Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Various Enterotoxin and Other Virulence Factor Genes Widespread Among Bacillus cereus and Bacillus thuringiensis Strains

  • Kim, Min-Ju (Department of Food Science and Technology, Chung-Ang University) ;
  • Han, Jae-Kwang (Department of Food Science and Technology, Chung-Ang University) ;
  • Park, Jong-Su (Department of Food Science and Technology, Chung-Ang University) ;
  • Lee, Jin-Sung (Department of Biological Sciences, Kyonggi University) ;
  • Lee, Soon-Ho (Nutrition Safety Policy Division) ;
  • Cho, Joon-Il (Division of Food Microbiology, The Korean Ministry of Food and Drug Safety) ;
  • Kim, Keun-Sung (Department of Food Science and Technology, Chung-Ang University)
  • Received : 2015.02.03
  • Accepted : 2015.03.15
  • Published : 2015.06.28
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Abstract

Many strains of Bacillus cereus cause gastrointestinal diseases, and the closely related insect 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 of 10 enterotoxigenic genes (hblA, hblC, hblD, nheA, nheB, nheC, cytK, bceT, entFM, and entS), one emetogenic gene (ces), seven hemolytic genes (hlyA, hlyII, hlyIII, plcA, cerA, cerB, and cerO), and a pleiotropic transcriptional activator gene (plcR). These genes encode various enterotoxins and other virulence factors thought to play a role in infections of mammals. Amplicons were successfully generated from the strains of B. cereus and B. thuringiensis for 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 B. thuringiensis strains as well as B. cereus strains.

Keywords

Introduction

The Bacillus cereus sensu lato group contains six valid species, including Bacillus cereus and Bacillus thuringiensis, which show a high degree of phenotypic similarity [21]. The classification of these species is based on pathogenicity to mammals or insects, plasmid content, and gross morphological characteristics [30,31]. The genetic similarity among members of the B. cereus group has been extensively studied by means of various molecular methods [17,19,28,30].

B. cereus is an opportunistic endospore-forming bacterium involved in a range of intestinal and extraintestinal infections [5,37]. It is a common soil inhabitant that is often implicated in food poisoning in humans [35,37]. The virulence of this bacterium has been ascribed to different extracellular factors. Two of these virulence factors, hemolysin BL (HBL) [4] and nonhemolytic enterotoxin (NHE) [14], are tripartite protein complexes. Other enterotoxigenic factors are single gene products encoded by the cytotoxin K (cytK), enterotoxin FM (entFM), enterotoxin S (entS), and enterotoxin T (bceT) genes [2,11,18,26]. The virulence of the emetic strains is related to cereulide, a thermostable cyclic dodecadepsipeptide synthesized by a nonribosomal peptide synthetase encoded by ces genes [10]. Products from other genes, such as hemolysin A (hlyA), hemolysin II (hlyII), hemolysin III (hlyIII), phosphatidylinositolspecific phospholipase C (plcA), cereolysin A or phospholipase C (cerA), cereolysin B or sphingomyelinase (cerB), cereolysin O (cerO), and their pleiotropic transcriptional activator (plcR), are also involved in the pathogenesis of B. cereus [3,20,32,35,37,38]. B. thuringiensis is a typical endosporeforming bacterium distinguished by the accumulation of polypeptides that form a crystalline parasporal inclusion during sporulation [34]. These crystal proteins may be toxic to insects, leading to the extensive use of B. thuringiensis as a biological insecticide for crop protection [8,34].

Recent studies have suggested that B. cereus and B. thuringiensis should be considered members of a single species [19,33,42]. However, other studies have shown sufficient genetic differentiation of B. cereus and B. thuringiensis [9,39]. Thus, at the present time, there is no consensus as to whether these two bacterial species should be considered separate species or specialized variants of a single species. Given such discordant taxonomic classification of B. cereus and B. thuringiensis strains and the introduction of high numbers of B. thuringiensis spores into the human food chain through vegetables treated with B. thuringiensis-based insecticides, we examined a panel of B. cereus and B. thuringiensis strains comprising mainly reference strains to assess the distribution of genes encoding the putative virulence factors mentioned above for B. cereus.

