Detection of emetic toxin (ces) gene and antimicrobial susceptibility of Bacillus cereus isolates from Iranian traditional dairy products


 Background

 Bacillus cereus is known as one of the major foodborne pathogens that often associated with the contamination of raw milk and dairy products. The aim of this study was to isolate the emetic toxin-producing Bacillus cereus (B. cereus) in unpasteurized traditional dairy products. Also, the antimicrobial resistance pattern was investigated in the isolates.
Methods

For this purpose, 150 samples of unpasteurized ice cream, cheese, doogh, curd and butter (prepared by traditional method) were randomly selected from the market from March 2019 to July 2020. Samples were evaluated for the presence of emetic toxin-producing B. cereus using culture and PCR method.
Results

Sixteen of 150 (10.66%) samples were contaminated with B. cereus. This pathogen was isolated from one (3.33%) sample of ice cream, 9 (30%) of doogh, 6 (20%) of curd in the mean contamination levels of 2.0 ± 0.0 and 4.4 ± 0.6, respectively. B. cereus was not isolated from cheese and butter samples. The contamination level of curd to this bacterium was significantly higher than other products (p < 0.05). PCR results showed that 14 (78.5%) of 16 isolates contained the ces gene. The results of the antibiogram test showed that B. cereus isolates were resistant to penicillin, cefazolin and co-trimoxazole (trimethoprim-sulfamethoxazole), semi-sensitive to vancomycin and erythromycin, and sensitive to gentamicin, ciprofloxacin and clindamycin. Also, 31.25% of isolates were resistant to six antibiotics simultaneously.
Conclusion

The findings of present study showed that traditional unpasteurized dairy products could provide a great potential for intoxication by antimicrobial-resistance and emetic toxin-producing strains of B. cereus.


Abstract
Background Bacillus cereus is known as one of the major foodborne pathogens that often associated with the contamination of raw milk and dairy products. The aim of this study was to isolate the emetic toxinproducing Bacillus cereus (B. cereus) in unpasteurized traditional dairy products. Also, the antimicrobial resistance pattern was investigated in the isolates.

Methods
For this purpose, 150 samples of unpasteurized ice cream, cheese, doogh, curd and butter (prepared by traditional method) were randomly selected from the market from March 2019 to July 2020. Samples were evaluated for the presence of emetic toxin-producing B. cereus using culture and PCR method.

Results
Sixteen of 150 (10.66%) samples were contaminated with B. cereus. This pathogen was isolated from one (3.33%) sample of ice cream, 9 (30%) of doogh, 6 (20%) of curd in the mean contamination levels of 2.0 ± 0.0 and 4.4 ± 0.6, respectively. B. cereus was not isolated from cheese and butter samples. The contamination level of curd to this bacterium was signi cantly higher than other products (p < 0.05). PCR results showed that 14 (78.5%) of 16 isolates contained the ces gene. The results of the antibiogram test showed that B. cereus isolates were resistant to penicillin, cefazolin and co-trimoxazole (trimethoprimsulfamethoxazole), semi-sensitive to vancomycin and erythromycin, and sensitive to gentamicin, cipro oxacin and clindamycin. Also, 31.25% of isolates were resistant to six antibiotics simultaneously.

Conclusion
The ndings of present study showed that traditional unpasteurized dairy products could provide a great potential for intoxication by antimicrobial-resistance and emetic toxin-producing strains of B. cereus.

