Prevalence and Virulence Characterization of Staphylococcus aureus Isolates from Chicken Meat and Ready to Eat Chicken Products to Assess Hygiene and Consumer Safety

Vijaya Singh Thakur1, Bhavana Gupta1,*, R.V. Singh1, Anju Nayak2, Sanjay Shukla2
1Department of Veterinary Public Health and Epidemiology, College of Veterinary Science and Animal Husbandry, Nanaji Deshmukh Veterinary Science University, Jabalpur-482 001, Madhya Pradesh, India.
2Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Nanaji Deshmukh Veterinary Science University, Jabalpur-482 001, Madhya Pradesh, India.
Background: Meat and meat products are good source of proteins and essential amino acids for health. These products are contaminated with various pathogens like S. aureus, Salmonella spp, E coli and Listeria spp., S. aureus is one of most important food borne pathogen associated with food products. The present study was carried out to estimate prevalence of S. aureus and their molecular characterization in raw chicken meat and ready-to-eat chicken products in Jabalpur city.

Methods: The samples were collected from different retails outlets and processed as per standard microbiological procedures for isolation of S. aureus and subjected further for molecular characterization and processed to assess the prevalence of S. aureus. A total of 100 raw chicken meat and 70 ready to eat chicken meat products (chicken samosa, momos, pattis, tikka, barbeque etc.) have been collected. 

Result: The overall prevalence of S. aureus was observed 38.82% with 38.00% in raw chicken meat and 40.00% in ready-to-eat chicken (RTE) products. The molecular study revealed that all the S. aureus isolates were positive for 16s ribosomal RNA (rRNA). Out of 66 isolates, 27 (71.05%) from raw chicken meat and 12 (42.85%) from ready to eat chicken meat products isolates were found to be positive for sea gene where as 3 (7.8%) isolates from raw chicken meat and 6 (21.42%) from ready-to-eat chicken products were found positive for nuc gene respectively.
Meat and meat products are one of the important sources of high quality nutrients with high biological value proteins and essential amino acids. World population is growing at a rate of 1.0% per year in developed countries and 2.5% per year in the developing countries. India stands at 5th rank (6.3 million tons) and accounts for 3% of the total world meat production (220 million tones) including poultry meat (Islam et al., 2016) and the per capita meat consumption is running at 5.0 to 5.5 kilograms (11 to 12 pounds) per year (FAO, 2011). As per the Ministry of Fisheries and Animal Husbandry of India, poultry is contributing nearly 47.86% of total meat production and per capita consumption of poultry meat is estimated at around 3.1 kg.

Meat consumption pattern in majority of the countries are culture dependent and in India, meat consumption pattern is controlled by customs, tradition and religious taboos. Of the various meats consumed in India, poultry meat occupies the major share among various sections because of its versatility, relatively low cost; no social and religious taboo associated with its consumption and is considered to be lean with low fat content (Bai et al,. 2022).

Staphylococcal food-borne disease (SFD) is a gastrointestinal illness with rapid onset caused by consuming foods contaminated with enterotoxins produced by the bacterium (Bousbia et al., 2018). Approximately 0.5 ng/mL concentration of staphylococcal enterotoxin (SEs) in contaminated food may cause a large outbreak. Staphylococcal enterotoxin A (sea) is one of the most important cause of gastroenteritis and more than 50% of food poisoning (FP) is caused by S. aureus. It is a Gram-positive, non-spore forming organism which can grow at optimum temperature of 30-37°C, pH 7-7.5 and NaCl concentration upto 15-20%. It is a commensal and opportunistic pathogen that can cause wide spectrum of infection from superficial skin infection to severe and potentially fatal invasive diseases. It is found on the skin and nose (about 25%) of healthy people and animals without causing illness but has ability to produce toxins which leads to food poisoning in human being and mastitis in animals (CDC, 2016). Although pathogen produces various toxins but the food poisoning prevalence is mainly due to 22 different enterotoxins (Hennekinne et al., 2012). Most of the S. aureus food poisoning occurrences are caused by A, B, C, D and E enterotoxins (Montville and Mathews, 2008 and FDA, 2012). More than 90% S. aureus strains producing enterotoxin are also producing coagulase and thermostable nuclease (Jay et al., 2005).

