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Mechanisms in Biofilm Formation, Tolerance and Control: 2nd Edition
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Mechanisms in Biofilm Formation, Tolerance and Control: 2nd Edition

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Microbiology".

Deadline for manuscript submissions: 20 June 2025 | Viewed by 9923

Special Issue Editors

Dr. Manuel Simões

grade E-Mail Website
Guest Editor
LEPABE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Rua Roberto Frias, s/n, 4200-465 Porto, Portugal
Interests: antimicrobial agents; emerging antimicrobial strategies; antimicrobial resistance; biofilms; plant secondary metabolites
Special Issues, Collections and Topics in MDPI journals
Dr. Lúcia Chaves Simões

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Guest Editor
CEB-Centre of Biological Engineering, Campus de Gualtar, University of Minho, 4710-057 Braga, Portugal
Interests: biofilm formation; biofilm control
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Research on biofilms has progressed rapidly in the last two decades. The scientific community has come to understand many things about the particular biology of microbial biofilms through a variety of microscopic, physical, chemical, and molecular techniques. Such advances provided fundamental insights for the management of biofilms by different approaches. For problematic biofilms, some studies seek to prevent biofilm formation, others aim to develop antimicrobial agents to treat existing biofilms, and others to disrupt the polymeric ties that bind the biofilms together. It is now clear that the study of biofilms requires a multidisciplinary approach, and that the knowledge on many of the aspects affecting biofilm formation and resistance remains superficial.

This Special Issue aims to collect the recent advances in biofilm research, particularly the mechanisms underlying biofilm formation and tolerance to antimicrobials. Studies on control approaches targeting specific biofilm mechanisms are within the aim of this Special Issue.

Dr. Manuel Simões
Dr. Lúcia Chaves Simões
Guest Editors

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Keywords

  • adhesion
  • antimicrobial action
  • antimicrobial resistance
  • biofilm development
  • biofilm matrix
  • biofilm structure
  • quorum sensing
  • motility
  • surfaces

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Related Special Issue

Published Papers (8 papers)

