Abstract
Bacteriophage is considered an alternative to antibiotics and
environmentally friendly approach to tackle antimicrobial resistance
(AMR) in aquaculture. Here, we reported isolation, morphology and
genomic characterizations of a newly isolated lytic bacteriophage,
designated pAh6.2TG. Host range and stability of pAh6.2TG in different
environmental conditions, and protective efficacy against a pathogenic
multidrug-resistant (MDR) Aeromonas hydrophila in Nile tilapia
were subsequently evaluated. The results showed that pAh6.2TG is a
member of the family Myoviridae which has genome size of 51,780
bp, encoding 65 putative open reading frames (ORFs), and is most closely
related to Aeromonas phage PVN02 (99.33% nucleotide identity).
The pAh6.2TG was highly specific to A. hydrophila and infected
83.3% tested strains of MDR A. hydrophila (10 out of 12) with
relative stability at pH 7 9, temperature 0 40 °C
and salinity 0 40 ppt. In experimental challenge, pAh6.2TG treatments
significantly improved survivability of Nile tilapia exposed to a lethal
dose of the pathogenic MDR A. hydrophila , with relative percent
survival (RPS) of 73.3% and 50% for phage multiplicity of infection
(MOI) 1.0 and 0.1, respectively. Significant reduction of bacterial
counts in rearing water at 3 h (6.7 ± 0.5 to 18.1 ± 6.98 folds) and in
fish liver at 48 h post-treatment (2.7 ± 0.24 to 34.08 ± 26.4 folds) was
observed in phage treatment groups while opposite pattern for bacterial
counts was observed in untreated control. Interestingly, the surviving
fish provoked specific antibody (IgM) against the challenged A.
hydrophila . These results might explain the higher survival in phage
treatment groups. In summary, the findings suggested that the lytic
bacteriophage pAh6.2TG is an effective alternative to antibiotics to
control MDR A. hydrophila in tilapia and possibly other
freshwater fish.
Keywords: Aeromonas hydrophila , alternative to
antibiotics, antimicrobial resistance, aquaculture, bacteriophage,
multidrug resistance
INTRODUCTION
The farming of carps, tilapias, and catfishes accounts for 35.84% of
world aquaculture production with revenue of 83 billion dollars in 2018.
They contribute not only great economic value but also food and global
nutrition security (FAO, 2020; Naylor et al., 2021). One of the
challenges for sustainable aquaculture is production loss due to
infectious diseases (Stentiford et al., 2020; Stentiford et al., 2017).Aeromonas hydrophila infection is considered one of the most
important bacterial diseases responsible for the loss of millions of
dollars in the global freshwater aquaculture industry (da Silva et al.,
2012; Hossain et al., 2014; Peterman & Posadas, 2019; Pridgeon &
Klesius, 2012). The control of this disease still heavily relies on
antibiotics, especially in low-middle income countries (LMICs).
Consequently, a global issue of concern of multidrug-resistant (MDR)A. hydrophila is becoming increasingly ubiquitous (Guz &
Kozinska, 2004; Patil et al., 2016; Stratev & Odeyemi, 2016).
Non-antibiotic approaches can minimize the requirement for
antimicrobials to tackle infectious diseases in both animals and human
health (Hoelzer et al., 2018). In the battle to combat A.
hydrophila infection in aquaculture system, bacteriophage is one of the
environmentally friendly approaches which replace or complement
chemotherapy to reduce the hazard of bacterial disease and antimicrobial
resistance in aquatic animals.
Lytic bacteriophages (also called phages) are unique viruses that can
infect and kill bacterial cells (Kutateladze & Adamia, 2010). Phage
therapy is a viable option to control bacterial infections due to their
unique advantages, including high host specificity, rapid
self-proliferation, and low intrinsic toxicity (Cao et al., 2021). For
instance, Luo et al. (2018) injected phage HN48 with multiplicity of
infection (MOI) = 1 (MOI represents the ratio of the numbers of virus
particles to the numbers of the host cells) against Streptococcus
agalactiae infection in Nile tilapia (Oreochromis niloticus )
with relative percent survival (RPS) of 60%. Feeding phage cocktails of
PVHp5 and PVHp8 showed protective effectiveness in turbot
(Scophthalmus maximus ) against Vibrio harveyi infection
with RPS from 38.6 to 79.5% (Cui et al., 2021). In addition,
intraperitoneal injection of phages FpV4 and FPSV-D22 showed protection
of rainbow trout (Oncorhynchus mykiss ) to Flavobacterium
psychrophilum with RPS of 53.8%, while feed-based and bath
administrations were not effective (Donati et al., 2021). Previous
studies have demonstrated that phages can be applied in aquaculture to
combat A. hydrophila infection (Anand et al., 2016; Cao et al.,
2020; Dang et al., 2021; Jun et al., 2013; Le et al., 2018). Hence,
strategy using phages for biocontrol of A. hydrophila has become
increasingly attractive. The earlier studies have analyzed phenotypic
and genotypic characterization, and evaluated protective effect of
phages against A. hydrophila , including Myoviridae pAh1-C
and pAh6-C (Jun et al., 2013); Podoviridae Ahp1 (Wang et al.,
2016); Myoviridae pAh-1 (Easwaran et al., 2017);Myoviridae CT45P and TG25P (Hoang et al., 2019);Podoviridae MJG (Cao et al., 2020), Myoviridae AHP-1
(Chandrarathna et al., 2020); Siphoviridae Akh-2 (Akmal et al.,
2020), Podoviridae LAh1-LAh6, Siphoviridae LAh7, andMyoviridae LAh10 (Kabwe et al., 2020); Myoviridae PVN-02
(Tu et al., 2020); Myoviridae AhyVDH1 (Cheng et al., 2021). In
this study, we isolated and characterized specific an A.
hydrophila phage from water sources in Mekong Delta, Vietnam.
