Parasitological and Microbiological Investigation of Meat and Offal Consumed in Istanbul

In this study, it was aimed to explore the presence of some important foodborne parasitological and microbiological parameters that may seriously risk the consumers’ health (total coliform, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Escherichia coli EHEC O157:H7, Salmonella spp., Salmonella enteritidis, Salmonella typhimurium and Toxoplasma gondii) in meat and offals that are sold in various sales points in different districts of Istanbul. For this purpose, 400 samples (100 mutton, 100 beef, 100 minced meat and 100 sheep brain) were collected and analyzed by using PCR procedures for aforementioned parameters. The data obtained, were interpreted by statistical methods and binary correlations among the parameters will be exposed. Besides, a risk alligment was formed among the sales points and the products. According to the results, Escherichia coli EHEC O157:H7 and Toxoplasma gondii were determined in any samples. However except the aforementioned parameters all the analyzed microbiological variables were exposed in different samples with different concentrations. The results were veried by real-time PCR procedures. Binary correlation analysis were applied to the positive determined microbiological parameters. The results showed that all the positive determined microbiological parameters were positively correlated with each other. The result of the study showed that applynig good hygien procedures is very important to supply qualied and safe food to the costumers is very improtant in very step of food production chain but especially in the sales points that sell ready to eat foods. Besides, it was concluded that the implementation of effective control / inspections and continious education programs which would be held by related government agencies would be very effective for decreasing the incidence of the foodborne pathogens. another study, the riskiest nutrients for Salmonella spp. were lungs (lights) (7.5%) and mombar (2.5%); for S. enteritidis (5%) and S. typhimurium (2.5%) was lungs (Abd-El-Malek and El-Khateib, 2017). In the study conducted by Ras et al. (2019), the risk ranking for S. aureus was found for cattle-sheep kidney (30%), buffalo-camel kidney (20%); buffalo liver (30%), cattle-camel liver (% 20) and sheep liver (0%). For E. coli, the buffalo and the camel kidney and liver had the same risk (20%). Risk was not present in beef liver-kidney and sheep liver (0%) (Ras, 2019). Both our study and other studies show that in order to prevent the high-risk level observed in terms of mentioned parameters, it is necessary to follow the necessary rules, good production practices, sanitation standard operating procedures and food safety systems during cutting and processing of animals due to contamination of offal.


Introduction
The strong connection between food consumption and human diseases was de ned by Hippocrates in ancient times and foodborne pathogens were reported as biological agents causing foodborne diseases (Bintsis, 2017). According to the World Health Report, 1/10 of the people worldwide get sick due to food contamination and 1.8 million people die from foodborne diseases every year (Wei and Zhao, 2021). Consumption of animal foods such as meat, milk and eggs is increasing due to globalization, rapid population growth, changing dietary habits and lifestyle. Consequently; Mass production and global movement of products occur, increasing the risk of contamination of foodborne pathogens at any stage of the chain from farm to fork (Abebe et al., 2020). Meats; tools, knives, hands, clothes and air can be easily contaminated during cutting, transportation, processing, packaging and distribution and cause biological, chemical, physical and especially microbial food hazards (Bantawa In this study, meat and products (mutton, beef, minced meat and sheep brain) offered to consumers in different types of retail businesses in the Istanbul region were studied. Some important food-borne microbiological and parasitological parameters, which may be serious risk factors for consumer health, were investigated by real time PCR (rt-PCR) method. In our study, in addition to other studies on red meat and offal, sheep brain, which is a type of offal that is frequently consumed by individuals, was also examined.

Material And Method
Selection of samples: Within the scope of the study, 400 sheep meat, beef, minced meat and sheep brain (total 100 samples for each different variety) were collected from different types of sales points in Istanbul that sell directly to the public, and T. gondii, Salmonella spp., Salmonella enteritidis (S. enteritidis), Salmonella typhimurium (S. typhimurium), Listeria monocytogenes (L. monocytogenes), S. aureus, E. coli and E. coli were analyzed for EHEC O157:H7 parameters.

