To the best of our knowledge, this is the first report of phage therapy in a mouse model against a multiple-antibiotic–resistant V. parahaemolyticus pandemic strain infection.

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Aeromonas spp. are opportunistic pathogenic bacteria associated with a multitude of diseases in ornamental fish. In this study, virulence properties and antibiotic resistance patterns of 43 Aeromonas strains isolated from 46 zebrafish were investigated. The isolates were identified as Aeromonas veronii biovar veronii (n = 26), A. veronii biovar sobria (n = 3), Aeromonas hydrophila (n = 8), A. caviae (n = 3), Aeromonas enteropelogenes (n = 2) and Aeromonas dhakensis (n = 1) by gyrB gene sequencing. The sequence divergence within and between the species ranged from 0-5·80% and 4·90-8·00%. Each species formed a distinct group in a neighbour-joining phylogenetic tree. The lipase production, biofilm formation, DNase activity, gelatinase production, caseinase production and β-hemolysis were phenotypically observed in 34 (79·07%), 33 (74·74%), 30 (69·77%), 25 (58·14%), 22 (51·18%) and 21 (48·84%) isolates. The virulence genes were detected by polymerase chain reaction (PCR) in following frequencies- aer (86·05%), hlyA (83·72%), gcaT (83·72%), lip (72·09%), act (67·44%), fla (65·12%), ascV (58·14%), ast (55·81%), ser (41·86%), ahyB (39·53%) and alt (25·58%). Every isolate was resistant to at least four antibiotics in disk diffusion test. The multiple antibiotic resistance (MAR) index values ranged from 0·22-0·50 among the isolates. Our study suggests that zebrafish can be a potential reservoir of virulent and multi-drug resistant Aeromonas spp. SIGNIFICANCE AND IMPACT OF THE STUDY: Aeromonas spp. are Gram-negative and facultative anaerobic bacteria which are ubiquitous in aquatic environments. Virulence properties and antibiotic resistance of ornamental fish-borne Aeromonas spp. are poorly understood. The virulence factors as well as multiple antibiotic resistance profiles of zebrafish-borne Aeromonas spp. were characterized for the first time in Korea. Most of the isolates were positive for phenotypic virulence traits and harboured several virulence genes revealing the virulence potential of zebrafish-borne Aeromonas spp. Additionally, the high multiple antibiotic resistance (MAR) index values displayed by the isolates highlight the necessity of responsible use of antibiotics in the ornamental fish industry.

Pet turtles are well-known to harbor an array of bacterial pathogens which can cause zoonotic infections in humans as well as opportunistic infections in the turtles itself. Essential oils are the natural plant extracts which have been traditionally used for disease treatment. In the present study, the essential oil of lavender (EOL) was examined for its antibacterial activity against thirty-eight strains of turtle-borne pathogenic bacteria belonging to seven species; Aeromonas hydrophila, A. caviae, A. dhakensis, Citrobacter freundii, Proteus mirabilis, Salmonella enterica and Pseudomonas aeruginosa. Antibacterial activity of EOL was tested by means of disk diffusion, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) tests. In addition, the antimicrobial susceptibility pattern of 11 commonly used antimicrobials was examined and the multiple antibiotic resistance (MAR) index was calculated. The results revealed that EOL was active against all tested turtle-borne pathogenic bacteria except P. aeruginosa. The range of MIC and MBC values of EOL against isolates except P. aeruginosa were recorded as 0.5-1% (V/V) and 0.5-2% (V/V), respectively. The MBC/MIC ratio was detected as <4, revealing that the tested EOL was bactericidal. Besides, most of the isolates were resistant to different antimicrobials in antimicrobial disk diffusion test. MAR index values of the tested strains were ranging from 0.27 to 0.91. The outcomes indicate that EOL has a potential to be used as an antibacterial agent against pathogenic bacteria isolated from pet turtles.

The antibacterial activity of lime (Citrus aurantifolia) essential oil (LEO) and limonene was tested against seven Gram-negative and nine Gram-positive fish pathogenic bacteria isolated from cultured olive flounder, Paralichthys olivaceus (Temminck & Schlegel) in Korea. Limonene was >99% concentrated and LEO consisted of eleven chemical compounds including 56.22% of limonene. Disk diffusion assay, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) tests were done. LEO and limonene inhibited the growth of both Gram-negative and Gram-positive bacteria. LEO and limonene (MBC/MIC= 2-8) were both bactericidal and bacteriostatic for the strains tested. In every fish pathogenic bacteria, the inhibition zone diameter (IZD) increased in proportion to the oil concentration and the maximum effect was found at 100% (V/V) concentrations of LEO and limonene. The antibiogram pattern indicated that all the bacterial strains, excluding three strains of S. iniae (S186, S530, and S131), showed resistance to one or more antibiotics. The percentage of the relative inhibition zone diameter (RIZD %) exhibited high values at higher concentrations of all the agents. Since antibacterial activities of LEO and limonene were considerably effective against fish pathogenic bacteria, they could be used as alternatives to treat bacterial infections in aquaculture.

To develop an alternative bio-control measure for multi-drug resistant pathogenic Aeromonas hydrophila, which causes motile Aeromonas septicemia in fish, novel virulent phage (AHP-1) was isolated from carp tissues. Morphological analysis by transmission electron microscopy revealed that AHP-1 belongs to Myoviridae family. AHP-1 displayed 81% of moderate adsorption by 25 min, and latent period of 40 min with burst size of 97 PFU mL −1 at an optimal multiplicity of infection (MOI) 0.1. AHP-1 was stable over a broad range of pH (4-11), temperature (4-50°C), and salinity (0.1-3.5%). Both time and MOI dependent in vitro A. hydrophila growth inhibition was observed with AHP-1. AHP-1 (10 MOI) showed higher growth inhibition against A. hydrophila than chloramphenicol (5 μg mL −1), and combined treatment was more promising than individuals. Immune gene expression analysis of zebrafish upon continuous bath exposure to AHP-1 resulted significantly higher (il-6 and sod-1) or slight induction (tnf-α, il1-β, il-10, and cxcl-8a) than controls at beginning of the phage exposure, but those lowered to basal level by day 12 post-phage exposure. It suggests no adverse immune responses have occurred for the AHP-1 dose that used, and have potential for the phage therapy. Further detailed in vivo studies are needed to confirm the protective efficacy of newly isolated AHP-1 against A. hydrophila infection.