Background
Bacillus cereus (B. cereus) belonging to the Bacillaceae family is a gram-positive, spore-forming, facultative anaerobe, motile and rod-shaped bacterium with the size of 1 × 3-4 µm that is arranged in singly or in short chains (Evelyn & Silva, 2015). It is an active hemolytic, catalase-positive, mannitolnegative and mesophilic bacterium, lacking rhizoid and containing phospholipase system (lecithinase). It has also the S-layer and beta-lactamase enzyme playing an important role in drug resistance (Dworkin et al. 2006). B. cereus strains are mostly saprophyte. They have a widespread in nature because of the resistance of their spores to various stresses and long survival in adverse environmental conditions (Messaoudi et al. 2010).
Bacillus cereus is known as one of the major foodborne pathogens that often contribute to the contamination of fast food and dairy products. It causes two types of food poisoning: diarrheal and emetic types (Le Lay et al. 2015). Diarrheal type of poisoning leads to abdominal pain and diarrhoea with the incubation period of 16 − 8 hours. This disease causes by enterotoxins which are produced during the proliferation of B. cereus in the small intestine (Le Lay et al. 2015). Another type of disease is the emetic type with the incubation period of 1-5 hours that causes vomiting and nausea-like the food poisoning This type of intoxication is caused through the ingestion of emetic toxin named "cereulide". Cereulide can't be destroyed easily by heat treatment and it is stable at 126 °C for 90 minutes. It is also resistant to acidic conditions of the stomach and proteolytic enzymes. Cereulide is encoded by ces gene. It binds to the 5-HT3 receptor and stimulates the vagal afferent nerve that causes nausea and vomiting. This effect leads to liver disorder. Recovery of histopathological changes and regeneration of hepatocytes occur after one week. Cereulide also causes cellular damages and inhibition of the natural killing activity of the In addition to being a pathogenic microorganism, this bacterium is considered as an important cause of food spoilage due to its proteolytic, lipolytic and amylolytic activity. Because of some psychotropic types of this bacterium that are able to grow at 1-6 °C, the food spoilage may also occur during storage at refrigerator temperatures. By the action of proteinase, lipase and phospholipases enzymes produced by B. cereus, some defects such as bad taste, the sweet and bitter clot can be observed in dairy products Since, B. cereus has important role in food poisoning and spoilage in dairy products and these products are the most consumed food in the human diet, the aim of this study was to determine the prevalence of this bacterium in traditional unpasteurized dairy products, compare the contamination level with standard limit and evaluate the frequency of ces gene and antimicrobial susceptibility pattern of isolates.

Methods
In the present study, 150 samples of unpasteurized dairy products including cheese, butter, curd, doogh (a savoury yogurt-based beverage in Iran) and ice cream (30 samples from each product) were randomly purchased from the markets in Tabriz city (Iran) between March 2018 to July 2019. They were transferred aseptically to the laboratory of food hygiene for detection and enumeration of B. cereus using culture method and PCR technique.
Dilutions of 10 − 1 , 10 − 2 and 10 − 3 of samples were prepared and cultured in MYP agar (Mannitol egg-yolk polymyxin phenol red agar) (Merck, Germany) according to the Iranian national standard. Large and pink colonies with a halo detected and enumerated as B. cereus. Isolates were also con rmed by hemolysis test and Gram staining (ISO 7932, 2004).
According to the Kirby-Bauer method, the antibiotic susceptibility pattern of isolates was studied using the disks of antibiotics which are currently used in the area. The sensitivity level and drug resistance were determined according to the CLSI standard (CLSI, 2016).
PCR test was performed to detect the ces gene in the isolates. For this purpose, DNA extraction was done by boiling method. Nanodrop spectrophotometer was used to evaluate the concentration and purity of extracted DNA samples. Ampli cation of 154 bp product of ces gene was performed using the designed primers (Table 1) Table 2. PCR program included an initial denaturation in 94 °C for 3 minutes, followed by 35 cycles of denaturation at 94 °C for 35 seconds, annealing at 55 °C for 35 seconds, extension at 72 °C for 10 seconds and 1 cycle of nal extension at 72 °C for 5 minutes.  In order to separate the ampli ed segments; the 1% agarose gel was prepared and DNA safe stain was added to it. The ladder was loaded into the rst well. The next wells were loaded with samples (mixed with loading buffer). A positive sample of B. cereus PTCC: 1015 was used as a positive control. Then, the lid of the electrophoresis tank was closed. After setting the electric voltage on 100V, the electrophoresis of PCR products was performed for 45 minutes. Afterward, the gel was removed slowly and results were visualized by ultraviolet radiation.
Results were analyzed by SPSS software (version 18) and presented using the indicators of the central tendency and dispersion. For comparison of the contamination level of dairy products with national standard limit (< 1 × 10 2 ) and the contamination level between products, one sample T-test and chi-square test at 95% con dence level and α = 5% were used, respectively.

