Literature Review: Isolation and Characterization of Bacteriophage Infecting Vibrio alginolyticusVibrio alginolyticus IntroductionVibrio alginolyticus is a facultative Gram-negative anaerobic bacterium, which was formerly regarded as an opportunistic pathogen causing vibriosis in marine fish and shellfish (Austin & Austin, 2007; Egidius, 1987). In recent year, V.
alginolyticus has been detected as a major threat to seawater aquaculture. The marine bivalve’s consumption is an increased alimentary habitat coastal region around the world. therefore any consumption of marine or aquatic life without cooking or after an insufficient process of cooking may be risk for human health because those animal are capable to retain pollutants and pathogenic bacteria as vector in their organisms (Matté et al., 1994). For Vibrio genus natural habitat are belong to the aquatic ecosystem and the most and studied Vibrio is Vibrio cholera, the causative agent of cholera.
However, there are also many other species be able to transmit intestinal or extra intestinal disease to human, especially for V. vulnificus, V. carchariae and V. alginolyticus that widely spread in the marine environment either in closed seas or in open ocean (Matté et al., 1994; Schmidt, Chmel, & Cobbs, 1979).
These bacteria can be found and isolated in water sample from marine environment, cockle, fish and their product. Until the end of the 1980, the human infection was correlated to the presence of these microorganisms mainly by consumption of aquatic organisms. Vibriosis in aquacultureVibrio is a group of bacteria that highly potential to cause serious illness called vibriosis (F. R. Chen, Liu, & Lee, 2000). Vibrios are facultative gram negative bacteria that are abundant in aquatic environment and marine ecosystem and also present as free-living in the water column parts of biofilm or can be associated with a host in the marine (Austin & Austin, 2007; Thompson, Iida, & Swings, 2004).
The number of Vibrio species has reported rise in number and more than nine of Vibrio has been associated with diseases in human, where they can cause gastrointestinal disorder and skin infection (Venkateswaran, Dohmoto, & Harayama, 1998). Even though, Vibrio has been reported and studied scientifically as one of the pathogenic bacteria and causal agents of diseases in aquatic organisms; surprisingly, other species of Vibrio such as Vibrio alginolyticus has been used as probiotic for shrimp production (Vandenberghe, Thompson, Gomez-Gil, & Swings, 2003). Generally, Vibriosis can be detected by the sign and symptoms shown by the aquatic organisms include lethargy, slow growth, tissue and appendage necrosis, slow metamorphosis, body malformation, muscle opacity, melanisation and bioluminescence (Aguirre-Guzmán, Ruíz, & Ascencio, 2004). In addition, infected fish by Vibrio show the presence of red necrotic lesion in the abdominal muscle, erythema (bloody blotches) at the bae of the fins, around the vent and within the mouth, and skin discoloration. However, Vibrio are classified as opportunistic bacteria because they only causing and infect the host organisms when the immune become suppressed or stress either due to the super intense culture or adverse environmental or both (Alderman & Hastings, 1998).
Control of Vibriosis in AquacultureThere is little common practice to control the vibriosis in aquaculture including the application of antibiotic via the oral route to groups of fish that share tanks or cages and feeding infected fish with antibiotic-medicated food (Defoirdt, Boon, Sorgeloos, Verstraete, & Bossier, 2007; Pridgeon, 2012). However, due to development of antimicrobial compound resistance in the pathogen due to frequent use, unrestricted use of antibiotics where there is presence of residual antibiotics in the commercialised of aquaculture products that lead to allergy and toxicity in human and massive use of antibiotics for livestock’s resulting in frequent rejection and ineffective approach to contol the Vibriosis towards marine life (Cabello, 2004). Therefore, the alternative approach of prophylactic approaches will be sustainable to solve and control Vibriosis was rise due to antibiotics issues because within the aquaculture system that culture the animals in several types of system and not always in their most optimal conditions. There are several prophylactic approaches has been developed and applied in aquaculture system that aim to control and prevent the Vibriosis including immunostamulation, vaccination, probiotics and quorum sensing of peptidoglycan (PG) to inhibit the virulence factors of Vibrios (Arala-Chaves & Sequeira, 2000; Duvic & Söderhäll, 1990; Natrah et al., 2011; Sajeevan, Philip, & Bright Singh, 2009; Vine, Leukes, & Kaiser, 2006).
Morphology of BacteriophageBacteriophage are specific bacterial viruses that are able to infect target bacterial host cells with great host specificity of species or strain level and consequently multiply, finally resulting in lysis and death of the target host cell. Normally phage is great host specificity, however there are a few phages do shows wide host range that being able to infect a broad subset of a given species or even a few species (J. Chen & Novick, 2009; Hagens & Loessner, 2010). To differentiated the bacteriophage species, it can be categorized by vary of both in size 24 to 400 mm in length and genome length. All bacteriophages consist of a head that that that part is an important feature of a bacteriophage that contain and store genomic material either in form DNA or RNA (Tavares, Zinn-Justin, & Orlova, 2012).
