Introduction:
Aeromonas species are opportunistic aquatic pathogens associated with disease outbreaks in fish and human infections. Assessing their virulence is essential for aquaculture health management and understanding zoonotic potential. This study investigated the virulence potential of aquatic Aeromonas isolates through an integrated approach combining in vitro phenotypic assays, in vivo infection in Galleria mellonella larvae, and genome sequencing.
Materials and methods:
Phenotypic characterization included assessment of hemolytic activity on 5% sheep blood agar, extracellular enzyme production (protease, DNase, amylase, and lipase), motility, and biofilm formation. For in vivo evaluation, G. mellonella larvae (~220 mg) were injected with 10⁴–10⁸ CFU of bacterial suspension and incubated at 22 °C and 37 °C for 120 hours. Larval mortality was recorded at 24-hour intervals to evaluate strain-specific, dose-dependent virulence. Whole-genome sequencing using Illumina and nanopore platforms was followed by hybrid assembly with Unicycler. Species identification was confirmed using the TYGS platform. Virulence and antimicrobial resistance genes was determined via Virulence Factor Database (VFDB) and the Comprehensive Antibiotic Resistance Database (CARD).
Results:
Phenotypic profiling revealed that most isolates exhibited strong lipolytic and proteolytic activity, while DNase and tributyrin lipase activity were absent. β-glucosidase activity was observed in only a few isolates. Larval infection assays revealed strong inter-strain and dose-dependent variability. Highly virulent strains such as A. hydrophila A-12, A. hydrophila A-13, A. dhakensis A-15 caused complete mortality within 24 hours at 10⁵ CFU, whereas others, such as Aeromonas sp. A-1, A-2, and A-4 produced delayed or reduced mortality. The in vivo assay differentiated high- and low-virulence strains based on larval mortality across bacterial doses. Genomic analysis showed that adherence-related virulence genes were most abundant (n=1,923), followed by secretion systems (n=685) and toxins (n=59). Genes associated with antiphagocytosis (n=26) and iron acquisition (n=17) were less prevalent. AMR profiling indicated broad-spectrum resistance, with beta-lactam resistance genes against penicillins (n=14), carbapenems (n=13), and cephalosporins (n=12) predominating. Resistance to tetracyclines and fluoroquinolones (n=11) was also common.
Conclusion:
This integrated approach revealed strain-specific virulence profiles shaped by phenotypic and genomic traits. The G. mellonella model, when used alongside genomic analysis, offers a rapid, ethical platform for high-resolution virulence screening in aquatic pathogens. These findings contribute to the characterization of virulence and resistance traits among aquatic Aeromonas spp. strains.
Acknowledgment: This research was supported by Scientific and Technological Research Council of Turkey (Project Number: 124Z735)