Now a days, the demand for chicken meat and processed chicken meat are at rise due to consumer’s preference for poultry meat over other animal proteins as it is ubiquitously accepted meat because of different religious and social taboos in eating of pork, water buffalo and beef meat and due to its relatively low cost. Meat may be excellent source for various type microorganism viz. virus, bacteria, parasites, toxins etc. In meat, the common bacteria leads to infections are Salmonella, Campylobacter, S. aureus, C. perfringens, pathogenic E. coli etc., so consumption of raw or improperly cooked or contaminated meat may results in various types of food borne infectious diseases (Kadariya et al., 2014). Pathogens may gain entry into meat due to unhygienic production, transportation or processing and some time, post processing contamination from various sources such as water, raw ingredients, environment and food handlers.
The present research work was performed out during the November 2017- June 2018 in the Department of Veterinary Public Heath, College of Veterinary Science and A.H., Nanaji Deshmukh Veterinary Science University, Jabalpur (M.P.), India.
Collection of samples
A total of 170 samples (100 raw chicken and 70 RTE chicken products consisting of chicken samosa, chicken pattis, chicken momos, chicken barbeque and chicken tikka) (Table 1) were collected from different retail outlets in Jabalpur city. The samples were collected in properly sterilized polythene bags and transferred to laboratory in chilled condition for bacteriological examination on ice and stored at 4°C till further processing. All samples were processed for isolation of S. aureus within 24 hrs of arrival in the laboratory.

Table 1: Distribution of samples raw chicken meat and ready to eat chicken (RTE) products collected from different sources.

Enrichment and selective media
One ml of diluted sample was inoculated in 5 ml Staphylococcal enrichment broth having 5.5% salt and mixed thoroughly and incubated overnight at 37°C. The enrichment inoculum (0.1 ml) was streaked on Baird Parker (egg yolk tellurite) agar plates and incubated at 37°C for 18-24 hrs. Colonies showing shiny, jet-black (halo around colony) were picked up and considered as presumptive S. aureus.
Morphological and biochemical identification of S. aureus isolates
The presumptive isolates of S. aureus were microscopically and biochemically characterized on the basis of colony morphology, Gram’s staining, coagulase , catalase, oxidase test, indole, MR, VP and thermonuclease tests according to method described by Cruickshank et al. (1975) and Agarwal et al. (2003).
Polymerase chain reaction for detection of 16SrRNA, nuc and sea genes of S. aureus
The template DNA was prepared by boiling and snap chilling method to detect 16S rRNA, nuc and sea genes of S. aureus. PCR amplification for the individual genes was setup in 25 µl of reactions. The PCR protocol was initially standardized by varying the annealing temperature (50-60°C) and cyclic conditions. The standardized amplification reaction started with initial denaturation at 94°C for 5 min, followed by 30 cycles each having denaturation at 94°C for 1 min, annealing at 50°C for 16S rRNA gene, 55°C for nuc gene for 30 sec and sea gene for 57°C for 2 min and extension at 72°C for 1 min, with final extension for 10 min at 72°C. Amplified products were analyzed by agarose gel (1.0%) electrophoresis and primers used to detect genes of S. aureus are listed in Table 2 (Fig 1-4).

Table 2: Details of primers used for PCR reaction.

Fig 1: Distribution of 16 S rRNA , nuc and sea genes in raw chicken meat and ready to eat chicken (RTE) products.

Fig 2: Agarose gel electrophoresis showing amplified PCR product of 16S rRNA of S.aureus isolates from raw chicken and its products (228 bp; L:2-9).

Fig 3: Agarose gel showing amplified PCR product of nuc of S. aureus isolates from raw chicken and its products (270 bp; L:1, 3 and 6).

Fig 4: Agarose gel showing amplified PCR product of sea of S. aureus isolates from raw chicken and its products(102 bp; L:1-9).

Out of 170 samples of raw chicken meat and its products examined, 66 samples were found positive for S. aureus as depicted in Table 3, Fig 1. The occurrence of S. aureus in raw chicken meat was 38% and in case of chicken meat products (RTE), the occurrence was 40.00% in chicken samosa (70%), chicken momos (50%), chicken pattis (30%), chicken tikka (20%) and chicken barbeque (10%).

Table 3: Prevalence of S. aureus isolates from raw chicken meat and ready to eat chicken (RTE) products.


Virulence characterization of S. aureus isolates by PCR
Presumptive isolates were selected on the basis of colony characteristics and biochemical testing and subjected for virulence characterization by PCR (Table 4). showing any illness (CDC, 2016).

Table 4: Virulence characterization of S. aureus in raw chicken meat and ready to eat chicken (RTE) products.

