Keywords

20.1 Introduction

Over the past few decades, the aquaculture sector has served the nutritional needs of the people throughout the globe. The contribution from Asian subcontinent was maximum, i.e. 89% of total volume and 79% of the total value of fish production globally (Bostock et al. 2010). However, there are several factors which continue to play a crucial role in limiting the aquaculture production such as infectious diseases, especially those of bacterial origin. As per an assessment of Lafferty et al. (2014), the bacterial infection alone accounts for about 34% of total outbreaks encountered in the aquaculture system. Additionally, the indiscriminate use of chemotherapeutics to mitigate the disease problem has caused the rise in antimicrobial resistance (AMR) strain and the situation can exaggerate by the emergence of superbugs. According to Van Boeckel et al. (2019), the application of chemical therapeutics, especially antibiotics, for rearing of the farm animals including aquatic animals, accounts for about 73% of all antibiotic usage throughout the globe. In the recent past, various chemical agents have been used either as a prophylactic treatment or as growth enhancers. This would have paved the way that, due to the emergence of drug-resistant aetiological agents, the pathological condition that was resolved easily earlier is becoming a major setback to aquaculture production (Gelband et al. 2015). Consequently, researchers all over the world have been engaged with the development of alternative treatment approaches. In light of the investigation for substitute, the biocontrol strategy via bacteriophages could be considered as a sustainable option. The phage therapy, however, is an aged approach but the latest developments in the identification of potential isolates and their multidimensional application strategies have also fuelled the investigations towards the use of bacteriophages as a biological tool for health management in aquaculture.

20.2 Brief About Bacteriophages

Bacteriophages are the viruses which are obligate intracellular parasites of bacteria; they ultimately kill or lyse the host cell and release new progenies (Al-Sum and Al-Dhabi 2014). Bacteriophages are informally called phages, which is derived from a Greek word “phagein” meaning “to devour”. They utilize the bio-machinery of the bacterial host for all kinds of metabolic support in order to survive (Al-Sum and Al-Dhabi 2014). As the natural environment is replete with loads of bacterial host, the occurrence of phages is natural and can flourish in soil up to 107–8 virions g−1 and in water approximately 107 virions mL−1 either in fresh or saline environment (Ninawe et al. 2020; Park et al. 2020). According to Abedon et al. (2011), the total count of bacteriophages on the earth is about 10 times the total bacterial host thriving in different environments, which accounts for about 1030–31. The International Committee on the Taxonomy of Viruses (ICTV) is responsible for the typing of phages and they have classified bacteriophages into 19 families, among which a few are well characterized including Microviridae, Myoviridae, Inoviridae, Podoviridae and Siphoviridae (Simmonds et al. 2017; Adriaenssens et al. 2018; Walker et al. 2019). The vast abundance and diversity of phages in the biosphere provides an already equipped resource to mine for the potential phages for a variety of purposes (Nikolich and Filippov 2020). Employment of precise killing capability of phages to control lethal bacterial pathogens is called as phage therapy or phagotherapy. The putative phages are composed of proteinous outer shell/capsid measuring about 24 to 200 nm in size, which contains proteins and nucleic acids (either DNA or RNA) ranging 17 and 700 kb in length (Ackermann 2003; Sharma et al. 2017). The majority of phages possess a tail (variable in size) in their structure with tail fibres on it which helps in the precise identification and adherence to the bacterial host (Kowalska et al. 2020).