Microbiological Analysis
Total Coliforms: Firstly, dilution and homogenization procedures were applied to the samples collected using sterile containers and delivered to the laboratory in compliance with the asepsis conditions, and then the standard spreading method was used instead of VRB (Violet Red Bile Agar) agar medium prepared beforehand and poured into petri dishes. After seeding, a second layer of VRB agar was added to the petri dishes. Petri dishes were incubated for 24 hours at 37ºC and the typical colonies formed at the end of the incubation period were counted (Manual, 2011). S. aureus: Dilution and homogenization procedures were applied to the samples, which were collected using sterile containers and delivered to the laboratory in compliance with the asepsis conditions, and then the standard smear method was used instead of the BPA (Baird -Parker Agar) medium prepared beforehand and poured into petri dishes. Petri dishes were incubated for 24 hours at 37ºC. At the end of the incubation period, typical colonies were transferred to DNAse Agar and DNAse plates were again incubated at 37ºC for 24 hours. After the typical colonies were subjected to the coagulase test, the identi cation procedure was completed (Bennett and Lancette, 2001 E. coli EHEC O157:H7: Petri dishes TBX (Tryptone Bile X-glucuronide) were incubated at 44ºC for 24 hours and the typical colonies formed at the end of the incubation period were counted. For EHEC O157: H7, after homogenization, the samples were transferred to MTSB (Modi ed Triptic Soy Broth) and incubated at 37ºC for 24 hours. Afterwards, a loopful sample from each medium was transferred to SMAC Agar (Sorbitol Mac Conkey Agar) and incubated again at 37ºC for 24 hours (Manual, 2011). Salmonella spp./S.ytphimurium/S.enteritidis: Incubation and selective enrichment procedures were performed. For the samples incubated for 24 hours at 37ºC, strains showing red color at the top, yellow color and blackening at the bottom on TSI agar (Triple Sugar Iron Agar), strains that grew as a purplish color at the bottom and bottom on LI agar (Lysine Iron Agar), and strains that gave a negative reaction on urea broth Salmonella spp. was accepted as positive. The motility of the suspicious colonies on the agar in question at 37ºC for 24 hours showed that Salmonella spp. evaluated positively. Salmonella spp. black "rabbit eye"-like colonies with a metallic bright zone around it were con rmed as S. enteritidis on Bismuth Sulphite Agar for samples that were positive, and bluish-gray/dark gray colonies were con rmed as S. typhimurium on Hektoen Enteric Agar (FDA, 2014).

Dna Extraction
T. gondii: Homogenization of 50 grams of meat followed by DNA extraction was performed according to the kit protocol (Macherey-Nagel, Nucleospin®). In the study, two separate DNA preparation procedures were applied for microbiological parameters. One of them is bacterial lysis by heat treatment. The other method is lysis resulting from the treatment of a mixture of lysozyme, proteinase K and SDS with phenol/chloroform extraction (Gutierrez et al., 2010).

PCR
The presence of Salmonella spp., S.typhimurium, S. enteritidis, L. monocytogenes, S. aureus, E. coli and E. coli EHEC O157:H7 was sought in all samples collected as planned by the PCR procedure. For this purpose, the PCR procedure de ned for each microbiological parameter was applied to the isolates that were evaluated as suspicious/positive as a result of the cultivation  (Table 1).

Results
Applied microbiological sows showed the presence of Salmonella spp. and S. aureus in meat and meat products, and this nding was con rmed by PCR. Fig. 1 shows PCR products of the invA gene con rming the presence of Salmonella spp., while Fig. 2 shows PCR products of nuc, coa and spa genes that indicate the presence of S. aureus (Fig. 1,Fig. 2).
Of the mutton obtained from butcher's 22% was positive for coliform, 17% for S. aureus, 11% for E. coli, 7% for L. monocytogenes, 3% for Salmonella spp, and of the beef 25% was positive for coliform, 21% for S. aureus, 16% for E. coli, 5% for L. monocytogenes, 4% for Salmonella spp. No sample was positive for E. coli EHEC O157:H7, S. typhimurium, S. enteritidis and T. gondii (0%). Of the sheep's brain obtained from the butcher's 12% was positive for S. aureus, 8% for coliform, 2% for E. coli and Salmonella spp. No sample was positive for E. coli EHEC O157:H7, S. typhimurium, S. enteritidis, L. monocytogenes and T. gondii (0%). Of the minced meat obtained from restaurant/kebab house/mobile vendor 37% was positive for coliform, 28% for S. aureus, 23% for E. coli EHEC O157:H7, 17% for Salmonella spp., 12% for L. monocytogenes, and 4% for S. typhimurium and S. enteritidis. No sample was positive for E. coli EHEC O157:H7 and T. gondii (0%). The product with the highest risk in terms of all parameters was minced meat, while the product with the lowest risk was sheep's brain (Fig. 3).

