Pseudomonas fragi or not? Beyond MALDI-TOF in the frontier of Pseudomonas diversity.

Advanced platforms, such as whole genome sequencing (WGS), should be employed to enhance and refine microbial identification compared to phenotypic methods, including miniaturized biochemical tests and MALDI-TOF. The application of WGS has led to the reclassification of clinical bacterial pathogens previously misidentified by phenotypic techniques. In this study, eight clinical isolates initially identified as Pseudomonas fragi by VITEK MS were subjected to WGS and bioinformatics analysis. The results revealed one strain as Pseudomonas lundensis, while average nucleotide identity and phylogenetic reconstruction suggested that the remaining seven strains represent novel species within the Pseudomonas fluorescens superclade. The strains harbored four antimicrobial resistance genes conferring resistance to β-lactams, fluoroquinolones, phenicols, and tetracyclines, yet in vitro assays indicated susceptibility to carbapenems and intermediate susceptibility to colistin. Additionally, the strains possessed virulence factor genes associated with alginate biosynthesis, flagellar formation, pilus assembly, and iron uptake, with Type III Secretion System (T3SS)-related genes detected only in P. lundensis. Notably, the isolated Pseudomonas spp., exhibited multiple haplotypes, a closely related pan-genome, and similar phenotypic characteristics. These findings underscore the necessity of integrating multiple approaches, including molecular methods such as 16S rRNA gene amplification, sanger sequencing, and WGS, alongside traditional phenotypic techniques, to improve the accuracy of microbial identification in clinical settings.