Characterization and protective effects of lytic bacteriophage pAh6.2TG against a pathogenic multidrug-resistant Aeromonas hydrophila in Nile tilapia ( Oreochromis niloticus )

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.

Non‐antibiotic approaches to combat motile Aeromonas infections in aquaculture: Current state of knowledge and future perspectives

“…The immersion of phage was also used to control MAS in tilapia culture reducing 50% mortality from 68% in the control group to 18% in the treatment group using Podoviridae ‐phage ɸZH1 370 . As a prophylactic agent, phages can be released directly to pond water as a water‐borne administration to decrease the concentration of pathogens in a disease season or reduce the infection when the temperature changes abruptly 364,371–375 . However, preparing of many phages for use in commercial ponds or open aquaculture systems is a crucial obstacle.…”

“…370 As a prophylactic agent, phages can be released directly to pond water as a water-borne administration to decrease the concentration of pathogens in a disease season or reduce the infection when the temperature changes abruptly. 364,[371][372][373][374][375] However, preparing of many phages for use in commercial ponds or open aquaculture systems is a crucial obstacle. Because of this, hitherto, only a few researchers have focused on phages as a prophylactic agent.…”

Isolation and characterization of the lytic Aeromonas phage HJ05,a new member of Caudovirales

“…Phagebased biocontrol strategies have been regarded as a promising and environmentally friendly alternative to antibiotics for the treatment of A. hydrophila infection. Recently, various phages have been identi ed and characterized to test their potential against A. hydrophila infection, which at least include Autographiviridae phage Ahy-yong1 [12], Podoviridae phage vB_ AhaP_PZL-Ah8 [13], Caudovirales phage AhMtk13a [14], Podoviridae phage ZPAH7 [15], Caudovirales phage AHP-1[16], Podoviridae phage MJG [17], Caudovirales phage CC2[18], Caudovirales phage PVN02 [19], Caudovirales phage Ahp2 [20], Podoviridae phage PZL-Ah152 [21], Podoviridae phages PZL-Ah1 and PZL-Ah8 [22], Caudovirales phage phiA8-29 [23], Caudovirales phage AhyVDH1 [24], Caudovirales phage vB-AhyM-AP1 [25], Caudovirales phages N21, W3 and G65[26], Podoviridae phages Y71 and Y81[26], Chaseviridae phage pAh6.2TG [27], Siphoviridae phage Akh-2[28], Caudovirales phage VTCCBPA6 [29], Caudovirales phages CF8, PS1, and PS2 [30]. Siphoviridae phages AhSzq-1 and AhSzw-1 [31].…”

Bacteriophage and ozone-nanobubble based approaches for combating multidrug-resistant aeromonas hydrophila in nile tilapia (oreochromis niloticus) culture system

“…The bacterial isolate, Aeromonas hydrophila BT14 and the Myoviridae phage pAh6.2TG used in this study were isolated and characterized in our previous studies (Dien et al, 2021a;Dien et al, 2021b). Prior to phage propagation, A. hydrophila BT14 was cultured in 15 mL of Tryptic Soy Broth (TSB; Becton Dickerson, USA) at 28 C for 24 h. Then, 100 µL of phage (10 8 PFU/mL) was mixed with 100 µL of bacteria in 3 mL of TSB supplemented with 0.5% agar.…”

“…The supernatant was filtered through a 0.2 μm filter. The filtrate was harvested and enumerated by a phage plaque assay as indicated previously (Dien et al, 2021a). Nanobubble system (Model: aQua+075MO, AquaPro Solutions Private Limited Company, Singapore) was set up as previously described with oxygen input of 2 L/min.…”