Genomic Characterization of Clinical Stenotrophomonas Strains from Thailand Reveals Five Novel Species and Extensive Functional Diversity

Background Stenotrophomonas spp. are increasingly recognized as opportunistic pathogens with high antimicrobial resistance (AMR) nowadays. Despite their clinical importance, genomic diversity within the genus remains underexplored, particularly in Thailand and Southeast Asia. Methods Ten Stenotrophomonas strains were isolated from diverse clinical specimens at Songklanagarind Hospital, Thailand, in 2023. The whole genome sequencing was performed using MGISEQ 2000 platform. Genome-based classification was assessed via average nucleotide identity (ANI), and digital DNA-DNA hybridization (dDDH). Functional annotation was conducted using RAST and COG databases. Pan-genome structure, AMR/virulence genes, and phenotypic traits (biofilm and hemolysis) were investigated. Results The genomes ranged from 4.11–5.05 Mb with 66.02–66.84% GC content. While 16S rRNA gene sequences showed high similarity to type strains, ANI and dDDH analyses clearly differentiated ten studied strains from validly published species and grouped them into five novel species. The pan-genome was open, with only 21.6% core, 1.5% soft-core and 76.9% accessory/unique genes, indicating high plasticity. Functional annotation revealed enrichment in genes related to regulation, metabolism, and cell envelope biogenesis, reflecting metabolic flexibility and environmental adaptability. AMR profiling showed conserved aminoglycoside-modifying enzymes, β-lactamase bla _L1 and efflux pumps across all strains. Phenotypically, all strains exhibited multidrug resistance but remained uniformly susceptible to cotrimoxazole, current choice of treatment. Virulence genes for adhesion, hemolysin, proteases, and biofilm were conserved, consistent with observed alpha-hemolysis and moderate-to-strong biofilm formation. Conclusion This study reveals substantial genomic and functional diversity among ten Stenotrophomonas strains, highlighting five putative novel species and underscoring the importance of continued surveillance and accurate identification for effective clinical management strategies.