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Keywords:

  • 16S;
  • Polymerase chain reaction;
  • Detection;
  • Bacillus cereus group;
  • Enterotoxic

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References

Recent investigations have shown that members of the Bacillus cereus group carry genes which have the potential to cause gastrointestinal and somatic diseases. Although most cases of diseases caused by the B. cereus group bacteria are relatively mild, it is desirable to be able to detect members of the B. cereus group in food and in the environment. Using 16S rDNA as target, a PCR assay for the detection of B. cereus group cells has been developed. Primers specific for the 16S rDNA of the B. cereus group bacteria were selected and used in combination with consensus primers for 16S rDNA as internal PCR procedure control. The PCR procedure was optimized with respect to annealing temperature. When DNA from the B. cereus group bacteria was present, the PCR assay yielded a B. cereus specific fragment, while when non-B. cereus prokaryotic DNA was present, the consensus 16S rDNA primers directed synthesis of the PCR products. The PCR analyses with DNA from a number of non-B. cereus confirmed the specificity of the PCR assay.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References

The Bacillus cereus group comprises six species, namely B. cereus, B. thuringiensis, B. anthracis, B. mycoides[1], B. pseudomycoides[2] and B. weihenstephanensis[3]. The differentiation between B. cereus and the other closely related species depends upon the absence of δ-endotoxin crystals (from B. thuringiensis), motility and hemolytic activity (from B. anthracis), non-rhizoid growth and motility (from B. mycoides) and growth at temperatures below 7°C (from B. weihenstephanensis). B. pseudomycoides is a genetically distinct group, also separable from B. cereus and B. mycoides by its fatty acid composition [2]. B. anthracis causes anthrax, while a number of B. cereus strains cause gastrointestinal diseases due to the production of an emetic toxin or different enterotoxins [4]. Several B. thuringiensis, B. mycoides and B. weihenstephanensis strains also possess genes and produce enterotoxins similar to that of B. cereus[5–7].

The species within the B. cereus group share many phenotypic and genotypic properties, but the status as separate species is still under debate [8–10]. The species have high degrees of DNA–DNA relatedness [11], high rDNA similarity [8,12] and indistinguishable 16S–23S rDNA intergenic spacers [5,13].

Different attempts have been done to develop DNA-based methods for detection of and differentiation between the species within the B. cereus group [14–20]. None of these methods have been shown unambiguously to detect all strains within one species or to differentiate between all six species within the B. cereus group. In contrast to this, Francis et al. [21] and Hsieh et al. [7] developed specific primer sets targeting a cold-shock protein and the sph gene encoding a sphingomyelinase, respectively, which could be used for the detection of all species within the group.

The objectives of this study were to design and test 16S rDNA-targeting primers for the specific detection of all six species within the B. cereus group.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References

2.1Bacteria and culture conditions

Bacterial strains are listed in Table 1. B. thuringiensis, B. cereus, B. mycoides, B. pseudomycoides and B. weihenstephanensis were cultivated on Luria–Bertani agar [22]. All other species were cultivated on agar media as recommended by DSMZ.

Table 1.  Bacterial strainsa analyzed by PCR
Strains belonging to the B. cereus groupNon-B. cereus group strainsb
  1. aATCC: American Type Culture Collection; AH: A.-B. Kolst?, University of Oslo, Norway; DBt: National Environmental Research Institute, Denmark; DSMZ: Deutsche Sammlung von Mikroorganismen und Zellkulturen; F: Central Public Health Laboratory, London, UK; HD: U.S. Department of Agriculture. Isolates B. cereus T01 176:C2, B. cereus IP 5832 and B. thuringiensis subsp. israelensis 4Q2-72 were from the Pasteur Institute, Paris, France; B. cereus B-4ac was kindly supplied by Dr. Norio Agata, Nagoya, Japan.

  2. bAll Non-B. cereus group strains are type strains.

  3. cStrain deviating from S-S-Bc-200-a-S-18 in three positions.

  4. dStrain deviating from S-S-Bc-470-a-S-18 in four positions.

