J. Microbiol. Biotechnol. (2015), 25(6), 872–879 http://dx.doi.org/10.4014/jmb.1502.02003 Research Article Review jmb Various Enterotoxin and Other Virulence Factor Genes Widespread Among Bacillus cereus and Bacillus thuringiensis Strains Min-Ju Kim1, Jae-Kwang Han1, Jong-Su Park1, Jin-Sung Lee2, Soon-Ho Lee3, Joon-Il Cho4, and Keun-Sung Kim1* 1Department of Food Science and Technology, Chung-Ang University, Ansung 456-756, Republic of Korea 2Department of Biological Sciences, Kyonggi University, Suwon 442-760, Republic of Korea 3Nutrition Safety Policy Division, and 4Division of Food Microbiology, The Korean Ministry of Food and Drug Safety, Cheongju 361-709, Republic of Korea Received: February 3, 2015 Revised: March 2, 2015 Many strains of Bacillus cereus cause gastrointestinal diseases, and the closely related insect Accepted: March 15, 2015 pathogen Bacillus thuringiensis has also been involved in outbreaks of diarrhea. The diarrheal diseases are attributed to enterotoxins. Sixteen reference strains of B. cereus and nine commercial and 12 reference strains of B. thuringiensis were screened by PCR for the presence First published online of 10 enterotoxigenic genes (hblA, hblC, hblD, nheA, nheB, nheC, cytK, bceT, entFM, and entS), March 20, 2015 one emetogenic gene (ces), seven hemolytic genes (hlyA, hlyII, hlyIII, plcA, cerA, cerB, and cerO), *Corresponding author and a pleiotropic transcriptional activator gene (plcR). These genes encode various Phone: +82-31-670-3032; enterotoxins and other virulence factors thought to play a role in infections of mammals. Fax: +82-31-675-4853; Amplicons were successfully generated from the strains of B. cereus and B. thuringiensis for E-mail: [email protected] each of these sequences, except the ces gene. Intriguingly, the majority of these B. cereus enterotoxin genes and other virulence factor genes appeared to be widespread among pISSN 1017-7825, eISSN 1738-8872 B. thuringiensis strains as well as B. cereus strains. Copyright© 2015 by The Korean Society for Microbiology Keywords: Bacillus cereus, Bacillus thuringiensis, enterotoxins, virulence factors, genes and Biotechnology Introduction by the cytotoxin K (cytK), enterotoxin FM (entFM), enterotoxin S (entS), and enterotoxin T (bceT) genes [2, 11, 18, 26]. The The Bacillus cereus sensu lato group contains six valid virulence of the emetic strains is related to cereulide, a species, including Bacillus cereus and Bacillus thuringiensis, thermostable cyclic dodecadepsipeptide synthesized by a which show a high degree of phenotypic similarity [21]. The nonribosomal peptide synthetase encoded by ces genes [10]. classification of these species is based on pathogenicity to Products from other genes, such as hemolysin A (hlyA), mammals or insects, plasmid content, and gross morphological hemolysin II (hlyII), hemolysin III (hlyIII), phosphatidylinositol- characteristics [30, 31]. The genetic similarity among members specific phospholipase C (plcA), cereolysin A or phospholipase of the B. cereus group has been extensively studied by C (cerA), cereolysin B or sphingomyelinase (cerB), cereolysin means of various molecular methods [17, 19, 28, 30]. O (cerO), and their pleiotropic transcriptional activator B. cereus is an opportunistic endospore-forming bacterium (plcR), are also involved in the pathogenesis of B. cereus [3, involved in a range of intestinal and extraintestinal 20, 32, 35, 37, 38]. B. thuringiensis is a typical endospore- infections [5, 37]. It is a common soil inhabitant that is often forming bacterium distinguished by the accumulation of implicated in food poisoning in humans [35, 37]. The polypeptides that form a crystalline parasporal inclusion virulence of this bacterium has been ascribed to different during sporulation [34]. These crystal proteins may be toxic extracellular factors. Two of these virulence factors, to insects, leading to the extensive use of B. thuringiensis as hemolysin BL (HBL) [4] and nonhemolytic enterotoxin a biological insecticide for crop protection [8, 34]. (NHE) [14], are tripartite protein complexes. Other Recent studies have suggested that B. cereus and enterotoxigenic factors are single gene products encoded B. thuringiensis should be considered members of a single June 2015 ⎪ Vol. 25⎪ No. 6 873 Kim et al. species [19, 33, 42]. However, other studies have shown (Pharmacia, Peapack, NJ, USA). sufficient genetic differentiation of B. cereus and B. thuringiensis Target genes for PCR detection. PCR analyses were carried out [9, 39]. Thus, at the present time, there is no consensus as to to detect 10 enterotoxigenic genes (hblA, hblC, hblD, nheA, nheB, whether these two bacterial species should be considered nheC, cytK, bceT, entFM, and entS), one emetogenic gene (ces), separate species