Bacteriological Evaluation of some meat products from different markets in El-Behera Governorate

Bacteriological Evaluation of some meat products from different markets in

El-Behera Governorate

Saber Ali Saad ¹, Eman Mahrous², Mohamed Abdelgawad Hassan ³ and  

Amal Mohamed El-Sayed⁴

¹ Damanhur branch, Animal Health Research Institute, ARC, Egypt;

²Bacteriology Department, Animal Health Research Institute, Agriculture Research   Center (ARC), Egypt

³ Damanhur branch, Animal Health Research Institute, ARC, Egypt

⁴Department of Food Hygiene and Control, Faculty of Veterinary Medicine, Aswan University, Egypt

Abstract

The study assessed the bacterial quality of some meat products; minced meat, Luncheon, and Sausage. One hundred and Fifty random samples of meat products including 50 samples each of minced meat, luncheon and sausage were randomly collected from different market at El Behera Governorate for bacteriological evaluation; Aerobic plate count, S. aureus, E. coli, Salmonella and Clostridium perfringens. In addition, detection of virulence genes in some isolated strains.  The obtained results revealed that prevalence of S. aureus, E. coli, Salmonella and C. perfringens was (40, 24, 20 and 30%), (1, 2, 0, 2), (28, 10, 18 and 36%) in the examined minced meat, luncheon and sausage, respectively. Most isolated salmonella were belonged to S. Typhimurium. Serogrouping of the isolated E. coli revealed O₁₁₁, O₂₆, O₁₄₆, O₁₂₆, O₁₁₄, O₁₅₈ and untyped strains. Some isolates were tested for virulence genes and (Pvl and Sea) and (invA and Stn) were detected in all tested S. aureus and Salmonella strains, respectively. eaeA was detected in all tested E. coli strains while Stx2 was detected in only one strain. Enterotoxin gene (cpe) and alpha-toxin (cpa) were in all tested C. perfringens isolates. The presence of S. aureus, E. coli, Salmonella, and C. perfringens indicating unhygienic measures during processing and handling of meat products. Therefore, strict application of hygienic practices should be adopted to improve the quality of such meat products.

Key words: C. perfringens , E. coli , Salmonella, S. aureus, Serovars and virulence genes

INTRODUCTION

Foodborne diseases (FBD) represent global public health issues resulting in considerable morbidity and mortality in all age groups (He et al., 2023). The most frequent bacterial causes of foodborne diseases worldwide are Salmonella, vibrio parhaemolytica, listeria monocytogenes, S. aureus and some other pathogens (Gallo et al. 2020).  Meat and meat products are an important source for protein, basic amino acids, vitamins, fats, minerals and other nutritive components for human (Abuelnaga et al., 2021). In contrast, it can serve as an ideal media for the growth of different organisms due to unhygienic practices during meat processing (Alahakoon et al., 2015)

1. coli is one of the major normal intestinal inhabitants in human and mammals; it is harmless to the host and can cause diseases only in the immune-compromised host or when it breaches the gastrointestinal barriers (Ibrahim et al., 2018).

Meanwhile, some E. coli strains can acquire specific viru­lence and represent primary pathogens with enhanced potential to cause many disease conditions (Morshdy et al., 2018).

 All over the world, Staphylococcal food poisoning is one of the most common food-borne diseases; resulting from ingestion of preformed staphylococcal enterotoxins in food by enterotoxigenic strains of S. aureus (Lika et al., 2021).

Salmonella is a vital microorganism responsible for FBDs mainly found in raw food of animal origin (Sun et al., 2021).  Salmonella annually causes 200 million to over 1 billion infections worldwide with 93 million cases of gastroenteritis and 155.000 deaths (Chlebicz et al., 2018). 

1. perfringensbacteria are one of the most common causes of food poisoning. CDC estimates that the bacterium causes nearly 1 million foodborne illnesses in the United States every year. C. perfringens makes spores, which are inactive forms of the bacterium that help it to survive heat, dryness, and other environmental conditions. C. perfringensspores can transform into active bacteria, which multiply in the food then produce a toxin (poison) that causes diarrhea (CDC, 2022). Therefore, the present study aimed to assess the bacterial contamination of some meat products such as Luncheon, minced meat and Sausage with further molecular analysis of isolates' virulence genes. In addition, evaluation of the suitability of these products for human consumption.

