Enterotoxigenic Genes of Staphylococcus aureus Isolated from Raw Milk and Milk Products in Mizoram, India

D. Paul1,*, E. Motina1, D. Deka1, R. Das2
1Department of Veterinary Public Health and Epidemiology, College of Veterinary Sciences and Animal Husbandary, Central Agricultural University, Selesih, Aizawl-796 014, Mizoram, India.
2Immunology Section, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly-243 122, Uttar Pradesh, India.
Background: Staphylococcal food poisoning (SFP) is one of the most common food-borne diseases worldwide resulting from the contamination of food by preformed S. aureus enterotoxins and milk is considered to be a major source of SFP. This study aimed at the PCR based detection of S. aureus and its enterotoxigenic genes in raw milk and milk products.

Methods: A total of 300 samples of raw milk and milk products were collected randomly from Aizawl district of Mizoram and were subjected to phenotypic and genotypic (nuc gene) detection of S. aureus and subsequent detection of enterotoxigenic genes namely, sea, seb, sec  and sed in S. aureus isolates.

Result: A proportion of 30.67% S. aureus isolates were found to be positive for species specific nuc gene and 28.26%, 2.17%, 4.34% and 5.43% S. aureus strains were positive for sea, seb, sec and sed genes, respectively. Most contamination of raw milk was observed with S. aureus harbouring highest proportion of sea gene. Combination of two enterotoxigenic genes, sea with another gene, was also possessed by the S. aureus isolates from milk and milk products.
Staphylococcus aureus is an opportunistic human and animal pathogen that colonizes the mucosal surfaces and the anterior nares are the primary reservoir sites of S. aureus. However, extra-nasal colonization of S. aureus also takes place in several locations such as skin, pharynx and gastrointestinal tract (Nair et al., 2014). Among the various food borne pathogens, S.aureus has grabbed the interest from food safety point of view because of their ubiquity and adaptability, inhabiting the skin and mucous membrane of humans, other mammals and birds. The factors contributing to the rise of this organism as a formidable pathogen involve multiple mechanisms of virulence including its acquired ability to resist antibiotics and evade host defences, production of an arsenal of virulence factors and secreted toxins such as enterotoxins (SEs) that are responsible for food poisoning (Joo et al., 2011). Staphylococcal enterotox ins are heat stable which are potent gastrointestinal exotoxins causing food borne intoxication. Classical SEs have been divided into five serological types (SEA through SEE) having emetic properties on the basis of their antigenicities (Argudin et al., 2010) and possess super-antigenic activity and are encoded by accessory genetic elements including plasmids, prophages, pathogenicity islands, vSa genomic islands or by genes located next to the staphylococcal cassette chromosome (SCC) implicated in methicillin resistance (Zhenzhen et al., 2018). The SEs are a major cause of food poisoning, which typically occurs after ingestion of different foods like processed meat and dairy products contaminated with S. aureus due to improper handling and subsequent storage at elevated temperatures. Food handlers carrying enterotoxin producing S. aureus in their noses or on their hands are regarded as the main source of food contamination through direct contact or through respiratory secretions (Argudin et al., 2010).  
Milk is considered to be a good medium for S. aureus growth and enterotoxin production and contaminated raw milk has been often involved in SFP (Asao et al., 2003). Mastitis by S. aureus is a serious problem in dairy production and milk from infected animals may contaminate bulk milk. Moreover, human handlers, milking equipment, environment, udder and teat skin of dairy animals may be other possible sources of bulk milk contamination and subsequently contaminate the milk products. Thus, enterotoxigenic S. aureus may be a potential risk of SFP after consumption of raw milk and milk products (Jorgensen et al., 2005). The present study was taken up for molecular detection of S. aureus and its enterotoxigenic genes from raw milk and milk products in Aizawl district of Mizoram, India.
The study was conducted in the department of Veterinary Public Health and Epidemiology, College of Veterinary Sciences and animal husbandary, Aizawl, Mizoram during 2020-21.
Collection of samples
A total of 300 samples, 90 raw cow milk, 30 pasteurised milk and 200 milk products including 50 curd, 50 rasmalai, 50 ice-cream and 30 paneer, were collected randomly from different unorganised farms, milk vendors, milk co operatives and sweet shops of Aizawl district, Mizoram.
Isolation and identification of S. aureus
The isolation and identification of S. aureus from raw milk and milk products was done following standard bacteriological method (Cowan and Steel, 1993). One ml or one gm of sample was mixed with 9 ml sterile 0.1% peptone water for enrichment and was centrifuged at 8000 rpm for 2-3 minutes for extrication of the sample in the broth and incubated for 24 hours at 37°C. A volume of 0.1 ml of broth culture showing cloudy discolouration was streaked aseptically on Baired Parker agar (BPA) plates and incubated at 37°C for 24-48 hours. The characteristic colonies on BPA plates were streaked on Mannitol Salt agar (MSA) plate and incubated at 37°C for 24 hours. The S. aureus isolates were phenotypically characterized based on morphological characteristics like colonies of jet black colour with halo zone on BPA and yellow colonies with yellow colouration of media on MSA and positive reactions in Gram stainingand coagulase test (Quinn et al., 1999).
Molecular detection of S. aureus and its enterotoxigenic genes
All the presumptive S. aureus isolates were processed for bacterial lysate preparation using boiling and snap chilling method. A purified colony of isolate was inoculated into one ml of Luria Bertani (LB) broth and incubated at 37°C for 24 hours and the cells were pelleted by centrifugation at 8000 rpm for 10 minutes at 4°C. The pellet was washed three times with sterile normal saline solution (0.85%) and finally re-suspended in 200 µl of nuclease free sterile distilled water. The cell suspension was heated in a boiling water bath for 5 minutes followed by immediate chilling. The cellular debris was sediment by centrifugation at 5000 rpm for 5 minutes and the supernatant was used as DNA template for PCR assay.
The DNA templates of S. aureus isolates were subjected for detection of species specific nuc gene by using published primers and subsequently all the nuc gene positive S. aureus strains were screened for enterotoxin producing genes, namelysea, seb, sec and sed using published primers (Table 1) as described by Blaiotta et al. (2004). The details of various thermal cycling conditions for species specific gene (nuc) and different enterotoxigenic genes are given in Table 2. The amplified PCR products were further analysed by agarose gel electrophoresis.

