Novel Aeromonas Phage Ahy-Yong1 and Its Protective Effects against Aeromonas hydrophila in Brocade Carp (Cyprinus aka Koi)

Abstract

Aeromonas hydrophila is a zoonotic pathogen and an important fish pathogen. A new lytic phage, Ahy-yong1, against multi-antibiotic-resistant pathogen A. hydrophila was isolated, identified, and tentatively used in therapy. Ahy-yong1 possesses a head of approximately 66 nm in diameter and a short tail of approximately 26 nm in length and 32 nm in width. Its complete dsDNA genome is 43,374 bp with a G + C content of 59.4%, containing 52 predicted opening reading frames (ORFs). Taxonomic analysis indicated Ahy-yong1 as a new species of the Ahphunavirus genus of the Autographiviridae family of the Caudoviricetes class. Ahy-yong1 was active only against its indicator host strain among the 35 strains tested. It is stable at 30-40 °C and at pH 2-12. Aeromonas phage Ahy-yong1 revealed an effective biofilm removal capacity and an obvious protective effect in brocade carp (Cyprinus aka Koi). The average cumulative mortality for the brocade carp in the blank groups intraperitoneally injected with PBS was 1.7% ± 2.4%;for the control groups treated with A. hydrophila (10⁸ CFU/fish) via intraperitoneal injection, it was 100.00%;and for the test group I, successively treated with A. hydrophila (10⁸ CFU/fish) and Aeromonas phage Ahy-yong1 (10⁷ PFU/fish) via intraperitoneal injection witha time interval of 2 hours, it was only 43.4% ± 4.7%. Furthermore, the cumulative mortality of the test group II, successively treated with Aeromonas phage Ahy-yong1 (10⁷ PFU/fish) and A. hydrophila (10⁸ CFU/fish), was only 20.0% ± 8.2%, and that of the test group III, simultaneously treated with Aeromonas phage Ahy-yong1 (10⁷ PFU/fish) and A. hydrophila (10⁸ CFU/fish), was only 30.0% ± 8.2%. The results demonstrated that phage Ahy-yong1 was very effective in the therapies against A. hydrophila A18, prophylaxis was more effective than rescue, and earlier treatment was better for the reduction of mortality. This study enriches knowledge about Aeromonas phages.

Keywords: Aeromonas hydrophila; brocade carp; genome; phage; therapy.

Figure 1

Visualization of Ahy-yong1 plaques and Aeromonas hydrophila A18 cultures. (A) Aeromonas phage Ahy-yong1 formed clear and circular plaques on A. hydrophila A18 lawns. Small plaques can be seen in 3 h, and the average diameter of the plaques reached 0.84 mm in 4 h. (B) Normal A. hydrophila A18 culture (left) and lysateofphageAhy-yong1-infected A. hydrophila A18 (right).

Figure 2

Transmission electron microscopy of negatively stained Aeromonas phage Ahy-yong1 and A. hydrophila A18 cell infected with Ahy-yong1. (A–C) Free intact-matureAhy-yong1 virion. Ahy-yong1 possesses a head with a diameter of 66 nm and a short non-contractile tail of 26 nm in length and 32 nm in width under a transmission electron microscope. (D) Immature phage particles being packaged within a viral factory in an infected A. hydrophila A18 cell.

Figure 3

The one-step growth curve under the MOI of 0.1 and the results of the temperature stability and pH stability test of Aeromonas phage Ahy-yong1. (A) The one-step growth curve demonstrated that the latent period of Aeromonas phage Ahy-yong1 was 10 min, followed by a burst period of 50 min. (B) Aeromonas phage Ahy-yong1 was very stable at 30 °C, maintaining constant production for over 120 min; relatively stable at 40 °C; not stable at 50 °C, 60 °C, and 70 °C. Phage Ahy-yong1 eventually became inactive when the temperature exceeded 50 °C. (C) Aeromonas phage Ahy-yong1 was found to be stable at pH 2 to 12, indicating that even extremes of pH could not affect infectivity of phage Ahy-yong1. All values represent the mean of triplicate measurements, and error bars represent the standard deviations (n = 3).