2-MATERIAL AND METHODS

2.1. Collection of Samples

        A total of One Hundred and Fifty random meat product samples including minced meat, luncheon and sausage (50 samples of each) were collected from different supermarkets at Damanhur city, each sample was collected in a separate sterile plastic bag and transferred in an ice box for bacteriological examination.

2.2. Bacteriological Examination of Meat Products

2.2.1. Sample preparation

        The samples preparation was done according to APHA (2001). Twenty Five grams of each sample were aseptically transferred into sterile blender flask containing 225 ml of sterile peptone water 1% and homogenized at 14000 rpm for 2.5 minutes.

2.2.2. Aerobic Plate Count

        Aerobic Plate Count (APC) was determined using the standard plate count following (ISO 4833-1, 2013).

2.2.3. Staphylococcus aureus count

     Using Baird-Parker Agar Plates according to Quinn et al. (2011). Suspected colonies were picked up for further morphological and biochemical identification according to (FDA, 2001).

2.3.4. Detection of C. perfringens

        Detection of C. perfringens was done according to (ISO 7937, 2004) using Tryptose Sulfite Cycloserine (TSC) media at 37 ± 1 ◦C under anaerobic conditions for 20 ± 2h.

2.3.3. Isolation and identification of E. coli according to Quinn et al. (2011)

      A loopful from enriched broth was streaked onto MacConkey's agar and Eosin Methylene Blue agar (EMB) and incubated at 37 ºC for 24h. The metallic green colonies were picked up and identified biochemically.

2.3.4. Serological identification of E. coli isolates according to Quinn et al. (2011)  

     The somatic (O) antigen of E. coli was detected by slide agglutination test as well as Flagellar (H) antigen was serotyped. Anti-O-sera were purchased from DENKA SEIKEN CO LTD Tokyo, Japan.

2.3.5. Isolation and identification of Salmonella according (ISO, 2002).  

      Salmonella Shigella agar and XLD agar plates were used. Suspected colonies were identified morphologically by Gram-stain and biochemically. All salmonella isolates were subjected to serological typing by slide agglutination test using standard polyvalent and monovalent salmonella antisera (Seiken, Japan).

Detection of virulence genes in isolated strains by PCR

DNA extraction: DNA extraction from three isolates from Staph. aureus, Salmonella species and E. coli and two isolates of C. perfringens was performed using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) with modifications from the manufacturer’s recommendations. Briefly, 200 µl of the sample suspension was incubated with 10 µl of proteinase K and 200 µl of lysis buffer at 56 ºC for 10 min. After incubation, 200 µl of 100% ethanol was added to the lysate. The sample was then washed and centrifuged following the manufacturer’s recommendations. Nucleic acid was eluted with 100 µl of elution buffer provided in the kit.

Oligonucleotide Primer: Primers used were supplied from Metabion (Germany) are listed in table (A).

PCR amplification: For PCR, primers were utilized in a 25-µl reaction containing 12.5 µl of Emerald Amp Max PCR Master Mix (Takara, Japan), 1 µl of each primer of 20 pmol concentration, 4.5 µl of water, and 6 µl of DNA template. The reaction was performed in an applied biosystem 2720 thermal cycler.

Analysis of the PCR Products: The products of PCR were separated by electrophoresis on 1.5% agarose gel (AppliChem, Germany, GmbH) in 1x TBE buffer at room temperature using gradients of 5V/cm. For gel analysis, 20 µl of the PCR products were loaded in each gel slot. Gene ruler 100 bp ladder (Fermentas, Thermo Scientific, Germany) was used to determine the fragment sizes. The gel was photographed by a gel documentation system (Alpha Innotech, Biometra) and the data was analyzed through computer software.

• Statistical analysis

        Data were expressed as mean ± SEM using Statistical data analysis and carried out using SPSS 17.0 for windows (SPSS Inc, Chicago, IL, USA) according to Feldman (2003).

