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Mechanisms in Biofilm Formation, Tolerance and Control

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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: closed (15 December 2022) | Viewed by 28722

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Dr. Lúcia Chaves Simões

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CEB-Centre of Biological Engineering, Campus de Gualtar, University of Minho, 4710-057 Braga, Portugal
Interests: biofilm formation; biofilm control
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Dr. Manuel Simões

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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
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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 remain superficial.

This Special Issue aims to collect the recent advances in biofilm research, particularly on 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. Lúcia Chaves Simões
Dr. Manuel Simões
Guest Editors

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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. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

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Keywords

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

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Mechanisms in Biofilm Formation, Tolerance and Control 2.0 in International Journal of Molecular Sciences

Published Papers (12 papers)

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Editorial

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3 pages, 205 KiB

Open AccessEditorial

Contribution to Understanding the Mechanisms Involved in Biofilm Formation, Tolerance and Control

by Lúcia Chaves Simões and Manuel Simões

Int. J. Mol. Sci. 2023, 24(11), 9475; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24119475 - 30 May 2023

Cited by 1 | Viewed by 781

Abstract

Biofilms constitute a protected mode of growth that allows the colonizing microbial cells to survive in hostile environments, even when an antimicrobial agent is present. The scientific community has come to understand many things about the growth dynamics and behavior of microbial biofilms. It is now accepted that biofilm formation is a multifactorial process that starts with the adhesion of individual cells and (auto-)coaggregates of cells to a surface. Then, attached cells grow, reproduce and secrete insoluble extracellular polymeric substances. As the biofilm matures, biofilm detachment and growth processes come into balance, such that the total amount of biomass on the surface remains approximately constant in time. The detached cells retain the phenotype of the biofilm cells, which facilitates the colonization of neighboring surfaces. The most common practice to eliminate unwanted biofilms is the application of antimicrobial agents. However, conventional antimicrobial agents often show inefficacy in the control of biofilms. Much remains to be understood in the biofilm formation process and in the development of effective strategies for biofilm prevention and control. The articles contained in this Special Issue deal with biofilms of some important bacteria (including pathogens such as Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) and fungi (Candida tropicalis), providing novel insights into their formation mechanisms and implications, together with novel methods (e.g., use of chemical conjugates and combinations of molecules) that can be used to disrupt the biofilm structure and kill the colonizing cells. Full article

(This article belongs to the Special Issue Mechanisms in Biofilm Formation, Tolerance and Control)

Research

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18 pages, 6402 KiB

Open AccessArticle

Enhancement of Inhibition of the Pseudomonas sp. Biofilm Formation on Bacterial Cellulose-Based Wound Dressing by the Combined Action of Alginate Lyase and Gentamicin

by Magdalena Charęza, Katarzyna Przygrodzka, Anna Żywicka, Bartłomiej Grygorcewicz, Peter Sobolewski, Sylwia Mozia, Marcin Śmiglak and Radosław Drozd

Int. J. Mol. Sci. 2023, 24(5), 4740; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24054740 - 01 Mar 2023

Cited by 2 | Viewed by 1836

Abstract

Bacterial biofilms generally contribute to chronic infections, including wound infections. Due to the antibiotic resistance mechanisms protecting bacteria living in the biofilm, they are a serious problem in the wound healing process. To accelerate the wound healing process and avoid bacterial infection, it is necessary to select the appropriate dressing material. In this study, the promising therapeutic properties of alginate lyase (AlgL) immobilised on BC membranes for protecting wounds from Pseudomonas aeruginosa infection were investigated. The AlgL was immobilised on never dried BC pellicles via physical adsorption. The maximum adsorption capacity of AlgL was 6.0 mg/g of dry BC, and the equilibrium was reached after 2 h. The adsorption kinetics was studied, and it has been proven that the adsorption was consistent with Langmuir isotherm. In addition, the impact of enzyme immobilisation on bacterial biofilm stability and the effect of simultaneous immobilisation of AlgL and gentamicin on the viability of bacterial cells was investigated. The obtained results showed that the AlgL immobilisation significantly reduced the amount of polysaccharides component of the P. aeruginosa biofilm. Moreover, the biofilm disruption by AlgL immobilised on BC membranes exhibited synergism with the gentamicin, resulting in 86.5% more dead P. aeruginosa PAO-1 cells. Full article