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20 pages, 8077 KiB  
Article
New Insights into the Mediation of Biofilm Formation by Three Core Extracellular Polysaccharide Biosynthesis Pathways in Pseudomonas aeruginosa
by Qianhui Liu, Qian Wu, Jiawen Liu, Tianming Xu, Jing Liu, Qin Wu, Pradeep K. Malakar, Yongheng Zhu, Yong Zhao and Zhaohuan Zhang
Int. J. Mol. Sci. 2025, 26(8), 3780; https://doi.org/10.3390/ijms26083780 - 17 Apr 2025
Viewed by 340
Abstract
Pseudomonas aeruginosa biofilms, driven by extracellular polysaccharides (EPSs), exacerbate pathogenicity and drug resistance, posing critical threats to public health. While EPS biosynthesis pathways are central to biofilm formation, their distinct contributions and regulatory dynamics remain incompletely understood. Here, we systematically dissect the roles [...] Read more.
Pseudomonas aeruginosa biofilms, driven by extracellular polysaccharides (EPSs), exacerbate pathogenicity and drug resistance, posing critical threats to public health. While EPS biosynthesis pathways are central to biofilm formation, their distinct contributions and regulatory dynamics remain incompletely understood. Here, we systematically dissect the roles of three core EPS pathways—Psl, Pel, and alginate—in biofilm architecture and function using multi-omics approaches. Key findings reveal Psl as the dominant regulator of biofilm elasticity and thickness, with its deletion disrupting chemotaxis, quorum sensing, and 3′,5′-Cyclic GMP (c-di-GMP)/amino acid metabolism. Pel redundantly enhances biofilm biomass, but elevates flagellar synthesis efficiency when Psl is absent. Alginate exhibited negligible transcriptional or metabolic influence on biofilms. These insights clarify hierarchical EPS contributions and highlight Psl as a priority target for therapeutic strategies to dismantle biofilm-mediated resistance. Full article
(This article belongs to the Special Issue Mechanisms in Biofilm Formation, Tolerance and Control: 2nd Edition)
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17 pages, 12673 KiB  
Article
Identification of Novel Staphylococcus aureus Core and Accessory Virulence Patterns in Chronic Rhinosinusitis
by Simon P. Goldie, Laurie C. Lau, Huw A. S. Jones, Philip G. Harries, Andrew F. Walls and Rami J. Salib
Int. J. Mol. Sci. 2025, 26(8), 3711; https://doi.org/10.3390/ijms26083711 - 14 Apr 2025
Viewed by 376
Abstract
Staphylococcus aureus (S. aureus) colonizes the nasal cavities of both healthy individuals and patients with chronic rhinosinusitis (CRS) with (CRSwNP) and without (CRSsNP) nasal polyps. Treatment-resistant S. aureus biofilms and intracellular persistence are common in CRS patients, requiring the expression of [...] Read more.
Staphylococcus aureus (S. aureus) colonizes the nasal cavities of both healthy individuals and patients with chronic rhinosinusitis (CRS) with (CRSwNP) and without (CRSsNP) nasal polyps. Treatment-resistant S. aureus biofilms and intracellular persistence are common in CRS patients, requiring the expression of specific virulence factor genes to transition into these forms. We hypothesized that S. aureus isolates from non-diseased controls, CRSsNP patients, and CRSwNP patients would exhibit distinct virulence factor patterns contributing to persistence and intracellular survival in CRS patients. Nasal swabs from seventy-seven individuals yielded S. aureus cultures in eight non-diseased controls, eight CRSsNP patients, and five CRSwNP patients. Whole-genome sequencing analyzed stress, antimicrobial resistance, and virulence genes, including plasmids and prophages. Four virulence factor gene patterns emerged: a core set (hlgA, icaC, hlgB, hlgC, hld, and aur) present in all isolates, and accessory sets, including the enterotoxin gene cluster (seo, sem, seu, sei, and sen) and a partial/complete invasive virulence factor set (splE, splA, splB, lukE, and lukD) (p = 0.001). CRSwNP isolates exhibited incomplete carriage of the core set, with frequent loss of scn, icaC, and hlgA (p < 0.05). These findings suggest that S. aureus has clusters of virulence factors that may act in concert to support the survival and persistence of the bacteria, resulting in enhanced pathogenicity. This may manifest clinically with resistant disease and refractoriness to antibiotics. Full article
(This article belongs to the Special Issue Mechanisms in Biofilm Formation, Tolerance and Control: 2nd Edition)
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14 pages, 3027 KiB  
Article
Antibiofilm Activities of Halogenated Pyrimidines Against Enterohemorrhagic Escherichia coli O157:H7
by Hyejin Jeon, Yong-Guy Kim, Jin-Hyung Lee and Jintae Lee
