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.
1. Patients (inpatients or outpatients) clinically diagnosed with urinary tract infection (UTI).
1. Patients who received systemic antibiotic therapy within the previous 48-72 hours prior to urine sample collection.
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.
Case-only
Prospective
chitosan nanoparticles
Other interventionChitosan 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.
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.
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.
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.
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.
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