Structurally, head of the bacteriophage also called as protein or lipoprotein capsid that contain and encapsulated a core nucleic acid that connected to a tail that interacts with the target bacterial surface receptors via the tip of the tail fibers. The interaction between tail of phage and bacterial receptor shows a compatibility of specificity of phages to acertain group of bacteria or even to a particular strain (Deresinski, 2009). Most of the pages consist of icosahedral shape of capsid and has the core function to protect the genetic material from the external environment. A connector that function as adaptor that lies in the between the head and tail of bacteriophage responsible to attach both structure of the phage. Genetic material passes thru from capsid to target host bacteria via tail of phage that consist of hollow tube that acts as passage way (Koch, Hertwig, Lurz, Appel, & Beutin, 2001). To transfer the genetic material between phage and target host bacteria, binding process must be occur by the help of the tail fiber and base plate that located at the end of the phage to the receptor of the bacteria (São-José et al., 2006).
Nature of BacteriophageBacteriophages can be classified as the most abundant organisms in the environment, their total number can be 10 times more than their bacterial host where approximately estimated to be between 1030 to 1031 (Abedon, Kuhl, Blasdel, & Kutter, 2011). Phage is natural killer of bacteria, self-replicating in their target bacterial host and self-limiting, and phage also can adapt to bacterial resistance (Fernandes et al., 2014; Jaiswal, Koley, Mitra, Saha, & Sarkar, 2014). Phage can be normally be found in enormous number wherever their bacterial host live whether in soil, in ocean, in deep thermal vent, in sewage or in animal (Karunasagar, Shivu, Girisha, Krohne, & Karunasagar, 2007; Kim, Rahman, Seol, Yoon, & Kim, 2012; Rong, Lin, Wang, Khan, & Li, 2014). Until now, most of the bacteriophage that found in the marine and that has been reported can be divided into three families which are Siphoviridae that contain filamentous non-contractile tail with icosahedral capsid, Myoviridae consist a helical contractile tail with icosahedral symmetrical head separated by neck and Podoviridae consist of very short non-contractile tail with icosahedral symmetrical head (Madhusudana Rao & Lalitha, 2015; Short & Suttle, 2005).
Genome and Genetic of BacteriophageA bacteriophage can be only consist either DNA or DNA as their genetic material that encapsulated by capsid in form of both single and double stranded. Phage genomic material structure is similar with other living organisms, where the polynucleotide chain consist a deoxyribose (DNA) or ribose (RNA) phosphate backbone that attach to a specific sequence of four basic nucleotides; adenine (A), thymine (T) or uracil (U), guanine (G) and cytosine (C). It is uncommon in single stranded phage where two complementary chain are paired together in a double helix (Birge, 2006)One of the complete genome sequences that have been reported is T4-like phage, vibrio-phage KVP40 discovered in Japan. This vibrio-phage has a prolate icosahedral capsid, contractile tail with associated baseplate and extended tail fibres and with double –stranded DNA genome sequence in length of 244 835 bp that belong to Myoviridae family. KVP40 is a broad host range bacteriophage that can be infecting several species of Vibrio including pathogenic species, Vibrio anguillarum, Vibrio parahaemolyticus and the non-pathogenic species, Vibrio natriegens.
Factors of the high flexibility in host range adaption of the phage is the presence of a few copies of genes responsible in encoding protein linked with either phage tail or tail fibres (Miller et al., 2003). Another genomic sequence that has been studied is on phage vB_VpaM_MAR found in non-treated seawater in Maxico. This phage is highly specific infecting host bacterial belongs to Myoviridae family that capable to lyse 76% of the Vibrio parahaemolyticus strain tasted. From the genomic analysis resulting the phage consist 51.3% of G+C content with 41 351 bp double-stranded DNA and encodes 63 open reading frames (ORFs) (Alanis Villa, Kropinski, Abbasifar, & Griffiths, 2012).
Another bacteriophage, SSP002, was isolated from the coastal area of the Yellow Sea of South Korea that specifically infects Vibrio vulnificus and belongs to Siphoviridae family. Genomic sequencing of both phages SSP002 and vB_VpaS_MAR10 are closely related and highly similar however, comparative genomic analysis showed differences among their tail-related genes and supporting different host ranges at the species level (Lee, Choi, Shin, Lee, & Choia, 2014). Newly, two broad host range phage (H1 and H7) were isolated from Danish fish farm and both belong to the Myoviridae family and has large genome size approximately 194 kb (Tan, Gram, & Middelboe, 2014). Performing genomic analysis can provide full genomic sequence that help in understanding vibriophage genome and detail characterisation on phage structure, phage host range and the interaction.
