotic in aquaculture pond cannot just be overlooked. Table 1 Actinobacteria reported from different habitats and their ecological role Actinobacterial Source/habitat Activities References isolates Streptomyces Coastal soil Chitinase Mukherjee and Sen (2006) venezuelae Marine Sediments Chitinolytic Pisano et al. (1986, 1992) actinomycetes Streptomyces spp. Sediments Fatty acid Das et al. (2007) Marine Sediments Cellulolytic Veiga et al. (1983) actinomycetes Chandramohan et al. (1972); Streptomyces sp. Sediments Cellulolytic Balasubramanian et al. (1979) Heald et al. (2001); Brando and Bull Rhodococus spp. Sediments Nitrile hydrolyzing (2003) Amylolytic, Ellaiah et al. (2002, 2004); Das Streptomyces spp. Sediments proteolytic and liplytic (2007) Secondly, the efficiency of probiotics over commercial antibiotics to control the bacterial population has also been proved. The use of kanamycin antibiotic can control the total bacterial population, but the bacterial population restores the original level once the effect of this antibiotic leaves after a few days. However, it is projected that the probionts never kill the native bacterial flora but suppress their growth (Fig. 6). Streptomyces release antibiotics in a sort of biochemical warfare to eliminate the competing microorganisms from the environment. These antibiotics are small molecules and interfere with gyrase protein, which assists in DNA replication. As a result, bacteria are not able to divide normally. However, Streptomyces protects itself from its own antibiotics by the production of efflux pumps (used against the influx of antibiotics), ribosomal protection proteins (protect ribosome and prevents interfering with

protein synthesis), and modifying enzymes (neutralize antibiotics by the production of acetyl or phosphate groups). Fig. 6 State of bacterial population a in the presence of Kanamycin antibiotics (after Maeda et al. 1997) and b in the presence of probiotics (projected) The mode of action of probiotics that was reviewed in the previous section may also be very applicable in the case of marine actinobacteria. The definition of probiotics states that a probiotic must be adherent and colonized within the intestinal tract, it must replicate to high numbers, it must produce antimicrobial substances, and it must withstand the acidic environment of the intestine based on the belief that a probiotic must become a permanent member of the intestinal flora. However, transient bacteria can also exert beneficial effects (Isolauri et al. 2004), and a probiotic may only possess one mode of action (Kesarcodi-Watson et al. 2008). Selection strategy for potential actinobacterial strain Sampling site selection The sampling sites to isolate probiont strains have been described by many authors. Maeda et al. (1997) suggested the following: natural seawater where larvae are growing, culture water where larvae are densely reared in the laboratory, and the digestive gut of fish. It is quite obvious to recommend the site from the native environment, which confirms the survivability of the strains. Microbial food webs are an integral part of aquaculture ponds and have a direct impact on productivity (Moriarty 1997). Therefore, the probiotic strains screened from the pond environment can play important roles in productivity, the nutrition of the cultured animals, disease control, water quality, and environmental impact of the effluent (Wang et al. 2008). Strategy for selecting suitable actinobacterial strains General rules of thumb for the selection of probiotic strains were described by Gomez-Gil et al. (2000). The steps are as follows: (1) collection of background information, (2) acquisition of potential probiotics, (3) evaluation of the ability of potential probiotics to out-compete pathogenic strains, (4) assessment of the pathogenicity of potential probiotics, (5) evaluation of the effect of the potential probiotics in the host, and (6) an economic costbenefit analysis. In support of the marine actinobacteria, in particular, Streptomyces, as probionts, the comprehensive screening procedure can be followed (Table 2). Streptomyces can be masscultured, harvested, and fortified to feed. However, the supernatant of the broth culture of the strain can also be collected, concentrated, and supplemented in the feed. In this case, the positive effect may be obtained (Kumar et al. 2006), but it may violate the definition of probiotics, as there is no addition of live microbial supplement. Table 2 The experiments and the observations to be conducted to select suitable marine Streptomyces as probiotic for shrimp Experiments Observations A. Pre1. Isolation: Isolation of marine 1. Actinomycetes colonies are recognized by experimental Streptomyces from shrimp farm on the presence of filamentous hyphae and/or

Experiments Starch Casein agar or Yeast Extract malt Extract Agar (ISP-2) media. 2. In vitro antagonism test: Cross streak assay and double agar layer method. 3. Extracellular enzymatic activity: Amylase and protease activity to test.

