Pleiotropic effects of arnT deletion on colistin resistance, virulence, and stress tolerance in Aeromonas hydrophila are associated with impaired outer membrane homeostasis

Aeromonas hydrophila is a widespread zoonotic and aquatic pathogen posing growing challenges in aquaculture and public health due to increasing antimicrobial resistance and virulence. The arnT gene has been regarded primarily as a mediator of colistin resistance via lipopolysaccharide modification. In this study, we show that deletion of arnT in A. hydrophila r esults in pleiotropic defects in antimicrobial resistance, pathogenicity, and environmental fitness, which are associated with impaired outer membrane homeostasis. Consistent with the known role of ArnT in lipid A modification, arnT deletion led to marked alterations in lipid A glycosylation, which correlated with dramatically increased colistin susceptibility (MIC from ≥ 256 to 1 µg/mL) and increased susceptibility to several other antibiotics tested in this study. Lipid A analysis revealed that arnT is required for normal lipid A modification. Phenotypic characterization showed that arnT deletion compromised outer membrane integrity, as evidenced by membrane structural abnormalities, increased permeability (confirmed by ALP, β-galactosidase, and NPN uptake assays), and elevated membrane potential. These membrane defects were accompanied by reduced biofilm formation, impaired motility, and decreased survival under hyperosmotic, high-temperature, oxidative, and pH stress conditions. In a mouse intraperitoneal infection model, the Δ arnT mutant exhibited attenuated virulence, with significantly reduced bacterial burdens in the liver, spleen, and kidney at 24 h post-infection, along with milder histopathological lesions and lower levels of pro-inflammatory cytokines. Collectively, these findings demonstrate that arnT contributes to both colistin resistance and multiple virulence-associated traits in A. hydrophila , and these phenotypes are associated with impaired outer membrane homeostasis. This study provides a foundation for understanding the relationship between resistance and virulence in bacterial pathogens and suggests that arnT may represent a potential target for further investigation in the context of anti-virulence and anti-resistance strategies.