Peptides panned from a phage-displayed random peptide library are useful for the detection of Bacillus anthracis surrogates B. cereus 4342 and B. anthracis Sterne

Recent use of Bacillus anthracis as a bioweapon has highlighted the need for a sensitive monitoring system. Current bacterial detection tests use antibodies as bio-molecular recognition elements which have limitations with regard to time, specificity and sensitivity, creating the need for new and improved cost-effective high-affinity detection probes. In this study, we screened a commercially available bacteriophage-displayed random peptide library using Bacillus cereus 4342 cells as bait to identify peptides that could be used for detection of Bacillus. The method enabled us to identify two 12-amino acid consensus peptide sequences that specifically bind to B. cereus 4342 and B. anthracis Sterne, the nonpathogenic surrogates of B. anthracis strain. The two Bacillus-binding peptides (named BBP-1 and BBP-2) were synthesized with biotin tag to confirm their binding by four independent detection assays. Dot-blot analysis revealed that the peptides bind specifically to B. cereus 4342 and B. anthracis Sterne. Quantitative analysis of this interaction by ELISA and fluorometry demonstrated a detection sensitivity of 10² colony forming U/ml (CFU/ml) by both assays. When the peptides were used in combination with Qdots, the sensitivity was enhanced further by enabling detection of even a single bacterium by fluorescence microscopy. Immunoblot analysis and protein sequencing showed that BBP-1 and BBP-2 bound to the S-layer protein of B. anthracis Sterne. Overall, our findings validate the usefulness of synthetic versions of phage-derived peptides in combination with Qdot-liquid nanocrystals as high sensitivity bioprobes for various microbial detection platforms.     

Identification of a Bacillus anthracis specific indel in the yeaC gene and development of a rapid pyrosequencing assay for distinguishing B. anthracis from the B. cereus group

Bacillus anthracis, the causative agent of anthrax, is a potential source of bioterrorism. The existing assays for its identification lack specificity due to the close genetic relationship it exhibits to other members of the B. cereus group. Our comparative analyses of protein sequences from Bacillus species have identified a 24 amino acid deletion in a conserved region of the YeaC protein that is uniquely present in B. anthracis. PCR primers based on conserved regions flanking this indel in the Bacillus cereus group of species (viz. Bacillus cereus, B. anthracis, B. thuringiensis, B. mycoides, B. weihenstephnensis and B. pseudomycoides) specifically amplified a 282 bp fragment from all six reference B. anthracis strains, whereas a 354 bp fragment was amplified from 15 other B. cereus group of species/strains. These fragments, due to large size difference, are readily distinguished by means of agarose gel electrophoresis. In contrast to the B. cereus group, no PCR amplification was observed with any of the non-B. cereus group of species/strains. This indel was also used for developing a rapid pyrosequencing assay for the identification of B. anthracis. Its performance was evaluated by examining the presence or absence of this indel in a panel of 81 B. cereus-like isolates from various sources that included 39 B. anthracis strains. Based upon the sequence data from the pyrograms, the yeaC indel was found to be a distinctive characteristic of various B. anthracis strains tested and not found in any other species/strains from these samples. Therefore, this B. anthracis specific indel provides a robust and highly-specific chromosomal marker for the identification of this high-risk pathogen from other members of the B. cereus group independent of a strain's virulence. The pyrosequencing platform also allows for the rapid and simultaneous screening of multiple samples for the presence of this B. anthracis-specific marker.     

Application of Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Rapid Identification of Neisseria Species

Atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry (AP-MALDI MS) was applied to develop a proteomics-based method to detect and identify Neisseria species. Heat-inactivated clinical isolate cell suspensions of Neisseria gonorrhoeae and strains belonging to five serogroups (A, B, C, W135, and Y) of Neisseria meningitidis were subjected to on-probe protein/peptide extraction and tryptic digestion followed by AP-MALDI tandem MS (MS/MS)-based proteomic analysis. Amino acid sequences derived from three protonated peptides with m/z values of 1743.8, 1894.8, and 1946.8 were identified by AP-MALDI MS/MS and MASCOT proteome database search analysis as belonging to neisserial acyl carrier protein, neisserial-conserved hypothetical protein, and neisserial putative DNA binding protein, respectively. These three peptide masses can thus be potential biomarkers for neisserial species identification by AP-MALDI MS.     

