Molecular Multilocus gyrB–rpoB–16S rRNA Gene Typing, MAR Index, Biofilm formation, and LD50 in Aeromonas hydrophila 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.