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BACTERIOPHAGE THERAPY IN AQUACULTURE – FRIEND OR FOE?
LISA ELLIOTT
AusPhage Pty Ltd
Australia
Introduction
Aquaculture is currently one of the fastest growing food producing industries in the world with an average growth rate
of 6.2% (2000-2012)(FAO, 2014). In 2000, farmed food fish contributed 25.7% to global total fish production,
increasing to 42.2% in 2012 with a total worth in excess of US$144 billion (FAO, 2014). The aquaculture and fisheries
sectors also provide significant nutritional requirements to people in developed and developing countries and a source
of income and livelihood to approximately 58.3 million people, equivalent to 10-12% of the world population (FAO,
2014). With a dramatic increase in population growth and an ever-increasing demand for seafood, aquaculture is an
increasingly important source of sustainable food production (WHO, 1999). The need for sustainable aquaculture has
lead to an increase in research and development across a range of areas such as nutrition, environmental impacts, good
management procedures and disease control, and consequently contributed to great improvements in these areas.
However the greatest threats to sustainable aquaculture are biological (infectious disease) and chemical (agrochemicals,
chemotherapeutants, contaminants, and organic pollutants)(OIE, 2011).
The growth of the aquaculture sector is extensively impeded by infectious disease, the effects of which have a
significant impact on the economy and health of many countries and people of the world (FAO, 2005). A report by
FAO (Food and Agriculture Organization of the United Nations) states, “A multitude of factors has contributed to the
health problems currently faced by aquaculture. Over the past three decades, aquaculture has expanded, intensified, and
diversified, based heavily on movements of animals and animal products such as broodstock, seed, and feed. Such
movements are now clearly recognized as having played a pivotal role in the introduction and spread of pathogens and
disease into aquaculture systems” (FAO, 2005). The report also identifies the impact of disease and associated
production losses on the livelihoods of communities by loss of employment and income and a subsequent decrease in
food availability.
Disease outbreaks (parasitic, viral and bacterial) in the past have resulted in losses amounting to billions of dollars to
the aquaculture industry (Leung et al., 2013; FAO, 2014). Advances in the understanding of how many of these
pathogenic organisms cause disease and implementation of ‘good management practices’ within the industry, have
helped in the control of many pathogens. Research and development of vaccines has significantly aided in the control of
many viral and bacterial pathogens and this has been extensively reviewed (Gudding and Van Muiswinkel, 2013).
Vaccines developed from inactivated bacterial pathogens have been generally successful in the control of bacterial
diseases caused by Vibrio spp., Aeromonas spp., Yersinia spp., Pasteurella spp., Edwardsiella spp., and
Flavobacterium spp., (Holvold et al., 2014). Yet, vaccines to control viral disease are vastly fewer and no vaccines have
been developed for parasites (Holvold et al., 2014). To date there have been no commercial vaccines developed for
invertebrates, however a review on this subject has recently been published with some promising results (Rowley and
Pope, 2012).
Despite advancements in good management practices and vaccine production, bacterial infections still pose major
problems in both hatcheries and grow-out, often resulting in mass mortalities (70-90%). These mortalities are typically
associated with pathogenic Vibrio spp., (Noriega-Orozoo et al., 2007; Chatterjee and Haldar, 2012; Alagappan et al.,
2013; Tran et al., 2013) Aeromonas spp., (Nielsen et al., 2001; Shayo et al., 2012) Pseudomonas spp., (Kumaran et al.,
2010; Shayo et al., 2012) and Streptococcus spp., (Bromage et al., 1999; Lahav et al., 2004; Agnew and Barnes, 2007;
Park et al., 2009; Haenen et al., 2013), all of which have global significance and an increasing number of which are
multi-drug resistant (MDR).
A variety of antimicrobial and chemical treatments have been used to control and treat bacterial disease in humans,
animals and production systems. However, the World Health Organisation report on global surveillance of
antimicrobial resistance states, “Existing antimicrobials are losing their effect. At the same time there is a decline in the
development of new antimicrobials. Similarly, there is insufficient new research into new diagnostics to detect resistant
microorganisms, and vaccines for preventing and controlling infections. If this trend continues, the arsenals of tools to
combat resistant microorganisms will soon be depleted.” (WHO, 2014).