Int. J. Mol. Sci. 2025, 26(3), 1386; https://doi.org/10.3390/ijms26031386 - 6 Feb 2025
Cited by 1 | Viewed by 644
Abstract
Enterohemorrhagic Escherichia coli (EHEC) is a significant public health concern due to its ability to form biofilms, enhancing its resistance to antimicrobials and contributing to its persistence in food processing environments. Traditional antibiotics often fail to target these biofilms effectively, leading to increased [...] Read more.
Enterohemorrhagic Escherichia coli (EHEC) is a significant public health concern due to its ability to form biofilms, enhancing its resistance to antimicrobials and contributing to its persistence in food processing environments. Traditional antibiotics often fail to target these biofilms effectively, leading to increased bacterial resistance. This study aims to explore the efficacy of novel antibiofilm agents, specifically halogenated pyrimidine derivatives, against EHEC. We screened pyrimidine and 31 halogenated pyrimidine derivatives for their antimicrobial and antibiofilm activities against EHEC using biofilm quantification assays, SEM analysis, motility, and curli production assessments. Our findings reveal that certain halogenated pyrimidine derivatives, notably 2-amino-5-bromopyrimidine (2A5BP), 2-amino-4-chloropyrrolo[2,3-d]pyrimidine (2A4CPP), and 2,4-dichloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (2,4DC5IPP) at 50 µg/mL, exhibited significant inhibitory effects on EHEC biofilm formation without affecting bacterial growth, suggesting a targeted antibiofilm action. These compounds effectively reduced curli production and EHEC motility, essential factors for biofilm integrity and development. qRT-PCR analysis revealed that two active compounds downregulated the expression of key curli genes (csgA and csgB), leading to reduced bacterial adhesion and biofilm formation. Additionally, in silico ADME–Tox profiles indicated that these compounds exhibit favorable drug-like properties and lower toxicity compared with traditional pyrimidine. This study highlights the potential of halogenated pyrimidine derivatives as effective antibiofilm agents against EHEC, offering a promising strategy for enhancing food safety and controlling EHEC infections. The distinct mechanisms of action of these compounds, particularly in inhibiting biofilm formation and virulence factors without promoting bacterial resistance, underscore their therapeutic potential. Full article
(This article belongs to the Special Issue Mechanisms in Biofilm Formation, Tolerance and Control: 2nd Edition)
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21 pages, 6495 KiB  
Article
The Coexistence of Klebsiella pneumoniae and Candida albicans Enhanced Biofilm Thickness but Induced Less Severe Neutrophil Responses and Less Inflammation in Pneumonia Mice Than K. pneumoniae Alone
by Pornpimol Phuengmaung, Chiratchaya Chongrak, Wilasinee Saisorn, Jiradej Makjaroen, Uthaibhorn Singkham-in and Asada Leelahavanichkul
Int. J. Mol. Sci. 2024, 25(22), 12157; https://doi.org/10.3390/ijms252212157 - 12 Nov 2024
Viewed by 1744
Abstract
Due to the possible coexistence of Klebsiella pneumoniae (KP) and Candida albicans (CA), strains of KP and CA with biofilm production properties clinically isolated from patients were tested. The production of biofilms from the combined organisms (KP+CA) was higher than the biofilms from [...] Read more.
Due to the possible coexistence of Klebsiella pneumoniae (KP) and Candida albicans (CA), strains of KP and CA with biofilm production properties clinically isolated from patients were tested. The production of biofilms from the combined organisms (KP+CA) was higher than the biofilms from each organism alone, as indicated by crystal violet and z-stack immunofluorescence. In parallel, the bacterial abundance in KP + CA was similar to KP, but the fungal abundance was higher than CA (culture method), implying that CA grows better in the presence of KP. Proteomic analysis was performed to compare KP + CA biofilm to KP biofilm alone. With isolated mouse neutrophils (thioglycolate induction), KP + CA biofilms induced less prominent responses than KP biofilms, as determined by (i) neutrophilic supernatant cytokines (ELISA) and (ii) neutrophil extracellular traps (NETs), using immunofluorescent images (neutrophil elastase, myeloperoxidase, and citrullinated histone 3), peptidyl arginine deiminase 4 (PAD4) expression, and cell-free DNA. Likewise, intratracheal KP + CA in C57BL/6 mice induces less severe pneumonia than KP alone, as indicated by organ injury (serum creatinine and alanine transaminase) (colorimetric assays), cytokines (ELISA), bronchoalveolar lavage fluid parameters (bacterial culture and neutrophil abundances using a hemocytometer), histology score (H&E stains), and NETs (immunofluorescence on the lung tissue). In conclusion, the biofilm biomass of KP + CA was mostly produced from CA with less potent neutrophil activation and less severe pneumonia than KP alone. Hence, fungi in the respiratory tract might benefit the host in some situations, despite the well-known adverse effects of fungi. Full article
