Detection of Escherichia coli O157:H7 and Shiga toxin 2a gene in pork, pig feces, and clean water at Jagalan slaughterhouse in Surakarta, Central Java Province, Indonesia

Background and Aim: The feasibility assessment of food products on the market becomes one of the milestones of food safety. The quality of food safety of animal origin especially pork need to get attention and more real action from the parties related and concerned. Since pork is also a source of transmission for the contagion of foodborne disease so that the study of the existence of several agents in the pork and its products become the benchmark of safety level. This study aimed to isolate, identify, and detect the Shiga toxin 2a (stx2a) gene from Escherichia coli O157:H7 in pork, pig feces, and clean water in the Jagalan slaughterhouse. Materials and Methods: A total of 70 samples consisting of 32 pork samples, 32 pig fecal samples, and 6 clean water samples were used to isolate and identify E. coli O157:H7 and the stx2a gene. Isolation and identification of E. coli O157:H7 were performed using culture on eosin methylene blue agar and Sorbitol-MacConkey agar media and confirmed molecularly with polymerase chain reaction to amplify the target genes rfbE (317 bp) and fliC (381 bp). The isolates, which were identified as E. coli O157:H7, were investigated for the stx2a gene (553 bp). Results: The results of this study show that of the total collected samples, E. coli O157:H7 was 28.6% in Jagalan slaughterhouse and consisted of 25% of pork samples, 31.25% of pig fecal samples, and 33.3% of clean water samples. The isolates that were identified to be E. coli O157:H7 mostly contained the stx2a gene, which was equal to 75%, and consisted of seven isolates from pork samples, seven isolates from fecal samples, and one isolate from clean water samples. Conclusion: E. coli O157:H7 was found in 28.6% of pork, pig feces, and clean water in Jagalan slaughterhouse and 75% of identified E. coli O157:H7 contained the stx2a gene.


Introduction
Escherichia coli O157:H7 is a zoonotic bacterium and causes foodborne disease. The infection by these bacteria initially causes non-bleeding diarrhea accompanied by abdominal cramps. Then, it may develop into bloody diarrhea and hemolytic uremic syndrome (HUS), which causes kidney failure in humans [1]. At the beginning of 1993, there was an E. coli O157:H7 outbreak in Washington, Idaho, California, and Nevada, causing 56 people to suffer from HUS and 4 others to die [2]. In 1996, E. coli O157:H7 was found in Louisiana. The number of cases was 8-20 cases per year. In 2015, an outbreak caused 15 students to suffer from diarrhea and abdominal pain [3]. It was also reported that E. coli O157:H7 was found (15.8%) in the feces of patients who had kidney failure at RSUP Sanglah Bali, Indonesia [4]. Humans are infected by E. coli O157:H7 in various ways, one of which is by ingesting contaminated meat. Bacterial contamination of meat can occur from the process of cutting until it is being consumed. The meat can become contaminated in slaughterhouses from fecal contamination and other contaminations during the skinning process [5].
Pork is a foodstuff that is widely consumed by the people of Surakarta City, Central Java. It is provided by a large number of pigs slaughtered in Central Java's slaughterhouse, the number of which continues to increase every year. The number of slaughtered pigs in this slaughterhouse in 2012 was 15,315, in 2013, it was 18,547, and in 2014, it was 18,443 [6]. Therefore, it is essential to conduct a research investigation on the detection of E. coli O157:H7 in pork, pig feces, and clean water at Jagalan slaughterhouse in Surakarta, Central Java, Indonesia. The results of this study may become a source of information for both veterinarians and the public, so pork can be processed, cooked, and consumed properly to prevent foodborne diseases. This research may also become the foundation or a reference for the government to improve the management of slaughterhouses and handling of pork in Jagalan slaughterhouse.
This study aimed to detect E. coli O157:H7 in pork, pig feces, and clean water in the Jagalan slaughterhouse and the Shiga toxin 2a (stx2a) gene from E. coli O157:H7 isolates isolated from pork, feces, and clean water from the Jagalan slaughterhouse.