Rapid Characterization of Spores of Bacillus cereus Group Bacteria by Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry

Matrix-assisted laser desorption-ionization (MALDI) time-of-flight mass spectrometry was used to characterize the spores of 14 microorganisms of the Bacillus cereus group. This group includes the four Bacillus species B. anthracis, B. cereus, B. mycoides, and B. thuringiensis. MALDI mass spectra obtained from whole bacterial spores showed many similarities between the species, except for B. mycoides. At the same time, unique mass spectra could be obtained for the different B. cereus and B. thuringiensis strains, allowing for differentiation at the strain level. To increase the number of detectable biomarkers in the usually peak-poor MALDI spectra of spores, the spores were treated by corona plasma discharge (CPD) or sonicated prior to MALDI analysis. Spectra of sonicated or CPD-treated spores displayed an ensemble of biomarkers common for B. cereus group bacteria. Based on the spectra available, these biomarkers differentiate B. cereus group spores from those of Bacillus subtilis and Bacillus globigii. The effect of growth medium on MALDI spectra of spores was also explored.     

Identification of Bacillus anthracis by Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and Artificial Neural Networks

This report demonstrates the applicability of a combination of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and chemometrics for rapid and reliable identification of vegetative cells of the causative agent of anthrax, Bacillus anthracis. Bacillus cultures were prepared under standardized conditions and inactivated according to a recently developed MS-compatible inactivation protocol for highly pathogenic microorganisms. MALDI-TOF MS was then employed to collect spectra from the microbial samples and to build up a database of bacterial reference spectra. This database comprised mass peak profiles of 374 strains from Bacillus and related genera, among them 102 strains of B. anthracis and 121 strains of B. cereus. The information contained in the database was investigated by means of visual inspection of gel view representations, univariate t tests for biomarker identification, unsupervised hierarchical clustering, and artificial neural networks (ANNs). Analysis of gel views and independent t tests suggested B. anthracis- and B. cereus group-specific signals. For example, mass spectra of B. anthracis exhibited discriminating biomarkers at 4,606, 5,413, and 6,679 Da. A systematic search in proteomic databases allowed tentative assignment of some of the biomarkers to ribosomal protein or small acid-soluble proteins. Multivariate pattern analysis by unsupervised hierarchical cluster analysis further revealed a subproteome-based taxonomy of the genus Bacillus. Superior classification accuracy was achieved when supervised ANNs were employed. For the identification of B. anthracis, independent validation of optimized ANN models yielded a diagnostic sensitivity of 100% and a specificity of 100%.Members of the genus Bacillus are rod-shaped bacteria that exhibit catalase activity and can be characterized as endospore-forming obligate or facultative aerobes. The genus Bacillus contains two important groups of bacteria named after B. subtilis and B. cereus. The best-characterized member of the former group is B. subtilis, a renowned model organism for genetic research. Other group members, like B. pumilis, B. licheniformis, B. atrophaeus, and B. amyloliquefaciens, exhibit a high degree of phenotypic similarity and are thus not easily distinguishable (15).The B. cereus group comprises a number of closely related bacteria, some of which interfere with human health. Bacteria