Bacteriophage applications in aquaculture

Introduction

Aquaculture is a rapidly growing industry worldwide, from which people derive about 50% of their animal protein requirements.¹ As the Food and Agriculture Organization reported in 2020, aquaculture production in 2030, which provides about 1/3 of the world’s aquaculture resources, is estimated to rise to 53%.² In addition to fish, species such as carp, tilapia, and trout and aquatic organisms such as oysters, clams, and shrimps are grown in aquaculture in fresh and marine waters.³

However, aquaculture suffers from heavy financial losses every year globally because of viral, fungal, parasitic, and bacterial disease outbreaks at any stage of the breeding process.^4–7 The same bacterial pathogens detected disease agents in aquatic organisms: Gram-negative Aeromonas salmonicida, A. hydrophila, Pseudomonas plecoglossicida, Edwardsiella tarda, E. piscicida, E. ictaluri, Vibrio spp. (V. harveyi, V. parahaemolyticus, V. anguillarum, V. splendidus, V. alginolyticus, V. coralliilyticus), Flavobacterium columnare, F. psychrophilum, Yersinia ruckeri and Gram-positive Lactococcus garvieae, Renibacterium salmoninarum, Streptococcus iniae, Mycobacterium species.^8,9 These bacterial pathogens that are easily transmitted through water in case of external stressors, including intensive stock densities, inadequate nutrition, build-up of toxic chemicals, poor water quality, and low oxygenation, can therefore infect many aquatic organisms.

Various strategies, including probiotics, prebiotics, immunostimulants, and vaccination, have increased fish defense and prevented bacterial diseases.^10,11 However, vaccine administration methods and routes vary depending on species, quantity, size of organisms, pathogens, temperature, and environment.^12,13 Therefore, vaccination becomes a tedious job for large-scale aquaculture systems. Also, vaccines may not be as effective against larvae and invertebrates without a robust immune system.¹⁴ Of the biocides, malachite green is used in the treatment of protozoal and fungal infections, while formaldehyde and formalin solutions are prophylactic disinfectants for eggs and larval development.

The use of amoxicillin, oxytetracycline, sulfonamides, tetracyclines, nitrofurans, fluoroquinolones, and florfenicol among antibiotics as a therapeutic agent is also the most preferred method to inhibit the growth of bacteria and stop heavy mortalities during outbreaks of infectious bacterial diseases in aquaculture and fisheries.^8,13,15 Although they are rapid, effective and commenly used for bacterial infection of aquaculture and agriculture, antibiotics generally target both pathogenic and non-pathogenic microflora of the environment. In additional, their long-term and heavy use caused a number of unfavourable impacts such as accumulation and toxicity in organisms, occurence of antibiotic-resistant bacterial strains and suppression of the immune response of the host, thus increasing the ineffectiveness of antibiotic treatments.^9,11,13,16 The above disadvantages of antibiotics have prompted the development of species-specific, eco-friendly and less expensive way to prevent bacterial infectious diseases in sustainable aquaculture.^3,7 In search of alternative tool or a possible solution, the use of bacteriophages seems to be very promising and appropriate strategy as approached “post-antibiotic era” as the World Health Organisation (WHO) announced in different diseases of animals.^17–22

This review mainly focuses on and summarizes extensive research literature in the last 40 years and future directions, with many different bacteriophages and its applications and outcomes as alternative ways, due to overuse of antibiotics, to prevent, control and treat diseases in aquatic organisms (such as crustaceans, molluscs and fish) and their environment.

Phage morphology

The bacteriophages, also known as phages (meant to imply “eat” or “devour” in Greek), are very small bacterial viruses that can range in size from 20 to 200 nm, are host-specific, and can only infect and kill targeted bacteria, without harming the surrounding microbiota and animal or plant cells.^23–25 Diverse phages can be found in all environments that are abundant in nature, such as river and seawater, sediments, soil, sewage, and food products, and were also readily detected in human and animal feces/urine.^26–28

The taxonomy and three-dimensional structure of typical phage morphology is well defined. It is classified according to their general morphology, the presence of outer envelope and lipid structures, and the type of genome that is in the form of ssRNA, dsRNA, ssDNA and dsDNA.^17,29–31 According to this approach, the International Committee for Taxonomy of Viruses (ICTV) identified 14 distinct and well-characterized phage families: Myoviridae, Podoviridae, Siphoviridae, Microviridae, Inoviridae, Herelleviridae, and Ackermannviridae, as shown in Table 1. Table 1 has been prepared according to information from Acherman^32,33 and Sharp et al.³⁴

Table 1.Morphology and genome characteristics of the fourteen phage families

The phage genome is enclosed in a protein capsid head (e.g., filamentous, helical, icosahedral, pleomorphic, and polyhedral), a tail with spiral sheath and tail fibers and surface receptors responsible for recognizing specific host bacterial molecules and attaches themselves to the cell’s surface.^35,36 These phages, which cannot perform their molecular replication under normal conditions, now use the host mechanism to reproduce themselves after their genome is injected into the bacterial host. They take over the bacterial biosynthesis control mechanism and command the bacterial host to produce different viral proteins and release progeny and phages that can continue to infect other hosts.

