Open Veterinary Journal, (2020), Vol. 10(3): 297–307 ISSN: 2226-4485 (Print) Original Research ISSN: 2218-6050 (Online) DOI: http://dx.doi.org/10.4314/ovj.v10i3.8 Submitted: 08/06/2020 Accepted: 25/08/2020 Published: 12/09/2020 Isolation of enterotoxigenic Staphylococcus aureus harboring seb gene and enteropathogenic Escherichia coli (serogroups O18, O114, and O125) from soft and hard artisanal cheeses in Egypt Ola Wagih Hegab*, Eman F. Abdel-Latif and Ashraf Ahmed Moawad Department of Food Hygiene and Control, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt Abstract Background: Soft and hard artisanal cheeses are regularly consumed in Egypt. These products are usually processed from raw milk which may harbor many pathogenic and spoilage microorganisms. Aim: To evaluate the safety of some artisanal cheeses in Egypt, such as Ras, Domiati, and Mish, through chemical and microbiological examination. Methods: One hundred and fifty random samples of traditional Ras, Domiati, and Mish cheeses (50 each) were microbiologically and chemically analyzed. Counts of total bacteria, presumptive coliform, staphylococci, yeast, and mold were estimated. Furthermore, isolation of Escherichia coli and Staphylococcus aureus was performed, followed by PCR confirmation; isolates of E. coli were examined for the presence of virulence genes; on the other hand, the detection of the five classical enterotoxin genes ofS. aureus was performed using multiplex PCR. Regarding chemical analysis, moisture, salt, and acidity content were measured. Correlations between chemical and microbial findings were investigated. Results: Mean counts of total bacteria, presumptive coliform, staphylococci, yeast, and mold were (2 × 108, 3 × 106 and 1 × 107 ), (3 × 105, 5 × 10 and 5 × 102), (1 × 106, 4 × 105and 1 × 105), (3 × 105, 1 × 105 and 5 × 105), and (7 × 103, 4 × 103 and 3 × 104) for Ras, Domiati and Mish cheeses, respectively. Serological identification of suspected E. coli revealed that E. coli O125 was isolated from Ras and Domiati samples, E. coli O18 was recovered from Ras samples, while E. coli O114 was isolated from Mish samples. PCR results revealed that all detected isolates of E. coli were positive for both iss (increased serum survival) and fimH (type 1 fimbriae) genes. Concerning isolated S. aureus, all examined products were harboring S. aureus enterotoxigenic strains, with seb and sed genes being the most common. The mean values of moisture, salt, and acidity were (30.03, 56.44, and 58.70), (3.30, 6.63, and 7.56) and (0.65, 0.68, and 0.50) for Ras, Domiati, and Mish cheeses, respectively. Conclusion: Enterotoxigenic S. aureus harboring seb gene and enteropathogenic E. coli (serogroups O18, O114, and O125) were frequently isolated from soft and hard artisanal cheeses in Egypt. Therefore, strict hygienic measures should be applied during their manufacture, handing, and distribution. Keywords: Domiati, E. coli virulence genes, Mish, Ras, S. aureus enterotoxins. Introduction areas. It is mainly made from raw milk, either cow’s Traditional Egyptian dairy products are an essential or a mixture of cow and buffalo milk, mostly without part of the daily diet in Egypt. Raw milk is the common adding a starter culture, and fermentation and ripening factor in making all traditional products, which usually occur only due to the native flora of raw milk, then it record high sensorial characteristics, as consumers is kept to ripen for 3–8 months till a sharp flavor is mostly choose their dairy products based on its acquired, similar to the Greek Kefalotyri cheese (Awad, sensory properties, regardless if it is made from raw or 2006; El-Fadaly et al., 2015). pasteurized milk (Awad, 2006; El-Ghaish et al., 2010; Domiati or Damietta cheese (Gebnah Domiata) is Ewida and Hussein, 2019). Some traditional products one of the most popular white, soft pickled cheeses. have been traced back to the pharaoh era of 4,000 It is named after the Egyptian seaport city “Dumyât”. BC, and is then handed down from one generation to Typically, it is salty, unlike other pickled cheeses, as the another. There are different types of traditional cheese salt is added directly to the raw milk at the proportion in Egypt and among them are Ras, Domiati, and Mish of 5%–14%, which varies according to season and (Benkerroum, 2012). ripening conditions (Robinson and Tamime, 1991). Ras cheese is a hard cheese commonly known in Egypt Mish is one of the oldest indigenous milk products in as “Romi”. It is one of the artisan type cheeses, which Egypt that has a sharp harsh flavor with a relatively is produced often in small factories located in rural higher salt content and is mostly yellowish brown color. *Corresponding Author: Ola Wagih Hegab. Department of Food Hygiene and Control, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt. Email: [email protected] 297 http://www.openveterinaryjournal.com O. W. Hegab et al. Open Veterinary Journal, (2020), Vol. 10(3): 297–307 Karish cheese is considered as the basic raw material Serological identification of