The role of uropathogenic Escherichia coli biofilms in antibiotic-resistant urinary tract infections: Nanoparticle-based, phage therapy, and quorum-sensing inhibitor approaches

Abstract

Background:

Urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC) pose a global health challenge, largely due to UPEC biofilms that drive persistent infections and antibiotic resistance.

Materials and methods:

To explore the role of UPEC biofilms in antibiotic-resistant UTIs and summarize emerging therapeutic strategies, this study conducted a systematic review adhering to PRISMA guidelines and registered in PROSPERO (CRD420251040212). A structured search of PubMed, Google Scholar, Scopus, and Web of Science identified English-language studies published up to 2024, with 57 eligible studies selected after three-stage screening and analyzed via thematic synthesis.

Results:

This study explored UPEC biofilms enhance resistance through extracellular matrix barriers, persister cell formation, efflux pump upregulation, and horizontal gene transfer; emerging therapies including bacteriophage therapy, quorum-sensing inhibitors, and nanoparticle-based drug delivery effectively target biofilms by penetration, signaling disruption, and improved drug efficacy. Additional approaches such as antibiofilm peptides, probiotics, and immunotherapy also demonstrate potential.

Conclusions:

The UPEC biofilms are key to chronic UTIs, and novel targeted therapies offer promising solutions, but clinical validation, regulatory hurdles, and combination therapy optimization are critical for translation to clinical practice.

1.  Introduction

Urinary tract infections (UTIs) are among the most prevalent infectious diseases globally and affect individuals across all age groups. They account for approximately 150 million cases annually and impose a substantial burden on health care systems worldwide. The clinical manifestations of UTIs range from uncomplicated cystitis to severe pyelonephritis with the potential for systemic involvement if not managed adequately.^[1]

The escalation of antibiotic resistance is a major concern in the treatment of UTIs. The widespread and often indiscriminate use of antibiotics has led to the emergence of multidrug-resistant bacterial strains, complicating treatment protocols and increasing morbidity and health care costs.^[2] In the United States alone, antibiotic-resistant infections surpass 2.8 million cases annually, resulting in over 35,000 deaths. Projections suggest that, by 2050, antimicrobial resistance could cause an additional 10 million deaths globally.^[3]

Escherichia coli (E. coli) is the predominant uropathogen, responsible for up to 95% of community-acquired UTIs and more than 50% of all catheter-associated UTIs. Among the various E. coli pathotypes, uropathogenic E. coli (UPEC) is particularly adept at colonizing the urinary tract. Uropathogenic E. coli possesses a repertoire of virulence factors that facilitate adhesion, invasion, and persistence within the host urinary epithelium. These include adhesins, toxins, iron-acquisition systems, and factors that confer resistance to host immune responses.^[4]

A critical aspect of UPEC pathogenicity is its ability to form biofilms—structured bacterial communities encased in a self-produced extracellular matrix. Biofilm formation on surfaces such as catheters, bladder walls, and within bladder epithelial cells provides a protective niche for UPEC, rendering them less susceptible to host immune defenses and antibiotic treatments.^[5] This biofilm mode of growth is associated with chronic and recurrent infections, because bacteria within biofilms can withstand higher concentrations of antibiotics and are shielded from phagocytosis. Uropathogenic E. coli biofilms exhibit a unique resistance pattern because of their ability to enter quiescent states and upregulate efflux pump activity, distinguishing them from other biofilm-forming pathogens such as Pseudomonas aeruginosa. Understanding the mechanisms underlying UPEC biofilm formation and maintenance is crucial, because it holds the key to developing novel therapeutic strategies aimed at eradicating persistent and antibiotic-resistant UTIs.^[6]

This study aimed to explore the role of UPEC biofilms in the persistence and antibiotic resistance of UTIs and to highlight emerging therapeutic strategies that offer potential solutions. Given the limitations of conventional antibiotics, alternative approaches, such as bacteriophage therapy, quorum-sensing inhibitors (QSIs), and nanoparticle-based drug delivery, have gained increasing attention. This review discusses these novel treatment modalities, their mechanisms of action, and their potential to overcome the challenges posed by UPEC biofilms, with an emphasis on their clinical applicability and prospects.

