Molecular detection of the genes encoding virulence factors of coagulase-positive Staphylococcus aureus isolated from restaurants

1. Isolation and identification of aureus using the classical methods from various regions of Erbil Governorate.
2. Detection of the same genes encoding virulence factors such as nuc, clfA, clfB, and coa genes using the PCR assay.
3. Evaluation of the meat and tools used in the restaurants hygienic.

Introduction

Staphylococci are significant pathogens transmitted through food that can cause important public health problems (1). They have been classified as the third most common cause of diseases internationally among foodborne diseases that have been determined (2), and their pathogenicity is based on several components of the surface of the bacteria (3). Staphylococcus (S.) aureus is a serious foodborne illness. It is a complex pathogen that affects sentient beings, causing many diseases, such as septicemia and pneumonia (4). Staphylococcus aureus is one of the most significant microorganisms that can contaminate or recontaminate the food prepared by workers' hands, tools, or scraps (5). Staphylococcus aureus is considered an important foodborne pathogen globally due to its ability to generate several extracellular toxins, which usually induce an abrupt onset of nausea, vomiting, and abdominal cramping (6). The capacity of certain strains of S. aureus to produce heat-stable enterotoxins that result in staphylococcal food poisoning (SFP), one of the most prevalent causes of gastroenteritis in the world, is especially pertinent for the food processing sector (7). Additionally, staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin-like (SEls) are the main cause of food-related diseases (8). These five traditional enterotoxins are said to be responsible for 95% of SFP cases, with an outstanding 5% of illnesses caused by newly recognized SEs (9). According to many earlier research, S. aureus was determined from a variety of foods, including meat (10), fish (11), cow's milk (12), and camel milk (13). Foods such as meat and its products, particularly ham, are linked to the emergence of staphylococcal occurrences poisoning (14). In recent decades, S. aureus has been found in domesticated animals raised for food, such as pigs, cattle, chickens, and other types of animals (15). Food containing a high protein content should be handled by hand, with care, usually in conjunction with improper heating and/or storage (16). In addition, meat is highly susceptible to spoiling and is commonly associated with the transmission of foodborne illnesses. Raw meat contamination is a major cause of foodborne diseases (17). Meat can be contaminated from the first stage on the farm during direct contact of animals with contaminated surfaces or in the slaughterhouse, which is considered a second stage of contamination of meat during slaughtering, evisceration, and storage; finally, meat will contaminate during transmission, processing, and cooking. Various methods were used to determine the S. aureus isolates, including traditional and molecular biology. The Conventional methods are based on biochemical testing and the structural features of S. aureus colonies. However, molecular techniques are quicker and more accurate, providing results for identifying S. aureus in three to five hours (18).

The objectives of our study are to identify the pathogen S. aureus in the different types of samples taken from restaurants in various parts of Erbil city and to identify the genes in S. aureus isolates that encode virulence factors by the PCR assay.

Materials and methods

Ethical approval

All samples were collected with the owners' agreement and utilized based on the ethical criteria issued by the Institutional Animal Care and Use Committee at Mosul University's College of Veterinary Medicine, using the approved ID of UM. Vet. 2024.047.

Samples Collection

Three hundred and fifty samples (knife, meat, table, hand, and machine) were collected from various areas in Erbil, Iraq (30-meter streets, 40-meter streets, 60-meter streets, and 100-meter streets). Studying began in August 2024 and ended in November 2024. All types of samples (knife, meat, table, hand, and machine) were collected in sterile swabs in sanitary containers before being sent immediately to the College of Veterinary Medicine's Health Laboratory at Mosul University. All peptone water containers were placed in an incubator and underwent a pre-enrichment process for 18 to 24 hours at 37°C. After streaking each sample on Mannitol salt agar, they were incubated at 37°C for a full day.

1. aureus Isolation and Characterization

Phenotypic examination, coagulase and catalase activity tests, and gram staining were employed to investigate and determine phenotypic characterizations of the S. aureus colonies in conformity with the conventional procedures used for isolating and identifying S. aureus colonies (19).

