Molecular detection of Staphylococcal enterotoxins and mecA genes products in food samples collected from different areas in Khartoum state

identication


Conclusion
There was a relatively moderate prevalence of Methicillin resistant staphylococcus aureus with very low frequency of enterotoxin B gene in different kinds of food samples which collected from Khartoum state. These ndings highlight the high potential risk for consumers of meat and dairy products especially in the absence of strict hygienic and preventive measures to avoid Staphylococcus aureus enterotoxins production in foods.

Background
Food borne diseases (FBD) remain one of the greatest concerns in public health and food safety, they are caused by a large variety of pathogens that contaminate food and food products (1). Many food sources may serve as substrate for many microorganisms which are transmitted during harvesting, storage or food processing and handling by multiple environmental sources such as: water, soil, insects, or even by the handlers (2).
Staphylococcal Food Poisoning (SFP) is an intoxication that results from the consumption of improperly prepared or stored foods containing su cient amounts of one or more preformed enterotoxins (3,4). A wide variety of foods support growth of Staphylococcus aureus and are ideal for enterotoxin production including: milk, raw milk, meat, meat products, dairy products, and ready-to-eat food (5,7).
Staphylococcus aureus may produce a large variety of enterotoxins but 95% of food poisoning outbreaks are caused by classical enterotoxins: A, B, C, D and E (6). These toxic proteins withstand exposure to 100 °C for several minutes, when ingested as preformed toxins in contaminated food, microgram amounts of toxin within a few hours can induce the symptoms of staphylococcal food poisoning: nausea, vomiting and diarrhea (8).
Because of the resistance of Staphylococcus aureus to the heat and drying, foods can easily become contaminated from food handlers or from the environment, it multiply and release toxin in uncooked or inadequately cooked foods, especially if the foods are unrefrigerated (9).
The occurrence of Staphylococcus aureus and MRSA in foods of animal origin and potential risk of transmission to humans through foods if consumed without maintaining adequate hygienic standards pose a serious threat to the well-being of humans due to uncountable clinical implications (10).
Non-hygienic handling practices, working conditions, and improper storage and refrigeration; all these can increase the opportunity for food contamination. So, it is important to follow the standard practices in food handling such as hand washing, proper cooking, proper storage and others to reduce or prevent food contamination (11,14). In our country, only few information is available about the occurrence of virulence genes among the staphylococci isolated from foods, so this study conducted to spot in enterotoxins and mecA genes in out of 84 S. aureus isolates were show an enterotoxin B gene product (both isolates were from cheese samples) ( Table 2), (additional Fig. 1).
Our result show that out of hundred raw meat samples examined 30 (30%) were identi ed as S. aureus (Table 3) of which 11(36.7%) isolated from raw beef and 19(63.3%) from restaurants meat. All isolates, 30 (100%) were sensitive to cipro oxacin. While 26(86.7%) were sensitive to gentamycin and 4 (13.3%) were resistant. Additionally, the sensitivity test results also revealed that 28(93.4%) out of the 30 S. aureus isolates were sensitive to oxacillin and 25 (83,3%) were sensitive to vancomycin (Table 4). All 30 S. aureus isolates investigated for the presence of enterotoxin genes by multiplex PCR; no enterotoxins genes were detected (additional Fig. 1).
In this research we nd that out of hundred cheese samples examined 20 (20%) isolates were identi ed as Staphylococcus aureus, 4(4%) were identi ed as bacteria others than Staphylococcus aureus while 76(76%) were show no growth ( Table 3). The antimicrobial sensitivity pattern of was examined in All of twenty cheese Staphylococcus aureus isolates. All isolates were sensitive to Gentamicin, Cipro oxacin. 17(85%) isolates were sensitive to Vancomycin and 3(15%) were resistant. However, 19(95%) were sensitive to oxacillin and 1(5%) was resistant ( Table 4). All 20 cheese Staphylococcus aureus isolates were investigated for the presence of enterotoxin genes by multiplex PCR, and seb was detected in only 2(10%) isolates (additional Fig. 1).
