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

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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.

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