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Microorganisms
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Microbial Biofilms: New Insights into Formation, Resistance and Control
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Microbial Biofilms: New Insights into Formation, Resistance and Control

A special issue of Microorganisms (ISSN 2076-2607). This special issue belongs to the section "Biofilm".

Deadline for manuscript submissions: closed (31 March 2026) | Viewed by 13376

Special Issue Editor

Dr. Marius Stefan

E-Mail Website
Guest Editor
BioActive Research Group, Faculty of Biology, University Alexandru Ioan Cuza of Iasi, Iasi, Romania
Interests: plant–microbial interactions; PGPR; biofertilizers; biopesticides
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Biofilms are structured microbial communities formed on different surfaces which play an important role for the protection and survival of microorganisms against environmental stresses. Adherent cells are well known for their resilience and resistance to different antimicrobial agents. Therefore, biofilms pose significant medical challenges when pathogenic bacteria or fungi adhere to medical devices and wounds, leading to chronic infections that are difficult and sometimes impossible to treat. Understanding biofilm formation, as well as their structure, may provide essential information for developing new effective strategies to prevent biofilm formation or eradicate existing biofilms, leading to innovative therapeutic solutions.

Authors are invited to submit their latest original findings on biofilm formation, structure, and communication of biofilm cells as well as strategies used to control microbial biofilms to this Microorganisms Special Issue entitled “Microbial Biofilms: New Insights into Formation, Resistance and Control”.

Dr. Marius Stefan
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 250 words) can be sent to the Editorial Office for assessment.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Microorganisms is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • biofilm formation
  • biofilm surfaces
  • biofilm structure
  • multi-species biofilms
  • microbial communities
  • biofilm communication
  • biofilm cells resistance
  • biofilm control
  • new antibiofilm agents

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Published Papers (4 papers)

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Research

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23 pages, 25297 KB  
Article
Bacterial Adhesion on Soft Surfaces: The Dual Role of Substrate Stiffness and Bacterial Growth Stage
by René Riedel, Garima Rani and Anupam Sengupta
Microorganisms 2025, 13(3), 637; https://doi.org/10.3390/microorganisms13030637 - 11 Mar 2025
Cited by 6 | Viewed by 4485
Abstract
The surface adhesion and stiffness of underlying substrates mediate the geometry, mechanics, and self-organization of expanding bacterial colonies. Recent studies have qualitatively indicted that stiffness may impact bacterial attachment and accumulation, yet the variation in the cell-to-surface adhesion with substrate stiffness remains to [...] Read more.
The surface adhesion and stiffness of underlying substrates mediate the geometry, mechanics, and self-organization of expanding bacterial colonies. Recent studies have qualitatively indicted that stiffness may impact bacterial attachment and accumulation, yet the variation in the cell-to-surface adhesion with substrate stiffness remains to be quantified. Here, by developing a cell-level force–distance spectroscopy (FDS) technique based on atomic force microscopy (AFM), we simultaneously quantify the cell–surface adhesion and stiffness of the underlying substrates to reveal the stiffness-dependent adhesion of the phototrophic bacterium Chromatium okenii. As the stiffness of the soft substrate, modeled using a low-melting-point (LMP) agarose pad, was varied between 20 kPa and 120 kPa by changing the agarose concentrations, we observed a progressive increase in the mean adhesion force by over an order of magnitude, from 0.21±0.10 nN to 2.42±1.16 nN. In contrast, passive polystyrene (PS) microparticles of comparable dimensions showed no perceptible change in their surface adhesion, confirming that the stiffness-dependent adhesive interaction of C. okenii is of a biological origin. Furthermore, for Escherichia coli, the cell–surface adhesion varied between 0.29±0.17 nN and 0.39±0.20 nN, showing a weak dependence on the substrate stiffness, thus suggesting that stiffness-modulated adhesion is a species-specific trait. Finally, by quantifying the adhesion of the C. okenii population across different timescales, we reported the emergent co-existence of weak and strongly adherent sub-populations, demonstrating diversification of the adherent phenotypes over the growth stages. Taken together, these findings suggest that bacteria, depending on the species and their physiological stage, may actively modulate cell-to-surface adhesion in response to the stiffness of soft surfaces. While the surface properties, for instance, hydrophobicity (or hydrophilicity), play a key role in mediating bacterial attachment, this work introduces substrate stiffness as a biophysical parameter that could reinforce or suppress effective surface interactions. Our results suggest how bacteria could leverage stiffness-dependent adhesion and the diversity therein as functional traits to modulate their initial attachment to, colonization of, and proliferation on soft substrates during the early stages of biofilm development. Full article
(This article belongs to the Special Issue Microbial Biofilms: New Insights into Formation, Resistance and Control)
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15 pages, 7584 KB  
Article
5-Fluorouracil Inhibits Bacterial Growth and Reduces Biofilm in Addition to Having Synergetic Effects with Gentamicin Against Pseudomonas aeruginosa
by Amani A. Niazy, May M. Alrashed, Rhodanne Nicole A. Lambarte and Abdurahman A. Niazy
Microorganisms 2024, 12(11), 2257; https://doi.org/10.3390/microorganisms12112257 - 7 Nov 2024
Cited by 13 | Viewed by 2473
Abstract
Pseudomonas aeruginosa is a multidrug-resistant pathogen known for chronic infections, mainly due to biofilm formation. This study aimed to explore the potential repurposing of 5-fluorouracil (5-FU), an anticancer drug, to treat P. aeruginosa infections. Firstly, we investigated the inhibitory effects of 5-FU on [...] Read more.
Pseudomonas aeruginosa is a multidrug-resistant pathogen known for chronic infections, mainly due to biofilm formation. This study aimed to explore the potential repurposing of 5-fluorouracil (5-FU), an anticancer drug, to treat P. aeruginosa infections. Firstly, we investigated the inhibitory effects of 5-FU on bacterial growth using the microdilution method. Secondly, the impact of 5-FU on biofilm formation and disassembly was assessed via biofilm biomass measurements with the crystal violet staining method and confocal microscopy analyses. Lastly, the potential synergy between 5-FU and the antibiotics gentamicin and meropenem was evaluated using a checkerboard assay. Results revealed that 5-FU inhibited bacterial growth in a dose-dependent manner, with 100% inhibition observed at concentrations of 25 µg/mL and higher. Also, 70% and 100% reductions in biofilm biomass were demonstrated at concentrations of 12 and 100 µg/mL, respectively. Controversy, these higher concentrations unexpectedly increased biofilm biomass in pre-formed biofilms. Synergistic interactions were observed between 5-FU and gentamicin in both growth inhibition (FICI 0.31) and biofilm inhibition (ZIP 14.1), while no synergy was found with meropenem. These findings highlight the potential of 5-FU as an adjunctive therapy for P. aeruginosa infections, especially in combination with gentamicin. However, further research is required to address 5-FU limitations against mature biofilms. Full article
(This article belongs to the Special Issue Microbial Biofilms: New Insights into Formation, Resistance and Control)
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Review