(This article belongs to the Special Issue Mechanisms in Biofilm Formation, Tolerance and Control: 2nd Edition)
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17 pages, 3258 KiB  
Article
BpfD Is a c-di-GMP Effector Protein Playing a Key Role for Pellicle Biosynthesis in Shewanella oneidensis
by Jean-Pierre Poli, Anne Boyeldieu, Alexandre Lutz, Amélie Vigneron-Bouquet, Amine Ali Chaouche, Marie-Thérèse Giudici-Orticoni, Michel Fons and Cécile Jourlin-Castelli
Int. J. Mol. Sci. 2024, 25(17), 9697; https://doi.org/10.3390/ijms25179697 - 7 Sep 2024
Cited by 2 | Viewed by 996
Abstract
The aquatic γ-proteobacterium Shewanella oneidensis is able to form two types of biofilms: a floating biofilm at the air–liquid interface (pellicle) and a solid surface-associated biofilm (SSA-biofilm). S. oneidensis possesses the Bpf system, which is orthologous to the Lap system first described in [...] Read more.
The aquatic γ-proteobacterium Shewanella oneidensis is able to form two types of biofilms: a floating biofilm at the air–liquid interface (pellicle) and a solid surface-associated biofilm (SSA-biofilm). S. oneidensis possesses the Bpf system, which is orthologous to the Lap system first described in Pseudomonas fluorescens. In the Lap systems, the retention of a large adhesin (LapA) at the cell surface is controlled by LapD, a c-di-GMP effector protein, and LapG, a periplasmic protease targeting LapA. Here, we showed that the Bpf system is mandatory for pellicle biogenesis, but not for SSA-biofilm formation, indicating that the role of Bpf is somewhat different from that of Lap. The BpfD protein was then proved to bind c-di-GMP via its degenerated EAL domain, thus acting as a c-di-GMP effector protein like its counterpart LapD. In accordance with its key role in pellicle formation, BpfD was found to interact with two diguanylate cyclases, PdgA and PdgB, previously identified as involved in pellicle formation. Finally, BpfD was shown to interact with CheY3, the response regulator controlling both chemotaxis and biofilm formation. Altogether, these results indicate that biofilm formation in S. oneidensis is under the control of a large c-di-GMP network. Full article
(This article belongs to the Special Issue Mechanisms in Biofilm Formation, Tolerance and Control: 2nd Edition)
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25 pages, 4067 KiB  
Article
Carbon Source and Substrate Surface Affect Biofilm Formation by the Plant-Associated Bacterium Pseudomonas donghuensis P482
by Magdalena Rajewska, Tomasz Maciąg, Magdalena Narajczyk and Sylwia Jafra
Int. J. Mol. Sci. 2024, 25(15), 8351; https://doi.org/10.3390/ijms25158351 - 30 Jul 2024
Viewed by 1419
Abstract
The ability of bacteria to colonize diverse environmental niches is often linked to their competence in biofilm formation. It depends on the individual characteristics of a strain, the nature of the colonized surface (abiotic or biotic), or the availability of certain nutrients. Pseudomonas [...] Read more.
The ability of bacteria to colonize diverse environmental niches is often linked to their competence in biofilm formation. It depends on the individual characteristics of a strain, the nature of the colonized surface (abiotic or biotic), or the availability of certain nutrients. Pseudomonas donghuensis P482 efficiently colonizes the rhizosphere of various plant hosts, but a connection between plant tissue colonization and the biofilm formation ability of this strain has not yet been established. We demonstrate here that the potential of P482 to form biofilms on abiotic surfaces and the structural characteristics of the biofilm are influenced by the carbon source available to the bacterium, with glycerol promoting the process. Also, the type of substratum, polystyrene or glass, impacts the ability of P482 to attach to the surface. Moreover, P482 mutants in genes associated with motility or chemotaxis, the synthesis of polysaccharides, and encoding proteases or regulatory factors, which affect biofilm formation on glass, were fully capable of colonizing the root tissue of both tomato and maize hosts. Investigating the role of cellular factors in biofilm formation using these plant-associated bacteria shows that the ability of bacteria to form biofilm on abiotic surfaces does not necessarily mirror its ability to colonize plant tissues. Our research provides a broader perspective on the adaptation of these bacteria to various environments. Full article