Discussion
Meat consumption increases worldwide in parallel with increasing challenges in meat hygiene and safety (Iyer et al., 2013). Infections of foodborne Salmonella spp. are important worldwide and are recognized as the second most common foodborne pathogen in the European Union (Bonardi, 2017 (Evangelopoulou et al., 2015). In our study, Salmonella spp. and S. aureus, ranked rst among foodborne pathogens that threaten public health reportedly by World Health Organization (WHO) and the European Union, have shown to have positive correlation and to stimulate their development ( Table 2). Since E. coli EHEC O157:H7 and T. gondii were not detected in any example, these parameters are excluded from evaluation.
Numerals written with bold characters are statistically signi cant (p<0.005).
The signi cance is that it stimulates the reproduction of the other for each binary parameter. That is, the presence of one of both parameters, which is signi cant, positively affects the reproduction of the other.
In a study conducted by Zarei et al. (2013); in a total of 210 samples, each 500 g collected from retail outlets and popular supermarkets, the prevalence of Salmonella spp. was 7.1% in lamb (n=70), 4.3% in beef (n=70) and 2.8% in buffalo meat (n=70) (Zarei et al., 2013). In our study, prevalence of Salmonella spp. was 12% in minced meat obtained from restaurants/kebab houses/vendors, 4% in beef, 3% in mutton, and 2% in sheep brain obtained from butcher's. Another study examined 60 meat samples obtained from hypermarkets (n=20), groceries (n=20) and butchers (n=20); the prevalence of Salmonella spp. was determined to be 45% in butchers, 25% in grocery stores and 5% in hypermarkets (Iyer et al., 2013). In a study conducted on 189 beef and 190 chickens, 45% of chicken samples and 20.2% of beef samples were found to be positive for Salmonella spp., and one of the most isolated serotypes in unpackaged products was S. enteritidis (Soltan Dallal et al., 2014). In another study conducted by Ristori et al. (2017) on 552 chilled meat products (138 hot dogs, 138 raw pork sausage, 138 raw minced meat, 138 raw chicken legs); in terms of Salmonella spp., 62.5% of the positive specimens were pork sausage and 37.5% of chicken legs. In addition, while in terms of Salmonella spp., 12.5% of the positive samples belonged to S. enteritidis and 28.1% belonged to S. typhimurium serotypes, in our study 4% of minced meat was positive for S. typhimurium and S. enteritidis, but mutton, beef and sheep brains were not positive in terms of mentioned serotypes (Ristori et al., 2017). In another study, Salmonella spp. was isolated in 38.06% of 155 poultry offal (Sanda Abdelkader et al., 2019). In a study of pig offal products, 21.8% of 370 samples were positive for Salmonella spp.; intestinal (20%), brain (21%), liver, heart (73%) and kidney (87%) samples were determined to be suitable for human consumption (Erickson et al., 2019). This shows that, with the appropriate use of adequate heat treatment of meat products, cooling, freezing processes, application of correct cooking and packaging techniques, outbreaks of these pathogens can be prevented as a result of the absence of cross-contamination of meat products with other products ready for consumption.
In our study, it was determined that the highest risk food in terms of all microbiological parameters was minced meat. The foods that are risky after minced meat for Coliform, E. coli EHEC 0157:H7, S. aureus and Salmonella spp. were beef, mutton and sheep brain, respectively. Salmonella spp. and E. coli are the main factors that can lead to infections with food poisoning. In a study conducted by Zafar et al. (2016); 30 pieces of minced beef, mutton and chicken meat were examined and the highest microbiological load in terms of Salmonella spp. was seen in minced beef (6.57-7.39), followed by chicken and mutton, respectively (Zafar et al., 2016). In another study, 8.34% of the meat collected from the market, 11.86% of mutton, 12.59% of pork and 13.53% of chicken meat were contaminated with Salmonella spp., in which the greatest risk was found to be in chicken meat (Tadesse and Gebremedhin, 2015). In the study conducted by Martínez-Chávez et al. (2015); risk factor for Salmonella spp. pathogen was 71% (±11) for minced meat, 39% (±10) for beef pieces and 18% (±6) for beef carcasses, whereas for E. coli it was found to be 100% for minced meat, 97% (±27) for beef carcasses and 84% (±8) for beef pieces. This is due to the fact that during the processing of raw meat in butchers, the microbial load on meat is transferred to minced meat and to beef pieces, and also , it was reported that there were risks in terms of E. coli (43.75%), S. aureus (37.5%) and Salmonella spp.; and also, meat was not separated from offal, sold openly for 5 hours, and butchers were not hygienic (Zerabruk et al., 2019). In another study, the riskiest nutrients for Salmonella spp. were lungs (lights) (7.5%) and mombar (2.5%); for S. enteritidis (5%) and S. typhimurium (2.5%) was lungs (Abd-El-Malek and El-Khateib, 2017). In the study conducted by Ras et al. (2019), the risk ranking for S. aureus was found for cattle-sheep kidney (30%), buffalo-camel kidney (20%); buffalo liver (30%), cattle-camel liver (% 20) and sheep liver (0%). For E. coli, the buffalo and the camel kidney and liver had the same risk (20%). Risk was not present in beef liver-kidney and sheep liver (0%) (Ras, 2019). Both our study and other studies show that in order to prevent the high-risk level observed in terms of mentioned parameters, it is necessary to follow the necessary rules, good production practices, sanitation standard operating procedures and food safety systems during cutting and processing of animals due to contamination of offal.