Extraction of DNA

The genomic DNA from S. aureus was analyzed, and the positive isolates were analyzed and cultured in a mannitol salt medium for over eight hours at 37°C. The Genomic DNA Extraction Kit (Addbio, Korea) utilized to isolate the DNA was processed following the manufacturer's guidelines for Gram-positive bacteria. Following this, the concentration of DNA was measured using Nanodrop (Jenway, UK). The DNA concentration of S. aureus isolates ranged from 18.5 μg/μl to 50 μg/μl; the extracted DNA was stored at -20°C.

Reaction of PCR

The nuc, clfA, clfB, and coa genes of S. aureus were discovered by the PCR technique. The molecular weight of the nuc gene was 166 bp (20), clfA was 288 bp (21), clfB was 203 bp (21), and coa was 674 bp (22). The PCR reaction required 25 μl in total volume, and the mixture was made in a 200 μl tube (Addbio, Korea). The resulting amplicons underwent a gel electrophoresis evaluation on a 2% agarose gel (Peqlab, Erlangen, Germany), utilizing a reference of a 100 bp ladder. The reaction mixture consisted of 6.5 μl of DNeasy-free water from Promega Corporation (USA), 4 μl of the S. aureus DNA template, 12.5 μl of the GoTaq Mix Master (2×) from addbio company (Korea), 1 μl of each primer 1 and 2 (Table 1).

Table 1: Various primers utilized in the PCR programs to detect the genes of S. aureus

PCR program: I: 35 times (94°C - 30s, 55°C - 30s, 72°C - 30s), II: 35 times (94°C - 30s, 60°C - 30s, 72°C - 30s)

Statistical analysis

All analyses were performed using JMP® 16.1 software (23). The Chi-square test was used to determine if there was a significant difference between the percentages of S. aureus isolated from different streets in Erbil city. The results were substantial, with a P<0.05.

Results

The S. aureus colonies that provided positive data appeared on Mannitol salt agar with a golden-yellowish color. Additionally, specific biochemical tests that verified the existence of S. aureus, such as the coagulase and catalase assays, produced positive results. The concentration of extracted DNA from S. aureus in this study was 18.5 mg/μl to 30 mg/μl. The PCR assay confirmed that all S. aureus isolates were positive (Figures 1-7).

Figure 1: Comparative prevalence of S. aureus isolated from restaurants in various areas of Erbil city.

Figure 2: Prevalence of S. aureus isolated from different samples

Figure 3: Visualization and comparative concentration of S. aureus whole genome DNA using agarose gel electrophoresis

Figure 4: A 2% agarose gel electrophoresis illustrating the typical amplicon of the nuc gene product of S. aureus isolates.

Figure 5: A 2% agarose gel electrophoresis illustrating the typical amplicon of the clfA gene product of S. aureus isolates.

Figure 6: A 2% agarose gel electrophoresis illustrating the typical amplicon of the clfB gene product of S. aureus isolates.

Figure 7: A 2% agarose gel electrophoresis illustrating the typical amplicon of the coa gene product of S. aureus isolates.

According to the present study, 43.4% (152/350) of S. aureus were determined to be prevalent in Erbil city. The high percentage isolated of S. aureus was 50% (40/80) on a 40-meter street. Subsequently, the prevalence rate of S. aureus in a 60-meter street was 48% (48/100). Although the lowest isolated S. aureus ratio observed in a 30-meter street and a 100-meter street was 38.8% (31/80) and 36.7% (33/90), respectively (Table 2). Statistical analysis revealed that the percentage of S. aureus isolated from various streets in Erbil city did not differ significantly (P<0.05).

Table 2: Number and percentage of S. aureus isolated from different streets in Erbil city

Furthermore, the present investigation appeared to indicate various incidence rates of S. aureus in different regions. In a 30-meter street, the incidence rate of S. aureus was 38.8% (31/80). The high incidence rate of S. aureus isolated in a 30-meter street from knives and meat was 43.8% (7/16). Subsequently, the incidence rate of S. aureus isolated from the hand and table was 37.5% (6/16). The low incidence rate of S. aureus isolated from machines was 31.3% (5/16) (Table 3).