E. coli was done using for such a product. Some other ingredients that may be DENKA SEIKEN CO., LTD. kits. The molecular included in Mish are sour milk, buttermilk, morta (a characterization, and virulence genes detection was byproduct of ghee), different types of pepper (green, performed using PCR, where DNA was extracted from red, and paprika), table salt, and a portion of an old the bacterial isolates using QIAamp® DNA Mini Kit Mish which acts as the starter culture. All ingredients (Catalogue no.51304) according to the instruction are brined under microaerophilic conditions mostly in manual. PCR was done for characterization of 5 genes earthenware containers for more than 1 year to obtain of E. coli using Emerald Amp GT PCR master mix, matured mish, which consists of Mish cheese and Mish Takara (Catalogue No. RR310A), using 5 different slurry (pickling medium), and both are consumed pair of primers (as uniplex PCR) as mentioned in Table (Tamime, 2006). 1. The PCR products were electrophoresed in 1.5% Raw milk used in the production of traditional dairy agarose according to that described in Sambrook et products and harbor a large number of native microflora al.’s (1989) study. that may participate in the development of a desirable Staphylococci count (CFU/g) and S. aureus isolation aroma and an appropriate flavor, but it may also contain were carried out according to the APHA guidelines pathogenic microorganisms such as Escherichia coli, (American Public Health Association) (2004). Staphylococcus aureus, and others that are considered Identification of suspected S. aureus strains as public health hazards, causing a serious food illness (microscopic examination, catalase activity test, and may produce lethal toxins in the food (Awad et al., coagulase test, thermostable nuclease, and anaerobic 2003; El-Ghaish et al., 2010). Moreover, undesirable utilization of mannitol) was carried out according to spoilage organisms may also be found that cause that described in Silva et al.’s (2019) study. Molecular defects and limits the shelf life of these products, characterization of S. aureus strains and enterotoxin rendering them unmarketable. Many other factors may genes detection was done as in E. coli. Six genes of cause an increase in the microbial load of traditional S. aureus (5 enterotoxins done by multiplex PCR and products, such as manufacture, handling, storage, and 23S rRNA) and six pairs (5 as multiplex and 1 pair as even marketing ways, which are still primitive and uniplex PCR) were used (Table 1). unhygienic (Osman et al., 2011). Yeast and mold counts (CFU/g) were conducted The current study was conducted to assess the safety of according to APHA’s guidelines (American Public some traditional cheeses in Egypt, such as Ras, Domiati, Health Association) (2004). and Mish, through chemical and microbiological Statistical analysis examination. The collected data were analyzed using Statistical Package for the Social Sciences Statistics 17 for Materials and Methods Windows. Samples collection Ethical approval One hundred and fifty random samples of Ras, In this study, no experimental live animals were used. Domiati, and Mish cheeses (50 each) were collected All cheese samples were collected directly from the from different Egyptian Governorates. The samples Egyptian local markets. were identified, placed in an ice box, and examined immediately. Results Chemical analysis Chemical analysis Moisture and acidity were analyzed according to the The chemical analysis of the examined samples, AOAC guidelines (Association of Official Analytical including moisture, salt, and acidity, is illustrated Chemists) (2000) and salt according to the APHA in Table 2; for Ras cheese samples, the mean values guidelines (American Public Health Association) were 30.03, 3.30, and 0.65, respectively. The mean (2004). values of Domiati cheese samples were 56.44, 6.63, Microbiological examination and 0.68, respectively, and were 58.70, 7.56, and 0.50, Preparation of food homogenate and decimal dilutions, respectively, for Mish samples. total bacterial count (TBC; CFU/g), presumptive Microbiological examination coliform count (MPN/g), and isolation of E. coli As reported in Table 3, the TBCs of Ras, Domiati, were carried out according to the APHA guidelines and Mish samples were 2 × 108, 3 × 106, and 1 × (American Public Health Association) (2004). 107, respectively, while coliforms were determined Biochemical identification of E. coli (microscopic in 44%, 48%, and 92% of that examined in Ras, examination, oxidase, urea, IMVC, TSI, L-Lysin Domiati, and Mish samples, with a mean values of 3 decarboxylase, carbohydrate fermentation tests, and × 105, 5 × 10, and 5 × 102, respectively. The examined confirmed tests for thermotolerant (fecal) coliforms) samples were positive for E. coli in 8% of Ras cheese was carried out according to Silva et al.’s (2019) samples and in 4% of both Domiati and Mish samples, description. respectively, as shown in Table 5. The serological 298 http://www.openveterinaryjournal.com O.