B. thuringiensis subsp. alesti HD-4B. agri DSMZ 6348
B. thuringiensis subsp. canadensis HD-224B. alcalophilus DSMZ 485d
B. thuringiensis subsp. dakota HD-932B. alginolyticus DSMZ 5050
B. thuringiensis subsp. darmstadiensis HD-146B. alvei DSMZ 29
B. thuringiensis subsp. entomocidus HD-9B. amyloliquefaciens DSMZ 7
B. thuringiensis subsp. finitimus HD-3B. amylolyticus DSMZ 3034
B. thuringiensis subsp. israelensis 4Q2-72B. badius DSMZ 23
B. thuringiensis subsp. israelensis HD-567B. brevis DSMZ 30
B. thuringiensis subsp. kurstaki DBt 514B. chondroitinus DSMZ 5051
B. thuringiensis subsp. kurstaki DBt F-H2B. circulans DSMZ 11
B. thuringiensis subsp. kurstaki DMU Bt 67RB. coagulans DSMZ 1
B. thuringiensis subsp. kurstaki HD-1B. fastidiosus DSMZ 91
B. thuringiensis subsp. kurstaki HD-73B. firmus DSMZ 12
B. thuringiensis subsp. kyoshuensis HD-541B. flexus DSMZ 1320
B. thuringiensis subsp. ostriniae HD-501B. fusiformis DSMZ 2898
B. thuringiensis subsp. pakistani HD-395B. glucanolyticus DSMZ 5162
B. thuringiensis subsp. roskildiensis DMU Bt 39B. gordonae DSMZ 5395
B. thuringiensis subsp. sotto HD-770B. insolitus DSM 5
B. thuringiensis subsp. toumanoffi HD-201B. laterosporus DSMZ 25d
B. thuringiensis DBt 248B. lautus DSMZ 3035
B. cereus AH 184B. lentus DSMZ 9
B. cereus AH 185B. licheniformis DSMZ 13
B. cereus ATCC 10876B. macerans DSMZ 24
B. cereus ATCC 4342B. macquariensis DSMZ 2
B. cereus ATCC 7064B. megaterium DSMZ 32
B. cereus ATCC 14579TB. pabuli DSMZ 3036
B. cereus ATCC 33018B. pantothenticus DSMZ 26d
B. cereus ATCC 6464B. pasteurii DSMZ 33
B. cereus B-4acB. polymyxa DSMZ 36
B. cereus F2038/78B. pumilus DSMZ 27
B. cereus F3502/73B. simplex DSMZ 1321
B. cereus F4433/73B. sphaericus DSMZ 28
B. cereus F4810/72B. subtilis DSMZ 10
B. cereus IP5832B. validus DSMZ 3037
B. cereus T01 176:C2Lactobacillus delbrueckii subsp. delbruckii DSMZ 20074c
B. mycoides DSMZ 299Staphylococcus aureus subsp. aureus DSMZ 20231c
B. mycoides DSMZ 303Staphylococcus auricularis DSMZ 20609c
B. mycoides DSMZ 307Staphylococcus warneri DSMZ 20316c
B. mycoides DSMZ 2048T 
B. pseudomycoides DSMZ 12442T 
B. weihenstephanensis DSMZ 11821T 

2.2Polymerase chain reaction (PCR)

PCR was performed as described previously [5] using denaturation at 94°C for 15 s, annealing at 63°C for 45 s, and extension at 72°C for 2 min for a total of 30 cycles. PCR products were analyzed by 1.5% agarose gel electrophoresis, using MW VI (Boehringer-Mannheim, GmbH) as molecular mass marker. Specific primers for the B. cereus group 16S rDNA were selected using the probe design software of the ARB software package [23]. The specificity of the primers was checked against all sequences in EMBL and GenBank using the BLAST algorithm [24]. Besides the specific B. cereus group 16S rDNA primers, universal 16S rDNA primers modified from Alm et al. [25]: S-*-Univ-518-b-S-18 (5′-GTA TTA CCG CGG CTG CTG-3′) and S-*-Univ-1492-b-A-19 (5′-GGT TAC CTT GTT ACG ACT T-3′) were used.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References

The ARB probe design program suggested a number of B. cereus 16S rRNA specific probes, of which two 18-nucleotide regions (positions 200–217 and 470–487 in the 16S rDNA; numbers refer to the B. cereus 16S rDNA sequence with GenBank accession number X55063.1) were selected for design of the B. cereus group specific 16S rDNA PCR-primers: S-S-Bc-200-a-S-18 (5′-TCG AAA TTG AAA GGC GGC-3′) and S-S-Bc-470-a-A-18 (5′-GGT GCC AGC TTA TTC AAC-3′). Comparisons of these two regions with the corresponding sequences of the 28 B. cereus group strains (11 B. cereus, 10 B. thuringiensis, six B. mycoides and one B. anthracis), appearing in the ARB database (comprising 6194 bacterial 16S rDNA sequences), revealed no sequence variation within region 470–487, while one B. cereus strain deviated in one position in the 200–217 region. Other strains having high homology to the two primers are included in the list of non-B. cereus group strains in Table 1. The selected primers were further aligned with sequences in EMBL and GenBank, which confirmed the specificity of the two primers.

To develop a PCR-mediated assay for the detection of B. cereus group strains, the primers S-S-Bc-200-a-S-18 and S-S-Bc-470-a-A-18 were initially tested with B. cereus ATCC 14579T and B. subtilis DSMZ 10T. The annealing temperature for the PCR assay was optimized in the interval 55–65°C. At an annealing temperature of 63°C, one distinct PCR product of 288 nucleotides was formed (Fig. 1) with B. cereus ATCC 14579T. No product was formed with B. subtilis DSMZ 10T. Under these conditions two products of approximately 1000 and 800 nucleotides of 16S rDNA were amplified using the universal primers: S-*-Univ-518-b-S-18 and S-*-Univ-1492-b-A-19 with both strains (Fig. 1). Sequence analyses showed the two products to be conformational variants (data not shown).