classified as B. cereus are occasionally associated with food poisoning (16, 28), while B. thuringiensis is primarily an insect pathogen because of its ability to produce toxins that have been widely used for the biocontrol of insect pests (28, 30). A third member of the B. cereus group, B. anthracis, is the causative agent of anthrax and is highly relevant to human and animal health. Other members of the B. cereus group are B. mycoides, B. pseudomycoides, and B. weihenstephanensis (4, 15).B. anthracis is a possible agent in biological warfare and bioterrorism. Its applicability as a biological warfare agent was made apparent by an accidental release from a Soviet military facility in Sverdlovsk (1, 10). Also, the well-publicized mailing of B. anthracis spores in the United States, which caused 18 confirmed cases of cutaneous and inhalational anthrax and an additional 4 suspected cases of cutaneous anthrax (3, 22), demonstrated that B. anthracis may become a threat from terrorist groups (10).Rapid detection of B. anthracis may be challenging because of its great genetic similarity to other species of the B. cereus group (10) and the difficulties of phenotypic differentiation of B. cereus group members (15). There is some controversy in the literature regarding the taxonomy of the B. cereus group. Indeed, some authors state that B. anthracis, B. cereus, and B. thuringiensis are one species with various virulence plasmids for the toxin pXO1 and the capsule pXO2 of B. anthracis and the insecticidal toxin of B. thuringiensis (10, 19). Other authors do not support this opinion and suggest the presence of even more species within the group (21).Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) intact-cell mass spectrometry (ICMS) has been suggested as a rapid, objective, and reliable technique for bacterial identification (8, 13, 23, 25, 38). As a proteomic technique, ICMS of whole bacterial cells, or cell lysates, relies on the reproducible detection of microbial protein patterns and thus delivers information complementary to genotypic or phenotypic test methods. With the pattern-matching approach, microbial identification is achieved by comparing experimental mass spectra with a collection of mass spectra of known organisms. This requires the compilation of large databases of bacterial reference spectra but has the advantage that an extensive knowledge of biomarker identities is not required. Another advantage of the pattern-matching approach is that genus- and species-specific procedures or consumables are not required, i.e., the same methodology can in principle be applied to all kinds of microorganisms (multiplex advantage).It is thus believed that ICMS offers the possibility to systematically investigate the diversity of bacterial subproteomes, complementing existing methodologies of bacterial characterization. This potential and the need for a rapid, objective, and reliable microbial identification technique that does not rely on nucleic acid detection and the availability of an MS-compatible inactivation protocol for highly pathogenic biosafety level 3 microorganisms and bacterial endospores (26) prompted us to systematically study the MALDI-TOF MS profiles of Bacillus strains and to establish a database of bacterial mass spectra. In the present work, we describe strategies of spectral analysis that allow the identification and validation of group- and species-specific sets of biomarkers. Using unsupervised hierarchical cluster analysis (UHCA) and supervised artificial neural network (ANN) analysis, we also demonstrate how microbial spectra can be employed to establish an MS-based methodology for rapid, objective, and reliable identification of the target species, B. anthracis.     

