Abstract

Fifty food samples were collected from 60 randomly chosen fast food and traditional restaurants in Al-Najaf, Iraq. These samples were incubated to encourage bacterial growth at 37°C for 24 to 48 hours, while fungi were cultured at 25°C for seven days. The analysis showed that out of the 17 samples isolated from the food, nine different bacterial genera and ten fungal genera were identified, the bacterial and fungal species isolated in this study included Staphylococcus aureus, Shigella sp., Escherichia coli, Streptococcus sp., Salmonella sp., Bacillus subtilis, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Penicillium expansum, Penicillium sp., Rhizopus sp., Cladosporium sp., Alternaria sp., and Fusarium sp. The findings revealed that Aspergillus niger was the most predominant species, followed by Penicillium sp., and then Aspergillus flavus and Alternaria sp., with their respective frequencies being 52%, 45%, 50%, and 31.3%. The occurrence rates in falafel were 80%, 75%, 73%, and 45% respectively. In the case of beef burgers, Aspergillus flavus was predominant, with a frequency and visible percentage of 36%, 53%, and 35% respectively. Cladosporium sp. showed the lowest frequency and appearance percentage in beef pizza, with values of 26% and 6.6% respectively.

Highlights:

1. Food samples tested: 9 bacterial, 10 fungal species identified.
2. Aspergillus niger predominant in falafel, beef burgers, and beef pizza.
3. Cladosporium sp. showed lowest frequency and appearance in beef pizza.

Keywords:   Street-Food , Fast Food, Staphylococcus aureus, Sandwiches

Introduction

Food is a vital substance that sustains life by providing essential nutrients necessary for growth, health maintenance, and disease prevention. However, if food is not properly sourced, transported, stored, prepared, cooked, and served in hygienic conditions, it can pose significant risks to organisms (Adams and Moss, 2000) . The connection between food contamination and various diseases, along with the resulting morbidity and mortality rates, is well-established and documented worldwide , contamination can occur at various stages of food production and preparation, as microbial pathogens, chemicals, and parasites may enter the food chain, potentially leading to illness, disability, or even death among consumers (Angelillo et al., 2000).

The WHO (Washington, 2009) has estimated that a significant proportion of deaths from diarrheal diseases globally is due to the consumption of contaminated food. While pinpointing the exact global incidence of foodborne illnesses is challenging, In 2000, an estimated 2.1 million people lost their lives to diarrheal diseases, many of which were associated with contaminated food and drinking water, according to the Centers for Disease Control. The World Health Organization (2009) further estimated that exposure to contaminated food and water contributes to about 1.5 billion cases of diarrhea and over three million deaths among children annually. Furthermore, records show that over 600 million cases of foodborne illnesses occur annually, meaning nearly one in ten people worldwide fall ill after consuming contaminated food (Baumgart et al., 2007).

In recent years, the majority of fast-food establishments have started offering salads and fresh greens, including lettuce, cabbage, carrots, cucumbers, onions, ketchup, and mayonnaise. Some of these items are cooked before consumption, while others are consumed raw. Pre-cut salad ingredients in sealed packages, sold in grocery stores and used in institutions, are an example of products that include both fresh and processed vegetables. While these products are generally considered fresh, contamination can occur during processing, and Changes in microbial growth during storage can alter the microbial composition of these products both in quantity and quality. The internal tissues of healthy plants and animals are typically devoid of microorganisms, The surfaces of raw vegetables and meats can be contaminated with various microorganisms, with the level of contamination influenced by factors such as the product's condition, handling methods, and storage duration and conditions (Bichai et al., 2008).

Microbial food safety has become a critical public health issue on a global scale. In the United States, it is estimated that approximately 76 million cases of foodborne illness occur each year Pathogens such as Campylobacter spp., non-typhoidal Salmonella, and pathogenic Escherichia coli, which are present in the gastrointestinal tracts of various wild and domesticated animals—especially those raised for human consumption—are often responsible for these illnesses). Contamination with these pathogens can happen at various stages of the food chain, including during production, processing, distribution, and retail (Chen et al. ,2007).

