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

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 5^th 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.


 
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).

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.