Bacteriocins of Aquatic Microorganisms and Their Potential Applications in the Seafood Industry

Bacteriocins are potent antimicrobial polypeptides and proteins produced by most lineages of Bacteria and, perhaps, by all members of Archaea (O'Connor & Shand, 2002; Riley & Wertz, 2002a, 2002b; Tagg et al., 1976). Although initially the focus of numerous biochemical, evolutionary, and ecological studies, more recently, their potential to serve in human and animal health applications has taken center stage (Gillor et al., 2008). The use of bacteriocins in probiotic applications, as preservatives, and, (most excitingly) as alternatives to classical antibiotics is being broadly explored (Abee et al., 1995; Einarsson & Lauzon, 1995; Gillor & Ghazaryan, 2007; Gillor et al., 2007).

. Competition for resources results in a "rock-paper-scissors"-like interaction of microorganisms (adapted from Kerr et al., 2002).
In contrast to traditional antibiotics, which are used in human health applications precisely because of their ability to kill a diversity of bacterial pathogens, bacteriocins generally target only members of their producing species and its closest relatives (although numerous exceptions abound) (Riley et al., 2003;Tagg et al., 1976). Riley et al. (2003) mapped the killing spectrum of bacteriocins from seven enteric species (Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacter freundii, Hafnia alvei, and Serratia plymuthica) onto the molecular phylogeny of the same species (Fig. 1). This study showed that bacteriocin producers tend to kill strains belonging to their same species. However, there are some exceptions, such as bacteriocins of E. coli that inhibit distantly related H. alvei (Fig. 1).

Bacteriocin naming
Bacteriocins were originally named based on the producer species such as colicins produced by Escherichia coli, pyocins of Pseudomonas aeruginosa (formerly named pyocyania), cloacins of Enterobacter cloacae, cerecins of Bacillus cereus, and pesticins of Yersinia pestis (Reeves, 1965). Fredericq (1957) created the first classification, and thus nomenclature, of bacteriocins focusing on the colicins of E. coli (Fredericq, 1957). Fredericq grouped colicins into 17 different types (colicins A, B, C, D, E, F, G, H, I, J, K, V, S1, S2, S3, S4, and S5) based on their receptor specificity. These colicins were then further subtyped (colicin E1, E2, and E3, etc.) based on their immunity patterns. In this scheme, all subtypes were recognized by the same receptor, but they possessed different immunity phenotypes (Fredericq, 1957). Later, the addition of the producer strain's name provided further differentiation of bacteriocins produced by strains of the same species (Daw & Falkiner, 1996). This scheme is still used today.

Bacteriocin classes
In general, bacteriocins are produced by Bacteria and studied based on the gram designation of their producing species (Gram-negative versus Gram-positive). Additionally, a relatively small number of bacteriocins from Archaeal species have also been characterized. A comprehensive review of bacteriocins from Bacteria and Archaea can be found elsewhere (O'Connor & Shand, 2002;Reeves, 1965;Riley & Gordon, 1999;Riley & Wertz, 2002a, 2002bTagg et al., 1976). Below are short descriptions of the bacteriocin classes of Bacteria and Archaea and examples of bacteriocins belonging to each class (  E3 Cascales et al., 2007Michel-Briand & Baysse, 2002Gillor et al., 2004Reeves, 1965 Heng et al., 2007Drider et al., 2006Field et al., 2007Maqueda et al., 2004 Class II Class IIa-antilisterial Class IIb-synergistic < 10

Bacteriocins of Gram-positive bacteria
Bacteriocins of Gram-positive bacteria are generally divided into four classes based on size, morphology, physical, and chemical properties (Lee & Kim, 2011). Class I bacteriocins are lantibiotics, which are small peptides (<5 kDa) (Field et al., 2007). They are posttranslationally modified, incorporating non-traditional amino acids such as dehydroalanine, dehydrobutyrine, methyl-lanthione, and lantionine (Cleveland et al., 2001). This class is subdivided into Type A, B, and C with the distinction being that members of Type A are positively charged, linear peptides whereas those in Type B are either neutrally or negatively charged rigid globular peptides. Members of Type C require synergistic activity of two peptides to be active. This class includes the well-studied bacteriocins nisin and lacticin (McAuliffe et al., 2001).
Class II bacteriocins are small (<10 kDa), heat-stable peptides that are not post-translationally modified (Heng et al., 2007). Class II is also subdivided into two subgroups. Class IIa are pediocin-like or Listeria-active peptides, which contain a conserved N-terminal sequences (YGNGVxCxxxxCxV). Class IIb bacteriocins require the synergistic activity of two peptides to be fully active (Nissen-Meyer et al., 1992). Class III bacteriocins are generally large (>10 kDa),