Food handling and preparation are essential factors in foodborne illness outbreaks. Epidemiological studies have shown that animal-derived foods are major sources of illnesses caused by foodborne pathogens In particular, raw or undercooked poultry and red meats are major carriers of these pathogens. Other sources of infection include contaminated produce, contact with farm animals and pets, and person-to-person transmission (Cosgrove et al., 2009).

Dangerous microorganisms can be present in soil, water, animals, and humans. They may transfer to food through contact with hands, wiping cloths, and utensils, particularly chopping boards. Even minimal contact can result in foodborne illnesses. Both fast and traditional foods can contain pathogens that lead to various human diseases Salmonella is a frequent cause of foodborne illness, commonly associated with undercooked chicken and eggs Additionally, Listeria spp. has been detected in retail foods, ready-cooked chicken, on food workers' hands, and in human feces (Clouditz et al., 2006).

Urban sewage and soil can harbor harmful microorganisms (These pathogens, including Campylobacter spp., Staphylococcus spp., Escherichia coli, Salmonella spp., Yersinia spp., and Listeria, can be found on various foods such as meat, seafood, vegetables, chicken shawarmas, both raw and Contamination has been reported in various food items, Microorganisms such as Mucor spp. have been identified on a variety of items, including raw and cooked foods, chicken, beef burgers, ready-to-eat salads, commercial mayonnaise, frozen chicken, poultry products, and even on the hands of food workers , Aspergillus fumigatus, Trichoderma, Neurospora crassa, and Aspergillus niger in raw vegetable salads (Dharmarha and Vaishali , 2009).

Fast food is usually high in refined sugar, white flour, trans fats, polyunsaturated fats, salt, and a range of additives, while being low in protein, vitamins, and fiber. Its consumption is associated with obesity and related health problems. Most fast food items are made using processed ingredients at a central facility. and then reheated or assembled at individual outlets, resulting in foods that are high in calories, saturated fat, and sodium. contributing to weight gain, clogged arteries, and increased blood pressure. Examples from McDonald's include chicken sandwiches, hamburgers, French fries, Egg McMuffins, premium salads, and yogurt parfaits (Farber and Peterkin, 2009).

To ensure food safety, it is important to cook food thoroughly to the proper temperature to eliminate harmful microorganisms. Cooking to 70°C is recommended, especially for poultry and minced meats such as hamburgers and sausages, to ensure safety, the center of the food should be heated to at least 70°C for a minimum of two minutes. Chilled ready-to-eat foods should be stored at temperatures under 5°C, while hot foods should be maintained at a temperature above 60°C before serving. Leftovers should be cooled quickly and refrigerated within two hours to prevent bacterial growth. Hot foods should be cooled before refrigeration to avoid raising the temperature of other items (Dykes and Dworaczek , 2002).

Elevated aerobic bacterial counts have been found in vegetables that come into contact with soil, Examples include lettuce, carrots, potatoes, cabbage, and falafel served with fresh vegetable salads, which also show high levels of microorganisms. The high acidity and sugar content in fruits often promote the growth of yeasts and molds, while the carbohydrate-rich environment of many vegetables supports lactic acid bacteria. Contamination of frozen vegetables is commonly linked to equipment that is hard to clean thoroughly, including choppers, slicers, conveyors, inspection hoppers, and filling machines. Belts can be particularly troublesome due to the persistence of microorganisms on certain surfaces and moisture absorption, which can facilitate microbial buildup (Feng et al., 2007).

Salmonella is a significant pathogen linked to eggs and egg products. For meat and poultry items, such as flame-seared beef patties and cooked beef, which are processed at relatively low temperatures, the final bacterial counts may include heat-resistant bacteria like enterococci, typically ranging from 10^3 to 10^4. Recontamination frequently occurs if cooked products are not promptly packaged while hot and frozen, with potential sources including equipment, food handlers, raw ingredients, or dust(Fotadar et al., 2005).

