1. Introduction

E. coli is a prominent member of the gastrointestinal tract of their hosts and are released into the environment by human and animal dejections when appropriate hygiene and sanitary practices are not implemented ^{[1, 2]}. It is estimated that up 4% of all cultivable bacteria in the colon are E. coli and the fact that these bacteria species are most often present in the microbiota of the environment they are therefore used as indicators of faecal pollution ^{[1, 2, 3]}. Despite the fact that a number of non-pathogenic E. coli strains are known to maintain the physiological state of the gastrointestinal tract, enhance digestion and defend the host against other enteric pathogens, some strains possess and express a variety of virulence determinants ^{[4, 5, 6]}. Consequently, a number of E. coli strains are currently known to cause diseases ranging from diarrhoea, haemolytic colitis (HC), haemorrhagic uraemic syndrome (HUS) and septicaemia in humans ^{[4, 5, 7, 8, 9]}. Enterohaemorrhagic E. coli (EHEC) are Shiga toxin-producing E. coli strains that possess unique pathogenic properties ^[4], and are characterized by certain seropathotypes that are frequently associated with outbreaks or sporadic cases of human infections ^{[10, 11, 12, 13, 14]}. The host range of EHEC strains are domestic animals and colonized animals usually shed the bacteria through their faeces ^[15]. This therefore increases the chances of contamination ^[16] and the consumption of contaminated food of animal origin is known to be a source of human EHEC infections ^{[17, 18]}. EHEC have thus been isolated from water, sewage, soil, manure, animal faeces, sprouts and fresh produce ^{[12, 15, 19, 20, 21, 22]}. Despite the fact that contaminated vegetables and sprouted seeds are considered the main vehicles for transmitting EHEC strains, meat and meat products have also been linked to some outbreaks caused by diarrheagenic E. coli in many parts of the world ^{[23, 24]}. In South Africa and the North West Province in particular, E. coli O157:H7 strains have been extremely investigated mainly due to its severe concern resulting from the serious clinical outcomes of infections caused by the pathogen [25-34]^[25].

Although E. coli serotype O104:H4 has rarely been associated with human diseases in the past ^[35], outbreaksof infections caused by thisstrain was responsible for the clinical signs of HUS and bloody diarrhoea in humans in Germany, France, UK, Canada and the USA [8,9,22,36-42]. Moreover, epidemiological analysis involving pulsed-field gel electrophoresis, semi-automated rep-PCR and optical mapping analysis revealed that isolates from the different countries had similar genetic profiles ^[39]. It is therefore suggested that the isolates from Germany might have been transmitted through food sources, water or by person-to-person contact to the other countries.

To the best of our knowledge, there is currently no available information on the occurrence of E. coli O104 strains in humans, animals, environmental samples and food products in South Africa. Given the fact that E. coli O104:H4 has been associated with diseases in countries with more advanced public health care facilities there is a need to screen for the presence of these pathogens in South African food products. In this study we present the first investigation on the occurrence of enterohaemorrhagic E. coli O104 strains in raw meat products. This was designed to determine whether these meat products may serve as a potential source for the transmission of the organisms to humans.

2. Materials and Methods

A total of 19 rawmeat samples including beef, mince, and lamb were purchased from butcheries in Mafikeng and local retail shops in Mabule and Legabane respectively. The samples were properly labeled and transported on ice to the laboratory for selective isolation of E. coli. Table 1 indicates the type and number of samples collected from the different areas.

Table 1.

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Upon arrival in the laboratory, 2 grams of each meat sample was washed in 10mL of 2% (w/v) peptone water. Ten-fold serial dilutions were prepared using sterile 2% (w/v) peptone water. Aliquots of 100µL from each dilution was spread-plated on sorbitol MacConkey agar and used for the detection of lactose-fermenting (red colonies) and non-lactose fermenting (white colonies).Plates were incubated aerobically at 37°C for 24 hours. Sixteen presumptive colonies from each plate were purified by sub-culturing on sorbitol MacConkey agar and the plates were incubated aerobically at 37°C for 24 hours. These isolates were stored at room temperature and used for bacteria identification tests.

The morphology of presumptive isolates was determined using standard methods ^[43]. Gram negativerod shaped bacteria were retained for further identification tests.

Triple Sugar Iron agar (Biolab, Merck Diagnostics, South Africa) was used to differentiate members of the Enterobacteriaceae from other Gram-negative bacteria. The test was used to access the ability of isolates to breakdown the three sugars glucose, lactose and sucrose that are present at concentrations of 0.1%, 1.0% and 1.0%. Pure cultures were stab-inoculated into the butt and streaked on the surface of the slant of the TSI agar. The tubes were then incubated at 37°C for 24 hours. Results were read and isolates were classified based on the ability to ferment the sugars with or without the production of gas, colour change from red to yellow and the production of hydrogen sulphide (H₂S) ^[44].

This test was performed using the Test Oxidase reagent^TM (PL. 390) as recommended by the manufacturer (Mast Diagnostics, Neston, and Wirral, UK). Positive results were recorded based on the production of a purple or blue colour and vice versa.