Although there is not enough data, undercooked and raw meat is considered to be the most important cause of T. gondii infections that are effective in muscle and nerve tissues in humans (Sroka et al., 2019). This parasite leads to clinical manifestations such as encephalitis, hepatitis, pneumonia, myalgia and myocarditis in immunosuppressed individuals (Ducrocq et al., 2021). In the study of raw and smoked sausage, ham, dried bacon and minced meat, 5.4% of the samples were positive for T. gondii. 45.1% of the positive samples were sausages, 27.4% were smoked meat products, 19.4% were minced meat and 8% were ham (Sroka et al., 2019). In a study conducted in Turkey, T. gondii was present in 20% of the ovine muscle, 19% of fermented sausage, 6% of the bovine muscle, 4.17% of the ovine brain and 2% of the bovine brain. With regard to this, it can be said that the risk of toxoplasmosis is high in uncooked/commercial meat and products (Ergin et al., 2009). In our study, on the contrary, no nutrient was positive for T. gondii. Based on these ndings, it can be said that further study is needed to determine risk factors in T. gondii infections (Hussain et al., 2017).
L. monocytogenes is a major pathogen known worldwide as the causative agent of listeriosis and contaminates with food. This pathogen is a bacterium that causes meningoencephalitis, cerebral abscesses, cerebritis, bacteremia, meningitis and sepsis, especially in immunosuppressed individuals and pregnant women, and has a high mortality rate (20 -30%) (Montero et al., 2015).
In a study conducted by Bouymajane et al. (2021) in Morocco, 520 food samples were examined. It was found that 15 (2.9%) of the analyzed samples were contaminated with L. monocytogenes. It was observed in 5.7% of raw minced meat and raw sausage, while was found in 1.9% of raw beef, poultry and raw sh samples. Additionally, it was observed that all strains detected carried the actA gene (Bouymajane et al., 2021). In another study, the prevalence of L. monocytogenes in consumption-ready products was examined and 783 delicatessen products were analyzed. The positive ratio of the products sold with vacuum packaging was 2.7%, while it was reported to be 8.5% in delicatessen meat products packaged in the store (Garrido et al., 2009 slaughterhouses and 112 swab samples from 5 unregistered animal slaughter points. Swabs were made before and after washing the carcasses. The total number of coliform bacteria in slaughterhouses varies between 5.0-6.3 kob/cm 2 before washing and 4.6-6.3 kob/cm 2 after washing. To ensure that coliform bacteria, and especially E. coli, the most prominent hygiene indicator, is not a threat to public health and access to reliable food, it is clear that it is necessary to provdie the hygiene conditions of production, equipment and personnel.
Our study found that 22% of mutton meat, 25% of beef and 8% of sheep brain samples were contaminated with coliform bacteria.
In E. coli analysis, the results obtained were as follows: 11% of mutton, 16% of beef and 2% of sheep's brain were contaminated with E. coli. As in the study of Guran et al. (2017), there was no E. coli O157:H7 in any sample. As highlighted in other studies, these ndings obtained in our study suggest that personnel, equipment, and operational hygiene and critical control points within the enterprise should be provided.

Conclusions
As a result, in our study, as well as other studies on red meat and offal, the sheep's brain, which is often consumed by individuals, has been studied. When the necessary conditions are not met, it seems that the sheep's brain is potentially at risk of microbiological and parasitological contamination and can therefore threaten public health. Even though signi cant progress has been made in recent years on food safety and public health, more steps must be taken. A multidisciplinary team, including public health experts, veterinarians and nutritionists, should focus on food hygiene to reduce the number of cases and deaths caused by nutritional pathogens in humans worldwide and this multidisciplinary team should ground on disease surveillance, consumer education, processing and marketing practices.

Declarations
Availability of data and materials Not applicable. All data sets from which conclusions of the manuscript have been drawn are presented in the paper.