Investigating the genome diversity of B. cereus and evolutionary aspects of B. anthracis emergence

Here we report the use of a multi-genome DNA microarray to investigate the genome diversity of Bacillus cereus group members and elucidate the events associated with the emergence of Bacillus anthracis the causative agent of anthrax—a lethal zoonotic disease. We initially performed directed genome sequencing of seven diverse B. cereus strains to identify novel sequences encoded in those genomes. The novel genes identified, combined with those publicly available, allowed the design of a “species” DNA microarray. Comparative genomic hybridization analyses of 41 strains indicate that substantial heterogeneity exists with respect to the genes comprising functional role categories. While the acquisition of the plasmid-encoded pathogenicity island (pXO1) and capsule genes (pXO2) represents a crucial landmark dictating the emergence of B. anthracis, the evolution of this species and its close relatives was associated with an overall shift in the fraction of genes devoted to energy metabolism, cellular processes, transport, as well as virulence.     

Identification and Validation of Specific Markers of Bacillus anthracis Spores by Proteomics and Genomics Approaches

Bacillus anthracis is the causative bacteria of anthrax, an acute and often fatal disease in humans. The infectious agent, the spore, represents a real bioterrorism threat and its specific identification is crucial. However, because of the high genomic relatedness within the Bacillus cereus group, it is still a real challenge to identify B. anthracis spores confidently. Mass spectrometry-based tools represent a powerful approach to the efficient discovery and identification of such protein markers. Here we undertook comparative proteomics analyses of Bacillus anthracis, cereus and thuringiensis spores to identify proteoforms unique to B. anthracis. The marker discovery pipeline developed combined peptide- and protein-centric approaches using liquid chromatography coupled to tandem mass spectrometry experiments using a high resolution/high mass accuracy LTQ-Orbitrap instrument. By combining these data with those from complementary bioinformatics approaches, we were able to highlight a dozen novel proteins consistently observed across all the investigated B. anthracis spores while being absent in B. cereus/thuringiensis spores. To further demonstrate the relevance of these markers and their strict specificity to B. anthracis, the number of strains studied was extended to 55, by including closely related strains such as B. thuringiensis 9727, and above all the B. cereus biovar anthracis CI, CA strains that possess pXO1- and pXO2-like plasmids. Under these conditions, the combination of proteomics and genomics approaches confirms the pertinence of 11 markers. Genes encoding these 11 markers are located on the chromosome, which provides additional targets complementary to the commonly used plasmid-encoded markers. Last but not least, we also report the development of a targeted liquid chromatography coupled to tandem mass spectrometry method involving the selection reaction monitoring mode for the monitoring of the 4 most suitable protein markers. Within a proof-of-concept study, we demonstrate the value of this approach for the further high throughput and specific detection of B. anthracis spores within complex samples.Bacillus anthracis is a highly virulent bacterium, which is the etiologic agent of anthrax, an acute and often lethal disease of animals and humans (1). According to the Centers for Disease Control and Prevention, B. anthracis is classified as a category A agent, the highest rank of potential bioterrorism agents (http://www.bt.cdc.gov/agent/agentlist-category.asp). The infectious agent of anthrax, the spore, was used as a bioterrorism weapon in 2001 in the United States when mailed letters containing B. anthracis spores caused 22 cases of inhalational and/or cutaneous anthrax, five of which were lethal (2). These events have emphasized the need for rapid and accurate detection of B. anthracis spores.Bacillus anthracis is a member of the genus Bacillus, Gram-positive, rod-shaped bacteria characterized by the ability to form endospores under aerobic or facultative anaerobic conditions (3). The genus Bacillus is a widely heterogeneous group encompassing 268 validly described species to date (http://www.bacterio.net/b/bacillus.html, last accessed on August 9^th 2013). B. anthracis is part of the B. cereus group which consists of six distinct species: B. anthracis, B. cereus, B. thuringiensis, B. mycoides, B. pseudomycoides, and B. weihenstephanensis (4, 5). The latter three species are generally regarded as nonpathogenic whereas B. cereus and B. thuringiensis could be opportunistic or pathogenic to mammals or insects (5, 6). B. cereus is a ubiquitous species that lives in soil but is also found in foods of plant and animal origin, such as dairy products (7). Its occurrence has also been linked to food poisoning and it can cause diarrhea and vomiting (6, 8). B. thuringiensis is primarily an insect pathogen, also present in soil, and often used as a biopesticide (9).B. anthracis is highly monomorphic, that is, shows little genetic variation (10), and primarily exists in the environment as a highly stable, dormant spore in the soil (1). Specific identification of B. anthracis is challenging because of its high genetic similarity (sequence similarity >99%) with B. cereus and B. thuringiensis (5, 11). The fact that these closely related species are