Isolated pure colonies were streaked on the slant and a stab inoculated into the butt of Simmons Citrate agar (Fluka, Biochemika).The tubes were incubated at 37°C for 24 hours and observed for colour change from green to blue which was recorded aspositive results.

Isolates wereinoculatedinto a test tube containing phenol red sorbitol broth and incubated at 37°C for 24 hours. Positive results were based on a colour change from red to yellow ^[45].

The isolates were examined for biochemical properties of E. coli using the API 20E system (Bio-Mérieux, Marcy l’Etoile, France) according to the manufacturer’s instructions. The 20 biochemical tests performed included o-nitrophenyl-β-D-galactosidase, lysine decarboxylase, ornithine decarboxylase, urease production, citrate utilization, deamination of phenylalanine, malonate utilization, Esculin hydrolysis, fermentation of arabinose, xylose, adonitol, rhamnose, cellobiose, melibiose, sucrose, trehalose, raffinose and glucose; production of indole and acetoin and testing for the production of cytochrome oxidase. Results were read with or without the addition of reagents and the API web software was used to determine the identities of isolates based on the indices that were generated.

Genomic DNA was extracted from all presumptive isolates using a modified cell boiling method ^[46]. To carry out the procedure 500μl of sterile water was placed in 1.5 mL microfuge tubes and pure cultures of the isolates were transferred into the tubes. The tubes were vortexed vigorously to prepare a homogenous suspension. The cell suspension was incubated at 100°C in a heating block (Biorad, Digital dry bath) for 15 minutes and this was followed by centrifugation for 2 minutes at 13500 rpm. After centrifugation, the tube was placed on ice for 5 minutes and the supernatant was transferred to a new tube. An aliquot of 5μL of this supernatant was used for PCR analysis.

A PCR-based protocol was used to screen isolates for the presence of enterohaemorrhagic E. coli O104 strains through the amplification of the wzx_O104 gene fragments ^[47]. Specific primer sequences O104F (TGTCGCGCAAAGAATTTCAAC) and O104R (AAAATCCTTTAAACTATACGCCC) ^[47] were utilized and amplifications were performed using a C1000 Touch ^TM Thermal Cycler (Bio-Rad, Johannesburg, South Africa. Standard 25 µL PCR mixtures that contained 12.5µl master mix (Thermo scientific PMM), 8.5µL of nuclease free water, 1.5µL of loading buffer, 0.5µL oligonucleotide both primers and finally, 2µL of DNA template were prepared. All the reagents were Fermentas, USA products and were obtained from Inqaba Biotec Ltd, Sunnyside- South Africa. PCR amplification was performed according to the following in-laboratory optimized conditions: initial denaturation at 95°C for 3 minutes, followed by 40 cycles of denaturation at 95°C for 30 seconds, annealing at 64°C for 1 minute, extension at 72°C for 1 minute 30 seconds and final extension at 72°C for 5 minutes. The PCR products were stored at 4°C before separation by electrophoresis.

The PCR products were resolvedby electrophoresis on a 2% (w/v) agarose gel. A horizontal Pharmacia biotech equipment system (model Hoefer HE 99X; Amersham Pharmacia biotech, Sweden) was used to carry out electrophoresis and this was run for 2h at 80V using 1x TAE buffer (40mM Tris, 1mM EDTA and 20mM glacial acetic acid, PH 8.0). Each gel contained a 100bp DNA molecular weight marker (Fermentas, USA). The gels were stained in ethidium bromide (0.001µg/ml) for 15 minutes and the amplicons were visualized under U.V light at a wavelength of 420nm ^[48]. A Gene Genius Bio Imaging System (Syngene, Synoptics; UK) was used to capture the image using GeneSnap (version 6.00.22) software. GeneTools (version 3.07.01) software (Syngene, Synoptics; UK) was used to analyze the images in order to determine the relative sizes of the amplicons.

3. Results and Interpretation

A total of nineteen meat samples that comprised samples from beef, mince, and lamb were collected from four butcheries in Mafikeng, four shops in Mabule and three shops in Legabane. Sixteen characteristic isolates from each sample were subjected to bacteria identification tests for E. coli (Gram staining, oxidase test, Citrate utilisation test, TSI and API 20E test). Detailed results of the number of isolates that were positive for the different tests are shown in Table 2 and Table 3. All the isolates were Gram negative rods and therefore satisfied that presumptive characteristic for E. coli. A large proportion (93%) of these isolates was oxidase negative; fermented the sugars in the TSI medium; utilized citrate and fermented the carbohydrate sorbitol. On the contrary, only a small proportion (27.3%) of the isolates produced hydrogen sulphide gas (Table 2). Based on patterns obtained for the biochemical profiles of the isolates 102 (33.6%) were positively identified as E. coli (Table 3).