The life cycle of bacteriophages can be categorized into two stages, first is lytic (virulent) and second, temperate. In the first lytic cycle, the phages adhere themselves to bacterial host followed by taking control of the host’s bio-molecular machinery to proliferate and ultimately kill the host bacteria, concurrently releasing its progeny phages. The lytic phages are responsible for the production of two specific proteins to kill the host, “holins and endo-lysins”. The protein, holins work in synergy with the endo-lysins and are responsible for the perforation on the bacterial cell followed by the destruction of cell wall after phage multiplication (Cisek et al. 2017). In the second temperate lysogenic stage, after the infection of bacterial host the phage genome shifts to dormant stage “prophage” which can exist within the host in the form of a plasmid and can last for many generations and can make its genes (including virulent genes) functional for the host bacterium. However, any sudden exposure or any triggering factor such as DNA damage, UV exposure and antibiotic treatment might lead the conversion of lysogenic phage to lytic stage (Letchumanan et al. 2016; Kowalska et al. 2020). Temperate phages are favourable to bacteria because they might encode for antibiotic resistance gene or some other potent genes; additionally, these lethal genes can be horizontally transferred to another bacterium in the residing environment (Lin et al. 2017). On the contrary, virulent lytic phages kill the bacterial cells directly where the possibility of any genes transfer is limited, which make lytic phages a desirable candidate for therapeutic bacteriophage therapy (Jassim and Limoges 2014; Letchumanan et al. 2016). However, according to the report of Freifelder (1987), the prevalence of lysogenic phage compared to lytic phages is as more as 90% in nature, which makes phage isolation a crucial state in development of phage therapy. There are few literature who vote for another third phage variant, a carrier state of the lysogenic stage termed as pseudolysogenic cycle, where the phage genetic material does not replicate but instead remains inactivated within the host till the occurrence of favourable condition (such as nutrient availability which hinders the bacteriophage gene expression). Once the favourable situation prevails, carrier state might be initiated with either the lytic cycle or the commencement of true lysogeny (Sieiro et al. 2020).

20.3 History of Bacteriophage Researches

Ernst Hankin in 1896 was the first one to demonstrate the presence of certain unidentified antimicrobial compounds against Vibrio cholera which are heat labile, filterable and transmissible, from the waters sample of the Ganges river system of India (Hankin 1896); however, he was not able to come to a conclusion regarding the reason behind anti-bacterial activity (Twort 1915; D’Hérelle 1917; Summers 2005). Later, in 1915, Frederick Twort, a British pathologist, was the first to demonstrate the presence of an “ultra-microscopic virus” that could affect bacteria; however, he also failed to explain the phenomenon, including the existence of virus (Summers 2005). Two years later in the year 1917, a French-Canadian microbiologist Felix d’Herelle observed a similar clear zone phenomenon in stool samples of bacillary dysentery patients. Unlike Twort, this time, d’Herelle was able to explain the presence of “invisible microbe”, a virus which he termed as “Bacteriophage” (Brunoghe and Maisin 1921). Later, during the 1920s, various clinical trials on phagotherapy were carried out in Eastern Europe and the Soviet Union, where therapy was used for the treatment of variety of diseases including bubonic plague and cholera in India (Nikolich and Filippov 2020). Despite encouraging initial success of the page therapy, their application as antimicrobial approach was declined because of the discovery of antibiotics in the mid-nineteenth century.

20.4 Bacterial Diseases in Aquaculture and Its Control Measures

Despite the fact that aquaculture is one of the fastest rising food-production sectors in the world, it is currently plagued by frequent and severe outbreaks of diseases. The sector is under threat from several groups of pathogen such as bacteria, fungi, viruses, and parasites. Among all these concerns, the bacterial pathogens can endure well in both fresh water and marine water aquatic ecosystem without their host; and the attribute favours them as major impediments to the aquaculture industry. The situation is further exaggerated by the adopted intensive culture practices and human anthropogenic activities which has led the foundation for the adulteration in the optimal physico-chemical quality of the aquatic environment (Pridgeon and Klesius 2012). Till now, about 13 bacterial genera have been identified as pathogenic to aquatic organisms including fish, which comprises both gram-negative pathogens (Edwardsiella, Aeromonas, Vibrio, Flavobacterium, Pseudomonas, Yersinia, Francisella, Piscirickettsia, Photobacterium and Tenacibaculum) and gram-positive (Renibacterium, Lactococcus and Streptococcus) (Pridgeon and Klesius 2012; Gui and Zhang 2018).

