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.
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
60
Chitosan nanoparticles will be applied to strong biofilm-forming UPEC isolates in vitro to evaluate their antibiofilm activity. Treatments include: * Chitosan nanoparticles alone at sub-inhibitory concentrations * Ciprofloxacin alone at sub-inhibitory concentrations * Combination of chitosan nanoparticles and ciprofloxacin Biofilm formation will be quantified using crystal violet staining in 96-well microtiter plates. The intervention also includes coating of urinary catheter segments with chitosan nanoparticles to assess biofilm inhibition.
Faculty of Medicine, Assiut University, Microbiology and Immunity department
Asyut, Assiut Governorate, Egypt
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.
Time frame: 24 hours after treatment of bacterial cultures in vitro.
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.
Time frame: 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.
Time frame: 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.
Time frame: Immediately after 24-hour treatment of bacterial cultures in vitro.
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