or specialized variants of a single species. seven hemolytic genes (hlyA, hlyII, hlyIII, plcA, cerA, cerB, and cerO), and a pleiotropic transcriptional activator gene (plcR) among Given such discordant taxonomic classification of B. cereus B. cereus and B. thuringiensis strains. Table 1 provides information and B. thuringiensis strains and the introduction of high concerning the primers used for the amplification of each gene in numbers of B. thuringiensis spores into the human food this study. chain through vegetables treated with B. thuringiensis- Conditions for PCR amplification. Twenty-five nanograms of based insecticides, we examined a panel of B. cereus and DNA was used for each reaction. Ultrapure water (Invitrogen, B. thuringiensis strains comprising mainly reference strains Life Technologies, Seoul, Korea) was used in all negative control to assess the distribution of genes encoding the putative reactions and for the preparation of the PCR mixture. All reaction virulence factors mentioned above for B. cereus. mixtures for amplification of sequences encoding toxins and putative virulence factors contained 5 µl of template DNA (25 ng), Materials and Methods 10 mM Tris-HCl (pH 8.3), 10 mM KCl, 0.2 mM each deoxynucleoside triphosphate, 2.5 mM MgCl2, 1 µM each primer, Bacterial Strains and Growth Conditions and 0.5 U of Taq DNA polymerase (Solgent Co., Daejeon, Korea). Five commercial B. thuringiensis strains isolated from five different PCR amplification was conducted using a model PTC-100 biopesticide products obtained from local retail establishments in Programmable Thermal Controller (MJ Research, Inc., MA, USA). rural areas of the Republic of Korea were screened in this study. A The optimized PCR conditions were as follows: a single strain of each of the following commercial B. thuringiensis subspecies denaturation step of 3 min at 95°C; 35 cycles of denaturation at was also screened: B. thuringiensis subsp. kurstaki, B. thuringiensis 94°C for 30 sec, annealing at 58°C for 45 sec, and extension at 72°C subsp. aizawai, B. thuringiensis subsp. israelensis, and B. thuringiensis for 1.5 min; and a final extension at 72°C for 5 min. subsp. tenebrionis. To validate the results, all PCR amplifications were performed a Twelve and 16 reference strains of B. thuringiensis and B. cereus, minimum of three times. After DNA amplification, PCR fragments respectively, were screened in this study. B. thuringiensis ATCC were analyzed by submarine gel electrophoresis, stained, and 33679, B. thuringiensis ATCC 35646, B. thuringiensis ATCC 19266, visualized under UV illumination. Suitable molecular size markers B. thuringiensis ATCC 19268, B. thuringiensis ATCC 13367, B. thuringiensis were included in each gel. To identify cases in which poor quality ATCC 13366, B. thuringiensis ATCC 11429, B. cereus ATCC 21366, of template DNA caused amplification failure, the quality of any B. cereus ATCC 21768, B. cereus ATCC 10876, B. cereus ATCC 21772, DNA extract that failed to amplify in a specific reaction was B. cereus ATCC 11778, B. cereus ATCC 10702, B. cereus ATCC 13061, examined by attempting amplification with a pair of universal B. cereus ATCC 14579, B. cereus ATCC 21769, and B. cereus ATCC primers designed to amplify a region of the 16S rRNA gene. 21771 were obtained from the American Type Culture Collection Negative controls were included with all amplifications. Suitable (Manassas, VA, USA). B. thuringiensis KCTC 1508, B. thuringiensis controls such as buffer, media, PCR mixtures, and template DNA KCTC 1510, B. thuringiensis KCTC 1511, B. thuringiensis KCTC were used to detect any false-positive or false-negative reactions. 1512, B. thuringiensis KCTC 1513, and B. cereus KCTC 1094 were obtained from the Korean Collection for Type Cultures (Daejeon, Results Korea). B. cereus KFRI 181 was obtained from the Korea Food Research Institute (Sungnam, Korea). B. cereus IFO 3514, B. cereus PCR Amplification of Virulence Genes Using Gene-Specific IFO 3563, B. cereus IFO 3001, and B. cereus IFO 3003 were obtained PCR Primers from the Institute for Fermentation (Osaka, Japan). All bacterial The approach used in this study relied on PCR strains were grown at 30°C on nutrient agar or in nutrient broth amplification of target virulence genes. Virulence genes with shaking for preparation of template DNA for PCR screening. targeted for PCR detection included a first set of genes encoding enterotoxins and emetic toxin and a second set of PCR Detection of Genes for Enterotoxins, Emetic Toxin, and genes encoding extracellular hemolysins considered to be Other Virulence Factors potential virulence factors of B. cereus and B. thuringiensis. Preparation of total DNA. Template DNA for PCR screening The first set included genes encoding hemolytic