Volume 29, Issue 9 (12-2022)                   RJMS 2022, 29(9): 31-40 | Back to browse issues page

Research code: 45897
Ethics code: IR.TUMS.VCR.REC.1398. 857
Clinical trials code: شامل این پژوهش نمی شود

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Soltan Dallal M M, Nabatchian F, Sharifi-Yazdi S, Haghighat Khajavi S, pourmoradian M. Isolation and Identification of Bacillus cereus of Dry Bulk and Packaged Vegetables by Culture and PCR. RJMS 2022; 29 (9) :31-40
URL: http://rjms.iums.ac.ir/article-1-6910-en.html
Professor, Food Microbiology Research Center/ Division of Food Microbiology, Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran , msoltandallal@gmail.com
Abstract:   (673 Views)
Background & Aims: Bacillus cereus is a Gram-positive, rod-shaped, facultative anaerobic, motile, beta-hemolytic, spore forming bacterium commonly The bacteria is commonly found in the environment, is often found in soil and vegetation, and can be present in foods. It can cause foodborne illness worldwide. B. cereus illness is related to many foods - beef, turkey, rice, beans, and vegetables. Specifically, the diarrheal illness is often related to meats, milk, vegetables, and fish. This product may be contaminated with many bacterial pathogens, including Bacillus cereus (4). There are several ways to store vegetables; one way is drying, which is important for several reasons: long-term storage, reducing the volume for the storage, and move products more easily and faster The emetic-type illness is most often associated with rice products, but it has also been associated with other types of starchy products such as potato, pasta, and cheese products (5). Some food-mixtures (sauces, puddings, soups, casseroles, pastries, and salads, have been associated with food-borne illness in general. There are two types of food-borne B. cereus illness. In the first, contaminated food (many types of food, often left at room temperature) makes its way to the small intestine where the toxin, in this case, a large-molecular-weight protein, is released. This can lead to diarrhea, cramps, and sometimes nausea. Usually, vomiting is not present in this form of illness (6, 7). Incubation for the first type is 6 to 15 hours. In the second type, affected food, most often starchy food, and classically, rice, contains a different type of toxin (cereulide, an ionophoric low-molecular-weight dodecadepsipeptide that is pH-stable and heat and protease-resistant). This toxin causes emetic-type B. cereus illness. The incubation period for this type is 30 minutes to 6 hours (9). Bacillus cereus is caused by the ingestion of food contaminated with either the enterotoxigenic B. cereus or with the emetic toxin. In non-gastrointestinal illness, reports of respiratory infections similar to respiratory anthrax have been attributed to B. cereus strains harboring B. anthracis toxin genes (10). 
The spore of this bacterium is resistant to severe environmental conditions including heat, freezing, drying, and radiation, and may be considered an infectious agent for this bacterium. Changes in diet and lifestyle in recent years and the growing trend of using ready-made products (4). In the gastrointestinal tract (small intestine), vegetative cells, ingested as viable cells or spores, produce and secrete a protein enterotoxin and induce a diarrheal syndrome, whereas emetic toxin, a plasmid-encoded cyclic peptide (cereulide), is produced in food products and ingested as a formed toxin. Dry vegetables it is suitable for the survival quickly multiply at room temperature (12). There are two main types of intestinal illnesses caused by B. cereus. One is diarrheal, and one leads more to nausea/vomiting. B. cereus has also been implicated in infections of the eye, respiratory tract, and in wounds. The pathogenicity of B. cereus, whether intestinal or nonintestinal, is intimately associated with the production of tissue-destructive exoenzymes. Among these secreted toxins are four hemolysins, three distinct phospholipases, an emesis-inducing toxin, and proteases. The specific name, cereus, meaning "waxy" in Latin, refers to the appearance of colonies grown on blood agar.Bacillus cereus is one of the most important causes of spore-bearing bacteria such as Bacillus (5, 7, 11). The aim of this study was to isolate and identify Bacillus cereus in bulk and packaged dried vegetables by culture and PCR.
Methods: This is a cross-sectional descriptive study from February 2019 to October 2020. In total 160 samples (80 from each of open and packed dried vegetables) were evaluated for the contamination of Bacillus cereus. 25 g of the sample was poured into 225 ml of peptone water and incubated at 37 ° C for 24 hours and then one ml of 0.1 dilutions of the suspension was inoculated into on the specific medium of Bacillus cereus (Scharlau, Spain). (MYP Agar) Mannitol Egg Yolk Polymyxin Agar. For the total count, 10 g of each dried vegetable was added into 90 ml of 0.1% peptone water and then 1 ml of dilutions (10-1, 10-2, 10-3), were inoculated into MYP Agar medium and incubated at 37 ° C for 24 hours.
Large pink colonies (no fermentation of mannitol) with a precipitated halo (due to lecithinase production) were considered suspicious colonies. The identification was carried out by catalase and biochemical tests. anaerobic conditions, growth at 45 ° C, lestinase C test, hemolysis B in blood agar with 5% sheep blood, MR-VP test, penicillin sensitivity 10 IU test,and nitrate reduction(13). For molecular analysis, the polymerase chain reaction for the internal transcribed spacer (ITS) gene was used to confirm Bacillus cereus. In order to extract DNA from a pure colony of bacteria, it was cultured in an LB medium and after incubation, the resulting culture was precipitated and the genome was extracted using phenol-chloroform. The quality and quantity of DNA extracted were evaluated by spectrophotometry and electrophoresis. The extracted DNA was frozen at -20 ° C for later use (14). The PCR program consisted of an initial temperature of 94 ° C for 5 min, then 35 cycles consisting of 94 ° C for 30 s, 52 ° for 30 s, and 72 ° for 30 s. PCR reaction was performed in Thermal Cycler (Primus 96, Peqlab Biotechnologie GmbH, Erlangen, Germany). The standard strain of 11778 Bacillus cereus was used as a positive control. The PCR product was electrophoresed in 1% agarose gel at a voltage of 100-80 volts and after staining with ethidium bromide 1 mg/ml was observed and photographed by a gel dock device. Initially, the PCR gradient for the gene was placed in the annealing temperature range of 50 to 60 degrees, which was finally selected at an optimum temperature of 52 degrees (15). Using SPS software, data were analyzed by chi-square test. p<0.05 was considered significant.
Results: The B. cereus contamination were found in 21 (26.25%) and 13 (16.25%) of open and packed dried vegetable samples respectively. There was no statistically significant difference (p>0.05) between contamination rate of B. cereus in open and packed dried vegetable samples. Also, contamination rate of B. cereus was not significantly different (p>0.05) among various kinds of vegetable samples. The results of this study showed that 34 (21.25%) out of 160 samples of dried vegetables were infected with Bacillus cereus by the phenotypic method. PCR results of the ITS gene showed that all 34 strains isolated by culture were also identified by molecular method. In total 7 (4.37%) dill, 5 (3.12%) tarragon, 5 (3.12%) parsley, 5 (3.12%) mint, 6 (3.75%) soup, 4 (2.5%) coriander and 2 (1.25%) turmeric were identified as Bacillus cereus respectively.
Conclusion: Our study showed that despite the supply of packaged dried vegetables, there is still the possibility of microbial contamination, especially by anaerobic bacteria. Although the rate of contamination of dried vegetables in open packaged (13.2%) was more than dried packaged (12.8%).This percentage of contamination probably indicates a lack of hygiene in the drying process in both traditional and industrial forms.
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Type of Study: Research | Subject: Microbiology

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