Abstract: The purpose of this study was to determine the presence of selected virulence genes in Escherichia coli isolated from diarrheic calves (Tehran province) and lambs (Ghume provinces) of Iran. We examined 40 isolates (29 from calves and 11 from lambs). All 40 isolates were tested for the presence of stx1, stx2, eae, espB and hly genes by multiplex polymerase chain reaction in two protocols. In the first protocol the isolates were tested with EC and hly primers and in the second protocol the isolates were examined with eae, stx1, stx2 and espB primers. Multiplex PCR showed that from 29 strains isolated from diarrheic calves, 4 of isolates (13.7%) were stx1 positive, 16 isolates (55.17%) carried stx2 and 2 isolates (6.89%) had espB gene. 1 isolate (3.44%) possessed eae. Among 11 strains isolated from diarrheic sheep, 9 isolates (81.81%) carried stx2 and 2 isolates (18.18%) had espB gene. Six isolates (54.54%) possessed eae and none of them was stx1 positive. The hly gene was not detected in any of the isolates. The findings of this study indicated that the stx2 may be widespread among pathogenic Escherichia coli in Iran.
INTRODUCTION
Shiga toxin-producing Escherichia coli (STEC) have emerged as food poisoning pathogens which can cause sever disease in humans, such as Hemorrhagic Colitis (HC) and Hemolytic Uremic Syndrome (HUS) (Armstrong et al., 1996; Paton and Paton, 1998). STEC are defined by production of shiga toxins (stx1 and stx2) or Vero toxins (Vt1 and Vt2), encoded by the stx genes of temperate, lambdoid bacteriophage that remain integrated in the E. coli chromosome (O’brain and Holmes, 1987). STEC may carry either stx subtype stx1, stx2 or both stx1 and stx2. In addition to toxin production, virulence-associated factor expressed by STEC is a protein called intimin (94-97 kDa protein) which is responsible for intimate attachment of STEC to the intestinal epithelial cells, causing attaching and effacing (AE) lesions in the intestinal mucosa. Intimin is encoded by the chromosomal eae gene which is located in the pathogenecity island termed the locus for enterocyte effacement (LEE) (Jerse et al., 1990; McDaniel et al., 1995; McDaniel and Kaper, 1997). The LEE encodes a type III secretion system, a series of protein secreted by this system called Esps, intimin and the receptor for intimin which is translocated into host cells (Frankel et al., 1998; Nataro and Koper, 1998). Secretion of the espB protein is essential for attachment and signal transduction in host cells and A/E lesions (Donnenberg et al., 1993; Foubister et al., 1994). The espB gene is located approximately 5 kb downstream of the eae gene (Donnenberg et al., 1993). In addition, one of the main agents of urinary tract infections in humans is E. coli which produces Hly (haemolysin) in urinary tract, causing release of iron for the bacteria.
Domestic ruminants especially cattle and sheep have been found to be the principal reservoir of STEC that are transmitted to human through foods contaminated with fecal material and cause human infections (Armstrong et al., 1996; Blanco et al., 1996; Blanco et al., 1997; Zschock et al., 2000; Cid et al., 2001; Osek and Gallien 2002).
The aim of this study was to detection of the STEC-associated virulence genes stx1, stx2, eae, espB and hly in Escherichia coli isolated from diarrheic calves and lambs, using multiplex PCR with the purpose of determining whether they can be a potential source of STEC pathogenic for human.
MATERIALS AND METHODS
Bacterial strains: Among 150 Escherichia coli isolated from diarrheic calves around Tehran (Iran) and 22 Escherichia coli isolated from diarrheic lambs during 2004-2005, 40 isolates (29 isolates from calves and 11 isolates from lambs) which were positive in serotyping with monovalent and polyvalent antiserums, were selected and tested for the presence of the selected genes. All isolates which had been stored at 4°C, were recultured on nutrient agar, then subcultured on blood agar, Mc Conkey agar, EMB agar and TSI agar (Merck) and incubated for 24 h at 37°C. Reference E. coli strains used as positive controls were Escherichia coli O157 (Strain No. 84-4, Tarbiat Modarres University) for the first protocol (eae, stx1, stx2, espB) and Escherichia coli ATTC 35218 and NTCC 11954 for the second protocol (alr, hly). Sterile dionised water was as negative control.