(This article belongs to the Special Issue Mechanisms in Biofilm Formation, Tolerance and Control)

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20 pages, 4145 KiB

Open AccessArticle

Real-Time Impedance-Based Monitoring of the Growth and Inhibition of Osteomyelitis Biofilm Pathogen Staphylococcus aureus Treated with Novel Bisphosphonate-Fluoroquinolone Antimicrobial Conjugates

by Parish P. Sedghizadeh, Philip Cherian, Sahar Roshandel, Natalia Tjokro, Casey Chen, Adam F. Junka, Eric Hu, Jeffrey Neighbors, Jacek Pawlak, R. Graham G. Russell, Charles E. McKenna, Frank H. Ebetino, Shuting Sun and Esmat Sodagar

Int. J. Mol. Sci. 2023, 24(3), 1985; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24031985 - 19 Jan 2023

Cited by 2 | Viewed by 1747

Abstract

Osteomyelitis is a limb- and life-threatening orthopedic infection predominantly caused by Staphylococcus aureus biofilms. Bone infections are extremely challenging to treat clinically. Therefore, we have been designing, synthesizing, and testing novel antibiotic conjugates to target bone infections. This class of conjugates comprises bone-binding bisphosphonates as biochemical vectors for the delivery of antibiotic agents to bone minerals (hydroxyapatite). In the present study, we utilized a real-time impedance-based assay to study the growth of Staphylococcus aureus biofilms over time and to test the antimicrobial efficacy of our novel conjugates on the inhibition of biofilm growth in the presence and absence of hydroxyapatite. We tested early and newer generation quinolone antibiotics (ciprofloxacin, moxifloxacin, sitafloxacin, and nemonoxacin) and several bisphosphonate-conjugated versions of these antibiotics (bisphosphonate-carbamate-sitafloxacin (BCS), bisphosphonate-carbamate-nemonoxacin (BCN), etidronate-carbamate-ciprofloxacin (ECC), and etidronate-carbamate-moxifloxacin (ECX)) and found that they were able to inhibit Staphylococcus aureus biofilms in a dose-dependent manner. Among the conjugates, the greatest antimicrobial efficacy was observed for BCN with an MIC of 1.48 µg/mL. The conjugates demonstrated varying antimicrobial activity depending on the specific antibiotic used for conjugation, the type of bisphosphonate moiety, the chemical conjugation scheme, and the presence or absence of hydroxyapatite. The conjugates designed and tested in this study retained the bone-binding properties of the parent bisphosphonate moiety as confirmed using high-performance liquid chromatography. They also retained the antimicrobial activity of the parent antibiotic in the presence or absence of hydroxyapatite, albeit at lower levels due to the nature of their chemical modification. These findings will aid in the optimization and testing of this novel class of drugs for future applications to pharmacotherapy in osteomyelitis. Full article

(This article belongs to the Special Issue Mechanisms in Biofilm Formation, Tolerance and Control)

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14 pages, 2817 KiB

Open AccessArticle

The Arylamidine T-2307 as a Novel Treatment for the Prevention and Eradication of Candida tropicalis Biofilms

by Angela Maione, Alessandra La Pietra, Antonietta Siciliano, Aldo Mileo, Maria De Falco, Elisabetta de Alteriis, Marco Guida and Emilia Galdiero

Int. J. Mol. Sci. 2022, 23(24), 16042; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232416042 - 16 Dec 2022