3. Results

3.1. Aerobic Plate Count

        Aerobic mesophilic bacteria were detected in all examined meat products samples with mean values of 1.70×10⁶±0.13×10⁶, 4.38×10⁴±0.38×10⁴ and 7.90×10⁵±0.15×10⁵ Cfu/g, in examined minced meat, luncheon and sausage; respectively (Table 1).

3.2. Staphylococcus aureus determination

3. aureus was detected at level of 40, 24, 20 and 30% in examined minced meat, luncheon and sausage with mean values of 3.15×10⁴±0.47×10⁴, 5.20×10²±0.41×10² and 1.85×10³±0.32×10³ Cfu/g; respectively (Table 2).

3.3. Isolation and identification of E. coli:

        The prevalence of E. coli in examined minced meat, luncheon and sausage were 36, 8 and 30%; respectively (Table 3)

3.5. Isolation and identification of Salmonella:

        The prevalence of Salmonella in minced meat, and sausage were 1 and 3 respectively. S. Typhimurium was the prevalent serotype (Table 4).

3.6. Detection of C. perfringens

2. perfringens were detected in examined minced meat, luncheon and sausage at a percentage of 28, 18 and 36 with mean values of 2.6×10³±0.24×10³, 1.75×10²±0.24×10² and 2.59×10³±0.24×10³ Cfu/g; respectively (Table 5).

3.7. Serological identification of E. coli strains.

Isolated E. coli strains were serogrouped into, O₁₁₁, O₂₆, O₁₄₆, O₁₂₆, O₁₁₄, O₁₅₈ and untyped strains (Table 6).

3.7.1. Detection of virulence genes in tested by PCR

        All tested S. aureus strains harbored pvl gene at 433bp gene and sea at 102bp by PCR (Fig 1). All tested E. coli Strain harbored eaeA gene (248bp) detected tested E. while Stx2 gene (779bp) detected only in one isolated E. coli strain (Fig 2). stn gene and invA were detected at 617 and 284bp in three Salmonella isolates (Fig 3). All tested C. perfringens strains possess enterotoxin gene (cpe) at 247bp and alpha toxin (cpa) at 402bp by PCR (Fig 4).

4. DISCUSSION:

Meat is favored by millions of people worldwide as a main source of animal protein. In the contrast, it is a suitable medium for the growth of different microorganisms, so it acts as a hygienic risk problem to the consumer (Elsayed et al., 2018). Meat and meat products are considered a major cause of foodborne pathogens human (Abuelnaga et al., 2021). Unhygienic practices of manufacture, storage and handling from production sites to the consumers. Therefore, it is crucial to use microbiological criteria to ensure the quality of those products (Abuzaid et al., 2020).

The obtained result in table (2) were higher than Hassanien (et al. 2018) who detected aerobic bacterial count in examined sausage, minced meat and luncheon as 3.3x10⁵, 3.3x10⁵ and 3.3x10⁵ Cfu/g; respectively. In addition, Shaltout et al. (2016) reported that mean value of aerobic plate count in examined minced meat, sausage and luncheon were 8.03×10⁴ ±0.12×10⁴, 6.74×10⁴ ±0.28×10⁴ and 5.85×10⁴ ±0.24×10⁴ Cfu/g; respectively. The total bacterial count reflects the bacterial contamination level and hygienic quality of meat products (Salem et al. 2018).

Concerning data in table (3), lower results obtained by Hassanien et al. (2018) as 0.2x10², 1x10² and 3x10² Cfu/g in minced meat, luncheon and sausage, respectively. In addition, lower prevalence of S. aureus was detected by Shaltout et al. (2016) as 8, 6, 20 and 25% in luncheon, sausage and minced meat, respectively.

Higher results of S. aureus in sausage were reported by Abd El-Latef (2014) as 2.8x10⁴ Cfu/g. S. aureus can be carried in nasal passage and on the hands. Most outbreaks of food borne disease were due to contamination following poor handling and production of heat stable toxins S. aureus. Refrigeration, proper cooking and sanitary food handling are recommended measure to prevent disease outbreak related to S. aureus (Morshdy et al., 2023).