Similar prevalence studies conducted by various workers have also reported S. aureus to be associated with variety of meat and meat products including chicken meat having varying percentage. Saikia and Joshi (2010), Hanson et al., 2011; Zargar et al., 2014 have found comparatively less prevalence in chicken meat 20%, 17.8% and 15.7%, respectively  whereas Kargirwar, 2004 and Abdulrahman et al., (2015) reported higher prevalence percentage than our study results, although Das and Mazumder (2016) found quite similar prevalence with present study at 48.57%. In case of ready-to-eat chicken meat products, Abd-El-Malek (2017) had reported 16.3% prevalence while Shafizi et al. (2016) observed 53.00% prevalence. Oguttu et al. (2014) conducted study in Tshwane South Africa and reported results in accordance to our study with 44.00% prevalence in ready-to-eat (RTE) chicken. Difference prevalence rate were reported by various workers might be due to variation in hygienic standards maintained at different steps in food chain starting from farm to table or due to variation in isolation protocol. The higher prevalence in ready-to-eat chicken meat products than raw chicken meat is indicative of post cooking contamination during handling or inappropriate cooking procedure.

Colony characteristics, Gram staining and different biochemical tests like oxidase, catalase used for presumptive identification of S. aureus were found quite effective in this study and enrichment media SB broth and selective media Baird Parker agar were  used quite effectively. All isolates were further subjected to detect staphylococcal enterotoxin genes A (sea) and 59.09% isolates were found to be positive for sea with high percent was observed in raw chicken meat (71.05%) than ready to eat chicken meat products (42.85%). In present study 6.06% (04) isolates found to possess both nuc and sea gene.

Madahi et al. (2014) reported in their study conducted in Iran, 33.33% of isolates fromchicken nuggets producing sea. Similarly Gupta et al., (2014) have also reported 42.22% prevalence of sea gene with 31.11% was from raw fish samples and 11.11% from RTE fish productsas found with present study.
Presence of nuc and sea positive strain in raw chicken as well as ready to eat chicken meat products (RTE) indicates unhygienic handling of meat and meat products and contamination of food items at the time of processing or post processing. As enterotoxins are heat stable and even though organism may be destroyed by various methods of cooking and processing, The presence of sea gene might be food intoxication due to already produced toxin in raw or in ready to eat products. The study revealed that the chicken meat and meat products in the study area were contaminated with S. aureus which indicates poor hygienic practices in slaughter house and cold chain failure at retail outlets.

  1. Abdulrahman, L.S., Adriana, S., Harrington, W. and  Mohamed, K.F. (2015). Isolation, virulence and antimicrobial resistance  of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin sensitive Staphylococcus aureus (MSSA) strains from Oklahoma retail poultry meats. International Journal of Environmental Research and Public Health. 12(6): 6148-6161.

  2. Abd-El-Malek, A.M. (2017). Cooked poultry meat and products as a potential source of some food poisoning bacteria. Journal  of Environmental Science. 11(6): 23-29.

  3. Abeer, A.A., Abdel, A., Bashandy, M.M., Yasin, M.H. and  Ibrahim, A.K. (2010). Assessment of conventional and molecular features of S. aureus isolated from bovine milk samples and contract dairy workers. Global Veterinaria. 4(2): 168-175.

  4. Agarwal, R.K., Bhilegaonkar, K.N., Singh, D.K., Kumar, A. and Rathore, R.S. (2003). Laboratory Manual for the Isolation and Identification of Foodborne Pathogens. 1st Edn., Jai Ambey Publishing Co.U.P. 

  5. Bousbia, A., Boudalia, S., Gueroui, Y., Belaize, B., Meguelati, S., Amrouchi, M., Ghebache, R., Belkheir, B.  and Benidir, M. (2018). Nutritional and hygienic quality of raw milk intended for consumption in the region of Guelma, Algeria.  Asian Journal of Dairy and Food Research. 37(3): 192-196.

  6. Bai, A., Ruban, S.W., Spandan, P.V., Barry, A.I.G., Kumar, S.N., Indresh, H.C. and Nagaraja, C. S. (2022). Carcass and meat quality characteristics of native chicken reared under backyard and farm setting in Karnataka. Asian Journal of Dairy and Food Research. 41(1): 111-115. 

  7. Brakstad, G.O., Aasbakk, K. and Maelanda, A.J. (1992). Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. Journal of Clinical Microbiology.  30: 1654-1660.