Phage life cycle

Phages can multiply and propagate by infecting bacteria in 2 paths: 1) lytic life cycle (virulent) and 2) lysogenic life cycle (temperate-dormant). The first phage cycle, which lasts between 20 minutes and 2 hours, begins when phages attach to the host, integrate their genetic material, and continue to multiply to produce viral progeny. The virulent phages will control the host’s protein. This cycle results in the secretion of lysins and holins enzymes by phages, lysis of the host bacterial cell membrane, and releasing the newly formed progeny virions into the environment. Afterward, the new progenies infect different host bacteria. The lysogenic (temperate) phages, in contrast, attach their genome to the host’s and remain in a dormant and stable stage for a long time until environmental conditions are favorable for the rapid growth of new prophages.^36–42 Therefore, lytic phages that proliferate exponentially and damage the pathogenic host in any case of antibiotic resistance status are more amenable to developing therapeutic intent.

Phage history and their potential applications

Phages therapy first came onto the scientific domain about a hundred years ago after finding by Twort⁴³ and d’Herelle,⁴⁴ respectively.^{23,40,45–48} Phage has been used successfully to treat severe hemorrhagic Shigella dysentery among French troops patients and against cholera by Vibrio cholera in India.^30,31,49,50 Bruynoghe and Maisin⁵¹ reported phage therapy treatment of staphylococcal skin disease. The first commercial phage in history was the anticholine phage, successfully used to control the epidemic that threatened the southeastern regions of the Soviet Union (SSCB) and then Georgia in 1931.⁵² In the 1930s and 1940s, the phage therapy application against mixed bacteria caused by Clostridium perfringens, Staphylococcus, Streptococcus, Escherichia coli and Proteus species were tested in Poland, Belarus, Georgia, Russia, Ukraine, and Azerbaijan.^23,53 Concurrent with the advent of commercial antibiotics in the 1940s, there was a huge decline in using phages as therapeutic agents in Western countries and the United States. However, in the period from the 1950s to late 1970s, the SSCB and in East Europe continued using the phage treatment against S. typhi and S. paratyphi and phage for prophylaxis in the fast spread of infections occurred such as military and schools.^54,55 In the 1980s, Smith et al.⁵⁶ showed that E. colidiarrhea in calves could be treated with phage, and this successful result then prompted the West to explore the possibility that phages could be used in human infections as well.

Because of the occurrence of multi-antibiotic-resistant bacteria, phages have been reappraised in the last two decades and are at the forefront again as therapeutic/prophylactic agents against human infectious diseases,^{18,42,57–60} aquaculture,^15,61–63 agriculture, animal and plant pathogens,^40,64,65 food^23,36,66; wastewater^31,67,68 and other subjects like biofilm removers^69,70 and biosensor.⁷¹ Multiple studies on using phages in animal agriculture have explored Salmonella, E. coli, Clostridium, and Campylobacter for the pig, chicken, cattle, and sheep industries.^22,72,73 Figure 1 summarizes phage applications in different areas.

Figure 1.Illustration of phage applications in human, aquaculture, food, agriculture, and environment

Phage applications in fish diseases and Aquaculture

The aquaculture and farmed fish industry have high mortality rates and considerable economic losses because of certain microbial infectious diseases. Widespread bacterial fish diseases are aeromonasis, hemorrhagic septicemia, furunculosis, vibriosis, edwardsiellosis, mycobacteriosis, ulcer disease, columnaris, lactococcosis, enteric red mouth disease, fry syndrome, and cold-water disease, respectively.⁸ Phages were used to control and prevent bacterial infections caused by antibiotic-resistant bacteria at the laboratory level or in small-scale trials for field applications in aquaculture.