Figure 4

The ability of A. hydrophila A18 to form biofilms and the ability of Aeromonas phage Ahy-yong1 to eliminate biofilm. (A,B) The OD₅₉₀ of values of the crystal-violet-stained biofilm and cells. (C,D) The wells stained with crystal violet. All values represent the mean of triplicate measurements, and error bars represent the standard deviations (n = 3). ** p < 0.01 and *** p < 0.001.

Figure 5

Genomic map of Aeromonas phage Ahy-yong1. From outside to inside, circle 1 shows the 52 predicted ORFs, circle 2 shows the size in pairs (kb), circle 3 displays the GC of the genome, and the innermost circle shows the GC skew plot (G − C)/(G + C). The direction of the arrow indicates the transcription direction of each gene. The color of each gene refers to the most similar phages, yellow stands for the gene similar to the Aeromonas phage and gray for the gene without similarity in the NCBI database.

Figure 6

Genome comparison of the Aeromonas phage Ahy-yong1 and the three closest relatives (Aeromonas phage LAh1, Aeromonas phage CF7, and Aeromonas phage Ahp1). The orientation of the arrows indicates the direction of gene transcription. The color of each arrow refers to the functional categories: blue indicates DNA replication and regulation; yellow indicates DNA packaging; red indicates lysis; orange indicates structure; purple indicates RNA polymerase; gray indicates hypothetical protein. The homologous regions are represented by green bars. Light green to dark green represent low to high homology between genes.

Figure 7

Proteomic tree based on the complete genome sequences of Aeromonas phage Ahy-yong1, 62 classified phages of Caudoviricetes class with shorter evolutionary distance from Ahy-yong1 in the original tree and unclassified Aeromonas phage LAh1 sharing the top highest homology with Ahy-yong1 in BLASTn scanning. Bacteriophage family assignments according to the official ICTV classification (March 2022) are provided with different color bars. The red star indicates phage Ahy-yong1. In the proteomic tree, Aeromonas phage Ahy-yong1 clustered with Aeromonas phages of the family Autographiviridae, especially closely related with Aeromonas phage LAh1, Ahp1, and CF7.

Figure 8

The internal organs and cumulative mortality curves of brocade carps (Cyprinus aka Koi) in the blank group, control group, and test groups. Each fish in the blank group was successively injected intraperitoneally twice with 0.01 M PBS. Each fish in the control group was successively injected intraperitoneally with A. hydrophila A18 (10⁸ CFU/mL) and 0.01 M PBS. Each fish in the test group I was successively injected intraperitoneally with A. hydrophila A18 (10⁸ CFU/mL) and Aeromonas phage Ahy-yong1 (10⁷ PFU/mL). Each fish in the test group II was successively injected intraperitoneally with Aeromonas phage Ahy-yong1 (10⁷ PFU/mL) and A. hydrophila A18 (10⁸ CFU/mL). The injection time intervals were 2 h and the injection volume was 100 µL. Each fish in the test group III was injected with 100 µL of Aeromonas phage Ahy-yong1 (10⁷ PFU/mL) immediately after the injection of 100 µL of A. hydrophila A18 (10⁸ CFU/mL). (A) The internal organs of brocade carps infected with A. hydrophila (control groups) were swollen and rotten and their eyes become cloudy relative to the blank groups and test groups. (B) The cumulative mortality of brocade carps in the blank groups was 1.7% ± 2.4%. The cumulative mortality of brocade carps in the test groups I, II, and III were 43.3% ± 4.7%, 20.0% ± 8.2%, and 30.0% ± 8.2%, respectively, which were significantly lower than those in the control groups, of which the cumulative mortality was 100.0%. All values represent the mean of triplicate measurements, and error bars represent the standard deviations (n = 3).

Figure 9

The dynamic curves of phage load and A. hydrophila A18 load in the muscles of the brocade carps. (A) The dynamic curves of phage load in the muscles of the brocade carps. (B) The dynamic curves of A. hydrophila A18 load in the muscles of the brocade carps. All values represent the mean of triplicate measurements, and error bars represent the standard deviations (n = 3).