Detection of virulence genes by multiplex PCR: For multiplex PCR amplification, 40 Escherichia coli isolates and positive control strains were cultured on LB agar for 24 h at 37°C. To extract bacterial DNA, 6 to 8 colonies of each culture were picked and suspended in 100 μL of sterile deionized water, incubated at 100°C for 10 min to release the DNA and centrifuged at 6000×g for 5 min. The supernatant was used in the PCR reaction as the template DNA. Base sequence and predicted size of amplified product for each oligonucleotide primers (CinnaGene Inc, Iran) used in this study are shown in Table 1. Primers were used in two different protocols. In the fist protocol, EC and hly primers and in the second protocol, stx1, stx2, eae and espB primers were included. EC primers confirmed the isolates as E. coli (for alanine racemase gene). Amplification reactions were performed in a 25 μL volume containing 2.5 μL of 10X PCR buffer, 1 μL of 50 mM MgCl2, 1.5 μL of 10 mM deoxynucleoside triphosphate (CinnaGene Inc, Iran), 1 μL of each primer, 0.5 μL of Taq DNA polymerase (CinnaGene Inc, Iran), 2 μL of the template DNA and 13.5 μL (9.5 μL in the second protocol) of sterile dionised water. Using a thermal cycler (Techne, UK), the conditions for the multiplex PCR were programmed as follows: 94°C for 10 min for initial denaturation of DNA followed by 30 cycles of 94°C for 1 min, 48°C for 1 min (64°C in the second protocol) and 72°C for 1 min.
The amplified products were visualized by gel electrophoresis using 10 μL of the final reaction mixture on a 1.2% agaros gel in TBE buffer. The samples were electrophoresed for 1 h at 100 V. Amplified DNA fragments of specific sizes were located by UV illuminator after staining with ethidium bromide. Molecular size markers (Gene ruler 100 bp DNA ladder plus, Fermentas) were included in each gel.
RESULTS
To determine the prevalence of selected virulence genes among ruminants Escherichia coli, we examined 40 isolates (29 from diarrheic calves and 11 isolates from diarrheic lambs) by multiplex PCR amplification (Table 2 and 3).
| Table 1: | Primers sequences used in multiplex PCR |
| Table 2: | Occurrence of virulence factors among different serotypes of E. coli isolated from diarrheic lambs |
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a Poly 2: O26, O55, O111, O119, O126; b Poly 3: O 86, O 114, O125, O127, O128; c Poly 4: O44, O112, O124, O142; + = Presence of gene; - = Absent of gene | |
| Table 3: | Occurrence of virulence factors among different serotypes of E. coli isolated from diarrheic calves |
a Poly 2: O26, O55, O111, O119, O126; b Poly 3: O 86, O 114, O125, O127, O128; c Poly 4: O44, O112, O124, O142; + = Presence of gene; - = Absent of gene | |
| Fig. 1: | Multiplex PCR of isolated Escherichia coli from calves and lambs, using primer set espB: 260 bp, stx1: 302 bp. stx2: 515 bp and eae: 775 bp. Lane M: 100 bp Marker (fermantase). Lane 1 to 6: Escherichia coli isolate from diarrheic lambs (6, 1, 8, 4, 12, 21 samples); Lane 8 to 13: Escherichia coli isolates from diarrheic calves (53, 83, 93, 112, 98, 143); Lane 14: Escherichia coli O157 (Strain No. 84-4, Tarbiat Modarres University) as positive control; Lane 15: negative control (Water) |
All isolates were positive with EC primer that confirmed the isolates as E. coli. From 11 ovine strains just one isolates was O157 positive and the others belonged to other serogroups. Totally 11 isolates, 9 isolates (81.81%) showed stx2 gene and none of them had stx1 (Fig. 1). In addition 6 isolates (54.54%) carried eae gene and two isolates (18.18%) possessed espB gene (Table 2).
Among 29 bovine strains 9 isolates belonged to O157 serogruop and the other were non O157. Totally 29 isolates, 12 isolates (41.37%) carried stx2 and 4 isolates (13.79%) had both stx1 and stx2. Furthermore 2 isolates (6.89%) showed espB and just one isolate (3.44%) had eae gene (Table 3).
None of the bovine or ovine isolates had hly (Heamolysis) neither on blood agar nor by multiplex PCR.
