Antibiofilm Activity of Chitosan Nanoparticles Against Uropathogenic Escherichia Coli

Evaluation of the Antibiofilm Activity of Chitosan Nanoparticles Against Uropathogenic Escherichia Coli Isolated From Assiut University Hospitals

1. Isolation of uropathogenic Escherichia coli (UPEC) and determination of their Antimicrobial sensitivity Patterns.
2. Evaluation of biofilm-forming capacity of UPEC isolates.
3. Assessment of the antibiofilm efficacy of chitosan nanoparticles alone and in combination with ciprofloxacin against UPEC isolates.
4. Examination of the effectiveness of chitosan nanoparticle coating in preventing biofilm formation by UPEC on urinary catheter surfaces.
5. Evaluation of the impact of chitosan nanoparticles on the expression levels of biofilm associated genes in UPEC.

Study Overview

• Status: Not yet recruiting
• Conditions: Urinary Tract Infections (UTIs); Catheter-Associated Urinary Tract Infections (CAUTIs); Biofilm Formation by Uropathogenic Escherichia Coli (UPEC)
• Intervention / Treatment: Other: chitosan nanoparticles

Detailed Description

Urinary tract infections (UTIs) remain among the most prevalent bacterial infections globally, causing substantial healthcare burden, with uropathogenic Escherichia coli (UPEC) responsible for the majority of cases in both community and hospital settings. Catheter-associated urinary tract infections (CAUTIs) represent a major proportion of healthcare-associated infections due to bacterial adhesion and biofilm formation on indwelling urinary catheters. Biofilm formation enables microorganisms to attach to abiotic surfaces and produce an extracellular polymeric matrix that enhances bacterial survival under adverse environmental conditions.

Biofilm-associated bacteria exhibit increased resistance to host immune responses and antimicrobial agents, contributing to chronic and recurrent infections. Cells embedded within biofilms may demonstrate markedly elevated antibiotic tolerance compared with planktonic bacteria, limiting therapeutic success. Although systemic antibiotics remain the mainstay of treatment, rising antimicrobial resistance among biofilm forming UPEC strains highlights the need for alternative antibiofilm strategies.

Chitosan, a naturally derived biopolymer, has attracted attention due to its biocompatibility, biodegradability, and intrinsic antimicrobial properties. Chitosan disrupts bacterial membranes and inhibits biofilm matrix formation and surface adhesion. Emerging evidence indicates that chitosan can downregulate biofilm-related gene expression involved in adhesion and extracellular polysaccharide synthesis. Furthermore, nanoparticle formulation enhances chitosan penetration into biofilms and improves antimicrobial efficiency compared with bulk polymer forms. Moreover, chitosan nanoparticles may enhance antibiotic diffusion and demonstrate synergistic antibiofilm activity when combined with ciprofloxacin.

Ciprofloxacin, a fluoroquinolone antibiotic widely used in UTIs, has demonstrated partial inhibition of biofilm formation through interference with bacterial DNA replication, initial bacterial adhesion to surfaces, reduction of expression of biofilm-related genes, quorum-sensing activity and decreases production of extracellular polymeric substances (EPS); however, its efficacy is reduced against mature biofilms. Consequently, chitosan nanoparticles may demonstrate enhanced antibiofilm activity when combined with ciprofloxacin.

Despite promising findings, limited studies have evaluated chitosan nanoparticle coatings on clinically relevant catheter surfaces against UPEC isolates. Therefore, investigating this strategy may offer an effective preventive approach for CAUTIs and support improved infection control practices.

• Study Type: Observational
• Enrollment (Estimated): 60

Contacts and Locations

• Study Contact: Name: Yasmin G Shazly, Demonstrator; Phone Number: +20 109 017 7354; Email: yasmin.g.shazly@aun.edu.eg

Study Locations

• Egypt
  • Assiut Governorate
    • Asyut, Assiut Governorate, Egypt
      • Faculty of Medicine, Assiut University, Microbiology and Immunity department

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: No

• Sampling Method: Non-Probability Sample

Study Population

This study will include UTI patients attending Assiut University Hospitals. Urine samples will be collected aseptically for isolation of uropathogenic E. coli. Only patients meeting inclusion criteria and providing consent will be included.

Description

Inclusion Criteria:

• 1. Patients (inpatients or outpatients) clinically diagnosed with urinary tract infection (UTI).

  2. Presence of significant bacteriuria in urine culture (≥10⁵ colony-forming units per milliliter (CFU/mL) for midstream urine, or as clinically indicated).

  3. Isolation of Escherichia coli as the sole or predominant pathogen from urine culture.

  4. Patients of any age and both sexes. 5. Patients who have not received antibiotic therapy within the previous 48-72 hours.

Exclusion Criteria:

• 1. Patients who received systemic antibiotic therapy within the previous 48-72 hours prior to urine sample collection.

  2. Urine cultures showing insignificant bacteriuria (<10⁵ CFU/mL for midstream urine, unless clinically justified).

  3. Polymicrobial growth in urine culture (mixed bacterial growth suggestive of contamination).

  4. Isolation of organisms other than Escherichia coli. 5. Improperly collected, leaking, or contaminated urine samples. 6. Patients unwilling to provide informed consent.

Study Plan

How is the study designed?

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Percentage of biofilm inhibition of UPEC isolates by chitosan nanoparticles alone and in combination with ciprofloxacin	The primary outcome is the reduction in biofilm formation of strong biofilm-producing uropathogenic Escherichia coli (UPEC) isolates after treatment with sub-inhibitory concentrations of chitosan nanoparticles alone or combined with ciprofloxacin. Biofilm biomass will be quantified using the crystal violet microtiter plate assay, and the percentage of inhibition will be calculated using optical density (OD) values according to the following formula: [(ODcontrol - ODTreated)/ODcontrol] × 100. Each experiment will be performed in triplicate and the mean values will be reported.	24 hours after treatment of bacterial cultures in vitro.

