Distribution of Staphylococcal Enterotoxin (SE) Genes among Different Staphylococcus Species Isolated from Bovine Mastitis

Staphylococci are one of the leading causes of mastitis in animals and food poisoning in humans. In dairy cows, mastitis is considered an economically important infectious disease and Staphylococcus aureus has long been identified as the predominant pathogen causing mastitis in bovine and small ruminants (Nesaraj et al., 2023; Doley Sharmita et al., 2017). These pathogens produce several toxins such as α, β, γ and δ toxins, toxic shock syndrome toxin (TSST-1), enterotoxins, nuclease, coagulase, catalase, hyaluronidase, phosphatase, lipase, staphylokinase and proteases which can have direct effects on mammary tissue and or lead to an uncontrolled host inflammatory response by acting as superantigens (Fayisa et al., 2023). These toxins are highly stable and are resistant to heat and most of the proteolytic enzymes such as pepsin and trypsin and are often considered as the most common bacterial toxins involved in food poisoning in humans (Abdallah et al., 2024; Narayan et al., 2023). Staphylococcus aureus is the world’s third most important cause of food-borne illness (Rahimi and Alian, 2013) and milk and milk products are considered to be the common vehicles for this species (De Buyser et al., 2001; Jorgensen et al., 2005). Among different species of Staphylococcus, the coagulase-producing (CPS) staphylococci were considered more pathogenic than the coagulase-negative species; however, several studies have proved that coagulase-negative species (CNS) as important cause of mastitis in ruminants (Seker et al., 2023; Deng et al., 2023) and many CNS have been isolated from human blood cultures and nosocomial infections (Akgul and Bora, 2023). 

Staphylococcal enterotoxins (SEs) are one of the toxins produced by enterotoxigenic Staphylococcus species. These SEs act as potent superantigens and help the organism to persist in the mammary gland resulting in recurring mastitis in the infected herds. There are at least five major classical types of SEs that include sea, seb, sec, sed and see. Among these classical types, sea and seb have frequently been detected in milk and milk products but recently, several other toxins like seg, seh, sei, sek, sel, sem, sen, seo, sep, seq, ser and seu, have also been detected in staphylococci isolated from cases of bovine mastitis (Wiśniewski et al., 2023). The pathogenicity of Staphylococcus is determined by its ability to produce different toxins and the potency of toxin to produce clinical forms of disease in animals and humans. Therefore, in the present study, we aimed to analyze the distribution of the different enterotoxins among CPS and CNS species isolated from bovine mastitis cases.

A total of 135 milk samples from bovine clinical mastitis cases were aseptically collected from Pondicherry and processed in the Department of Veterinary Microbiology, Rajiv Gandhi Institute of Veterinary Education and Research, Pondicherry, India. About one milliliter of milk sample was centrifuged at 6000 rpm for 5 min and the sediment was streaked onto Sheep Blood Agar plates and incubated at 37°C for 48 h. The characteristic colonies were stained with Gram’s staining. Different species of staphylococci were identified and differentiated from other cocci by a series of biochemical reactions that included catalase production, oxidase production, nitrate reduction, oxidation-fermentation test, sugar fermentation (lactose, maltose, mannitol, mannose, sucrose and trehalose), ability to produce coagulase and urease and Voges-Proskauer test. CPS and CNS were differentiated based on, colony pigments, the ability to produce coagulase and acetoin production.

DNA was extracted from isolates as per the protocol (Chomczynski and Rymaszewski, 2006). Briefly, 2 to 3 fresh staphylococcal colonies were dissolved in 500 µl of alkaline polyethylene glycol (AL-PEG) reagent and the suspension was held in a water bath set at 60°C and kept for 10 min. The lysed suspension was centrifuged at 10,000 rpm for 5 min and the obtained supernatant was diluted with nuclease-free water at a 1:10 ratio and was used as a DNA template. PCR for Staphylococcus genus-specific, S. aureus species-specific and enterotoxins were carried out using the primers listed in Table 1.

Isolation and identification of different Staphylococcus species from milk samples
 
About 74.07% (100 isolates) of samples were found positive for Staphylococcus species based on cultural identification, Gram staining and biochemical tests. The isolates were further confirmed at the genus level by polymerase chain reaction amplifying 756 bp fragment of 16s rRNA (Fig 1a). Staphylococcus aureus species were identified by amplifying the nuc gene fragment (Fig 1b). The coagulase test revealed that 44 isolates belonged to coagulase test-positive Staphylococcus (CPS) and 56 were coagulase-negative Staphylococcus (CNS). Out of 44 CPS, 42 isolates were identified as Staphylococcus aureus and two isolates were Staphylococcus intermedius.



CNS further characterized up to the species level by series of biochemical tests and they were identified as Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus simulans, Staphylococcus hominis, Staphylococcus xylosus, Staphylococcus caprae, Staphylococcus capitis, Staphylococcus sciuri, Staphylococcus lugdenensis, Staphylococcus saccharolyticus, Staphylococcus lentus, Staphylococcus auricularis, Staphylococcus hemolyticus and Staphylococcus arlettae.
 