 

Materials and Methods

Bacterial Strains and Growth Conditions

Five commercial B. thuringiensis strains isolated from five different biopesticide products obtained from local retail establishments in rural areas of the Republic of Korea were screened in this study. A strain of each of the following commercial B. thuringiensis subspecies was also screened: B. thuringiensis subsp. kurstaki, B. thuringiensis subsp. aizawai, B. thuringiensis subsp. israelensis, and B. thuringiensis subsp. tenebrionis.

Twelve and 16 reference strains of B. thuringiensis and B. cereus, respectively, were screened in this study. B. thuringiensis ATCC 33679, B. thuringiensis ATCC 35646, B. thuringiensis ATCC 19266, B. thuringiensis ATCC 19268, B. thuringiensis ATCC 13367, B. thuringiensis ATCC 13366, B. thuringiensis ATCC 11429, B. cereus ATCC 21366, B. cereus ATCC 21768, B. cereus ATCC 10876, B. cereus ATCC 21772, B. cereus ATCC 11778, B. cereus ATCC 10702, B. cereus ATCC 13061, B. cereus ATCC 14579, B. cereus ATCC 21769, and B. cereus ATCC 21771 were obtained from the American Type Culture Collection (Manassas, VA, USA). B. thuringiensis KCTC 1508, B. thuringiensis KCTC 1510, B. thuringiensis KCTC 1511, B. thuringiensis KCTC 1512, B. thuringiensis KCTC 1513, and B. cereus KCTC 1094 were obtained from the Korean Collection for Type Cultures (Daejeon, Korea). B. cereus KFRI 181 was obtained from the Korea Food Research Institute (Sungnam, Korea). B. cereus IFO 3514, B. cereus IFO 3563, B. cereus IFO 3001, and B. cereus IFO 3003 were obtained from the Institute for Fermentation (Osaka, Japan). All bacterial strains were grown at 30℃ on nutrient agar or in nutrient broth with shaking for preparation of template DNA for PCR screening.

PCR Detection of Genes for Enterotoxins, Emetic Toxin, and Other Virulence Factors

Preparation of total DNA. Template DNA for PCR screening was prepared by processing 5 ml of culture grown for 18 h at 30℃, using a QIAamp DNA Mini Kit (Qiagen, Inc., Chatsworth, CA, USA). The presence, concentration, and purity of total DNA in the prepared samples were detected by measuring the absorbance at 260 and 280 nm using an Ultraspec 3000 spectrophotometer (Pharmacia, Peapack, NJ, USA).

Target genes for PCR detection. PCR analyses were carried out to detect 10 enterotoxigenic genes (hblA, hblC, hblD, nheA, nheB, nheC, cytK, bceT, entFM, and entS), one emetogenic gene (ces), seven hemolytic genes (hlyA, hlyII, hlyIII, plcA, cerA, cerB, and cerO), and a pleiotropic transcriptional activator gene (plcR) among B. cereus and B. thuringiensis strains. Table 1 provides information concerning the primers used for the amplification of each gene in this study.

Table 1.a HBL: Hemolysin BL b NHE: Nonhemolytic enterotoxin

Conditions for PCR amplification. Twenty-five nanograms of DNA was used for each reaction. Ultrapure water (Invitrogen, Life Technologies, Seoul, Korea) was used in all negative control reactions and for the preparation of the PCR mixture. All reaction mixtures for amplification of sequences encoding toxins and putative virulence factors contained 5 µl of template DNA (25 ng), 10 mM Tris-HCl (pH 8.3), 10 mM KCl, 0.2 mM each deoxynucleoside triphosphate, 2.5 mM MgCl2, 1 µM each primer, and 0.5 U of Taq DNA polymerase (Solgent Co., Daejeon, Korea). PCR amplification was conducted using a model PTC-100 Programmable Thermal Controller (MJ Research, Inc., MA, USA). The optimized PCR conditions were as follows: a single denaturation step of 3 min at 95℃; 35 cycles of denaturation at 94℃ for 30 sec, annealing at 58℃ for 45 sec, and extension at 72℃ for 1.5 min; and a final extension at 72℃ for 5 min.

