Detection of Escherichia coli O157 and Escherichia coli O157:H7 by the immunomagnetic separation technique and stx1 and stx2 genes by multiplex PCR in slaughtered cattle in Samsun Province, Turkey

Enterohemorrhagic Escherichia (E.) coli strains are a subset of the Shiga toxin-producing E. coli (STEC) that cause diseases in humans and pose a threat to public health worldwide [19, 20]. Many environmental and food sources have caused E. coli O157 or E. coli O157:H7 infections, but they are primarily attributed to consumption of food of animal origin, especially cattle [36], or to direct or indirect contact with cattle or other farm animals [24]. Human infection by E. coli O157:H7 has been reported in over 30 countries, and cattle appear to be the chief source of infection. Indeed, many outbreaks have been linked to beef consumption [8, 10, 26]. Cattle and other ruminants have been established as major natural reservoirs of E. coli O157 [32] and play a significant role in the epidemiology of human infections [20]. Specifically, between 1% and 35.8% of cattle in the United Kingdom, and the United States were estimated to be contaminated with E. coli O157 [8, 15, 25, 34].

E. coli O157 and E. coli O157:H7 are present in the intestines of cattle as a component of the native microbiota and they can contaminate both the meat and the slaughterhouse environment. As a result, contamination of both carcasses and the environment by E. coli O157 and O157:H7 from the intestinal contents of cattle during slaughter is one of the most significant risk factors in transmission to humans [26, 31]. Hide removal operations potentially constitute another very important source of cattle carcass contamination. Therefore, feces and hide removal are considered to be the main sources of E. coli O157 and E. coli O157:H7 contamination of carcasses during slaughter [12, 25]. Contamination of carcass meat with E. coli O157 and E. coli O157:H7 can occur during dressing, primarily during the skinning, but also during the evisceration phase. Once the E. coli is transferred to the carcass surface, handling and trimming operations can spread the pathogen to the beef trimmings [17].

The pathogenicity of E. coli O157 and E. coli O157:H7, including STEC, is associated with several virulence factors. The main factor contributing to their pathogenicity is their capacity to produce two potent phage-encoded cytotoxins called Shiga-toxins (namely, Stx1 and Stx2). Shiga toxins cause diseases such as hemorrhagic colitis and hemolytic uremic syndrome through cytopathic effects on the vascular endothelial cells of the kidneys, intestines, central nervous system and other organs [16]. In addition to the production of toxins, another virulence-associated factor expressed by STEC is a protein called intimin, which is encoded by the eae gene and responsible for the intimate attachment of STEC to the intestinal epithelial cells. The role of other virulence genes through the production of enzymes such as enterohemolysin, an extracellular serine protease, and a catalase/peroxidase in causing infection appears to be minor [23].

According to studies reported from various parts of the world, cattle carcasses carry a potential risk of the presence of E. coli O157 and E. coli O157:H7 through fecal contamination [5, 7, 22, 25, 35, 38, 39]. There have been many studies conducted to determine E. coli O157 and E. coli O157:H7 in only carcass or rectal samples at abattoirs worldwide, including Turkey [5, 7, 22, 41]. However, to the best of our knowledge, there has been no attempt to detect E. coli O157 and E. coli O157:H7 in both rectal and carcass samples from the same animal. Therefore, this study was conducted to investigate the presence of both E. coli O157 and E. coli O157:H7 and to detect the presence of the stx1 and stx2 genes in isolates from both cattle carcasses and their rectal samples obtained from two commercial abattoirs located in the Samsun Province of Turkey.

In this study, a total of 200 swab samples obtained from 100 slaughtered cattle were tested to investigate the presence of E. coli O157 and E. coli O157:H7, as well as to detect the presence of stx1 and stx2 genes in the isolates. Swab samples were taken from 100 cattle carcasses and their rectal contents at two commercial abattoirs located in Samsun Province, Turkey between December 2007 and March 2008. Samples were collected immediately after removal of the hide (dressing step) from the carcass. Each carcass surface sample consisted of two pooled neck and rump subsamples. An area of 200 cm² of the neck and rump regions of the carcass was swabbed (100 cm² - 10 × 10 cm - of each region) using two different sterile cotton swabs [37]. For isolation, the swabs were placed in a modified tryptone soy broth (mTSB-Oxoid-CM 989; Basingstoke, England) supplemented with novobiocine (20 mg/L, N1628; Sigma, USA) in 10 mL tubes and then incubated at 41.5℃ for 24 h [35]. The two tubes from one carcass were mixed and evaluated as one carcass sample. A total of 100 rectal samples were collected and processed in the same manner as the carcass samples. Immunomagnetic separation (IMS) was conducted using immunomagnetic beads coated with an anti-E. coli O157 antibody (Dynabeads anti-E. coli O157; Dynal A.S., Norway) according to the manufacturer's instructions. The 50 µL IMS bead complex finally recovered was spread onto tellurite (2.5 mg/L) - cefixime (0.05 mg/L) - sorbitol MacConkey (TC-SMAC) agar (Oxoid-CM 813, Supl.SR 172 E; Basingstoke, England) and incubated at 37℃ for 24 h. Up to five colonies exhibiting typical presumptive positive characteristics of E. coli O157 colonies were identified, subcultured onto yeast extract-tripticase soy agar (Oxoid-CM 131-L21; Basingstoke, UK), incubated for 24 h at 37℃ and subjected to the confirmatory tests described below:

Determination of virulence genes (stx1 and sxt2)

Multiplex PCR was conducted to detect the presence of the shiga-toxin genes (stx1 and stx2) in E. coli O157 and E. coli O157:H7 isolates were trated according to a modified version by using multiplex PCR assays [14, 30]. The primers and PCR conditions used in this study are shown in Table 1. E. coli O157:H7 ATCC 43895 was used as a reference strain.

Table 1
Oligonucleotide primers sequences used in multiplex PCR for amplification of stx1 and stx2 genes

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DNA extractions of strains were conducted using the boiling method. Briefly, each positive colony was inoculated on TSA and incubated for 24 h at 37℃. Two colonies were then selected and suspended separately in 500 µL of sterile distilled water in microcentrifuge tubes, after which they were incubated at 95℃ for 10 min in a water bath (Memert, Germany). The tubes were then centrifuged (Hettich-Universal-320R unit) at 9,503 × g for 10 min, after which the supernatant containing the DNA to be used as the template DNA was transferred into Dnase/Rnase-free microcentrifuge tubes. The extracted DNA samples were stored at -20℃ until use.

To detect the stx1 and stx2 genes, 10 µL of extracted DNA was used as a template in a reaction mixture with a final volume of 50 µL that contained 200 mM of each deoxynucleoside triphosphate (dNTP), 250 nM stx1 primer, 250 nM stx2 primers, 1U of Taq DNA polymerase in 1× PCR buffer and 2 mM of MgCl₂. The amplification of DNA (MJ Mini; BioRad, USA) was conducted as follows using a thermocycler (MJ Mini-PTC-1148; BioRad, USA): initial denaturation at 94℃ for 10 min, 35 cycles of denaturation at 94℃ for 1 min, annealing at 60℃ for 1 min, extension at 72℃ for 1 min, and final extension at 72℃ for 7 min [14].

For gel electrophoresis, the 20-µL amplicon mixtures were supplemented with 4-µL of loading dye and loaded onto a 2.0% agarose gel containing ethidium bromide (Gene choice). Electrophoresis (Power Pac-Basic; Bio-Rad, USA) was then conducted at 90 V for 1.5 h. A 100~1,000 bp molecular weight marker was used to identify the amplified products, which were visualized by UV illumination (WiseUV-Wuv-L50; Daihan Scientific, Korea). The nucleotide sequences and predicted product sizes of the primers are shown in Table 1.

In the present study, E. coli O157 or O157:H7 was detected in 52 of 200 samples tested (49 E. coli O157 and 3 E. coli O157:H7). The E. coli O157 strain was isolated from 24 carcasses and 25 rectal samples, while the E. coli O157:H7 strain was isolated from two carcasses and one rectal sample.

Of the 49 samples that contained E. coli O157, 32 were from the rectal and carcass samples of the same animal, while the other 17 isolates were all from different cattle. Specifically, eight were from carcasses and the remaining nine were from rectal samples. Two of the E. coli O157:H7 isolates were obtained from the carcass and rectal swabs of the same animal, while the remaining isolate was obtained from a carcass sample (Table 2).

Table 2
Isolation of E. coli O157 and E. coli O157:H7 strains from slaughtered cattle and detection of stx1 and stx2 genes

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The results of multiplex PCR for the detection of stx1 and stx2 genes are shown in Fig. 1. While both the stx1 and stx2 genes were detected in 35 E. coli O157 isolates from 17 carcass and 18 rectal samples, they were detected in only one E. coli O157:H7 isolate from a carcass sample. The stx2 gene alone was detected in only two E. coli O157 isolates and the stx1 gene was not detected alone in any isolates. Neither of these genes was detected in the 12 E. coli O157 and two E. coli O157:H7 isolates from carcass and rectal samples (Figs. 1 and 2).

