Background: Cytotoxic necrotizing factor type 1 (CNF1), a cytoplasmic protein presents in certain diarrheagenic and uropathogenic strains of Escherichia coli (UPEC), is a member of a family of bacterial toxins that target the Rho family of small GTP-binding proteins in mammalian cells contributing to inflammation and tissue damage, particularly in ascending urinary tract infections. Aim: Comparatively evaluate the Phenotypic expression of crude cytotoxic necrotizing factor type 1 (CNF1) produced by hemolytic and non-hemolytic uropathogenic E.coli isolates using diverse tissue culture cell lines. Methodology: Eight well identified uropathogenic E.coli which isolated from prostate and bladder cancers Iraqi patients and all harboring the cnf1 gene. These isolates were tested for their hemolytic ability by culturing on blood agar media. Cytotoxic necrotizing factor 1 preparations were made for chosen hemolytic and non-hemolytic isolates by cell lysis through harvesting the bacterial cells and sonicate the suspension at 10 μm peak amplitude for 5 min in an ice bath. The obtained cell lysate centrifuged at 6000 × g, 4 °C for 15 min. then the supernatant was collected and sterilized by Passing through a 0.22 μm filter to remove any remaining bacterial cells. The toxin concentration was determined using Bradford method. The cytotoxic effects were determined using MTT assay on three tumor cell lines including HeLa cells (human cervical carcinoma), Du145 cells (prostate cancer), and a normal cell line, HDF (human dermal fibroblast) at various dilution ratios, and cell viability was measured spectrophotometrically (OD Absorbance was quantified at 570 nm utilizing an ELISA reader values) after 24 hours of exposure. cells were stained with Giemsa solution after fixation. The stained cells were examined under a light microscope (40× objectives), and representative images were captured for analysis. Results: Three UPEC isolates were hemolytic and five were non-hemolytic from total eight. Only two isolates were used to determine phenotypic expression of CNF1 (one hemolytic and the other non-hemolytic). The protein concentrations of crude CNF1 from both isolates after extraction were 0.4 and 0.25 µg/ml respectively. Results of cytotoxic activity of CNF1 on the three tumor cell lines, on HDF cells, hemolytic E. coli caused a marked decline in viability, showing the lowest O.D value at 1:2 dilution (0.18), indicating strong cytotoxicity while non-hemolytic isolate showed less effect, with the highest OD at 1:8 (0.5). Similarly, HeLa cells exposed to hemolytic E. coli exhibited a decrease in OD (~0.28–0.3 at 1:4 and 1:8), while non-hemolytic isolates showed minimal cytotoxicity (OD ~0.48 at 1:8). In DU-145 cells, hemolytic E.coli isolates demonstrated the greatest cytotoxic effect at 1:2 dilution with OD (0.34), whereas non-hemolytic E.coli isolates showed higher OD (0.49 at 1:8), indicating lower toxicity. Conclusion: The data indicate a significant effect of crude cell lysate of both hemolytic and non-hemolytic UPEC on HDF cell lines , OD values generally increase with hemolytic UPEC, reaching the lowest values at 1:2(0.18), reflecting higher cytotoxicity, In contrast, non-hemolytic UPEC exhibited a high increase in OD at higher dilutions, with the highest at 1:8 (0.5) suggesting lower cytotoxic effects.The morphological changes of hemolytic UPEC ranging between sever damage cosist with apoptotic /necrotic featutre in dilution ration 1:1 to very mild or non near to control in dilution ration 1:8. The effect of non-hemolytic UPEC morphological changes ranging between stress with prominent apoptotic/ necrotic feature in dilution rate 1:8.
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How to cite this article: Rehan ES, Al-Mudallal NHA, Phenotypic Detection of Crude Cytotoxic Necrotizing Factor Type 1 (CNF1) of Hemolytic and Non-Hemolytic Uropathogenic E. coli Isolates Using Different Tissue Culture Cell lines..Int J Drug Deliv Technol. 2026;16 (1s): 397-411; DOI: 10.25258/ijddt.16. 397-411