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Cited by 34Bacterial biofilms and quorum sensingAntifungal resistance and susceptibilityAntimicrobial agents and applications

The DOI matches its Crossref record; 291 of the 293 references it cites were found in a registry or an index, and 2 are not checked yet; none of the 331 sources its claims cite is checked for support yet.

Understanding Pseudomonas aeruginosa Biofilms: Quorum Sensing, c-di-GMP Signaling, and Emerging Antibiofilm Approaches

Open access
Elbehiry, Ayman, Marzouk, Eman, Edrees, Husam M., Ibrahem, Mai, Alzahrani, Safiyah, Anagreyyah, Sulaiman, Abualola, Hussain, Alghamdi, Abdulaziz, Alzahrani, Ahmed, Jaber, Mahmoud, Abu-Okail, Akram·2026·Microorganisms
DOI 10.3390/microorganisms14010109PMC PMC12844471
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Abstract

Pseudomonas aeruginosa (P. aeruginosa) forms biofilms that are difficult to eliminate. The matrix protects the cells, efflux pumps reduce intracellular drug levels, and dormant subpopulations survive treatment. Routine minimum inhibitory concentration (MIC) testing does not account for these features, which helps explain why infections often continue even when therapy appears appropriate. This review describes how quorum-sensing (QS) and cyclic di-guanosine monophosphate (c-di-GMP) regulate matrix production, efflux activity, and dormancy within P. aeruginosa biofilms. Important matrix components, including Psl, Pel, alginate, and extracellular DNA, slow the movement of antimicrobial agents. Regulatory proteins such as sagS and brlR increase the activity of the MexAB-OprM and MexEF-OprN efflux systems, further reducing intracellular drug concentrations. Oxygen and nutrient limitation promote persister cells and viable but nonculturable cells, with both having the ability to survive antibiotic levels that would normally be lethal. These defenses explain the gap between MIC values and biofilm-specific measurements, such as the minimum biofilm inhibitory concentration and the minimum biofilm eradication concentration. This review also summarizes emerging antibiofilm strategies. These include QS inhibitors, compounds that lower c-di-GMP, such as nitric oxide donors, nanoparticles, depolymerases, bacteriophages, and therapies that are directed at host targets. Modern diagnostic tools, such as confocal laser scanning microscopy, optical coherence tomography, and Raman spectroscopy, improve detection and guide treatment planning. A staged therapeutic approach is presented that begins with the dispersal or loosening of the matrix, continues with targeted antibiotics, and concludes with support for immune clearance. Viewing these strategies within a One Health framework highlights the role of biofilms in clinical disease and in environmental reservoirs and supports more effective surveillance and prevention.

Checked 2026-11-15 · 2 references not checked yet

Overview

CoChat Summary

TL;DR

This review describes how quorum sensing (QS) and c-di-GMP signaling coordinate *Pseudomonas aeruginosa* biofilm matrix production, efflux, dormancy, and antimicrobial tolerance, explaining why MIC testing often fails to predict biofilm treatment outcomes. It then surveys emerging antibiofilm strategies—QS inhibitors, c-di-GMP modulators, nanoparticles, phages/depolymerases, and host-directed therapies—and argues for biofilm-aware diagnostics and a One Health approach.

Key findings

  • *P. aeruginosa* biofilms resist treatment through matrix components (Psl, Pel, alginate, eDNA), efflux pumps such as MexAB-OprM and MexEF-OprN, and dormant persister/VBNC subpopulations; MBIC and MBEC values often exceed planktonic MIC values by large margins.
  • The PAO1 genome is about 6.3 megabases, and c-di-GMP turnover involves approximately 41 proteins: about 17 GGDEF-only, 5 EAL-only, and 16 dual GGDEF–EAL proteins, with additional HD-GYP proteins annotated.
  • A thermosensory diguanylate cyclase increases its catalytic rate more than one hundred-fold across a ten-degree temperature change, linking environmental cues to biofilm lifestyle.
  • A MALDI-TOF feature of approximately 5.45 kilodaltons corresponding to PA2146 increases during *P. aeruginosa* biofilm growth on biopsy channel polymers and is proposed as a biofilm-stage marker.