2.  Materials and methods

This literature review followed a systematic approach, adhering to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines to ensure a comprehensive and unbiased selection of relevant studies related to UPEC biofilms and antibiotic-resistant UTIs. The review protocol was prospectively registered with the International Prospective Register of Systematic Reviews under the registration number CRD420251040212. The complete protocol is available at https://www.crd.york.ac.uk/PROSPERO/view/CRD420251040212.

2.1.  Search Strategy

A structured literature search was conducted using four major electronic databases: PubMed, Google Scholar, Scopus, and Web of Science. The search encompassed peer-reviewed articles, systematic reviews, and clinical studies published until 2024. The search strategy employed Boolean operators as follows:

• (“Uropathogenic E. coli” OR “UPEC”) AND (“Biofilms” OR “Biofilm formation”)

• (“Antibiotic resistance” OR “Multidrug resistance”) AND (“Urinary tract infections” OR “UTIs”)

• (“Bacteriophage therapy” OR “Phage therapy”) AND (“Biofilm disruption” OR “Biofilm eradication”)

• (“Quorum sensing inhibitors” OR “QSIs”) AND (“Biofilm control”)

• (“Nanoparticles” OR “Nanotechnology”) AND (“Antibiofilm strategies”)

To refine the search, filters were used to include only articles published in English, with full-text availability, and studies that focused on UPEC biofilms in UTIs. Additional relevant articles were identified through manual searches of the reference lists of key review papers and meta-analyses.

2.2.  Inclusion and exclusion criteria

The selection process involved applying strict inclusion and exclusion criteria.

Inclusion criteria:

1. Studies published in peer-reviewed journals

2. Research focused explicitly on UPEC biofilms in the context of UTIs

3. Articles exploring mechanisms of antibiotic resistance in UPEC biofilms

4. Studies evaluating emerging therapeutic strategies (bacteriophages, QSIs, nanoparticles, and probiotics, etc.)

5. Experimental, clinical, and systematic review studies

Exclusion criteria:

1. Studies not in English

2. Articles focused on general bacterial biofilms without specific reference to UPEC

3. Conference abstracts, book chapters, and gray literature

4. Studies lacking sufficient methodological details or sample size

2.3.  Data extraction and synthesis

The retrieved articles were screened in 3 stages: title screening, abstract review, and full-text evaluation. A total of 129 articles were initially retrieved. After eliminating duplicates and assessing their relevance, 57 unique studies met the inclusion criteria and were analyzed. The selection process is summarized in the PRISMA flow diagram (Fig. 1).

Figure 1.

This figure illustrates the PRISMA flow diagram. PRISMA = Preferred Reporting Items for Systematic Reviews and Meta-Analyses; UPEC = uropathogenic Escherichia coli; UTIs = urinary tract infections.

The key data extracted included study objectives, methodology, findings, and conclusions, with a focus on biofilm-formation mechanisms, antibiotic-resistance pathways, and novel treatment strategies. Thematic synthesis was employed to categorize the findings into major themes, enabling a structured discussion of biofilm-associated UPEC infections and potential therapeutic interventions.

3.  UPEC and biofilm formation

Uropathogenic E. coli is the primary causative agent of UTIs and is responsible for over 80% of the cases of UTIs globally. The pathogenesis of UPEC is multifaceted and involves a suite of virulence factors that facilitate its colonization, invasion, and persistence within the urinary tract. These factors include adhesins such as type 1 and P fimbriae, which mediate attachment to uroepithelial cells, enabling bacteria to resist urinary flow and establish infections.^[7] In addition, UPEC produces toxins like α-hemolysin, which contribute to tissue damage and immune evasion. Iron-acquisition systems, including siderophores, are also critical for UPEC to thrive in the iron-limited environment of the urinary tract.^[8]

