Rapid Detection of Enterotoxigenic Staphylococcus aureus Isolated from Raw Cow Milk in Sharkia Governorate, Egypt

| Staphylococcus aureus (S. aureus) produces various types of toxins, that cause food poisoning. This study was designed for a rapid investigation of enterotoxigenic S. aureus from raw milk. One hundred raw milk samples were collected from different farms in Sharkia governorate and examined for genome corresponding to enterotoxin genes (Sea, Seb, Sec, Sed, and See) of S. aureus using multiplex polymerase chain reaction (M-PCR). A reverse passive latex agglutination assay (RPLA) was also used for screening the enterotoxin production. The presence of S. aureus was detected in 20% of the examined samples. The antimicrobial susceptibility patterns showed sensitivity to ciprofloxacin (100%), followed by vancomycin (96%). Maximum resistance was reported for oxacillin (100%), ampicillin (95%) and amoxicillin (90%). Staphylococcal enterotoxin D gene (Sed) was detected in all isolates, while Seb and Sec genes were not detected. Though the results of PCR and RPLA had congruence, a few discrepancies were also reported for some of the isolates. The study concludes the detection of enterotoxigenic S. aureus (Sed and Sea gene) in raw milk samples, and therefore, ascertain necessary interventions to avoid food poisoning.

Staphylococcal enterotoxins (SEs) proteins are about 26.900 -29.600 Kilo Dalton, These are resistant to heat and the pro teolytic enzymes in the gastrointestinal tract (Martın et al., 2004). These are not affected by pasteurization, For instance, Staphylococcal enterotoxin A (SEA) keeps some activity even after exposure to heat at 121°C for 28 min (Rall et al., 2008). A consumption of staphylococcal enterotoxin SEA is the most frequent cause of food poisoning (Stewart, 2003), and several outbreaks are attributed accordingly (Asao et al., 2003). It is the leading cause of staphylococcal food poisoning outbreaks in the developed countries where it represents about 75% of outbreaks followed by SED, SEB, and SEC (Normanno et al., 2005).
Multiplex PCR is required for the simultaneous identification of S. aureus enterotoxin genes. However, gene existence does not confirm the enterotoxigenic properties of a strain. Therefore, an expression of the gene also should be measured (Van Belkum, 2003).
Recently enzyme-linked immunosorbent assays were employed for direct detection of SE in food. However, these methods are time consuming and do involve a high cost. On the other hand, reverse passive latex agglutination (SET-RPLA) is considered the best method for the identification of SE in bacterial culture (Morandi et al., 2007) that can detect enterotoxin protein using specific antibodies (Sergeev et al., 2004). The present work aimed to identify enterotoxin genes Ses (Sea, Seb, Sec, Sed and See) among multidrug-resistant S. aureus strains using multiplex polymerase chain reaction (PCR), and SEs protein production using the RPLA technique.

SamplIng
During the period from March to October 2018, one hundred raw milk samples were randomly collected from farms in Sharkia Governorates, Egypt. Milk samples were incubated at 37°C for 24 h, centrifuged at 3000 rpm for 20 minutes, and the sediment was used for bacteriological cultivation.

bactErIologIcal ExamInatIon
All samples were subjected to the routine preliminary phenotypic characterization of S. aureus isolates on the basis of standard microbiological techniques (Baird,1996).

EntErotoxIn gEnES
A total of ten S. aureus isolates were used for genome extraction that showed the highest multidrug-resistant. DNA extraction was performed using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH). Briefly, 200 μl of the sample suspension was incubated with 10 μl of proteinase K and 200 μl of lysis buffer at 56 o C for 10 min. After incubation, 200 μl of 100% ethanol was added to the lysate. The sample was then washed and centrifuged following the manufacturer's recommendations. Nucleic acid was eluted with 100 μl of elution buffer. The primer sequences, their corresponding genes and the respective molecular sizes of PCR amplified products are listed in Table 1. PCR amplification reactions were performed in a final volume of 25 μL containing 12.5 μL of DreamTaq TM Green Master Mix (2X) (Fermentas, Inc. Hanover, USA), 0.1 μL of 100 pmol of each primer (SigmaAldrich, Co., St. Louis, USA), and 2 μL of S. aureus DNA template. The volume of the reaction mixture was completed to 25 μL using DNase/ RNase-free water. The cycling condition was carried out using a PTC-100 TM programmable thermal cycler (MJ Research Inc., Waltham, USA). PCR was performed with an initial denaturation at 94˚C /5 min, followed by 35 cycles of each of 94°C for 30 sec, 57°C for 40 sec, and 72°C for 40 sec, and a final extension of 10 min at 72°C (Mehrotra et al., 2000).

