Integron architecture and virulence-associated gene profiles in extensively drug-resistant high-risk Pseudomonas aeruginosa ST309 and ST233

José Luis Fernández-Vázquez, Ma Dolores Jarillo-Quijada, Santiago Castillo‐Ramírez, María Luisa Hernández-Medel, Manuelita Zavala-Pineda, Mónica Osorio-Guzmán, José Ignacio Santos-Preciado, Marı́a Dolores Alcántar-Curiel

Background Pseudomonas aeruginosa is a major cause of healthcare-associated infections and exhibits considerable genomic plasticity, which allows the accumulation and structural organization of resistance determinants while retaining virulence-associated traits in some high-risk clones (HRCs). HRCs such as Sequence Type (ST) 309 and ST233 are increasingly associated with extensively drug-resistant (XDR) and pandrug-resistant (PDR) phenotypes worldwide. However, the integron architecture and virulome associated with XDR P. aeruginosa have not been fully characterized. This study aimed to characterize the resistome and virulome of highly resistant ST309 and ST233 isolates and to analyze the structural variability of chromosomal resistance regions. Methods Whole-genome sequencing and comparative genomic analyses were performed on four bloodstream P. aeruginosa isolates recovered from two tertiary-care hospitals in Mexico. Results Three isolates (Psa/12, Psa/19, and Psa/21) were classified as XDR and belonged to ST309, whereas one isolate (Psa/20) was PDR and assigned to ST233. The ST233 isolate carried the highest number of resistance genes, including the carbapenemase gene bla VIM–2 . ST309 isolates carried between 11 and 12 resistance genes and shared a conserved class 1 integron-associated chromosomal resistance region flanked by int1 and insertion sequences, with structural differences among the analyzed isolates. In contrast, ST233 carried a multidrug resistance region highly similar to transposon Tn6061. The in silico virulome analysis identified a large, conserved repertoire of genes (203–209 genes), with 199 genes shared across all isolates. Notably, 14 virulence-associated genes differed between the ST309 and ST233 isolates, including differential carriage of type III secretion system effectors ( exoU in ST309 and exoS / exoY in ST233), phenazine biosynthesis genes, and type IV pili components. Conclusion XDR high-risk P. aeruginosa ST309 and ST233 isolates carried distinct chromosomal resistance regions and showed differences in virulence genes. The analyzed isolates combined extensive antimicrobial resistance with a largely conserved set of virulence-associated genes, highlighting the persistence of clinically relevant high-risk clones in Mexican hospitals.