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Antimicrobial Resistance in Biofilm-Associated Infections

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A special issue of Antibiotics (ISSN 2079-6382). This special issue belongs to the section "Antibiofilm Strategies".

Deadline for manuscript submissions: closed (30 April 2026) | Viewed by 16402

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Special Issue Editors

Dr. Fazlurrahman Khan

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Guest Editor

Ocean and Fisheries Development International Cooperation Institute, Pukyong National University, Busan, Republic of Korea
Interests: probiotics; bacteria; food biochemistry; biofilm; natural and synthetic compound; antimicrobial; antibiofilm; nanomaterials
Special Issues, Collections and Topics in MDPI journals

Dr. Nazia Tabassum

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Guest Editor

Marine Integrated Biomedical Technology Center, The National Key Research Institutes in Universities, Pukyong National University, Busan, Republic of Korea
Interests: biofilm; antibiofilm; antivirulence; microbial pathogenesis; antimicrobial resistance; bacteria-fungi interaction; nanoparticles

Prof. Dr. Young-Mog Kim

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Guest Editor

Department of Food Science and Technology, Pukyong National University, Busan, Republic of Korea
Interests: antibiofilm drugs; antivirulence drugs; secondary metabolites; microbial pathogenesis; synthesis of nanocomposites; natural antimicrobial products
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Biofilms, organized colonies of microorganisms enclosed in a self-produced matrix of extracellular polymeric substances, are common in natural and artificial settings. They are known to play an important role in chronic infections and are notoriously hard to treat because of their intrinsic resistance mechanisms. Biofilm-related infections are common in a variety of clinical settings, including persistent wound infections, dental plaque, and infections linked with medical implants and devices. These infections are particularly difficult to treat because of their strong resistance to traditional antimicrobial therapy. The biofilm's physical barrier, together with the changed microenvironment and microbial phenotype, greatly reduces the effectiveness of antibiotics.

In this Special Issue, we welcome submissions encompassing cutting-edge research and reviews that investigate the multidimensional nature of antimicrobial resistance in biofilms. The papers should emphasize recent developments in understanding the molecular and cellular processes that contribute to pathogen persistence and resistance in biofilms. Key themes include the following:

Mechanisms of Resistance: How biofilm formations and microbial populations contribute to antimicrobial resistance. Studies should show the importance of efflux pumps, altered metabolic states, and the protective biofilm matrix.
Diagnostic and Treatment Strategies: Improvements in diagnostic tools for detecting biofilm-associated infections, innovative antimicrobial drugs, and physical modalities such as ultrasound or photodynamic therapy.
Emerging Therapies: The development of novel treatment strategies, such as bacteriophage therapy, quorum sensing inhibitors, and procedures (natural, synthetic, and nanomaterials), for disrupting biofilm formation and improving drug penetration.
Clinical Implications. The real-world consequences of biofilm-associated antimicrobial resistance, which include case studies and clinical experiences, highlighting the need for innovative preventative and treatment measures.

As the burden of biofilm-associated diseases expands, the research described in this Special Issue is an important step toward understanding and resolving the challenges faced by antibiotic resistance. Combining these findings will accelerate the development of more effective therapies while improving patient outcomes.

Dr. Fazlurrahman Khan
Dr. Nazia Tabassum
Prof. Dr. Young-Mog Kim
Guest Editors

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. Antibiotics 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 2900 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

biofilms
antimicrobial resistance
chronic infections
biofilm matrix
virulence factors
antibiotic efficacy
pathogen persistence
biofilm formation
medical implants
quorum sensing

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Published Papers (6 papers)

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Research

20 pages, 14008 KB

Open AccessArticle

The Antimicrobial Peptide CRAMP-34 Eradicates Escherichia coli Biofilms by Interfering with the kduD-Dependent Network

by Hongzao Yang, Jing Xiong, Sisi Su, Zhuo Yang, Wu Yang, Lianci Peng, Suhui Zhang, Jinjie Qiu, Yuzhang He and Hongwei Chen

Antibiotics 2026, 15(1), 83; https://doi.org/10.3390/antibiotics15010083 - 14 Jan 2026