To control bacterial disease outbreak in an aquatic system, feeding fishes with drug-medicated feed, especially antibiotics, is a general practice. At present, the addition of various kinds of nutraceuticals or functional food is very well accepted to remediate the situation either as a prophylactic or therapeutic agent (Pridgeon and Klesius 2012). However, the approach is usually expensive and maybe ineffective for therapeutic purposes as infection-weaken fish do not accept any kind of feed especially medicated feed. Additionally, frequent and sub-therapeutic level of chemical additives or drugs over an extended period led the base for the development of AMR among pathogens (Cunha 2009). Substitutes for antimicrobial agents with similar or enhanced protection are therefore urgently needed to provide robust protection against variety of bacterial aetiological agents in target organisms. At present, the application of various kinds of vaccines, immunostimulant of natural or chemical origin is very well accepted in commercial aquaculture farms, along with several biocontrol strategies such as application of probiotic, bacteriophages and symbiotic. Among these alternative strategies, phagotherapy emerges as a sustainable substitute to chemical therapeutics, since phage application has the potential to not only eliminate the virulent pathogens precisely but can also to help in the creation of homeostasis in aquatic environment by minimizing the application of chemicals and other remedial drugs to achieve the goals of “One Health” approach of WHO.

20.5 Research on Bacteriophage Therapy in Aquaculture

Although bacteriophages were discovered way back at the beginning of the nineteenth century, however, the focus of research on its therapeutic potential against bacterial diseases was limited to a certain part of the world because of the poor understanding of phage life cycle and bacteria-phage interactions (Almeida et al. 2009). Furthermore, with the discovery of antibiotics, the application of phages remains underexplored. However, in some places such as Eastern Europe and in the Soviet Union, they successfully demonstrated several clinical trials on human patients which laid the foundation to the future work (Park et al. 2020). Moreover, the emergence of multi-drug resistant bacteria has substantially encouraged researchers to explore the potential of phagetherapy; because, phages can be employed as bioagents against wide range of bacterial pathogens. Owing to the specificity of phages to their host, the probability of disrupting natural microflora of aquatic environment or host inhabiting beneficial bacteria will be null which is very unlikely with the administration of common broad-spectrum antibiotics (Fortuna et al. 2008). The very first attempt to employ phage therapy in aquaculture was made in the year 1981 in Taiwan against Aeromonas hydrophila in loach (Misgurnus anguillicaudatus) (Wu et al. 1981). Nowadays, work associated with the phagotherapy against bacterial pathogens in aquaculture has been accepted worldwide and encouraging researchers to explore the application and efficacy of phage therapy in different circumstances under various culture conditions (Table 20.1).

Table 20.1 Isolation and application of bacteriophage in aquaculture
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20.6 Phage-Based Products for Therapy in Aquaculture

The potential and efficacy of phages have encouraged some private companies/institutes to develop phage-based product for commercial application to treat bacterial diseases in aquaculture which is tabulated below (Table 20.2).

Table 20.2 Phage-based products for therapy in aquaculture
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20.7 Strategic Guideline for the Development of Phage Therapy in Aquaculture

For the development of bacteriophages therapy in aquaculture, a set of standard protocols need to be followed (Nakai and Park 2002; Choudhury et al. 2017) (Fig. 20.1). This includes isolation and characterization of phage (Fig. 20.2), in vivo and in vitro therapeutic potentiality testing, safety testing and regulatory approval, etc.