Observations formation of tough, leathery colonies that adhered to the agar surface. 2. Strains showing good antagonism against shrimp pathogens to be selected. 3. Strains showing broad spectrum enzymatic activities to be selected.

screening

B. Experimental screening

4. One day after the injection, survival 4. Evaluation of pathogenicity: should be monitored and samples of Pathogenicity test on experimental hemolymph and gut to be plated on marine aquatic organism (Penaeus agar to see whether the defense systems of monodon) under normal and/or the shrimps are able to cope with the stress conditions. This can be done intrusion as well whether the strain is by injection challenges, by bathing reaching to the digestive system. Because, the host in a suspension or adding the production of inhibitory compounds in the strains in culture. sufficient amounts is of no relevance if the strain is not ingested by the host. 5. The growth of nonmotile Streptomyces is 5. Mass scale culture: Mass scale generally observed on the surface of the culture of Streptomyces strain can be medium in case of static condition. Harvest done into ISP-2 liquid medium. the cell by centrifugation and store at 4 C. Alternatively, the antibiotic production of the actinomycetes The supernatant culture of strains in the strain can be done in the marine broth medium can be concentrated in actinomycetes growth medium and vacuum evaporator and can be stored at 4 C. the supernatant of the broth may be used. 6. In vivo evaluation of putative probiotic effects: The powdered strain or the supernatant extract can 6. To monitor be added to the host or its ambient environment by following ways (1) addition to the artificial diet Growth and health (2) addition to the culture water Survival (3) bathing Water quality parameters (4) addition via live food Interaction with phytoplankton Growth, survival, water quality parameters should be in optimum condition and the strain should not have algicidal effect. 7. Colonization or adhesion: The 7. It is to observe that whether the strain

C. Postexperimental screening

Experiments Observations ability of strain to colonize in the gut should either be supplied on a regular basis or an external surface of the host to or be able to colonize and persist in the host be monitored by isolation of strain or its ambient environment. periodically on agar plate. 8. In vivo challenge test: In vivo 8. It is to observe whether the growth or the challenge test involves experimental activity of the pathogen is really destroyed infection with a representative by probiotics or simply delayed due to some pathogen, e.g., Vibrio harveyi and competition for nutrients. It is to further simultaneous addition of observe whether the probiotics enhances experimental strain. immune response to the host. 9. Dose optimal: The effective way 9. Streptomyces is nonmotile aerobic form of introduction and optimum dose to which grow as mat in broth (in static be determined. Since Streptomyces condition) and as clump (in shaker culture). is a very slow grower, the lag period Cells can be harvested by centrifugation and and the doubling time would also be packed. determined. 10. Identification of the potent actinomycetes strain should be done by chemotaxonomical [genus affiliation, (Lechevalier and Lechevalier 1970)] and conventional [species affiliation, Nonomura (1974)] approach. Further, confirmation could also be done by molecular identification (gene sequencing and 16S rRNA phylogenetic analysis). 11. Purification and identification of antibacterial compound may be carried out (for further research) following purification by fermentation, silica gel separation and TLC, and identification by spectral analyses (IR, UV and NMR). 12. Parameters for mass scale culture and optimum culture conditions to be studied determined since the metabolic processes are controlled by sources of carbon, nitrogen, phosphorous, metals, induction, feedback regulation, growth rate, and enzyme decay (Demain 2006). Finally, a costbenefit analysis will also have to be carried out.

The final selected actinobacterial probiotic strain should have certain properties, as described by Verschuere et al. (2000): it should not be harmful to the host it is desired for (i.e., nonpathogenic to the host), it should be accepted by the host (e.g., through ingestion and potential colonization and replication within the host), it should reach the location where the effect is required to take place, it should actually work in vivo as opposed to in vitro findings, and it should preferably not contain virulence resistance genes or antibiotic resistance genes. In addition, the selected strain should also be beneficial to or should not hamper the growth of microalgae in the aquaculture environment. Present status and future research Research to date