Methods

  • Design: narrative review; no primary experiments, systematic search protocol, or meta-analysis are reported.
  • Data/sample: synthesis of published literature on *P. aeruginosa* biofilms, QS, c-di-GMP signaling, antimicrobial tolerance, antibiofilm therapies, diagnostics, and One Health, with references numbered 1–302.
  • Main analysis: thematic synthesis organized around biofilm architecture, QS and c-di-GMP regulatory networks, tolerance/persistence mechanisms, emerging therapeutic strategies, diagnostic tools, and translational barriers.

Limitations

  • The paper states that resistance can emerge to QS inhibitors and phage-based therapies, that there is currently no general biofilm producer database across ESKAPE pathogens, and that ISO 10993-5 cytotoxicity results show substantial interlaboratory variability.
  • Evident from the text: as a narrative review, it does not report systematic inclusion/exclusion criteria, search strategy, or risk-of-bias assessment, and it presents no primary experimental data.
  • Evident from the text: many therapeutic and diagnostic claims are based on cited preclinical or in vitro studies, with limited clinical validation described for several approaches.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.3390/microorganisms14010109

Cites

291 of 293 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Figures

Figure 1 The structural code of P. aeruginosa biofilms. A conceptual overview that summarizes how biofilms form, strengthen, and disperse. The panel on the left presents the staged progression from reversible attachment to maturation and active dispersal. These stages are guided by genetic checkpoints that include sadB, pel, psl, algD, and bdlA. The central panel illustrates EPS architecture, which contains Psl that mediates cell-to-cell and cell-to-surface adhesion, Pel that contributes to cohesion and tolerance, alginate that forms the mucoid matrix, CdrA that crosslinks with Psl, and eDNA that provides mechanical reinforcement. The panel on the right shows how oxygen and nutrient gradients create stratified physiology. These gradients support phenazine-linked redox activity and favor the development of drug-tolerant persister and VBNC subpopulations.
Figure 1 The structural code of P. aeruginosa biofilms. A conceptual overview that summarizes how biofilms form, strengthen, and disperse. The panel on the left presents the staged progression from reversible attachment to maturation and active dispersal. These stages are guided by genetic checkpoints that include sadB, pel, psl, algD, and bdlA. The central panel illustrates EPS architecture, which contains Psl that mediates cell-to-cell and cell-to-surface adhesion, Pel that contributes to cohesion and tolerance, alginate that forms the mucoid matrix, CdrA that crosslinks with Psl, and eDNA that provides mechanical reinforcement. The panel on the right shows how oxygen and nutrient gradients create stratified physiology. These gradients support phenazine-linked redox activity and favor the development of drug-tolerant persister and VBNC subpopulations.CC BY · PubMed Central
Figure 2 c-di-GMP network dynamics in P. aeruginosa. Environmental and host-related signals that include nitric oxide, temperature changes, and hypoxia or redox-linked cues sensed by PAS domain regulators converge on diguanylate cyclases that include WspR, sadC, and RoeA, and on phosphodiesterases such as BifA and RbdA. These enzymes set intracellular c-di-GMP levels and shape biofilm behavior. High c-di-GMP promotes matrix assembly. PelD activates Pel polysaccharide synthesis, and Alg44 promotes alginate polymerization. Low c-di-GMP, produced through PDE activity or nitric oxide-mediated reduction, favors motility and detachment. The schematic reads from left to right and shows inputs, enzymes, the c-di-GMP pool, and downstream effectors or phenotypes. The figure also highlights therapeutic intervention points at DGCs, PDEs, and c-di-GMP-dependent effectors. Abbreviations: DGC, diguanylate cyclase; PDE, phosphodiesterase; EPS, extracellular polymeric substance.