 All tested S. aureus strains harbored pvl gene at 433bp and sea at 102bp by PCR (Fig 1). Similar results were obtained by Morshdy et al. (2018) and Saif et al. (2019) who detected Sea gene in all tested S. aureus strains form meat samples.

  Prevalence of E. coli shown in table (4) was similar to El-Shabrawy (2015) who detected E. coli in 8% of luncheon samples. Shaltout et al. (2016) and Eltanani and Arab (2021) reported lower prevalence of E. coli in minced meat (22.9 & 16 %) and in sausage (17.1 & 20%) respectively. Moreover, lower prevalence (5.7%) in luncheon was reported by (Shaltout et al. 2016).

In addition, Abd El-Tawab et al. (2015) cited that the prevalence of E. coli in sausage was 9%. Meanwhile, higher prevalence of E. coli in luncheon was 36 and 28% reported by Ashraf (2016) and Ramadan (2015).  

Escherichia coli was serologically identified as O26, O126, O111, O158, O146, and untyped E. coli as shown in Table 7. E. coli O26 was the most prevalent serogroup in the current study like Abuelnaga et al. (2021) but Moawad et al. (2017) in Egypt identified E. coli O158 mainly in beef meat.

Detection of virulent genes in some E. coli isolates obtained in Fig (2) and agreed with Abd El-Tawab et al. (2019) detected eaeA gene (248bp) in all tested E. coli strains from meat products. In contrary, Hassan et al. (2020) reported that E. coli isolates from minced meat and sausage were negative for Stx2 gene.

Prevalence of Salmonella in table (4) agreed with Edris et al. (2011) who didn’t detect Salmonellae in the examined luncheon samples.

In contrary, Eltanani and Arab (2021) isolated Salmonella from luncheon at prevalence rate 12%. Similar prevalence of Salmonella in minced meat 2.8% was reported by Filliol et al. (2008) while, higher prevalence in minced meat 24% was reported by Eltanani and Arab (2021). Similar prevalence of Salmonella in sausage 4% was reported by Abd El-Atty and Meshref (2007) while, higher results 24% were reported by Hassanien et al. 2018).

In the present study, Salmonella Typhimurium was the prevalent serovar (table, 4).

In Egypt, similar results obtained by Moawad et al. (2017) and Aboelnaga et al. (2021) who indicated that, S. Typhimurium was the most dominant serovar in the examined meat and meat samples. In addition, invA and Stn gene were detected in all tested isolates at 617 and 284bp PCR amplified fragment, respectively (Fig, 3). The results were agreed with (Ezzat et al. 2020) who mentioned that all isolated Salmonella from meat products harbored invA gene at 284bp. In addition, Sallam et al. (2014) confirmed the presence of Stn gene in all identified Salmonella isolates form meat products. 

Tabulated C. perfringens prevalence in table (7) was lower than that obtained by (Khalafalla et al., 2020) in sausage and luncheon samples as 60 and 52%; respectively. In addition, Kamber et al. (2007) and Younis et al. (2018) reported higher prevalence in minced meat which was 55 and 93.3%; respectively. 

Prevalence of C. perfringens  in minced meat may be attributed to poor hygienic measures during processing as well as spores could be separately added to meat products during cutting, wrapping and handling (Younis et al. 2018). 

The C. perfringens ability to produce alpha toxin (cpa) and enterotoxins (cpe) which are responsible for possible food poisoning has remained of public health relevance (Farag et al. 2023). In addition, Fig (4) declared that two tested C. perfringens strains possess enterotoxin gene (cpe) at 247bp and alpha toxin (cpa) at 402bp by PCR. These results agreed with (Younis et al. 2018) who found that all tested C. perfringens type A possess cpa at 204bp and cpe at 247bp.

5. CONCLUSION

        Results in the present study proved that meat products were highly contaminated indicating improper handling during processing.

Consequently, strict maintenance of good practices during processing, maintaining the cold chain during transport, distribution and carcass commercialization is very important to ensure both public health and food quality. In addition, obligatory implementation of strict food safety regulations in restaurants that sell meat products should be imposed and checked by regulatory agencies in Egypt for food safety.