MOLECULAR DETECTION AND SEQUENCE ANALYSIS OF THE CNF1 GENE IN UROPATHOGENIC ESCHERICHIA COLI ISOLATED FROM URINARY TRACT INFECTION PATIENTS

Abstract

Background: Urinary Tract Infection (UTI) is a huge burden in Pakistan with multidrug resistant (MDR) Uropathogenic Escherichia coli (UPEC) being a major contributor to UTIs. Cytotoxic Necrotizing Factor 1 (cnf1) is the main virulence factor contributing to damage of cells and invasion of tissues, but little is known about its distribution in low resource countries.

Objectives: This study was designed to assess the prevalence of cnf1 in UPEC isolates and to determine if there was correlation with hemolysis and antibiotic resistance as well as distribution in phylogenetic groups with an accessible workflow.

Methodology: 100 isolates from UTI patients to UPEC were tested. Hemolysis was noted on blood agar and antibiotic susceptibility was done using Kirby-Bauer disk diffusion. Cnf1 gene was found by conventional PCR and PCR-RFLP was optimized for the assignment of the phylogenetic clade (A, B2, D, F). Chi-square and t-tests were used to validate statistical associations.

Results: cnf1 was found in 25% (25/100) of the isolates. There was a good correlation between cnf1 positivity and β-hemolysis (p < 0.0001), with 84% of the cnf1-positive strains being hemolytic. Significant association was not found with MDR (p = 0.4173) or patient age (p = 0.9068) but was found with ciprofloxacin resistance, with 72% of the cnf1-positive isolates being resistant. RFLP analysis showed that all DNA sequences were 98.81% similar, and were grouped mainly into (56%, n = 11) clade B2, and (24%, n = 5) clade D.

Conclusions: cnf1 is prevalent in hemolytic UPEC strains, is associated with fluoroquinolone resistance and is mainly associated with the virulent B2 lineage. The workflow of the PCR-RFLP technique shows that it can be a powerful molecular surveillance tool that can be carried out in low-resource laboratory settings.