PCR assay a new approach for detection of enterotoxins

INTRODUCTION

The genus Bacillus contains 51 species and is divided into three groups based on the morphology of spores and sporangia (Groups 1-3). Group 1 is subdivided into 1A and 1B based on the cell size and the presence of poly – β – hydroxybutyrate in the cytoplasm. The organisms belong to Group 1A (B. cereus group) are; B. cereus, B. anthracis, B. thuringiensis, B. mycoides, B. psuedomycoides and B. weihenstephanensis. Enterotoxin production is linked to Bacillus induced disease and a majority members of Group 1A produce enterotoxin (Bhunia, 2008).

Bacillus cereus is Gram – positive, rod shaped, spore forming bacterium and a human opportunistic pathogen, which can cause diarrheal and emetic types of food poisoning (Arnsen et al.,2008). B. cereus is commonly found in a wide variety of different foods and environments (Duc et al., 2005; Bartoszewicz et al., 2008).

The enterotoxins that have been associated with diarrheal type food poisoning are hemolysin BL (HBL), non-hemolytic enterotoxin (NHE), cytotoxin K (CytK) and enterotoxin FM (Lund et al., 2000; Sergeev et al., 2005). However, enterotoxins have already been comparatively well characterized at the molecular level (Lund et al., 2000) and in immunochemical assay (Dietrich et al., 2005).

Additionally, emetic type food poisoning is caused by an emetic toxin (cereulide). This type of food poisoning is small, heat and acid stable circular dodecadepsipeptide with the following stereochemistry: [D-O-Leu-D-Ala-L-O-Val-L-Val]. (Ehling-Schulz et al., 2005).

However, since the first case of human food poisoning by Bacillus group was detected, B. cereus group has been implicated in various outbreaks worldwide (Stenfors et al., 2008; Bennett et al., 2013).

Moreover, B. cereus group is considered as potential problem since it can contaminate many dairy products in several countries (Sadek et al., 2006; Reyes et al., 2007; Bartoszewicz et al., 2008; Zhou et al., 2010; Mohamed et al., 2016). This group is able to cause two types of syndroms i.e. diarrheal and emetic. Also, B. cereus group is an important spoilage organism to many foods (Tan et al., 1997; Fermanian, 1997).

Traditionally, B. cereus group have been associated with the spoilage of food products, however, recently they have been linked to potential food poisoning issues (Rodriguez-Lozano et al., 2010).

Recently, several investigators used Triple-primer polymerase chain reaction (PCR) assay to detect diarrheal and emetic toxins producing by B. cereus group strains (Zhou et al., 2010; Kim et al., 2013).

Therefore, the aim of the current study was to used a reliable and accuracy method for detection enterotoxic genes by PCR technique in B. cereus group strains.

MATERIALS AND METHODS

Gel preparation (1% agarose gel)

 Agarose gel 1% is prepared by adding 1g. of agarose to 100 ml. TBE. The solution was boiled to dissolve the agarose in microware oven for 1-3 min., and cooled down to 45˚C, 3µL of ethidium bromide (1%) was added and left for solidification at room temperature.

PCR master mix

GoTaq Green Master Mix, is a premixed ready-to-use solution containing bacterially drived Taq DNA polymerase, dNTPs, Mg Cl₂ and reaction buffers at optimal concentration for efficient amplification of DNA templates by PCR.

Genomic DNA isolation Kit

 Total genomic DNA was extracted from ten isolates according to manufacture instructions using Zymo Research. Fungal/ Bacterial DNA Mini Prep^TM Kit (catalog No. D6005 ZR CORP, India). The Kits were purchased from Sigma Company, Egypt. PCR primers and annealing temperature used for the detection of emetic and diarrheal toxins:

The three primer sets which used in the present study are illustrated in Table 1.

Table1. PCR primers and annealing temperature for the detection of emetic and diarrheal toxin producing B. cereus group.

AT= Annealing Temperature.

Amplified fragments visualization

Agarose gel electrophoresis 1.5% (wt/vol) was used for migrating the amplified DNA fragments. Gel was stained with ethidium bromide (0.5 µL/mL) and directly loaded on the gel, DNA ladder was also loaded on the gel for fragment size comparison and visualized under UV light using electrophoresis machine (Mupid-exU, Mupid, Tokyo, Japan). Also, the concentration of the extracted DNA was measured by using UV- spectrophotometer (Model UV-1700, Shimadzu, Tokyo, Japan).

Tested Bacillus group strains

Ten identified Bacillus cultures were used in the present study, six isolates belonging to Bacillus cereus, while the rest four cultures were belonging to Bacillus mycoides. However, all the tested cultures were isolated from different dairy products.

DNA extraction from Bacillus group strains   

The DNA was extracted according to manufacture instructions by using DNA extraction kit (Wizard Genomic DNA purification kit, Promega, Madison, WI, USA).

