1. Introduction
Anthrax affects mostly cattle and sometimes humans, causing respiratory distress and bleeding. This disease can also be potentially transferred from warm-blooded animals to man, hence acting as vectors for human infection. Anthrax is caused by the bacterium Bacillus anthracis, an aerobic spore-forming bacillus. Spores are highly resistant to heat, cold, desiccation, radiation, and disinfectants, thus enabling the bacterium to persist in otherwise inhospitable environments [1].
The three disease forms denote the sites of infection: dermal (skin), pulmonary (lung), and intestinal. Pulmonary and intestinal infections are often fatal if untreated. Spores are taken up by macrophages and become internalized into phagolysosomes (membranous compartment) whereupon germination starts. Bacteria are released into the bloodstream once the infected macrophage lyses, whereupon they rapidly multiply, spreading throughout the circulatory and lymphatic systems, a process that results in septic shock, respiratory distress and organ failure. The spores of this pathogen have been used as a terror weapon. Virulence factors that set Bacillus anthracis apart from Bacillus cereus are encoded in two plasmids, pXO1 (anthrax toxin) and pXO2 (capsule genes) [2].
The capsule protects against phagocytosis once the vegetative bacterium enters the bloodstream. The anthrax toxin consists of three components: a protective antigen (PA), a lethal factor (LF) [3], and an edema factor (EF) [4]. A binary combination of these protein complexes, i.e., PA/LF and PA/EF, is internalized by host cells, where the LF (metalloprotease) and EF (calmodulin-dependent adenylate cyclase) causes edema and cell death in the host. At high levels, the LF induces cell death and release of the bacterium, while the EF increases the host susceptibility to infection and promotes fluid accumulation within cells [5].
Over 10 Bacillus anthracis genomes have been sequenced to date and 20 other genomes are being assembled and have been deposited in public databases such as the J. Craig Venter Institute and the National Center for Biotechnology Information. In the genome, approximately 35%–35.5% of the bases are guanine and cytosine. The prevalence of A + T means that this DNA has a lower melting temperature than that of many other bacteria [6].
Bacillus anthracis genome contains approximately 5.5 Mb and an average of 5700 protein-coding genes have been identified; there are 33 ribosomal RNA genes (23S, 16S and 5S) [7]. The chromosome represents about 95% of the genome, and it also contains two plasmids: pXO1 (181,600 bp) and pXO2 (94,800) [8].
Recent studies have focused on finding differences between subspecies of Bacillus anthracis and their phylogenetic relationship. Several methods have been developed for the detection and classification of Bacillus anthracis species and many more are still in the development phase. Those detection assays can be classified into three types: (a) whole organism; (b) bacterial antigen; and (c) nucleic acid detection. Five methods for detecting Bacillus anthracis are available: (1) Culture-based conventional method; (2) Immunological detection; (3) Nucleic-acid detection; (4) Ligand-bases detection; and (5) Biosensors [9].
An ideal detection system should be able to detect a very low number of copies in a variety of organisms (sensitivity), with no cross-reactivity (specificity), in a short time and a cost-effective manner.
DNA microarrays have become a powerful tool for the fast detection of bacteria and together with massive parallel sequencing are essential for genomic analysis. As demonstrated by our group by hybridizing target nucleic acid molecules with arrays of probes bound onto a surface and then analyzing the resulting virtual hybridization patterns, sequences can be comparatively analyzed to detect mutations and identify microorganisms. It also has been useful in gene expression profiling and verification of sequencing data. Microarray-based techniques would enable the rapid and reliable detection and identification of microorganisms (genus, species and strains), species within a given genus, new species, and would be useful in basic biochemical, genetic, and ecological research as well as in medical and industrial applications [10].
Our team recently designed a strategy for the in vitro identifying and studying bacteria, called Universal Fingerprinting Chip (UFC) [11]. The Virtual Hybridization (VH) approach uses the thermodynamic parameters of Santa Lucia, for calculation of the stability of DNA duplexes [12]. Our group has performed studies based on the experimental analysis of 16S rRNA genes and Virtual Hybridization. In this study an array of probes designed to identify several Pseudomonas and Bacillus strains was made and the duplexes formed with the PCR product of each strain were revealed. A strong correlation between the in silico hybridization and the experimental data was demonstrated, being able to identify both the mutations and the microorganisms.
UFC is an in silico microarray composed of 15,264 13-mer probe sequences which hybridize randomly and uniformly with whole genome sequences to produce highly informative fingerprints. In this study, we analyze a DNA microarray to discriminate between highly similar Bacillus anthracis genomes. Virtual hybridization is a powerful tool based on DNA microarrays that can discriminate between highly similar strains (up to 99% similarity). The 13-mer probes set hybridizes with genomes, revealing the exact position and stability of the duplex formed, thus creating a genomic fingerprint unique to each organism. This can then be used to calculate genomic distances between organisms to construct phylogenomic trees [13].
Other studies designed Influenza Probe Set (IPS, consisting in 1249 probes with a length 9-mer, extracted from sequence alignment zones with maximum entropy within the full viral genome of over 5000 viruses reported, considering almost all viral subtypes of Influenza A [14].