rather omnipresent in the environment further complicates identification of B. anthracis. The main difference among these three species is the presence in B. anthracis of the two virulence plasmids pXO1 and pXO2 (1), which are responsible for its pathogenicity. pXO1 encodes a tripartite toxin (protective antigen (PA), lethal factor (LF), and edema factor (EF)) which causes edema and death (1), whereas pXO2 encodes a poly-γ-d-glutamate capsule which protects the organism from phagocytosis (1). B. anthracis identification often relies on the detection of the genes encoded by these two plasmids via nucleic acid-based assays (12–14). Nevertheless, the occasionally observed loss of the pXO2 plasmid within environmental species may impair the robustness of detection (1). In addition, in recent years a series of findings has shown that the presence of pXO1 and pXO2 is not a unique feature of B. anthracis. Indeed, Hu et al. have demonstrated that ∼7% of B. cereus/B. thuringiensis species can have a pXO1-like plasmid and ∼1.5% a pXO2-like plasmid (15). This was particularly underlined for some virulent B. cereus strains (i.e. B. cereus strains G9241, B. cereus biovar anthracis strains CA and CI) (16–20).Because of these potential drawbacks, the use of chromosome-encoded genes would be preferable for the specific detection of B. anthracis. Such genes (rpoB, gyrA, gyrB, plcR, BA5345, and BA813) have been reported as potential markers (21–25), but concerns have also been raised about their ability to discriminate B. anthracis efficiently from closely related B. cereus strains (26). Ahmod et al. have recently pointed out, by in silico database analysis, that a specific sequence deletion (indel) occurs in the yeaC gene and exploited it for the specific identification of B. anthracis (27). Nevertheless, a few B. anthracis strains (e.g. B. anthracis A1055) do not have this specific deletion and so may lead to false-negative results (27).In the last few years, protein profiling by MS, essentially based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF MS), has emerged as an alternative (or a complement) to genotypic or phenotypic methods for the fast and efficient identification of microorganisms (28, 29). Such an approach is based on the reproducible acquisition of global bacterial protein fingerprints/patterns. The combination of MS-based protein patterns and chemometric/bioinformatic tools has been demonstrated to efficiently differentiate members of the B. cereus group from other Bacillus species (30). However, the specific discrimination of B. anthracis from the closely related B. cereus and B. thuringiensis remains difficult (30). This study of Lasch and coworkers, performed on vegetative cells, identified a few ribosomal and spore proteins as being responsible for this clustering (30). Closer inspection of the data revealed that B. anthracis identification was essentially based on one particular isoform of the small acid-soluble spore protein B (SASP-B)¹ (30–34), which is exclusively expressed in spores, as the samples were shown to contain residual spores. However, the specificity of SASP-B has recently been questioned as the published genomes of B. cereus biovar anthracis CI and B. thuringiensis BGSC 4CC1 strains have been shown to share the same SASP-B isoform as B. anthracis (35). Altogether these results underline that the quest for specific markers of B. anthracis needs to be pursued.Mass spectrometry also represents a powerful tool for the discovery and identification of protein markers (36, 37). In the case of B. anthracis, this approach has more commonly been used for the comprehensive characterization of given bacterial proteomes. For example, the proteome of vegetative cells with variable plasmid contents has been extensively studied (38–40), as the proteomes of mature spores (41, 42) and of germinating spores (43, 44). Only one recent study, based on a proteo-genomic approach, was initiated to identify protein markers of B. anthracis (45). In this work, potential markers were characterized but using a very limited number of B. cereus group strains (three B. cereus and two B. thuringiensis). Moreover, this study was done on vegetative cells, whereas the spore proteome is drastically different. To our knowledge, no study has characterized and validated relevant protein markers specific to B. anthracis spores, which constitute the dissemination form of B. anthracis and are often targeted by first-line immunodetection methods (46).Here we report comparative proteomics analyses of Bacillus anthracis/cereus/thuringiensis spores, undertaken to identify proteoforms unique to B. anthracis. Preliminary identification was performed on a limited set of Bacillus species both at the peptide (after enzymatic digestion) and protein levels by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a high resolution/high mass accuracy LTQ-Orbitrap instrument. The pertinence of 11 markers was further demonstrated using proteomics and genomics approaches on a representative larger set of up to 55 different strains, including the closely related B. cereus biovar anthracis CI, CA, and B. thuringiensis 9727. Lastly, as a proof-of-concept study, we also report for four B. anthracis markers the implementation of a targeted LC-MS/MS method using selected reaction monitoring (SRM), based on the extension of a previous one focused on SASP-B (35). Preliminary results regarding method usefulness for the high throughput and accurate detection of B. anthracis spores in complex samples were also obtained and will be reported herein.     

