Toxigenic largest group of endospore-forming bacteria and

Toxigenic Profiling of Enterotoxin-Producing Bacillus cereus, Isolated from Raw Chicken Meat, and Human subjects Swab samples by Triplex and Multiplex PCR
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ABSTRACT

Bacillus cereus comprises the largest group of endospore-forming bacteria and can cause emetic and diarrheal food poisoning. A total of 42 B. cereus strains isolated from various sources (Raw chicken meat, Human subjects Swab samples) were assessed by a Triplex and multiplex PCR for the presence of enterotoxin genes. The detection rate of nheB, hblA, hblD, and cytK, nheA, CER, hblC, entFM enterotoxin genes among all B. cereus strains was 83.33%, 80.95%, 69.04%, 21.42%, 47.61%, 0%, 61.90%, and 92.85%, respectively. Enterotoxigenic profiles were determined in enterotoxin-producing strains (19 patterns). The results provide important information on toxin prevalence and toxigenic profiles of B. cereus from various sources. The study revealed that B. cereus must be considered a serious health hazard and greater potential concern to food safety among all B. cereus group members. Also, there is need for intensive and continuous monitoring of products embracing both emetic toxin and enterotoxin genes.
Keywords: Bacillus cereus, enterotoxigenic profiling, triplex PCR, multiplex PCR
Introduction
Members of the genus Bacillus are well known for their great diversity and widespread distribution in nature (Oh et al., 2012). Of these bacteria Bacillus cereus group (B. cereus sensu lato) consisting of the genetically closely related species B. cereus, Bacillus thuringiensis, Bacillus anthracis, Bacillus mycoides, Bacillus pseudomycoides, and Bacillus weihenstephanensis is widely recognized as the causative agent of foodborne illness (Helgason et al., 2000; Kim et al., 2012; Jim´enez et al., 2013). Due to its wide distribution in the environment and ability to produce spores, the Gram-positive, rod-shaped, opportunistic human pathogen grow well in wide adverse environmental condition, the pH ranges from 4.5 to 9.3, with high water activity 0.92 and the temperature ranges for growth from 4ºC to 50ºC (Kramer and Gilbert, 1989). Foodborne illness resulting from consumption of B. cereus-contaminated food may result in diarrheal or emetic type syndromes (Kim et al., 2010; Sandra et al., 2012).
The diarrheal food poisoning is caused by heat-labile protein enterotoxins produced during vegetative growth of B. cereus in the small intestine with non-haemolytic enterotoxin (nhe), enterotoxin FM (entFM), Haemolysin BL (hbl) and cytotoxin K (cytK) are of the highest importance and therefore often used for the detection of enterotoxic strains (Kim et al., 2012). Symptoms including diarrhea and abdominal pain usually occur 8 to 16 h after ingestion of contaminated food which can be occasionally misdiagnosed with Clostridium perfringens food poisoning (Park et al., 2009).
The emetic food poisoning occur due to emetic toxin (cereulide) is a small cyclic peptide (dodecadepsipeptide), which is heat and acid stable, induces swelling of mitochondria in Hep-2 cells, respiratory distress, and occasional loss of consciousness possibly leading to coma and ultimately death of individual (Ladeuze et al., 2011). The heat stable emetic toxin can withstand temperatures of up to 121°C for 90 min. The symptoms usually include nausea, vomiting and stomach pain, which occur 1-5 h after food ingestion and can easily be misdiagnosed with Staphylococcus aureus food poisoning (Kim et al., 2011).
B. cereus is present in starch reach foods such as rice, nutrient enriched foods of animal origin as like meat, milk, dairy products and chicken meat and desserts etc (Jay, 2005). In India, occurrence of B. cereus has been reported from foods like milk (Garg et al., 1977; Chopra et al., 1980), meat (Bacchil and Negi, 1984; Bacchil and Jaiswal, 1988), chicken meat (Tahmasebi et al., 2014; Aklilu et al., 2016) and various other foods (Kamat et al., 1989; Meena et al., 2000). Therefore, it is of great food safety, food quality, medical, and economic importance, and several methods have been developed for its detection. These methods include a wide range of approaches including but not limited to conventional culture methods (Fricker et al., 2008), biochemical and morphological tests (Fernandez-No et al., 2011), and last but not least a variety of molecular approaches using toxin or other appropriate genes of B. cereus group species as target (Kim et al., 2012). Recently, molecular diagnostic tools mainly focus on the toxin genes since they are broadly distributed within the B. cereus group (Wehrle et al., 2010) and the toxin gene profiles might be better than the exact species for the outbreak investigations (Ehling-schulz and Messelhausser, 2013). Also, assessment of toxin genes prevalence in wild B. cereus isolates would help controlling these potential pathogens in food industry (McKillip, 2000). Furthermore, such assessments would be valuable to determine the genetic diversity of the species leading to a broader ecological view (Oh et al., 2012). Therefore, there have been some studies investigating the toxigenic diversity of B. cereus isolated from different sources. There is restricted information available in comparison to the enterotoxic strains. accordingly, the reason of this study became to: (a) estimate the pathogenic capacity of B. cereus through determining the toxigenic profiles of lines strains; (b) investigate the relationship between isolation source and toxigenic profiles, compare toxigenic patterns, and compare the results with previous studies to have a better insight on the topic.
Materials and Methods
Bacterial strains
B. cereus strains used in this study were obtained from the raw chicken meat and human subjects swab samples. A total of 42 strains isolated were used consisting of 0 emetic toxin- and 42 enterotoxin-producing strains. The detailed source of strains was as follows: Thigh muscle (8 strains), Breast muscle (4 strains), Wing muscle (4 strains), Rib muscle (2 strains), Neck muscle (4 strains), Heart portion (3 strains), Liver portion (4 strains), Gizzard portion (1 strains), Chicken meat handler (5 strains), Butchers knife (3 strains), and Chopping board (4 strains).
DNA extraction
All B. cereus strains were grown on nutrient agar (HiMedia Pvt. Ltd.) slants at 37 °C for 24 h. A single colony was inoculated in Luria-Bertani broth (HiMedia) and incubated at 37 °C for 24 h. After recommended incubation, the total genomic DNA of individual was extracted by using mericon DNA Bacteria plus Kit (Qiagen) according to the manufacturer’s instructions with some modification. The quality, purity and concentration of isolated DNA were determined by agarose gel electrophoresis and NanoDrop™2000/2000c Spectrophotometers. The extracted genomic DNA did not show any shearing during gel electrophoresis, which indicated its superior quality. The DNA samples were analysed by measuring its absorbance at 260 and 280 nm. All the samples showed A260/A280 ratio more than 1.8 and found suitable for PCR amplification. This observation was in agreement with the method described by Sambrook and Russel (2001).

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