Integrative Genomic and Structure-Based Prioritization of Molecular Targets in Multidrug-Resistant Salmonella enterica from Brazilian Poultry

Background/Objectives: Multidrug-resistant Salmonella enterica in poultry production poses a persistent challenge for antimicrobial stewardship and food safety. This study integrated phenotypic susceptibility testing, whole-genome sequencing, structural bioinformatics, molecular docking, and cheminformatics to prioritize resistance- and virulence-associated proteins in S. Heidelberg and S. Minnesota. Methods: Five multidrug-resistant poultry-associated isolates—SH16.3, SH23.6, SM23.7, SM23.8, and SM23.9—were analyzed. Resistome and virulome profiles guided the selection of CTX-M-2, CMY-2, Sul2, AcrB, and SpvC. Experimental protein structures or a high-confidence ColabFold model were used for GNINA docking. AcrB- and Sul2-directed analogs were generated with RDKit/BRICS and ranked by docking score, physicochemical properties, structural similarity, and PLIP interaction profile. Results: The SH23.6, SM23.7, SM23.8, and SM23.9 assemblies supported comparative genomic analysis, whereas SH16.3 was highly fragmented and restricted to target-level interpretation. Independent integrity checks recovered complete blaCTX-M-2 and spvC coding sequences with 100% coverage and identity to curated references, although their assignment to SH16.3 remains limited by the absence of raw-read and orthogonal confirmation. CMY-2, Sul2, SpvC, and AcrB showed 99.81–100% sequence identity and full coverage relative to the selected experimental structures. Sul2 analog generation did not surpass the best parental compounds. By contrast, several AcrB analogs achieved more favorable docking scores than the reference ligands. ACRB_BRICS_00124 yielded a GNINA score of −10.80 kcal/mol and formed six hydrogen bonds, three hydrophobic contacts, and one salt bridge. Conclusions: The integrated workflow identified AcrB as the most tractable molecular target and ACRB_BRICS_00124 as the leading candidate for subsequent molecular-dynamics simulations, synthetic-feasibility assessment, and experimental validation.