Computational Biomarker Pipeline from Discovery to Clinical Implementation: Plasma Proteomic Biomarkers for Cardiac Transplantation

Recent technical advances in the field of quantitative proteomics have stimulated a large number of biomarker discovery studies of various diseases, providing avenues for new treatments and diagnostics. However, inherent challenges have limited the successful translation of candidate biomarkers into clinical use, thus highlighting the need for a robust analytical methodology to transition from biomarker discovery to clinical implementation. We have developed an end-to-end computational proteomic pipeline for biomarkers studies. At the discovery stage, the pipeline emphasizes different aspects of experimental design, appropriate statistical methodologies, and quality assessment of results. At the validation stage, the pipeline focuses on the migration of the results to a platform appropriate for external validation, and the development of a classifier score based on corroborated protein biomarkers. At the last stage towards clinical implementation, the main aims are to develop and validate an assay suitable for clinical deployment, and to calibrate the biomarker classifier using the developed assay. The proposed pipeline was applied to a biomarker study in cardiac transplantation aimed at developing a minimally invasive clinical test to monitor acute rejection. Starting with an untargeted screening of the human plasma proteome, five candidate biomarker proteins were identified. Rejection-regulated proteins reflect cellular and humoral immune responses, acute phase inflammatory pathways, and lipid metabolism biological processes. A multiplex multiple reaction monitoring mass-spectrometry (MRM-MS) assay was developed for the five candidate biomarkers and validated by enzyme-linked immune-sorbent (ELISA) and immunonephelometric assays (INA). A classifier score based on corroborated proteins demonstrated that the developed MRM-MS assay provides an appropriate methodology for an external validation, which is still in progress. Plasma proteomic biomarkers of acute cardiac rejection may offer a relevant post-transplant monitoring tool to effectively guide clinical care. The proposed computational pipeline is highly applicable to a wide range of biomarker proteomic studies.     

Coherent pipeline for biomarker discovery using mass spectrometry and bioinformatics

Background  

Robust biomarkers are needed to improve microbial identification and diagnostics. Proteomics methods based on mass spectrometry can be used for the discovery of novel biomarkers through their high sensitivity and specificity. However, there has been a lack of a coherent pipeline connecting biomarker discovery with established approaches for evaluation and validation. We propose such a pipeline that uses in silico methods for refined biomarker discovery and confirmation.     

Strategy for Identification of Bacillus cereus and Bacillus thuringiensis Strains Closely Related to Bacillus anthracis

Bacillus cereus strains that are genetically closely related to B. anthracis can display anthrax-like virulence traits (A. R. Hoffmaster et al., Proc. Natl. Acad. Sci. USA 101:8449-8454, 2004). Hence, approaches that rapidly identify these “near neighbors” are of great interest for the study of B. anthracis virulence mechanisms, as well as to prevent the use of such strains for B. anthracis-based bioweapon development. Here, a strategy is proposed for the identification of near neighbors of B. anthracis based on single nucleotide polymorphisms (SNP) in the 16S-23S rRNA intergenic spacer (ITS) containing tRNA genes, characteristic of B. anthracis. By using restriction site insertion-PCR (RSI-PCR) the presence of two SNP typical of B. anthracis was screened in 126 B. cereus group strains of different origin. Two B. cereus strains and one B. thuringiensis strain showed RSI-PCR profiles identical to that of B. anthracis. The sequencing of the entire ITS containing tRNA genes revealed two of the strains to be identical to B. anthracis. The strict relationship with B. anthracis was confirmed by multilocus sequence typing (MLST) of four other independent loci: cerA, plcR, AC-390, and SG-749. The relationship to B. anthracis of the three strains described by MLST was comparable and even higher to that of four B. cereus strains associated with periodontitis in humans and previously reported as the closest known strains to B. anthracis. SNP in ITS containing tRNA genes combined with RSI-PCR provide a very efficient tool for the identification of strains closely related to B. anthracis.     

Comparison of Minisatellite Polymorphisms in the Bacillus cereus Complex: a Simple Assay for Large-Scale Screening and Identification of Strains Most Closely Related to Bacillus anthracis

