Novel lytic bacteriophage AhFM11 as an effective therapy against hypervirulent Aeromonas hydrophila

Abstract

Several farmed fish species, including carps, tilapia, salmon, and catfish, have experienced significant economic losses in aquaculture due to motile Aeromonas septicemia caused by Aeromonas hydrophila. In the present study, a novel lytic bacteriophage infecting hypervirulent Aeromonas hydrophila (vAh) was isolated and characterized. This is the first report of a phage against vAh. Phage AhFM11 demonstrated lytic activity against both vAh strains and the A. hydrophila reference strain ATCC 35654. The AhFM11 genome was sequenced and assembled, comprising 168,243 bp with an average G + C content of 41.5%. The genome did not harbor any antibiotic resistance genes. Genomic information along with transmission electron microscopy revealed that phage AhFM11 belongs to the Straboviridae family. Therapeutic application of monophage AhFM11 in fish showed 100% survival in injection, 95% in immersion and 93% in oral feeding of phage top-coated feed. Fish and chicken meat spiked with A. hydrophila and phage showed significant reduction of A. hydrophila. These findings support that phage AhFM11 can be used as a biocontrol agent against vAh as an alternative to antibiotics.

Keywords: Antibiotic resistance genes and biocontrol; Aquaculture; Bacteriophage therapy; Hypervirulent Aeromonas hydrophila; Lytic phage.

Figure 1

In vitro growth characteristics of phage AhFM11 (A) one-step growth curve showing the latent period and burst size of the phages (B) adsorption efficacy. This experiment was carried out at MOI 0.001. Error bars represent the Mean ± SD (n = 3).

Figure 2

Phage (AhFM11) characteristics on agar plate, soft overlay method and transmission electron microscopy. (A) Spot tests for phage AhFM11 on hypervirulent Aeromonas hydrophila HypAh-20 and the spot 4 (black dotted rectangle) was used for further passage to get the titer value for AhFM11. (B,C) Plaque morphology of AhFM11 on TSA soft agar plates showing 1–3 mm plaque diameter. (D) Transmission electron microscopy image negatively stained with 2% uranyl acetate showing single phage AhFM11 (Bar = 50 nm) with its icosahedral head along with sheathed tail tube and its branched tail fibers.

Figure 3

Circular genome map of phage AhFM11 against the hypervirulent Aeromonas hydrophila. The ORFs are marked with arrows indicating the direction of transcription. The ORFs encoding putative proteins for which functions could be predicted are color-coded and labelled on the figure.

Figure 4

Comparative analysis of four phages was conducted using Easyfig. Coding domain sequence (CDS) are shown as arrows to indicate the direction of transcription and are mentioned in the bottom legend in accordance with their predicted functions. The percentage of sequence similarity is shown as the intensity of the gray to black color.

Figure 5

Phylogenetic tree of (A) MCP gene of phages constructed using the Maximum-likelihood method with a bootstrap value of 1000 replicates. A total of 28 nucleotide sequences were included for the analysis including AhFM11 (1-Present study), A. hydrophila phage (11), A. salmonicida (4) and Enterobacteriaceae phages (12). (B) Phylogenetic tree of whole-genome sequences of phages were aligned using MAFFT v7.3, the tree was constructed using online software by interactive tree of life (iTOL) and the bolded ON42478.1 indicates the phage AhFM11 isolated from the present study.

Figure 6

Therapeutic protective effects of phage AhFM11 in vivo. (A) injection, (B) immersion and (C) phage impregnated feed groups. Mortality was recorded daily for 15 days, and cumulative percent survival rate was calculated.