Figure 2 c-di-GMP network dynamics in P. aeruginosa. Environmental and host-related signals that include nitric oxide, temperature changes, and hypoxia or redox-linked cues sensed by PAS domain regulators converge on diguanylate cyclases that include WspR, sadC, and RoeA, and on phosphodiesterases such as BifA and RbdA. These enzymes set intracellular c-di-GMP levels and shape biofilm behavior. High c-di-GMP promotes matrix assembly. PelD activates Pel polysaccharide synthesis, and Alg44 promotes alginate polymerization. Low c-di-GMP, produced through PDE activity or nitric oxide-mediated reduction, favors motility and detachment. The schematic reads from left to right and shows inputs, enzymes, the c-di-GMP pool, and downstream effectors or phenotypes. The figure also highlights therapeutic intervention points at DGCs, PDEs, and c-di-GMP-dependent effectors. Abbreviations: DGC, diguanylate cyclase; PDE, phosphodiesterase; EPS, extracellular polymeric substance.CC BY · PubMed Central
Figure 3 Quorum-sensing and c-di-GMP networks reinforce antimicrobial tolerance in P. aeruginosa biofilms. QS hierarchies that include Las, Rhl, and Pqs, together with the second messenger c-di-GMP, function as coordinated regulatory hubs that strengthen tolerance in biofilm communities. Elevated c-di-GMP acts as a master lifestyle regulator and promotes EPS synthesis through Psl, Pel, and alginate, and increases eDNA accumulation, which restricts antibiotic penetration and buffers cationic agents. In parallel, QS and c-di-GMP enhance the expression of efflux regulators brlR and sagS, and RND pumps mexAB-oprM and mexEF-oprN, which improve intracellular detoxification. These combined interactions favor the formation of persister and VBNC populations and help maintain horizontal gene transfer and plasmid stability within the matrix. Together, this layered regulatory architecture produces a biofilm tolerance phenotype in which MBIC and MBEC values rise well above conventional MIC thresholds.
Figure 3 Quorum-sensing and c-di-GMP networks reinforce antimicrobial tolerance in P. aeruginosa biofilms. QS hierarchies that include Las, Rhl, and Pqs, together with the second messenger c-di-GMP, function as coordinated regulatory hubs that strengthen tolerance in biofilm communities. Elevated c-di-GMP acts as a master lifestyle regulator and promotes EPS synthesis through Psl, Pel, and alginate, and increases eDNA accumulation, which restricts antibiotic penetration and buffers cationic agents. In parallel, QS and c-di-GMP enhance the expression of efflux regulators brlR and sagS, and RND pumps mexAB-oprM and mexEF-oprN, which improve intracellular detoxification. These combined interactions favor the formation of persister and VBNC populations and help maintain horizontal gene transfer and plasmid stability within the matrix. Together, this layered regulatory architecture produces a biofilm tolerance phenotype in which MBIC and MBEC values rise well above conventional MIC thresholds.CC BY · PubMed Central
Figure 4 Mechanistic overview of network disruption strategies targeting the P. aeruginosa biofilm code. Four complementary therapeutic modules converge to dismantle biofilms and resensitize bacteria to antibiotics. Quorum-sensing inhibition uses small-molecule antagonists, signal-degrading enzymes, and CRISPR interference that targets lasI, rhlI, and pqsR to mute virulence and community signaling. Furthermore, c-di-GMP modulators, including PDE activators, DGC inhibitors, and nitric oxide donors, lower intracellular c-di-GMP and promote dispersal with a shift toward motile states. Matrix disruption relies on nanoparticles, depolymerases such as PslG, and mesoporous silica carriers to degrade EPS and improve antibiotic penetration. Host-directed and immunomodulatory approaches employ monoclonal antibodies such as MEDI3902, vaccines, and immune modulators to enhance clearance. Together, these strategies collapse the biofilm tolerance architecture and restore the effectiveness of conventional antibiotics.
Figure 4 Mechanistic overview of network disruption strategies targeting the P. aeruginosa biofilm code. Four complementary therapeutic modules converge to dismantle biofilms and resensitize bacteria to antibiotics. Quorum-sensing inhibition uses small-molecule antagonists, signal-degrading enzymes, and CRISPR interference that targets lasI, rhlI, and pqsR to mute virulence and community signaling. Furthermore, c-di-GMP modulators, including PDE activators, DGC inhibitors, and nitric oxide donors, lower intracellular c-di-GMP and promote dispersal with a shift toward motile states. Matrix disruption relies on nanoparticles, depolymerases such as PslG, and mesoporous silica carriers to degrade EPS and improve antibiotic penetration. Host-directed and immunomodulatory approaches employ monoclonal antibodies such as MEDI3902, vaccines, and immune modulators to enhance clearance. Together, these strategies collapse the biofilm tolerance architecture and restore the effectiveness of conventional antibiotics.CC BY · PubMed Central