To validate the results, all PCR amplifications were performed a minimum of three times. After DNA amplification, PCR fragments were analyzed by submarine gel electrophoresis, stained, and visualized under UV illumination. Suitable molecular size markers were included in each gel. To identify cases in which poor quality of template DNA caused amplification failure, the quality of any DNA extract that failed to amplify in a specific reaction was examined by attempting amplification with a pair of universal primers designed to amplify a region of the 16S rRNA gene. Negative controls were included with all amplifications. Suitable controls such as buffer, media, PCR mixtures, and template DNA were used to detect any false-positive or false-negative reactions.

 

Results

PCR Amplification of Virulence Genes Using Gene-Specific PCR Primers

The approach used in this study relied on PCR amplification of target virulence genes. Virulence genes targeted for PCR detection included a first set of genes encoding enterotoxins and emetic toxin and a second set of genes encoding extracellular hemolysins considered to be potential virulence factors of B. cereus and B. thuringiensis. The first set included genes encoding hemolytic (hblA, hblC, and hblD) and nonhemolytic (nheA, nheB, and nheC) enterotoxin complexes, cytotoxin K (cytK), various putative enterotoxins (bceT, entFM, and entS), and cereulide (ces) as shown in Fig. 1. The second set included genes encoding a number of hemolytic factors (hlyA, hlyII, hlyIII, plcA, cerA, cerB, and cerO) and their pleiotropic transcriptional activator, PlcR (plcR), as shown in Fig. 2.

Fig. 1.Representative PCR amplicons to detect enterotoxigenic and emetogenic genes among B. cereus and B. thuringiensis strains. Lane M, 100 bp DNA ladder; lane 1, hblA gene; lane 2, hblC gene; lane 3, hblD gene; lane 4, nheA gene; lane 5, nheB gene; lane 6, nheC gene; lane 7, cytK gene; lane 8, entFM gene; lane 9, bceT gene; lanes 10 to 13, entS gene (TY123/TY124, TY123/TY125, TY123/TY126, and TY123/TY127); and lane 14, ces gene.

Fig. 2.Representative PCR amplicons to detect other putative virulence genes among B. cereus and B. thuringiensis strains.

The gene-specific PCR primers successfully amplified each target virulence gene, except the ces gene, from total DNA of B. cereus and B. thuringiensis strains. All primers produced amplicons of the expected sizes from their respective target virulence genes, as representative PCR amplicons were shown in Figs. 1 and 2. Using three DNA templates independently prepared from each test strain as described above, the PCR amplification results in triplicate experiments were 100% reproducible for each target gene.

Distribution of Bacillus Enterotoxigenic and Emetogenic Genes Among B. cereus and B. thuringiensis Strains

The distribution of the genes encoding the two major Bacillus tripartite enterotoxins among the tested B. cereus and B. thuringiensis strains is shown in Table 2. Eight of the 12 B. thuringiensis reference strains possessed all three genes encoding the enterotoxic HBL complex (hblA, hblC, and hblD), whereas only two of the 16 B. cereus reference strains harbored all three genes. The remaining B. thuringiensis strains, except one commercial isolate and one reference strain, possessed at least one of the three genes encoding the complex, whereas four B. cereus reference strains had none and the remaining 10 B. cereus reference strains possessed at least one of the three genes. All three genes encoding the nonhemolytic enterotoxin (nheA, nheB, and nheC) were detected in 12 (57%) of the 21 B. thuringiensis strains, including all four known B. thuringiensis subspecies, and in nine (56%) of the 16 B. cereus reference strains. Whereas all B. thuringiensis strains possessed at least two of the NHE genes, two B. cereus reference strains harbored only one gene and two other strains lacked all three genes.

Table 2.Occurrence of enterotoxigenic hbl and nhe genes in B. cereus and B. thuringiensis.