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Bacteriocins of Aquatic Microorganisms and Their Potential Applications in the Seafood Industry 307 heat-labile peptides. They are subdivided into two subtypes. Type IIIa are bacteriolysins, which are bacteriolytic enzymes. Lysostaphin is the most studied bacteriocin in this subtype (Schindler & Schuhardt, 1964). Type IIIb are non-lytic bacteriocins. Helveticin J (37 kDa) produced by Lactobacillus helveticus belongs to this type (Joerger & Klaenhammer, 1986). Finally, Class IV bacteriocins have unique structural characteristics. The first and last amino acids of these bacteriocins are covalently bonded, thus they have cyclic structures. Enterocin AS-48 produced by Enterococcus faecalis subsp. liquefaciens S-48 was the first characterized bacteriocin belonging to this class (Maqueda et al., 2004).

Bacteriocins of Archaea
The Archaea also produce unique types of bacteriocin-like antimicrobial compounds, called archaeocins (Shand & Leyva, 2007). They have been much less studied than the bacteriocins of Bacteria. Thus far, two major types of archaeocins have been identified: halocins of halobacteria and sulfolobicins of Sulfolobus species. Halocins can be peptides (< 10 kDa) and/or proteins (>10 kDa) (Shand & Leyva, 2007). According to Torreblanca and Meseguer (1994), halocin production is a universal feature of halobacteria. Halocins are located on megaplasmids (or minichromosomes). Halocins H4 and S8 are located on ~300 kbp and ~200 kbp plasmids, respectively (Cheung et al., 1997;Price & Shand, 2000). Their activity is usually detected at the late exponential to early stationary growth phase (Cheung et al., 1997;Price & Shand, 2000). There is not much known about sulfolobicins. Prangishvili et al. (2000) screened sulfolobicin production from Sulfolobus islandicus isolated from volcanic vents throughout Iceland. This study predicted that sulfolobicin activity is membraneassociated and is not released from the cell. Sulfolobicins are also associated with membranous vesicles ranging in size from 90 to 180 nm in diameter (Prangishvili et al., 2000). Like many bacteriocins, they are thermostable and sensitive to protease treatment. Their mode of action is still unknown (Ellen et al., 2011).