Molecular screening for staphylococcal enterotoxin genes
 
All Staphylococcal isolates were subjected to molecular screening by PCR for detection of 9 different Staphylococcal enterotoxins (SE) encoding genes namely sea, seb, sec, sed, see, seg, seh, sei and sej. Overall, PCR detected 7 SE genes (sei, sea, seg, sec, sej, sed and seb) in CPS and CNS isolates (Fig 2) however, none of these isolates revealed see and seh. Among CPS isolates, sei gene was found the highest number of isolates, followed by sea, seg, sec, sej, sed and seb while, in CNS isolates, sei was found in the highest number of isolates followed by sea, seg, sed, seb, sej and sec. The genes encoding SE production were detected in 84.09% and 96.42% of CPS and CNS isolates respectively. The distribution of SE among different species is detailed in Table 2. The overall distribution of SE genes was higher in CNS than in CPS isolates and a maximum of six SE genes (sea, seb, sed, seg, sei and sej) were detected in a CNS isolate belonging to S. caprae.





Staphylococci species are reported to be a major pathogen causing mastitis in dairy cattle and are also associated with a widespread spectrum of infections and food poisoning in humans (Waseem et al., 2020). Coagulase-positive S. aureus is considered the most pathogenic strain of its genus and is an etiological factor for various clinical manifestations including food poisoning (Yiğin-Akın et al., 2018). Staphylococcal food poisoning is a kind of foodborne intoxication that occurs due to ingestion of preformed SEs and is characterized by vomition, gastroenteritis, diarrhea painful contraction of gastrointestinal smooth muscles with a short incubation period usually 30 minutes to 8 h (Le et al., 2003, Podkowik et al., 2013).  S. aureus was considered the only species of its genus capable of producing enterotoxins however, recent reports have shown the involvement of CNS species in hospital-acquired infections (Podkowik et al., 2013). CNS are capable of producing various virulence factors similar to S. aureus including enterotoxins (Podkowik et al., 2012). Moreover, CNS such as S. simulans, S. chromogenes are now considered as emerging pathogens of bovine mastitis (Pyorala and Taponen, 2009). Therefore, it is important to understand the distribution of the SEs in both CPS and CNS isolated from the bovine mastitis milk. In the present study, sixteen species of staphylococci from bovine mastitic milk were isolated and identified by a series of biochemical tests. Biochemical tests most commonly used tool for the differentiation of different Staphylococci (Thorberg et al., 2009). S. aureus was most prevalent followed by S. chromogens, S. epidermidis, S. simulans and S. caprae. Both CPS and CNS are capable of producing enterotoxins; however, the pattern of toxin-producing genes is highly variable.  Out of nine SEs screened, seven different enterotoxin genes namely sei, sea, seg, sec, sej, sed and seb were detected by PCR and their prevalence was higher among CNS than in CPS. The number of SE genes per species was also highest in S. caprae, a CNS species with six SE encoding genes (sea, seb, sed, seg, sei and sej). The predominant SE encoding genes detected were sei, sea and seg. Park et al., (2011) reported that the distribution of the super antigens such as seb, seln and selq were highest among the S. chromogenes, S. xylosus, S. haemolyticus, S. sciuri subsp. carnaticus, S. simulans and S. succinus which were CNS isolated from the bovine mammary infections. Similarly, de Freitas Guimaraes et al., (2013) reported that 66% of CNS and 35% of CPS isolated from subclinical mastitis cases possessed the SE genes with a high frequency of sea, seb  and sec. Among different SE encoding genes, sea genes lesser extent sei genes are mainly linked to staphylococcal food poisoning worldwide (Kwon et al., 2004; Argudin et al., 2011). Studies have also reported that all the seg producing staphylococci consistently carried sei gene but not vice-versa (Omoe et al., 2002) which was further confirmed in the present study as well. The high prevalence of SE-encoding genes in CNS isolates of mastitis origin may pose a potential risk of food poisoning as these toxins are highly heat resistant even at pasteurization temperature.

The CNS that was once considered less pathogenic is becoming more pathogenic with increased enterotoxin-producing abilities. Although the mere presence of SE encoding genes does not confirm the toxin-producing ability, their presence suggests their potential to produce toxins under suitable conditions. Therefore, it is imperative to screen raw milk obtained from healthy udder, processed and stored milk and ready to eat milk products for different SE encoding genes to safeguard human health. Personal hygiene and hygienic practices during milking, transportation and storage might reduce the chances of infection and contamination; but, routine screening of milk and milk products for different pathogenic microbes and their toxins is essential to formulate suitable control strategies. Nevertheless, the present findings indicate a potential public health hazard and underscore the need to establish both effective bovine mastitis control programs and diagnostic methods to limit staphylococcal food poisoning.

We thank the Dean, Rajiv Gandhi Institute of Veterinary Education and Research, (RIVER) Puducherry for providing the necessary facilities and financial support to complete this work. We sincerely thank the clinicians at Veterinary Teaching Hospital, RIVER and Veterinary Dispensaries, Puducherry for their kind cooperation in sample collection.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

We declare that none of the authors has a financial or personal relationship that could inappropriately influence or bias the manuscript’s contents.