The distribution of other Bacillus enterotoxigenic and emetogenic genes among the tested B. cereus and B. thuringiensis strains is shown in Table 3. The cytK gene was frequently detected in the B. thuringiensis (91%) and B. cereus strains (81%). The bceT gene was also frequently detected in the B. thuringiensis (95%) and B. cereus strains (88%). The entFM gene was detected in 10 (83%) of the 12 B. thuringiensis reference strains and 12 (75%) of the 16 B. cereus reference strains, but the gene was not detected in any of the five commercial B. thuringiensis isolates or the four known B. thuringiensis subspecies strains. PCR fragments were successfully amplified in all of the B. thuringiensis and B. cereus strains using at least one of the four primer sets targeting the entS gene (TY123/124, TY123/125, TY123/ 126, and TY123/127). The primer set targeting the ces gene (cesF1/cesR1) failed to amplify PCR fragments in any of the B. thuringiensis and B. cereus strains tested in this study.

Table 3.Occurrence of other enterotoxigenic and emetogenic genes in B. cereus and B. thuringiensis.

Distribution of Other PutativeBacillus Virulence Factor Genes Among B. cereus and B. thuringiensis Strains

The distribution of other putative Bacillus virulence factor genes among the tested B. cereus and B. thuringiensis strains is shown in Table 4. The hlyA gene was detected in 19 (90%) of the 21 B. thuringiensis strains, including all five commercial B. thuringiensis isolates and the four known B. thuringiensis subspecies strains, and in all (100%) of the 16 B. cereus reference strains. The hlyII gene was detected in three (60%) of the five commercial B. thuringiensis isolates and two (50%) of the four known B. thuringiensis subspecies strains but only one (8%) of the 12 B. thuringiensis reference strains. This gene was also detected in four (25%) of the 16 B. cereus reference strains. Both primer sets targeting the hlyIII gene (bchem1/4 and bchem2/3) failed to amplify PCR fragments in three (8%) of the tested strains: one commercial B. thuringiensis isolate and two known B. thuringiensis subspecies strains. At least one of the two primer sets successfully amplified a PCR fragment in the remaining B. thuringiensis and B. cereus strains. The plcA gene was detected in 20 (95%) of the 21 B. thuringiensis strains, including all five commercial B. thuringiensis isolates and the four known B. thuringiensis subspecies strains, and in 12 (75%) of the 16 B. cereus reference strains. The cerA gene was detected in all (100%) of the five commercial B. thuringiensis isolates and three (75%) of the four known B. thuringiensis subspecies strains but only six (50%) of the 12 B. thuringiensis reference strains. This gene was also detected in six (38%) of the 16 B. cereus reference strains. The cerB gene was detected in 18 (86%) of the 21 B. thuringiensis strains and in nine (56%) of the 16 B. cereus reference strains. The cerO gene was detected in 16 (76%) of the 21 B. thuringiensis strains, including all five commercial B. thuringiensis isolates, and in eight (50%) of the 16 B. cereus reference strains.

Table 4.Occurrence of hemolytic genes and pleiotropic transcriptional activator gene in B. cereus and B. thuringiensis.

 

Discussion

B. cereus is known to produce a number of toxins that are involved in its ability to cause gastrointestinal and somatic diseases. These toxins include at least two enterotoxins, one emetic toxin, one cytotoxin, two hemolysins, and three enzymes involved in the degradation of phospholipids [20]. B. thuringiensis, like the foodborne and opportunistic human pathogen B. cereus, belongs to the B. cereus sensu lato group. B. thuringiensis and B. cereus are closely related and are indistinguishable phenotypically and genetically, except that the former harbors insecticidal plasmids and produces crystal toxin inclusions during sporulation [37]. Thus, measuring the toxins and monitoring the toxin-producing capabilities of a strain may be more important than identifying the species using other criteria. The virulence factors produced by members of the B. cereus sensu lato group include enterotoxins, phospholipases, and delta endotoxins.