Ethical approval
This study did not involve living creatures, either both animals and humans as research subjects, so it does not require ethical clearance approval.

Sample collection
Sampling was performed at the Jagalan slaughterhouse Surakarta, Central Java Province, Indonesia. The total samples taken were 70 and consisted of 32 samples of pork, 32 samples of pig feces, and 6 samples of clean water from a water reservoir. Sampling was performed aseptically. Pork and pig feces were put into sterile plastic tubes while the water was put in a sterile bottle. The samples were taken to the Laboratory of Gadjah Mada University using a cool box for conventional testing of the culture on eosin methylene blue agar (EMBA) and Sorbitol-MacConkey agar (SMAC) media followed by molecular testing (polymerase chain reaction [PCR]).

Meat, feces, and clear water sample
The bacterial isolation procedure in this study referred to Suardana et al. [4]. Ten grams of meat and fecal samples and 10 ml of the water sample were added to 90 ml of buffered peptone water (Conda CAT: 1403.00) 0.1%; then, the meat was mashed using a stomacher, while the feces and water samples were homogenized. Furthermore, as much as, 100 µl of diluted samples were spread on the surface of the EMBA media (Oxoid CM0069) using sterile bent glass, incubated at 37°C for 24 h. The growing colonies that showed a green metallic color with black spots in the middle counted as E. coli colonies and tested positive (+).
Positive results on EMBA media from the three types of samples were cultured on SMAC selective media (Oxoid CM 0813) and were incubated at 37°C for 24 h. Colonies identified as E. coli O157 show characteristics of transparent colonies or colorless colonies, or non-sorbitol fermenting colonies. Colorless colonies were then cultured on brain heart infusion (BHI) media (Conda CAT: 1400.00) (800 µl), incubated at 37°C for 24 h and 200 µl of glycerol was added and stored at −20°C until needed for further examination. Positive bacteria growing on BHI media are characterized by turbidity in the media as shown in Figure-1. In this study, positive control of E. coli O157:H7 was used from the Inter-University Center Universitas Gadjah Mada.

Isolation of bacterial genomic DNA
Bacterial DNA isolation was done according to the Zymo Research supplier instructions. First, E. coli O157 isolates were harvested by centrifugation (12,000 rpm for 5 min), the supernatant was removed, and the cell pellets were mixed with 200 µl of the elution buffer DNA and vortexed. Then, the solution was mixed with 200 µl of biofluid and cell buffer and 20 µl of proteinase K. Following that, the mixture was vortexed and incubated in a water bath for 10 min at 55°C. Next, one volume (420 µl) of the Genomic Binding Buffer was added and vortexed for 10-15 s. Then, it was transferred into the Zymo-Spin IIC-XL Column in the collection tube, centrifuged at 12,000 rpm for 1 min, and the collection tube was discarded. After that, 400 µl of prewash buffer DNA was added into the spin column in a new collection tube, centrifuged at 12,000 rpm for 1 min; then, the liquid was removed (the collection tube was not removed). Next, 200 µl of gDNA wash buffer was added to the spin column and centrifuged at 12,000 rpm for 1 min. Then, the liquid and collection tube were removed. The spin column was transferred to the microcentrifugation tube, and 50 mL of elution buffer DNA was added. Following this, the solution was incubated for 5 min at room temperature and then centrifuged at maximum speed for 1 min to reach DNA elution. The spin column was then removed, and bacterial DNA was kept in the microcentrifugation tube. Electrophoresis was then performed and observed using a UV transilluminator to see the thickness of the extracted DNA, as shown in Figure-2. The pure DNA to be used immediately was stored at 4°C. Otherwise, it was stored in a −20°C freezer until needed [4].
The PCR reaction for the detection of the rfbE and fliC genes was carried out at a total volume of 25 µl containing 12.5 µl Bioline mixture, 8.5 µl ddH 2 O, 2 µl template DNA, 1 µl primer (F), and 1 µl primer (R). Amplification was carried out on Thermal Cycler Model TC25/H machines with pre-denaturation conditions at 94°C for 5 min, followed by 40 cycles with denaturation reaction conditions at 94°C for 45 s, annealing at 56°C for 30 s, and polymerization at 72°C for 45 s. At the end, polymerization was added at 72°C for 10 min. After the amplification was complete, 5 µl of PCR product was mixed with 1 µl of loading dye and was electrophoresed on a 2% agarose gel filled with cyber safe dyes, along with a 100 bp DNA Ladder marker. Electrophoresis was performed at a voltage of 100 V for 55 min. The visualization of the band was done by employing a UV transilluminator and then using a digital camera to take a picture [4].

