Isolation and Identification of Human Pathogenic Bacteria and Related Toxicity Potential to Different Cell Line

Abstract

Objectives : The present study was undertaken to investigate three different virulence mechanisms (adherence, invasion and cytotoxicity) of isolated Escherichia coli from urine and stool specimens and the relation between these different mechanisms and the ability of isolated Escherichia coli to induce apoptosis in Vero cells. Methodology: Different clinical samples have been collected from some Egyptian hospitals ( Suez canal authority hospital, Benha university hospital and Ismailia university hospital ). Bacterial strains were isolated and identified by morphological and biochemical analysis using biolog plates for selecting Escherichia coli strains from clinical samples which resulted in 30 Escherichia coli strains. The In-vitro antimicrobial sensitivity of the E.coli isolates were tested against 11 antimicrobial drugs used in human treatment. Results: Five E.coli isolates were selected from five groups of E.coli isolates which were classified according to antibiotic sensitivity similarity. These five selected Escherichia coli isolates were screened for adherence after 1h , invasion after 3h , 5h and cytotoxicity against Vero cells. All the tested Escherichia coli isolates were able to adhere to Vero cells by variable degrees. Concerning invasion, the tested Escherichia coli isolates were found to have the highest levels of invasion reaching at 3 h and the invasion deacresed after 5h of this assay. on screening for cytotoxicity by the MTT assay, the tested Escherichia coli isolates with highly adherence and invasion showed highly cytotoxic effects to Vero cells after 6 h indicating a relation between adherence and cytotoxicity. using Annexin V/PI staining by flow cytometry, highly cytotoxic tested Escherichia coli isolates showed high percentage of apoptotic Vero cells . Upon screening for activation of caspase 3, highly cytotoxic strains showed highly activation in caspase 3 which plays a central central role in driving the apoptotic pathways indicating a relation between cytotoxic effect and induction apoptosis in Vero cells. Conclusions: These results suggest that the tested Escherichia coli strains exhibit different capacity to adhere to Vero cells and invasion is a post adherence effect and it's preferable time is 3 h because after this peroid Vero cells were ruptured. The cytotoxicity of the isolated Escherichia coli is associated with higher adherence and invasion to Vero cells. This demonstrates that Escherichia coli strains induce apoptosis of Vero cells at different degrees . Induction of apoptosis in Vero cells is associated with higher adherence ,invasion and cytotoxicity Escherichia coli of to Vero cells. The induction of apoptosis is correlated with the virulence of Escherichia coli.