Introduction

Meat from cow (beef) is a major source of protein all over the world. In parts of Nigeria where protein is a short supply [1], cow meat serves as the main source of animal protein for most families. Proper handling of meat poses problems to both. Exposure during sales could result in contamination by a wide range of bacteria. The poor hygienic situation of markets in developing countries like Nigeria could expose meat to bacteria including Staphylococcus spp, Bacillus spp, Vibrio spp, Salmonella spp and Listeria spp (Wei-wei et al., 2018). These organisms affect the quality of meat and are potentially harmful to humans, the consumer.

Staphylococcus aureus is a major foodborne pathogen, growing at temperature 15⁰C-45⁰C and NaCl concentration above 15%. [2]. The organism is a facultative anaerobic, Gram positive, non-motile, non-spore forming toxic bacteria [3]. Meat and meat products have been identified as reservoir for Staphylococcus aureus [4].

Presently, the genus Staphylococcus has 62 species and 30 sub-species of which Staphylococcus aureus is the most important foodborne pathogen [5]. Food poisoning could result from the production of heat-stable enterotoxins by Staphylococcus aureus [6]. There is a need therefore to improve the quality of meat sold in markets by reducing contact with bacteria and other food spoilage organisms.

Materials and Methods

Study Area

The study was carried out in Yenagoa (4⁰55’N, 6⁰16’E), Bayelsa Sate, Nigeria. Samples for the study were collected from three markets;

1. Swali market
2. Opolo market
3. Tombia market

Sample Collection and Preparation

Samples were collected randomly from three sellers from each of the selected markets. The samples were labelled, packed in polythene bags and transported in ice to the laboratory for preparation and analysis. Collected meat samples were ground using mortar and pestle and 1g of the samples weighed into 9mL of sterile distilled water (1:10 w/v) [7]. This was homogenized using a Stomacher blender. 1ml of the obtained homogenate was measured using a sterile pipette and diluted into 9mL of sterile distilled water (10^-1). The serial dilutions were repeated to obtain 10^-6 dilutions and used for further analysis.

Nutrient agar (Oxoid) and Mannitol Salt agar (Oxoid) used for cultivation of bacteria were prepared as described by Cheesbrough (1985) [8]. Slants were prepared using sterilized specimen bottles.

Sample Inoculation

Prepared aliquots of the sample (0.1 ml) were inoculated onto Nutrient Agar and Mannitol salt agar in duplicates using the spread plated technique [9] and incubated at 37⁰C for 24-48 hrs.

Characterization and Isolation

After 24-48 hrs of incubation, the plates were observed for bacterial growth. Colonies were counted, described and recorded. The observed colonies were then transferred to agar slants using wireloop and flame sterilization and incubated at 37⁰C for 24 hrs for further observation, identification and subculture on Mannitol Salt agar.

Total Bacterial Count (TBC) was determined using the formula;

TBC (CFU/g) =

Confirmation of Staphylococcus aureus

Standard biochemical tests and molecular techniques were used for the identification and confirmation of Staphylococcus aureus. Biochemical tests used included Gram staining, Coagulase test, Catalase test, Methyl red test and Citrate utilization test [10-12].

Molecular Identification

Bacterial isolates sub-cultured on Luria Bertami broth for 24 hrs were subjected to molecular identification using PCR based multiplex assay [13] using target genes that encode for bacterial subunits (16s). The BigDye Terminator kit was used for sequencing and similar sequences were downloaded from the National Center for

Biotechnology Information (NCBI) data base using Basic Local Alignment Search Tool Nucleotide (BLASTN) [14]. The evolutionary history was inferred using the Neighbour-Joining method in MEGA 6.0 (Saitou and Nei, 1987) and evolutionary distances were computed using the Jukes-Cantor method [15].

Results and Discussion

Total Bacterial Count (TBC)

After 24 hrs of incubation, the plates were observed for bacterial growth and the total bacterial counts recorded. The results of the total bacterial counts are shown in Table 1. The results indicated that samples from Tombia market had the highest no of bacterial cultures in Colony Forming Units (CFU) while those from Opolo market had the least. The highest total bacterial count of 1.03 x 10⁹ CFU/g was recorded for C2 and the least of 1.3 x 10⁸ CFU/g was recorded for B1. The results obtained from inoculation on Mannitol salt agar was almost similar.