It has been estimated that approximately 23,000 people die annually in the USA as a direct result of infections due to
MDR bacteria and many more die due to complications associated with these infections (Frieden, 2013). The estimated
total health care cost related to these infections is as much as US$26 billion annually (Congress, 2011). These figures

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do not take into consideration the economic impacts of complications associated with MDR infections such as lost
workdays and productivity, or human suffering and the affects on livelihoods. In addition to this, production losses and
suffering due to emergent MDR bacteria in livestock (including aquaculture) has a devastating affect on the economy of
developing countries and animal health and welfare. These problems are of great concern to organisations such as OIE
(World Organisation for Animal Health), WHO (World Health Organisation) and FAO all of which have expressed that
there is an urgent need for the development of sustainable alternative approaches to the control of MDR bacteria.
With the rise in antimicrobial resistance and chemical residues in food, and the tightening of regulations surrounding
the use of chemotherapeutics, bacteriophage may provide a natural, sustainable solution to successfully address this
need.
Bacteriophage
Since their discovery almost 100 years ago, bacteriophages have been investigated extensively and a plethora of
literature reviewing these studies exists (Ellis and Delbruck, 1938; Carlton, 1999; Sulakvelidze et al., 2001; Skurnik and
Strauch, 2006; Loc-Carrillo and Abedon, 2011; Abedon, 2011; Ormala and Jalasvuori, 2013). Bacteriophages
(commonly called phage) are a group of naturally occurring antibacterial agents (viral in nature) that infect only
bacterial cells. It is estimated that in excess of 1030
phage exist, making them the most abundant entities on earth. In all
known environments, phages exist as part of a complex microbial ecosystem. They can be part of a free-living
environment such as soils, vegetation and oceans, or as part of a microbial environment within a macro organism such
as an animal system. Phages play a crucial role in the regulation of nutrient cycling, as sources of diagnostic and genetic
tools and as novel therapeutic agents. To date, phages have been used in a number of areas of biotechnology and
medical science including rapid bacterial detection and diagnosis of disease (phage typing), prevention of bacterial
disease (phage vaccine), treatment (phage therapy) and biocontrol (Haq et al., 2012).
Bacteriophages are highly specific and can only infect bacterial cells that present cell surface receptors matching those
of the phage (similar to a lock and key mechanism)(Lindberg, 1973; Kutter and Sulakvelidze, 2005). Without the
matching receptors, phages are unable to multiply and can quickly be degraded in the environment. Phages can either
multiply via the lytic cycle (virulent phage) or lysogenic cycle (temperate phage). While virulent phages kill the cells
they infect (lytic cycle), temperate phages can establish a persistent infection of the cell without killing it (lysogenic
cycle).
Virulent phages are effective at controlling bacterial populations with no known side effects to human, animal or plant.
The method by which virulent phages kill their specific host bacterium is called ‘lysis’ (Kutter and Sulakvelidze, 2005;
Sulakvelidze, 2011). Virulent phages attach to receptors on the surface of bacteria, insert their genetic material through
the bacterial membrane and take over the bacterium’s transcription and translation machinery to synthesize many new
phages. Finally, the bacterial cell wall is destroyed (lysed), releasing large numbers of newly assembled phage to the
environment, where they can attack new bacteria (Figure 1). This entire process can take as little as 25 minutes (Gill
and Hyman, 2010). Virulent phages have been intensely investigated for their bactericidal properties and are
particularly suitable for applications that require destruction of the host bacterium such as biological control and phage
therapy, making them an attractive treatment alternative to antibiotics.
Conversely, phages that replicate without immediately killing their host bacteria are termed temperate phage. These
phages can either multiply via the lytic cycle (cell death) or enter a dormant state in the cell (lysogeny)(Table 2). In
most cases the phage DNA integrates into the host chromosome (termed prophage) and is replicated along with the host
bacterium, being passed on to the daughter cells (Figure 2)(Kutter and Sulakvelidze, 2005). The host bacterium
continues to replicate without adverse affects to the host until host conditions become unfavourable. At this point the
lytic cycle is initiated and the host cell is destroyed, releasing progeny phage. A bacterial cell that harbors a prophage
may occasionally carry genes that are expressed in the cell and present new properties to the bacteria such as host
virulence (toxin production), increased pathogenicity or antimicrobial resistance (Skurnik and Strauch, 2006; Kutter et
al., 2010; Gill and Hyman, 2010). Temperate phages have various applications and are particularly suited to purposes
that require the transport or expression of genes such as phage display, phage typing and phage vaccines (Haq et al.,
2012). Due to the largely non-lytic nature of temperate phage and their ability to exchange genes, these phages are not
good candidates for therapeutic applications that require immediate destruction of the host cell, such as in the treatment
or control of disease (Haq et al., 2012).