Table 1: Oligonucleotide primers used for PCR detection of enterotoxigenic genes of S. aureus.

Table 2: Thermal cycling conditions for nuc and enterotoxigenic genes of S. aureus.

The statistical analysis of data was carried out as per the method of Snedecor and Cochran (1994).
Phenotypic and molecular detection of S. aureus
Out of 300 raw milk and milk product samples analysed, 51.33% samples revealed jet black colonies with halo zone in BPA and subsequently 46.00% samples revealed yellow colonies on MSA. The bacterial colonies showing positive Gram reaction and catalase reaction are presumptively identified as S. aureus.  A total of 30.67% S. aureus isolates were confirmed  by amplification of species specific nuc gene (279 bp) (Fig 1) from 138 (46.00%) presumptively identified S. aureus isolates distributed in raw milk (43.33%), rashmalai (34.00%), ice cream (46%), paneer (23.33%) and pasteurized milk (20.00%) (Table 3). Wide variations in the prevalence of S. aureus in milk and milk products from different places were reported by Siriken et al. (2016) (56%), Sutejo et al. (2017) (97%) and Haque et al. (2018) (19.44%) from Turkey, Indonesia and Bangladesh,  respectively. Kandil et al. (2018) reported the prevalence of S. aureus as 80% and 40% in raw milk and ice-cream, respectively from Egypt. In India, Sudhanthiramani et al. (2015), Hamid et al.(2017), Begum et al. (2018) and Bhati et al.(2018) reported 39.09%, 21.21%, 92.31% and 63.80% S. aureus in raw milk and milk products from Tirupati, Jammu and Kashmir, Chennai and Rajasthan, respectively. Although pasteurization of milk is an important food safety phenomenon, the occurrence of S. aureus in pasteurized milk is not uncommon and reported in variable rates which might be due to cross contamination during storage and selling along with raw milk, local conditions, sample sizes, sample sources and geographic location (Dai et al., 2019). The abundance of S. aureus in the air, dust, waste, water, milk, food or food equipment, environmental surfaces, humans or animals including cow’s udders may increase the occurrence of the organism in milk and milk products during their unhygienic production, processing, storage and selling. Ojokoh (2006) and Jadhav and Raut (2014) reported that hands, skin and clothing of handlers and droplets produced by coughing, talking and sneezing can settle on food and may act as possible sources of this organism in milk products.

