Hayfaa Jumaah Hasan Alhasnawi, Basima Jasim Mohammed and
Zahira A. Al-Zuhairi

Department of Public Health, College of Veterinary Medicine, University of Al-Qadisiyah, Diwaniyah, Iraq.

Abstract

Staphylococcus aureus still resembles one of the primary causes of food poisoning induced specifically by milk and its products. Recent studies from neighbor countries of Iraq showed the presence of this bacterium in the milk of cattle, sheep, and goat. The current study aimed to genetically identify S. aureus that was cultivated from 16 raw milk, 20 cheese, and 13 cream samples. The results show positive isolation of the bacterium in 6 (37.5%), 8 (40%), and 3(23%) samples respectively. Then, the isolates were subjected to multiplex polymerase chain reaction (mPCR) technique targeting enterotoxin genes, sea, seb, and seg. Interestingly, the milk samples showed amplification of these genes in 3 (33.3%), 1 (16.6%), and 4 (66.6%) isolates respectively. the cheese results revealed amplification of the genes in 4 (50%), 2 (25%), and 5 (62.5%) isolates respectively. Moreover, the cream indicated amplification of seg gene only in 1 (33.3%) isolate. Finally and to genotype the bacterium, the accessory gene regulator (agr) was employed to detect the bacterium nucleotide polymorphism. The results place the isolated S. aureus in different genotype groups but mostly in Group I and Group III. The current study results determine S. aureus as a pathogenic organism that thrives the milk and its products and might be responsible for many cases of food poisoning in the city.

Keywords: Staphylococcus aureus, food poisoning, dairy products, genotyping.

Introduction

Food poisoning caused by ingestion of contaminated dairy products with pathogenic bacteria such as staphylococcus aureus is considered to be one of the major concerns that affect people life because milk and its products are important ingredients of everyday-people food such as raw milk, cheese, and cream. These products are used in food industries and that is what could make the problem of food poisoning even worse (1). Results from a recent study by (2) have shown the high presence of this bacterium in the raw milk in Egypt. The presence of S. aureus in raw milk or its products could occur due to accidental contamination by handlers (3). In the same regards subclinical mastitis is considered as a source of S. aureus that induces food poisoning (4). In a study from Egypt by (5) have detected that S. aureus isolates were found in 40% of the bovine mastitis tested samples that most of them are resistant to wide range of antibacterial drugs. The problem of antibiotic resistance enhances transforming into subclinical cases and causes passing of S. aureus and or its enterotoxins through milk to consumers (6,7). Diagnosis and classification of the bacterium need advanced tools and methods to help controlling the problem of food poisoning, in which genetic procedures could add major differences to the field due to their high specificity and sensitivity (8,9). S. aureus secrets various toxins that are responsible of food poisoning in humans such as SEA that belongs to SEs group of toxins (10,11). Using these enterotoxin genes to genetically diagnose and genotype strains of S. aureus is substantial to improve food industries and control food poisoning caused by contaminated milk and its products. To the best of my knowledge, there have been no studies conducted using these genetic tools to classify strains of S. aureus present in raw milk and its products in Iraq and more specifically in Al-Diwaniyah city, ~ 180Km south to Baghdad, and that is why this study was performed.

Materials and Methods

Sample collection
A total number of 49 raw milk and milk product samples (16 raw milk, 20 cheese, and 13 cream) were collected from different local market in Al-Diwaniyah city, Iraq. Samples were placed into clean containers and immediately transferred to a laboratory in the College of Veterinary Medicine, University of Al-Qadisiyah, Diwaniyah, Iraq. The samples were then stored in 4 ̊C until bacterial cultivation.

Bacterial cultivation
For primary enrichment, the samples were inoculated into Brain Heart Infusion Broth (BHIB) and incubated at 37°C overnight. After that, the bacterial growth was inoculated into mannitol salt agar (MSA) and incubated at 37°C overnight to purely isolate Staphylococcus aureus.