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Citations

Cites

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Research works printed but not linked: 2

  1. Jia, J. (2021). Spatiotemporal Mapping of Quinolone and Phenazine Secretion in Pseudomonas aeruginosa Biofilms by Confocal Raman Imaging. [Ph.D. Thesis, University of Notre Dame].
  2. Pleguezuelos-Manzano, C., Beenker, W.A., van Son, G.J., Begthel, H., Amatngalim, G.D., Beekman, J.M., Clevers, H., and den Hertog, J. (2023). Establishment and characterization of a new Pseudomonas aeruginosa infection model using 2D airway organoids and dual RNA sequencing. bioRxiv. · DOI · Paper page

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Metadata

Authors
Elbehiry, A., Marzouk, E., Edrees, H. M., Ibrahem, M., Alzahrani, S., Anagreyyah, S., Abualola, H., Alghamdi, A., Alzahrani, A., Jaber, M., & Abu-Okail, A.
Affiliations
  • Qassim University (SA)
  • University of Tabuk (SA)
  • King Khalid University (SA)
  • Family Medicine Department, King Fahad Armed Hospital, Jeddah 23311, Saudi Arabia
  • Home Health Care Department, King Fahad Armed Forces Hospital, Jeddah 23311, Saudi Arabia
  • Prince Sultan Military College of Health Sciences (SA)
Corresponding author
Elbehiry, Ayman
ORCID
Venue
Microorganisms
Publisher
MDPI AG
Published
2026-01-04
Volume
14
Issue
1
Pages
109
ISSN
2076-2607 (ISSN-L)
DOI
10.3390/microorganisms14010109
PMC
PMC12844471
OpenAlex
W7118168642
Semantic Scholar
e7f25d8a025e648f1a1aed31f52cfb4aac2076b4
Cited by
34
References
291
Citations by year
2026: 34
FWCI
16.94
Type
journal article
Language
en
Open access
Licence
Locations
  • Microorganisms (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Qassim University (QU- APC-2026)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bacterial biofilms and quorum sensing
  • Health Sciences › Medicine › Infectious Diseases › Antifungal resistance and susceptibility
  • Life Sciences › Immunology and Microbiology › Applied Microbiology and Biotechnology › Antimicrobial agents and applications
Keywords
Pseudomonas aeruginosa, biofilm, quorum sensing, c-di-GMP signaling, biofilm matrix, efflux pumps, MexAB-OprM, MexEF-OprN, persister cells, viable but non-culturable bacteria, antibiotic tolerance, minimum biofilm eradication concentration, QS inhibitors, nitric oxide donors, bacteriophages, host-directed therapy, biofilm dispersal, One Health

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CoChat. (2026). Citation check: Understanding Pseudomonas aeruginosa Biofilms: Quorum Sensing, c-di-GMP Signaling, and Emerging Antibiofilm Approaches. https://app.cochat.ai/papers/10.3390/microorganisms14010109
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Elbehiry, A., Marzouk, E., Edrees, H. M., Ibrahem, M., Alzahrani, S., Anagreyyah, S., Abualola, H., Alghamdi, A., Alzahrani, A., Jaber, M., & Abu-Okail, A. (2026). Understanding Pseudomonas aeruginosa Biofilms: Quorum Sensing, c-di-GMP Signaling, and Emerging Antibiofilm Approaches. Microorganisms. https://doi.org/10.3390/microorganisms14010109

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