In our research we examine a total of 100 sh samples of which 50 were salted sh samples while the rest were collected from raw sh. The result nd that Staphylococcus aureus was isolated in 24 (24%) sh samples while the rest 84 (76%) were other bacteria ( Table 3). All isolated S. aureus showed complete sensitivity to cipro oxacin and gentamicin (100%). While Vancomycin and oxacillin were showed e cacy rate of 92% against S. aureus isolates and therefore the resistance rate was 8% both mentioned antibiotic ( Table 4). All 24 isolated S. aureus were con rmed by detection of the presence of 16Shousekeeping gene and Enterotoxin genes A, B, C, D and E were also investigated in the con rmed S. aureus but there was no enterotoxin gene detected (additional Fig. 1).
In this research a total of 100 milk samples were collected from different areas in Khartoum state. 63(63%) of isolates were identi ed as S. aureus, 26(26%) were identi ed as others than S. aureus and 11(11%) were show no growth ( Table 3). The antimicrobial susceptibility test of isolated S. aureus was showed high sensitivity rate to Cipro oxacin 98.4%, 87.3% to Gentamicin, 84% to Oxacillin and 65% to Vancomycin ( Table 4). The results for enterotoxins genes reveal that there is no S. aureus isolate produce such gene (additional Fig. 1).     (3) this nding is greatly exciding our results. Those great discrepancies between our nding and other studies results may be due to variation in foods, habits, cooking behaviors, food keeping hygiene in addition to environmental factor like the weather temperature and moist which affect greatly the bacterial growth in food materials.
Resistance gene (mecA) in S. aureus witch responsible for resistance to β-lactam antimicrobials was detected by using PCR among only 84 randomly selected S.aureus Isolates, and we nd that 17% of S. aureus positive for mecA gene while 69 (83%) were negative. Most previous mecA gene detection studies results showed a signi cant higher prevalence than our nding as in study done by Khayri in Makkah city he found that about 44.4% of his S. aureus isolates were positive for mecA gene (4). Similarly, Papadopoulos and his colleagues found that 81.3% of their isolates were positive for the mecA gene (3). This variation could be due to the different in the antibiotic protocol that applied by the doctors for their patients or due to the extensive usage of methicillin antibiotic in their communities or by doctors to treat infection which could cause by S. aureus in these countries and eventually lead to high prevalence rate of MRSA.
In contrary, in study conducted by Novak et al in 2000 the mecA gene prevalence was lower than our nding 11.4% (5). Other studies done Kamal et al in Egypt, Rizek in Brazil and wang et al in china found that the prevalence of mecA gene was 5.1%, 9% and 7.9% respectively (6)(7)(8). The variation in the results may be due to variation of source of samples and usage different molecular techniques in different countries for detection of mecA gene product.
In this study one hundred raw meat samples were obtained from different supermarkets and restaurants in Khartoum state, and examined for presence of S. aureus. Thirty (30%) samples were found to be contaminated with S. aureus. These ndings highlight the high potential risk for consumers of meat and dairy products especially in the absence of strict hygienic and preventive measures to avoid Staphylococcus aureus enterotoxins (SEs) production in foods. In other comparative studies similar results were presented by Kelman et al. (23) in Washington who reported that the prevalence of S. aureus in meat samples was 29.0%. However, in the present study results are higher than that obtained by Ramatla et al. in south Africa who reported that S. aureus was in 26.5% (24), and lower than those obtained by Das et al. in India who reported that out of 65 samples S. aureus incidence was in 46.1% (25). The variation in the prevalence may be due to the variation in community hygiene in certain countries and variation in the environment especially the moisture and temperature which affect the growth of S. aureus.
In our study none of the meat S. aureus isolates was resistant to cipro oxacin and 13.3% were resistant to gentamicin. Das et al in India found that 16.66% of S. aureus meat isolates were resistant to cipro oxacin (25) and Pu S et al., in Louisiana found that 13.0% were resistant to cipro oxacin. Also, in contrast to our ndings Pu S et al. in Louisiana found that 3.0% were resistant to gentamicin (26). Vancomycin resistant Staphylococcus aureus (VRSA) is a type of antibiotic resistant S. aureus which have developed a resistance and can no longer be treated with vancomycin. This study showed that 16.6% of the meat isolates were resistant to vancomycin, which suggest that the contamination may be coming from VRSA carrier's food handlers and processors, however Das et al found that 3.33% of the isolates were resistant to vancomycin (VRSA) which is low compared with our ndings (25). Methicillin-resistant S. aureus (MRSA) strains have acquired a gene that makes them resistant to nearly all beta-Lactam antibiotics, animal-adapted MRSA strains also exist although it's in small percentage but it's of a clinical importance and may cause serious problems to immunocompromised individuals as well as healthy ones (carriers). In this study 6.6% of meat isolates were resistant to oxacillin, this nding were high compared to Inge et al results whom found that 2.5% of S. aureus meat isolates were resistant to oxacillin (27), and low compared to Das et al results whom found that 23.3% of S. aureus isolates were resistant to oxacillin (25).
