Characterization by PCR of Escherichia coli from Beef and Chicken Used in Restaurants in Yaoundé Cameroon

Meat constitutes the main source of protein and occupies an important place in our diet. Indeed, the production of poultry and beef has increased. How-ever, when PCR and microbial culture were used to assess for E. coli in beef and a non-statistically significant difference was observed in the case of chicken meat. Also, a statistically significant difference was noticed with the different transport conditions, but this wasn’t the case with the supplier source as well as the storage conditions where a non-statistically significant difference was seen. This study revealed that PCR-based methods are fast and reliable in the identification and characterization of Escherichia coli in meats (beef and chicken) as well as in assessing the prevalence of pathogenic E. coli, in Cameroon.


Introduction
Eating is a primary need for everyone. The foods we eat must be healthy and balanced to provide the body with the nutrients necessary for physical and mental well-being. About 25% of the population still suffers from chronic undernourishment in sub-Saharan Africa [1]. Malnutrition is generally rife in poor countries because of nutritional deficiencies. Meat constitutes the main source of protein and occupies an important place in our diet. It has a high protein content and contains all the essential amino acids, iron, zinc and vitamins A, B12, B6, D and E in particular [2]. In addition, meats are rich in lipids and carbohydrates essential for growth and development [3]. Meat can be part of a balanced diet providing important nutrients for health. Fortunately, the production of poultry and beef has increased [4]. World production of bovine meat was estimated at 67.7 million tons and that of poultry meat at 107.0 million tons in 2013 [5]. Meat production in Cameroon reached 344,000 tons in 2016 with around 118,267.2 tons of poultry and over 7.4 million head of cattle [6]. Cattle and poultry meats are marketed in Cameroon in fresh or processed forms (roast, shawarma, beefsteak, kebabs, sausages, etc.). Unfortunately, the hygienic quality of meat is not always guaranteed [7] [8]. Almost one out of ten people in the world fall ill after consuming food contaminated with pathogens and contaminants (bacteria, viruses, parasites, toxins and chemicals) [9]. Foodborne diseases are a global public health problem; apart from the toxi-infections that they can cause very quickly after consumption, they can also cause long-term illnesses, such as cancer, kidney or liver failure and brain or nervous disorders. These diseases can be more serious in children, pregnant women, the elderly or those with a weakened immune system. Microorganisms such as Escherichia coli, Staphylococcus aureus and Salmonella were found in the meat carcasses analyzed which could cause diarrhea, dysentery, various infections, food poisoning, gastroenteritis, or typhoid fever [10].
E. coli is a Gram-negative bacterium belonging to the Enterobacteriaceae fam-ily. They are short rods shaped and motile by means of peritrichous flagella. Moreover, they are facultative anaerobes, non-sporulating and 2 -4 μm in length and approximately 0.6 μm in diameter, E. coli is one of the fecal coliforms normally found in the digestive microflora of humans and many animals such as cattle and poultry. Many E. coli species are safe for humans and animals. However, some strains such as E. coli O157: H7 are pathogenic for humans. Humans can be healthy carriers of these bacteria, with no visible symptoms. However, ingestion of a few hundred bacteria may be sufficient to reach the infective dose [11]. Their main clinical sign is diarrhea, which is more often hemorrhagic and can be associated with abdominal pain and sometimes vomiting. The incubation phase depends on the virulence of the strain, the dose ingested and the resistance of the patients. Humans can become infected by eating undercooked meats. In effect, E. coli is very abundant in the intestinal flora but does not persist for long in the environment and thus, the best indicator of faecal contamination. In the meat industry, the main source of E. coli contamination is the intestinal tract of animals. Their presence in meat indicates a technical slaughter defect or cross contamination. It can also be due to contamination from people handling meat [12]. In sub-Saharan Africa, cases of foodborne illness are enormous, but their estimates are largely underestimated by health authorities and their origins are rarely elucidated [13]. In Cameroon, studies carried out so far have shown the presence of pathogenic microorganisms in some meat samples [7] [14] [15].
Thus, the present study was designed to characterize Escherichia coli from beef and chicken meat used in restaurants in Yaoundé, Cameroon.

Sample Collection
A total of 105 meat samples (n = 105) were collected randomly following a convenience sampling method without repetition of meats. Sixty (60) and forty-five

Microbial Culture
In the laboratory, 10 g of each sample was introduced aseptically into a sterile stomacher bag and macerated in 90 mL of sterile diluent (0.1% peptone and 0.8% sodium chloride from DIFCO LABORATORIES Detroit MI USA). Serial dilutions were carried out by introducing 1 mL of the meat suspension into 9 mL of sterile diluent which was then used for the cultivation of micro-organisms.

DNA Extraction
A loop full of bacteria from the culture plate was placed into a 1.5 mL micro -centrifuge tube and mixed with 200 μL of 5% Chelex-100 resin (Bio-Rad) and 2 μL of Proteinase K (20 mg/ml, NEBiolabs). After incubation at 56˚C for an hour and then at 95˚C for 10 minutes, the sample was mixed and then centrifuged at 13,000 rpm for 5 minutes to completely separate the layers. The DNA-containing supernatant was used as a template in PCR reactions.

