Comparative Genomics of a Hemolymph-Derived Pseudomonas sp. Reveals Metabolic Versatility and Multidrug Resistance

Flacherie is a major bacterial disease compromising silkworm health and cocoon productivity; however, the diversity and genomic features of larva-associated pathogens remain poorly characterized. In the present study, we isolated and characterized a Gram-negative bacterium, designated Pseudomonas sp. RAC1, from the hemolymph of diseased silkworm larvae. The isolate exhibited clear swimming motility, indicating an active flagellar system. Biochemical profiling using the VITEK-2 platform revealed broad metabolic capabilities, including utilization of multiple organic acids and amino-acids, related enzymatic activities, highlighting its adaptability under nutrient-variable host conditions. Chemotaxonomic analysis using fatty acid methyl ester (FAME) profiling further supported its identity within the genus Pseudomonas. Whole-genome sequencing showed a 4.49 Mb GC-rich genome encoding diverse functional pathways related to central metabolism, aromatic compound degradation, and carbohydrate-active enzymes, suggesting strong ecological flexibility. Importantly, genomic screening identified multiple virulence-associated genes and a large repertoire of antimicrobial resistance determinants, dominated by efflux pumps and outer membrane permeability factors, which correlated with phenotypic resistance to all tested antibiotics. In addition, RAC1 contained several mobile genetic elements and three prophage regions, reflecting a highly plastic genome. Pangenome analysis across related Pseudomonas strains indicated an open pangenome, driven largely by accessory and cloud genes. Overall, Pseudomonas sp. RAC1 represents a multidrug-resistant and genomically dynamic strain with traits that may contribute to persistence and pathogenicity in the development stage of the silkworm ecosystem.