شناسایی باکتری ‏های اشریشیا کلی وروتوکسیژن جداشده از ماهیان سرد آبی به‏ وسیله واکنش زنجیره‌ای پلیمراز چندگانه در استان چهارمحال و بختیاری

Introduction

Escherichia coli live commensally in the gastrointestinal tract of most mammals and incidence of this organism is very rare in fishes. However, some serotypes of E.coli are human pathogens and have been implicated in foodborne illness.

Verotoxigenic Escherichia coli (STEC) is an important group that may cause gastrointestinal disease in humans, particularly since these infections may result in life- threatening sequel such as the hemolytic- uremic syndrome (HUS) (1). STEC produces one or both of two major types of Shiga toxin, designated Stx1 and Stx2; production of the latter is associated with an increased risk of developing HUS (2). Other putative accessory virulence factors produced by subsets of STEC include the capacity to produce attaching and effacing lesions on intestinal mucosa, as well as megaplasmid- encoded factors such as the enterohemolysin (3 and 4). One of the most important serotypes with increasing frequency over the last 2 decades is O157: H7 which first time identified as a human pathogen in 1982 has emerged as a major cause of both sporadic cases and outbreaks of bloody diarrhea in North America. Most of E.coli O157: H7 infections are food- borne. Food of bovine origin have been identified as the principal vehicle in most outbreaks (3). Also, the non- O157: H7 serotypes that produce verotoxin, A/E lesions and enterohemolysin have been reported (5 and 6). DNA- based assays for identifying Stx genes, eae and enterohemolysin (ehly) genes do not seem to discriminate the non- O157 E.coli from E.coli O157: H7 serotypes. This makes accurate diagnosis of non- O157 EHEC more difficult especially in developing countries where advance techniques are not available in most laboratory facilities. EHEC appears to be transmitted primarily through the ingestion of fecal contaminated foods, particularly undercooked beef (4). However, a large number of outbreaks of EHEC have also been associated with consumption of contaminated drinking water or contact with recreational water (7).

This study was carried out to identify the verotoxigenic E.coli and other virulence factors isolated from cold water fishes in ‍Chaharmahal va Bakhtiari province, Iran.

Materials and methods

Collecting the samples

A total of 100 fecal samples and 100 skin and meat samples of theRainbow trout fishes individually were collected. The samples were chosen randomly (cluster random sampling) from 10 producing pools located in the south of the Chaharmahal va Bakhtiari province at the final step of production in spring and summer 2012.

Isolation of E.coli

MacConkey and SMAC agar (Merck) were used to detection of the E.coli and EHEC colonies (8). A swab of fecal samples and suspension preparing from skin and meat (25 g of sample in 225 ml of LB broth) was cultured on MC and SMAC agar and incubated at 37 ^oC for 18 to 24 h. Five Non- Sorbitol fermenting (NSF) (colorless) colonies from SMAC agar and five red colonies from MC agar were transferred individually to fresh Lurian- Bertani (LB) broth and incubated at 37^oC for 18 h. Complete biochemical and differentiated tests were used to be certain that strains belonged to the E. coli species (8).

Serology

Isolated E.coli was examined by O157 and H7 antisera (MAST), to identify O157 or O157: H7 serotypes.

Detection of virulence genes using PCR

PCR primers

The PCR primers were chosen from each of the verotoxins (Stx1 and Stx2), eae and hly from published sequences (Table 1). The primers were selected to have similar melting temperatures and resulted in products of different sizes that could be easily distinguished in agarose gels (9).

PCR

Each test was performed in a volume of 25 ul containing the following PCR components: dNTP (200 umol/liter of each), PCR buffer 10x, MgCl₂ (final concentration 5 mmol/liter), various concentrations of each primer set (10 pmol/ul), 2 U of Taq DNA polymerase, and 2 ul of DNA template. A whole- cell preparation of isolates was tested as crude DNA templates and was prepared by resuspending 1 bacterial colony in 25 ul of sterile DNase- free, RNase- free deionized water. Before being added to the PCR mixture, to control purposes and isolated strain, DNA was isolated and purified from the reference E.coli O157: H7 using a genomic DNA isolation Kit (Fermentase). For the PCR amplifications, a total 50 ng of the isolated DNAs was used. PCR amplifications were performed in a thermal cycle under the following conditions: an initial DNA denaturation step at 94 ^o C for 5 min following by 35 cycles beginning with 1 min of denaturation at 94 ^o C, 1 min of primer annealing at 50 ^o C, and 1 min of extension at 72^o C. The final extension step was performed at 72 ^o C for 7 min. The amplified PCR products (10- ul aliquots) were analyzed by electrophoresis in 2% agarose gels in Tris acetate (TAE) buffer at 100 V for 45 min. The gels were stained with ethidium bromide and photographed under ultraviolet light using a commercial documentation system.

Optimization of PCR conditions.

To standardize the conditions for the multiplex PCR test, the concentration of each of the 4 primer pairs, the optimal annealing temperature, and Mg²⁺ concentration were determined using the DNA (50 ng) of isolated strains as templates. Initially, the same concentration (100 nmol/ liter) of each set of primers was used, but this approach resulted in uneven intensities or even lack of some of the amplified products. To overcome this problem, the concentration of each primer pair was adjusted without changing the optimal annealing temperature (50 ^oC) and the concentration of Mg²⁺ (5 mmol/liter). The best concentrations of each primer set that yielded 4 distinct PCR bands were 10 pmol.

Results

Identification of virulence genes

Out of the overall E.coli strains (18), 14 strains were isolated from skin and 4 strains were isolated from feces, but none of them reacted positively with O157 and H7 antiseras. The results of the multiplex PCR revealed that 8 (57%) of 14 strains isolated from skin and 2 (50%) of 4 strains isolated from feces contain virulence genes (Stx1, Stx2 and eae) and none of the isolates contain hly gene (Table 2).

Table 1- Primers used in the multiplex PCR for stxI, stxII, Intimin genes

Table 2- Virulence genes of E.coli isolated from feces, skin and meat of Rainbow trout fish

Fig 1- Multiplex PCR for detection of Stx1 (555 bp), Stx2 (118 bp) and eae (425 bp)

1: DNA Ladder, 2 Control positive, 7 Control negative. 3, 4, 5 and 6 positive samples