Protease-sensitive antibacterial activity from tilapia gut-derived Enterobacter ludwigii is associated with a colicin V-like genomic candidate

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Fish-associated intestinal bacteria are promising sources of antimicrobial compounds that may reduce reliance on conventional antibiotics in aquaculture. Here, 64 intestinal bacterial isolates from apparently healthy tilapia were screened against bacterial indicators, and isolate A1 was selected because its cell-free supernatant (CFS) showed antibacterial activity. Neutralization and catalase treatment did not abolish activity, and heat treatment reduced but did not eliminate the inhibitory effect, whereas trypsin and proteinase K abolished detectable inhibition. This profile supports a protease-sensitive, partially heat-tolerant bacteriocin-like inhibitory substance (BLIS). Ammonium sulfate precipitation and dialysis enriched the active fraction, increasing specific activity to 10.604 AU \(μ\) g \(⁻¹\) against the Gram-positive bacterium Listeria monocytogenes and 9.643 AU \(μ\) g \(⁻¹\) against the Gram-negative bacterium Aeromonas hydrophila . Whole-genome sequencing yielded a curated 4.96-Mb draft genome with 61 contigs, 54.31% GC content, 100% coverage breadth, and a weighted mean depth of 392.6-fold. Average nucleotide identity (ANI) assigned A1 to Enterobacter ludwigii , with 98.8048% ANI to the type strain EN-119. Gene prediction and antimicrobial-domain mining identified ELUD_A1_000009_42 , encoding a 162-amino-acid colicin V/microcin V-like protein, as the strongest candidate determinant. The candidate showed a Proteins family (Pfam) Colicin_V hit and full-length similarity to publicly available colicin V proteins. These results associate a protease-sensitive antibacterial phenotype with a specific, testable genomic candidate.