Table 2. Proportion of isolates that were positive for E. coli characteristics based on the preliminary biochemical tests

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Table 3. Number of isolates that were positively identified as E. coli O104 based on specific PCR analysis

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A total of 304 E. coli isolates were screened for characters of enterohaemorrhagic E. coliO104 strains by specific PCR amplification of the wzx_O104 genetic marker. Table 3 indicates the number of isolates that were positive for the target gene from the different sampling areas. Overall a total of 52 isolates were positively identified based on the presence of the wzx_O104. Despite this, not all samples were positive for this pathogen. However the detection of the recently emerged enteroaggregative E. coli O104 strain in raw meat samples in the North West Province, South Africa was a cause for concern. On the basis of the findings obtained in our study, E. coli O104 strains were isolated from raw meat samples and these food products may serve as potential vehicles for transmitting these pathogens to humans if the food products are consumed undercooked. Figure 1 indicates a 1% (w/v) agarose gel of the wzx_O104 gene fragments amplified.

Figure 1. Agarose gel of wzx_O104gene fragments amplified from E. coliO104 isolates. Lane M= 100bp marker; Lanes 1-9= wzx_O104 gene fragments amplified from E. coliO104 isolates obtained from meat samples

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4. Discussions

It has been established that domestic and wildlife animals are the natural reservoirs of STEC and EHEC and the presence of these bacteria in the environment results through the uncontrolled release of faeces ^[15]. Therefore, these bacteria pathogens have the potential to contaminate food and water bodies if proper hygiene is not implemented. Bacterial foodborne zoonotic infections are the most common cause of human intestinal disease in many countries. This explains the need for enhanced research efforts and surveillance programs to enforceappropriate control measures by the food industry and also awareness to consumers ^[49]. Shiga toxin-producing E. coli are currently considered an important group of foodborne zoonotic pathogens due to the fact that they are frequently associated with diarrhoea, haemorrhagic colitis (HC) and the life threatening haemolytic uraemic syndrome (HUS) in humans in many countries worldwide ^[50]. Domestic animals especially pigs and cattle have been reported as the major reservoirs of STEC in South Africa ^{[30, 31, 33]} and other parts of the world ^[51]. There is currently no report describing the detection of non E. coli O157:H7 strains from food products in the North West Province of South Africa.

The objective of this study was to isolate E. coli O104 strains in meat samples collected from different supermarkets in the Mafikeng area and to identify these isolates using Gram staining, oxidase test, Triple Sugar Iron test, citrate utilization, Sorbitol fermentation tests, API 20E biochemical profiles and PCR analysis. In this study, E. coli O104 was detected and isolated from raw meat products that are sold in some supermarkets. The E. coli O104 strains were identified using both preliminary microbiology identification methods and a PCR assay. Most of the studies conducted on these species employed PCR assays only for their detection from samples ^[19]. Generally, there are two different approaches that are currently used to detect STEC in foods ^{[52, 53, 54, 55, 56, 57]}. Serogroup-independent techniques rely on the prevalence of genes and the characteristics of contaminating STEC strains ^{[52, 54]} while serogroup-dependent methods target a subset of E. coli serogroups that frequently implicated in human diseases and outbreaks and may be directed at E. coli O26, O45, O91, O103, O111, O121, O145 and O157 strains ^{[53, 55, 56, 57]}. Consequently, other E. coli serotypes such as O113:H21, O174:H21 and the newly emerging O104:H4 serotype that are less frequently associated with human infections, but can still cause cases of HUS and can contaminate food products will therefore be missed ^[58]. In addition most of the protocols designed to detection of Shiga toxin-producing Escherichia coli (STEC) in food and water sources ^{[27, 28, 29, 30, 32, 59]} typically focus on E. coli O157:H7 ^{[27, 28, 29, 32, 59]}. Against this background there is need to develop species specific assays to constantly and affectively monitor the occurrence of these non-O157 strains in food and water.

In France cattle was not a reservoir for the highly virulent E. coli O104:H4 strains ^[60]. However, other related E. coli O104 strains have been isolated from contaminated milk in the USA and sprouts in Germany ^[20]. EHEC strains are present in the gastrointestinal tract of domestic animals and during the removal of intestinal contents, faecal contamination or contact between the hide and carcass can facilitate the transmission of pathogenic E. coli strains to the meat ^{[15, 61]}. Moreover in most developing countries hygiene conditions are highly compromised ^[62] and therefore these increase the chances of contamination ^[16]. The implementation of routine pathogen surveillance strategies would great reduce human infections. This study reveals the presence of E. coli O104 strains in raw meat samples obtained from some supermarkets in the North West Province, South Africa. There is need to identify virulence determinants that would serve as definitive markers for determining the pathogenicity of the current strains.

5. Conclusion

In the present study E. coli O104 strains were successfully isolated from raw meat products and these isolates could have severe health complications on humans if thefood products arenot properly cooked before consumption. Given the complications posed by this pathogen in some European countries recently, coupled with the present baseline data it is suggested that a full scale study should be conducted to determine the occurrence of this organisms in a number of food products. An investigation of their virulence gene determinants and the genetic relationships of isolates from different sources will be of great epidemiological value.

Acknowledgement

We gratefully acknowledge Mr. B.J Morapedi for his assistance during the collection of samples and Mrs Huyser Rika for technical assistance during this study. This study was financially supported by Department of Biological Sciences, North West University and the North West University Postgraduate Merit Bursary.

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