Distribution of Toxin Genes and Enterotoxins in Bacillus thuringiensis Isolated from Microbial Insecticide Products

Abstract

Bacillus thuringiensis microbial insecticide products have been applied worldwide. Although a few cases of B. thuringiensis foodborne illness have been reported, little is known about the toxigenic properties of B. thuringiensis isolates. The aims of this study were to estimate the pathogenic potential of B. thuringiensis selected from microbial insecticide products, based on its possession of toxin genes and production of enterotoxins. Fifty-two B. thuringiensis strains selected from four kinds of microbial insecticide products were analyzed. PCR assay for detection of toxin genes and immunoassay for detection of enterotoxins were performed. The hemolysin BL complex as a major enterotoxin was produced by 17 (32.7%), whereas the non-hemolytic enterotoxin complex was detected in 1 (1.9%) of 52 B. thuringiensis strains. However, cytK, entFM, and ces genes were not detected in any of the tested B. thuringiensis strains. The potential risk of food poisoning by B. thuringiensis along with concerns over B. thuringiensis microbial insecticide products has gained attention recently. Thus, microbial insecticide products based on B. thuringiensis should be carefully controlled.

Introduction

Bacillus cereus has been recognized as the causative agent of diarrheal and emetic food poisoning associated with various categories of foods [5]. Food poisoning is induced by enterotoxins and emetic toxins such as hemolysin BL (HBL), non-hemolytic enterotoxin (NHE), enterotoxin FM (EntFM), cytotoxin K (CytK), and cereulide (emetic toxin) [34]. Concerns over B. cereus food poisoning are growing in the food industry [24]. Bacillus thuringiensis is commonly isolated from the environment, such as water, soil, insects, vegetables, and foods [1,3,12,29,39], and has been widely applied worldwide as a microbial insecticide to reduce amounts of chemical pesticides [22] based on its advantages, such as no harmful effects on humans or ecological environment, low impact on non-target organisms, and a narrow spectrum of lepidopteran targets [28].

B. cereus and B. thuringiensis cannot be discriminated by using genetic and phenotypic assays [25,32], as they share high 16S rRNA gene sequence similarity (>99%) [31]. These reports suggest that these two strains are one species or at least originate from a common ancestor [21]. The distinguished characteristics between B. cereus and B. thuringiensis are the presence or absence of plasmid-encoded virulence properties, such as crystal proteins (δ-endotoxin) encoded by cry genes, which show insecticidal activity in B. thuringiensis [32]. Thus, an enumeration method for B. cereus, except B. thuringiensis cell counts, was established in the Korea Food Code.

On the other hand, cry genes encoded in the plasmid show high potential for horizontal gene transfer between B. thuringiensis and B. cereus [20,41]. B. thuringiensis, which lacks the cry gene, is indistinguishable from B. cereus [18]. These reports indicate that B. thuringiensis might be a foodpoisoning bacterium, as a few cases of food poisoning caused by B. thuringiensis have been reported [19,37]. Thus, it is necessary to evaluate the toxigenic potential of B. thuringiensis applied as microbial insecticides, even though little is known about the toxigenic properties of B. thuringiensis microbial insecticide products.

The objectives of this study were to identify B. thuringiensis selected from microbial insecticide products and to estimate its pathogenic potential based on possession of toxin genes and production of enterotoxins.

 

Materials and Methods

Samples

Four kinds of microbial insecticide products based on B. thuringiensis were purchased from Gyeonggi-do and the Internet from 2011 to 2013. Each microbial insecticide product was named as A, B, C, and D, respectively.

Biochemical Identification of B. thuringiensis

Each microbial insecticide product (1 g or 1 ml) was mixed with 10 ml of buffered peptone solution (Oxoid Ltd., Basingstoke, UK) and homogenized by a Vortex-Genie 2 mixer (Scientific Industries Inc., Bohemia, NY, USA) for 1 min. After vortexing, 1 ml of homogenate was serially diluted (10-fold) in 0.85% saline, and 100 μl of each diluent was spread onto Mannitol-Egg Yolk-Polymyxin agar (MYP; Difco, Detroit, MI, USA) and incubated at 30°C for 20 h. Thirteen colonies showing a pink color on MYP agar inoculated from each microbial insecticide product (total of 52 colonies) were randomly selected for further culture on tryptone soy agar (TSA; Oxoid Ltd) at 30°C for 20 h. Biochemical identification of the selected strains was carried out using the Vitek-II system with a BCL card (bioMérieux, Inc., Marcy l’Etoile, France) according to the manufacturer’s directions.