Bacteriocin genetics
Bacteriocins can be encoded on chromosomes, plasmids, and other transposable elements. For example, the colicins of E. coli and halocin H4 are plasmid-encoded while the pyocins of P. aeruginosa are chromosomal (Chavan & Riley, 2007;Cheung et al., 1997;Michel-Briand & Baysse, 2002). Lacticin 481 has been shown to reside on the transposon Tn5721 (Dufour et al., 2000) and some bacterial species such as Serratia marcescens possess both plasmid and chromosomally encoded bacteriocins (Riley & Wertz, 2002b). Just as we see differences in the function of Gram-negative, Gram-positive, and Archaeal bacteriocins, we can also trace these distinctions to the genetic level.
In general, the full function of bacteriocins produced by Gram-negative bacteria is encoded via three tightly linked genes, the toxin, immunity, and lysis genes ( Fig. 2A). However, there are significant differences in the genetics of colicins, colicin-like bacteriocins, phage-tail-like bacteriocins, and microcins (Fig. 2). For example, the colicin gene cluster consists of the three bacteriocin-related genes in close proximity, whereas colicin-like pyocin S3 does not possess a lysis gene ( Fig. 2A-B). Two representative phage-tail like bacteriocins, R and F-type pyocins, R2 and F2, are encoded in a large gene cluster that spans more than 40 kb (Nakayama et al., 2000) and includes 44 open reading frames (Nakayama et al., 2000). The open reading frames include regulatory, lysis, R, and F pyocin genes (Fig. 2C). A-Genetic organization of colicin gene cluster. PSOS and Pimm represent promoter regions of the SOS and immunity genes. Gene caa encodes colicin A; cai encodes the immunity gene; and cal encodes the lysis gene (adapted from Cascales et al., 2007). B-Genetic organization of colicin-like gene cluster. P-box refers to the binding site for transcriptional regulator (PrtN); pyoS3A gene encodes pyocin S3; pyoS3I encodes the immunity gene (adapted from Duport et al., 1995). C-Genetic organization of phage-tail like bacteriocin gene cluster. prtN and prtR encode transcriptional activator (PrtN) and repressor (PrtR), respectively. PRF 9 and PRF24-26 encodes the lysis genes; PRF10-23 and PRF28-43 encode the R2 and F2 pyocin structural genes, respectively (adapted from Nakayama et al., 2000). D-Genetic organization of lantibiotic gene cluster. P nisA , P nisRK , and P nisF encode promoter genes for nis A, nisRK, and nisFEG, respectively; nisA encodes nisin A precursor; nisR and nisK encode proteins for nisin biosynthesis; nisB, nisC, nisT, and nisP encode proteins for nisin processing and translocation; nisI, nisF, nisE, and nisG encode immunity proteins (adapted from Kuipers et al., 1993;Mierau & Kleerebezem, 2005). E-Genetic organization of archaeocin gene cluster. Sulfolobicin is composed of SulA and SulB proteins, encoded by sulA and sulB genes (adapted from Ellen et al., 2011).
The Gram-positive bacteriocins are more complicated genetically, with genes that encode post-translational modification of the toxin. The genetic organization varies between the Gram-positive bacteriocin classes as well -such as the requirement of two peptides for the full activation of Class II bacteriocins. An example is provided by the nisin gene cluster (Fig.  2D), which includes 11 genes (nisABTCIPRKFEG) encoding functions such as synthesis of the nisin precursor (nisA), regulation of nisin biosynthesis (nisRK), the processing and translocation of nisin (nisBCTP), and immunity (nisIFEG) (Kuipers et al., 1993;Mierau & Kleerebezem, 2005).
The genetic organization of archaeocins is relatively unknown in comparison to other bacteriocins. A recent study showed that sulfolobicin of Sulfolobus acidocaldarius is comprised Bacteriocins of Aquatic Microorganisms and Their Potential Applications in the Seafood Industry 309 of two proteins, SulA and SulB, which are encoded by sulA and sulB genes, respectively (Fig.  2E). These two proteins are associated with membrane vesicles in the extracellular medium (Ellen et al., 2011). The gene organization of archaeocins is also unique. Sun et al. (2005) showed that the gene halC8 encoded both halocin C8 and its immunity protein HalI (Sun et al., 2005). It differs from other bacteriocins in that typically separate genes encode bacteriocin toxin and immunity proteins.

Bacteriocin biosynthesis
Bacteriocins are often produced under stress conditions, such as nutrient limitation and overpopulation (Riley & Gordon, 1999). The biosynthesis of Gram-negative bacteriocins is regulated by the host; often involving the SOS system. The SOS system is comprised of RecA and LexA proteins. LexA is a transcriptional repressor, which binds to the bacteriocin promoter and prevents its transcription. In stress conditions such as DNA damage and UV exposure expression of the recA gene is induced. RecA binds to LexA and therefore prevents the repression of bacteriocin expression (Cascales et al., 2007). The expression of colicin-like S-pyocins and phage-tail like RF pyocins also depends on RecA, except these genes possess a P-box in their promoter region instead of an SOS box (Nakayama et al., 2000;Sano et al., 1993). Further, there is no LexA-dependent repression of bacteriocin expression. Activated RecA cleaves the PtrR transcriptional repressor protein, which leads to expression of the transcriptional activator ptrN gene. PtrN binds to the P-box and induces the expression of the pyocin genes Michel-Briand & Baysse, 2002). Alternatively, Grampositive bacteria possess bacteriocin-specific biosynthesis pathways. For example, nisin regulates its own biosynthesis in cell-density dependent conditions (Eijsink et al., 2002). Finally, there is much less known about archaeocins. Regulation of their biosynthesis is still under investigation.