Viewed by 789

Abstract

Background/Objectives: Bacterial biofilms formed by Escherichia coli pose a significant challenge in veterinary medicine due to their intrinsic resistance to antibiotics. Antimicrobial peptides (AMPs) represent a promising alternative. AMPs exert their bactericidal activity by binding to negatively charged phospholipids in bacterial membranes via electrostatic interactions, leading to membrane disruption and rapid cell lysis. Methods: In vitro assays including MIC determination, biofilm eradication testing (crystal violet, colony counts, and CLSM), swimming motility, and EPS quantification were performed. CRISPR/Cas9 was used to construct and complement a kduD mutant. A transposon mutagenesis library was screened for biofilm-defective mutants. In an in vivo murine excisional wound infection model treated with the mouse cathelicidin-related antimicrobial peptide (CRAMP-34), wound closure and bacterial burden were monitored. Gene expression changes were analyzed via RT-qPCR. Results: CRAMP-34 effectively eradicated pre-formed biofilms of a clinically relevant, porcine-origin E. coli strain and promoted wound healing in the murine infection model. We conducted a genome-wide transposon mutagenesis screen, which identified kduD as a critical gene for robust biofilm formation. Functional characterization revealed that kduD deletion drastically impairs flagellar motility and alters exopolysaccharide production, leading to defective biofilm architecture without affecting growth. Notably, the anti-biofilm activity of CRAMP-34 phenocopied aspects of the kduD deletion, including motility inhibition and transcriptional repression of a common set of biofilm-related genes. Conclusions: This research highlights CRAMP-34 as a potent anti-biofilm agent and unveils kduD as a previously unrecognized regulator of E. coli biofilm development, which is also targeted by CRAMP-34. Full article

(This article belongs to the Special Issue Antimicrobial Resistance in Biofilm-Associated Infections)

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18 pages, 4278 KB

Open AccessArticle

Attenuation of Staphylococcus aureus Biofilms and Virulence by 3-Fluorocatechol

by Taehyeong Kim, Nazia Tabassum, Aqib Javaid and Fazlurrahman Khan

Antibiotics 2025, 14(12), 1240; https://doi.org/10.3390/antibiotics14121240 - 8 Dec 2025

Cited by 1 | Viewed by 1016

Abstract

Background/Objectives: Staphylococcus aureus is a well-known opportunistic pathogen that causes a wide range of infections, from cutaneous blemishes to potentially fatal systemic diseases. The increasing prevalence of antibiotic-resistant bacteria highlights the critical need for alternative therapeutic methods that target virulence factors rather than growth. Methods: The antibacterial activity of 3-fluorocatechol (3-FC) against bacterial and fungal pathogens (e.g., Candida albicans) was determined by broth microdilution to establish the lowest inhibitory concentration. The antibiofilm impact of 3-FC against S. aureus was evaluated using crystal violet staining and viable colony counts, followed by scanning electron microscopy to visualize the biofilm architecture. The methanol extraction method was used to quantify staphyloxanthin synthesis in S. aureus cells. Furthermore, in silico molecular docking was used to evaluate 3-FC binding interactions and provide mechanistic insight into its impacts on S. aureus biofilms and virulence-associated factors. Results: Although the study showed that 3-FC exhibits weak antibacterial activity against S. aureus (MIC > 2048 µg/mL), it shows effective inhibition of up to 86.5% at sub-inhibitory doses during the initial stage of biofilm formation. The CFU enumeration also confirms the significant reduction of viable cell count of S. aureus in the presence of sub-MIC of 3-FC. The SEM analysis confirms disruption of the S. aureus biofilm architecture in the presence of a sub-MIC of 3-FC. Furthermore, the eradication of mature S. aureus biofilm at a sub-MIC dose of 3-FC was 60.6%. 3-FC significantly reduced staphyloxanthin formation, a vital antioxidant pigment that contributes to bacterial pathogenicity, with a maximal suppression of 66.3% at 2048 µg/mL. Molecular docking analyses provide further insight into the molecular basis of 3-FC activity, revealing strong binding affinities with numerous S. aureus virulence regulators and enzymes, suggesting interference with quorum-sensing, adhesion, and oxidative-stress response pathways. Conclusions: Collectively, our findings indicate that 3-FC has antibiofilm and antivirulence properties against S. aureus. Furthermore, this study suggests 3-FC as a viable structural scaffold for the development of a novel anti-infective agent to treat chronic staphylococcal infections. Full article

(This article belongs to the Special Issue Antimicrobial Resistance in Biofilm-Associated Infections)

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16 pages, 843 KB

Open AccessArticle

α-Amylase-Mediated Antibiotic Degradation and Sequestration in Pseudomonas aeruginosa Biofilm Therapy

by Robert K. Murray, Allison E. Martin, Sarah Zipkowitz, Nusrat Jahan, Tony D. Davis and Whitni K. Redman

Antibiotics 2025, 14(9), 941; https://doi.org/10.3390/antibiotics14090941 - 18 Sep 2025