A pivotal aspect of UPEC pathogenicity is its ability to form biofilm-structured bacterial communities encased in a self-produced extracellular matrix. Biofilm formation is initiated when UPEC adheres to surfaces such as bladder epithelium or indwelling catheters.^[9] Following attachment, the bacteria proliferate and produce extracellular polymeric substances (EPSs), including polysaccharides, proteins, and extracellular DNA, which constitute the biofilm matrix. This matrix anchors bacteria to surfaces and provides protection against environmental stresses, including antibiotic exposure and host immune responses.^[10]

Biofilm formation by UPEC is an important factor in the persistence and recurrence of UTIs. Within biofilms, UPEC cells can enter a quiescent state, rendering them less susceptible to antibiotics that target actively dividing cells. Moreover, the biofilm matrix impedes the penetration of antimicrobial agents and shields bacteria from phagocytosis.^[11] This protective environment allows UPEC to survive in hostile conditions within the urinary tract, leading to chronic infections and frequent relapses. Understanding the mechanisms underlying UPEC biofilm formation and maintenance is crucial for developing effective therapeutic strategies to combat persistent antibiotic-resistant UTIs.^[5]

4.  Antibiotic-resistance mechanisms in UPEC biofilms

Uropathogenic E. coli biofilms exhibit multifaceted resistance to antibiotics, which complicates the treatment of UTIs. One primary mechanism involves the EPS matrix, which acts as a physical barrier that limits antibiotic penetration, thereby reducing drug efficacy. This matrix not only impedes antimicrobial agents but also restricts the diffusion of oxygen and nutrients, creating microenvironments that can further diminish antibiotic activity.^[12] The EPS matrix is composed of polysaccharides, proteins, and extracellular DNA, which together form a dense and protective scaffold around bacterial cells. This complex structure not only hinders the diffusion of antibiotics but also sequesters antimicrobial agents, reducing their effective concentrations at the site of bacterial cells.^[13] In addition, the matrix can interact with antibiotics, leading to their inactivation or degradation before they reach their targets. Thus, the physical and chemical properties of the EPS matrix play a crucial role in the resilience of UPEC biofilms to antibiotic treatment.^[14]

Within these biofilms, a subpopulation of bacteria, known as persister cells, can enter a dormant state, rendering them tolerant to antibiotics that typically target active cellular processes. These persister cells are not genetically resistant but can survive antibiotic treatment, leading to infection recurrence.^[15] Formation of persister cells is a survival strategy employed by UPEC to withstand hostile conditions, including antibiotic exposure. These cells exhibit a transient, nonheritable tolerance to antibiotics, which allows them to persist in the presence of antimicrobial agents. Upon the removal of antibiotic pressure, persister cells can resuscitate and repopulate the biofilm, leading to chronic and recurrent infections.^[16] Persister cells within UPEC biofilms exhibit metabolic dormancy, allowing them to evade conventional antibiotic action and repopulate the biofilm, leading to recurrent infections. The mechanisms underlying persister cell formation involve the modulation of various cellular processes, including toxin-antitoxin systems, energy production, and stress responses. Understanding the biology of persister cells is essential to develop strategies to eradicate biofilm-associated infections.^[17]

Efflux pumps, which actively expel antibiotics from bacterial cells, are another important factor involved in biofilm-associated resistance. In UPEC biofilms, the expression of these pumps is often upregulated, further decreasing the intracellular antibiotic concentrations and contributing to treatment failure.^[18] Efflux pumps are membrane proteins that transport a wide range of substrates, including antibiotics, out of bacterial cells. Overexpression of efflux pumps in biofilm cells reduces the intracellular accumulation of antibiotics, thereby decreasing their efficacy. Moreover, the activity of efflux pumps can contribute to the development of multidrug resistance, because they can expel multiple classes of antibiotics.^[19] Regulation of efflux pump expression in biofilms is complex and involves various genetic and environmental factors. Targeting efflux pump activity represents a potential strategy for enhancing the effectiveness of antibiotic treatments against UPEC biofilms.^[20]