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Figure 1. Schematic representation of the lytic cycle of a virulent bacteriophage. (1) Attachment (host specific) – Penetration
and Insertion - Phage attaches to a specific host bacterium and inserts its genetic material, (2) Bacterial chromosome is
degraded, (3) Transcription - disrupting the bacterial genome and killing the bacterium, (4) Replication - taking over the
bacterial DNA and protein synthesis machinery to make phage components. (5) Assembly - the assembly of new phage, and
(6) Lysis - the lysis of the bacterial cell wall to release hundreds of new copies of phage.
Figure 2. Schematic representation of the lytic and lysogenic cycles of a temperate bacteriophage. (1) Attachment (host
specific) - Penetration and Insertion - Phage attaches to a specific host bacterium and inserts its genetic material, (2) The
phage DNA integrates into the host chromosome. This integrated state of phage DNA is termed prophage, (3) Lysogenic
bacterium replicate normally and phage DNA is replicated along with the host chromosome and passed on to the daughter
cells, (4) In the event that host conditions become unfavorable, the lytic cycle is initiated and the host cell is destroyed.

4. elisabethelliott53@gmail.com
Bacteriophage Therapy
Phage therapy can be largely described as the use of bacteriophages to control specific pathogenic or problematic
bacteria. In human and animal health sectors, phage therapy has been practiced in regions of Eastern Europe with
proven success for more than 60 years (Kutter et al., 2010). Early phage trials often produced unreliable and
inconsistent results due to a poor understanding of phage biology and quality control during the preparation of phage
therapeutic formulations. As phage therapy was gaining momentum in 1930-1940, antibiotics were discovered and
western countries all but forgot about phage. Due to the isolation of many Eastern European countries from the
advancements in antibiotic production during this time, a number of countries continued to develop and perfect phage
treatments (Chanishvili, 2012). Today phage therapy is a widespread form of treatment in a number of Eastern
European countries such as Russia, Poland and Georgia (Chanishvili, 2012).
Due to the specificity of phages, virulent phage can be considered a natural and effective way to target difficult or
problem bacteria, without affecting normal beneficial bacteria and without negatively affecting the environment.
Importantly, phages are able to infect bacteria regardless of their susceptibility to antibiotics and are capable of
penetrating biofilms (Sulakvelidze et al., 2001; Kutter et al., 2010). As described above, virulent phages kill their
bacterial hosts and liberate large numbers of progeny, which are able to infect neighboring susceptible bacteria and start
the cycle again. This replication continues until the phage can no longer find the specific targeted bacterial cells,
significantly reducing bacterial biomass. It is for this reason that virulent only phages are used in phage therapies.
The use of bacteriophage preparations has advantages and challenges, the critical points being high bacterial specificity,
transference of virulence or toxin genes, appropriate administration of phage preparations and the development of phage
resistant bacteria (Table 1).
Table 1: Comparison of advantages and disadvantages of bacteriophage in phage therapy.
Advantages Comments Challenges Comments
High specificity. Minimal disruption of normal beneficial
microflora.
Do not contribute to resistance in the
beneficial microflora such as seen with
antibiotics.
High specificity. The disease causing bacterium must be
positively identified before phage therapy
can be successfully initiated. However
phage can be successfully used in
combination with other antimicrobials.
Virulent phages are
bactericidal agents.
The target bacteria are killed and are
unable to develop resistance to phage or
other antimicrobials.
Temperate phages
and can transfer
genes between
bacteria.
Phages have two life cycles, virulent
(lytic) and temperate (lysogenic). For
phage therapies only obligately virulent
phages are used that do not possess toxin
or antibiotic resistance genes or virulent
factors. They kill the host bacteria.
Low inherent
toxicity and low
environmental
impact.
Phages are protein-encapsulated nucleic
acids thus are inherently nontoxic to
plant, animal or environment.
May interact with
the immune
response.
There is little evidence of detrimental
immune responses from phage
themselves. However it is crucial that
protocols are developed resulting in
highly purified preparations to avoid
contamination with bacterial components.