pcr product analySIS
The PCR products were separated by electrophoresis on 1.5% agarose gel (Applichem, Germany, GmbH) in 1x TBE (Tris-borate-EDTA) buffer at room temperature using gradients of 5V/cm. The fragment sizes of PCR products were determined with a 100 bp DNA ladder (Qiagen, Germany), photographed, and the data was analyzed through computer software.

StapHylococcal EntErotoxIn (SE) dEtEctIon by tHE rpla mEtHod
The detection of enterotoxin production (SEA, SEB,

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Advances in Animal and Veterinary Sciences 2020 | Volume 8 | Special Issue 1 | Page 13 SEC, SED) from broth cultures of S. aureus isolates were detected by commercially available kit (SET-RPLA,TD 900, Oxoid, UK) according to manufacturer's instructions. Out of 100 raw milk samples, 30 of them (30%) were positive for Staphylococcus spp. However, the coagulase test illustrated only 20 isolates (20 %) were coagulase positive (S. aureus) and, therefore, considered as pathogenic strains.

antImIcrobIal SuScEptIbIlIty tEStIng
Antimicrobial susceptibility patterns of all coagulase positive (S. aureus) isolates are illustrated in Figure 1. Analysis of methicillin resistance confirmed that all the recovered isolates were MRSA. All tested MRSA isolates were susceptible to ciprofloxacin (100%) and vancomycin (96%). However, higher frequency levels of resistance were obtained for ampicillin (95%), followed by amoxicillin (90%) clindamycin (80%). All the isolates were considered resistant to three or more of the antimicrobial agents of three different groups and therefore taken as multiple drug resistance (MDR).

S. aureuS EntErotoxIgEnIc gEnES
S. aureus isolates from raw milk samples were examined by multiplex PCR, where ten isolates showed their genomic potential to produce enterotoxin genes, as shown in Table  2 and Figure 2. All isolates produced the Sed enterotoxin gene followed by Sea and See at percentages of 70% and 50%, respectively. On the other hand, seven strains had Sea and Sed enterotoxin genes, three isolates harbored Sea, See and Sed enterotoxin genes, and two isolates had gene corresponding to Sed and See enterotoxin genes. Seb and Sec genes were not detected in any of the study isolates.

S. aureuS EntErotoxIgEnIc protEIn
Enterotoxins, extracted from 10 enterotoxigenic S. aureus broth culture, revealed varying results. All isolates produced SED, six strains produced SEA, while none of the strains produced SEB and SEC. Six isolates produced SEA in combination with SED (Table 2). Interestingly, except for SEE, which was not found associated with See gene (P=0.068), we found that all S. aureus enterotoxin proteins were significantly associated with S. aureus enterotoxin genes (P= 0.014).This can be evidenced in  (Figure 3).