In this study the PCR results showed no enterotoxin genes in meat S. aureus Isolates, Larsen et al in Denmark nearly found a similar results which showed that only 0.2% of isolates were enterotoxin gene possessing S. aureus (28), meanwhile in contrast to our ndings Lis et al., in China found that the prevalence of enterotoxin gene possessing S. aureus was 46.0% (29), and Bergdoll, found that the percentage of enterotoxigenic strains of S. aureus is estimated to be around 25% (30). Moreover, most of S. aureus food isolates are not SEs producers, thus considerable research effort is still required for better understanding of the interactions between S. aureus and the food matrix and of the mechanism of SEs production in food stuffs (31). The data obtained in this study probably underestimated the enterotoxigenic properties of the analyzed strains, since the possible presence of newly described SEs was not considered and sample size was too small to represent S. aureus contaminated meat effectively. However, there is always the possibility of mutation at the level of the corresponding gene, leading to the absence of its detection. Therefore, a positive PCR shows the presence of the enterotoxin genes but a negative PCR does not point the absence of the corresponding operon (31).
In this study One hundred sample white cheese were collected from different retailers in Khartoum State to detect the presence of staphylococcus aureus. In which 20 (20%) isolates were detected; that was higher than the results obtained by Shanehbanali et al., in Iran and Katsuda et al., in Japan, whom isolated Staphylococcus aureus from white cheese with percentage of 16% and 13.3% respectively (32, 33), and lower than the results obtained by Gucukoglu et al., in Turkey who reported Staphylococcus aureus 37.5% in white cheese (34). This study was disagreed with Mohamed et al., who reported absence of Staphylococcus aureus in white cheese in Khartoum (35).
All of the twenty cheese S. aureus isolates were sensitive to gentamicin and cipro oxacin (100%) this nding was disagreed with Seguin et al., in U.S.A, who estimated 75% resistant to gentamicin (36) and Jaber et al., who measured 25% resistant to cipro oxacin in Iraq (37). However, among cheese S. aureus isolates only one sample (5%) was resistant to oxacillin, which lower than result reported by Alshammary and Galfoori in Iraq which showed higher resistance to oxacillin observed in 6 isolates (20%) (38). Additionally, 17 (85%) out of 20 cheese S. aureus isolates were sensitive to vancomycin these result lower than result obtained by Valsangiacomo et al., in Switzerland who reported 100% sensitivity to vancomycin (39, 40) The molecular detection of Staphylococcus aureus enterotoxins (A, B, C, D and E); among cheese isolates resulted in the detection of seb gene in 10% of the 20 isolates, that was lower than the results obtained by Salheen in Sudan who reported 20% of seb gene was detected in cheese (41). The variation of these results among researcher could be due to several factors such as source of sampling, geographical origin, sensitivity of identi cation methods and sample size can affect the outcomes.
In this study out 108 sh samples, 22% of samples were contaminated with S. aureus which lower than result demonstrated by Mohammed (47). The variation of these results among researcher could be due to several factors such as source of sampling, geographical origin, sensitivity of identi cation methods and sample size can affect the outcomes.
The molecular analysis of the enterotoxins genes among sh isolated S. aureus gave no results for all genes which in agreement with result obtained by Arsland and Ozdemer, in Turkey and Pu et al.,in Louisiana (46,51), whom found no enterotoxins B, C and E genes. The current study was slightly lower than that obtained by Ali in Tanzania who detected enterotoxin B and C genes in 0.3% of his study samples, with the absence of enterotoxin A gene in all samples (52), while our result is signi cantly lower than that of Arsland and Ozdemer in Turkey whom reported enterotoxins A and D genes in 10.5% of their samples (46). The variation of these results among researcher could be due to several factors such as source of sampling, geographical origin.
