INTRODUCTION

Escherichia coli strains that cause extraintestinal infections 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 creation of pores in the target cell membrane and affects erythrocytes, leukocytes ^[3], and renal tubular cells ^[2]. Its activity 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 leads 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 Isolates:

A 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 Testing:

Antibiotic susceptibility test was determined for these isolates to routinely used antibiotics such as ampicillin, amoxicillin, amikacin, norfloxacin, ceftazimide, cefota-xime, ciprofloxacin and gentamicin, imipenem as by Kirby Bauer disc diffusion method^[7].

Detection of hlyA Gene in E.coli:

Escherichia 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 Mgcl₂0.2mM dNTP mix and 0.8µM of hlyA gene with 0.5U of Taq polymerase (New England Biolabs) in a 1x PCR buffered reaction. A positive control of E.coli with hlyA gene was also included in this study. PCR amplification was carried out using thermal cycler (Eppendorf) with the following cycling condition. Initial denaturation at 98^oC for 5 min and 30 cycles for 30s, 70^oC for 30s and 68^oC for 60s, followed by a final extension of 6 min at 75^oC. PCR products were resolved in 2% agarose gel. A 100bp ladder was including in all the gel analysis^[8].

Table 1: Gene sequencing of hlyA gene

Primer	Primer sequence	Product size
hlyA	AAC AAG GAT AAG CAC TGT TCT GGCT ACC ATA TAA GCG GTC ATT CCC GTC A	172 bp

RESULTS:

Sample wise distribution of clinical isolates of E.coli:

Of the 20 clinical isolates of E.coli, 12/20 (60%) were from acute urinary tract infections and 8/20 (40%) were from chronic urinary tract infections. Figure 1 depicts the sample wise distribution of clinical isolates of E.coli.

Figure 1: Sample wise distribution of urinary isolates of E.coli

Antibiotic Susceptibility Testing:

In our isolates, we have found increased percentage 14/20 (70%) of isolates showed sensitivity to amikacin followed by gentamicin, which showed sensitivity of 9/20 (45%). 80- 90% of E.coli isolates showed resistance to cephalosporin group of drugs. 6/20 (30%) were found to be resistant to imipenem. However, we have observed an elevated level of resistance to other routinely used antibiotics. The detailed resistant pattern of E.coli isolates is shown in table 2.

Table 2: Showing antibiotic sensitivity pattern of E. coli

Antibiotics	Sensitivity (20) (%)	Intermediate (20 (%)	Resistant (20) (%)
Ampicillin	5	0	95
Amoxicillin	5	0	95
Ceftazidime	10	10	80
Cefotaxime	5	5	90
Amikacin	70	10	20
Gentamicin	45	20	35
Norfloxacin	15	15	70
Ciprofloxacin	20	5	75
Imipenem	70	0	30

Result of hlyA gene in E.coli:

12/20 (60%) clinical isolates of urinary isolates of E.coli was found to harbor hlyA gene.

Figure 2: Representative gel picture showing positive for hlyA gene

DISCUSSION:

The most common extra intestinal infections caused by E. coli are usually urinary tract infections. The -hemolysin is present in about 25 to 56% of isolated strains from urinary tract infections ^[9], similar to the previous studies we also got increased percentage of E. coli isolates from urinary tract. Study done by Kerenyi and coworkers found that, the presence of the sheA gene in normal fecal strains is higher (85.4%) than in the isolates from urinary tract infection (47.1%). In his study, the occurrence of hlyA gene in normal fecal isolates was 7.3%, similar to that previously reported ^[10]. Similarly we also found 12/20 (60%) of our uropathogenic E.coli were showed positive for hlyAgene. Though it is accepted that the -hemolysin is an important virulence factor to the pathogenic profile of E. coli, the role played by the silent hemolysin PSheA in disease is unknown.

CONCLUSION:

From our study we have observed the presence of hlyA gene encodes for alpha hemolysin production. There are several virulent factors are associated with urinary tract infections, production of alpha hemolysin also plays a crucial role in pathogenesis of UTI. To conclude this more number of isolates with different sets of genes for other hemolysins needs to be detected.

ACKNOWLEDGMENT:

We thank Dr. Kalyani, Professor and Head of the Department of Microbiology, Saveetha Medical College, Chennai for kindly providing the clinical isolates to carry out our research work fruitfully.

REFERENCES:

1. Welch RA, Forestier C, Lobo A, Pellett S, Thomas W, Rowe J. The synthesis and function of the Escherichia coli hemolysin and related RTX exotoxins. FEMS Microbiol Immunol. 5; 1992:29-36.

2. Keane,Welch WFR, Gekker G, and Peterson PK. Mechanism of Escherichia coli alpha-hemolysin-induced injury to isolated renal tubular cells. Am J Pathol. 126;1987:350-357.

3. Bhakdi, Greulich SS, Muhly M, Eberspacher B, Becker.H, Thiele A, and Hugo F. Potent leukocidal action of Escherichia coli hemolysin mediated by permeabilization of target cell membranes. J Exp Med 169; 2989:737-754.

4. Konig, Ludwig BA, Goebel W, and Konig W. Pore formation by the Escherichia coli alpha- hemolysin: role for mediator release from human inflammatory cells. Infect Immun. 62; 1994:4611-4617.

5. Bhakdi, S, and Martin E. Superoxide generation by human neutrophils induced by low doses of Escherichia coli hemolysin. Infect Immun. 59; 1991:2955–2962.

6. Felmlee, Pellett TS, and Welch RA. Nucleotide sequence of an Escherichia coli chromosomal hemolysin. J Bacteriol. 163; 1985 : 94–105.

7. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk Tests; Approved Standards; Document M2-A9, 2015,9^th ed., Vol 26. Wayne, PA: CLSI.

8. Kaczmarek A, Budzynska A, Gospodarek E. Prevalence of genes encoding virulence factors among Escherichia coli with K1 antigen and non-K1 E. coli strains. Journal of Medical Microbiology 61; 2012:1360–1365.

9. Minshew, Jorgensen BHJ, Swanstrum M, Grootes-Reuvecamp GA, and Falkow S. Some characteristics of Escherichia coli strains isolated from extraintestinal infections of humans. J Infect Dis. 137;1978:648–654

10. Johnson JR. Virulence factor in Escherichia coli urinary tract infection. Clin Microbiol Rev. 4;1991:80–128.

Received on 25.06.2016 Modified on 16.07.2016

Research J. Pharm. and Tech 2016; 9(10):1629-1631.

DOI: 10.5958/0974-360X.2016.00325.5