Subsequently, we evaluated its protective effects for juvenile Nile
tilapia challenged with a pathogenic MDR A. hydrophila .
MATERIALS AND METHODS
Bacterial isolates
All bacterial strains used in this study are listed in Table 1. The
isolates of Aeromonas , Streptococcus , andEdwardsiella were cultured in Tryptic Soy Broth (TSB; Becton
Dickerson, USA) at 28 °C while Lactobacillusisolates were cultured in De man, Rogosa, and Sharpe (MRS, HiMedia,
India) broth at 37 oC. All laboratory isolates ofAeromonas were previously isolated from diseased fish using
selective medium, Rimler-Shotts agar (RS, HiMedia, India) supplemented
with Novobiocin (Oxoid, UK), identified by PCR and sequencing ofgyr B housekeeping gene (Navarro & Martínez‐Murcia, 2018).
Multidrug-resistant strains of A. hydrophila (Table S1) were
identified based on the method proposed by Magiorakos et al. (2012).
Phage isolation and morphology
Preparation of host strain
The MDR A. hydrophila BT09 (Tables 1 and S1) was chosen as a
bacterial host for phage isolation. Prior to phage isolation, prophage
induction using Mitomycin C (Sigma-Aldrich, USA) was carried out as
described by Walker et al. (2009) to ensure that the host cells do not
contain prophage. Briefly, 100 μL of bacterial cells suspended in normal
saline solution (OD600 = 0.6) was added into each of 10
mL of TSB supplemented with 250, 500, and 1,000 ng/mL of Mitomycin C.
All cultures were incubated at 28 °C for 8 h. The induced phage
production using Mitomycin C was evaluated by the Plaque Drop Assay
(Adams, 1959).
Phage isolation
Water samples were collected from striped catfish culture ponds in Tien
Giang Province, Vietnam. The samples were enriched to increase phage
concentration according to Van Twest and Kropinski (2009) and isolated
by Plaque Assay method described by Jun et al. (2013). Briefly, the
samples were centrifuged at 4,500 x g , 4 oC for
30 min, and the supernatant was filtered through a 0.2 μm filter (Merck
Millipore, USA) to remove residual bacteria cells. Then, 10 mL filtrate
was mixed with 10 mL of A. hydrophila BT09 in TSB supplemented
with 1.0 mM CaCl2 and 0.5 mM MgSO4(MTSB). The mixture was cultured at 28 °C for 24 h with 50 rpm shaking.
The mixture was then centrifuged at 10,000 x g , 4oC for 15 min, and the collected supernatant was
serially diluted (10-1 to 10-4). A
volume of 100 μL of each dilution was transferred to a tube containing
3.0 mL of TSA 0.5% agar supplemented with 1.0 mM CaCl2and 0.5 mM MgSO4 (MTSA), together with 100 μL ofA. hydrophila . The mixture was vortexed lightly and poured onto a
plate of TSA 1.5% agar. The plates were incubated at 28oC for 16 h and the growth of phages was observed
(clear plaque on the plate). The individual clear plaque was picked and
aseptically transferred to 200 μL of SM buffer (100 mM NaCl, 10 mM
MgSO4, 50 mM Tris-HCl, pH 7.5). The mixture was vortexed
vigorously and kept in 4 oC refrigerator overnight.
The phages in SM buffer were obtained by filtering the supernatant
through a 0.2 μm filter after centrifugation at 10,000 x g for 10
min. The filtrate was propagated four times continuously using the same
protocol mentioned above for purification of the obtained phages. The
isolated phages were stored in SM buffer supplemented with 30% glycerol
at -80 oC until used.
Examination of phage morphology
The structure and size of the phage were determined by Transmission
Electron Microscope (TEM). The specific procedure was as follows; the
phage solution (3 mL) was centrifuged twice at 200,000 x g for 90
min. The pellets were resuspended in sterile distilled water. A volume
of 50 μL of 1% glutaraldehyde (g/vol) was then added to immobilize the
sample and rinsed with 0.1 M of cacodylate before proceeding with the
dye. The samples were coated with 0.1% Poly-Lysine solution onto the
surface of the 200-mesh carbon-coated grids to increase the adhesion of
phages on the mesh. A volume of 10 μL of the phages was added to the
grid and allowed to dry naturally for 5 min. The samples were dyed with
1% uranyl acetate sterilized with a 0.2 μm filter. The samples were
washed with distilled water, allowed to dry for 5 min and imaged with a
TEM-JEOL 1010 (Japan) with light projected through the grid for about 5
s at 80 kV. Phage morphology was classified according to the guideline
of International Committee on Taxonomy of Viruses (ICTV) and Ackermann
(2007).