Despite the source of several novel antibiotics, marine actinobacteria has been given no attention for use a probiotic in aquaculture. However, it was recommended that marine actinomycetes are promising candidates to be utilized in marine aquaculture (You et al. 2005, 2007). You et al. (2005) described the potential of actinomycetes against shrimp pathogenic Vibrio spp. and recommended marine actinomycetes as potential probiotic strains due to their ability to degrade macomolecules, such as starch and protein, in culture pond water; the production of antimicrobial agents; and the formation of heat- and desiccation-resistant spores. More recently, there were a few studies on the possible use of marine actinomycetes in disease prevention against aquatic pathogens. Das et al. (2006b) reported a preliminary study on the use of Streptomyces on the growth of black tiger shrimp. Kumar et al. (2006) extracted the antibiotic product from marine actinomycetes and incorporated it into feed to observe the in vivo effect on white spot syndrome virus in black tiger shrimp. You et al. (2007) reported the activity of marine actinomycete as a potential organism against biofilms produced by Vibrio spp. and recommended the use of actinomycetes to prevent the disease caused by Vibrio spp. All of this research indicated the importance of marine actinobacteria in aquaculture. Actinobacteria as probiotics in aquaculture: possible setbacks The possible setbacks in marine actinobacterial research have been outlined in detail by Das et al. (2008b). These include sporadic distribution in the marine environment, difficulties in culture methods, standardized media and inhibitory compounds, slow growth rate, and tedious laboratory procedures in culture-dependent studies for identification. A potential debate for using actinobacteria in aquaculture as probiotics may be the risk of lateral gene transfer of antibiotic-resistance genes that produce efflux pumps, ribosomal protection proteins, and modifying enzymes (described earlier), by which Streptomyces protects itself from its own antibiotics. It is expected that the probiotic strains should preferably not contain virulence resistance genes or antibiotic resistance genes because of the emergence of multidrugresistant pathogens in aquaculture. Extensive use of antibiotics in aquaculture has caused antibiotic-resistant bacteria to be widespread (Furushita et al. 2003), leading to the conclusion that the aquatic environment may serve as a reservoir of antibiotic resistance (Biyela et al. 2004). However, the question is what happens if the antibiotic resistance genes are naturally occurring in the organism for its own defense? Are they passed on to other organisms naturally? Chopra and Roberts (2001) reported that the presence of ribosomal protection proteins and antibiotic modifying enzymes (antibiotic resistance genes) likely originated from Streptomyces or any other antibiotic producing microbes by lateral gene transfer. Therefore, lateral gene transfer occurs naturally even when Streptomyces was not used as probiotic! In addition, most studies to date suggested that the probable cause for antibiotic resistance patterns found in aquatic microorganisms was the increased use of synthetic antibiotics and drugs (e.g., Dang et al. 2008). Thus, a likely lower chance of lateral gene transfer led us to recommend the use of actinobacteria as probiotics in aquaculture. Further in-depth studies may bring out significant contentions in the future. The other possible negative aspect of using marine actinobacteria as probiotics in aquaculture is the production of odors such as geosmin (trans-1,10,-dimethyl-trans-(9)-decalol) and MIB or 2-

methylisoborneol (exo-1,2,7,7-tetramethyl-[2.2.1]heptan-2-ol) in freshwater aquaculture (Klausen et al. 2005), and even in the recirculatory system (Guttman and van Rijn 2008). These two compounds are semivolatile terpenoid compounds and impart an earthymusty taste and odor to the water, as well as to the cultured fish. Geosmin and MIB, when released into the water, are absorbed through the gills, skin, or gastrointestinal tract by lipid-rich fish tissues and reduce the quality of fish in freshwater aquaculture and, thereby, lower the commercial value of the fish (Howgate 2004). However, actinomycetes are not only responsible for the production of these two compounds; planktonic cyanobacteria, bacteria, and several genera of fungi also produce geosmin and MIB (Wood et al. 2001). Therefore, studies on the production of odors in the marine environment by actinomycetes and their possible role in lowering the quality of fish are yet to be confirmed. Conclusions The stability of probiotics is influenced by various factors, including the species, strain biotype, water activity, temperature, hydrogen-ion concentration (pH), osmotic pressure, mechanical friction, and oxygen. Consequently, special attention must be paid during the process of actinobacterial probiotic selection, as in probiotic research, screening experiments involve a large number of tests to obtain a promising strain (Kesarcodi-Watson et al. 2008). In the light of the functional food concept, the enzymes produced from marine actinobacteria provide an important niche for probiotics, prebiotics, or their combination (synbiotics) approaches in aquaculture with the ever increasing demand for the use of both probiotics and prebiotics. Acknowledgement An Endeavour Research Fellowship to one of the authors (S.D.) by the Department of Education, Employment and Workplace Relations, Australian Government, to carry out Postdoctoral research at the University of Tasmania is gratefully acknowledged.

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