Bacteriophages as biocontrol agents of bacterial contaminants of bivalves and their microalgal feeds

Bivalves and associated diseases

Compared to many other cultured sea products bivalves feed primarily on natural phytoplankton and require minimum husbandry as a result they are perceived to be well-suited for aquaculture.^1–4 A recent case study⁵ related to regenerative and restorative aquaculture noted that seaweed and bivalve mollusc production in aquaculture are major sectors globally, each product contributing more than 97 and 89% respectively of the total production from marine aquaculture and marine wild harvest combined.⁵ In 2020, global cultivation of algae dominated by marine macroalgae, known as seaweed, grew by 0.5 × 10⁶ tonnes within 1 year.³ Simultaneously, total global aquaculture production of molluscs, mostly bivalves, was 17.7 × 10⁶ tonnes equalling A$46.7 billion in value.³ Co-culture-farming of these species side by side or co-located nearby at the same site has also gained importance.^4–6 Bivalve hatcheries, however, have been constantly threatened by bacterial pathogens that caused high economic losses to the industry.^7,8 The agents that cause diseases in marine bivalves include viruses, bacteria, fungi, protozoans, helminths and parasitic crustaceans.^8,9 Vibrio species, however, are one of the major bacterial groups identified as agents that caused the outbreaks^8–15 (Table 1). Particularly in hatcheries, Vibrio infections have occurred during almost all phases of the bivalve life cycles.¹⁵ For example, Vibrio aestuarianus caused 30% mortality among Pacific oyster (Crassostrea gigas) mortality on an oyster farm in France¹⁴ as well as V. splendidus, V. brasiliensis, V. harveyi and V. alginolyticus causing nearly 37% mortality rate in adult Pacific oysters (C. gigas) in experimental challenge trials in China.¹² Vibrio strains were linked to mortality in oyster hatcheries in Tasmania (Australia) as well.¹⁶ Moreover, these pathogenic bacteria have become resistant to antibiotics that are used to control microbial diseases of bivalves in aquaculture. Antibiotic resistant Vibrio species frequently are isolated from oysters. Examples include research of Kang et al.,¹⁷ where 100% V. alginolyticus isolated (n = 15) from shellfish farming areas in South Korea showed resistance to ampicillin, vancomycin and erythromycin. In their review, Albini et al.¹⁸ highlighted the existence of antibiotic-resistant bacteria in marine oysters referring to data collected from studies all over the world (Asia, n = 54; Europe, n = 27; America, n = 16; Africa, n = 2; Oceania, n = 1; and intercontinental and multicentre, n = 3).

The major potential sources of Vibrio infections in oyster hatcheries are broodstock (parent) oysters, microalgae used as food for oyster larvae (which is usually cultured on-site), and the seawater used within the hatchery for larval rearing despite prior treatment by filtration or often bactericidal UV exposure.¹⁹ Lewis et al.²⁰ indicated that high bacterial concentration (2.0 × 10⁶ colony-forming units mL^–1) in microalgae fed to oyster larvae could cause disease. Accordingly, considerable effort had to be directed toward reducing or eliminating bacterial contamination of microalgae cultures before they are used as a larval food source.

Bacteriophage treatments

Bacteriophages have been used as biocontrol agents to eliminate pathogenic bacteria in both medical and environmental settings. Their use has been shown to be an environmentally friendly solution to control pathogenic Vibrio species including aquaculture settings. Examples of such uses are listed in Table 2.

In a recent study conducted at the University of the Sunshine Coast (UniSC) bacteriophages (Fig. 1) successfully reduced cell numbers of the Vibrio species on oyster larvae as well as on the microalgae (Shellfish Diet 1800 and Pavlova 1800) used to feed these oysters,^25–28 thus significantly reducing pathogenic bacterial numbers both on the product and its feed (Table 3). These phages were isolated using a local Vibrio species (USC-26005) but were also active against V. harveyi (ATCC 14126) and V. alginolyticus (ATCC 17749), as well as against another local Vibrio isolate (USC-26004) from Sunshine Coast, Qld, showing cross infectivity that provided superior power toward elimination of the targeted bacteria.

Fig. 1.

Transmission electron micrographs of the Vibrio bacteriophages isolated against Vibrio spp. (a) Φ-1, (b) Φ-2, (c) Φ-3, (d) Φ-4, (e) Φ-5, (f) Φ-6.²⁶

Conclusions

The increasing prevalence of antibiotic resistance of bacteria, and the problems of antibiotic residues ending up in pristine environments where aquaculture farming happen indicate the need for alternative approaches to control bacterial diseases of bivalves. Bacteriophage therapy may be a viable alternative for controlling bacterial pathogens associated with hatcheries or farms for both bivalves and their algal feeds.^25–28 However, to date, only a few studies on actionable phage-based therapies have been reported in the literature that targeted phage application in laboratories, and even fewer have been successfully translated from laboratory settings to farm environments. The feasibility of the phage therapy approach on an industrial scale has yet to be established and is not widely approved for bacterial treatment due to unknowns related to the behaviour and safety of bacteriophages in natural environments.^25–28

Bivalves are filter-feeding aquatic species, and thus considered a bacterial reservoir, and high levels of bacteria can reach production sites, resulting in contamination of the surrounding water.¹⁵ The UniSC study clearly indicates that phages are effective prevention agents against bacteria and might be easily applied at farm sites by routine applications.^25–28

The effectiveness of the phage therapy approach to control pathogenic agents depends on different factors such as phage/bacteria ratio, environmental conditions (temperature, pH, UV, salinity), neutralisation of their antibacterial activity due to antibodies, dose and phage administration and bacterial resistance to phages. Thus, before initiating commercial level application, trials in hatcheries must be conducted to determine the safety of the phage approach. Risks of phage-mediated gene transfer and the development of phage-resistant bacteria also need to be investigated in ‘real-life’ situations.^25–28