Bacteriocins of marine bacteria
Bacteriocins produced by marine bacteria have generated a great deal of excitement due primarily to their potential to serve as probiotics and antibiotics in the seafood industry (Galvez et al., 2008;García et al., 2010;Pilet & Leroi, 2011). A recent antimicrobial screen of 258 bacterial strains isolated from water and sediment in the Yucatan peninsula revealed that 46 strains belonging to the genera Aeromonas, Bacillus, Burkholderia, Photobacterium, Pseudomonas, Serratia, and Stenotrophomonas possessed antimicrobial activity. Approximately fifty percent of this antimicrobial activity was due to bacteriocins or BLIS (De la Rosa-Garcia et al., 2007). Further, Wilson et al. (2000) investigated surface-attached bacteria isolated from Sydney Harbor, Australia. He showed that approximately 10% of surface-attached marine bacteria possess antibacterial activity. Proteinase K treatment showed that this inhibitory activity was associated with proteinaceous substances such as bacteriocins or BLIS (Wilson et al., 2010). Given the fact that bacteriocins and BLIS have been characterized in most culturable bacterial species, it is tempting to speculate about the diversity of new substances the marine environment will reveal.
The first bacteriocin isolated from marine microorganisms was detected in Vibrio harveyi (formerly Beneckea harveyi). McCall and Sizemore (1979) screened a total of 795 Vibrio spp. strains isolated from Galveston Island, Texas for bacteriocin production (McCall & Sizemore, 1979). This study revealed that approximately 5% of the Vibrio spp. possessed a high molecular weight bacteriocin-like killing agent. Further investigation revealed that the source of the bacteriocin-like killing was a plasmid. It was also determined that the killing range was limited to strains of B. harveyi. This bacteriocin was named harveyicin (McCall & Sizemore, 1979).
In 1982, Hoyt and Sizemore investigated the chemical nature of harveyicin. They showed that it is a proteinaceous substance, sensitive to protease, trypsin, and papain treatment, and resistant to pepsin and lipase treatment. Furthermore, it is stable at room temperature and -20 C for several weeks and several months, respectively. However, the protein did lose killing activity after heat treatment at 55 C for 4 hours (Hoyt & Sizemore, 1982). Hoyt and Sizemore (1982) also investigated the role of harveyicin in bacterial competition in enteric and planktonic environments at various temperatures (4-39C), pH's (5-9.5), and salt concentrations (1.75% and 3.5%). They also performed a competition assay on nonluminous bacteriocinogenic (SYLum-) and luminous bacteriocin sensitive strains (SYcured) in equal concentrations (10 6 cells/ml). The ratio of harveyicin producer strain (SYLum-) to the harveyicin sensitive strain (SYcured) peaked in an enteric environment (25 C, high salinity (3%), and alkaline pH (pH 9.5)). The harveyicin producer strain also outcompeted the harveyicin sensitive strain at an acidic pH (pH 5.0) (Hoyt and Sizemore, 1982). Given the fact that the natural habitat of these species is in the guts of fish (an acidic environment), bacteriocin production may serve as a competitive advantage to the bacteriocin producer strain in this environment.
The relatively few characterized marine bacteriocins are primarily isolated from Carnobacterium species, which are ubiquitous, Gram-positive lactic acid bacteria isolated from marine organisms (such as fish and marine sponges), from cold and temperate environments, as well as from terrestrial environments including Canadian winter soil, permafrost ice, composite piles, and horse manure (Leisner et al., 2007). C. divergens and C. maltaromaticum (formerly known as C. piscicola) are the most-studied species from this genus.
The bacteriocins and BLIS isolated from marine microorganisms are diverse. However, they do share common characteristics with bacteriocins from Bacteria and Archaea (Fig. 3). They can be small peptides (5-10 kDa) like microcins of Gram-negative bacteria, microhalocins of halobacteria, and class I and II bacteriocins of Gram-positive bacteria. They can also be larger in size (10-90 kDa) like colicins and colicin-like bacteriocins of Gram-negative bacteria. The majority of marine BLIS have been tested against a number of proteases including trypsin, proteinase K, and pronase A, which are commonly used to identify bacteriocin activity. Some have unique characteristics. For example, BLIS-IW1, BLIS-BC1, and BC2 from Vibrio species possess a carbohydrate moiety while BLIS VIB 571 from V. harveyi has a lipid moiety (Shehane & Sizemore, 2002). It is not clear if these moieties are Fig. 3. Shared and unique characteristics of bacteriocins and BLIS from marine Bacteria with bacteriocins of terrestrial Bacteria and Archaea involved in BLIS activity. The mode of actions of these BLIS is unknown and their killing ranges are variable; either narrow -like bacteriocins of Gram-negative bacteria -or quite broad -like that observed in bacteriocins of Gram-positive bacteria (Bhugaloo-Vial et al., 1996;Moro et al., 1997;Sugita et al., 1997;Suzuki et al., 2005;. Further, the stability of BLIS activity varies. BLIS IW1 is inactive after cold storage at -70C; like phage-tail-like bacteriocins. Others, such as BLIS-BC1, BLIS-BC2, and BLIS-400, are still active after cold treatment, as is observed in colicin and colicin-like bacteriocins. Some BLIS are also resistant to treatment with organic solvents. For example, BLIS-400 is resistant to organic chemicals including ethanol, methanol, acetone, and chloroform (Carraturo et al., 2006). This feature is similar to some bacteriocins isolated from Gram-positive bacteria and Archaea (Carraturo et al., 2006). Finally, most BLIS from marine bacteria are produced during the stationary phase of growth, similar to bacteriocins from Gram-positive bacteria (Pinto et al., 2009;Tahiri et al., 2004).

