Introduction
Meat serves as an excellent source of protein in human diets; however, due to its biochemical components , it is particularly vulnerable to microbial contamination. This contamination often results in significant spoilage and foodborne illnesses. Pathogens associated with meat can be readily transferred from the animal’s gastrointestinal tract, the surrounding environment, and the hands of those handling the meat, particularly in unsanitary conditions. Recent research has shown that consumers are increasingly seeking healthier and more nutritious meat options (Papatsiros et al., 2020).
Bacterial pathogens present in meat include Bacillus cereus, Clostridium botulinum, Clostridium perfringens, Salmonella, Escherichia coli, and Staphylococcus aureus. Contamination of meat can arise from unsanitary slaughtering, handling, and processing practices, the hands of operators, unclean abattoirs, or from the natural micro-flora found in the tissues of animals, as well as from the surrounding air and environment. Various microbes are introduced at different stages of meat processing following slaughter, leading to contamination of the meat. The existence of pathogenic microbes negatively impacts the hygienic quality of meat. Therefore, the microbiological quality of meat and its products can be assessed based on their hygienic standards. Additionally, microbial contamination of food can result from improper food handling. Consumers also play a role in the chain of food-borne bacterial illnesses through inadequate storage and cooking of meat and meat products. Pathogens such as B. cereus, C. jejuni, E. coli, L. monocytogenes, S. aureus, and Y. enterocolitica are recognized for causing food-borne infections and intoxications in humans (Pal Mahendra et al., 2018).
While muscles in healthy birds are sterile, various microbiotas inhabit the digestive tract, lungs, skin, feathers, and other areas. In slaughterhouses, bacteria are found on surfaces, in the air (aerosols), and in liquids. Consequently, carcasses and cuts can become contaminated by microbiota from both the animals and the slaughterhouse environment after the animals are killed. Although there are differences between large-scale commercial slaughterhouses and small-scale facilities, the primary steps in poultry slaughtering remain consistent. When comparing the slaughtering of poultry to that of mammals, notable differences include (i) the use of a water bath (either hot, or chilled) at various stages; (ii) the feather removal process, which is mechanical and differs from the skin removal of mammals; and (iii) the smaller size of birds (in contrast to cattle , or sheep), which affects the ease of carcass handling and the mechanization of certain processes. Throughout these steps, bacterial contamination can arise from equipment surfaces, water, and animal microbiota. Airborne bacteria and environmental contaminants can also affect broiler meat. The skin of poultry carcasses and cuts is in direct contact with air and equipment surfaces, making it susceptible to contamination. In fresh meat, bacteria are typically found on the surface rather than within the meat itself. However, in processed products, such as marinated items, bacteria can penetrate into the muscles. (Rajendran et al., 2017).The aim of study was isolation and identification of bacteria contaminating local and imported red meat.
Material and Methods
Collection of Samples:
The study included 20 samples of red meat imported meats) Jordanian , Saudi , Indian and Turki) and Local (cane shops) collected from 8 Different areas in Alanbar province are (Haditha, Altaamim, Aliskan, 5-kilo , Hay Aladel, 20th Street, Almalaab and 17th Street(, 100 grams of meat were taken according to sterile equipment and placed in sterilized tubes that were sealed on 20/12/2020, and they were transferred to the refrigerator at 4 C, then work began on the next day in the laboratories of the almaarif university college on 21/12/2020.Results and Discussion
Table 1: Origin of imported meats
| Number of sample | Origin |
| 2 | Jordanian |
| 2 | Saudi |
| 2 | Indian |
| 2 | Turky |
Table 2: Origin of local meats
| Number of sample | Origin |
| 2 | Alsofiya |
| 2 | Altaamim |
| 1 | Aliskan |
| 1 | 5-kilo |
| 1 | Hay Aladel |
| 1 | 20th Street |
| 2 | Almalaab |
| 2 | 17th Street |
Cultivation of samples:
Do the implant according to the method (Ranjan, K.D.,2007). As follows:
- The samples were tested directly in the laboratory but this could not be kept in the refrigerator to complete the work on the second day.
- The meat samples were chopped with the therm machine twice and mixed samples until they were mixed.
