Molecular Multilocus <em>gyrB–rpoB–16S rRNA</em> Gene Typing, MAR Index, Biofilm formation, and LD50 in <em>Aeromonas hydrophila</em> from Farmed Gourami in East Java, Indonesia

Translating resistance, biofilm, and virulence readouts into actionable control requires accurate species delimitation and fine-scale phylogrouping of A. hydrophila from gourami aquaculture. Nine isolates were sequenced at 16S (~1505 bp), rpoB (~558 bp), and gyrB (~1103 bp); species calls required ≥98.7% identity and ≥95% coverage for 16S plus ≥95% identity in ≥1 housekeeping gene vs type strains. ML (UFBoot=1000) and Bayesian (BPP) trees used locus partitioning. Susceptibility followed CLSI VET03/VET04 (2020); MAR = resistant/total agents. Biofilm (24 h) used crystal-violet OD. A conspecific LD50 challenge in juvenile Osphronemus goramy estimated LD50 (95% CI) by Reed–Muench. The bench-marks showed a stable hierarchy, gyrB (95–100% concordance; median UFBoot 95–100; BPP 0.98–1.00; 3–5 subclades; 0–1/N corrections) ≥ rpoB (90–97%; 93–100; 0.98–1.00; 3–4; 0–1/N) » 16S (70–85%; 70–90; 0.85–0.98; 1–2; 1–3/N); concatenation (gyrB + rpoB) corrected 16S misplacements. Ciprofloxacin remained fully active; doxycycline was variably active; several older classes were largely ineffective, yielding high MAR. Biofilm attachment varied by gyrB-defined phylogroup. The gourami challenge yielded LD50 ≈3–5 × 10⁵ CFU/mL with dose-dependent mortality. A “gyrB-first, rpoB-reinforced, concatenation-adjudicated” pipeline balances accuracy and throughput for diagnostics, while linking phylogroups to MAR, 24 h biofilm, and gourami LD50 supports control strategies in gourami aquaculture.