Applications and implications of marine bacteriocins
The international seafood industry is one of the world's most profitable commodities, worth more than $75 billion per year (Food and Agriculture Organization of the United Nations [FAO], 2006). Fish and seafood are major proteins in some areas of the world. In 2006, of the 143 million tons of total fishery production (including fish, crustacean, and mollusks), 110 million tons was for direct human consumption (Pilet & Leroi, 2011). To meet this demand, we have seen a marked rise in aquaculture (the farming of aquatic plants and animals) in the last decade (FAO, 2006). Recently, there have also been dramatic changes in the seafood industry due to technological advances, consumer habits, and globalization of the food market (Galvez et al., 2008). In particular, there has been an increase in consumer preference for foods that are minimally processed or preserved (especially those that claim health-promoting benefits). Consumers are also demanding that these foods be fresh tasting and ready-to-eat (Galvez et al., 2008). The demands on the industry to provide fresh, minimally preserved products in the ever-growing globalized food market is requiring a longer and more complex food-chain and increasing the risk of microbial contamination and spoilage (García et al., 2010).

Challenges in the seafood Industry: Spoilage and disease
With the expansion of the seafood industry and the rise in seafood consumption, spoilage and disease in fish are the main challenges the industry faces (Gram & Dalgaard, 2002;Toranzo et al., 2005). Microorganisms are the major cause of spoilage and diseases in the seafood industry. It is estimated that nearly 25% of all the seafood produced is lost to microbial spoilage (Baird-Parker, 2000). Microbes cause changes in the sensory properties of the seafood (smell, taste, color), which make it less appealing and often dangerous to eat (Gram & Dalgaard, 2002). Disease severely affects the production and trade of farmed seafood, creating high economic impacts for many countries (Bondad-Reantaso et al., 2005).

Common microbial diseases in aquaculture
Fear of disease, as well as climate change, are acting as deterrents to aquaculture farming (Bondad-Reantaso et al., 2005). It has been shown that bacteria exhibit greater pathogenesis at higher temperatures, leading to greater and more virulent disease in aquaculture (Desriac   (Noga, 2010). Nonmotile, psychrophilic Aeromonas salmonicida is the causative agent of furunculosis in salmonids (O'Brien et al., 1994). Besides furunculosis in salmonids, it causes erythrodermatitis in carp and ulcers in trout (Noga, 2010). Furthermore, motile mesophilic Aeromonas hydrophila and Aeromonas veronii are the causative agents of MAS in carp, tilapia, perch, catfish, and salmon. They also cause ulcer disease in catfish, cod, carp, and goby (Noga, 2010).
Vibrio species cause systemic infections in fish (Vibriosis). The common symptoms are loss of appetite and skin ulcers, which are associated with septicemia. Vibrio anguillarum and Vibrio salmonicida are the causative agents of vibriosis and cold water vibriosis in salmon and cod, respectively (Colwell & Grimes, 1984). Further, Vibrio vulnificus causes hemorrhagic septicemia in Japanese eels and V. damsela causes skin ulcers (Toranzo et al., 2005).
Columnaris disease affects the skin and gills of freshwater fish including salmonids, catfish, striped bass, and tilapia (Noga, 2010). Infection causes degradation of the cartilage tissue, the major cause of death. Cytophaga columnaris as well as Bacillus columnaris, Flexibacter columnaris, and Flavobacterium columnare are the causative agents of columnaris disease (Noga, 2010).
Streptococcosis is a fish disease caused by Streptococcus species. Some of the clinical symptoms of streptococcosis are anorexia, loss of orientation, erratic swimming, lethargy, and hemorrhaging (Noga, 2010). Streptococcus iniae is the causative agent of streptococcosis in at least 27 species of farmed finfish. The worldwide impact of streptococcosis was estimated globally to be around $100 million (USD) (Agnew &Barnes, 2007).
Pasteurellosis (pseudotuberculosis) is one of the most important diseases in Japanese aquaculture affecting commercial yellowtail, ayu, black sea bream, red sea bream, and hybrid striped seabass (Noga, 2010). Pasteurella piscidium is the causative agent of this disease (Romalde, 2002). Hemorrhaging around the gills and lesions in the skin, liver, and kidneys are common symptoms in acute and chronic forms of this disease (Noga, 2010).
Mycobacteriosis (or fish tuberculosis) can be observed in nearly 200 species including salmonids, seabass, turbo, cod, and halibut (Noga, 2010). Mycobacterium marinum is the most common Mycobacterium species causing tuberculosis. Greyish-white nodules in the spleen, liver, and kidney, and hemorrhagic lesions are among the symptoms of this disease (Jacobs et al., 2009).