Cited by 1 | Viewed by 1271

Abstract

Candida tropicalis is an emerging pathogen with a high mortality rate due to its virulence factors, including biofilm formation, that has important repercussions on the public health system. The ability of C. tropicalis to form biofilms, which are potentially more resistant to antifungal drugs and the consequent increasing antimicrobial resistance, highlights an urgent need for the development of novel antifungal. The present study analyzed the antibiofilm capacity of the arylamidine T-2307 on two strains of Candida tropicalis. Antimicrobial activity and time-killing assays were performed to evaluate the anticandidal effects of T-2307, the antibiofilm ability on biomass inhibition and eradication was evaluated by the crystal violet (CV) method. Furthermore, in Galleria mellonella infected larvae an increased survival after pre—and post- treatment with T-2307 was observed. The MTT test was used to determine the viability of immortalized human prostate epithelial cells (PNT1A) after exposure to different concentrations of T-2307. Levels of interleukin IL-4, IL-8, IL-10 were quantified after Candida infection of PNT1A cells and treatment. Active doses of T-2307 did not affect the viability of PNT1A cells, and drug concentrations of 0.005 or 0.01 µg mL⁻¹ inhibited the secretion of inflammatory cytokines. Taken together, these results provide new information on T-2307, indicating this drug as a new and promising alternative therapeutic option for the treatment of Candida infections. Full article

(This article belongs to the Special Issue Mechanisms in Biofilm Formation, Tolerance and Control)

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18 pages, 3359 KiB

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Assessment of the Antibiofilm Performance of Chitosan-Based Surfaces in Marine Environments

by Marta Lima, Luciana C. Gomes, Rita Teixeira-Santos, Maria J. Romeu, Jesus Valcarcel, José Antonio Vázquez, Miguel A. Cerqueira, Lorenzo Pastrana, Ana I. Bourbon, Ed D. de Jong, Jelmer Sjollema and Filipe J. Mergulhão

Int. J. Mol. Sci. 2022, 23(23), 14647; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232314647 - 24 Nov 2022

Cited by 4 | Viewed by 2386

Abstract

Marine biofouling is a natural process often associated with biofilm formation on submerged surfaces, creating a massive economic and ecological burden. Although several antifouling paints have been used to prevent biofouling, growing ecological concerns emphasize the need to develop new and environmentally friendly antifouling approaches such as bio-based coatings. Chitosan (CS) is a natural polymer that has been widely used due to its outstanding biological properties, including non-toxicity and antimicrobial activity. This work aims to produce and characterize poly (lactic acid) (PLA)-CS surfaces with CS of different molecular weight (Mw) at different concentrations for application in marine paints. Loligo opalescens pens, a waste from the fishery industry, were used as a CS source. The antimicrobial activity of the CS and CS-functionalized surfaces was assessed against Cobetia marina, a model proteobacterium for marine biofouling. Results demonstrate that CS targets the bacterial cell membrane, and PLA-CS surfaces were able to reduce the number of culturable cells up to 68% compared to control, with this activity dependent on CS Mw. The antifouling performance was corroborated by Optical Coherence Tomography since PLA-CS surfaces reduced the biofilm thickness by up to 36%, as well as the percentage and size of biofilm empty spaces. Overall, CS coatings showed to be a promising approach to reducing biofouling in marine environments mimicked in this work, contributing to the valorization of fishing waste and encouraging further research on this topic. Full article

(This article belongs to the Special Issue Mechanisms in Biofilm Formation, Tolerance and Control)

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20 pages, 6159 KiB

Open AccessArticle

The Assessment of Activity of Antiseptic Agents against Biofilm of Staphylococcus aureus Measured with the Use of Processed Microscopic Images

by Grzegorz Krasowski, Paweł Migdał, Marta Woroszyło, Karol Fijałkowski, Grzegorz Chodaczek, Joanna Czajkowska, Bartłomiej Dudek, Joanna Nowicka, Monika Oleksy-Wawrzyniak, Bartłomiej Kwiek, Justyna Paleczny, Malwina Brożyna and Adam Junka

Int. J. Mol. Sci. 2022, 23(21), 13524; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232113524 - 04 Nov 2022