Table 2 shows a comparison of the number of bacterial colonies obtained from Nutrient agar and Mannitol salt agar. The colony counts on Nutrient agar were far more than that on Mannitol salt agar. Mannitol salt agar is a selective media. The correlation coefficient using Pearson’s correlation coefficient showed that the results were significant at p(0.05) with r_(cal) (0.529)(tab)(0.666)

Morphological Characteristics of Bacteria

The morphological description of the cultures observed after incubation on Nutrient agar are presented in Table 3. The morphological characterization of the observed cultures indicates the growth of five types of bacteria on Nutrient agar. R1 showed golden-yellow colonies typical of Staphylococcus aureus. R2 were white, discrete, pancake colonies typical of species of Streptococcus spp. R3 were colonies that are white and serrated typical of species of Bacillus spp. R4 had white, flat colonies that are generally representative of Micrococcus spp and R5 showed white, shiny colonies that could indicate Enterococcus spp.

The results of the bacterial isolates sub-cultured on Mannitol salt agar are presented in Table 4. Results showed the growth of the isolates on the selective media with neutral red indicator. R1 showed the ability to ferment mannitol. This is a confirmation of Staphylococcus aureus. R4 showed growth with no colour change indicating Staphylococcus spp. unable to ferment mannitol typical of Staphylococcus epidermidis. R3, R4 and R5 had inhibited growth.

Biochemical Characterization of Bacteria

The results obtained from the biochemical identification of the isolated bacteria are presented in Table 5. Results from Gram staining and observation with the oil immersion lens indicated that R1, R2 and R4 are Gram positive cocci in clusters while R3 are Gram positive rods and R5 are Gram negative rods.

For the coagulase test for the presence of the enzyme Coagulase, the results showed that R1 and R4 are coagulase positive while the others (R2, R3 and R5) are coagulase negative i.e. they do not possess coagulase.

The catalase test showed that four of the isolates (R1, R2, R4 and R5) are catalase positive while one (R3) is catalase negative. Catalase breakdown hydrogen peroxide into hydrogen gas and water vapour (effervescence observed). The combination of the results of the various biochemical tests confirm R1 and R4 as Staphylococcus aureus, which are Gram positive, coagulase positive and catalase positive.

Molecular Identification of Bacteria

Uploading the DNA sequence of the extracted and amplified 16s DNA for R1 and R4 to the NCBI database produced a match. Both had a 100% match with Staphylococcus aureus strain UA899. The BLAST technique compared the 16s DNA sequence obtained for the isolated sample with similar sequence found in the database. The Neighbor-joining method was used to infer the evolutionary history of the isolated organism.

Conclusion

The results obtained from the study indicate that meat obtained from evening markets within Yenagoa had significant incidence of Staphylococcus aureus. Significant amount of the bacteria in the selected samples is of public health significance due to the ability of the organism to cause a wide range of infections. Higher levels of the bacteria in samples obtained from Tombia market is indicative of the socio-economic activities within the vicinity. Occurrence of Staphylococcus aureus in the samples may be due to contact with anthropogenic sources. Exposure of meat to the surrounding environment from the time of slaughter in the morning to the evening when they are sold will result in significant exposure to the bacteria [16].

Non-hygienic practices by buyers, sellers and passers-by could also result in exposure to the bacteria which is a normal flora of humans inhabiting the skin and the nasal tract. Recent evidence has shown that Staphylococcus aureus develops antibiotic resistance [6,17] and this could result in the presence of toxigenic strains of the bacteria in food that could result in serious public health consequences.

The presence of the bacteria in the samples may not indicate immediate health risks since meat and meat products are cooked before consumption. Occurrence of Staphylococcus aureus in meat and meat products was also reported by [16,18]. However, the presence of Coagulase positive strains calls for caution. There have been reported cases of Staphylococcal food poisoning in cooked food, preserved food and Ready-to-Eat food [19]. Food safety begins with production and continues to handling and finally consumption.

Sellers of meat and meat products should adopt better hygienic practices to ensure food safety. Meat bought from markets should be thoroughly cleaned, washed and cooked before consumption to prevent incidence of Staphylococcal food poisoning. The use of specially designed table and sheds with nets for the sale of meat and meat products to prevent unnecessary exposure of the products. Further studies on the implication of the bacteria in the selected areas should be carried out.

Tables

References

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