Detection of stx2a gene
To detect the stx2a gene, we used primer (F): (5′-CGAGGGCTTGATGTCTATCAG.-3′) and (R): (5′-TCAGTATAACGGCCACAGTCC-3') (product of PCR 553 bp), as previously conducted by Park et al. [9]. The PCR reaction was carried out on a total volume of 25 µl containing a 12.5 µl Bioline mixture, 8.5 µl ddH 2 O, 2 µl template DNA, 1 µl primary (F), and 1 µl primary (R). Amplification was performed on Thermal Cycler Model TC25/H machines with pre-denaturation conditions at 94°C for 5 min, followed by 40 cycles with denaturation reaction conditions at 94°C for 45 s, annealing at 56°C for 30 s, and polymerization at 72°C for 45 s. At the end, polymerization was added at 72°C for 10 min. After the amplification was complete, 5 µl of PCR product was mixed with 1 µl of loading dye and was electrophoresed on a 2% agarose gel filled with cyber safe dyes, along with a 100 bp DNA Ladder marker. Electrophoresis was carried out at a voltage of 100 V for 55 min. The visualization of the band was done by employing a UV transilluminator and then using a digital camera to take a picture [4].

Statistical analysis
The data of the conventional and molecular bacterial isolation and identification, along with the observational results, were provided in the form of tables and figures. These results were then analyzed descriptively.