Cited by 4 | Viewed by 2226

Abstract

Staphylococcal biofilms are major causative factors of non-healing wound infections. Their treatment algorithms recommend the use of locally applied antiseptic agents to counteract the spread of infection. The efficacy of antiseptics against biofilm is assessed in vitro by a set of standard quantitative and semi-quantitative methods. The development of software for image processing additionally allowed for the obtainment of quantitative data from microscopic images of biofilm dyed with propidium iodine and SYTO-9 reagents, differentiating dead cells from live ones. In this work, the method of assessment of the impact of antiseptic agents on staphylococcal biofilm in vitro, based on biofilms’ processed images, was proposed and scrutinized with regard to clinically relevant antiseptics, polyhexanide, povidone–iodine and hypochlorite. The standard quantitative culturing method was applied to validate the obtained data from processed images. The results indicated significantly higher activity of polyhexanide and povidone–iodine than hypochlorite against staphylococcal biofilm. Taking into account the fact that in vitro results of the efficacy of antiseptic agents against staphylococcal biofilm are frequently applied to back up their use in hospitals and ambulatory units, our work should be considered an important tool; providing reliable, quantitative data in this regard. Full article

(This article belongs to the Special Issue Mechanisms in Biofilm Formation, Tolerance and Control)

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16 pages, 1611 KiB

Open AccessArticle

Serratiopeptidase Affects the Physiology of Pseudomonas aeruginosa Isolates from Cystic Fibrosis Patients

by Marco Artini, Gianluca Vrenna, Marika Trecca, Vanessa Tuccio Guarna Assanti, Ersilia Vita Fiscarelli, Rosanna Papa and Laura Selan

Int. J. Mol. Sci. 2022, 23(20), 12645; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232012645 - 20 Oct 2022

Cited by 5 | Viewed by 1658

Abstract

Pseudomonas aeruginosa is frequently involved in cystic fibrosis (CF) airway infections. Biofilm, motility, production of toxins and the invasion of host cells are different factors that increase P. aeruginosa’s virulence. The sessile phenotype offers protection to bacterial cells and resistance to antimicrobials and host immune attacks. Motility also contributes to bacterial colonization of surfaces and, consequently, to biofilm formation. Furthermore, the ability to adhere is the prelude for the internalization into lung cells, a common immune evasion mechanism used by most intracellular bacteria, such as P. aeruginosa. In previous studies we evaluated the activity of metalloprotease serratiopeptidase (SPEP) in impairing virulence-related properties in Gram-positive bacteria. This work aimed to investigate SPEP’s effects on different physiological aspects related to the virulence of P. aeruginosa isolated from CF patients, such as biofilm production, pyoverdine and pyocyanin production and invasion in alveolar epithelial cells. Obtained results showed that SPEP was able to impair the attachment to inert surfaces as well as adhesion/invasion of eukaryotic cells. Conversely, SPEP’s effect on pyocyanin and pyoverdine production was strongly strain-dependent, with an increase and/or a decrease of their production. Moreover, SPEP seemed to increase swarming motility and staphylolytic protease production. Our results suggest that a large number of clinical strains should be studied in-depth before drawing definitive conclusions. Why different strains sometimes react in opposing ways to a specific treatment is of great interest and will be the object of future studies. Therefore, SPEP affects P. aeruginosa’s physiology by differently acting on several bacterial factors related to its virulence. Full article

(This article belongs to the Special Issue Mechanisms in Biofilm Formation, Tolerance and Control)

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21 pages, 3847 KiB

Open AccessArticle

The Medium Composition Impacts Staphylococcus aureus Biofilm Formation and Susceptibility to Antibiotics Applied in the Treatment of Bone Infections

by Justyna Paleczny, Malwina Brożyna, Ruth Dudek-Wicher, Karolina Dydak, Monika Oleksy-Wawrzyniak, Marcin Madziała, Marzenna Bartoszewicz and Adam Junka

Int. J. Mol. Sci. 2022, 23(19), 11564; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms231911564 - 30 Sep 2022

