Occurrence and associated risk factors of Bacillus cereus in fast foods in Jammu

Introduction

Bacillus cereus is a motile, facultative anaerobic spore-forming, rod shaped bacterium that belongs to the genus Bacillus (Kamat et al., 1989) [16] . Strains of Bacillus cereus are of great significance being ubiquitous in nature and with ability to occur in a wide range of foods (Ehling-schulz et al., 2004; Oguntoyinbo and Reyes et al., 2007) [7,26] . B. cereus is the most prevalent pathogenic species found in food and other industrial processes (Prittijarvi et al., Since people are adapting fast foods practices now a day and the processing practices of fast foods are of low standards that may lead to contamination which in turn can predispose people to food borne pathogens including B. cereus. Foodborne illnesses that are caused by consumption of contaminated food with toxic substances or infectious agents pose significant morbidity, mortality and economic losses, (Havelaar et al. 2015) [15] . Food poisoning caused by B. cereus is primarily diagnosed by isolating the pathogen from the samples of fast foods, from hands and utensils of vendors, faeces, vomitus etc. Foods sold in markets are often prepared in unsanitary environments, and with the use of contaminated containers that expose the food to multiple potential contaminants raises serious concerns in respect of safety of the food. In the UT of Jammu and Kashmir infectious agents are of paramount concern in the changing scenario of life style of common people. More to understand India is adjusting to these ready to eat foods just like other developed and developing nations. Few studies have been conducted on the incidence of food-borne pathogens with emphasis on Bacillus cereus (Yusuf et al., 1992; Desai and Varadaraj, 2009) [36,4] . When the food hygiene practices are not followed and food venders are not fully trained in adoption of hygienic principles during food processing and serving, food borne disease outbreaks cannot be neglected (Sahota et al., 2008) [28] . Keeping in view the role of B. cereus in food borne infection and intoxication as an emerging pathogen it was realized to assess the safety and quality of fast foods served in and around Jammu city.

Materials and Methods

A total of 105 samples comprising, Momos (25), Fried rice (25), Spring roll (18), Pasta (16), and Noodles (21), were collected randomly from Jammu city. The samples were transported to the laboratory in sample containers with ice packs and were processed immediately within 4 hours for isolation and identification of B. cereus. The reference strain of B. cereus (ATCC 14579) used in the current study was procured from Hi-media (Mumbai). The pure strain was maintained on nutrient agar slants by sub-culturing fortnightly.

Isolation and identification of B. cereus

For the selective isolation of B. cereus, Polymyxin-Pyruvate-egg yolk-Mannitol-Bromothymol blue agar (PEMBA) media was used. The samples were processed as per the protocol described by Shinagawa (1990) [30] and Tallent et al. (2012) [32] with suitable modifications. Briefly, 25 gm of sample was homogenized in 225 ml of brain heart infusion broth (BHIB) containing polymyxin B (10,000 U per 100 ml). The samples were then enriched at 35 ℃ for 16-18 hours. Following enrichment, a loopful was streaked onto PEMBA plates and then incubated at 37 ℃ for 24 hours. The plates showing crenate to fimbriate peacock blue-colored colonies of approximately 3-5 mm size, surrounded by a blue zone of egg yolk hydrolysis against a green background were purified and taken on nutrient agar slants and characterized further for identification of B. cereus).

Identification and confirmation of B. cereus

All the presumptive colonies of B. cereus and closely related species were collected and subjected to morphological and biochemical tests for identification. The morphological and biochemical tests conducted include Gram's staining, cell morphology, spore production, motility, reduction of nitrate, Christensen's citrate utilization, tyrosine decomposition, Voges-proskauer reaction, aerobic and anaerobic utilization of glucose, mannitol fermentation, and hemolysis. The tests and identification of species were carried out as described in the Bacteriological Analytical Manual of the United States Food and Drug Administration (Rhodehamel and Harmon 2001) and Bergey's Manual of Systemic Bacteriology (Clause and Berkeley 1986) [3] .

Study of Associated Risk Factors of Bacillus cereus

Questionnaire survey for Street vendors/hawkers (Section-I) At the time of sample collection, street vendors were interviewed randomly using a semi-structured questionnaire to collect data for assess the knowledge, attitude and practices related to food processing and services to conumers, to identify the factors associated with occurrence of B. cereus. The information collected is based on four modules comprising of four sections. Sections-I, deals with general questions and socio-demographic characteristics of food vendors, Sections-II, explains knowledge of venders about food safety issues, Sections-III, explains, attitude of food vendors towards ensuring food safety and Sections-IV, is related to the food safety practices of food vendors.

Descriptive Statistics

Descriptive statistics were calculated for each variable of interest, in term of percentage to simply the understanding of every one.

Results and Discussion

Overall Occurrence of B. cereus in fast food products A total of 105 fast foods samples were collected and processed to isolate the Bacillus cereus and 27 samples were found positive showing an overall occurrence of 25.71%. of

B. cereus

The present study revealed maximum contamination of rice (32%), noodles (28.57%), momos (28%) followed by pasta (18.75%) and spring roll (16.66%). The finding of this study was in agreement with similar incidence reported by Gdoura Ben Amor et al. (2018) [11] , who reported an incidence of 27.8% in different food stuffs (cereals, pastry, cooked poultry meat, fresh cut vegetables, spices and dairy products). These finding were lower than those mentioned by Yu et al. (2020) [35] , who isolated B. cereus 35% from the retail ready to eat food samples from different regions of china. But our results were higher than Mostafa et al. (2022) [ 20] who observed 11.1% B. cereus strain from meat products (luncheon, kofta, minced meat, beef burger and sasuage).

