Molecular screening of Staphylococcus aureus enterotoxins associated with samples of meat / Iraq

Staphylococcus aureus secreted many types of toxins accompanying Intestinal poisoning resulting from eating food contaminated with bacteria or their toxins. Five hundred meat samples were collected from local markets, including fresh, frozen, canned, sausage and hamburger to investigate their contamination with S.aureus and then determined their ability of these isolates to secrete enterotoxins by using polymerase chain reaction. The results showed that the ratio of isolated S.aureus is 30 (6%) and the percentage of encoding genes for toxins is 30(100%), 0(0%), 3(10), 0(0%),0(0%),3(10), 2(6.7), 1(3.3), 0(0%) and 3(10) to sea, seb, sec, sed, see, seg, see, sei, sej and sel respectively.The result shows the S.aureus isolated from contamination meat able to produce different type to enterotoxins sea, sec, seg, see, sei, and sel and present the sea toxin is the most prevalence type of staphylococcus enterotoxins.

Foodborne diseases result from eating food contaminated with pathogenic bacteria, and Staphylococcus bacteria is considered one of the main pathogens causing foodborne diseases in hospitals (Touimi et al., 2020).

S.aureus produce various types of intestinal toxins
including enterotoxin A, B, C, D, E, H, G and I which cause food poisoning as a result of food contamination with bacteria (Johler et al., 2016).
The molecular method, for example, Conventional PCR, multiplex PCR is recommended for the discovery of S. aureus enterotoxins genes instead of phenotypical strategies because the serological kits can not recognize all the serotypes (Saly et al., 2018).
The research was aimed to isolation and identi ication of S.aureus from meat and meat product and molecular study for the enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, sej, sel) responsible of food intoxication produced by S. aureus isolated.

MATERIALS AND METHODS
Using traditional culture media to isolated S.aureus from the 500 meat samples isolated from different butcher shop and supermarket in Hilla city. Approximately 5 grams of shipped meat samples were inoculated into 10 ml of heart infusion broth, then streaking on Mannitol Salt Agar. The positive colony further con irmatory by cultivated on Staphylococcus chromogenic media (Sundararaj et al., 2019).
To validate the diagnosis of the bacteria, Coagulase assay was performe by using MAST plasma coagu-lase kit (Subramanian et al., 2017). DNA was extracted from the probable S. aureus isolates from broth cultures utilizing phenol /chloroform DNA extraction method (Akkou et al., 2018;Hassan et al., 2019).
There are 35 cycles of PCR performed to obtain large number copies of a gene, by using the primers listed in (Table 1), for detection the S. aureus toxins genes.

RESULTS AND DISCUSSION
S. aureus is part of the normal lora found in the mucous layer, of the skin and mouth, But when the number of bacteria increases lead to the contamination the food and food poisoning as a result of eating this food (Silva et al., 2018;Pal et al., 2020).
The S.aureus isolated form yellow golden colony when growth on mannitol salt agar with mannitol fomentation, a purple colony on staphylococcus chromogenic media ( Figure 1).    The results indicated that 30(6%) isolates belong to the S.aureus bacteria out of 500 meat sample. The highest percentage was obtained from fresh meat was 14(2.8%). The lowest percentage from canned meat was 1(0.4%), while the percentage of S.aureus isolated from Frozen meat, Sausage and hamburger were 10(2%), 1(0.2%) and 4(0.8%) respectively ( Figure 2).
When comparing the results of this study with other studies, it was found that the isolation rate is lower than the result of other researchers, In Duhok city Abdulrahman (2020) showed that 28 (28%) of 100 local chicken and 80 (80%) of 100 imported frozen chicken were found to be positive with S. aureus using conventional methods, Maestro et al. (2020) collected 48 sample from farm animals in Algeria present 41 (85,4%) belong to S. aureus. Dashen and Cirfat1 (2020) also found the percentage of S.aureus isolated from ready-to-eat meat samples higher than the percentage in this study. The difference in the isolation ratios of the bacteria may be attributed to the extent of application of the health conditions used everywhere, the nature of production, location of sample collection, and the technical development of the country or its backwardness and through this study, it observed the underdeveloped reality of slaughtering and our distance from the simplest health ingredients.
The result showed that all the S.aureus isolated contain sea gene 30(100%), a similar pattern of results was obtained by Saly et al. (2018)  This result was also similar to the indings of other researchers Dehkordi et al. (2019) Who is found that sea were the most prevalent types in percentage (38.7%) (Figures 3 and 4).
But other researchers Saleh et al. (2016) and Babu et al. (2017) not detection the sea genes in S. aureus isolated.
In Karbala city, Mohammed and Alwan (2017) obtain 57 isolates of S. aureus from meats found the percentage of the sea was 20(35%) by using PCR assay, 12 (37.5%) from frozen and 8 (32%) from fresh meat samples. Adikwu et al. (2019) found the percentage of the sea was 17.5%.
Another researcher collected 100 MRSA and 100 MSSA present the prevalence of sea was (45.0%) among MRSA and the prevalence of sea 42.0% in MSSA (Arabestani et al., 2018).
The result shows the number of S.aureus contained seg toxin only 3 from 30, while Dehkordi et al. (2019) showed that seg was the most prevalent Staphylococcus enterotoxins types (Figures 3  and 6).
The result showed all S. aureus isolated not contain see and sej toxin gene (Figure 3), while the anther result show The prevalent see gene was (22.6%) isolated from cooked meat samples (Asgarpoor et al., 2018).
The percentage of sec was 3(10%) Li et al. (2018) found The percentage of enterotoxin sec gene was (6.9%). Rehab et al. (2016) found the predominant of sei gene was (51.4%), But in this study, found only 3.3% of S.aureus isolated contain this gene (Figures 3 and 5).
The current results demonstrated that all the S.aureus isolated from meat sample contain one or more type of toxin gene (  (Rehab et al., 2016).
The results of the study showed the type of toxin secreted by S.aureus isolated from fresh meat, was the sea, sec, seg, sel, she, frozen meat was sea, seg, seh, sei, the hamburger was sea, sel, sausage canned meat only sea type (Table 2).
If the workers in the restaurants do not follow the rules of hygiene, which lead to contamination of food in bacteria. It found 10 bacteria per gram of food suf icient to produce intestinal toxins by staph bacteria (Saly et al., 2018).
From the results, it is clear the widespread prevalence of detected SE genes may indicate a risk of food poisoning resulting from eating animal meat contaminated with bacteria and its toxins. The increased prevalence of this MRSA means increased resistance to antibiotics, which cause serious public health problems (Mashouf et al., 2015).

CONCLUSIONS
Staphylococcus aureus secreted many types of toxins contaminated by eating food. From 500 hundred meat samples were collected from local markets, including fresh, frozen, canned, sausage and hamburger obtain 30 S.aureus isolated able to produce different type to enterotoxins sea, sec, seg, see, sei and sel and present the sea toxin is the most prevalence type of staphylococcus enterotoxins.