PCR amplification and PCR reaction condition

The PCR amplification reactions were prepared in a total volume of 20 μL contained 30 pM of each primer, 20 ng of DNA template, 10 mM Tris-HCl, 1.5 mM MgCl₂, 30 mM KCl, 250 μM dNTP mixture and 1 U Top DNA polymerase (Bioneer, Daejeon, Korea).

The PCR conditions were as follows

Per-denaturation at 94 ˚C for 10 min., followed by 35 cycles of 94˚C for 1 min., annealing temperature was tested at 54 ˚C and 72 ˚C for 1 min. and a final elongation cycle at 72 ˚C for 5 min.

RESULTS AND DISCUSSION

Nowadays, molecular techniques have been increased and successfully applied to detect diarrheal and emetic toxins producing by Bacillus cereus group strains (Kim et al. 2013). Thus, polymerase chain reaction (PCR) assay using B. cereus group specific triple – primer CER, CES and groEL primers was carried out.

In this respect, a total genomic DNA was extracted from the ten tested isolates by using Zymo Research Fungal / Bacterial DNA Prep^TM Kit. Also three species specific primer pairs were used. Results from Table 1 showed that after testing each of the three primer set, groEL-F-1 / groEL-R-1 was highly specific for detection of gene groEl.

Results of amplified PCR fragments for ten tested Bacillus strains are shown in Figure 1. It is very clear from the results obtained that product of Bacillus cereus Dc 8-1 which isolated from Domiati cheese scored 236 bp in lan 1 by using primer pair (groEL F-1/groEL R-1). In addition, strains Bacillus cereus Rc 5-1 and Rc6-1 that were isolated from Ras cheese yielded amplified fragment of 236 bp in lan (3 and 4) by using primer (groEL F-1/groEL R-1).

Moreover, it is of interest to notice that both strains belonging to B. mycoides, named Rc 2-1 and Dc 7-1, isolated from Ras and Domiati cheese, respectively, showed amplified product of 236 bp in lan (5 and 6) by using the same primer (groEL F-1/groEL R-1).

In contrast, none of rest tested strains either B. cereus or B. mycoides yielded any amplified product in this PCR reaction as shown in Figure 1 and Table 1.

Figure 1. Gel electrophoresis of PCR products amplified with triple-primer on emetic and diarrheal toxin producing B. cereus and B. mycoides strains. M: DNA ladder 100 bp from promega. Lane 1: B. cereus  Dc8-1, Lane 2: B. mycoides Dc1-1, Lane 3: B. cereus Rc5-1, Lane 4: B. cereus Rc6-1, Lane 5: B. mycoides Rc2-1, Lane 6: B. mycoides Dc7-1, Lane 7: B. mycoides Rm5-2, Lane 8: B. cereus Rc5-2, Lane 9: B. cereus Dc10-1, Lane 10: B. cereus Dc10-2. 

Results of Table (2) showed that all the diarrheal enterotoxin producing B. cereus strains showed the presence of groEL gene but CER and CES genes were not present in any of the B. cereus strains tested in this study. Results also, clearly indicated that the 3 B. cereus strains out of 6 strains from Domiati cheese (one strain), namely B. cereus Dc8-1 and Ras cheese (2 strains), namely B. cereus Rc5-1 and B. cereus Rc6-1 gave positive with groEL gene only and gave negative with CER and CES genes.

Table 2. PCR assays of emetic and diarrheal enterotoxins producing by B. cereus strains.

It could be extracted from Table (3) that, all the diarrheal enterotoxin producing B. mycoides strains showed the presence of groEL gene but CER and CES genes were not present in any of the B. mycoides strains tested in this study. Results also, indicated that the 2 B. mycoides strains out of 4 strains from Ras cheese (one strain), namely B. mycoides Rc2-1 and Domiati cheese (one strain), namely B. mycoides Dc7-1 gave positive with groEL gene only and gave negative with CER and CES genes.

 Table (4) also, shows that 50% of tested B. cereus strains from both Ras cheese and Domiati cheese samples were toxigenic and showed the presence of groEL gene but CER and CES genes were not present in any of the B. cereus strains tested. The same behavior was noticed with 50% of tested B. mycoides strains which showed the presence of groEL gene but CER and CES genes were not present in any of the B. mycoides strains tested CONCLUSION

The obtained results in this study revealed that the tested strains of B. cereus and B. mycoides showed the presence of groEL genes, while CER and CES genes were completely absent. B. cereus group had an ability to produce diarrheal enterotoxin, but the tested strains of all B. cereus and B. mycoides had a limited ability to produce emetic toxin.

Table 3. PCR assays of emetic and diarrheal enterotoxins producing by B. mycoides strains.

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Table 4. Enterotoxin genes in B. cereus and B. mycoides strains.