image

Figure 1. PCR analysis of a B. cereus and a B. subtilis using either the universal primer set, the B. cereus group specific primer set, or the two primer sets in combination. The actual primers are given in the parentheses. Lane 1: B. cereus (S-*-Univ-518-b-S-18 and S-*-Univ-1492-b-A-19); lane 2: B. subtilis (S-*-Univ-518-b-S-18 and S-*-Univ-1492-b-A-19); lane 3: B. cereus (S-S-Bc-200-a-S-18 and S-S-Bc-470-a-A-18); lane 4: B. subtilis (S-S-Bc-200-a-S-18 and S-S-Bc-470-a-A-18); lane 5: B. cereus (S-*-Univ-518-b-S-18 and S-*-Univ-1492-b-A-19 together with S-S-Bc-200-a-S-18 and S-S-Bc-470-a-A-18); lane 6: B. subtilis (S-*-Univ-518-b-S-18 and S-*-Univ-1492-b-A-19 with S-S-Bc-200-a-S-18 together with S-S-Bc-470-a-A-18); lane M: size marker.

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However, PCR with both primer sets yielded only the 288-nucleotide product with B. cereus ATCC 14579T, while two products were formed with B. subtilis DSMZ 10T, corresponding in size to the products formed by the universal primers. Thus, at these specific PCR conditions, a product corresponding to the specific primer set was produced in the presence of a B. cereus target, while two products corresponding to the universal primers were formed in the absence of this target (Fig. 1).

To test the specificity of the developed PCR-assay for detection of B. cereus group strains, the assay with all four primers was conducted with 41 B. cereus group strains, 34 non-B. cereus group Bacilli, three Staphylococcus strains and one Lactobacillus strain (Table 1). A product corresponding to 288 nucleotides was formed with all B. cereus group strains, while products corresponding to 800 and 1000 nucleotides was formed by the remaining strains. Additionally, the specificity of the PCR procedure has been tested with 91 clinical food and environmental isolates from the B. cereus group, confirming the specificity of the B. cereus group PCR assay (data not shown).

The sensitivity of the B. cereus group PCR assay for detecting cells in food was tested with artificial contaminated boiled rice. B. thuringiensis subsp. kurstaki HD-1 was inoculated to boiled rice (0–5 CFU/ g−1 rice), incubated overnight at room temperature, and DNA was extracted by boiling. The lowest level of CFU g−1 boiled rice after incubation yielding the B. cereus specific PCR product was 150 CFU g−1.

4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References

Enterotoxigenic B. cereus, which causes acute gastroenteritis after the consumption of contaminated foods, have been known to produce emetic and diarrheal toxin types [4]. Strains from the other B. cereus group species, B. thuringiensis, B. mycoides and B. weihenstephanensis, are also potentially enterotoxic, as they possess genes and produce enterotoxins [5–7]. Thus, easy methods for an unequivocal determination of strains and for the detection of B. cereus group cells in food samples are important. We therefore wanted to develop a rapid and reliable assay for the differentiation of B. cereus group species from other Bacillus species.

Ideal targets for PCR primers for specific detection of the B. cereus group are the rRNA genes. We have developed a 16S rDNA-targeting primer set, specific for B. cereus group strains. Comparisons to all accessible 16S rDNA sequences reveal that both primers are unique for the B. cereus group. A distinct PCR product of the expected size was produced when DNA from B. cereus group cells was used as a target, while DNA from bacteria not affiliated to this group did not produce the product. Thus, a highly specific PCR assay for the detection of B. cereus group cells has been developed. This assay has a potential for the direct detection of B. cereus group cells in food without enrichment or culturing.

Two major limitations of PCR assay as a diagnostic tool are that false negative reactions can occur due to unsuccessful DNA extraction from the bacteria, and due to inhibition of the PCR reaction by inhibitory substances co-extracted with the target DNA. To avoid false-negative results, a universal primer set was included in the PCR assay. At the selected optimal annealing temperature, amplification of the B. cereus specific product competed out the amplification of the universal products. Thus, a single product is yielded when both primer sets are present.

Yamada et al. [16] and Hsieh et al. [7] also developed primer sets which can be used for the specific detection of B. cereus group cells. These primers were directed against genes for a cold-shock protein and a sphingomyelinase, respectively. Both showed positive results with all B. cereus group cells tested. This is indicative for the importance of these genes for the activity for B. cereus group cells. However, the average substitution rate for protein-encoding DNA has been estimated as 0.7−0.8% per million years, while the rate for 16S rDNA gene is 1% per 50 million years [26]. Thus, the probability for false-negative results with primer sets directed against protein-encoding genes is larger than for RNA-encoding genes. Furthermore, the 16S rDNA directed primers have potential to be probes for detection of vegetative B. cereus group bacteria by in situ hybridization.

Acknowledgments

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References

This work was supported by a grant from the Research Center for Environmental Health, The Danish Ministry of Health. Bente Rose Hansen is thanked for skilled technical assistance.

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  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References
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