DISCUSSION
Verotoxins or shigatoxins are cytotoxins produced by some enterohemorrhagic Escherichia coli (EHEC or STEC). VT1 (Stx1) and VT2 (Stx2) are two major types of Verotoxins that have been recognized. There is little information concerning the isolation and characterization of STEC strains and virulence gene in Iran. In the other countries there were some studies about virulence genes in E. coli especially by using multiplex PCR (Parriera et al., 2002; Call et al., 2001; Yamamoto et al., 1995).
Several reports have indicated the presence of verotoxins in E. coli which were isolated from ruminants like cattle (or calves) and sheep (or lambs) (Monserrat et al., 2003).
The eae gene encodes a protein named intimin which is responsible for intimate attachment of E. coli to the enterocytes causing attaching and effacing (A/E) lesions in the intestinal mucosa (Agin and Wolf, 1997). The carriage of eae gene sequence has been detected in E. coli isolates from ruminant sources in recent study. The EspB protein which is encoded by espB gene also helps bacteria to attach to the enterocytes (McDaniel and Kaper, 1997). Both eae and espB genes are part of a pathogenicity island termed the locus for enterocyte effacement (LEE). The presence of espB has been demonstrated in attaching and effacing Escherichia coli (AEEC) strains isolated from humans and animals (Cid et al., 2001; Orden et al., 2003).
China et al. (1999) found that among 191 E. coli isolates from diarrheic calves, 48% were EHEC and 44% were eae positive. Cid et al. (2001) detected eae and espB genes in 50 strain of 398 strains isolated from lambs. None of the 398 isolates carried stx genes. In Australia, a survey of 505 dairy cattle at a slaughterhouse found only 4 STEC O157 isolates which did not have stx gene and all of them carried eae gene (Hallaran and Sumner, 2001). In Spain, Rey et al. (2003) detected STEC O157: H 7 in 1% and non-O157STEC in 35% of 253 sheep samples. They showed with PCR that 43% of strains carried stx1 gene, 4% possessed stx2 and 53% had both stx1 and stx2 genes. Furthermore, eae gene was detected in 4% of STEC strains. Osek (2003), detected stx gene in 12.5% of 202 bovine isolates using multiplex PCR. 10 isolates were STEC O157. In addition 20 isolates carried eae gene. In India, 62 bovine and human STEC O157 isolates which 19% carried stx2 and 36.5% possessed stx1. 44.5% had both stx1 and stx2. Furthermore, just 6.6% had eae gene which was contributed with stx1. In Switzerland, all of the 11 STEC O157:H45 carried eae gene, none of them possessed stx gene.
In this study totally 10 isolates (25%) of 40 isolates were O157 serogroup in serotyping which nine of them were isolated from diarrheic calves and one was from diarrheic lamb. From 10 O157 isolates, 6 isolates (60%) carried stx2 gene and 3 isolates (30%) presented both stx1 and stx2. Furthermore, just two isolates (20%) carried eae gene which was contributed with stx2 and one (10%) isolate present espB. No isolates had hly.
These data indicate that domestic animals and birds constitute a natural reservoir of attaching and effacing Escherichia coli strains and some of these are known as human pathogens. Numerous researchers have underlined the strong association between the carriage of eae gene and the capacity of stx-positive strains to cause severe human diseases specially Hemolyitc Uremic Syndrome (HUS). In some studies, although the eae gene was present only in minimal portion of animals, (Rey et al., 2003; Osek 2003; Khan et al., 2002), in this study eae gene was found in 7(28%) isolates. The stx-positive isolates especially in ovine stx-positive isolates (66.6%) indicating that it is probably some of these strains in Iran show high virulence for human. However, production of intimin is not essential for pathogenesis, because a number of sporadic cases of HUS are caused by eae-negative non-O157 STEC strains.
The production of different types of hemolysins has been frequently contributed to E. coli from intestinal and extra intestinal diseases (Yamamoto et al., 1995; Rey et al., 2003). It causes the release of the ferro from cells, providing iron for the bacteria. Epidemiological studies have shown that α-hemolysin correlates with E. coli isolates associated with uropathogenic infection and sepsis (Yamamoto et al., 1995). However, most studies have reported the animal E. coli as non-hemolytic (Call et al., 2001; Emery et al., 1992; Fantinatti et al., 1994; Anjum et al., 2003) that corresponds with the present findings.
ACKNOWLEDGMENTS
We are grateful to the Ministry of Science, Researches and Technology, Research Council of University of Tehran and Research Council of Faculty of Veterinary Medicine of Tehran University for funding project No. 7504003.6.2.