Staphylococcus aureus and Staphylococcal enterotoxin detection in raw milk and cheese origin coagulase positive isolates

Yildirim, T, et al. International Journal of Science Letters. 2019. 1(1): 30-41. Research Article Staphylococcus aureus and Staphylococcal enterotoxin detection in raw milk and cheese origin coagulase positive isolates Tuba Yildirim1,2 * , Farid Sadati2,3 , Berna Kocaman2 , Belgin Siriken4 1 Department of Biology, Faculty of Art and Science, Amasya University, Amasya/Turkey 2 Amasya University, Central Research Laboratory, Amasya/Turkey 3 Department of Molecular Biology and Genetics, Faculty of Science, Bilkent University, Ankara/Turkey 4 Department of Water Products Diseases, Faculty of Veterinary Medicine, Ondokuz Mayis University, Samsun/Turkey Abstract Article History In the present study, a total 110 (60 cheese and 50 cows’ raw milk) samples was analyzed for coagulase-positive staphylococci (CPS) according to Food Drug Administration (FDA, 2001) previously. After the isolation, to confirmation of the isolates, catalase test, microscopic examination, coagulase test in tube and glucose-mannitol fermentation tests were applied. After the tests, we obtained 97 CPS isolates, and they were used as a material. We aimed that, 16S rRNA, nuc gene, and SEs genes in the S.aureus isolates were determined by using Polymerase Chain Reaction (PCR) assay. For the confirmation of the isolates being Staphylococci species, 16S rRNA was detected by using PCR assay. For the detection of the CPS isolates being S. aureus, nuc gene detected in the CPS isolates using PCR assay. The 16 S rRNA was detected in a total 97 (35 milk origin and 62 cheese origin) isolates. Therefore, these isolates were evaluated as (CPS). The nuc gene was detected in 50 out of 97 CPS isolates. So, the 50 (18 cheese origin and 32 milk origin) isolates were evaluated as S. aureus. However, none of the staphylococcal enterotoxin genes (SE A,B,C,D,E,G,H,I,J) was detected in 97 CPS or 50 S. aureus isolates. Received 03.07.2019 Accepted 08.08.2019 Keywords Bacterial toxin, CPS, Food contamination, nuc gene 1. Introduction Staphylococcal food poisoning is one of the leading foodborne illnesses in humans worldwide and is associated with contaminated foods of animal origin, such as milk and dairy products and other protein rich animal origin foods such as ice heavy cream, meat, poultry and fish (Tasci et al., 2011; Janstova et al., 2012). Several studies have shown that 15% to 1 Correspondence: 30 80% of the S. aureus isolated from various sources is able to produce enterotoxin (SE) (Toubar et al., 2018). SEs are single polypeptides of approximately 26.900-29.600 kDa. To date, 23 SEs have been reported in literature (Ono et al., 2015). SEA is the enterotoxin most frequently associated (Argudin et al., 2010) with staphylococcal foodborne outbreaks followed by SED. In Korea, about 90% of food poisoning isolates were reported to contain the sea gene (Cha et al., 2006). SEA also was the most common SE associated to SFP in Japan (Shimizu et al., 2000). In this country, an extensive outbreak that occurred in 2000 was attributed to low-fat milk containing SEA (Asao et al., 2003). The SEA is produced throughout the log phase, while SEB, SEC, and SED are produced primarily during the transition from the exponential to the stationary phases of growth. Expression of SEB, SEC, and SED is affected by 70 accessory gene regulator (agr), while SEA expressed simultaneously with the σ -like factor (Toubar et al., 2018). The amount of enterotoxin necessary to cause intoxication is very small about 94-184 ng. The importance of the enterotoxins comes due to their heat stability and their resistance to inactivation by gastrointestinal proteases like pepsin (Rall et al., 2008). SEs are resistant to inactivation by gastrointestinal proteolytic enzymes, such as trypsin and pepsin. The enterotoxins are quite heat resistant and the heat stability is very important property of SEs in terms of food poisoning (Le Loir et al., 2003; Presscott et al., 2012; Toubar et al., 2018). Although Staphylococcus can be killed at normal cooking temperature, the toxins remain active (Le Loir et al., 2003). They retain their biological activity even after pasteurization; staphylococcal enterotoxin A (SEA), for example, keeps some activity after 28 min at 121°C (Rall et al. 2008). The presence of a S. aureus enterotoxigenic strain places in the nasopharyngeal or oropharyngeal tract of a food handler (Todd et al., 2010; Gallina et al., 2013). Food poisoning occurs after the ingestion of food contaminated with enterotoxins produced by S. aureus; the onset of symptoms occurs a few hours (2–8) after ingestion of the contaminated food and improper preparation, handling, or storage (Schelin et al., 2011). Nausea, vomiting, abdominal cramp, and diarrhea are the most relevant symptoms (Riva et al. 2015); the disease severity depends on the amount of the ingested toxin and health of the consumer. In most 31 cases after 24 h, there is remission of symptoms; only a few cases of intoxications, ranging from 0.03% to 4.4%, are fatal in children and in the elderly (Doyle et al., 2007). S. aureus is one of the ubiquitous microorganisms in the environment and can be found in the air, water, humans and animals. In 2015, 16 Member States (MS) reported 434 foodborne outbreaks caused by staphylococcal toxins (EFSA and ECDC, 2016). It is also one of the major causes of bovine mastitis and therefore, raw milk and subsequently raw milk products may be contaminated with S. aureus (Waage et al., 1999). About 10% of cheeses in Europe are made from raw milk, which presents a considerable potential threat to human health (Beuvier et al., 2004). For example, in Scotland, S. aureus was found to be the most frequent pathogen of raw milk cheeses in Scotland (Williams et al., 2010), and in France, a study of foodborne disease outbreaks showed that S. aureus was one of the most common causative pathogens associated with milk-related outbreaks (De Buyser et al., 2001). S. aureus may be introduced to bulk milk either by direct excretion from the udder of a cow with clinical or subclinical staphylococcal mastitis or by fecal contamination (Callon et al., 2008). S.aureus may be introduced to bulk milk either by direct excretion from the udder of a cow with clinical or subclinical staphylococcal mastitis or by fecal contamination (Callon et al., 2008). The other possible explanations of the higher S. aureus level in some raw milk samples may be their contamination from the milking equipment or personnel involved in production (Rola et al., 2016). Therefore, the aims of the study were to detect S. aureus and staphylococcal enterotoxin types in coagulase positive staphylococci isolates originated from cows’ raw milk and cheese origins. 2. Materials and Methods In the present study, a total 110 (60 cheese and 50 cows’ raw milk) samples, consumed in Amasya province, Turkey, were analyzed for CPS according to Food Drug Administration (FDA, 2001) previously. After the isolation, to conf (...truncated)