Genetic adaptations within UPEC biofilms also play crucial roles in antibiotic resistance. The close proximity of cells within a biofilm facilitates horizontal gene transfer (HGT), allowing rapid dissemination of resistance genes among bacterial populations.^[21] Genetic exchange can occur through mechanisms such as conjugation, transformation, or transduction, leading to the acquisition of new resistance traits and emergence of multidrug-resistant strains. The biofilm environment provides an ideal setting for HGT, because the dense cell population and presence of extracellular DNA promote genetic exchange.^[22] In addition, stress conditions within biofilms can induce the expression of mobile genetic elements, further enhancing the potential for gene transfer. The acquisition of resistance genes through HGT can lead to the emergence of UPEC strains with enhanced survival capabilities in the presence of antibiotics.^[23]

Therefore, the clinical implications of biofilm-associated antibiotic resistance are profound. Biofilm-forming UPEC strains are often implicated in chronic and recurrent UTIs because their heightened resistance to standard antibiotic therapies leads to persistent infections. This necessitates prolonged or repeated treatment courses, increasing the risk of adverse effects and contributing to the broader issue of antibiotic resistance.^[24] Moreover, biofilms on indwelling medical devices, such as catheters, can serve as reservoirs for infection, posing major challenges in health care settings. The resistance of biofilm-associated infections to antibiotic treatment often results in prolonged hospital stays, increased health care costs, and higher morbidity and mortality rates.^[25] Although bacteriophage therapy shows promise, regulatory challenges and the need for strain-specific phages limit its immediate clinical implementation. The difficulty in eradicating biofilm-associated infections underscores the need for alternative therapeutic strategies and development of novel antimicrobial agents capable of penetrating biofilms and effectively targeting resident bacterial cells.^[26]

5.  Emerging therapeutic strategies against UPEC biofilms

The persistent challenge of UPEC biofilms in UTIs has necessitated the development of innovative therapeutic strategies beyond conventional antibiotics. Among the emerging approaches, bacteriophage therapy, QSIs, and nanoparticle-based drug-delivery systems have garnered substantial attention for their potential to disrupt UPEC biofilms and mitigate the associated antibiotic resistance.^[5,27]

Bacteriophage therapy employs viruses that specifically infect and lyse bacterial cells, offering a targeted approach for biofilm eradication. Phages can penetrate the extracellular matrix of biofilms and infect the embedded bacteria. Upon infection, phages replicate within bacterial cells, leading to cell lysis and subsequent dispersion of the biofilm structure.^[28] This mechanism is particularly advantageous over traditional antibiotics because phages can evolve alongside bacteria, potentially overcoming resistance mechanisms. Moreover, phages exhibit specificity toward their bacterial targets, minimizing their impact on beneficial microbiota. However, challenges such as the narrow host range of phages, the potential for bacterial resistance development, and regulatory hurdles in clinical applications necessitate further research to optimize phage therapy for UPEC biofilms.^[29]

Quorum sensing (QS) is a bacterial communication system that regulates gene expression in response to cell density and plays a crucial role in biofilm formation and maintenance. In UPEC, QS regulates the expression of virulence factors and biofilm-associated genes.^[30] Quorum-sensing inhibitors aim to disrupt these signaling pathways, thereby inhibiting biofilm development and rendering bacteria more susceptible to antimicrobial agents. Potential QSIs include small molecules, enzymes that degrade signaling molecules, and antibodies that target QS receptors.^[31] Although promising, the clinical application of QSIs faces challenges, such as the redundancy and complexity of QS systems, potential off-target effects, and the need for effective delivery mechanisms to the site of infection.^[32]

Nanoparticle-based drug-delivery systems have emerged as novel approaches for targeting UPEC biofilms. Nanoparticles can be engineered to deliver antimicrobial agents directly to biofilms, thereby enhancing drug penetration and efficacy. Various types of nanoparticles, including metallic (e.g., silver and gold), lipid-based, and polymeric nanoparticles, have been investigated for their anti-biofilm properties.^[33] These systems offer advantages, such as controlled drug release, protection of therapeutic agents from degradation, and the ability to functionalize surfaces for targeted delivery. Studies have demonstrated that nanoparticle-antibiotic combinations can effectively reduce biofilm biomass and viability. Although nanoparticle-based approaches are promising, their toxicity and biocompatibility remain major concerns that require further preclinical validation. Considerations of the biocompatibility, toxicity, and stability of nanoparticles are critical for their translation into clinical settings.^[34,35]