Mycotoxicoses, resulting from fungal toxins, can affect various food products. Numerous mycotoxins have been found in foods and animal feeds These toxins are typically identified in moldy foods and feeds, Storage fungi from the genera Aspergillus, Penicillium, and Fusarium are particularly troublesome. Additionally, molds found on bread and viruses like hepatitis A can also lead to foodborne illnesses

Canning, which prevents oxygen from reaching the food, controls aerobic organism growth and preserves food. However, anaerobic conditions can promote the growth of microorganisms like Clostridium botulinum, leading to botulism, a rare but serious foodborne illness associated with improperly preserved home-canned foods (Heaton and Jones , 2008) .

This study aims to identify pathogenic bacteria, fungi, and yeasts in fast food and traditional foods from various restaurants. It aims to investigate the relationship between microbial contamination and variables such as food type, temperature, and season. The research is designed to offer insights into preventive measures for foodborne diseases and strategies for managing microbial contamination.

Method

1. This study involved collecting fifty samples from fast food and traditional restaurants in Najaf and Al-Kufa. The samples included beef burger meat, beef shawarma, beef pizza, chicken shawarma, chicken burger, chicken pizza, falafel, and salads (lettuce, cucumbers, tomatoes). They were placed in sterile plastic bags, kept in an icebox, and transported to the Advanced Mycology Laboratory at the Faculty of Science, Kufa University, for analysis, following the procedures outlined by Chessbrough (1984).

2. For food sample preparation, 1 gram of each sample was aseptically weighed, macerated, and mixed with 9 ml of sterile distilled water. Serial dilutions were prepared using sterile distilled water as the diluent. A 1 ml aliquot from each dilution was plated using the pour plate method, as described by Swanson et al (1992).

3. Bacterial Isolation: The samples were cultured on different media and incubated at varying temperatures. Pure cultures were identified using the standard methods outlined by Barrow and Feltham (1993). The identification process involved Gram staining, biochemical tests, pigment analysis, and colony morphology. The streak plate technique was employed to isolate various bacterial species, with plates incubated at 37°C for 24 to 48 hours. The bacterial isolation media included:

- MacConkey Agar Medium: MacConkey agar is a differential medium that distinguishes between lactose fermenters (such as Klebsiella and Escherichia coli) and non-lactose fermenters (like Pseudomonas aeruginosa, Salmonella species, and Proteus mirabilis) (Oxoid, 1992).

1. Salmonella-Shigella Agar Medium: This medium was used to isolate Salmonella and Shigella, with incubation carried out at 35°C for 24 to 48 hours (Feng et al., 2007).

2. Violet Red Bile Agar: This medium was utilized for identifying coliform bacteria, whereas Eosin-Methylene Blue (EMB) Agar was used to isolate Escherichia coli (Oxoid, 1992).

3. Mannitol Salt Agar: This selective and differential medium is used to isolate Staphylococcus aureus. It contains mannitol, which is fermented by Staphylococcus aureus, causing the medium to turn yellow (Finegold and Martin, 1982).

4. Staphylococcus Medium (No. 110): This medium was used to isolate Staphylococcus spp. and Micrococcus spp., both of which are Gram-positive bacteria (Matthews et al., 1997).

B-Fungi

Sabouraud Dextrose Agar (SDA) was prepared according to the manufacturer's guidelines by dissolving 64 grams of SDA in 1000 ml of distilled water. The solution was then sterilized in an autoclave at 15 psi and 121°C for 15 minutes. The media were supplemented with 100 mg/L of amoxicillin. After sterilization, the media were poured into disposable Petri dishes, incubated at 25±2°C, and stored at 4°C until needed (Nrior, 2017).

Isolation and Identification of microbial isolate from foods:-

1.Bacteria

Bacterial isolates were identified by examining their cultural morphology and biochemical properties. The Gram staining method was employed for classification, as described by Barrow and Feltham (1993), Inglis et al. (1994), and Bergey’s Manual (1989).

2.Fungi

Fungi were isolated from food samples by cutting pieces of each sample, measuring 0.5–1 cm, and placing them in Petri dishes with SDA containing 100 mg/L of amoxicillin. Each Petri dish was inoculated with five pieces of food and incubated at 25±2°C for 5 days (Dou et al., 2021).