In our study, genes encoding enterotoxins were more frequently found in the B. thuringiensis strains than in the B. cereus strains. The presence of enterotoxin-encoding genes in commercial B. thuringiensis strains was also found in previous studies [12,18,19,23,27]. Since all commercial B. thuringiensis strains harbor genes for three known enterotoxins, HBL, NHE, and CytK, there is a risk that high levels of these organisms may cause human disease. Taking this enterotoxigenic potential into account, as well as the fact that B. thuringiensis cannot be separated from B. cereus at the chromosomal level, vegetable producers and authorities responsible for food safety should consider the amount of B. thuringiensis insecticide residue left on products after harvest. The European Food Safety Authority has recommended that processors ensure that the numbers of B. cereus do not reach 103 to 105 per gram at the stage of consumption. The Korean Ministry of Food and Drug Safety has specified that processed foods should contain fewer than 103 to 104 B. cereus per gram, depending on the food type. We suggest that this guideline should also apply to residues of commercial, enterotoxin-encoding B. thuringiensis strains.

B. thuringiensis is one of the leading biological insecticides for use on crops. B. thuringiensis insecticidal sprays, which contain mixtures of spores and insecticidal crystals, are chosen by organic farmers to meet guidelines for using strictly nonsynthetic materials. Many Korean local governments and the Korean Ministry of Agriculture, Food and Rural Affairs do not oppose the use of biological insecticides to produce organic agricultural products. B. thuringiensis spore/crystal formulations must be safe and effective, must be easy to use, and should have a long shelf-life. The spore/crystal mixture in commercial formulations is more effective and is cheaper to obtain than the crystals alone. Because some strains of B. thuringiensis have the potential during their vegetative growth to produce various toxins that may produce symptoms in mammals, the production process must be closely controlled and monitored to ensure that these exotoxins are not present in the spore/crystal formulations at levels that can cause significant adverse health effects. Some features of B. thuringiensis spore/ crystal-based biopesticides limit their use in insect control. First, the biopesticides must be ingested by the target insect. Second, the timing of B. thuringiensis sprays is critical to attaining economic levels of insect control. B. thuringiensis is usually applied when early instar larvae are present, as older larvae are more tolerant. Third, B. thuringiensis sprays persist for only a few days on the leaf surface. Both leaf surface proteases and sunlight contribute to the degradation of crystal proteins.

In B. thuringiensis strains harboring toxin-encoding genes, vegetative cells can produce various toxins if they proliferate to the point of toxin production. However, the spores of such strains cannot produce toxins at all, as long as they remain dormant. Although B. thuringiensis spores can germinate in rat gastrointestinal tracts, their growth can be inhibited by indigenous gastrointestinal microbial communities. Thus, the resulting vegetative cells cannot proliferate to the point of toxin production [6,7,40,41]. Here, the indigenous gastrointestinal microbial communities are important to inhibit the outgrowth of germinated B. thuringiensis spores. To date, the health effects of B. thuringiensis spore/crystal-based biopesticides have not been demonstrated in mammals in any infectivity or pathogenicity study. In field trials, no outbreaks have been reported in humans, although additional investigations are needed to determine whether B. thuringiensis toxin-encoding genes are expressed in the human gut after ingestion of the spores. Despite the ubiquitous presence of genes encoding enterotoxigenic and other virulence factor proteins and their expression in vegetative cells in vitro and in vivo, B. thuringiensis spore/crystal-based biopesticides do not appear to pose a real food poisoning risk. This is supported by public health studies that have failed to link exposure to the spore/crystal spray products with an increased incidence of gastroenteritis, even in urban areas after largescale aerial application or under intense exposure conditions in greenhouses [15,22,36]. Nevertheless, the presence of genes encoding enterotoxigenic and other virulence factor proteins continues to fuel negative public perceptions and could ultimately lead to restricted use of B. thuringiensis in pest control.

In summary, we detected the distribution of various enterotoxin genes and other virulence factor genes considered unique to B. cereus among strains of B. cereus and B. thuringiensis. These two species are members of the B. cereus sensu lato group and are indistinguishable phenotypically and genetically, except that B. thuringiensis produces crystal toxins during sporulation. B. cereus enterotoxins and other virulence factors produced during vegetative growth have been implicated in numerous food-poisoning outbreaks in humans, while B. thuringiensis spore/crystal complexes have been extensively used for crop protection against insects. B. cereus enterotoxin genes and other virulence factor genes appear to be widespread among B. thuringiensis strains as well as B. cereus strains.

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