Cited by 2 | Viewed by 1983

Abstract

Background: As of 2022, 80% of all documented microbial infections are biofilm-associated: communities of microorganisms adhered to a surface and enclosed in a complex extracellular polymeric substance (EPS). The EPS acts as a physical barrier protecting the bacteria from antimicrobial agents and host immune responses. To combat this hurdle, the application of glycoside hydrolases (GH) has been investigated due to their ability to cleave particular structural polysaccharides within the EPS, thus breaking down the protective barrier and improving antibiotic clearance. While various studies demonstrate the capacity of GHs to improve antibiotic efficacy against biofilms in combination, there is clear differential success between these treatments depending on the GH and antibiotic chosen. Due to the overlap of GH targets and antibiotic structures, it is imperative to ensure that the antibiotics in combinatorial treatments are not degraded by the GH. Methods: This study aimed to screen the GH α-amylase produced from Aspergillus oryzae (AO) and Bacillus subtilis (BS), combined with various antibiotics from different classes, charges, and mode of actions by determining MICs. against the bacterium Pseudomonas aeruginosa (PA) of 6 antibiotics with or without α-amylase and treat 2-day PA biofilms with antibiotics with or without GHs. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) stability assays and Differential Scanning Fluorimetry (DSF) were conducted to determine antibiotic and GH degradation as well as antibiotic sequestration. Results: Increased MICs in the presence of GHs as well as decreased antibiotic clearance against 2-day biofilms were suggestive of antibiotic degradation. LC-MS/MS stability assays of tetracycline and ciprofloxacin in the presence and absence of α-amylase further demonstrated the α-amylase-mediated antibiotic sequestration. Differential scanning fluorimetry (DSF) assays confirmed α-amylase-antibiotic interactions. Conclusions: This study suggests that α-amylase is capable of degrading and sequestering a variety of antibiotics, and the degree to which these phenomena occur varies depending upon the source of the GH. As a potential treatment for biofilm-associated infections, it is imperative that the GH + antibiotic combinations are determined compatible prior to clinical use. Full article

(This article belongs to the Special Issue Antimicrobial Resistance in Biofilm-Associated Infections)

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16 pages, 4134 KB

Open AccessArticle

Oral Administration of Heat-Killed Multi-Strain Probiotics Confers Durable Protection Against Antibiotic-Resistant Primary and Recurrent Urinary Tract Infections in a Murine Model

by Bo-Yuan Chen, Zhen-Shu Liu, Yu-Syuan Lin, Hsiao Chin Lin and Po-Wen Chen

Antibiotics 2025, 14(7), 634; https://doi.org/10.3390/antibiotics14070634 - 21 Jun 2025

Cited by 2 | Viewed by 3630

Abstract

Background/Objectives: Alternative therapies for urinary tract infections (UTIs) have been explored, but their efficacy remains inconsistent. With rising antibiotic resistance, this study aimed to evaluate simplified postbiotic formulations derived from heat-killed probiotics for long-term protection against primary and recurrent UTIs in a murine model. Methods: We compared a multi-strain (seven-strain) versus a single-strain postbiotic in preventing Escherichia coli-induced UTIs and recurrent polymicrobial UTIs, assessed protection persistence after treatment discontinuation, and established a novel sustained UTI model via intravesical co-inoculation of three uropathogens. Mice were allocated to three experimental groups: a placebo group (PBS), Postbiotic I group (a seven-strain heat-killed probiotic formulation), and Postbiotic II group (a single-strain heat-killed probiotic). After two weeks of treatment, mice were challenged with uropathogenic E. coli (UPEC) and treated for seven days. Following a 14-day washout and bacterial clearance, they were rechallenged with multidrug-resistant UPEC, Klebsiella pneumoniae, and Staphylococcus pseudintermedius. Results: Both postbiotics significantly accelerated bacterial clearance in primary UTIs (p < 0.05). In recurrent UTIs, placebo-treated mice exhibited persistent bacteriuria, while Postbiotic I maintained a significantly higher sterile urine rate (50–80%, p < 0.01) post-treatment. Histopathological analysis confirmed reduced bladder and kidney inflammation (p < 0.05) with Postbiotic I. Conclusions: These findings demonstrate the superior efficacy of Postbiotic I in mitigating UTIs, with sustained protection post-treatment, supporting its potential as a long-term, non-antibiotic strategy. Additionally, our reproducible chronic UTI model, achieved through the co-inoculation of three uropathogens, provides a valuable tool for future research on chronic UTI pathogenesis and treatment. Full article