The diagnosis of isolated Microbial was carried out as follow:

A- Morphological characteristics of the growth colonies, including color, texture, margin, colony reverse, and pigment production, were observed.

B- Microscopic examination was conducted to observe the shapes of fungi and bacteria, as well as spores, conidia, and mycelium using a light microscope.

Evaluation of the Frequency and Visibility of the Isolated fungi were determined using the following formula:

Figure 1.

Result and Discussion

3.1 Occurrences bacteria in foods

Table 1 displays the occurrence of bacteria in the food samples analyzed, which include beef burger meat, beef shawarma, beef pizza, chicken shawarma, chicken burger, chicken pizza, falafel, and salads (lettuce, cucumbers, tomatoes), based on microbiological examination.

3.2.Microorganisms isolated from sandwiches fast and traditional foods

All food samples, including those from street and fast food, were collected and subjected to microbial detection for the following:

3.1.1.Bacteria

Analysis of 50 food samples from 60 restaurants in Al-Najaf and Kufa identified 17 bacterial species from seven genera and ten fungal genera. These restaurants were selected due to their high exposure to pathogens. The samples revealed the presence of several bacteria, including Staphylococcus aureus, Shigella spp., Escherichia coli, Streptococcus spp., Salmonella spp., Bacillus subtilis, and Enterobacter. Staphylococcus aureus and E. coli were the most frequently found, with Salmonella being the second most common, particularly in falafel, beef shawarma, and salads (see Table 1).

The growing urban population has led to an increase in street food vendors, with about 2.5 billion people consuming street food globally. These foods, often prepared and sold in public places, typically lack adequate storage, refrigeration, and cooking facilities, which raises the risk of microbial contamination. Reports highlight the high levels of coliform and pathogenic bacteria in street-vended foods, contributing to various foodborne illnesses, including cholera, typhoid, salmonellosis, campylobacteriosis, shigellosis, amoebiasis, and Escherichia coli infections.

Figure 2.Growth of bacteria isolated from different foodstuffs in various culture media at 37 C° for 24-48 hours.

Figure 3.A) On MSA agar, Gram-positive cocci are observed in all the mentioned food samples. ( B;D) Differential agar displays Gram-positive cocci. ( C ) On MacConkey agar, Gram-negative bacilli are seen in samples of beef burger, falafel, and salads (lettuce, cucumbers, tomato).

fungi:-

Tables 2, 3, and 4 present the results of fungal contamination across various food samples. The ten isolated fungal species include Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Penicillium expansum, Penicillium spp., Rhizopus spp., Cladosporium spp., Alternaria alternata, and Fusarium spp.

All these fungi were found in falafel, with the exception of Cladosporium spp. Other notable contaminants in the food samples were Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, Fusarium spp., Alternaria spp., and Rhizopus spp.

Figure 4.

Figure 5.Fungal genus isolated from different foods in petri dishes (A)Asp.aculeatus ( B) Asp. terreus (C )P.expansium (D)Alteraria alternata (F P) Asp. flavus (J)Pencillium sp.(K)Cladosporium sp.(L) Asp. fumigates (N)Rhizopus sp. O( A. niger )PAsp.parasiticus)

Figure 6.Shows the shapes of the Mycelium and conidia of different fungi (A)micro; macroconidia of Cladosporium sp(B) Alteraria alternata (C) Penicillium sp.(D)Bipolaris sp.

The results showed that Aspergillus spp. was the most prevalent, with Penicillium spp. being particularly prominent, reaching visibility and frequency percentages of 80%, 75%, and 52.5% in falafel. Other genera had lower frequency and visibility percentages, ranging from 73%, 53%, 46%, 45%, 50%, 36%, 31.8%, and 31.3%. Cladosporium spp. had the lowest appearance percentages, at 6.6% and 26% in beef pizza. These findings are consistent with Alfattli (2010). Refer to Figures 1 and 2 for visual representation.