- Add 1 g of meat sample to 10 ml of Physiological salt solution mixed with blender at 2000 cycles Per minute for 3 minutes.
- Pull one ml by pipette of the first dilution It was added to the tube, which contains 9 ml of solution. Physiological mitigation to get the desired mitigation.
- Transfer 1 ml from the sample and at the conditions Sterile to a petri dish and repeating for each dilution. the dilution tubes 1, 2, 3 , 4,6, 7, 8, 9 and 10 were neglected and the dilution tube 5 was implanted from each sample as the average dilution
- Add 1ml from tube 5 to the medium in sterile conditions and mix by moving the dish to front, back, right and north circularly, then leave up Hardening the medium.
- incubated the dishes upside down in the incubator at a degree of 37 m For 24 hours.
Subculture of samples:
Secondary transplant samples were implanted to nutrient agar blood agar, MacConkey agar and SS agar to differentiate between positive and negative gram bacteria, for identification of bacterial isolates.
Identification of bacterial isolates
Manual methods:
Cultural Characteristics:
The nature of the growth of isolation on the solid nourishing medium was determined after a 24-hour incubation on Temperature of 30 m which includes observing the following qualities of bacterial colonies (textures and color viscosity, shape and edge).
Microscopic Characteristics:
The shape of the cells, the way they combine and respond to a gram dye have been identified by examining isolation Installed on the glass slide with a optical microscope.
Biochemical Tests:
Chemical tests were conducted, which included: Citrate utilization, Iodole test, VP (Voges Proskauer) ,MR (Methyl red)
VITEK2 Compact:
Use VITEK 2 Compact to confirm the diagnosis of elected bacterial isolation In the current study after the conduct of preliminary chemical tests, where the device consists of a holder Cassette and Reagent cards container 64 holes each represent the base material or In the middle of the test, plastic pipes as well as densiChek device and input and output unit Information.
Determination of thermal death degree:
1-Add 5 ml of distilled water to sterile glass tubes
2- Using a loop, we took from the culture medium that contains the bacteria and mixed them in the glass tube
3- Each sample was divided into three tubes, that was done in a sterile condition, according to sterile tools
4- The temperature was fixed at 100C and the time was changed to three times (10,15and20) minutes
5- The glass tubes containing the bacteria were placed in the water bath in three batches according to the time
6- After the expiration of the time for each batch, the bacteria were implanted from the glass tubes in nutrient agar media to detect heat-resistant bacteria and bacteria that did not resist heat and thus died.
Results and Discussion
Sample collection:
The meat samples were grown on the nutrient medium by the dilution method and the dilution tubes 1, 2, 3 and 4 were neglected due to the increased density of the sample, as well as the neglect of the dilution tubes 6, 7, 8, 9and 10 due to the low density of the sample and the dilution tube 5 was implanted From each sample as the average dilution and the growth of the sample is moderate, after cultivating it on the nutrient medium, bacterial colonies appeared to us as shown in the Figure (1)
Figure 1: Bacteria sample in nutrient agar
As shown above in figure (1) The meat sample is found to be contaminated with bacteria due to local meat sources. This contamination can arise from water or equipment surfaces, including knives or the hands of butchers, as well as from animal microbiota. Additionally, bacteria present in the air and surrounding environment can also lead to meat contamination. The skin of meat carcasses and cuts comes into direct contact with air and equipment surfaces, making it particularly susceptible to contamination. In the case of imported meat, the contamination observed in the analyzed sample is attributed to the meat’s origin and the health protocols implemented during production, which adhere to international health standards. However, inadequate transportation, storage, and manual handling practices, along with prolonged storage conditions that deviate from health regulations, are significant contributors to contamination. Furthermore, the methods of slaughter, whether conducted in accordance with Islamic law or not, also play a role in the overall contamination risk(Morshdy et al.,2025).
Subculture of samples:
Secondary transplant samples were implanted to nutrient agar, blood agar and MacConkey agar, to obtain pure and single colonies, the result is the samples grown in all the dishes (nutrient, blood and MacConkey agar) , so all bacteria are gram-negative due to their growth on all MacConkey agar because the MacConkey agar is selective and differential medium , selective for gram stain it allowed growth only for gram-negative bacteria and differentiate the gram-negative organisms based on their lactose metabolism, as shown in figure (2):
Figure2:Bacteria on Blood and Macconkey agar
Identification of bacterial samples
Biochemical examination
Biochemical examinations were conducted on the bacterial isolates, which are represented by the IMVC and gram stain as shown in the table (3).