PCR Confirmation of E. coli Isolates
The isolated organisms that were phenotypically identified as E. coli were then confirmed by PCR using primers specific to E. coli 16S rRNA gene (Table 1). PCR was performed following the procedure described by Schippa et al., with slight modification [16]. 25 μL reaction containing nuclease free water, 10× thermopol buffer, 10 mM dNTPs (200 μM of each deoxyribonucleotide), 20 pmol of each primer and 5 U/μL Taq polymerase and 3 ng of DNA. After initial incubation at 95˚C for 3 min, a 30-cycle amplification protocol was followed at 94˚C for 45 s, 58˚C for 45 s and 72˚C for 60 s, and a final extension step of 72˚C for 3 min. Electrophoresis of the PCR products was done using 2% agarose gel. After electrophoresis, the gel was stained for 10 minutes in ethidium bromide for visualization and the expected band size was observed at 585 bp under a UV lamp using a UV transilluminator.

Data Analysis
Data was entered into excel sheets and statistical analyses were performed using EPI INFO version 7.2.2.16. Using Pearson's Chi-square (χ 2 ) test difference among the variables was calculated. P values less than 0.05 were considered as significant.

Cultural and PCR Detection of E. coli
The isolates upon 24 hours of incubation at 44˚C produced blue colored colonies on TBX (Tryptone Bile X-glucuronide) agar. The isolated E. coli were subjected to PCR using E. coli specific EC-F and EC-R primers. Upon electrophoresis, a 585 bp on agarose gel was characteristic of the presence of E. coli by PCR examination ( Figure 1). A total of 105 meat samples were examined while 77 (73.33%) were positive for E. coli following microbial culture, and 35 (33.33%) were positive for E. coli following molecular examination. the difference was statistically significant (P < 0.05). Concerning the type of meat samples, PCR examination had detected E. coli less than microbial culture examination. In beef samples, there was a statistically significant difference between phenotypic and genotypic identifications. However, in chicken samples, there was no statistically significant difference between both methods (Table 2).

Prevalence of E. coli under Different Handling Conditions
The study was also focused on the prevalence of E. coli in different suppliers' sources, storage conditions and transport conditions. According to this study, the prevalence of E. coli was low in all parameters. The difference was statistically significant among different transport conditions but was not statistically significant among different suppliers' sources and storage conditions (Table 3).

Discussion
Escherichia coli is considered as the most important serotype of the Enterohemorrhagic E. coli and plays an important role in the incidence of ulcerative colitis, thrombotic thrombocytopenic purpura, and hemolytic uremic syndrome [17]. Hemolytic uremic syndrome occurs in 2% -7% of patients and results in death in 3% -5% of cases [18]. Although it has been proved that this bacterium is found in all geographical areas and can be isolated from different kinds of foods, its occurrence varies in different regions depending on the type of food, the sampling season and the methods of isolation [19] [20]. Journal of Biosciences and Medicines  demonstrated that out of the 105 samples, 35 cases were identified as E. coli.
These results confirmed the importance of molecular methods in detecting this pathogen. This method not only can save time and labour but also is very cost-effective. Therefore, it is suggested as a superior procedure for isolating E. coli from foods. In this regard, previous research from Iran declared that the PCR procedure is a fast method to detect the E. coli in the pharmaceutical, food, and water industries [21]. In another study in 2009 in Iran, researchers have proved that PCR-based methods are fast and reliable methods to detect and identify as few as 100 cells of E. coli, Vibrio cholerae, and Salmonella typhimurium [22]. This study revealed the presence of 33.33% E. coli in meat (beef and chicken), as well as the prevalence of E. coli in different parameters. According to this study prevalence of E. coli was low in supplier source, storage and transport conditions. Similar studies around the world have also reported a low prevalence of E. coli in meat. Akomoneh et al. [23] reported a prevalence rate of 10.9% in Buea Cameroon, Luga et al., [24] reported a prevalence rate of 9% in neighbouring Nigeria and Hiko et al., [25] and Bekele et al. [26] respectively reported 8% and 10.2% in Ethiopia. Callaway et al. [27] reported a prevalence rate of 11.3%, in the United States, Synge et al. [28] and Omisakin et al. [29] reported prevalence rates of 8.6% and 7.5% respectively in the United Kingdom while Hashemi et al. [30] had a prevalence rate of 8.3% in Iran. According to Mouafo et al. [31], the prevalence of E. coli in meat is 60% in Ngaoundéré, Cameroon and Kimassoum et al. [32] showed that the prevalence of E. coli in meat was 55% in Yaoundé, Cameroon and 70% in N'Djamena, Chad. These differences might be due to the differences in the methodology employed in these studies. Mouafo et al. [31] and Kimassoum et al. [32] described their findings based on microbial culture and biochemical analysis. However, in this study, in addition to traditional techniques, PCR was employed for the confirmatory identification of E. coli from sample meat.

Conclusion
The study revealed that PCR-based methods are fast and reliable methods to detect and identify as few as 100 cells of pathogenic E. coli, as well as in the determination of the prevalence of pathogenic E. coli, in Cameroon. Considering the circulation of this pathogen and human risks, we recommend that restaurant and abattoir workers should be trained on the basic concepts and requirements of food and personal hygiene as well as those aspects particular to the specific food-processing operations including waste disposal.

Conflicts of Interest
Authors declare no conflict of interest.