Bacterial genomic DNA extraction
Bacterial genomic DNA was extracted from Staphylococcus aureus isolates by using PrestoTM Mini gDNA Bacteria Kit (Geneaid, USA). Briefly, 1ml of overnight bacterial growth on BHIB was placed into 1.5ml microcentrifuge tubes and centrifuged at 10000 rpm for 1 minute. The supernatant was then discarded, and the bacterial cells pellets were used to extract the DNA according to the manufacturer protocol. Finally, the extracted gDNA was checked for quantity and quality using NanoDrop spectrophotometer. The DNA was stored in -20 ̊C to perform other assays later.

Molecular virulence characterization
Characterization of Staphylococcus aureus isolates were determined by detection of some enterotoxin genes (sea, seb, and seg) using mPCR technique. This technique protocol was followed according to (12). The primers were designed using NCBI database and primers 3 plus and deposited in GenBank as sea: GQ859135.1, seb: AY852244.1, and seg: AF064773.1. The primers were purchased from Bioneer Company, South Korea.

Then, mPCR Master mix was prepared using AccuPower® Multiplex PCR PreMix kit (Bioneer Company, South Korea). The process was performed following the manufacturer’s instructions.

After that, the mastermix components mentioned above were added to other principle components such as the DNA polymerase, dNTPs, and Mg++ that are required to perform the reaction. The reaction was generated in a thermocycler (Mygene Bioneer, Korea) using the following conditions: initial denaturation temperature of  95 °C for 5 min which was followed by 30 cycles of denaturation 95 °C for 30 Sec, annealing at 60 °C for 30 Sec, and extension at 72 °C for 1min. Then, the final extension at 72 °C for 5 min was done. The PCR products were examined by electrophoresis using 1% agarose gel that was stained with ethidium bromide to be visualized under UV transilluminator.

Agr-system based Genotyping
Genotyping of the isolated Staphylococcus aureus was generated using agr gene to detect locus nucleotide polymorphism. PCR was employed to fulfill this goal and then to sort out these isolate into certain groups of I, II, III, IV. The process was carried out according to (13), and the primers from the same reference were followed to purchase from Bioneer Company, South Korea.

Then, PCR Master mix was prepared using AccuPower® PCR PreMix (Bioneer, South Korea). The manufacturer instructions were followed to generate the mix.

Later, the mix was added to other components that are needed to perform the PCR reaction using a thermocycler (Mygene, Bioneer, South Korea). The following conditions were used: initial denaturation temperature of 95 °C for 5 min and followed by 30 cycles of denaturation 95 °C for 30 Sec, annealing 60 °C for 30 Sec, and extension 72 °C for 1min. The final extension was at 72 °C for 5 min. The PCR products were tested by electrophoresis using a 1% agarose gel which had been stained with ethidium bromide to be visualized under UV transilluminator.

Results

Bacterial cultivation
The cultivation of the bacteria showed that out of 16 raw milk, 20 cheese, and 13 cream samples were positive for S. aureus in 6 (37.5%), 8 (40%), and 3 (23%) samples respectively.

MPCR-enterotoxin genes
The S. aureus isolates were subjected to mPCR technique to target the enterotoxin genes, sea, seb, and seg. Interestingly, the milk showed amplification of these genes in 3 (33.3%), 1 (16.6%), and 4 (66.6%) isolates respectively. The Cheese revealed amplification of the genes in 4 (50%), 2 (25%), and 5 (62.5%) isolates respectively. Finally, the cream indicated amplification of seg only in 1 (33.3%) isolate as shown in figure 1.

Fig. 1. Shows amplification of enterotoxin genes, sea, seb, and seg at product sizes of 504, 272, and 150bp respectively. Ladder is 1000-50bp

Genotyping based on agr system
Table 1 reveals the results of the agr-system based genotyping in details. The raw milk isolates were genotyped as 3 in Group I, 2 in Group III, and 1 in Group IV. While the cheese isolates were grouped as 4 in Group I, 1 in Group II, 2 in Group III, and 1 in Group IV. For the cream, the isolates were genetically classified as 2 in Group I, and 1 in Group III. Figure 2 shows these results of the agr locus nucleotide polymorphism.