The essential step in aquatic phage therapy is identifying the fish disease agent and then detecting and isolating the phage that can effectively infect the host bacteria. The phage and bacteria interactions are essential to inactivate possible aquatic pathogens. Phages can interact with hosts bacteria to the lytic cycles, replicate their genome, and produce new phages that let out bacterial cell lysis into ponds, lakes, seas, rivers, and sewage, infecting new bacterial cells. New phages are exponentially replicated, and the number of bacteria decreases and disappears. Figure 2 is an overview of the steps of phage application in aquaculture.

Figure 2.Schematic steps on phage application in aquaculture.

1. Phages are isolated from ponds or aquatic animals 2. Phages are isolated, purified, and identified 3. Phage is added to the farm water/fish/hatchery 4. After adding phages to water, they attach and contact the infected bacterial pathogen and then undergo a lytic cycle. Phage DNA would penetrate the host bacteria and replicate, transcription, and translation. Then the phage would assemble, the fish pathogen would be lysed, and phages would be released from the pathogen bacteria 5. Lysis of fish pathogen bacteria 6. Results of phage therapy in fish diseases.

Our literature review screened numerous google scholar publications from 1997 to 2022 when typed in “Bacteriophages, aquaculture, and twelve fish pathogens” as the keywords (Figure 3). And as shown in the bar graph, the number of research studies on phage therapy applications in aquaculture has gradually increased worldwide for twelve bacterial agents.

Figure 3.Publication related to phage therapy in aquaculture over 25 years.

The bar indicates the number of the Web of Science search for publications related to phage therapy and genomes associated with the most important fish pathogens A. hydrophila, A. salmonicida, E. tarta, V. harveyi, V. parahaemolyticus, V. anguillarum, V. alginolyticus, F. columnare, F. psychrophilum, Y. ruckeri, L. Garviae, and S. iniae in the last 25 years.

There is a selection of studies on the genetic/morphologic identification, characterization of phages, and the effectiveness of different phages for biocontrol and treatment in eggs, larvae, juveniles and adult fish and shrimp, other aquatic organisms, and aquaculture food products.

In addition, numerous reviews have reported that the most studied phage families are Myoviridae, Podoviridae, and Siphoviridae as virulence and control tool against a wide variety of pathogens, A. salmonicida, A. hydrophila, E. tarda, Y. ruckeri, V. harveyi, V. parahaemolyticus, V. anguillarum, V. alginolyticus, F. columnare, F. psychrophilum, L. garviae and S. iniae, in vitro or in vivo.^{9,11,15,21,30,41,74–79}

A list of reported phage applications and outcomes against the most significant bacterial pathogens in aquaculture from 1981 to 2022 is shown in Table 2.

The result of summarized studies in Table 2 using phages specific to 12 fish diseases agents as direct or suspensions of single or cocktail, oral administration, injections, or as aquafeed^174–176 recommend that phages could be beneficial to prevent and treat bacterial infections of aquatic animals. Even though different methods are used, the literature indicates that the most prophylactic impact appears when administered intraperitoneally (Americo et al., 2020). Additionally, in recent years, the other practical way was using commercial phages developed and used against some pathogens of aquaculture.^41,177 For example, Intralytix and Phage Biotech Ltd have developed phages to destroy Vibrio spp. in oyster and shrimp aquaculture,^178,179 Phage named BAFADOR® registered by Proteon pharmaceutical against Aeromonas spp. and Pseudomonas spp.^180,181 ACD Pharma has developed phage solutions against Y. ruckeri.¹⁸² Fixed Phage Ltd. has developed aquafeed-phage pellets.¹⁸³ Mangalore Biotech Laboratory has also developed LUMI-NIL MBL to control pathogens in shrimp.¹⁸⁴

The first research on phage therapy used in aquaculture was notified by Wu and co-workers in 1981 as the pathogenicity loss of A. hydrophila to loach (M. anguillicaudatus), in which Ah1 phage infected the pathogen.⁸⁸ Numerous studies have declared the accomplished use of more than 35 A. hydrophila phages aiming to control motile Aeromonas and septicemia from 1981 to 2022 (Table 2).^{40,90,92,93,96–105,175,185,186}

Around 22 phages with treatment activity against A. salmonicida (caused furunculosis) have been identified and characterized from farm fish (Table 2).^26,80–87

Four phages named PLgY-16, PLgY-30, and PLgW-1 were used to treat L. garvieae infection in yellowtail (S. quinqueradiata)^{150,151(p1998)} and P. plecoglossicida infection in ayu (P. altivelis)^61,152 in the 1990s.

