Detection of hly-a gene encoding alpha hemolysin in urinary isolates of escherichia coli from tertiary care hospital in kancheepuram dist

Abstract

Alpha hemolysin is a toxin secreted by Escherichia coli causes cell death by binding with the outer membrane, with subsequent oligomerization of the toxin monomer and waterfilled channels. These are responsible for osmotic phenomena, cell depolarization, and loss of vital molecules, leading to its demise. A total of 20 clinical isolates of E. coli were screened for the presence of hlyA gene by PCR. We have observed 60% positivity among our isolates. There are several virulent factors that are associated with urinary tract infections, production of alpha hemolysin one amongst those factors also play a crucial role in pathogenesis of UTI. INTRODUCTIONEscherichia coli causes different infections that possess numerous virulence factors, including hemolysin production. E. coli α-hemolysin (HlyA) produces large, clear zones of hemolysis around colonies on blood agar. The hemolysin is present in cell-free filtrates and is the best characterized member of the RTX (repeat in toxin) toxin family [1] . HlyA lyses cells by the creating pores in the target cell membrane and affects erythrocytes, leukocytes [3] , and renal tubular cells [2] . It also acts on polymorphonuclear granulocytes liberates leukotrienes, histamine, and ATP [4] and is neutralized by specific antiserum.Sublytic concentrations of this toxin induce various reactions in eukaryotic target cells which lead to cellular dysfunction [5] . The hly operon required for synthesis and extracellular secretion of E. coli hemolysinwhich contains four structural genes arranged in the order hlyC, hlyA, hlyB, and hlyD [6] . Gene hlyA encodes 110-kDa hemolysin protein (pro-HlyA) which represents an inactive precursor of the mature toxin. The conversion of pro-HlyA to the hemolytically active hemolysin (HlyA) takes place in the cytoplasm of E. coli and is mediated by HlyCgene [2] . With this background our aims to detect the presence of hlyA gene among urinary isolates of E. coli. MATERIALS AND METHODS Bacterial isolatesA total of 20 non repetitive urinary isolates of Escherichia coli were collected from Saveetha Medical College and Hospitals, Chennai. They were processed for a battery of standard biochemical tests and confirmed. Isolates were preserved in semisolid trypticase soy broth stock and were stored at 4 ºC until further use. Antibiotic susceptibility testingAntibiotic susceptibility test was determined for these isolates to routinely used antibiotics such as ampicillin, amoxicillin, amikacin, norfloxacin, ceftazimide, cefotaxime, ciprofloxacin and gentamicin, imipenem as by Kirby Bauer disc diffusion method [7]. Detection of hlyA gene in E.coliEscherichia coli isolates were detected for the presence of hlyA gene by PCR analysis. Detection of the gene was carried out using primer as depicted in table 1. Bacterial DNA was extracted by boiling lysis method. 1 µL of DNA extract was used as template for PCR reaction. The reaction mixture contained 1mM of Mgcl 2 0.2mM dNTP mix and 0.8µM of hlyA gene with 0.5U of Taq polymerase (New England Biolabs) in a 1x PCR b...