  8. CDC, (2016). Staphylococcal Food Poisoning Online https://www. cdc. gov/foodsafety/diseases/staphylococcal.htm.

  9. Cruickshank, R., Duguid, J.P., Marmion, B.P. and   Swain, R.H.A.  (1975). Medical Microbiology, 12th Edn., Crurchill Livingstone.,  Edinburgh. Pp 207-215.

  10. Das, P. and  Mazumder, P.B. (2016). Prevalence of S. aureus in raw meat samples in Southern Assam, India. Journal of Agriculture and Veterinary Science. 9: 23-29.

  11. FAO. (2011). India’s meat consumption on the rise online http://

  12. FDA. (2012). Bad Bug Book Foodborne Pathogenic Microorganisms and Natural Toxins Handbook. 2nd Edn. 87-92. 

  13. Gupta, B., Ghatak, S. and Gill, J.P.S. (2014). Prevalence and characterization of S. aureus isolates from fresh fish and ready to eat fish products. Environmentand Ecology. 32(3): 1163-1167.

  14. Hanson, B.M., Dressler, A.E., Harper, A.L., Scheibel, R.P., Wardyn, S.E., Roberts, L.K., Kroeger, J.S. and  Smith, T.C. (2011). Prevalence of Staphylococcus aureus and methicillin- resistant Staphylococcus aureus (MRSA) on retail meat in Iowa. Journal of Infection and Public Health. 4(4): 169-74.

  15. Hennekinne, A.J., Buyser, D.M. and   Dragacci, S. (2012). Staphylococcus  aureus  and its food poisoning toxins characterization and outbreak investigation. FEMS Microbiology Review. 36(4):  815-836.

  16. Islam, M.M., Anjum, S., Modi, R.J. and  Wadhwani, K.N. (2016). Scenario of livestock and poultry in India and their contribution  to national economy. International Journal of Science. Environment and Technology. 5(3): 956-965.

  17. Jay, J.M., Martin J.L. and David, A.G. (2005). Modern Food Microbiology.  Springer publishing Co., 7thEdn. Ch.11, pp  241-244.

  18. Kadariya, J., Smith, T.C. and Thapaliya, D. (2014). Staphylococcus aureus and staphylococcal food-borne disease: An ongoing  challenge in public health. Bio Medical Research International.  USA. Pp 1-9.

  19. Kargirwar, S.V. (2004). Studies on microbial quality of traditional meat products. M.V.Sc. thesis, (Veterinary Public Health and Epidemiology), Maharashtra Animal and Fishery Sciences University, Nagpur.

  20. Madahi, H., Rostami, F., Rahimi, E. and Dehkordi, S.F. (2014). Prevalence of enterotoxigenic Staphylococcus aureus isolated from chicken nugget in Iran. Jundishapur Journal Microbiology. 7(8): 10237. 

  21. Mehrotra, M., Wang, G. and Johnson M.W. (2000). Multiplex PCR for detection of genes for Staphylococcus aureus enterotoxins,  exfoliative toxins, toxic shock syndrome toxin and methicillin  resistance. Journal of Clinical Microbiology. 38(3): 1032-1035.

  22. Montville, T.J. and  Matthews, K.R. (2008). Food Microbiology: An Introduction. 2nd Edn., ASM Press, Washington D.C. 

  23. Oguttu, W.J., McCrindle, E.M.C., Makita, K. and Grace, D. (2014). Investigation of the food value chain of ready-to-eat chicken and the associated risk for staphylococcal food poisoning in Tshwane Metropole, South Africa. Food Control. 45: 87-94.

  24. Saikia, P. and Joshi, S.R. (2010). Retail market poultry meats of North-East India-A microbiological survey for pathogenic contaminants. Research Journal of Microbiology. 5(1): 36-43.

  25. Shafizi, A.W., Mohammad Ridzuan, M.S., Ubong, A., New, C.Y., Mohhiddin, O., Toh, P.S., Chai, L.C. and Son, R. (2016). Assessing Staphylococcus aureus in ready to eat (RTE) food and risk assessment of food premises in Putrajaya. International Food Research Journal. 23(4): 1761-1766. 

  26. Zargar, S.H.M., Doust, H.R. and Mobarez, M.A. (2014). Staphylococcus  aureus enterotoxin a gene isolated from raw red meat and poultry in Tehran, Iran. International Journal Enteric Pathogens. 2(3): 16085.

Editorial Board

View all (0)