From 2000 to 2022, more lysogenic phages named PLgT-1, PLgY-30, PLG-II, and WWP-1 were involved in L. garvieae infection colonizing marine fish, S. quinqueradiata, S. dumerili, S. lalandi, O. mykiss (Table 2).^149–155

Several phages for controlling F. columnare, which causes Columnaris disease in fish like Clarias batrachus and O. Mykiss, were isolated genetically and characterized.¹⁶⁶

Application of columnar phages (FCP1, FCP9, and FCP1) to infected fish resulted in the resolution of disease symptoms and stopped infection.^160,161 Four phages infecting F. columnare were isolated from fish farms during columnaris outbreaks. The zebrafish (100%) and the rainbow trout (50%) survived after the phage treatment.^162,163 Furthermore, several studies reported isolation and application for F. psychrophilum phages that could be used for biocontrol of the fry syndrome and cold water disease in S. salar, O. Mykiss, and P. altivelis. Lytic phages against F. psychrophilum strains provided the future potential in the treatment of this disease (Table 2).^167–173

The therapeutic effects of six S. iniae lytic phages with dsDNA were studied against Streptococcosis infection in P. olivaceus at 25°C for 2 weeks (Table 2).¹⁵⁶

Y. ruckeri is the causative bacterium of yersiniosis, known as enteric red mouth disease in freshwater salmonid fish. Yer A41, ɸ 2, ɸ 3, ɸ 3, ɸ 9 and φNC10 phages were tested as a combined or single intraperitoneal injection to treat Y. ruckeri and antibody production was reported in phage-treated fish (Table 2).^157–159

Two phages, named PT2 and phiKMV, were obtained from sewage, identified, and treated for P. aeruginosa infection at the surface of C. gariepinus. It was observed that the number of infective lesions decreased after 8-10 days in phage-treated fish.¹⁴⁸

Similarly, phages, namely PPpW-3 and PPpW-4, were used to treat bacterial hemorrhagic ascites disease caused by P. plecoglossicida in P. altivelis.^61,147

There are a few studies reported that specific phages of E. tarda, E. İctaluri, and E. piscicida stop especially growth of bacteria and reduce edwardsiellosis in vitro in D. rerio, P. hypophthalmus, A. japonica and S. maximus.^94,106–113

Various Vibrio species, such as V. harveyi, V. vulnificus, V. anguillarum, V. parahaemolyticus, V. alginolyticus, V. coralliilyticus, V. splendidus, and P. damselae (formerly V. damselae) are the cause of vibriosis have also been controlled by vibriophages which is biocontrol agents in fish (S. maximus L., S. salar, D. labrax, D. rerio, G. morhua L.) and P. monodon, L. vannamei, P. ornatus, H. laevigata, A. japonicus.¹⁸⁷ Studies have reported that approximately 60 bacteriophages were morphologically identified and genome sequenced and applied against Vibrio strains with no side effects shown in Table 2.^{10,114–118,121,127–131,134,138,140,145,146,165}

Conclusion

Despite good management practices, chemotherapeutic and prophylactic applications such as vaccines and various antibiotics for producing fish, crustaceans, and mollusks in many countries where aquaculture is a vital economic resource, bacterial diseases still stay a severe problem as they cause high mortality rates. An additional problem of bacterial infection is nowadays not only bacterial resistance to antibiotics but also the use of all known antibiotics in the treatment. Therefore, phage therapy has been shown as a perfect and valid option for antibiotic treatment. It is also an environmentally friendly, relatively rapid, simple administration, and inexpensive approach to disease prevention and control in aquaculture. Furthermore, therapeutic and prophylactic phage applications in aquaculture can effectively inactivate and eliminate pathogenic bacteria without harming useful microbiota and are easy to apply at various stages of vertebrates or invertebrates. Most of the aquaphage studies of the last 40 years, which we reviewed in this article, showed us that phage therapy has a general protective effect and can be substituted for possible antibiotics (Table 2). From the author’s perspective, although phage applications provide an optimistic view of future benefits for disease prevention and treatment in the world aquaculture sector, caution is required as the potential evolution of phage resistance against bacterial agents may also be present. In addition, more field applications should be made with large-scale cultivation and long-term preservation, and standardized methods and formulations should be developed. Furthermore, new commercially patented aquaphage products must be developed for future aquaculture practice. More research and improvement in phage therapy will play a significant role in sustainable aquaculture globally.

Author contributions

Conceptualization: Ifakat T. Çağatay (Lead). Investigation: Ifakat T. Çağatay (Lead). Writing – review & editing: Ifakat T. Çağatay (Lead).