In this study S. aureus was isolated in 63% raw milk samples, which closes on with results obtained by de Oliveira et al., in Brazil 68% (53) and Chye et al., in Malaysia 60% (54). However, our nding was lower than results reported by Ekici et al., in Turkey 75% (55) and Agban and Ahmad in Egypt 82% (56). On another hand the present study was higher than AL-Kafaje, in Baghdad, and Mustafa in Basrah, whom recorded that S. aureus was isolated from clinical and subclinical mastitis in cows in percentages of 53.33%, 43.5% respectively (57,58). however, our result is dramatically higher than levels of contaminations which were reported by Yagoub et al. in Sudan and Abdel-Hameed and El-Malt in Egypt whom isolated S. aureus from raw milk with percentage of 30%, 24.8% respectively (59,60). The variation of these results among researcher could be due to several factors such as source of sampling, geographical origin, sensitivity of identi cation methods and sample size can affect the outcomes.
In relation to antimicrobial susceptibility testing, this result showed high sensitivity rate for milk S. aureus isolates to cipro oxacin (98.4%) which was agreed with Islam et al., ndings in Bangladesh whom reported a sensitivity rate of 93.3% (61). While lower level of cipro oxacin potency was detected by Jahan et al., in Bangladesh and Thaker et al., in India whom recorded an 83.3% and 80.0% sensitivity rate for S. aureus raw milk isolates (62,63). Gentamicin sensitivity test showed 87% potency that was disagreed with that obtained by Yagoub et al., in Sudan whom recorded that 47.6% sensitivity (59). While slightly similar to that obtained by Beyene, in Ethiopia, and Thaker et al., in India that both showed 90% of isolated S. aureus were sensitive to gentamicin (64,63). Reports from other researchers was indicated higher level of sensitivity to gentamicin as 100% reported by Abraha et al.,in Ethiopia (65). Vancomycin sensitivity showed 65%. Which incompatible with results reported by Idbeis, in Basrha (Iraqi), AL -Marsomy, and Bendahou et al., in North Morocco whom mentioned that S. aureus isolated from raw milk and milk product showed sensitivity to vancomycin 100% (66,67,68). In contrast Abraha et al., in Ethiopia and Alsaady, in Baghdad reported that the resistant rate of isolated S. aureus from raw milk is 100% to vancomycin (65,69). The isolated S. aureus were sensitive to oxacillin (84%), which slightly higher than result achieved by Thaker et al., in India 70% (63) and Jahan et al., in Bangladesh showed complete sensitivity (100%) to oxacillin that was conversed to this result (62). The variation of these results among researcher could be due to several factors such as source of sampling, geographical origin, sensitivity of identi cation methods and sample size can affect the outcomes.
In this study no enterotoxigenic milk staphylococci were detected that was similar to that obtained Peles et al., in Hungary whom recorded that the isolated S. aureus from dairy milk farms were showed no enterotoxins (70). The variation of these results among researcher could be due to several factors such as source of sampling, geographical origin, sensitivity of identi cation methods and sample size can affect the outcomes.

PCR Detection of Staphylococcal Enterotoxins Genes
Multiplex PCR, ampli cation was done using (CLASSIC K960, UK) thermo cycle. PCR ampli cation of staphylococcal enterotoxins (SE) genes, namely (sea, seb, sec, sed and see) was performed using Maxime PCR Premix kit (iNtRON, Korea) and speci c primers listed in Table 5 The PCR assay was carried out in a total volume of 25 μL of mixture containing 2 μL Maxime PCR Premix, 0.5 μL of each of the toxin gene-speci c primers (5 μL), 2 μL of template DNA and 16 μL of double distilled water. The ampli cation conditions included three steps: initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 45sec, annealing at 50°C for 45sec, and extension at 72°C for 45 sec; and the nal extension at 72°C for 7 min (14).

PCR Detection of mecA Gene
Primers were used for detection of mecA gene, showed in table 5. DNA ampli cation was done using Maxime PCR Premix kit (iNtRON, Korea), The PCR assay was carried out in a total volume of 20 μL of mixture containing 2 μL Maxime PCR Premix, 0.5 μL of each of the gene-speci c primers (5 μL), 2 μL of template DNA and 13 μL of double distilled water. The ampli cation conditions included three steps: initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 45sec, annealing at 52°C for 45sec, and extension at 72°C for 45 sec; and the nal extension at 72°C for 7 min. Availability of data and materials The datasets analyzed during the current study are available from the corresponding author on reasonable request.