Figure 7.Percentages of the Visible(%) of Fungi Isolates from the Different Types foods

This study's key findings indicate that microbiological contamination in fast food samples is both extensive and significant. Contamination levels were higher in foods containing fresh vegetables and salads compared to those that are cooked, such as chicken shawarma and beef burgers. Certain bacteria can survive and grow at temperatures up to 50°C. Contamination sources include raw ingredients, polluted irrigation water, improper handling, and contaminated containers. Frequent consumption of high-fat, high-calorie fast foods can lead to liver damage and inflammation. To mitigate foodborne illnesses, it is crucial to manage pH levels, water activity, and temperature.

Research on street and fast foods has highlighted the health risks posed by pathogenic microorganisms, mainly due to inadequate hygiene during cooking and handling. This study detected pathogens like Staphylococcus aureus, Shigella spp., Escherichia coli, Streptococcus spp., Salmonella spp., and Bacillus subtilis in both fast and traditional foods.

Fungal contamination is widespread because fungi can produce numerous resilient spores that easily spread through the air and enter food storage areas. Fungi, especially Aspergillus species, thrive in a broad range of temperatures, from 5-45°C or higher (Moubasher et al., 1982; Hocking & Pitt, 1997).

Conclusion

This study reveals that fast foods sold near the BAU campus are often contaminated with common indicator bacteria. Many restaurant vendors lack proper training in food safety, leading to issues such as poor personal hygiene, improper food handling, extended storage at room temperature, and the sale of leftover food from previous days. To mitigate these risks, it is essential to provide food safety education and training for restaurant vendors. Additionally, consumers should be educated about hygiene issues related to fast foods and encouraged to insist on adherence to food safety standards.

Regular monitoring by health authorities, along with strict enforcement of hygiene regulations, is crucial for improving fast food practices. Preventing contamination requires a collaborative approach from all parties involved in food production. Essential practices include washing and drying hands before handling food, thoroughly cleaning food preparation areas and equipment, and maintaining overall kitchen hygiene.

