Pan-genome analysis reveals novel chromosomal markers for multiplex PCR-based specific detection of Bacillus anthracis

Abstract

Background: Bacillus anthracis is a highly pathogenic bacterium that can cause lethal infection in animals and humans, making it a significant concern as a pathogen and biological agent. Consequently, accurate diagnosis of B. anthracis is critically important for public health. However, the identification of specific marker genes encoded in the B. anthracis chromosome is challenging due to the genetic similarity it shares with B. cereus and B. thuringiensis.

Methods: The complete genomes of B. anthracis, B. cereus, B. thuringiensis, and B. weihenstephanensis were de novo annotated with Prokka, and these annotations were used by Roary to produce the pan-genome. B. anthracis exclusive genes were identified by Perl script, and their specificity was examined by nucleotide BLAST search. A local BLAST alignment was performed to confirm the presence of the identified genes across various B. anthracis strains. Multiplex polymerase chain reactions (PCR) were established based on the identified genes.

Result: The distribution of genes among 151 whole-genome sequences exhibited three distinct major patterns, depending on the bacterial species and strains. Further comparative analysis between the three groups uncovered thirty chromosome-encoded genes exclusively present in B. anthracis strains. Of these, twenty were found in known lambda prophage regions, and ten were in previously undefined region of the chromosome. We established three distinct multiplex PCRs for the specific detection of B. anthracis by utilizing three of the identified genes, BA1698, BA5354, and BA5361.

Conclusion: The study identified thirty chromosome-encoded genes specific to B. anthracis, encompassing previously described genes in known lambda prophage regions and nine newly discovered genes from an undefined gene region to the best of our knowledge. Three multiplex PCR assays offer an accurate and reliable alternative method for detecting B. anthracis. Furthermore, these genetic markers have value in anthrax vaccine development, and understanding the pathogenicity of B. anthracis.

Keywords: Bacillus anthracis; Anthrax; PCR; Pan-genome; Specific genes.

Fig. 1

Pan-genome analysis of B. anthracis and its closely related species, B. cereus and B. thuringiensis, with Heap’s law estimation. A The pan-genome of 115 B. anthracis strains and the corresponding Heap’s law estimation. B The pan-genome of 142 B. cereus strains, along with its Heap’s law estimation. C The pan-genome of 93 B. thuringiensis strains with Heap’s law estimation. Each Heap’s law estimation was conducted with 1000 permutations. The blue dots in the Heaps law graphic represent the observed number of unique genes as genomes are incrementally added. The red line represents the fitted Heap’s law curve based on the observed data. The x-axis represents the number of genomes. The y-axis represents the count of unique genes. k is intercept, representing the initial number of unique genes when only a few genomes are considered. γ is an exponent parameter that characterizes the rate at which new genes are added as more genomes are included in the analysis.If γ is closer to 1, it indicates an open pangenome, meaning the number of unique genes continues to increase significantly with additional genomes. Conversely, if γ is closer to 0, it suggests a closed pangenome, where the most genetic diversity is captured with the current dataset

Fig. 2

The pan-genome analysis illustrates the clustered presence or absence of genes and the distribution of accessory genes among the 151 Bacillus strains

Fig. 3

The localization of genes identified from pan-genome analysis on the chromosome of B. anthracis Ames. Red lines indicate the genes identified in this study, while blue boxes represent the lambda prophage regions described in a previous study [27]. An additional new region was identified between the coordinates 1,596,297 and 1,605,500

Fig. 4

Undefined specific gene region in the B. anthracis chromosome. B. anthracis Ames represents the Ban group; B. cereus ATCC14579 and B. thuringiensis ATCC10792 represent the Bce1 group; while B. cereus G9241 and B. thuringiensis 97-27 represent the Bce2 group, as categorized in this study. Green arrows indicate genes that are common among the bacterial species and red arrows indicate genes exclusive to B. anthracis

Fig. 5

MPCR-1 targets BA1698, pag, and capA. M is a 100 bp DNA ladder used as a marker. Lanes 1–10, and A1-A7 are amplicon results of B. anthracis strains (Strain ID in Table 4, 1–10, A1-A7), lane NC no-template negative control. Lanes 11–55 are amplicon results of non-B. anthracis strains (Strain ID in Table 4, 11–55). We used 1.5% agarose gel

Fig. 6

MPCR-2 targets BA5354, pag, and capA. M is a 100 bp DNA ladder used as a marker. Lanes 1–10, and A1-A7 are amplicon results of B. anthracis strains (Strain ID in Table 4, 1–10, A1-A7), lane NC no-template negative control. Lanes 11–55 are amplicon results of non-B. anthracis strains (Strain ID in Table 4, 11–55). We used 1.5% agarose gel

Fig. 7

MPCR-3 targets BA5361, pag, and capA. M is a 100 bp DNA ladder used as a marker. Lanes 1–10, and A1-A7 are amplicon results of B. anthracis strains (Strain ID in Table 4, 1–10, A1-A7), lane NC no-template negative control. Lanes 11–55 are amplicon results of non-B. anthracis strains (Strain ID in Table 4, 11–55). We used 1.5% agarose gel

Fig. 8

Sensitivity of multiplex PCRs analyzed by agarose gel electrophoresis. M is a 100 bp DNA ladder used as a marker. Lanes 1–7 are amplicon results of B. anthracis CZC5 strain (640, 320, 160, 80, 40, 20, 10 pg/μl), lane NC no-template negative control