Abstract
Aeromonas salmonicida is a major pathogen in aquaculture, and its ability to form biofilms contributes significantly to antibiotic resistance and chronic infections. This study investigated the effects of four antibiotics—ampicillin, amoxicillin, oxytetracycline, and doxycycline—at various concentrations on bacterial growth, biofilm formation, and gene expression related to antibiotic resistance and quorum sensing (QS) in two subspecies: A. salmonicida subsp. masoucida (ASM) and A. salmonicida subsp. salmonicida (ASS). Bacterial isolates from Atlantic salmon were identified using 16S rRNA and vapA gene sequencing. Growth inhibition was more pronounced in ASS than ASM under high antibiotic concentrations. Conversely, sub-inhibitory concentrations (sub-MICs) enhanced biofilm formation in both subspecies, particularly in ASM. PCR results showed that tetA and tetE resistance genes were present only in ASM. qRT-PCR analysis revealed that expression of QS-related genes (ahyI and ahyR) was generally downregulated under tetracycline treatment, while litR expression varied across antibiotic conditions and strains. Some isolates showed increased litR expression alongside elevated biofilm formation, suggesting involvement of additional regulatory mechanisms. These results highlight the potential for sub-MIC antibiotic exposure to promote biofilm development and modulate gene expression, emphasizing the need for careful antibiotic use in aquaculture and providing insight into alternative pathogen control strategies.