References

1. M. R. Adams and M. O. Moss, "Food Microbiology," R. Soc. Chem. Sci. Park, Cambridge, 2000, p. 447.
2. A. A. Adesiyun, "Prevalence of Listeria spp. Campylobacter," 1993.
3. I. F. Angelillo, N. M. Viggiani, L. Rizzo, and A. Bianco, "Food Handlers and Foodborne Disease: Knowledge, Attitudes, and Reported Behavior in Italy," Journal of Food Protection, vol. 63, pp. 381-385, 2000.
4. R. M. Atlas, "Handbook of Microbiological Media," L. C. Purks, CRC Press, Boca Raton, Ann Arbor, London, Tokyo, 1993, pp. 666, 798, 196.
5. AVI Biopharma, "Antisense Antibacterial Method and Compound," World Intellectual Property Organization, 2008.
6. J. A. Barnett, R. W. Payne, and D. Yarrow, "Yeasts: Characteristics and Identification," 2nd ed., Cambridge University Press, Cambridge, United Kingdom, 2000.
7. G. I. Barrow and R. K. A. Feltham, "Cowan and Steel's Manual for the Identification of Medical Bacteria," 3rd ed., Cambridge University Press, Cambridge, 1993, p. 331.
8. M. Baumgart, B. Dogn, M. Rishniw, et al., "Culture-Independent Analysis of Ileal Mucosa Reveals a Selective Increase in Invasive Escherichia coli of Novel Phylogeny Relative to Depletion of Clostridiales in Crohn's Disease Involving the Ileum," ISME Journal, vol. 1, no. 5, pp. 403-418, 2007.
9. "Bergey's Manual of Determinative Bacteriology," Williams and Wilkins Co., Baltimore, London, 1989.
10. F. Bichai, P. Payment, and B. Barbeau, "Protection of Waterborne Pathogens by Higher Organisms in Drinking Water: A Review," Canadian Journal of Microbiology, vol. 54, no. 7, pp. 509-524, 2008.
11. M. Cheesbrough, "Microbiological Examination of Specimens and Biochemical Testing of Microorganisms," Medical Laboratory Manual of Tropical Countries, 1st ed., vol. 2, Tropical Health Technology, Butterworth Heinemann Ltd., Cambridge, 1984, pp. 26-39, 57-69.
12. L. Chen, et al., "Consumption of Dairy Products and Risk of Parkinson's Disease," American Journal of Epidemiology, vol. 165, no. 9, pp. 998-1006, 2007.
13. A. Clouditz, A. Resch, K. P. Weiland, A. Peschel, and F. Gotz, "Staphylococcus Plays a Role in the Fitness of Staphylococcus aureus and Its Ability to Cope with Oxidative Stress," Infection and Immunity, vol. 74, no. 8, pp. 4950-4953, 2006.
14. S. E. Cosgrove, G. A. Vigliani, M. Campion, et al., "Initial Low Dose Gentamicin for Staphylococcus aureus Bacteremia and Endocarditis Is Nephrotoxic," Clinical Infectious Diseases, vol. 48, no. 6, pp. 713-721, 2009.
15. Y. Dou, F. Francesca, M. Ziga, and M. Luigi, "A New European Land Systems Representation Accounting for Landscape Characteristics," 2021.
16. Vaishali Dharmarha, "A Focus on Listeria monocytogenes," National Agricultural Library, Food Safety Research Information Office, 2009.
17. G. A. Dykes and M. Dworaczek, "Influence of Interactions Between Temperature, Ferric Ammonium Citrate, and Glycine Betaine on the Growth of Listeria monocytogenes in a Defined Medium," Letters in Applied Microbiology, vol. 35, no. 6, pp. 538-542, 2002.
18. J. M. Farber and P. I. Peterkin, "Listeria monocytogenes: A Foodborne Pathogen," Microbiology Reviews, vol. 55, pp. 476-511, 2009.
19. P. Feng, S. Weagant, and M. Grant, "Enumeration of Escherichia coli and the Coliform Bacteria," Bacteriological Analytical Manual, 8th ed., FDA/Center for Food Safety and Applied Nutrition, 2007.
20. S. M. Finegold and W. J. Martin, "Enterobacteriaceae and Non-Fermentative Gram-Negative Bacilli," in Bailey and Scott's Diagnostic Microbiology, 6th ed., C. V. Mosby Company, St. Louis, Toronto, London, 1982, pp. 199-239, 249-265.
21. U. Fotadar, P. Zaveloff, and L. Terracio, "Growth of Salmonella at Elevated Temperatures," Journal of Basic Microbiology, vol. 45, no. 5, pp. 403-404, 2005.
22. M. J. Gray, R. N. Zadoks, E. D. Fortes, B. Dogan, S. Cai, Y. Chen, V. N. Scott, et al., "Listeria monocytogenes Isolates from Foods and Humans from Distinct but Overlapping Populations," Applied and Environmental Microbiology, vol. 70, pp. 5833-5841, 2004.
23. J. C. Heaton and K. Jones, "Microbial Contamination of Fruits and Vegetables and the Behavior of Enteropathogens in the Phyllosphere," The Applied Microbiology, vol. 104, no. 3, pp. 613-626, 2008.
24. V. Inglis, R. J. Roberts, and N. R. Bromage, "Bacterial Diseases of Fish Farming," Gbeni Sodipo Press Ltd., Lagos, 1994, p. 83.
25. C. I. Liu, G. Y. Liu, Y. Song, F. Yin, M. E. Hensler, W. Y. Jeng, V. Nizet, A. H. Wang, and E. Oldfield, "A Cholesterol Biosynthesis Inhibitor Blocks Staphylococcus aureus Virulence," Science, vol. 31, no. 5868, pp. 391-394, 2008.
26. Nrior, "Biodegradation Potential of Aspergillus Niger and Rhizopus Arrhizus Isolated from Crude Oil Spilled Site in Rivers State," 2017.
27. P. Washington, "Final Rule Issued by the FDA to Reduce Salmonella Contamination in Eggs," 2009.