Administration of
phages can be oral,
aerosols,
immersion,
injection, in feed or
topically.
Phage preparations can be made into
tablets, liquid or powder and can be
viable for many years in some
preparations.
Diseased animals
may not feed.
Injections of large
numbers of animals
(e.g. fish) may be
problematic.
Phage released into the water from
uneaten treated feed can also act as an
immersion treatment. Advancements in
vaccine delivery technologies offer
relevant methods for vaccination of large
numbers of animals.
Selecting new
phages is a
relatively rapid and
cost effective
process.
Evolutionary arguments support the idea
that virulent phages can be selected
against every antibiotic-resistant or phage
resistant bacterium by the ever-ongoing
process of natural selection.
Strictly virulent
phages only must be
selected and
purified.
Advances in molecular biology and phage
biology have reduced the time and cost to
select for virulent phage.
Replicate at the site
of infection, ‘auto
dosing’.
The exponential growth of phages at the
site of infection may require less frequent
phage administration in order to achieve
the optimal therapeutic effect.
.
Bacteria that have
become resistant to
one phage continue
to be susceptible to
other phages.
Selecting new phages is a relatively rapid
and cost effective process. The
development of phage cocktails
significantly reduces the appearance of
phage resistant bacteria.

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Phages are active
against antibiotic
resistant bacteria.
Phages do not contribute to antibiotic
resistance and possess different receptors
to antibiotics.
With advances in technologies and better understanding of bacteriophage biology, these challenges can all be
addressed. For example, virulent phages are very good candidates for phage therapy, whereas temperate phages are not.
With rigorous testing, favorable virulent phages can be selected and safe phage preparations for therapeutic use can be
developed. It is crucial that the perceived challenges associated with phage therapy do not impede the advancement of
this technology in the Western world.
The use of phage products in the food industry, human medicine, agriculture and aquaculture has gathered momentum
recently. A range of products have been approved by the FDA (Food and Drug Administration), US Environmental
Protection Agency (EPA) and FSANZ (Food Standards Australia and New Zealand) for the control of Listeria
monocytogenes, Salmonella, pathogenic E. coli and Pseudomonas putida. This is primarily due to the increase in MDR
bacteria, antimicrobial and chemical residues in food and the environment, and the decline in research to develop new
antibiotics. An increased understanding of phage biology, a long history as therapeutics and an urgent need, as defined
by the above agencies, to find alternatives to overcome antibiotic resistance in traditional medicine have also aided in
the acceptance of bacteriophage products in the human food chain. This is evidence of the gathering acceptance of
phage as alternative antibacterial treatments.
Bacteriophage Therapy in Aquaculture
The use of bacteriophage in aquaculture has recently been reviewed with a recommendation for further research in this
area due to its positive therapeutic potential (Oliveira et al., 2012). The therapeutic potential for the use of phage in the
control of bacterial disease in aquaculture has been reported for finfish (Park et al., 2000; Nakai and Park, 2002; Park
and Nakai, 2003)) and prawns (Vinod et al., 2006; Karunasagar et al., 2007) with promising results. However the vast
majority of publications focus on isolating and characterizing phage capable of reducing the biomass of bacterial
pathogens associated with aquaculture species in vitro. There is an urgent need for studies to be undertaken in vivo to
fully prove the advantages of using phage therapy as a control measure for antibiotic resistance organisms in particular.
For nearly a decade, we have played a major role in the development and testing of phage preparations in aquaculture
systems and animals with outstanding results. In 2008, a project was undertaken in collaboration with James Cook
University, Australia (Elliott and Owens, 2008) to develop a phage preparation able to reduce the biomass of
pathogenic V. harveyi. A total of 17 virulent phages were isolated, purified and tested against greater than 100 different
Vibrio strains, with a combined phage susceptibility of 77% of bacterial strains. Several phage cocktails were prepared
and toxicology trials were undertaken using Penaeus monodon postlarvae (PL5) under controlled laboratory conditions.
The trial was conducted over 5 days with no significant unfavorable affects on survival reported.
In response to a disease outbreak in 2010 caused by Streptococcus agalactiae in Central America, eight virulent phages
were isolated and purified with 80% of the S. agalactiae strains susceptible to the phage preparation (Elliott, 2010).
More recently (2013), as part of a preceding project conducted by James Cook University Townsville, we report on the
successful use of bacteriophage therapy in the control of pathogenic Aeromonas hydrophila in a Redclaw crayfish
hatchery (Elliott and Valverde, 2013).
