Abstract
ABSTRACT
Pseudomonas aeruginosa
is a major multidrug-resistant pathogen whose biofilm formation complicates treatment.
R
ugose
s
mall
c
olony
v
ariants (RSCVs) exhibit enhanced biofilm production and resistance, posing significant clinical challenges; however, their formation mechanisms in clinical strains remain incompletely understood. Paired clinical isolates AR8023-1 (wild type) and AR8023-2 (RSCV) were chronologically collected from urine samples of a neurosurgery inpatient. Genome comparison via Breseq and plasmid complementation constructed the complemented strain AR8023-2
RifR
::
wspA
AR8023-1
. All strains were examined for RSCV phenotype, biofilm formation, motility, transcriptomic changes, c-di-GMP quantification, and antimicrobial susceptibility under planktonic and biofilm conditions. Both isolates were identified as ST3420. AR8023-1 exhibited typical smooth morphology, while AR8023-2 displayed an RSCV phenotype with a single SNP—a glutamine deletion at position 289 (ΔQ289) in WspA. Complementation restored wild-type morphology, motility, and biofilm formation. Transcriptomics revealed significant upregulation of c-di-GMP metabolic genes in RSCVs (
P
P
P. aeruginosa
PAO1 did not recapitulate the RSCV phenotype, indicating that strain background contributes to the phenotypic outcome. This study confirms and extends the role of a clinical WspAΔ289Q mutation that elevates c-di-GMP, driving RSCV formation and biofilm-mediated resistance.
IMPORTANCE
This study further characterizes a specific WspAΔ289Q mutation in clinical
Pseudomonas aeruginosa
that is associated with activation of the c-di-GMP pathway, driving rugose small colony variant (RSCV) formation and hyper-biofilm phenotypes. The mutation markedly enhances biofilm-mediated β-lactam resistance, which is consistent with treatment failure. Complementation restores wild-type traits, supporting the importance of WspA structural integrity as a key regulator of colony morphology and antibiotic tolerance. These findings provide a molecular biomarker for RSCV surveillance and highlight c-di-GMP signaling as a therapeutic target against biofilm-associated chronic infections, although the phenotype is strain-dependent and the mutation occurs in a previously identified WspA hotspot.