Cytotoxic Effects of Cytolethal Distending Toxin-Producing Escherichia coli on Normal Human Fibroblasts: Pilot Study

Abstract

     Cytolethal Distending Toxin (CDT) is a genotoxin produced by Escherichia coli and encoded by the cdt gene. It consists of three subunits: CDT-A, CDT-B, and CDT-C. As a virulence factor, CDT induces DNA damage in host cells, leading to cell death and potentially contributing to the development of cancer. This is a pilot study with the aims of investigating the impact of CDT-producing E. coli on normal cells and determining the prevalence of cdtB genes among clinical E. coli isolates. A total of 120 specimens were collected from various sources) urine, wound, blood, stool, and colorectal cancer tissue (CRC)) and biochemical tests coupled with Polymerase Chain Reaction (PCR) identified 60 samples as positive for E. coli. The PCR results for detecting the cdtB gene revealed that only 3 out of 60 isolates ( one from CRC and two from stool) contained the cdtB gene. Moreover, Quantitative Real-Time PCR was conducted on three isolates to detect the cdtB gene expression. The results revealed that the expression of the isolate from stool was higher than that of CRC, followed by another stool isolate. The phenotypic effects of CDT were assessed by treating Normal Human Fibroblasts (NHFs) with cdt+ E. coli isolates. To evaluate the impact of CDT, an apoptosis estimation assay was employed using (propidium iodide/acridine orange), with fluorescence intensity serving as the measure of DNA damage and cell death. The results showed that NHF cells exposed to the E. coli CRC isolate had a significantly higher percentage of dead cells (81.46 ± 0.99%), followed by the urine isolate (74.11±1.52%), compared to the control group (4.67±1.11%). Whole genome sequencing revealed that the E. coli CRC isolate carries various virulence factors that may facilitate CDT-mediated DNA damage and apoptosis. These findings suggest possible DNA damage and cell death, which may reflect the genotoxic and cytotoxic potential of CDT.