Molecular Architecture of Biofilm Formation in Extensively Drug-Resistant Pseudomonas aeruginosa Clinical Isolates: Transcriptomic, Proteomic, and Genomic Integration from Nigerian Burn Wound Infections

Biofilm formation by Pseudomonas aeruginosa in chronic burn wound infections dramatically reduces antibiotic penetration and immune cell access, constituting a primary driver of treatment failure in extensively drug-resistant (XDR) strains. Deciphering the molecular architecture governing biofilm developmental stages in XDR clinical isolates is a prerequisite for anti-biofilm therapeutic target identification. This dissertation presents an integrated transcriptomic, proteomic, and genomic investigation of biofilm formation mechanisms in XDR P. aeruginosa clinical isolates from burn wound patients at National Orthopaedic Hospital Enugu and University of Nigeria Teaching Hospital (UNTH) Enugu, Southeast Nigeria. Forty XDR P. aeruginosa isolates were collected and subjected to minimum biofilm eradication concentration (MBEC) assays. Three phenotypically distinct biofilm-forming capacity groups were identified. RNA-seq (Illumina NovaSeq) and quantitative proteomics (data-independent acquisition mass spectrometry) were performed on planktonic and biofilm states at 6-hour, 24-hour, and 72-hour timepoints. Pangenome analysis was performed across all 40 genomes using Roary. Transcriptomic analysis identified 847 differentially expressed genes (DEGs) between planktonic and mature biofilm states, with the GacS/GacA two-component system and Pel/Psl exopolysaccharide biosynthesis gene clusters among the most significantly upregulated. Proteomic data confirmed elevated abundance of PelB, AlgD, and PilY1 in mature biofilm fractions. Three genomic islands unique to hyper-biofilm-forming isolates were identified, carrying homologs of recently described biofilm-regulatory genes absent in PAO1 reference. The integrated multi-omics model resolves a biofilm regulatory hierarchy in XDR clinical strains and identifies PelB and GacA as primary anti-biofilm target candidates for the Nigerian clinical context. Keywords: Pseudomonas aeruginosa, biofilm, multi-omics, XDR, burn wound infection.