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33 pages, 1475 KB  
Review
Understanding Pseudomonas aeruginosa Biofilms: Quorum Sensing, c-di-GMP Signaling, and Emerging Antibiofilm Approaches
by Ayman Elbehiry, Eman Marzouk, Husam M. Edrees, Mai Ibrahem, Safiyah Alzahrani, Sulaiman Anagreyyah, Hussain Abualola, Abdulaziz Alghamdi, Ahmed Alzahrani, Mahmoud Jaber and Akram Abu-Okail
Microorganisms 2026, 14(1), 109; https://doi.org/10.3390/microorganisms14010109 - 4 Jan 2026
Cited by 4 | Viewed by 2720
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Microbial Biofilms: New Insights into Formation, Resistance and Control)
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42 pages, 2218 KB  
Review
A Collection and Analysis of Simplified Data for a Better Understanding of the Complex Process of Biofilm Inactivation by Ultraviolet and Visible Irradiation
by Martin Hessling, Wendy Meulebroeck and Beatrix Alsanius
Microorganisms 2025, 13(9), 2048; https://doi.org/10.3390/microorganisms13092048 - 3 Sep 2025
Cited by 1 | Viewed by 2154
Abstract
Biofilms are communities of microorganisms that pose a problem in many areas, including the food industry, drinking water treatment, and medicine, because they can contain pathogens and are difficult to eliminate. For this reason, the possibility of biofilm reduction by ultraviolet (UV) or [...] Read more.
Biofilms are communities of microorganisms that pose a problem in many areas, including the food industry, drinking water treatment, and medicine, because they can contain pathogens and are difficult to eliminate. For this reason, the possibility of biofilm reduction by ultraviolet (UV) or visible light was investigated using data from published reports. Results for different applications, spectral ranges, and microorganisms were compared by performing MANOVA tests. Approximately 140 publications were found that dealt with the irradiation of water or surfaces for biofilm reduction or reduction in biofilm formation. Irradiation of surfaces with UV or visible light in the spectral range 200–525 nm had a positive effect on biofilm reduction and reduction in biofilm formation, although the results for irradiation of water were conflicting. Most investigations were carried out on P. aeruginosa biofilms, but other Gram-positive and Gram-negative bacteria, as well as some fungi and their biofilm sensitivities to irradiation, were also analyzed. Limited data were available for the UVB (280–315 nm) and UVA (315–400 nm) range. Most experiments to date have been carried out in the UVC (100–280 nm) or in the visible violet/blue spectral (400–500 nm) range, with the UVC range being 2–3 orders of magnitude more efficient in terms of applied irradiation dose. Other quantitative statements were difficult to make as the results from the different working groups were highly scattered. Irradiation can reduce the microorganisms in biofilms but does not completely remove biofilms. New biofilm formation can at least be delayed by surface irradiation. Whether it is also possible to prevent the formation of new biofilms in the long term is open to question. Which irradiation wavelengths are optimal for anti-biofilm measures is also still unclear. Full article
(This article belongs to the Special Issue Microbial Biofilms: New Insights into Formation, Resistance and Control)
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