Results and Discussion
E. coli O157:H7 is one of the strains of enterohemorrhagic E. coli that was first identified as a pathogenic bacterium in humans in 1992. This pathogen is associated with the occurrence of foodborne disease outbreaks, namely, hemorrhagic colitis (bloody diarrhea) [5]. Cows are animals that are considered the main reservoir of E. coli O157:H7 [2], but these bacteria are also found in other animals including sheep, goats, horses, dogs [10], and pigs [11].
The results of cultured samples showed that all samples (100%) were positive on EMBA media and consisted of 32 pork samples, 32 pig fecal samples, and 6 clean water samples. Positive results on EMBA media were characterized by the growth of green metallic sheen colonies (Figure-3). A total of 29 samples (11 pork samples, 15 fecal samples, and 3 clean water samples) were positive on SMAC media characterized by the growth of transparent colonies or colorless colonies (Figure-4) The confirmation results using the PCR method showed that there were 28.6% E. coli O157:H7 (20/70) in the slaughterhouse with 25% of pork samples, 31.25% of fecal samples, and 33.33% of clean water samples. The results were confirmed by the presence of the rfbE and fliC genes. Samples   Table-1 and Figures-5 and 6. The results of this study showed that E. coli O157:H7 was found in 31.25% of pig feces taken from Jagalan slaughterhouse, Surakarta, Central Java Province, Indonesia (Table-1). The results of this study were lower than the ones reported by Ateba and Mbewe [12], which stated that there was E. coli O157:H7 in 43.1% of pig feces (56/130) in the Northwest Province, South Africa. It was reported that the prevalence of E. coli O157:H7 in pigs in Chile was 10.8%, while in cattle, it was 2.9% [13]. Besides that, it was also reported that E. coli O157:H7 was found in 1.89% of cattle stool at a slaughterhouse in Ethiopia [14]. In Surakarta, Central Java, Indonesia, there have been no reports on the prevalence of E. coli O157:H7 in both pigs and cattle, but in Bali, Indonesia, it was reported by Suardana et al. [4] that the prevalence of E. coli O157:H7 in cattle feces was 5%.
The number of positive results found for E. coli O157:H7 in pig feces indicates that the infection is high in the pig population studied. The results of this study might be able to estimate the level of E. coli O157:H7 infection in a larger pig population in Surakarta, Central Java. However, it is quite challenging to retrace the origin of slaughtered pigs in the Jagalan slaughterhouse.
The results of this study also found E. coli O157:H7 in 25% of pork and 33.3% of clean water taken from the water reservoir in Jagalan, Surakarta (Central Java) ( Table-1). The positive results of this study were higher than the results reported by Enabulele and Uraih [15]. They stated that there was E. coli O157:H7 in 6.94% of beef samples taken from a slaughterhouse in Benin City, Nigeria. However, it was also reported by Bouvet et al. [16] that the prevalence of verotoxigenic E. coli in pig carcasses in three slaughterhouses in France was higher than 80%.
The level of carcass and water contamination at Jagalan (slaughterhouse) in this study was quite high, at 25% and 33%, respectively. This result can also be correlated with the high positive results of E. coli O157:H7 found in 31.25% of pig feces. Similarly, Elder et al. [17] reported that E. coli O157:H7 was found in 17% of beef in the United States, which had a strong correlation between feces and meat contamination. The meats that were contaminated by E. coli O157:H7 in the slaughterhouses might be caused by various factors including fecal contamination on the meats, contaminated tools that were not sterilized anyhow, improper personal hygiene practices, and both air and rodent contamination [18].
Contamination by E. coli O157:H7 in water in slaughterhouses might be attributed to the condition of the reservoirs, which were not covered so that bacterial contamination could occur from pig feces. Contamination might also occur due to contact with the hands of butchers (workers) and anything else from the environment since the reservoir is open.
The pathogenicity of E. coli O157:H7 is associated with its ability to produce Shiga toxins, both Shigatoxin1 (stx1) and Shigatoxin2 (stx2). Bardasi et al. [19] stated that E. coli O157:H7 can produce both stx1 and stx2 or just one of the two toxins. The toxin prototype produced by E. coli O157:H7 has been found. Therefore, stx1 is called stx1a and stx2 is called stx2a. Two stx1a variants have been found, namely, stx1c and stx1d, while the stx2a variants that have been found are stx2c, stx2d, stx2e, stx2f, and stx2g [19].  Available at www.veterinaryworld.org/Vol.12/October-2019/11.pdf Twenty isolates, which were identified with E. coli O157:H7, were followed by the detection of the stx2a gene. The results showed that 75% of E. coli O157:H7 isolates contained stx2a, with 87.5% isolates from pork samples, 70% isolates from fecal samples, and 50% isolates from clean water samples. Positive samples containing the gene stx2a are indicated by the formation of a band at the 553 bp position (Figure-7), as shown in Table-2. E. coli O157:H7 isolates found in this study mostly contained gene stx2a, which was 75% of the sample (15/20) (Table-2). The results of this study are almost the same as those reported by Ateba and Mbewe [12], who said that the prevalence of stx2 produced by E. coli O157:H7 isolates was 70.96% of pig fecal samples (22/31). Avery et al. [20] also reported that 79.2% of 24 E. coli O157:H7 isolates (19/24) isolated from beef carcasses produced stx2, 8.3% of the isolates (2/24) produced stx1 and stx2, and the other 12.5% (3/24) did not produce stx1 nor stx2. Then, 51.85% of E. coli isolates isolated from  raw fish and fish that were ready to eat in Ludhiana, Punjab, produced stx2 (28/54) [21]. According to Melton-Celsa [22], stx2a is more toxic than stx1a. This toxicity difference is proved by endothelial cells, which, when exposed to HUS, are more sensitive to stx2a than stx1a. In addition, Frank et al. [23] also stated that the outbreak of HUS disease in Germany in 2011 was closely related to stx2a.

Conclusion
Based on the results of isolation and identification, it can be concluded that there was a 28.6% contamination of E. coli O157:H7 found in Jagalan slaughterhouse, Surakarta city. The contamination was found in pig feces, pork, and water at 31.25%, 25%, and 33.3%, respectively. Of the 20 isolates of E. coli O157:H7 detected in the slaughterhouse, 75% of them contained gene stx2a.