Detection of Crystal Proteins

Microscopy observation was conducted to confirm the crystal proteins (δ-endotoxins; crystal shaped) produced by the selected B. thuringiensis strains. An optical microscope (Axioskop 2 plus; ZEISS, Jena, Germany) was used to observe the crystal proteins. The selected strains were cultured on TSA at 30°C for more than 120 h [9], after which cells were stained with TB carbol-fuchsin ZN (Difco) by using a simple staining procedure. The crystal proteins were observed with an oil immersion lens.

Immunoassay for Detection of Enterotoxins

The selected B. thuringiensis strains were cultured in tryptone soy broth (Oxoid Ltd) at 30°C for 24 h, after which 1 ml of each culture was centrifuged for 10 min at 10,000 ×g. The supernatants were applied for detection of enterotoxins, using immunoassay kits according to the manufacturer’s directions. The hemolysin BL enterotoxin was determined using an enterotoxin-reversed passive latex agglutination (BCET-RPLA) kit (Oxoid), and non-hemolytic enterotoxin was detected with a Bacillus diarrheal enterotoxin visual immunoassay (BDE-VIA) kit (Tecra Diagnostics, Reading, UK). B. cereus ATCC 14579 and F4810/72 were used as reference strains.

DNA Extraction

All the B. thuringiensis strains selected, and the B. cereus ATCC 14579 and F4810/72 reference strains, were cultured in tryptone soy broth at 35°C for 18 h, after which 1 ml of the culture broth was centrifuged at 10,000 ×g for 10 min at 4°C (Mega 17R; Hanil Science Industrial, Incheon, Korea). The pellet was suspended in 500 μl of sterilized distilled water, centrifuged under the same conditions as before, and resuspended again in 500 μl of sterilized distilled water. The suspended pellet was boiled for 10 min and centrifuged at 10,000 ×g for 10 min at 4°C. The supernatants were stored at -20°C until analysis for templates.

PCR Assay for Detection of Toxin Genes

The primers for detection of toxin genes are presented in Table 1. PCR amplification with a final volume of 20 μl was conducted using a thermal cycler (Mastercycler Gradient S; Eppendorf, Germany) with the reaction mixtures (AccuPower PCR PreMix; Bioneer, Daejeon, Korea) containing 2 μl of template DNA. The PCR assay for detection of cytK was 95°C for 1 min, followed by 30 cycles of 95°C for 60 sec, 48°C for 60 sec, and 72°C for 60 sec, and a final extension at 72°C for 7 min. For the entFM gene, PCR assay was carried out at 95°C for 3 min, followed by 35 cycles of 95°C for 30 sec, 60°C for 30 sec, and 72°C for 60 sec, and a final extension at 72°C for 5 min. The PCR program for detection of the ces gene was 95°C for 15 min, followed by 25 cycles of denaturation at 95°C for 60 sec, extension at 72°C for 50 sec, and a final extension at 72°C for 5 min. The annealing condition was at 58°C for 75 sec. The amplified products were separated by an automated capillary electrophoresis system (QIAxcel; Qiagen, Hilden, Germany) with a QIAxcel DNA high resolution kit and Qiaxcel DNA size marker (100 bp). Electrophoresis was conducted by the OM400 method (sample injection voltage 5 kV for 10 sec, separation voltage 6 kV for 400 sec), and the gel image was visualized with Bi°Calculator 3.0 (Qiagen). B. cereus ATCC 14579 and F4810/72 were used as reference strains.

Table 1.Primer sequences for detection of toxin genes.

 

Results and Discussion

Confirmation of B. thuringiensis

Biochemical identification and microscopic observation were conducted to confirm the B. thuringiensis. The 13 strains randomly selected from each microbial insecticide product based on B. thuringiensis (total of 52 strains) were identified as B. cereus/B. thuringiensis using the Vitek-II system with a BCL card. The genetic and phenotypic properties between B. thuringiensis and B. cereus are rarely distinguishable [25,32], and Helgason et al. [11] reported that these two strains might be one species. The Vitek-II system cannot discriminate between B. cereus and B. thuringiensis. The distinguished characteristics of B. thuringiensis are the presence of an insecticidal crystal protein (δ-endotoxins; crystal shaped) [28,32]. Microscopic observation of crystal-shaped proteins was conducted to confirm the B. thuringiensis, strains. The results of crystalshaped protein observation are shown in Table 2. Visible crystal-shaped proteins were detected in all strains selected from microbial insecticide products (Fig. 1). Thus, all strains selected from microbial insecticide products were identified as B. thuringiensis, based on biochemical identification and microscopic observation for crystal-shaped proteins.

Table 2.aB. cereus enterotoxin-reversed passive latex agglutination (BCET-RPLA) kit was used to detect hemolysin BL (HBL). bBacillus diarrheal enterotoxin visual immunoassay (BDE-VIA) kit was used to detect non-hemolytic enterotoxin (NHE). cBacillus cereus 14579 as enterotoxic reference strain and Bacillus cereus F4810/72 as emetic reference strain.