Redclaw crayfish (Cherax quadricarinatus) farming in Australia has typically been classified as ‘extensive’ rather than
‘intensive’ farming and the industry has not grown over the past several years. In recent years an Australian farmer
adapted an egg incubation process from Europe and developed a Redclaw hatchery process that has enabled a high level
of control over the life cycle of C. quadricarinatus. This has opened many new possibilities and is dramatically
changing the face of the Redclaw industry in Australia. Grow-out farmers are now able to stock selectively breed
animals with exceptional health status of exact quantities of the same life stage. The results of this have shown an
increase in grow-out production of up to 30% (per com) and, as the industry gains experience in using “craylings” from
the hatchery, expectations are that production will continue to increase.
Whilst major progress in hatchery techniques has been achieved, sporadic bacterial infections suspected as Aeromonas
hydrophila, resulted in mortalities in the order of 80-90% in Stage 2 larvae (S2L). With a substantial increase in
demand for hatchery reared "craylings" disease management in the hatchery has become crucial. To address this
problem and provide a natural antibiotic alternative, a bacteriophage therapy was developed and trialed in vivo over a
full hatchery season (approximately 4 months).

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Samples were obtained from various locations within the hatchery and the pathogen was confirmed as A. hydrophila.
Several phages were isolated, purified and tested against the pathogen using techniques suitable for the selection of
virulent phage. A bacteriophage preparation was prepared and used to treat the incubators, twice daily over the course
of the hatchery season.
Within 1 week a dramatic decrease in mortalities from greater than 80% to less than 5% of S2L was noted. In addition
to this, the biofilm growing on the internal surfaces of the incubators making cleaning difficult, was considerably
reduced making the system more efficient. Continued treatment over the course of the hatchery season resulted in a
constant survival rate of >80% and the pathogen remained under control. The hatchery is now able to produce at full
capacity without bacterial impediments.
The use of a bacteriophage preparation offered an effective antibiotic alternative treatment in a commercial setting
resulting in a survival improvement of >80%. With much research yet to be undertaken by the Redclaw industry in a
variety of areas including disease, nutrition and genetics to name a few, the ability to control specific pathogens so these
research areas can be successfully undertaken is fundamental. The ability to control disease in the hatchery and thus
increase the numbers of healthy "craylings" available to the industry will enable the industry to grow beyond the current
‘extensive’ farming practices
This project was in response to an emergency disease outbreak and therefore, full on-farm scientific rigor was not
applied i.e. no replication or control groups. The bacteriophages used in this study were isolated, purified, screened for
virulent phage only, and all phage not meeting the selection criteria appropriate for phage therapy as described above
were excluded. The preliminary results achieved from this short trial has facilitated government funding and a full
research project aimed at fully investigating the use of phage therapy in the control of bacterial infections in Redclaw
hatcheries is due to begin in 2014.
Conclusions
There is no doubt that sustainable aquaculture production is crucial to the future demands for seafood globally.
However, one of the biggest threats to the aquaculture industry is infectious disease. Whilst fish vaccinology has shown
remarkable developments in recent years, and major improvements have been made in good management practices, the
emergence of antimicrobial resistant bacteria has become a global problem. The consequences associated with these
infections are widespread and have a significant impact on the economy, livelihood, health and welfare (human and
animal) of entire communities and countries. The World Health Organization (2014) states, “Increasingly, governments
around the world are beginning to pay attention to a problem so serious that it threatens the achievements of modern
medicine. A post-antibiotic era—in which common infections and minor injuries can kill – far from being an
apocalyptic fantasy, is instead a very real possibility for the 21st
century.”
Bacteriophage have been investigated, tested and extensively used in human and animal medicine for more than 60
years in Europe. Although early studies were often inconclusive, modern technology, methods and a greater
understanding of phage and pathogen biology have provided an excellent basis for development of improved
preparations, overcoming many of the perceived disadvantages of phage therapy. Virulent phages are natural,
sustainable antimicrobials that are nontoxic and, when correctly selected and prepared, do not pose any risk to plant,
animal or the environment. Future research and development of bacteriophage preparations as therapies will contribute
to environmental, social and economical sustainability in global aquaculture and should be fully embraced and
supported by government, researchers and farmers.

7. elisabethelliott53@gmail.com
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