J Bacteriol Virol.  2008 Jun;38(2):61-75. 10.4167/jbv.2008.38.2.61.

Usefulness of Bacteriological Tests and sspE PCR for Identification of Bacillus cereus Group

• Kang SH
• Kim KJ
• Kim WY
• Chung SI

• KMID: 1483976
• DOI: http://doi.org/10.4167/jbv.2008.38.2.61

Abstract

The Bacillus cereus group includes B. anthracis, B. cereus, B. thuringiensis, B. mycoides, B. weihenstephanensis, B. pseudomycoides. The members of B. cereus group shares strong degree of DNA sequence similarity. Even though the biochemical test and bacteriological test have been used to identify the B. cereus group, an accurate identification system of the B. cereus group is required. We have developed a highly specific PCR-based assay for the B. cereus group chromosome using a sequence motif found within a spore structural gene (sspE). Using the assay, we were able to discriminate B. anthracis from the other members of B. cereus group. We also tried to find a new system for the B. cereus group identification. Five bacteriological tests (hemolysis, motility, penicillin susceptibility, rhizoid growth, toxic crystal formation), API system (API 50CHB & API 20E), MLST and sspE PCR were performed on 28 strains of the B. cereus group. The dendrogram generated from API system and bacteriological tests revealed that B. cereus and B. thuringiensis are grouped into the same cluster. In combination of sspE PCR and bacteriological tests, the dendrogram showed that 4 strains of B. cereus clustered within the same group. B. thuringiensis formed the subgroup in the same cluster. All strains of B. mycoides were encompassed together. Another cluster only included B. anthracis. The best system was determined to be sspE PCR and bacteriological tests. It is concluded that sspE PCR and bacteriological tests could be used for rapid discrimination and identification of B. anthracis and provided an effective means of differentiation between the B. cereus group.

Keyword

Bacillus cereus group; Identification; sspE PCR; Bacteriological tests

MeSH Terms

Bacillus
Bacillus cereus
Base Sequence
Discrimination (Psychology)
Penicillins
Polymerase Chain Reaction
Social Identification
Spores
Subacute Sclerosing Panencephalitis
Penicillins

Figure

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  Figure 1. Dendrogram of 28 strains of B. cereus group constructed by UPGMA based on the bacteriological and API System (API 50CHB & API 20E). The clades are indicated by roman numerals. Clade II encompassed 4 strains of B. anthracis. The scale bars indicate the percentage of missmatches between biological and biochemical properties.

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  Figure 2. MLST Dendrograms were generated by UPGMA method from genetic distances. The scale bar indicate the percentages of allelic distances between STs for MLST. The MLST dendrogram shows that B. thuringiensis BGSC 4AJ1, 4AB1, 4CC1 were the most proximate to B. anthracis.

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  Figure 3. Agarose gel electrophoresis analysis of sspE gene polymerase chain reaction (PCR) on 28 strains of B. cereus group. Lane: M, 100-bp DNA size ladder; 1, B. anthracis Sterne 34-F2; 2, B. anthracis Pasteur No.2; 3, B. anthracis ATCC 14185; 4, B. anthracis ATCC 14186; 5, B. thuringiensis BGSC 4AB1; 6, B. thuringiensis BGSC 4AJ1; 7, B. thuringiensis BGSC 4AQ1; 8, B. thuringiensis BGSC 4AS1; 9, B. thuringiensis BGSC 4CC1; 10, B. thuringiensis DSM 2046; 11, B. cereus KCTC 3624; 12, B. cereus BGSC 6E1; 13, B. cereus KCTC 1014; 14, B. cereus KCTC 1094; 15, B. cereus KCTC 3062; 16, B. mycoides ATCC 10206; 17, B. mycoides ATCC 21929; 18, B. mycoides ATCC 23258; 19, B. mycoides KCCM 40260; 20, B. mycoides KCTC 3453; 21, Bacillus spp. IB; 22, Bacillus spp. 003; 23, Bacillus spp. 9727; 24, Bacillus spp. IV; 25, Bacillus spp. III; 26, Bacillus spp. III BL; 27, Bacillus spp. III BS; 28, Bacillus spp. Rho; 29, B. subtilis ATCC 6051; 30, negative water control (no template DNA).

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  Figure 4. Dendrogram of 28 strains of B. cereus group constructed by UPGMA based on the bacteriological and sspE gene PCR. The clades are indicated by roman numerals. Clade III encompassed 4 strains of B. anthracis. The scale bars indicate the percentage of missmatches between biological and sspE gene PCR results.

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  Figure 5. The Identification scheme of Bacillus cereus group. (First step) Gram stain, (Second step) Differenciation of Bacillus cereus group by sspE gene PCR, (Third step) Additional bacteriological tests and final identification

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