A Sequence-Guided Protease Gene Approach for Differentiating Closely Related Aeromonas Species, Revealing Aeromonas dhakensis

Accurate species-level identification of Aeromonas remains challenging because of the limitations of 16S rRNA sequencing and phenotypic variability. This study aimed to develop protease gene-targeted PCR primers for differentiating closely related Aeromonas taxa. Homologous eprA ₁ protease gene sequences from A. hydrophila and A. caviae were aligned to identify conserved and species-variable regions for primer design. The primers were tested on a wastewater-derived Aeromonas isolate (PP542541.1), followed by sequencing and phylogenetic analysis. The F–R ₂ primer pair specifically amplified a 1,016 bp fragment from the isolate after optimization with Pfu polymerase. Sequencing showed 99.9% identity with A. dhakensis sequences, and the sequence was deposited in NCBI (OR546345.1). Phylogenetic analysis placed the isolate within the A. dhakensis clade, distinct from A. hydrophila . The proposed protease gene-targeted assay provides a reproducible and sequence-informed approach for resolving closely related Aeromonas taxa. This method offers improved resolution for environmental monitoring, bacterial identification, and taxonomic studies.