Table 3: Number and percentage of S. aureus isolated from different samples in 30-Meter Street

Additionally, the present study declared that the rate of occurrence of S. aureus isolated on a 40-meter street was 50% (40/80). The high occurrence of S. aureus strains obtained from the table was 62.5% (10/16). The low occurrence rate of S. aureus detected in meat samples was 37.5% (6/16). Meanwhile, the occurrence of S. aureus isolated from the hand, machine, and knife was 50% (8/16) (Table 4).

Table 4: Number and percentage of S. aureus isolated from different samples on 40-Meter Street

The incidence rate of S. aureus in 60-Meter Street was 36.7% (33/90). The high incidence rate of S. aureus from meat was 55.6% (10/18). The low incidence rate of S. aureus detected from a knife was 22.2% (4/18). Subsequently, the incidence of S. aureus isolated from the hand, table, and machine was 33.3% (6/18), 33.3% (6/18), and 38.9% (7/18) (Table 5).

Table 5: Number and percentage of S. aureus isolated from different samples on 60-Meter Street

Finally, the occurrence rate of S. aureus isolated in a 100-meter street was 48% (48/100). The high occurrence rate of S. aureus isolated from the hand was 55% (11/20). The low occurrence rate of S. aureus in meat was 40% (8/20). Subsequently, the occurrence rate of S. aureus isolated from the table, machine, and the table was 45% (9/20), 50% (10/20), and 50% (10/20), respectively (Table 6).

Table 6: Number and percentage of S. aureus isolated from different samples on 100-Meter Street

Furthermore, the high prevalence of S. aureus detected in restaurants from hand, table, and meat was 44.3% (31/70). The low prevalence of S. aureus obtained in restaurants from knives was 41.4% (29/70), and machines 42.9% (30/70) (Table 7). The statistical analysis showed no significant difference between the types of samples in 30, 40, and 100 meters of the street (P<0.05). Still, there was a substantial difference between knives and meat (P<0.05) in 60 meters of the street.

Table 7: Comparative of the percentage of S. aureus isolated from different streets in Erbil city

Based on Table 8, the results of the PCR assay supported the conclusions attained from traditional methods, confirming that the nuc and coa genes were present 100% (40/40) of S. aureus (Figures 4 and 7). Furthermore, our study showed that 95% (38/40) of S. aureus isolates possess the clfA, and 92.5% (37/40) possess the clfB genes (37/40) (Figures 5 and 6).

Moreover, the results of the current study showed that S. aureus was split into two different gene profiles according to the presence of various genes in each isolate (Table 9). The S. aureus isolates showed that the most frequent gene profile I (nuc + mecA + clfA + clfB + coa) was 34/40 (85%), the gene profile II (nuc + clfA + coa) was 2 (5%), the gene profile III (nuc + clfB + coa) was 3 (7.5%), and the genes profile IV (nuc + coa) was 1 (2.5%).

Table 8: The number and percentage of genes found in S. aureus isolates

Table 9: Types of gene profiles of S. aureus isolates (n = 40)

Discussion

It is a well-known truth that the primary way that harmful bacteria spread is through foodborne contamination. It is one of the leading causes of death and health complications, as well as enteric diseases in less economically developed countries (24). Meat is regarded as the most significant food source in the world since it gives customers the necessary amino acids, iron, phosphorus, and B complex vitamins, among numerous other nutrients (25). S. aureus is one of the most frequently identified organisms in meat processing plants, including contact and non-contact surfaces, raw materials, and various types of products (26). frequently, the incidence of S. aureus in the current study was high at 43.4% (152/350), as well as the incidence rate of S. aureus isolated from various areas in Erbil city was different, and this could be attributed to a variety of factors such as contamination the during transport of animals from farms to slaughterhouses, prolonged contact between healthy animals and infected animals in the abattoir, evisceration caused by both intestinal contents and the water applied to wash and rinse carcasses, and inadequate hygiene during the handling, transportation, and storage of carcasses for later use (27). In restaurants, the workers play an important role in causing cross-contamination between hands and meat while handling contaminated meat; for example, not wearing gloves, not washing their hands frequently, and wearing unclean clothes can lead to elevated bacterial contamination (28,29). However, minimizing the amount of S. aureus and other harmful bacteria in restaurants can be achieved by thoroughly washing and sanitizing equipment and tools before and after using them with meat (30,31).