Fig 1: Agarose gel electrophoresis plate showing PCR amplicons of nuc gene (279bp); L5: 100 bp ladder; L2 and L8: Positive control; L1, L3, L4, L6 and L9: Positive sample; L7: Negative control.

Table 3: Phenotypic and molecular detection of S. aureus isolates from milk and milk products in Aizawl district, Mizoram.

Detection of enterotoxigenic genes in S. aureus strains
From 92 (30.67%) nuc positive S. aureus strains from milk and milk products, 40.22% strains were found to be positive for different enterotoxigenic genes contributing to 71.79% in raw milk, 17.65% in rasmalai,  13.04% in ice-cream, 14.28% in paneer, 33.33% in pasteurized milk and absent in curd. The prevalence of enterotoxigenic genes was significantly (P<0.05) higher in raw milk than pasteurized milk and other milk products (Table 4) (Fig 2). Begum et al. (2018) and Haque et al. (2018) confirmed S. aureus by detecting the nuc gene of S. aureus in milk and milk products as 92.31% and 24. 66% from Chennai (India) and Mymensingh district of Bangladesh, respectively.

Table 4: Detection of enterotoxigenic genes of S. aureus strains isolated from milk and milk products.

Fig 2: Agarose gel electrophoresis plate showing PCR amplicons of enterotoxigenic genes(sea, seb, sec andsed); L5: 100bp ladder; L1to L4: Positive control of (seb, sec, sedandsea), respectively; L6, L7, L8and L9: Positive sample of (sea, sed, secandseb), respectively; L10: Negative control.

Among the enterotoxigenic genes, the prevalence of sea gene (28.26%) was significantly (P<0.05) higher than seb (2.08%), sec (4.34%) and sed (5.43%) genes. The sea gene was recorded in S. aureus isolates from raw milk and all milk products except curd with highest occurrence in strains from raw milk (53.85%). The seb, sec and sed genes occurred in lower variable rates or absent (Table 4). Enterotoxin A is the most frequently encountered staphylococcal enterotoxin among staphylococcal food poisoning cases. Similar to the present findings, Normanno et al. (2007) reported that the sea gene is the most commonly reported enterotoxigenic gene in S. aureus isolates obtained from different types of foods. Afifi et al. (2011) also isolated S. aureus in 50% of raw milk and 35% of ice-cream samples from Egypt with major classical enterotoxin sea (29.30%) followed by sec (16.10%) and sed (10.10%). Saka and Tergi (2018) revealed 5(41.60%) sea, 2(16.60%) sec and 1 (8.30%) sed in raw milk and milk product from Turkey. Gandhale et al. (2017) isolated 17 S. aureus strains with enterotoxigenic genes viz.seb (1.92%), sec (21.15%), sed (7.69%) and see (1.92%) in bovine milk from Satara district of Maharashtra, India. Sharma et al. (2017) revealed that out of 30 isolates of S. aureus from bovine raw milk, 30% were positive for sec, 10% for sea and 3.30% for seb in Rajasthan, India.
Out of 92 nuc gene positive S. aureus strains, 10 (18.87%) numbers of strains were found to be positive for combination of two numbers of virulence genes, 21.42% strains in raw milk, 15.38% strains in rasmalai and 33.33% in pasteurized milk (Table 5). Normanno et al. (2007), Rall et al. (2008) and Afifi et al. (2011) revealed that S. aureus strains from bovine milk and milk products synthesized enterotoxins in different combination from Italy, Brazil and Egypt, respectively. Mansour et al. (2017) also detected S. aureus genotypically (nuc gene) in 16.30% of raw milk samples in Egypt and three strains expressed single and combination of two and three enterogenic genes and absence of enterotoxin A and D.

Table 5: Detection of enterotoxin genes of S. aureus in combination from milk and milk products.

To conclude, enterotoxigenic S. aureus in raw milk and dairy products is an important public health problem especially in traditional dairy products.  Staphylococcal enterotoxins may cause food poisoning due to consumption of raw or unpasteurized milk products. Present study provide the information regarding the genetic background of S. aureus strains isolated from raw and pasteurized milk and milk products samples in Mizoram, a hilly North Eastern state of India and it is the first molecular approach to demonstrate virulence properties of S. aureus isolates from milk and milk products which is very important from staphylococcal food poisoning point of view in consumers. These isolated S. aureus strains had virulence potential as which half of them carried enterotoxin genes either singly or in combination of two. 

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