Phenotypic and Genetic Characterization of Aeromonas Hydrophila


Phage therapy can be an effective alternative to standard antimicrobial chemotherapy for control of Aeromonas hydrophila infections in aquaculture. Aeromonas hydrophila-specific phages AhMtk13a and AhMtk13b were studied for basic biological properties and genome characteristics. Phage AhMtk13a (Myovirus, 163,879 bp genome, 41.21% CG content) was selected based on broad lytic spectrum and physiologic parameters indicating its lytic nature. The therapeutic potential of phage AhMtk13a was evaluated in experimental studies in zebrafish challenged with A. hydrophila GW3-10 via intraperitoneal injection and passive immersion in aquaria water. In experimental series 1 with single introduction of AhMtk13a phage to aquaria water at phage–bacteria ratio 10:1, cumulative mortality 44% and 62% was registered in fish exposed to phage immediately and in 4 h after bacterial challenge, correspondingly, compared to 78% mortality in the group with no added phage. In experimental series 2 with triple application of AhMtk13a phage at ratio 100:1, the mortality comprised 15% in phage-treated group compared to the 55% in the control group. Aeromonas hydrophila GW3-10 was not detectable in aquaria water from day 9 but still present in fish at low concentration. AhMtk13a phage was maintained in fish and water throughout the experiment at the higher concentration in infected fish.

1. Introduction

The aquaculture industry has been expanding significantly worldwide in the past decades. In 2018, total fish production, trade, and consumption reached an all-time record equaling 114.5 million tons in live weight [1]. Aquaculture is one of the fastest growing and developing sectors in South Caucasus region, including Georgia. Rainbow trout is the most common cultured fish species in Georgia, comprising 61% of the total fish production [2].
One of the main challenges for the development of aquaculture worldwide and also in Georgia are bacterial infections that may lead to massive death in fish and, correspondingly, to large financial losses. Efforts to keep farmed fish free of disease are important for both fish welfare and fish farmers. The problem related to occurrence and spread of infections is especially acute for fish hatcheries and larvae producing facilities, and also juvenile fish ponds, where mortality rates usually are significantly higher compared to adult fish. The freshwater fish bacteriosis in the natural environment and in fish farms is mainly caused by a number of Aeromonas species [3]. Aeromonas hydrophila, a Gram-negative, rod-shaped facultatively anaerobic bacterium, is a natural inhabitant of fresh and brackish waters. Aeromonas hydrophila frequently causes disease outbreaks in wild and cultured fish leading to Aeromonas septicemia and ulcerative infections [4]. Recent studies have reported isolation of multidrug resistant (MDR) A. hydrophila from diseased fish and aquatic systems worldwide [5,6].
Various antimicrobials—chemicals, dyes, and antibiotics—have been used in aquaculture for treatment of sick fish and for prevention of bacterial infections. Use of antibiotics to control fish bacteriosis, including Aeromonas infections, is still the most widely used approach that guarantees reduction in morbidity and mortality, and contributes to significant advances in the health of the population. The public health hazards related to use of antimicrobials in aquaculture include development and spread of antimicrobial-resistant (AMR) bacteria and resistance genes, along with the presence of antimicrobial residues in aquaculture products and the environment [7]. The rising drug resistance among aquatic bacteria and adverse effects of antibiotics lead to a common understanding that use of antibiotics for prevention of fish diseases should remain low and should not be a primary treatment option in fish farming practices [8]. The use of antibiotics in aquaculture is under strict control in Europe and fish farmers are advised to use other complementary control strategies [9,10,11]. To follow the principle “prevention is better than treatment”, different types of vaccines, predominantly killed and live attenuated vaccines have been used for prevention of bacterial infections in farmed fish. Application of probiotics and prebiotics is a considerably new approach [12,13]. Prophylactic treatments are mostly confined to the hatchery, the juvenile or larval stages of aquatic animal production, and are thought to be more common in small-scale production units [13].
Biological control of diseases, including phage therapy and prophylaxis, is currently considered as a best approach for aquaculture [14,15]. Application of therapeutic phages in aquatic animals is promising due to several reasons: (i) bacteriophages are viruses that infect only bacteria and are highly specific (mainly species, or even strain specific); (ii) phage reproduction inside the organism (human, animals, etc.) takes place only when host bacteria are present, so they do not accumulate in the organism and in the environment; (iii) phages have ability for fast propagation and high burst size that leads to lysis of bacterial host; (iv) phages are immunomodulators and can promote specific mechanisms of bacterial clearance; (v) high natural abundance of phages in aquatic environment (ranging from 104 to 108 mL−1) makes the phage therapy in fish more tolerable than other approaches; (vi) regulations in aquaculture concerning use of biopreparations are considerably milder than in medicine that simplifies the implementation of phage-based treatments and products in aquaculture. Perhaps one of the earliest phage therapy applications in aquaculture was described in 1980s by Wu et al., when the strongly lytic phage AH1 was used to treat A. hydrophila-infected loaches [16]. Numerous in vitro assays and in vivo studies evaluated the potential of bacteriophages (individual phages and phage mixtures) and phage lytic enzymes to effectively combat fish pathogenic bacteria including multidrug resistant A. hydrophila [17,18,19,20,21,22].
In the last decade there has been a growing interest in Europe toward use of biological preparations, particularly phages, in aquaculture. The same trend in Georgia can be evidenced by the increased inquiry from aquaculture companies to the G. Eliava Institute of Bacteriophages, regarding development of aquaculture-targeted phage products. Most frequently this applies to fish bacterial infections, caused by Aeromonas spp. Currently, no commercial phage preparation is produced for aquaculture needs. Well-designed experimental studies with customized phage preparations are of high demand in order to demonstrate effectiveness and reproducibility of phage-based treatment.
This study aimed at the detailed characterization of two new A. hydrophila specific phages and evaluation of their potential for infection control in aquaculture. In order to assess the antibacterial efficacy of lytic phage AhMtk13a to combat A. hydrophila infection and to reduce the fish mortality, the experimental studies were conducted on the model of zebrafish challenged with A. hydrophila GW3-10, a virulent isolate from the sick trout.