Fig. 1
Amplification products of stx1 and stx2 genes in E. coli O157 and O157:H7 identified by multiplex PCR. Lane M: Marker (100~1,000 bp).

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Fig. 2
The determination of stx1 and stx2 genes of E. coli O157 and O157:H7 by multiplex PCR. Lane M: Marker (100~1,000 bp), Lane K: positive control (E. coli O157:H7 ATCC 43895).

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In Turkey and other parts of the world, although there have been many studies conducted to determine the presence of E. coli O157 and E. coli O157:H7 in either carcass or rectal samples at abattoirs, the authors of the present study are unaware of any attempts to detect E. coli O157 and E. coli O157:H7 in both the rectal and carcass samples of the same animal. Therefore, the present study was conducted to investigate the contamination of carcasses and their rectal contents with E. coli O157 and E. coli O157:H7 at two abattoirs in Samsun Province of Turkey because the majority of food-borne E. coli O157 and E. coli O157:H7 infections in humans occur after the consumption of contaminated beef and cattle products [31].

Isolation rates of E. coli O157 and E. coli O157:H7 from bovine carcasses and feces ranging from low (0.39%) to high (17.0%) have been reported for Mexico, Ireland, Belgium, England, France, Poland, Germany, the United States, Turkey and other countries. E. coli O157 or E. coli O157: H7 isolates obtained from cattle carcasses or feces have also been found to contain at least one of the stx1, stx2, eaeA, hylA and fliC_h7 genes [5, 7, 22, 25, 29, 35, 38, 39, 41]. In contrast to other countries, there has been only one study of carcasses reported in Turkey [21]. The results of that study revealed that 3.9% and 2.4% of the bovine carcasses were contaminated by E. coli O157 and E. coli O157:H7, respectively. There have been a few studies of cattle feces conducted in Turkey [1, 3, 9, 41], the results of which revealed that E. coli O157 or E. coli O157:H7 were present in 1.28% and 13.6% of the samples, respectively. It has also been reported that at least one virulence gene (stx1, stx2 or eae genes) was detected in the E. coli O157 or E. coli O157:H7 isolates from feces samples [1, 3, 41].

In the present study, the E. coli O157 strain was isolated from 24 carcasses (24%) and 25 rectal samples (25%), while the E. coli O157:H7 strain was isolated from two carcasses (2%) and one rectal sample (1%). These values for E. coli O157 were higher than those of previously conducted studies. However, previously conducted studies show a wide range of isolation ratios for E. coli O157 and E. coli O157:H7. This variation may be due, at least in part, to the sensitivity of the method, diverse geographical origins of cattle, numbers of cattle, study design, number of herds and cattle, sex and age of cattle, season, abattoir conditions and treatment with antimicrobial substances during the process [6, 7, 39]. It has also been reported that the prevalence of E. coli O157 and E. coli O157:H7 varies with the seasons, generally increasing in the warm months of March-September in the northern hemisphere [4, 6, 7, 25, 39]. Another important factor influencing the identification of individual strains of E. coli is the isolation method. Indeed, the detection of E. coli O157:H7 from cattle fecal samples is known to be very difficult due to their low concentration. Therefore, direct inoculation of samples onto plates is not sensitive enough. Several enrichment culturing methods and isolation methods have been developed to counter this problem [38]. One of the more sensitive methods is the IMS technique [28, 38, 40], which is why enrichment/IMS procedures were employed in the present study. Another study found that the IMS technique was superior to the classic culture technique for Salmonella isolation (unpublished data). In the present study, swab samples were collected from the rectum of the cattle instead of direct feces samples, because the recto-anal junction mucosa has been identified as the primary site of E. coli O157:H7 colonization in cattle [18, 33, 35]. In attempting to manage E. coli O157 and E. coli O157:H7 contamination in abattoirs, it is crucial to consider cross contamination during slaughter. E. coli O157 and E. coli O157:H7 have been reported to spread easily onto carcass surfaces from the hide or during evisceration [2, 12, 21]. The results of the present study support that contention, with many rectal and carcass samples of the same animal being positive.

Epidemiological studies in cattle indicate that the horizontal transmission of E. coli O157 or E. coli O157:H7 occurs in groups of animals, and that contaminated water may facilitate its spread and persistence within herds [13, 15]. Therefore, control of the spread of E. coli O157 and E. coli O157:H7 at the farm level becomes very important. Moreover, cross contamination may occur during the slaughter of cattle and other processes at abattoirs.

The results of the present study indicate that meat from cattle poses a risk to human health in Turkey because of potential E. coli O157 and E. coli O157:H7 contamination. To minimize the risk to public health, implementation of the HACCP system in abattoirs is recommended.