Fig. 1.Photomicrographs showing crystal proteins (δ-endotoxin) of Bacillus thuringiensis from microbial insecticide products.

Detection of Enterotoxins in B. thuringiensis

HBL and NHE are the major virulence factors among the HBL, NHE, CytK, EntFM, and cereulide (emetic toxin) produced by B. cereus [23,37]. In order to estimate the potential risk of food poisoning by the B. thuringiensis strains selected from microbial insecticide products, we investigated for the production of HBL and NHE, and the results are shown in Table 2. The HBL and NHE enterotoxins were produced by 17 (32.7%) and 1 (1.9%) out of 52 B. thuringiensis selected strains, respectively. Only the D1 strain produced both HBL and NHE simultaneously. These results indicate that the strains tested in this study were from different microbial insecticidal products such as A, B, C, and D. The production of HBL enterotoxin composed of B, L2, and L1 subunits depends on the expression of all three genes; namely, hblC, hblD, and hblA [30]. The NHE enterotoxin complex comprises proteins NheA, NheB, and NheC encoded by nheA, nheB, and nheC [33]. The NHE is only produced when all three NHE enterotoxin complexes are present [30]. Rosenquist et al. [29] reported that HBL and NHE are expressed in 36 (90.0%) and 40 (100%) out of 40 B. cereus-like organisms, respectively, and 81–94% of B. cereus isolated from clinical and food samples [4,15]. The much lower results of HBL (32.7%) and NHE (1.9%) production rates of B. thuringiensis are in contrast to previous studies where HBL-positive rates were present in 24 (85.7%) of 28 B. cereus-like organisms that possessed crystal protein [29], and all of the 59 B. thuringiensis strains (100%) were HBL positive [42]. NHE production rates were reported in 4 (30.7%) out of 13 B. thuringiensis isolates from rice products and 15 (36.6%) out of 41 B. thuringiensis isolates [10,24]. B. thuringiensis from human fecal samples produced enterotoxins and presented similar DNA fingerprints as microbial insecticide products based on B. thuringiensis without gastrointestinal symptoms [13]. This report indicates that human illness is not directly related with microbial insecticide products based on B. thuringiensis. However, there are concerns over B. thuringiensis microbial insecticide products based on the HBL enterotoxin production in the present study. Thus, we should ensure that B. thuringiensis strains in microbial insecticides cannot produce enterotoxins. The enumeration method for B. cereus in the Korea Food Code, which excludes B. thuringiensis cell counts, will be reconsidered to reduce food safety concerns.

Detection of Toxin Genes in B. thuringiensis

The distributions of cytK, entFM, and ces genes among B. thuringiensis strains selected from microbial insecticide products are presented in Table 2. The cytK, entFM, and ces genes were not detected in any of the B. thuringiensis strains. The cytK gene-encoded enterotoxin causing cytotoxic disease has been implicated in three deaths in France and is associated with severe foodborne outbreak of hemolysis [17,21]. Oh et al. [24] reported the cytK gene in 5 out of 13 (38.5%) B. thuringiensis strains from rice products. The distribution of cytK gene ranges from 0% to 4.7% in B. thuringiensis isolated from cooked rice (0 out of 20 strains), milk, and soil (1 out of 21 strains) [2,14]. The variable distribution of cytK gene is in the range of 13% to 73% of B. cereus isolated from the food, environment, and patients [8,15,36]. The entFM gene-encoded enterotoxin is thought to be a cell wall peptidase that participates in biofilm formation, adhesion, and virulence [40]. Prabhakar and Bishop [26] demonstrated that all nine Antarctic B. thuringiensis isolates carry the entFM gene. Kim et al. [15] demonstrated that B. cereus isolated in Korea carries the entFM gene (65%), and another study also detected the entFM gene in all B. cereus isolates from Sunsik [4]. The emetic toxin cereulide has a molecular mass of 1.2 kDa and is an acid-stable cyclic peptide ([D-O-Leu-D-Ala-L-O-Val-L-Val]3) [6]. Cereulide also resists different proteolytic enzymes and has remarkable heat stability [27]. The ces gene encoding cereulide synthetase was not detected in any of the strains tested in this study, which is consistent with previous reports of the ces gene in only 0.05% of B. cereus isolated in a dairy production chain [38] as well as reports that emetic strains are rare [16]. The cytK, entFM, and ces genes were not detected in B. thuringiensis in this study, which suggests that HBL enterotoxin was the most frequent toxin.

In conclusion, these results indicate that there is a potential risk of food poisoning by B. thuringiensis, raising concerns over B. thuringiensis microbial insecticide products. Thus, more study into the toxigenic properties of different B. thuringiensis strains is needed, and microbial insecticide products based on B. thuringiensis should be carefully controlled.