Polymorphism of five tandem repeats that are monomorphic in Bacillus anthracis was investigated in 230 isolates of the B. cereus group and in 5 sequenced B. cereus genomes in search for markers allowing identification of B. cereus and B. thuringiensis strains most closely related to B. anthracis. Using this multiple-locus variable number of tandem repeat analysis (MLVA), a cluster of 30 strains was selected for further characterization. Eventually, six of these were characterized by multilocus sequence type analysis. One of the strains is only six point mutations (of almost 3,000 bp) away from B. anthracis and was also proposed to be closest to B. anthracis by MLVA analysis. However, this strain remains separated from B. anthracis by a number of significant genetic events observed in B. anthracis, including the loss of the hemolysin activity, the presence of four prophages, and the presence of the two virulence plasmids, pXO1 and pXO2. One particular minisatellite marker provides an efficient assay to identify the subset of B. cereus and B. thuringiensis strains closely related to B. anthracis. Based on these results, a very simple assay is proposed that allows the screening of hundreds of strains from the B. cereus complex, with modest equipment and at a low cost, to eventually fill the gap with B. anthracis and better understand the origin and making of this dangerous pathogen.     

Bacillus anthracis physiology and genetics

Bacillus anthracis is a member of the Bacillus cereus group species (also known as the “group 1 bacilli”), a collection of Gram-positive spore-forming soil bacteria that are non-fastidious facultative anaerobes with very similar growth characteristics and natural genetic exchange systems. Despite their close physiology and genetics, the B. cereus group species exhibit certain species-specific phenotypes, some of which are related to pathogenicity. B. anthracis is the etiologic agent of anthrax. Vegetative cells of B. anthracis produce anthrax toxin proteins and a poly-d-glutamic acid capsule during infection of mammalian hosts and when cultured in conditions considered to mimic the host environment. The genes associated with toxin and capsule synthesis are located on the B. anthracis plasmids, pXO1 and pXO2, respectively. Although plasmid content is considered a defining feature of the species, pXO1- and pXO2-like plasmids have been identified in strains that more closely resemble other members of the B. cereus group. The developmental nature of B. anthracis and its pathogenic (mammalian host) and environmental (soil) lifestyles of make it an interesting model for study of niche-specific bacterial gene expression and physiology.     

Homoduplex and Heteroduplex Polymorphisms of the Amplified Ribosomal 16S-23S Internal Transcribed Spacers Describe Genetic Relationships in the “Bacillus cereus Group”

Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus pseudomycoides, Bacillus thuringiensis, and Bacillus weihenstephanensis are closely related in phenotype and genotype, and their genetic relationship is still open to debate. The present work uses amplified 16S-23S internal transcribed spacers (ITS) to discriminate between the strains and species and to describe the genetic relationships within the “B. cereus group,” advantage being taken of homoduplex-heteroduplex polymorphisms (HHP) resolved by polyacrylamide gel electrophoresis and silver staining. One hundred forty-one strains belonging to the six species were investigated, and 73 ITS-HHP pattern types were distinguished by MDE, a polyacrylamide matrix specifically designed to resolve heteroduplex and single-strand conformation polymorphisms. The discriminating bands were confirmed as ITS by Southern hybridization, and the homoduplex or heteroduplex nature was identified by single-stranded DNA mung bean nuclease digestion. Several of the ITS-HHP types corresponded to specific phenotypes such as B. anthracis or serotypes of B. thuringiensis. Unweighted pair group method arithmetic average cluster analysis revealed two main groups. One included B. mycoides, B. weihenstephanensis, and B. pseudomycoides. The second included B. cereus and B. thuringiensis, B. anthracis appeared as a lineage of B. cereus.     

Identification of a Bacillus anthracis specific indel in the yeaC gene and development of a rapid pyrosequencing assay for distinguishing B. anthracis from the B. cereus group