6.  Other potential strategies for UPEC biofilm control

The persistent challenge posed by UPEC biofilms in UTIs has driven researchers to explore a variety of alternative strategies aimed at controlling biofilm formation and mitigating infection recalcitrance.^[36] In addition to conventional antibiotic treatments, several innovative approaches have emerged, including the use of antibiofilm peptides and enzymes, probiotics with microbiome-modulation capabilities, immunotherapeutic interventions, and vaccines.^[37] These strategies seek not only to disrupt the biofilm structure but also to target the underlying mechanisms that facilitate UPEC persistence and antibiotic resistance.

Antibiofilm peptides and enzymes have gained substantial attention because of their ability to interfere with critical components of biofilm architecture. Antibiofilm peptides are short sequences that can disrupt bacterial cell-to-cell communication and prevent the assembly of biofilm matrices.^[38] For instance, some peptides have been shown to inhibit the formation of amyloid fibers and curli structures, which are essential for maintaining the structural integrity of UPEC biofilms. These peptides bind to the precursors of biofilm matrix components, thereby interrupting the polymerization process necessary for robust biofilm formation.^[39]

Simultaneously, enzymes such as DNases, dispersin B, and proteases, have demonstrated efficacy in degrading the EPS matrix that encases biofilm-embedded bacteria. By enzymatically cleaving the polysaccharides, proteins, and extracellular DNA that constitute the EPS matrix, these agents facilitate the dispersion of biofilms and expose bacterial cells to both the host immune response and conventional antibiotics.^[40] Recent studies have highlighted the synergistic effects of combining antibiofilm peptides with enzymatic treatments, offering a promising multipronged approach to dismantle biofilms and reduce the bacterial load.^[41]

Another promising strategy for controlling UPEC biofilms involves the use of probiotics and microbiome modulation. The urinary tract, which is traditionally considered a sterile environment, harbors a diverse microbiome that is involved in health and disease.^[42] Probiotic strains, particularly those belonging to the genus Lactobacillus, have been extensively studied for their potential to restore microbial balance and inhibit the colonization of uropathogens.^[43] These beneficial bacteria can competitively exclude UPEC by adhering to uroepithelial cells, thereby reducing the number of available pathogen-binding sites. Moreover, probiotics produce bacteriocins and biosurfactants that inhibit the growth of UPEC and interfere with their ability to form biofilms.^[44]

Biosurfactants, for example, reduce surface tension and prevent the initial adhesion of bacterial cells to urinary tract surfaces, which is a critical first step in biofilm formation. In addition, probiotic interventions have been shown to modulate local immune responses and enhance the host’s natural defense against infections.^[45] Recent clinical studies and meta-analyses have provided encouraging evidence that probiotic supplementation reduces the incidence of recurrent UTIs, likely by maintaining a healthy urinary microbiome and mitigating biofilm-related pathogenicity.^[46]

Immunotherapy and vaccine development represent other frontiers in the fight against UPEC biofilms. Immunotherapeutic strategies are designed to harness and augment the host immune system to target specific bacterial antigens associated with biofilm formation.^[47] One approach involves the use of monoclonal antibodies that bind to key adhesins and virulence factors, thereby neutralizing the ability of bacteria to adhere and form biofilms. For example, antibodies targeting the FimH adhesin, a critical component of UPEC attachment to uroepithelial cells, have shown promise in preclinical studies. These antibodies can inhibit the binding of UPEC to the bladder epithelium and prevent the initial stages of biofilm formation.^[48]

Vaccine-development efforts have focused on eliciting robust immune responses against a range of UPEC antigens. Candidate vaccines aim to induce both humoral and cellular immunity, thereby offering protection against planktonic bacteria and cells residing within biofilms.^[49] Recent advances in vaccine-adjuvant technology and antigen-delivery systems have improved the immunogenicity of these vaccine candidates, bringing them closer to clinical application. Despite these advances, challenges remain, including the heterogeneity of UPEC strains and the complexity of biofilm-associated antigens, which can complicate the design of a broadly effective vaccine.^[50] Nevertheless, ongoing clinical trials and experimental studies continue to refine these strategies, providing hope for future immunotherapeutic interventions that can effectively prevent and control UPEC biofilm-related infections.^[51]

