wspA mutation mediated Pseudomonas aeruginosa rugose small colony variant and its hyper-biofilm

Pseudomonas aeruginosa is a major multidrug-resistant pathogen whose biofilm formation complicates treatment. P. aeruginosa rugose small colony variants (RSCVs), characterized by enhanced biofilm production and resistance, pose significant clinical challenges. However, the formation mechanisms remain unclear in clinical strains. Paired clinical P. aeruginosa isolates AR8023-1 (wild-type) and AR8023-2 (RSCV) were collected from urine samples of a neurosurgery inpatient chronologically. The genome comparison was performed using Breseq, and plasmid complementation was performed to construct complemented strain (AR8023-2RifR::wspAAR8023-1). Then, all the aforementioned strains were examined RSCV phenotype, biofilm formation, and motility, and gene expression differences induced by the wspA mutation were analyzed via transcriptome sequencing, followed by quantification of c-di-GMP. In addition, we assessed antimicrobial susceptibility of the strains under both planktonic and biofilm conditions. The results showed that both AR8023-1 and AR8023-2 were identified as ST3420. AR8023-1 exhibited typical smooth morphology, while AR8023-2 displayed a RSCV phenotype. A single SNP difference was identified between the two strains, characterized by a deletion of glutamine at position 289 (CAG triplet deletion) in the wspA gene of strain AR8023-2. The complement isolates restored wild-type morphology, motility, and biofilm formation. Transcriptomics revealed a significant upregulation of c-di-GMP metabolic genes in RSCVs (P < 0.001), indicating that the WspA{Delta}289Q mutation activates diguanylate cyclase (DGC) activity, thereby elevating c-di-GMP synthesis. Intracellular c-di-GMP levels were significantly higher in RSCVs than those in WT and complement isolates, respectively (P < 0.001). Consequently, biofilm susceptibility testing demonstrated the {beta}-lactams MBIC of RSCVs was [&ge;]8-512-fold higher than those of WT and complement isolates. This study identifies a clinical WspA{Delta}289Q mutation that elevates c-di-GMP, driving RSCV formation and biofilm-mediated resistance. IMPORTANCEP. aeruginosa is a leading multidrug-resistant pathogen whose biofilm formation capability greatly complicates treatment outcomes. This study identifies a novel clinical mutation in the wspA gene (WspA{Delta}289Q) that drives the emergence of rugose small colony variants (RSCVs), a phenotype linked to heightened biofilm production and antimicrobial resistance. We demonstrate that this mutation upregulates cyclic di-GMP synthesis, leading to increased biofilm formation and markedly reduced susceptibility to {beta}-lactam antibiotics under biofilm conditions. These results provide important insights into the genetic basis of RSCV development in clinical settings and highlight the role of c-di-GMP signaling in biofilm-associated resistance. Our findings underscore the need to target c-di-GMP pathways as a potential strategy for combating persistent P. aeruginosa infections.