Integrated Phenotypic, Proteomic (MALDI-TOF MS), and Genomic (WGS) Investigation of a Prolonged Hospital Outbreak of Pseudomonas aeruginosa with High Biofilm-Forming Capacity

<b>Background/Objectives</b>: Hospital outbreaks of <i>Pseudomonas aeruginosa</i> are difficult to control due to the pathogen's extensive repertoire, including its ability to form biofilms, adapt and persist in diverse environments, and develop multidrug resistance, all of which contribute to prolonged outbreaks. This study integrates the phenotypic, proteomic, and genomic characterization of a nosocomial outbreak comprising 38 clinical isolates and one environmental isolate recovered from the intensive care unit (ICU) of Hospital IESS Quito Sur. <b>Methods</b>: Clinical data were collected, antimicrobial susceptibility was assessed by minimum inhibitory concentration (MIC), carbapenemase genes were detected by multiple PCR and immunochromatographic assays, and the biofilm formation index (BFI) was determined. In addition, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used for species identification and clustering based on spectral similarity. Twelve representative isolates underwent whole genome sequencing (WGS) to characterize the resistome and virulome and to compare phylogenetic relationships with proteomic clustering defined by MALDI Biotyper Compass Explorer software. <b>Results</b>: All isolates were identified as <i>P. aeruginosa</i>, and phenotypic antimicrobial susceptibility classified most isolates as multidrug resistant, including 32 CRPA strains. The <i>bla</i>_VIM gene was detected in 22 isolates, while BFI analysis showed that all isolates formed moderate to strong biofilms. Genomic analysis revealed that most isolates belonged to ST111 and ST253, and both conserved and heterogeneous resistome and virulome profiles, with a broad distribution of determinants related to biofilm formation, stress tolerance, and persistence. Comparison between MALDI-TOF MS and WGS showed predominant concordance in clustering, mainly within subclusters but disagreement at the cluster level. <b>Conclusions</b>: The detection of carbapenemases, biofilm-forming ability, and virulence determinants associated with prolonged persistence highlights the need for integrated molecular tools, such as MALDI-TOF MS with MALDI Biotyper Compass Explorer software, to support epidemiological surveillance and to inform strategies aimed at mitigating prolonged hospital outbreaks caused by <i>P. aeruginosa</i>.