Table 3: Biochemical examinations
| Number of Sample | Indole | MR(Methyl Red) | Vp(voges-proskauer) | Citrate | Gram stain |
| 1 | _ | _ | + | + | _ |
| 2 | _ | _ | + | + | _ |
| 3 | _ | + | _ | + | _ |
| 4 | + | + | _ | _ | _ |
| 5 | + | _ | _ | + | _ |
| 6 | + | + | + | + | _ |
| 7 | _ | + | + | + | _ |
| 8 | _ | + | + | + | _ |
| 9 | _ | _ | _ | + | _ |
| 10 | + | _ | _ | + | _ |
| 11 | _ | + | + | + | _ |
| 12 | + | _ | _ | + | _ |
| 13 | + | + | _ | _ | _ |
| 14 | _ | _ | + | + | _ |
| 15 | _ | _ | _ | + | _ |
| 16 | + | _ | _ | + | _ |
| 17 | _ | + | + | + | _ |
| 18 | _ | _ | _ | + | _ |
| 19 | _ | _ | + | + | _ |
| 20 | _ | + | _ | + | _ |
The data presented in table (3) revealed a mix of samples that were negative for citrate and some that were positive, while the other IMVIK tests displayed a variation in results, in contrast to the Gram stain. All samples were confirmed to be gram-negative. The positive citrate result indicates the sample’s capability to utilize citrate as an energy source. During the metabolism of citrate, ammonium salts are converted to ammonia, leading to an increase in alkalinity. This pH shift causes the bromthymol blue indicator in the medium to change from green to blue when the pH exceeds 7.6. Conversely, the negative citrate result indicates the sample’s inability to utilize citrate for energy, as illustrated in figure (3). In the indole test, the sample yielded a positive result due to its ability to break down the amino acid tryptophan to produce indole. In contrast, negative results indicate the sample’s lack of ability to split tryptophan to form indole, as depicted in figure (4). The positive and negative outcomes in the VP (Voges-Proskauer) test depend on whether the bacteria can generate acetylmethyl carbinol from glucose fermentation, as shown in figure (5). Meanwhile, the (MR) methyl red test results are based on the organism’s ability to produce and sustain stable acid end products from glucose fermentation, as illustrated in figure (6).
Figure 3: Result of Citrate Utilization Test positive results with blue color and negative results with green color
Figure 4: Indole Test; positive results with red color and negative results with no color
Figure 5: Voges–Proskauer (VP) Test; positive results with pink-red color and negative results with yellow color
Figure 6: Methyl Red (MR) Test; positive results with bright red color and negative results with yellow color
SS (salmonella, shigella) agar:
Samples of bacteria were transplanted from (blood and MacConkey agar) dishes into the SS agar, which is the selective and differential medium for Salmonella and shigella, to see if they contained Salmonella or Shigella, the results showed no growth for all samples except sample number 7 and 20 showed growth for Salmonella.
Figure 7:Salmonella on SS agar
Figure 8:Shigella on SS agar
As shown above sample (3,7and 20) salmonella grow in SS agar medium because it was appear as colorless, transplant, with a black center,as shown in figure(7), and the sample(4 and 13) shigella grow in SS agar it was appear as clear, colorless, transparent. as shown in figure (8).