Current practices in the seafood industry to combat spoilage and disease
The seafood industry employs numerous techniques to eliminate microorganisms from their products. The oldest and still widely used form of seafood preservation is drying/salting. Besides keeping the seafood chilled in cold-water or on ice, this is the most low-tech preservation technique. There are many variations to this method such as wet-salting or additional acidification, but most achieve the same results. In this method, the fish is dried (with or without salt), which creates an environment devoid of vast nutrients (and possibly with high salinity). This prevents most bacterial growth, but spoilage can still occur due to filamentous fungi growth or insect infestation. Yeast is also able to grow in heavily wetsalted fish (Gram & Dalgaard, 2002).
Other preservation methods include washing with disinfectants, including chlorinated water, iodophores, salts, organic compounds, aldehydes, hydrogen peroxide, quaternary ammonium compounds, and antiseptic dyes (Calo-Mata et al., 2007;Shao, 2001). Disinfectants are mostly used to kill fungi and parasites. However, they may also select for antibiotic resistance in bacteria (Murray et al., 1984). Seafood can also be marinated in an acidic solution to prevent bacterial growth. Vacuum-packing and preservatives such as sorbate and benzoate have also been employed to prevent microbial growth (Einarsson & Lauzon, 1995;Gram & Dalgaard, 2002). Recently, complex solutions such as carbon-dioxide packing, spray-drying of antimicrobials, and radiofrequency heating have been applied to fight these ever-present problems of spoilage and contamination (Calo-Mata et al., 2007;Galvez et al., 2007;Gram & Dalgaard, 2002).

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Bacteriocins of Aquatic Microorganisms and Their Potential Applications in the Seafood Industry

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The use of vaccines and antibiotics in aquaculture is aimed at preventing the initial colonization by microorganisms. The means of administration of these prophylactics is simple, they are either added to the water or feed, or given by injection (Shao, 2001). A large body of research in the 1970s resulted in vaccines against numerous seafood pathogens, primarily species of Vibrio (Shao, 2001). Although vaccines are effective (and cost-effective), there are still no vaccines against shrimp and mollusc pathogens (Subasinghe, 2009).
An alternative to vaccination is the use of antibiotics to prevent bacterial infections. Tetracycline has become one of the most popular antibiotics in aquaculture due to its lowcost, low-toxicity, and high efficacy. Further, florfenicol, sulfadimethoxine/ormetoprim, oxytetracycline, and sulfonamides, are used to treat common bacterial infections (Table 4). However, the rampant use of antimicrobials in this industry has created massive selective pressure for bacteria to develop resistance (World Health Organization [WHO], Fact sheet 194). While these drugs are effective in killing bacteria, they also play a much more nefarious role in aquaculture. Antibiotics have varying half-lives, meaning they degrade at different rates. Some antibiotics degrade slowly and thus proliferate in the aquatic environment (Cabello, 2006). Worse still, the drugs flow into open waterways, sewage systems, sediments, and can even remain in the flesh of the farmed seafood (Benbrook, 2002;Cabello, 2006). These antibiotics continue to impose selective pressures leading to resistance until they are eventually degraded. This resistance is not only seen in the bacteria that inhabit the seafood produced in antibiotic-using aquaculture facilities, but in the animals neighboring the facilities as well (Benbrook, 2002). It has also been shown that these resistant bacteria are able to horizontally transfer their resistance-conferring genes to other human pathogens (Benbrook, 2002). For these reasons, governing agencies, such as the US Food and Drug Administration (FDA) and Environmental Protection Agency (EPA), strictly regulate the use of antibiotics in aquaculture.
Due to the problems associated with antibiotic use, the seafood industry is exploring the use of probiotics to promote the growth of healthy microflora in the seafood that can combat infecting pathogens. Probiotics are live microbial feed supplements that beneficially affect the host animal by improving its intestinal health (Fuller, 1989). While this definition needs a bit of tweaking to fit the seafood industry, it does still apply.
One area of active research in seafood aquaculture is the utilization of bacteriocins as antimicrobials. Bacteriocins have a long history of use in dairy or meat applications and there is an increasing number of studies on the effect of bacteriocins as antimicrobials in the seafood industry (Table 5) (Aasen et al., 2003;Al-Holy et al., 2004;Budu-Amoako et al., 1999;Einarsson & Lauzon, 1995;Elotmani & Assobhei, 2004;Luders et al., 2003;Neetoo et al., 2008;Nilsson et al., 1997;Nykanen et al., 2000;Szabo &Cahill, 1999;Tahiri et al., 2009;Tsironi & Taoukis, 2010;Zuckerman & Ben Avraham, 2002). These studies have focused largely on the effects of nisin, a Gram-positive bacteriocin that has been generally recognized as safe (GRAS) by the FDA. Early studies of nisin indicated that it delayed growth of L. monocytogenes in cold-smoked salmon. Later research revealed that the addition of CO 2 atmospheric packing significantly increased the effectiveness of nisin against Listeria (Nilsson et al., 1997).
There has also been encouraging research into nisin-coated packaging. Neetoo et al. (2008) investigated the effect of nisin-coated plastic films on the survival of L. monocytogenes on vacuum-packed cold smoked salmon. This study showed that nisin-coated plastic films  Table 5. Examples of bacteriocin trials in seafood products (Adapted from Galvez et al. 2008).
reduced the number of L. monocytogenes by 3.9 log CFU/cm 2 at 4 o C and 10 o C after 56 and 49 days of incubation, respectively. Further, this study also showed that nisin-coated plastic films suppressed the growth of other aerobic and anaerobic spoilage microorganisms in a concentration-dependent manner (Neetoo et al., 2008).
The combination of nisin with heat has also been shown as an effective method to prevent L. monocytogenes contamination. Budu-Amoako et al. (1999) applied nisin along with moderate heating on cold-packed lobster and showed a reduction of L. monocytogenes by 3-5 logs in comparison to nisin and/or heat treatment alone (Budu-Amoako et al., 1999). Further, Al-Holy et al. (2004) used a radio-frequency heating method to apply heat treatment in conjunction with nisin. The combination of nisin and radio-frequency heating caused reduction of L. innocua by 100% (Al-Holy et al., 2004). With an industry moving away from traditional preservation techniques, bacteriocins (such as nisin) offer a promising alternative as antimicrobials in the seafood industry.