Additionally, the incidence of S. aureus isolated from meat was 44.3% (31/70). The results of the analysis of this research were higher than in other studies, which appeared to show that the frequency of S. aureus in meat was 16.4% in the USA (32) and in African countries 33.1% (33). While earlier studies found that the prevalence of S. aureus in beef meat was higher than the results of the present study, in China, it was 50% (34), and in the USA, it was 65.6% (35). There are reasons why the prevalence of S. aureus varied between this study and other studies, including the sampling strategy, isolation techniques, location of carcass sampling, various cuts of meat, contamination before and after slaughter, meat storage, and meat processing (36). While the prevalence of S. aureus isolated from hands and tables was 44.3% (31/70), machines were 42.9% (30/70), and knives were 41.4% (29/70). In addition to comparison with studies outside Iraq, in Thailand, the prevalence of S. aureus contamination in restaurants in the hands of food preparation workers was 78%, on tables was 26%, on plates was 23%, and on knives was 16% (37). The symbiotic bacterium S. aureus has been detected in the gastrointestinal tract, nose, and human skin. According to a prior study on nasal carriers operating in the production of food contexts, the Staphylococcus carrier can cause food contamination when in contact with respiratory secretions, which results in food poisoning due to Staphylococcus bacteria (38). Staphylococcus aureus can produce heat-stable toxins that are not destroyed by cooking (39). 

In this investigation, a PCR assay was applied to identify the various types of genes, such as the nuc gene, which was used to identify S. aureus isolates. Based on numerous research studies, the PCR assay is a very sensitive and reliable method for identifying coagulase-positive S. aureus in contrast to coagulase-negative Staphylococcus species (40,41). In addition, the current study appeared to show that S. aureus possesses the nuc gene, and the results of this study agree with the results of the previous studies that declared that all coagulase-positive S. aureus have the nuc gene (31,42). Furthermore, this investigation revealed that the coa gene was 100% in all S. aureus isolates, indicating that this gene is essential pathogenicity of the bacterium. The results of this study were consistent with other studies that found that all S. aureus isolates possessed the coa gene with different molecular weight ranges of 514 bp to 802 bp (43). However, a different study found that the coa gene was present in every S. aureus isolate with the same molecular weight of 580 bp (44). In addition, adhesion-encoding genes, such as the clfA and clfB genes, were highly detected in S. aureus isolates. Early studies showed that clfA and clfB were detected in S. aureus isolates at 76% and 76.67%, respectively (45). According to studies, the clfA gene was found in 19-100% of S. aureus isolates, whereas the clfB gene was found in 91.8-92.9% of isolates (46-50).

Conclusions

It was a long study conducted in various geographic areas of Erbil City, Iraq. Staphylococcus aureus was isolated from meat in restaurants, which indicated that meat had been contaminated from slaughterhouses by handling, transport, and storage under unsanitary conditions. In addition, S. aureus was isolated from utensils used in restaurants that infected all utensils to help spread S. aureus and contaminated meat with pathogenic bacteria. Furthermore, all restaurants may use unpackaged meat or store it in non-refrigerated temperatures that help to provide suitable conditions for the growth and amplification of bacteria and cause food poisoning for consumers. Furthermore, S. aureus exceeds the utensils used in restaurants, which means that they do not wash, clean, and sterilize the utensils frequently, leading to meat contamination through S. aureus, which causes a huge problem for people. S. aureus possessed various types of gene encoding virulence factors, meaning a difference in sequence types of S. aureus was isolated in this study.

Acknowledgments

We extend our heartfelt gratitude to the College of Veterinary Medicine at the University of Mosul for providing the indispensable resources that made this research possible.

Conflict of interest

The author confirms no conflicts of interest in the preparation or analysis of the manuscript.