(This article belongs to the Special Issue Mechanisms in Biofilm Formation, Tolerance and Control: 2nd Edition)
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16 pages, 1925 KiB  
Article
Study of the Resistance of Staphylococcus aureus Biofilm, Biofilm-Detached Cells, and Planktonic Cells to Microencapsulated Carvacrol Used Alone or Combined with Low-pH Treatment
by Samah Mechmechani, Jina Yammine, Sakhr Alhuthali, Majededdine EL Mouzawak, Georgia Charvourou, Adem Gharsallaoui, Nour Eddine Chihib, Agapi Doulgeraki and Layal Karam
Int. J. Mol. Sci. 2024, 25(13), 7222; https://doi.org/10.3390/ijms25137222 - 29 Jun 2024
Viewed by 2142
Abstract
Microbial biofilms pose severe problems in the medical field and food industry, as they are the cause of many serious infections and food-borne diseases. The extreme biofilms’ resistance to conventional anti-microbial treatments presents a major challenge to their elimination. In this study, the [...] Read more.
Microbial biofilms pose severe problems in the medical field and food industry, as they are the cause of many serious infections and food-borne diseases. The extreme biofilms’ resistance to conventional anti-microbial treatments presents a major challenge to their elimination. In this study, the difference in resistance between Staphylococcus aureus DSMZ 12463 biofilms, biofilm-detached cells, and planktonic cells against microcapsules containing carvacrol was assessed. The antimicrobial/antibiofilm activity of low pH disinfection medium containing the microencapsulated carvacrol was also studied. In addition, the effect of low pH on the in vitro carvacrol release from microcapsules was investigated. The minimum inhibitory concentration of microencapsulated carvacrol was 0.625 mg mL−1. The results showed that biofilms exhibited greater resistance to microencapsulated carvacrol than the biofilm-detached cells and planktonic cells. Low pH treatment alone, by hydrochloric acid addition, showed no bactericidal effect on any of the three states of S. aureus strain. However, microencapsulated carvacrol was able to significantly reduce the planktonic cells and biofilm-detached cells below the detection limit (no bacterial counts), and the biofilm by approximatively 3 log CFU mL−1. In addition, results showed that microencapsulated carvacrol combined with low pH treatment reduced biofilm by more than 5 log CFU mL−1. Thus, the use of microencapsulated carvacrol in acidic environment could be a promising approach to combat biofilms from abiotic surfaces. Full article
(This article belongs to the Special Issue Mechanisms in Biofilm Formation, Tolerance and Control: 2nd Edition)
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8 pages, 1085 KiB  
Opinion
What Is a Biofilm? Lessons Learned from Interactions with Immune Cells
by Paweł Krzyżek
Int. J. Mol. Sci. 2024, 25(21), 11684; https://doi.org/10.3390/ijms252111684 - 30 Oct 2024
Viewed by 1118
Abstract
Biofilms are unique, multicellular life forms that challenge our understanding of the microbial functioning. The last decades of research on biofilms have allowed us to better understand their importance in the context of both health and various pathologies in the human body, although [...] Read more.
Biofilms are unique, multicellular life forms that challenge our understanding of the microbial functioning. The last decades of research on biofilms have allowed us to better understand their importance in the context of both health and various pathologies in the human body, although many knowledge gaps hindering their correct comprehension still exist. Biofilms are classically described as mushroom-shaped structures attached to the substrate; however, an increasing body of evidence shows that their morphology in clinical conditions may differ significantly from that classically presented. Although this may result partly from the unique physicochemical conditions within the host, the interaction between microbes and immune cells during development of a biofilm should not be underestimated. The current Opinion confronts the classical view on biofilms with the latest scientific research describing the vitality of interactions with immune cells as a modulator of the biofilm phenotype and behavior in clinical conditions. Full article
(This article belongs to the Special Issue Mechanisms in Biofilm Formation, Tolerance and Control: 2nd Edition)
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