Bacteriocin potential in the seafood industry
There are a number of factors that play a significant role in the potential to use bacteriocins as probiotics and/or bio-preservatives in the seafood industry. The natural microbiota of the seafood needs to continue to be surveyed for its sensitivity to bacteriocins. This information should be incorporated into the guidelines for bacteriocin use in order to use these proteins prudently against relevant pathogens. The environmental conditions, such as pH and temperature, during seafood growth and processing could also affect the activity of applied bacteriocins and requires further investigation (Galvez et al., 2007).
Despite these factors, research on aquatic microorganisms has shown that bacteriocin production and diversity in aquatic environment is abundant (Bagenda et al., 2008;Bhugaloo-Vial et al., 1996;Carraturo et al., 2006;Hosseini et al., 2009;Hoyt & Sizemore, 1982;Longeon et al., 2004;McCall & Sizemore, 1979;Messi et al., 2003;Metivier et al., 1998;Moro et al., 1997;Nilsson et al., 2002;Pinto et al., 2009;Pirzada et al., 2004;Prasad et al., 2005;Rajaram et al., 2010;Selvin et al., 2004;Shehane & Sizemore, 2002;Stoffels et al., 1992;Sugita et al., 1997;Suzuki et al., 2005;Tahiri et al., 2004;Valenzuela et al., 2010;Zai et al., 2009). Bacteriocins have numerous qualities that make them attractive as alternatives to antibiotics. They have been shown to be non-toxic to eukaryotic cells and are GRAS, making them a safe alternative to traditional antimicrobials (Galvez et al., 2008). It has also been shown that purified bacteriocins do not effect the sensory qualities of seafood and that they are stable up to a salinity concentration of 10%. Additionally, the relatively narrow killing spectrum of bacteriocins compared to traditional antibiotics limits the selective pressure for bacteria to evolve resistance to these antimicrobials and thus reduces the incidence of drug-resistant pathogens.
Because of the above stated reasons, some have suggested that bacteriocins should be applied to foods by spray-drying as either dried bacteriocins or probiotic bacteriocinogenic strains (Calo-Mata et al., 2007;Galvez et al., 2007). For example, Brillet et al. (2005) has shown that bacteriocin producer Carnobacterium divergens V41 can be used as a biopreservative to inhibit the growth of Listeria monocytogenes in cold smoked salmon (Brillet et al., 2005). This study showed that spray application of C. divergens V41 on commercial smoked salmon did not affect the sensory qualities of the salmon (Brillet et al., 2005). Additionally, Schobitz et al. (1999) directly applied a BLIS from Carnobacterium piscicola into vacuum-packed meat, which inhibited the growth of L. monocytogenes in the vacuum-packed meat after 14 days of storage at 4 o C (Schobitz et al., 1999). These studies aid in the argument that bacteriocins should be used as a biopreservation technique in the seafood industry. This technology has already emerged in the terrestrial food industry as we see with nisin (an FDA approved food additive) and Microgard TM (a milk-based BLIS).
It has also been suggested that bacteriocins could be combined with current methods of antimicrobial treatment and preservation to produce synergistic effects, such as incorporating bacteriocins into bio-active packaging (Calo-Mata et al., 2007;Galvez et al., 2007;Pilet & Leroi, 2011). For example, bacteriocins can be impregnated into gel coatings and/or polyethylene films and can be applied to seafood during packaging (Neetoo et al., 2008). The application of bacteriocins on packaged seafood is steadily being seen as a very promising biopreservation method (Aasen et al., 2003;Al-Holy et al., 2004;Budu-Amoako et al., 1999;Einarsson & Lauzon, 1995;Elotmani & Assobhei, 2004;Luders et al., 2003;Neetoo et al., 2008;Nilsson et al., 1997;Nykanen et al., 2000;Szabo &Cahill, 1999;Tahiri et al., 2009;Tsironi & Taoukis, 2010;Zuckerman & Ben Avraham, 2002). In fact, immobilization of bacteriocins on coating materials for biopreservation may actually reduce the cost of packaging due to the reduced amount and cost of the antibacterial needed to attach to the film (Galvez et al., 2008). Creating combinations of bacteriocins and current methods used in the seafood industry has the potential to increase the guarantee of freshness by assuring the inhibition of spoilage causing microorganisms.
One trouble that the industry was having was the scale-up of these bacteriocins to levels that were high enough for use in pilot studies and/or on the industrial scale. However, there is technology in the pipeline that will make this an issue of the past (Galvez et al., 2008). As the exploration of the aquatic environments of our planet increases, we are sure to find new and exciting bacteriocins, which could play a vital role in antimicrobial effects and biopreservation in the seafood industry.