That information is important in order to overcome the drawback of phage therapy. Bacteriophage life cycleBacteriophage are highly specific towards host cell, phage can only infect host bacteria if the cell surface receptor match with the phage receptor this similar to a lock and key mechanisms. If the phage do not has any matching receptors and unable to infect any host cell and the phage will be immediately killed by the environment. If the bacteria successfully infect the host bacteria, phage will be either multiplies by the lytic cycle (virulent phage) or lysogenic cycle (temperate phage). For lytic cycle, virulent phage kill the infected host bacteria as phage progenies are release from lysis and in lysogenic cycle, temperate phage can establish a persistent infection of the bacteria without killing it as the temperate phage DNA is integrated into bacterial host chromosome and replicate as with cell division (Lindberg, 1973; (Kutter & Sulakvelidze, 2004)). Virulent phages in lytic cycle are competent at controlling the bacterial population without effect to animal, plant or human because the phage are specifically kill their host bacteria (lysis). Virulent phage bind and attach to the host bacteria cell receptor via it surface receptor and phage inject the nucleic acid into the host bacteria and lead to produce large number of phage progeny.
Those progeny then release to the environment by a fatal lysis of the cell and attend to attack new bacteria (Kutter & Sulakvelidze, 2004; Sulakvelidze, 2011). The whole process only takes 25 minutes to complete. Based on the investigation on virulent phages show that their bactericidal properties are particularly suitable for the biological control and phage therapy when it require host bacterium destruction and making them one of the best alternative treatments to antibiotics. In vitro conform that where the lytic phages clears the bacteria lawn in Patri dish plate and form plaques (Gill & Hyman, 2010).On the other hand, phages also can be replicate without immediately killing the host bacteria and this is known as temperate phage (lysogenic cycle). Temperate phage can be either multiply by the lytic cycle or stay in dormant phase in the cell. When the nucleic acid of the phage enters the bacteria, it will integrate with host genome and reproduce genetic material (prophage) in the host cell. The progeny will keep replicate and multiply along the host bacterium replication without adverse effect until the host condition become unfavourable.
Physiological stressor is one of the factors to trigger the host bacterium to switch into lytic cycle and destroy host cell, releasing progeny phage (Haq, Chaudhry, Akhtar, Andleeb, & Qadri, 2012).In general, there are 5 important steps for phage replication which are adsorption, penetration of nucleic acid, replication, maturation and bacteria lysis. During adsorption, the phage will attach to the target host bacteria via bacterium receptor in order to transfer and infect genetic material into the host bacteria. Penetration is where the actual infections of phage genetic material infect and disturb the host cell genetic material. In replication, the phage genetic material keeps replicate by take over the host cell metabolic machinery.
In order to become mature phage progeny all the phage compartment must be assemble and before it become infectious state (maturation) and release its progeny through cell lysis. Cell destruction occurs when the phage particle release lytic enzyme (Lysin) and the cell wall become compromise and weak enough for the matured phage breakthrough it. However, lysogenic phage will integrate into the host cell genome rather than being lytic (Haq et al., 2012).
Bacteriophage Therapy in AquacultureDue to phage specificity and immediate cell lysis, phage can be use as huge potential alternative against bacterial infection, compare to non-specific antibiotics. Therefore by using bacteriophage against target host bacterial theoretically could not be harm and effect to beneficial bacteria or normal flora, thus reducing the chances of opportunistic infection (Fortuna, Mi?dzybrodzki, Weber-D?browska, & Górski, 2008). Based on the studies that has been reported, the phage can be used to prevent infection or to inactivate different fish pathogenic bacteria (Crothers-Stomps, Høj, Bourne, Hall, & Owens, 2010; Merino, Camprubi, & Tomas, 1990; Munro, Oakey, Bromage, & Owens, 2003; T.
Nakai et al., 1999; Stevenson & Airdrie, 1984). From the reported results with the marine animal sample that has been demonstrated the therapeutic phage therapy against disease caused by Aeromonas salmonicida, Enterococcus seriolicida, Lactococcus garvieae, Photobacterium damselae subsp. piscicida, Pseudomonas aeruginosa, Pseudomonas plecoglossicida, Vibrio anguillarum, Vibrio harveyi, and Vibrio parahaemolyticus. The animal model include the seabass (Dicentrarchus labrax), larval stages of shrimp (Penaeus monodon), Ayu (Plecoglossus altivelis), rainbow trout (Oncorhynchus mykiss), yellowtail (Seriola quinqueradiata), Atlantic salmon (Salmo salar), and seabream (Sparus aurata) (Higuera, Bastías, Tsertsvadze, Romero, & Espejo, 2013; Karunasagar et al., 2007; T.
Nakai et al., 1999; Toshihiro Nakai & Park, 2002; Tanji et al., 2005; Vinod et al., 2006). From advance studies has discover the potential specific bacteriophage successfully control pathogen concentration and reduce fish mortality rate.
In some case, V. anguillarum and vibriophage cause the larvae mortality in the infected and treated group was similar to normal levels but significantly lower than the infected (Mateus et al., 2014). Recently, there is another successful bacteriophage therapy in controlling the pathogenic Aeromonas hydrophila in Redclaw crayfish hatchery in Australia (Elliott and Valverde, 2013). Another study found that lytic phage A3S and Vpms1 also successfully reduce the larvae mortality cause by V. parahaemolyticus (Lomelí-Ortega & Martínez-Díaz, 2014).
There are another trail treatment by using bacteriophage therapy that cause improving in larvae survival and decline in V. harveyi density in hatchery tank (Vinod et al., 2006).