Study of Associated Factors of Bacillus cereus

A Questionnaire based survey was conducted among 20 food vendors to study the factors associated with occurrence of Bacillus cereus. The results obtained were presented under the following heads:

Social characteristics of respondents Age and sex distribution of respondent

Twenty males' respondents were interviewed by selecting randomly during study. The average age of all the respondents was 31-45 years and maximum of respondent (9) were in age of 18-30 years, eight were in age of 31-45 years, followed by (2) in the age group of less than 18 . However, only one candidate was found in the category of greater than 45 years of age.

Distribution according to Education status

The educational attainment of respondents was evaluated in terms of different level of education, 35% were illiterate, 25% of the respondents had acquired primary education. 20% were matriculate, whereas, 20% of the respondents had acquired education up to higher secondary school level. It revealed that mostly 18-30 years age group of unemployed youth having primary educations were engaged in fast food services. They may not be practicing hygienic practices as a consequence there are chances of contamination of food items.

Distribution of respondents according to work experience

Majority of respondent (45%) were having 5-9 years of experience in this job. 30% were having less than 5 years, whereas, 15% were having 10-14 years of experience.

Others details of food vendors

Some of the food vendors surveyed were having their own small shops and some were having moveable street cabins for food processing and selling. Selling fast food was the only source of income of majority of respondents. Majority of the food vendors got training from their friend and relatives followed by their parents. None of the food vendors had received any formal training from any specialized source. Out of the 20 food vendors surveyed no one had additional job. None of the food vendors knew about Bacillus cereus infection or other food borne pathogens. So lack of knowledge was one contributory factor in spread of food borne pathogens. The finding of this study reveals the comparable results with the study of Loukieh et al. (2018) [18] .

Awareness level of food vendors regarding food safety and transmission of food borne pathogens

The survey conducted revealed that 70% of vendors had no knowledge about the food borne pathogens transmitted through contaminated food. However, 30% believed that unhygienic practices contaminate the food. Regarding personal hygiene 55% respondents had no knowledge about personal hygienic practices, whereas, 45% agreed that personal hygiene is important. Almost all food vendors agreed that use of whole some water for all purposes in processing and serving food is essential. The other parameters of assessing the level of awareness are tabulated in table-1.

Attitude of food vendors towards ensuring food safety

Regarding training on food hygiene 25% food vendors agreed that training is essential whereas, 50% had no idea about it and 25% vendors believe that training is not essential. Similarly, 25% of food venders agree on the importance of personal hygiene. 45% have no idea whereas, 30% disagree the statement. The other parameter of attitude of food venders are mentioned in the table-2 below. Based on the results from this study, the vendors have positive attitude towards preparation of nutritious and healthy meals, majority of food vendors (90%) recognize the importance of providing safe food as a job of responsibility. This attitude is in the line with another study in the United Kingdom where, 97% of respondents thought that it was necessary to produce health food than tasty food (Worsfold et al. 2004) [38] . Regarding the importance of personnel hygiene practices such as washing of hands, touching of body parts, use of detergents after toilets, handling money and engaging in activities that may contaminate food etc for maintaining food safety, 50% agreed with the statement. Thus there is a strong linkage between positive attitudes of food vendors in preparing, and offering healthy street meals is in agreement with another study Tuglo et al. (2021) [34] . The hands are the most important vehicle for the transmission of organisms from sources to food (WHO, 1989; Rane, 2011) [37,25] . Food handler should be trained supervised by government health agencies to ensure personnel hygienic practices and good sanitation procedures (Sneed et al., 2004) [31] . The majority, of workers (85%) agree that properly cooked food is free of contamination, while 15% have no opinion on the matter. The other important parameters of attitude are as in Table-2.

Food safety practices of food vendors

Regarding personal habits 80% of food vendors always handle money, unwashed plates, glass etc during food service. It means they do not know the importance of personal hygiene and routs of disease transmission and 20% never handle money, unwashed utensils during food service time. During food serving majority of responded (40%) were not eating or drinking during serving, 20% usually eat while working. Whereas (25%) vendors responded that they sometime eat during working, 15% rarely eat while working. Similarly, 55% venders responded that they always follow personal hygienic practices, 25% usually and 20% follow sometime. The other results related to the food safety practices of the food venders are briefly enlisted in table-3. In the present study, 90% of the vendors did not use any personal protective equipment/clothing as well as safety procedures. The poor socio-economic condition with lacking proper safety knowledge among the street vendors is one of the potential reason for their lower use of protective equipment (Gadaga et al. 2014) [10] . These finding were quite similar in Kenya (87.7%) and with the studies undertaken in Haiti and Uganda, where, 40% and 45% food vendor used personal hygiene ( [21,22,29] . Majority of the vendors had food that was already prepared when they set up shop to sell it. Food that has been cooked and left at room temperature has a higher potential to harbours dangerous bacteria. It was also found that very few of the vendors regularly cleaned the area where food was producessed and only few of the venders correctly covered their prepared food. Such set of practices were in agreement of the studies conducted on other parts of globe. Thus foods should ideally be handled with clean tongs, forks, spoons, or disposable gloves to lower the danger of crosscontamination, according to the WHO. 6)30% (4)20% Hand washing before handling food reduces the risk of food borne infections.

(8)40% (3)15% (9)45% Vendors should not touch the money or dirty utensils during food processing or distribution _ (8)40% ( 12)60% The uses of paper/polythene packs for food packaging are not safe.

(1)5% ( 16)80% (3)15% Use of masks and gloves is important in reducing risk of food borne pathogens (1)5% ( 9)45% (10)50%

Acknowledgement

AcknowledgementAuthors wish to thank the Dean, faculty of Veterinary Science and Animal Husbandry, R.S. Pura, SKUAST-Jammu for the facilities provided.

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