2. Results

2.1. Isolation and Characterization of A. hydrophila Specific Bacteriophages

2.1.1. Isolation of A. hydrophila Specific Bacteriophages

For the isolation of A. hydrophila-specific phages, water samples from the Mtkvari River (in the suburbs of Tbilisi, Georgia) were enriched with A. hydrophila GW3-10. The number of phage particles in the primary phage lysate was amplified on the host strain by use of soft agar overlay method [23]. After propagation of the primary phage lysate (that often can be a natural mixture of phages) on the A. hydrophila GW3-10, two morphologically different phage plaques were observed on the bacterial lawn: (i) predominating larger size (2.5–3 mm diameter) negative colonies with clear centers surrounded by narrow halozon; (ii) the smaller, turbid plaques with diameter 1–1.5 mm. For each of these two Aeromonas phages with different plaques, the pure lines were obtained using five cycles of purification to ensure a single phage-strain population. The phage forming larger negative colonies was given the name vB_AhMtk13a and the phage with small size plaques was named as vB_AhMtk13b.

2.1.2. Phage Virion Morphology

The two Aeromonas phages after propagation to high titers were examined for phage virion morphology by transmission electron microscopy (TEM). The study showed that both phages by morphological characteristics belong to the order Caudovirales (Figure 1a,b). The phage AhMtk13a has the elongated icosahedral head (118 ± 3 nm × 86 ± 3 nm) and contractile tail (123 ± 3 nm × 23 ± 4 nm) and attributed to the Myoviridae morphotype. AhMtk13b phage has symmetrical icosahedral head (68 ± 2 nm × 68 ± 2 nm) and long noncontractile tail (227 ± 4 nm × 11 ± 2 nm), and belongs to the Siphoviridae morphotype.

Read More