{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T13:53:49Z","timestamp":1790949229816,"version":"4.1.0"},"reference-count":43,"publisher":"American Society for Microbiology","license":[{"start":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T00:00:00Z","timestamp":1790899200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T00:00:00Z","timestamp":1790899200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      <jats:italic toggle=\"yes\">Pseudomonas aeruginosa<\/jats:italic>\n                      is a major multidrug-resistant pathogen whose biofilm formation complicates treatment.\n                      <jats:underline>R<\/jats:underline>\n                      ugose\n                      <jats:underline>s<\/jats:underline>\n                      mall\n                      <jats:underline>c<\/jats:underline>\n                      olony\n                      <jats:underline>v<\/jats:underline>\n                      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\n                      <jats:sup>RifR<\/jats:sup>\n                      ::\n                      <jats:italic toggle=\"yes\">wspA<\/jats:italic>\n                      <jats:sup>AR8023-1<\/jats:sup>\n                      . 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\u2014a glutamine deletion at position 289 (\u0394Q289) in WspA. Complementation restored wild-type morphology, motility, and biofilm formation. Transcriptomics revealed significant upregulation of c-di-GMP metabolic genes in RSCVs (\n                      <jats:italic toggle=\"yes\">P<\/jats:italic>\n                      &lt; 0.001), suggesting that WspA\u0394Q289 increases diguanylate cyclase activity and elevates c-di-GMP synthesis. Intracellular c-di-GMP levels were ~3-fold higher in RSCVs than in wild-type and complemented strains (\n                      <jats:italic toggle=\"yes\">P<\/jats:italic>\n                      &lt; 0.001). Biofilm susceptibility testing demonstrated that \u03b2-lactam MBICs for RSCVs were 8- to 512-fold higher than those of wild-type and complement isolates. Introduction of the same WspA\u0394Q289 allele into\n                      <jats:italic toggle=\"yes\">P. aeruginosa<\/jats:italic>\n                      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 WspA\u0394289Q mutation that elevates c-di-GMP, driving RSCV formation and biofilm-mediated resistance.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        This study further characterizes a specific WspA\u0394289Q mutation in clinical\n                        <jats:italic toggle=\"yes\">Pseudomonas aeruginosa<\/jats:italic>\n                        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 \u03b2-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.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.00530-26","type":"journal-article","created":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T13:01:16Z","timestamp":1790946076000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["A\n                    <i>wspA<\/i>\n                    mutation in a clinical isolate of\n                    <i>Pseudomonas aeruginosa<\/i>\n                    results in rugose small colonies and enhances biofilm formation"],"prefix":"10.1128","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-3633-137X","authenticated-orcid":true,"given":"Qing","family":"Yu","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00a2xv884","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Infectious Diseases, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine","place":["Hangzhou, China"]},{"name":"Key Laboratory of Microbial Technology and Bioinformatics of Zhejiang Province","place":["Hangzhou, China"]},{"id":[{"id":"https:\/\/ror.org\/00a2xv884","id-type":"ROR","asserted-by":"publisher"}],"name":"Regional Medical Center for National Institute of Respiratory Diseases, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine","place":["Hangzhou, 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