7.  Future directions and challenges

The development of biofilm-targeted therapies for UTIs presents significant translational challenges, particularly in bridging the gap between laboratory research and clinical applications. One major obstacle is the complexity of biofilm biology. Biofilms exhibit a heterogeneous structure composed of bacterial cells embedded within the EPS matrix, which impedes the penetration of therapeutic agents.^[52] This structural complexity necessitates the design of treatments that can effectively disrupting the EPS to enhance drug delivery. In addition, the presence of dormant or persister cells within biofilms contributes to their resilience against conventional antibiotics, because these cells can evade antimicrobial action and later repopulate the biofilm.^[53] Addressing these challenges requires a comprehensive understanding of biofilm physiology and the development of strategies that can target both active and dormant bacterial populations.

Regulatory hurdles further complicate the development of biofilm-targeting therapies in clinical practice. The approval process for new antimicrobial agents is stringent and requires extensive evidence of their safety and efficacy through well-designed clinical trials.^[54] However, conducting such trials for biofilm-associated infections poses a unique challenge. The standardization of biofilm models in clinical settings is challenging because of the variability in biofilm formation among different bacterial strains and patient conditions.^[55] Moreover, the assessment of treatment efficacy is complicated by the difficulty of quantifying biofilm reduction and correlating it with clinical outcomes. These factors contribute to the complexity of designing and implementing clinical trials that meet regulatory standards.^[56]

The potential of combination therapies in UTI management offers a promising avenue for overcoming the limitations of monotherapy. Combining agents that target different aspects of biofilm biology, such as EPS disruption, quorum-sensing inhibition, and antimicrobial activity, may enhance treatment efficacy.^[57] For instance, the use of dispersal agents to degrade the biofilm matrix, coupled with antibiotics to eradicate the released bacteria, has shown synergistic effects in preclinical studies. In addition, integrating novel therapies such as bacteriophages or antimicrobial peptides with traditional antibiotics could allow a multifaceted attack on biofilms, thereby reducing the likelihood of resistance development.^[16] However, the optimization of such combination therapies requires careful consideration of dosing regimens, potential interactions, and the specific characteristics of the biofilm-forming bacteria involved.

8.  Conclusions

In conclusion, the persistence of E. coli biofilms in UTIs presents a substantial clinical challenge, contributing to antibiotic resistance, chronic infections, and high recurrence rates. This review highlights the multifaceted mechanisms through which UPEC biofilms enhance bacterial survival, including extracellular matrix protection, genetic adaptation, and persister cell formation. Conventional antibiotics are often ineffective against biofilm-associated infections, necessitating the development of alternative therapeutic strategies. Emerging approaches such as bacteriophage therapy, QSIs, and nanoparticle-based drug delivery offer promising avenues for biofilm disruption and improved treatment outcomes. In addition, novel interventions, including antibiofilm peptides, probiotics, and immunotherapeutic strategies, have the potential to enhance host defense mechanisms and prevent recurrent infections. Despite these advancements, several translational challenges remain, including regulatory hurdles, the complexity of biofilm-targeting clinical trials, and the need for optimized combination therapies. Clinical validation of QSIs in catheter-associated UTIs is a key research priority. Further studies should focus on evaluating the long-term safety and efficacy of nanoparticle-based treatments in human trials. Addressing these challenges is critical for integrating biofilm-targeted therapies into routine UTI management, which will ultimately reduce the global burden of antibiotic-resistant UTIs.

Acknowledgments

None.

Statement of ethics

The review protocol followed PRISMA guidelines and was prospectively registered with the International Prospective Register of Systematic Reviews under the registration number CRD420251040212.

Conflict of interest statement

The authors declare that they have no competing interests.

Funding source

None.

Author contributions

The author was solely responsible for the entire scope of a project, from conception and design to analysis, interpretation, and writing.

Data availability

Data sharing not applicable to this article as no data-sets were generated or analyzed during the current study.