Conclusions and future research
Bacteriocins have been the focus of an extensive number of studies for the past sixty years due to their important role in nature and more recently, their potential for use as therapeutics and probiotics. Most studies of bacteriocins initially focused on phenotypic and molecular characterization of these toxins. However, due to their high potency and relatively narrow killing spectrum, they were quickly recognized as a natural alternative to antibiotics.
Knowledge regarding bacteriocins and their potential applications from terrestrial bacteria is vast. Gram-negative bacteriocins such as colicins and microcins of enteric bacteria and pyocins of P. aeruginosa have great promise in human and veterinary medicine. In addition, Gram-positive bacteriocins such as nisin, pediocin, and lacticin have been developed for use as food preservatives.
The focus of this chapter was to explore the bacteriocin and BLIS activity characterized from marine microorganisms and assess their potential applications in aquaculture. The bacteriocins and BLIS from marine microorganisms are under-studied relative to their terrestrial counterparts. Thus far, most studies are limited to the identification of BLIS activity and characterization of its killing breadth. Some studies have further characterized these proteinecous killing agents and classified them based on similarities to known bacteriocins. However, the abundance and diversity of bacteriocins in marine microorganisms remains to be fully explored. We predict that a wealth of interesting bacteriocin proteins can be easily identified as the screening efforts proceed.
Further, there is increased interest in the use of bacteriocins as alternatives to classical antibiotics in aquaculture. Bacteriocins are highly potent against marine pathogens and environmentally safe, due to the fact that they do not create intensive selection pressures for antibiotic resistance. Clearly, bacteriocins could prove extremely beneficial to the seafood industry and more research should be dedicated to exploring their potential applications as probiotics and therapeutics. Given the fact that all species of bacteria have the potential to produce bacteriocins, and only a handful have thus far been identified from marine microorganisms, we are confident that existing studies have exposed only the tip of the iceberg, in terms of bacteriocin diversity and potential use in the seafood industry.
Interactions of the bacteriocins sakacin P and nisin with food constituents.