Cited by 3 | Viewed by 2206

Abstract

The biofilm-associated infections of bones are life-threatening diseases, requiring application of dedicated antibiotics in order to counteract the tissue damage and spread of microorganisms. The in vitro analyses on biofilm formation and susceptibility to antibiotics are frequently carried out using methods that do not reflect conditions at the site of infection. To evaluate the influence of nutrient accessibility on Staphylococcus aureus biofilm development in vitro, a cohesive set of analyses in three different compositional media was performed. Next, the efficacy of four antibiotics used in bone infection treatment, including gentamycin, ciprofloxacin, levofloxacin, and vancomycin, against staphylococcal biofilm, was also assessed. The results show a significant reduction in the ability of biofilm to grow in a medium containing elements occurring in the serum, which also translated into the diversified changes in the efficacy of used antibiotics, compared to the setting in which conventional media were applied. The differences indicate the need for implementation of adequate in vitro models that closely mimic the infection site. The results of the present research may be considered an essential step toward the development of in vitro analyses aiming to accurately indicate the most suitable antibiotic to be applied against biofilm-related infections of bones. Full article

(This article belongs to the Special Issue Mechanisms in Biofilm Formation, Tolerance and Control)

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16 pages, 5166 KiB

Open AccessArticle

Boron Derivatives Accelerate Biofilm Formation of Recombinant Escherichia coli via Increasing Quorum Sensing System Autoinducer-2 Activity

by Huan Chen, Cheng-Hai Yan, Yu-Fan Zhan, Li-Tian Geng, Lin-Lin Zhu, Lu-Chan Gong and Jun Wang

Int. J. Mol. Sci. 2022, 23(15), 8059; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23158059 - 22 Jul 2022

Cited by 6 | Viewed by 1847

Abstract

Boron is an essential element for autoinducer-2 (AI-2) synthesis of quorum sensing (QS) system, which affects bacterial collective behavior. As a living biocatalyst, biofilms can stably catalyze the activity of intracellular enzymes. However, it is unclear how boron affects biofilm formation in E. coli, particularly recombinant E. coli with intracellular enzymes. This study screened different boron derivatives to explore their effect on biofilm formation. The stress response of biofilm formation to boron was illuminated by analyzing AI-2 activity, extracellular polymeric substances (EPS) composition, gene expression levels, etc. Results showed that boron derivatives promote AI-2 activity in QS system. After treatment with H₃BO₃ (0.6 mM), the AI-2 activity increased by 65.99%, while boron derivatives increased the biomass biofilms in the order H₃BO₃ > NaBO₂ > Na₂B₄O₇ > NaBO₃. Moreover, treatment with H₃BO₃ (0.6 mM) increased biomass by 88.54%. Meanwhile, AI-2 activity had a linear correlation with polysaccharides and protein of EPS at 0–0.6 mM H₃BO₃ and NaBO₂ (R² > 0.8). Furthermore, H₃BO₃ upregulated the expression levels of biofilm formation genes, quorum sensing genes, and flagellar movement genes. These findings demonstrated that boron promoted biofilm formation by upregulating the expression levels of biofilm-related genes, improving the QS system AI-2 activity, and increasing EPS secretion in E. coli. Full article

(This article belongs to the Special Issue Mechanisms in Biofilm Formation, Tolerance and Control)

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14 pages, 4146 KiB

Open AccessArticle

Bioadhesion on Textured Interfaces in the Human Oral Cavity—An In Situ Study

by Ralf Helbig, Matthias Hannig, Sabine Basche, Janis Ortgies, Sebastian Killge, Christian Hannig and Torsten Sterzenbach

Int. J. Mol. Sci. 2022, 23(3), 1157; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23031157 - 21 Jan 2022

