Preliminary in vitro characterization and lytic dynamics of three novel siphovirus-like bacteriophages targeting extensively drug-resistant Vibrio alginolyticus V79

The rapid growth of global aquaculture has led to a rise in bacterial diseases, threatening food security and economies in developing countries. Vibriosis, caused by Vibrio bacteria, is the most common and devastating disease in shrimp farming, a crisis severely exacerbated by the emergence of multidrug-resistant pathogens. This study isolates and characterizes three novel bacteriophages (P18, P24, and P25) targeting an extensively drug-resistant (XDR) Vibrio alginolyticus strain (V79) isolated from diseased Litopenaeus vannamei in Vietnam. The host strain V79 was confirmed by PCR using species-specific collagenase and 16S rRNA genes. V79 showed high multidrug resistance, resistant to 9 of 10 antibiotics, with a MAR index of 0.9. Transmission electron microscopy (TEM) classified all three isolated phages displayed a sipho morphotype, characterized by icosahedral capsids and long, non-contractile tails. In environmental tolerance assays, all three phages were completely inactivated at pH 3.0 and showed a precipitous decline in titers at 56°C, but remained highly stable across pH 5.0–9.0 and temperatures of 4.0–40.0°C (>108 PFU/mL). Notably, at pH 12.0, all three isolates showed remarkable alkaline resilience; Phage P18 exhibited significantly greater stability (p<0.05), retaining a titer of (2.66±0.37)×10 9 PFU/mL compared to P24 and P25. In 24-hour in vitro killing assays, Phage P25 demonstrated outstanding lytic efficacy, achieving a >1.5 log 10 reduction in bacterial load and maintaining sustained population suppression, whereas P18 and P24 were limited by the emergence of resistant small colony variants (SCV). Our findings suggest that, while all three phages exhibit high environmental resilience, Phage P25 is the most promising candidate for further genomic safety validation, serving as a vital biological component in therapeutic phage cocktail formulations to manage XDR vibriosis in sustainable aquaculture.