Results
In the present study, 150 samples of traditional dairy products were evaluated of which 16 samples were contaminated with B. cereus. The contamination level of each unpasteurized dairy product is shown in Table 3. The results of the chi-square test showed a signi cant difference between dairy products about the contamination of B. cereus. The highest contamination level was detected in curd and the lowest level of contamination was found in cheese and butter (p < 0.001). The results of t-test showed that the contamination level of curd has a signi cant difference with the national standard limit of Iran (1 × 10 2 ) (p < 0.05) (Fig. 1).
The results of the antibiogram test (Table 4) showed that most of the isolates were resistant against penicillin, cefazolin and co-trimoxazole, sensitive against gentamicin, cipro oxacin and clindamycin and semi-sensitive to vancomycin and erythromycin, respectively. The results of the PCR test showed that 14 of 16 isolates were contained the ces gene. As shown in

Discussion
Milk and dairy products are the most consumed and important resources in the daily diet of human. They also provide a considerable part of the public's needs for nutrients because of having a variety of vitamins and minerals including calcium.
The attitude of consumers about the side effects of additives in the dairy industry increases their tendency toward the consumption of milk, traditional dairy products and products made from unprocessed or semi-processed milk. Because of the possible presence of human pathogenic microorganisms, consumption of products produced from raw milk can be harmful (Verraes et al. 2015).
In this study, from a total 150 samples of dairy products, 16 samples ( From 30 traditional cheeses that were evaluated in this study, no positive results were found in the samples. However, according to the ndings of Williams et al. (2010) in Scotland, 28% of cheese samples produced from raw milk was contaminated with B. cereus. Since B. cereus can't tolerate the salt concentration more than 7.5%, this contradiction may be due to high salinity in Lighvan cheese (11%), which is known as a type of salty high-fat cheese.
In the present study, 6 (20%) of 30 curd samples were contaminated with B. cereus. Most of the production process of traditional curd is performed manually using traditional equipments. It also airdried in sunlight. So, all of these processes can increase the risk of product contamination by spoilage microorganisms especially B. cereus which can easily contaminate the product by dust. cereus expressed ces gene for production of emetic toxin. Cui et al. (2016) reported that from 9.8% of B. cereus isolates in raw milk samples, 1.1% contained ces gene. Since there is no similar study about the presence of ces gene in curd and doogh, the possible reason for the high content of this gene in these products can also be associated with sanitary conditions in their production.
The study of antibiotic susceptibility pattern of isolates showed that most of them were resistant against penicillin, cefazolin and co-trimoxazole. But they were sensitive against gentamicin, cipro oxacin and clindamycin and semi-sensitive against vancomycin and erythromycin. The ndings of Jalalpour & Abusaeedi (2009) were somewhat similar to results of present study. They found that all strains were resistant against the selected antibiotic against Bacillus infections, which was penicillin. However, they have a signi cant sensitivity against vancomycin, tetracycline and erythromycin. It should be noted that the antibiotic selective stress in each area could be the reason for similarities or differences in antibiotic pattern. In addition, excessive use of antibiotics may lead to resistant strains in the environment.

Conclusion
The ndings of this research indicate that the contamination of unpasteurized dairy products with B. cereus. Regarding high antibiotic resistance and detection of ces gene, there is a potential of intoxication by these products, especially by curd and doogh. So, the preparation and production of these products should carry out carefully. In order to reduce the contamination level to this bacterium which is highly resistant to environmental conditions, it is better to replace unpasteurized dairy products with pasteurized ones.

Declarations
Availability of data and materials All quantitative data generated or analysed during this study are included in this article.
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Competing Interests
The authors declare that they have no con ict of interest. Figure 1 Page 13/13

Figures
The results of T-test, comparison of the contamination level of dairy products with national standard level (1×102)