Cited by 3 | Viewed by 1828

Abstract

Extensive biofilm formation on materials used in restorative dentistry is a common reason for their failure and the development of oral diseases like peri-implantitis or secondary caries. Therefore, novel materials and strategies that result in reduced biofouling capacities are urgently sought. Previous research suggests that surface structures in the range of bacterial cell sizes seem to be a promising approach to modulate bacterial adhesion and biofilm formation. Here we investigated bioadhesion within the oral cavity on a low surface energy material (perfluorpolyether) with different texture types (line-, hole-, pillar-like), feature sizes in a range from 0.7–4.5 µm and graded distances (0.7–130.5 µm). As a model system, the materials were fixed on splints and exposed to the oral cavity. We analyzed the enzymatic activity of amylase and lysozyme, pellicle formation, and bacterial colonization after 8 h intraoral exposure. In opposite to in vitro experiments, these in situ experiments revealed no clear signs of altered bacterial surface colonization regarding structure dimensions and texture types compared to unstructured substrates or natural enamel. In part, there seemed to be a decreasing trend of adherent cells with increasing periodicities and structure sizes, but this pattern was weak and irregular. Pellicle formation took place on all substrates in an unaltered manner. However, pellicle formation was most pronounced within recessed areas thereby partially masking the three-dimensional character of the surfaces. As the natural pellicle layer is obviously the most dominant prerequisite for bacterial adhesion, colonization in the oral environment cannot be easily controlled by structural means. Full article

(This article belongs to the Special Issue Mechanisms in Biofilm Formation, Tolerance and Control)

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15 pages, 4079 KiB

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Using Knock-Out Mutants to Investigate the Adhesion of Staphylococcus aureus to Abiotic Surfaces

by Christian Spengler, Friederike Nolle, Nicolas Thewes, Ben Wieland, Philipp Jung, Markus Bischoff and Karin Jacobs

Int. J. Mol. Sci. 2021, 22(21), 11952; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222111952 - 04 Nov 2021

Cited by 4 | Viewed by 1932

Abstract

The adhesion of Staphylococcus aureus to abiotic surfaces is crucial for establishing device-related infections. With a high number of single-cell force spectroscopy measurements with genetically modified S. aureus cells, this study provides insights into the adhesion process of the pathogen to abiotic surfaces of different wettability. Our results show that S. aureus utilizes different cell wall molecules and interaction mechanisms when binding to hydrophobic and hydrophilic surfaces. We found that covalently bound cell wall proteins strongly interact with hydrophobic substrates, while their contribution to the overall adhesion force is smaller on hydrophilic substrates. Teichoic acids promote adhesion to hydrophobic surfaces as well as to hydrophilic surfaces. This, however, is to a lesser extent. An interplay of electrostatic effects of charges and protein composition on bacterial surfaces is predominant on hydrophilic surfaces, while it is overshadowed on hydrophobic surfaces by the influence of the high number of binding proteins. Our results can help to design new models of bacterial adhesion and may be used to interpret the adhesion of other microorganisms with similar surface properties. Full article

(This article belongs to the Special Issue Mechanisms in Biofilm Formation, Tolerance and Control)

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Review

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Biofilms in Diabetic Foot Ulcers: Impact, Risk Factors and Control Strategies

by Ana C. Afonso, Diana Oliveira, Maria José Saavedra, Anabela Borges and Manuel Simões

Int. J. Mol. Sci. 2021, 22(15), 8278; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22158278 - 31 Jul 2021

Cited by 45 | Viewed by 7431

Abstract

Diabetic foot ulcers (DFUs) are a serious complication from diabetes mellitus, with a huge economic, social and psychological impact on the patients’ life. One of the main reasons why DFUs are so difficult to heal is related to the presence of biofilms. Biofilms promote wound inflammation and a remarkable lack of response to host defences/treatment options, which can lead to disease progression and chronicity. In fact, appropriate treatment for the elimination of these microbial communities can prevent the disease evolution and, in some cases, even avoid more serious outcomes, such as amputation or death. However, the detection of biofilm-associated DFUs is difficult due to the lack of methods for diagnostics in clinical settings. In this review, the current knowledge on the involvement of biofilms in DFUs is discussed, as well as how the surrounding environment influences biofilm formation and regulation, along with its clinical implications. A special focus is also given to biofilm-associated DFU diagnosis and therapeutic strategies. An overview on promising alternative therapeutics is provided and an algorithm considering biofilm detection and treatment is proposed. Full article

(This article belongs to the Special Issue Mechanisms in Biofilm Formation, Tolerance and Control)

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