The prc gene as a high-resolution proxy for Aeromonas genus-wide clonal genealogy, phylogenomic validation and superiority over traditional MLST.

Aeromonas spp. are critical aquatic pathogens affecting global aquaculture and human health, yet their taxonomy is frequently confounded by persistent horizontal gene transfer and high recombination rates. While whole-genome sequencing offers definitive resolution, many laboratories remain dependent on biochemical profiling or multi-locus sequence typing (MLST) due to cost and infrastructure constraints. To address this diagnostic gap, a two-phase phylogenomic study to identify a robust single locus genetic marker was conducted. In the discovery phase, we analyzed 22 high-quality complete Aeromonas type strain genomes to quantify the evolutionary impact of recombination and selection of new marker candidate, followed by a validation phase involving high-quality 374 genus-wide assemblies. Our results indicate that an elevated recombination-to-mutation ratio (r/m ≈ 1.70) is associated with reduced phylogenetic congruence of standard MLST loci, including gyrB, groL, and recA. In contrast, the prc gene exhibited the highest congruence with the recombination-filtered clonal genealogy (score = 0.94). Within the 95-96% ANI species boundary, the prc gene identity consistently remained more than 97%. This observed prc stability may be associated with functional constraints related to its predicted role in the periplasm. We therefore propose that a prc gene phylogeny and sequence identity threshold of > 97% could serve as a rapid and cost-effective marker for preliminary species-level assignment and epidemiological surveillance of Aeromonas spp., particularly in settings where a whole-genome sequencing is not readily available.