A novel multiplex PCR discriminates Bacillus anthracis and its genetically related strains from other Bacillus cereus group species

Abstract

Anthrax is an important zoonotic disease worldwide that is caused by Bacillus anthracis, a spore-forming pathogenic bacterium. A rapid and sensitive method to detect B. anthracis is important for anthrax risk management and control in animal cases to address public health issues. However, it has recently become difficult to identify B. anthracis by using previously reported molecular-based methods because of the emergence of B. cereus, which causes severe extra-intestinal infection, as well as the human pathogenic B. thuringiensis, both of which are genetically related to B. anthracis. The close genetic relation of chromosomal backgrounds has led to complexity of molecular-based diagnosis. In this study, we established a B. anthracis multiplex PCR that can screen for the presence of B. anthracis virulent plasmids and differentiate B. anthracis and its genetically related strains from other B. cereus group species. Six sets of primers targeting a chromosome of B. anthracis and B. anthracis-like strains, two virulent plasmids, pXO1 and pXO2, a bacterial gene, 16S rRNA gene, and a mammalian gene, actin-beta gene, were designed. The multiplex PCR detected approximately 3.0 CFU of B. anthracis DNA per PCR reaction and was sensitive to B. anthracis. The internal control primers also detected all bacterial and mammalian DNAs examined, indicating the practical applicability of this assay as it enables monitoring of appropriate amplification. The assay was also applied for detection of clinical strains genetically related to B. anthracis, which were B. cereus strains isolated from outbreaks of hospital infections in Japan, and field strains isolated in Zambia, and the assay differentiated B. anthracis and its genetically related strains from other B. cereus group strains. Taken together, the results indicate that the newly developed multiplex PCR is a sensitive and practical method for detecting B. anthracis.

Fig 1. BA_5031 and hBC/BT primer annealing sites on genome sequences of Bacillus cereus group strains.

Alignment of B. anthracis, B. cereus, B.thuringiensis, B. toyonensis, B. cytotoxis and B. weihenstephanensis BA_5031 (A) and BACI_c47770 (B) sequences from the database and primer sequences in this study. The rectangles shaded with grey correspond to BA_5031 and BACI_c47770 primer sequences. GENETYX-MAC Network Version 15.0.5 software was used for multiple sequence alignment. Expected amplification was simulated in silico using Amplifx 1.7.0 software.

Fig 2. Comparison of the specificity of multiplex PCR and those of existing PCR methods.

The expected DNA fragments corresponding to the characteristics of samples were amplified in all samples by the multiplex PCR assay established in this study (uppermost panel) and by three single PCRs, two duplex PCRs and PCR using a commercial kit (lower panels). Template DNA was extracted from B.anthracis-infected tissues and B.anthracis harboring or lacking virulent plasmids. Lane M, 100-bp DNA ladder; lane 1, DNA from B.anthracis-infected elephant blood; lane 2, DNA from B.anthracis-infected hippopotamus tissue; lane 3, DNA from B.anthracis strain CZC5; lane 4, DNA from B.anthracis strain M1; lane 5, DNA from B.anthracis strain L158–1; lane 6, DNA from B.anthracis strain CZC5 clone1; lane 7, DNA from B.anthracis vaccine strain Sterne 34F2; lane 8, B.anthracis vaccine strain Sterne 34F2 clone2; lane 9, distilled water as a negative control.

Fig 3. Sensitivity of multiplex PCR for B. anthracis cultured in liquid media.

DNA fragments were amplified by the multiplex PCR in the extracted DNA from B. anthracis strain CZC5 cultured in LB broth containing 5% (v/v) sheep blood (lanes 1 to 7). All 5 bands were simultaneously detected at the template concentration of 2.99 × 10³ CFU/ml (lane 3). Lane M, 100-bp DNA ladder; lanes 1 to 7, serially 10-fold diluted B. anthracis strain CZC5. Lane 3, 2.99 × 10³ CFU/ml; lane 4, 3.83 × 10² CFU/ml; lane 5, 2.0 × 10 CFU/ml; lanes 6 and 7, 0 CFU/ml; lanes 8, distilled water as a negative control. CFU/ml in lanes 1 and 2 were not determined because of uncountable colonies.

Fig 4. Multiplex PCR for Bacillus field isolates and clinical strains genetically related to B. anthracis.

Field isolates in Zambia (left panel) and clinical outbreak strains in Japan (right panel), which were genetically related to B. anthracis, and field isolates in Japan (right panel) were subjected to the multiplex PCR assay developed in this study and Ba813 PCR [8]. Lane M, 100-bp DNA ladder; lane 1, B. cereus strain LZ48–5; lane 2, B. cereus strain LZ136–1; lane 3, B. cereus strain LZ136–2; lane 4, B. cereus strain LZ77–1; lane 5, B. cereus strain LZ77–2; lane 6, B. cereus strain LZ78–7; lane 7, B. cereus strain LZ78–8; lane 8, B. cereus strain GTC02891; lane 9, B. cereus strain GTC02896; lane 10, B. cereus strain GTC02916; lane 11, B. cereus strain GTC02917; lane 12, B. cereus strain GTC03222; lane 13, B. cereus strain BC_CZC1; lane 15, B. cereus strain BC_CZC2; lane 15, B. pseudomycoides strain BP_CZC1; lane 16, B. pseudomycoides strain BP_CZC2; lane NC, distilled water as a negative control; lane BC, B. cereus strain GTC02826T; lane BA, B. anthracis strain CZC5 as a positive control.