WITHDRAWN: Isolation and Characterization of the Lytic Bacteriophages and Its Application in Combination with Amoxicillin against Aeromonas dhakensis

Abstract

Aeromonas dhakensis is the most virulent Aeromonas species in tropical and subtropical areas and causes a variety of human diseases. Owing to its resistance against some antibiotics, there is an urgent need for new strategies against this pathogen. This is the first study to isolate and characterize lytic phages against A. dhakensis. Of Aeromonas isolates, only isolate AM could be used to isolate phages using the enrichment technique. In silico detection of antimicrobial resistance genes and virulence factors corresponding to the main bacterial virulence determinants were observed in A. dhakensis AM, which was then used as the host for phage isolation. Four lytic phages, designated vB_AdhS_TS3, vB_AdhM_TS9, vB_AdhM_DL, and vB_AdhS_M4, were isolated. Transmission electron micrographs showed that vB_AdhS_TS3 and vB_AdhS_M4 belonged to Siphoviridae family, whereas vB_AdhM_TS9 and vB_AdhM_DL belonged to Myoviridae family. The pre-treatment (co-inoculation of A. dhakensis and phage) with individual phages and phage cocktails reduced bacterial numbers in the range of 2.82-6.67 and 5.19-5.43 log CFU/mL, respectively, after 6 h of incubation. In post-treatment, the maximum inactivation was achieved with the log reduction of bacterial number in the range of 3.06-5.25 and 4.01-6.49 log CFU/mL after 6 and 12 h of incubation, respectively. A combination of phage cocktail with amoxicillin at sub-MIC showed complete inactivation in pre-treatment and post-treatment The complete genome and G+C content of phages vB_AdhS_TS3, vB_AdhM_DL, and vB_AdhM_TS9 were 115,560, 61,429, and 115,503 bp, respectively. This study demonstrated using phages as an adjuvant with a sublethal concentration of antibiotics as an effective therapeutic strategy.