dIScuSSIon
Cli nically, S. aureus is one of the most significant bacteria that cause food poising worldwide. In this study,20% of raw milk samples from Sharkia Governorate were found contaminated with S. aureus. This rate of prevalence has previously been documented by a number of other studies (Ekici et al., 2004;Santana et al., 2010;Tambekar and Bhutda, 2010;Ammar et al., 2016). They found analyzed samples contaminated with S. aureus in a varying rate of percentages that ranged from 17.34% to 18.80%. Some other studies reported significantly higher levels of contamination ranging from 40% to 61.7 % (Guven et al., 2010;Lingathurai and Vellathurai, 2010;Zakary et al., 2011). Similarly, other studies reported low contamination from 6.2% to 7.3% (Fagundes et al., 2010;Thaker et al., 2013). This is not surprising because milk can be contaminated internally through the production of milk from a diseased animal or externally by an infected person or the surrounding envi ronment (Serraino et al., 2004). Antimicrobial susceptibility testing of S. aureus against nine different antibiotics showed higher sensitivity rates to ciprofloxacin and vancomycin. These observations are in accordance with those stated previously by others (Aydin et al., 2011). The highest resistance rates were observed for ampicillin, amoxicillin, clindamycin, and gentamycin. With a negligible difference, these observations correlate with those reported previously by others (De Oliveira et al., 2000;Guérin-Faublée et al., 2003;Ammar et al., 2016).
All S. aureus isolates were completely resistant to oxacillin (100%), which contradicts previous findings (Thaker et al., 2013), who found only 20% of S. aureus isolates resistant to oxacillin. High percent isolation of MRSA in this study was considered an extension of previous studies conducted by other researcher groups in Egypt (Ammar et al., 2016) and Bangladesh (Nusrat et al., 2015).
Our findings showed that all the isolates were resistant to at least three antibiotics which is not consistence to previously reported result (Ombui et al., 2000) and contradict several studies which found high levels of MDR in MRSA isolates (Kérouanton et al., 2007;Ge et al., 2017;Sahibzada et al., 2017;Abdi et al., 2018;Suleiman et al., 2018).
S. aureus has the ability to produce enterotoxins that make the potential risk to public health. Our results demonstrated the detection of Sed (100%) gene among all tested S. aureus isolates from raw milk followed by Sea (70%) and See (50%). Sec or Seb genes were not detected in examined isolates. Similar findings were documented previously by others (Carfora et al., 2015).
In another study, a frequent occurrence of See gene was detected in all isolates of SE (100%) followed by a similar presence of Seb and Sec (33.3%), while none of the isolatehadSea or Sed genes (Mansour et al., 2017). Another study revealed Sea gene the most frequent one (61.8%) followed by See (33.1%), Sed (17.5%), Sec (15.9%), and Seb (13.9%) in the S. aureus (Mathenge et al., 2015). We found a significant association between Sea and Sed enterotoxin genes, which is in agreement with findings reported previously (Serraino et al., 2004;Normanno et al., 2005;Morandi et al., 2007).
Multiplex PCR assay can identify the presence of enterotoxin genes but didn't evaluate their expression. Therefore, in this study, we compared the multiplex PCR results and RPLA (SEA-SED) for toxin type. The RPLA is usually used as a qualitative assessment for S. aureus enterotoxin with a sensitivity of 0.25-0.5 ng/mL concentration of toxins in examined solutions (Rose et al.,

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Advances in Animal and Veterinary Sciences 2020 | Volume 8 | Special Issue 1 | Page 15 1989;Cretenet et al., 2011). We found that the frequency of SEA and SED enterotoxins were inconsistent with their corresponding genes. Previous studies indicated SEA as the top contributor (80%) to staphylococcal food poisoning outbreaks, followed by SEE, SED, SEB, and SEC. On the contrary, some researchers found a higher expression of SEC and SEB than SED and SEA (Mathenge et al., 2015).
Varying results among PCR and RPLA are not surprising because similar observation for both assays has previously been documented. For instance, a <5% difference in the results of the RPLA and the PCR assays were observed already by others ( Jørgensen et al., 2005;Pinto et al., 2005). Ranging from 15 to 32%, marked discrepancies in results of multiplex PCR and RPLA assays, especially for SEA, have been reported (Zouharova and Rysanek, 2008). Such a discrepancy could be explained by the production of enterotoxin in a quantity that was below the detection limit of the RPLA test.
The RPLA results detected some enterotoxin proteins; however, their enterotoxin genes were not detected by multiplex PCR for the same isolate. This finding is in agreement with the observations illustrated previously (Mansour et al., 2017).
Two strains produced SEA enterotoxin, while its encoding gene was not detected using multiplex PCR. Our findings were justified by others (Fraser and Proft, 2008;Argudín et al., 2010) who reported that, though genes more commonly found in groups, enterotoxin genes may found alone, in the large mobile segments of DNA called mobile genetic elements (MGEs) such as transposons, S. aureus pathogenicity islands (SaPIs), plasmids, prophases and the enterotoxin gene clusters (egc).

concluSIon
This study reported that SED and SEA are common enterotoxins in S. aureus isolates originating from raw milk samples. Therefore, the consumption of raw milk should be considered a potential public health hazard. Rapid and efcient detection of enterotoxigenic S. aureus is necessary for consumer safety, and RPLA is recommended for the detection of S. aureus enterotoxin. As presence of enterotoxin genes in S. aureus isolates not mean production of enterotoxin proteins.