VITEK2 system
After the biochemical tests and for more specific identification we used the VITEK2from the (blood and MacConkey agar) dishes , this device use system uses a fluorogenic methodology for organism identification and a turbidimetric method for susceptibility testing using a 64 well card, it is considered a modern and accurate technique for identifying bacteria, the result of VITEK2 diagnosed bacteria at probability with 96% in local meat samples in table (4), and the imported meat in table (5) below:
Table 4: Results of local meat samples
| Sample | Replicate |
| Aeromonas sobria | 2 |
| Raoultella ornithinolytioa | 2 |
| Pseudomonas aeroginosa | 3 |
Table 5: Results of imported meat samples
| Samples | Replicate |
| Pantoia spp | 1 |
| Pantoia agglomerans | 3 |
| Aeromonas hydrophila | 1 |
| Serratia liquefaciens | 2 |
| Aeromonas caviae | 1 |
Bacterial contamination in slaughterhouses can arise from various mechanisms and multiple points of vulnerability(Guergueb et al.,2014;Rouger et al.,2017) . During slaughter operations, inter contamination phenomena may result in the proliferation of bacterial pathogens on initially healthy carcasses. Possible sources of contamination include the alimentary tracts of the birds, water, packaging, utensils, and handlers )Guergueb et al.,2014).The slaughterhouse environment itself, encompassing surfaces and air, can also harbor bacteria that may contaminate carcasses and cuts. Moreover, equipment surfaces and the air within the facility are significant sources of contamination, along with animal microbiota (Rouger et al.,2017). Vulnerabilities in the slaughter process can occur at several stages. During defeathering, rubber fingers used to remove feathers can harbor bacteria, even when new, contributing to contamination. Conveyor belts can also transmit bacteria between carcasses. While scalding aims to reduce bacterial presence, it can inadvertently facilitate contamination as the high water temperature causes feather follicles to open and relax the skin, allowing bacteria to transfer from feathers to the skin. The evisceration process is another critical point, as the intestinal tract may be a source of contamination. Puncturing the viscera or leaving behind feces can result in cross-contamination of other carcasses. Additionally, workers’ hands and evisceration tools can further disseminate bacteria throughout the process (Guergueb et al.,2014;Moraret al.,2008;Zhao et al.,2020 ).
Detection Thermal Death Degree
The thermal death degree process is an important process to know the temperatures at which the types of bacteria die and the degrees of their resistance, this process depends on time and temperature, in our experience we fixed the temperature and changed the time to 3 times (10, 15 and 20) and the results were as shown in the table (6).
Table 6:Results of thermal death degree
| Bacterial isolate | Temperature at | Time (min) | Status of bacteria |
| 10 | Died | ||
| Pantoia spp | 100 | 15 | Died |
| 20 | Died | ||
| 10 | Died | ||
| Pantoia agglomerans | 100 | 15 | Died |
| 20 | Died | ||
| 10 | Died | ||
| Salmonellae | 100 | 15 | Died |
| 20 | Died | ||
| 10 | Died | ||
| Shigella | 100 | 15 | Died |
| 20 | Died | ||
| 10 | Died | ||
| Aeromonas sobria | 100 | 15 | Died |
| 20 | Died | ||
| 10 | Died | ||
| Aeromonas hydrophila | 100 | 15 | Died |
| 20 | Died | ||
| 10 | Died | ||
| Serratia liquefaciens | 100 | 15 | Died |
| 20 | Died | ||
| 10 | Died | ||
| Raoultella ornithinolytioa | 100 | 15 | Died |
| 20 | Died | ||
| 10 | Died | ||
| Pseudomonas aeroginosa | 100 | 15 | Died |
| 20 | Died |
As shown above all the bacterial samples are dead in 100 C in the three times (10,15 and 20)minutes, these bacterial sample cannot resist the 100C degree , most of these bacteria dead in range (40 to 60C) degree but we do this experience. Some strains of these bacteria maybe resist 100C, Even at temperatures (40-60) cause danger, because some people did not cook food properly and correctly and it is half cooked. Also, it cause danger, because some pets raised at home or stray animals eat raw meat and cause diseases such as typhoid fever, bacteremia, septicemia, and respiratory infection in humans.
Although numerous bacteria are eliminated at temperatures between 40-60°C (104-140°F), certain strains can endure higher heat levels. Additionally, vulnerable bacteria can present a threat if food is not adequately cooked. Food that is not cooked properly, even when it falls within the “danger zone” (40-140°F), may still contain harmful bacteria. Moreover, animals that eat raw meat can spread diseases to humans, underscoring the necessity of proper food handling and preparation( Ramirez-Lopez,2009;Hayes,2013 ).
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Acknowledgment
Non .
Conflicts Interest
Non.
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