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Minimum Inhibitory Concentration (MIC) of chitosan nanoparticles and ciprofloxacin against UPEC isolates	The MIC of chitosan nanoparticles and ciprofloxacin will be determined for strong biofilm-forming UPEC isolates using the broth microdilution method in sterile 96-well microtiter plates. Two-fold serial dilutions of each agent will be prepared in tryptic soy broth (TSB) and inoculated with standardized bacterial suspensions. The MIC will be recorded as the lowest concentration showing no visible bacterial growth compared with growth controls.	18-24 hours after inoculation
Effectiveness of chitosan nanoparticle coating in preventing UPEC biofilm formation on urinary catheter segments	This outcome evaluates the ability of chitosan nanoparticle coating to inhibit biofilm formation on 1-cm urinary catheter segments. Coated segments will be incubated with standardized UPEC suspensions for 18 hours. Biofilm biomass will be quantified by crystal violet staining, solubilization with ethanol, and optical density (OD) measurement at 595 nm. Percentage of biofilm inhibition will be calculated as [(ODcontrol - ODTreated)/ODcontrol] × 100. Experiments will be performed in duplicate.	18-24 hours after bacterial incubation on coated catheter segments.
Relative gene expression of biofilm-associated genes (fimH and luxS) in UPEC isolates after chitosan nanoparticle treatment	The secondary outcome is the change in expression levels of fimH and luxS genes in strong biofilm-forming UPEC isolates following treatment with chitosan nanoparticles. Ribonucleic acid (RNA) will be extracted, converted to complementary DNA (cDNA), and analyzed using SYBR Green quantitative real-time polymerase chain reaction (qRT-PCR). Gene expression will be normalized to the housekeeping gene rpoD and calculated using the 2-ΔΔCt method. Each reaction will be performed in triplicate, and mean values will be reported.	Immediately after 24-hour treatment of bacterial cultures in vitro.

Collaborators and Investigators

• Sponsor: Assiut University

Publications and helpful links

General Publications

• Oliveira MCF, Canellas ALB, Berbert LC, Cardoso AM, Silva VA, Garutti SST, et al. Assessment of antimicrobial resistance and virulence of biofilm-forming uropathogenic Escherichia coli from Rio de Janeiro. Antibiotics (Basel). 2025;14(9):869.
• Wiegand I, Hilpert K, Hancock REW. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc. 2020;15(6):1957-1978.
• Obaid NA, Alzahrani AM, Alaryni BA, Almegrin FF, Alsubhi RS, Alzahrani RS, et al. Effectiveness of chitosan coating catheter in preventing catheter-associated urinary tract infection (CAUTI). J Pharm Res Int. 2022;34(19A):6-19.
• Fattah RAFA, Fathy FEZY, Mohamed TAH, Elsayed MS. Effect of chitosan nanoparticles on quorum sensing-controlled virulence factors and expression of LasI and RhlI genes among Pseudomonas aeruginosa clinical isolates. AIMS Microbiol. 2021;7(4):415-430.
• Yao H, Liu J, Jiang X, Chen F, Lu X, Zhang J. Analysis of the clinical effect of combined drug susceptibility to guide medication for carbapenem-resistant Klebsiella pneumoniae patients based on the Kirby-Bauer disk diffusion method. Infect Drug Resist 2021; 14: 79-87.
• Hooton TM, Gupta K. Urinary tract infections and asymptomatic bacteriuria in adults. N Engl J Med. 2021;384(11):1028-37.
• Singh S, et al. Nanotechnology-based coatings for prevention of catheter-associated infections. Nanomedicine. 2024; 52:102640.
• Yan J, Bassler BL. Surviving as a community: biofilm tolerance mechanisms. Cell Host Microbe. 2022;30(1):15-26.
• Flemming HC, Wuertz S. Bacteria and archaea on Earth and their biofilms. Nat Rev Microbiol. 2021;19(4):247-60.
• Klein RD, Hultgren SJ. Urinary tract infections: microbial pathogenesis and host response. Nat Rev Microbiol. 2020;18(4):211-26.
• Sahariah P, Masson M. Antimicrobial chitosan nanoparticles: applications and mechanisms. Biomacromolecules. 2021;22(2):363-380.

Study record dates

Study Major Dates

• Study Start (Estimated): May 1, 2026
• Primary Completion (Estimated): May 1, 2027
• Study Completion (Estimated): June 1, 2027

Study Registration Dates

• First Submitted: March 15, 2026
• First Submitted That Met QC Criteria: March 15, 2026
• First Posted (Actual): March 19, 2026

Study Record Updates

• Last Update Posted (Actual): March 27, 2026
• Last Update Submitted That Met QC Criteria: March 23, 2026
• Last Verified: March 1, 2026

More Information

Terms related to this study

• Keywords: Biofilm; Ciprofloxacin; Uropathogenic Escherichia coli (UPEC); Chitosan Nanoparticles; Catheter-Associated Urinary Tract Infection (CAUTI); Antibiofilm Activity; Urinary Catheter Biofilm; quantitative real-time polymerase chain reaction (qRT-PCR) gene expression
• Additional Relevant MeSH Terms: Urogenital Diseases; Male Urogenital Diseases; Urologic Diseases; Female Urogenital Diseases; Female Urogenital Diseases and Pregnancy Complications; Infections; Urinary Tract Infections
• Other Study ID Numbers: not assigned yet

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No