(This article belongs to the Special Issue Antimicrobial Resistance in Biofilm-Associated Infections)

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21 pages, 7339 KB

Open AccessEditor’s ChoiceArticle

In Vitro Analysis of Interactions Between Staphylococcus aureus and Pseudomonas aeruginosa During Biofilm Formation

by Julia Scaffo, Rayssa Durães Lima, Cameron Dobrotka, Tainara A. N. Ribeiro, Renata F. A. Pereira, Daniela Sachs, Rosana B. R. Ferreira and Fabio Aguiar-Alves

Antibiotics 2025, 14(5), 504; https://doi.org/10.3390/antibiotics14050504 - 14 May 2025

Cited by 8 | Viewed by 4915

Abstract

Staphylococcus aureus and Pseudomonas aeruginosa are classified as ESKAPE pathogens that present a significant challenge to treatment due to their increased resistance to a considerable number of antimicrobial agents. Background/Objective: Biofilms exacerbate treatment challenges by providing enhanced antimicrobial and environmental protection. Mixed-species biofilms further complicate treatment options through numerous complex interspecies interactions, leading to potentially severe adverse clinical outcomes. Methods: This study assessed the interaction between clinical S. aureus and P. aeruginosa isolates during biofilm formation using microplate biofilm formation assays, scanning electron microscopy, and confocal microscopy. Results: We identified a competitive relationship between P. aeruginosa and S. aureus, where both pathogens exhibited a reduction in biofilm formation during mixed-species biofilms compared with monocultures, although P. aeruginosa outcompeted S. aureus. Furthermore, we found that the cell-free conditioned media (CFCM) of P. aeruginosa significantly reduced the S. aureus biofilms. Using fractioned CFCM, we identified that the anti-staphylococcal activity of the >10 kDa fraction was almost identical to the non-fractioned CFCM. Our confocal microscopy results suggest that P. aeruginosa CFCM depolarize S. aureus membranes and reduces the biofilm burden. Conclusions: These findings contribute to our understanding of the mechanisms underlying the interactions between these pathogens, suggesting that there is an antagonistic relationship between S. aureus and P. aeruginosa in a biofilm setting. Full article

(This article belongs to the Special Issue Antimicrobial Resistance in Biofilm-Associated Infections)

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18 pages, 5416 KB

Open AccessArticle

Bacteria-Inspired Synthesis of Silver-Doped Zinc Oxide Nanocomposites: A Novel Synergistic Approach in Controlling Biofilm and Quorum-Sensing-Regulated Virulence Factors in Pseudomonas aeruginosa

by Abirami Karthikeyan, Manoj Kumar Thirugnanasambantham, Fazlurrahman Khan and Arun Kumar Mani

Antibiotics 2025, 14(1), 59; https://doi.org/10.3390/antibiotics14010059 - 9 Jan 2025

Cited by 8 | Viewed by 2854

Abstract

Multidrug-resistant Pseudomonas aeruginosa infections pose a critical challenge to healthcare systems, particularly in nosocomial settings. This drug-resistant bacterium forms biofilms and produces an array of virulent factors regulated by quorum sensing. In this study, metal-tolerant bacteria were isolated from a metal-contaminated site and screened for their ability to synthesize multifunctional nanocomposites (NCs). Rapid color changes in the reaction solution evidenced the biotransformation process. The potent isolated Bacillus cereus SASAK, identified via 16S rRNA sequencing and deposited in GenBank under accession number MH885570, facilitated the microbial-mediated synthesis of ZnO nanoparticles and silver-doped ZnO NCs. These biogenic nanocomposites were characterized using UV-VIS-NIR spectroscopy, FTIR, XRD, zeta potential, HRTEM, FESEM, and EDX analyses. At a sub-MIC concentration of 100 µg/mL, 2% Ag-ZnO NCs effectively inhibited virulent factor production and biofilm formation in P. aeruginosa without affecting bacterial growth. Notably, there was a significant reduction in violacein pigment (96.25%), swarming motility, and pyocyanin concentration (1.87 µg/mL). Additionally, biofilm formation (81.1%) and EPS production (83.9%) using P. aeruginosa were substantially hindered, along with reduced extracellular protease activity, as indicated by zone formation (from 2.3 to 1.8 cm). This study underscores the potential of Ag-ZnO NCs as promising agents for combating quorum sensing-mediated virulence in chronic infections caused by multidrug-resistant P. aeruginosa. Full article

(This article belongs to the Special Issue Antimicrobial Resistance in Biofilm-Associated Infections)

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