Isolation and Characterization of the Lytic Bacteriophages and Its Application in Combination with Amoxicillin againstAeromonas dhakensis

Abstract

ABSTRACTAeromonas dhakensisis the most virulentAeromonasspecies 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 againstA. dhakensis. OfAeromonasisolates, only isolate AM could be used to isolate phages using the enrichment technique. This strain was identified via biochemical tests, 16S rDNA sequencing, and whole-genome analyses. All results confirmed that strain AM wasA. dhakensis. In silicodetection of antimicrobial resistance genes and virulence factors corresponding to the main bacterial virulence determinants were observed inA. dhakensisAM, 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 toSiphoviridaefamily, whereas vB_AdhM_TS9 and vB_AdhM_DL belonged toMyoviridaefamily. Host-range determination demonstrated that all phages were capable of infecting onlyA. dhakensis. Three phages, vB_AdhS_TS3, vB_AdhM_TS9, and vB_AdhM_DL, were selected since they had a shorter latency period and larger burst sizes. All phages were resistant to a wide range of pH values and remained relatively stable after a 60-minute incubation at 4 °C, 25 °C, 30 °C, and 37 °C but were sensitive to higher temperatures. The pre-treatment (co-inoculation ofA. dhakensisand 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 in a volume of 200 µL; however, an incomplete inhibition was observed in post-treatment in the volume of 20 mL but still decreased by about 1.2-1.7 log CFU/mL when compared to the control and other individuals used after incubation for 48 h. 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, with G+C contents of 41.10%, 61.7%, and 35.34%, respectively. This study demonstrated using phages as an adjuvant with a sublethal concentration of antibiotics as an effective therapeutic strategy.IMPORTANCEAeromonas dhakensisis widely distributed in the environment and can cause a variety of infections both in human and animals and is often misidentified with other members of theAeromonasfamily,A. hydrophila, A. veronii, orA. caviaeusing the commercial test kits which has led to challenges in treating this pathogen. Hence, our study isolated phages against this bacterial strain and extensively characterized their efficacy with common antibiotics used to treat the pathogen.