Fig. 20.1
figure 1

Strategies for bacteriophage therapy in aquaculture

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Fig. 20.2
figure 2

Zone of lysis by bacteriophages and plaques formed by bacteriophage

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20.8 Dose and Mode of Application for Phage Therapy

There are several modes of application of phage therapy reported by many researchers since its discovery. However, the application of phage in the aquaculture system includes direct release of phages in the culture system, injection through intramuscular or intraperitoneal mode, immersion, oral administration through feed, anal intubation, etc. Among all these reported modes, release of phages directly into the culture system is the most preferred method (Shivu et al. 2007; Choudhury et al. 2017; Silva et al. 2016). In recent times, various combination of phage as “cocktail” has gained a lot of interest among researchers as futuristic bacteriophage approach. Cocktail of diverse combinations such as phage-phage, phage-probiotic, phage-immunostimulant and phage-antibiotic are demonstrated in the literature (Fischetti et al. 2006; Chan et al. 2013; Choudhury et al. 2019). There are advantages and disadvantages to each mode of application; which often depends on the nature of the bacterial pathogen (Martinez-Diaz and Hipólito-Morales 2013; Richards 2014).

For effective phage therapy, it is important to know the exact dose of application. Various doses have been reported by researchers for both laboratory and field condition. However, in most cases, the dose of application depends on the type of pathogen, state of phage, multiplicity of infection (MOI) of phage or lytic capability, etc. For effective phage therapy, researchers may attempt to isolate phage with a high replication rate, broad host range with high lytic capacity at lower doses (Choudhury et al. 2017).

20.9 Positives and Negatives of Phage Therapy

Several well-established advantages of phage treatment include (Barrow et al. 1998; Nakai 2010):

  1. 1.

    Because of the natural abundance, phage isolation is comparatively easy and cheap.

  2. 2.

    Bacteriophages have narrow host range indicating that phages are very specific to host and do not harm the endemic intestinal or environmental microflora.

  3. 3.

    No inherent toxicity and environment friendly.

  4. 4.

    Self-replicating capability eliminates the necessity of multiple administrations.

  5. 5.

    Effective against biofilm-forming bacteria.

  6. 6.

    Bacteriolytic capability of phages allows them to eliminate MDR (multi-drug resistant) bacteria.

  7. 7.

    Because of the high specificity, phages do not contribute to the development of resistance among pathogens.

  8. 8.

    Administration of phages can be very feasible because of the multimodal application such as oral, aerosols, immersion, injection, and topical.

Bacteriophage application has an immense potential but even then, the feasibility, accessibility and field efficacy still remains a concern, which roots to several drawbacks in phage therapy:

  1. 1.

    Because of the high specificity of phages, the pathogenic bacteria must be identified before therapy, which may prove to be a realistic and practical challenge in the field condition.

  2. 2.

    Difficult to extrapolate in vivo efficacy in comparison to in vitro results.

  3. 3.

    Temperate phages can transfer lethal or toxic genes to harmless bacteria.

  4. 4.

    Because of the robust nature of the host bacteria phage resistance can be developed by bacteria.

  5. 5.

    Contradictory opinion on interaction with the immune responses of fish/shellfish.

  6. 6.

    There might be practical difficulties, e.g. injecting large numbers of animals, acceptance of phage mediated feed to diseased fish.

  7. 7.

    Conversion of lytic phage to lysogenic state is still a mystery among phage experts and may be a concern prior to application.

20.10 Conclusion

Bacteriophage therapy has been reintroduced in the system after the rise of drug-resistant bacteria and to cater the necessity of finding an alternative to chemotherapeutic application. Owing to the host specificity of phage and lytic capability, it can prove to be an attractive approach in that it provides a ray of hope against AMR. At present, the potential phagotherapy has established its efficacy in preventing or controlling the bacterial infections in both freshwater and marine water in various target species of fish and shellfish origin. Bacteriophage therapy has been intensively researched and developed against various clinical conditions in the area of biomedical application. However, in aquaculture, the therapy is not yet fully investigated. The lack of in vitro and in vivo research on optimization and efficacy in different culture condition existing in diverse aquatic environments has led to the challenge we are facing today, with the development of effective field-based formulation. It is high time that attempts are made to address the concerns that have arisen over time, and research efforts should therefore be conceptualized and aimed at establishing sustainable phage therapy.