Fig. 2. Shows amplification of agr locus nucleotide polymorphism. Ladder is 1000-50bp

Table 1. Shows the groups of the isolates based on the agr system genotyping

Total percent Group  I Group  II Group  III Group  IV
Milk 3/6 (50%) 0/6 (0%) 2/6 33.3%) 1/6 (16.6%)
Cheese 4/8 (50%) 1/8 (12.5%) 2/8 (25%) 1/8 (12.5%)
Cream 2/3 (66.6%) 0/3 (0%) 1/3 (33.3%) 0/3 (0%)
Total percent 9/17(52.9%) 1/17 (5.7%) 5/17(29.4%) 2/17(12%)

 

Discussion

Food poisoning induced by the ingestion of contaminated milk or its products with poisonous amount of S. aureus and or its enterotoxin is valued as a big obstacle facing food industries and public health (11,14). In the current study, isolation of S. aureus from milk, cheese, and cream indicates that this bacterium and its enterotoxin might play a major role in food poisoning in Al-Diwaniya city, where the samples were collected from. In a recent study in India by (15), S. aureus isolates were relatively as low as 12% of the collected cow milk samples. While in the current study, S. aureus isolates were higher, 37.5%, in the milk samples. In Greece, (16) have found that S. aureus isolates were 40% of the milk samples which is close to what the present study has recovered. Cultivation of bacteria from cheese indicated higher presence of S. aureus, 87%, in Serbia (17) than that in the current study, 40%. About 5.6% of S. aureus had been recovered from cream samples collected from markets in Iran (18) which is less than that in the present study, 23%. The risk factor of increasing the incidence of food poisoning comes from the milk products such as cheese and cream. These products could get contaminated from handlers and factory workers (19). The authors mentioned the isolation of the bacterium from workers in a restaurant, Italy, where cases of food poisoning had happened. Moreover, the places where these products such as cheese are processed might introduce contamination by S. aureus and cause food poisoning as was shown by (20).

In the current study, the mPCR results bring the attention that S. aureus isolates were from the virulent strains. The enterotoxin genes, sea, seb, and seg used for this purpose give high evidence that milk and its products are responsible for many cases of food poisoning in Al-Diwaniyah city. In the present study, Milk isolates of S. aureus contained all the three enterotoxin genes with highest percentage of seg gene 66.6%. However, (21) found that sea gene was the dominant one in milk isolates in China. In contrast, stool samples that were collected from hospital near to Al-Diwaniyah city showed high frequency of seg gene 65% (22). This increases the awareness that the strains isolated from milk in the current study might have had a link with those isolated from the stool samples that mentioned above. Cheese isolates showed high percentages of sea and seg genes 50% and 62.5% respectively. While (23,24) have recognized high frequencies of seh and seb genes respectively.

The present genotyping procedure using the agr system has placed the S. aureus isolates mostly in Group I and Group III. However, S. aureus strains isolated from bovine mastitis in Brazil were detected to be more frequent in Group II (25). Relatively similar to the current study results, all strains isolated from bovine mastitis in some regions in China were also genotyped to be in Group I (26). Like the present findings, studies from Iran have shown that most of the strains were classified into Group I and III (27,28).

Conclusion

The current study highlights the problem of the presence of S. aureus in the raw milk, cheese, and cream in Al-Diwaniyah city, Iraq. The study indicates that the isolates were enterotoxin-secreted strains that were proved by the high occurrence of seg gene in these strains. The genotyping study using the agr system classified the strains mostly in Group I and Group III. Further future studies are required to test and find proper drugs that target these genes and or their products to control food poisoning in the city, the country, and the world.

Acknowledgement

I would like to appreciate and thank University of Al-Qadisiyah and the College of Veterinary Medicine in Al-Diwaniyah City, Iraq.

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