Bacillus anthracis, the causative agent of anthrax, is a potential source of bioterrorism. The existing assays for its identification lack specificity due to the close genetic relationship it exhibits to other members of the B. cereus group. Our comparative analyses of protein sequences from Bacillus species have identified a 24 amino acid deletion in a conserved region of the YeaC protein that is uniquely present in B. anthracis. PCR primers based on conserved regions flanking this indel in the Bacillus cereus group of species (viz. Bacillus cereus, B. anthracis, B. thuringiensis, B. mycoides, B. weihenstephnensis and B. pseudomycoides) specifically amplified a 282 bp fragment from all six reference B. anthracis strains, whereas a 354 bp fragment was amplified from 15 other B. cereus group of species/strains. These fragments, due to large size difference, are readily distinguished by means of agarose gel electrophoresis. In contrast to the B. cereus group, no PCR amplification was observed with any of the non-B. cereus group of species/strains. This indel was also used for developing a rapid pyrosequencing assay for the identification of B. anthracis. Its performance was evaluated by examining the presence or absence of this indel in a panel of 81 B. cereus-like isolates from various sources that included 39 B. anthracis strains. Based upon the sequence data from the pyrograms, the yeaC indel was found to be a distinctive characteristic of various B. anthracis strains tested and not found in any other species/strains from these samples. Therefore, this B. anthracis specific indel provides a robust and highly-specific chromosomal marker for the identification of this high-risk pathogen from other members of the B. cereus group independent of a strain's virulence. The pyrosequencing platform also allows for the rapid and simultaneous screening of multiple samples for the presence of this B. anthracis-specific marker.     

Use of high-resolution melting and melting temperature-shift assays for specific detection and identification of Bacillus anthracis based on single nucleotide discrimination

Single nucleotide polymorphisms (SNPs) are important diagnostic markers for the detection and differentiation of Bacillus anthracis. High-Resolution Melting (HRM) and Melting Temperature (Tm)-shift methods are two approaches that enable SNP detection without the need for expensive labeled probes. We evaluated the potential diagnostic capability of those methods to discriminate B. anthracis from the other members of the B. cereus group. Two assays targeting B. anthracis-specific SNPs in the plcR and gyrA genes were designed for each method and used to genotype a panel of 155 Bacilli strains. All B. anthracis isolates (n = 65) were correctly and unambiguously identified. Assays also proved to be appropriate for the direct genotyping of biological samples. They could reliably detect B. anthracis in contaminated organs containing as little as 10³ CFU/ml, corresponding to a few genome equivalents per reaction. The HRM and Tm-shift applications described here represent valuable tools for specific identification of B. anthracis at reduced cost.     

Comparative analysis of the virulence of invertebrate and mammalian pathogenic bacteria in the oral insect infection model Galleria mellonella

Infection of Galleria mellonella by feeding a mixture of Bacillus thuringiensis spores or vegetative bacteria in association with the toxin Cry1C results in high levels of larval mortality. Under these conditions the toxin or bacteria have minimal effects on the larva when inoculated separately. In order to evaluate whether G. mellonella can function as an oral infection model for human and entomo-bacterial pathogens, we tested strains of Bacillus cereus, Bacillus anthracis, Enterococcus faecalis, Listeria monocytogenes, Pseudomonas aeruginosa and a Drosophila targeting Pseudomonas entomophila strain. Six B. cereus strains (5 diarrheal, 1 environmental isolate) were first screened in 2nd instar G. mellonella larvae by free ingestion and four of them were analyzed by force-feeding 5th instar larvae. The virulence of these B. cereus strains did not differ from the B. thuringiensis virulent reference strain 407Cry⁻ with the exception of strain D19 (NVH391/98) that showed a lower virulence. Following force-feeding, 5th instar G. mellonella larvae survived infection with B. anthracis, L. monocytogenes, E. faecalis and P. aeruginosa strains in contrast to the P. entomophila strain which led to high mortality even without Cry1C toxin co-ingestion. Thus, specific virulence factors adapted to the insect intestine might exist in B. thuringiensis/B. cereus and P. entomophila. This suggests a co-evolution between host and pathogens and supports the close links between B. thuringiensis and B. cereus and more distant links to their relative B. anthracis.     

Genome Differences That Distinguish Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis

The three species of the group 1 bacilli, Bacillus anthracis, B. cereus, and B. thuringiensis, are genetically very closely related. All inhabit soil habitats but exhibit different phenotypes. B. anthracis is the causative agent of anthrax and is phylogenetically monomorphic, while B. cereus and B. thuringiensis are genetically more diverse. An amplified fragment length polymorphism analysis described here demonstrates genetic diversity among a collection of non-anthrax-causing Bacillus species, some of which show significant similarity to B. anthracis. Suppression subtractive hybridization was then used to characterize the genomic differences that distinguish three of the non-anthrax-causing bacilli from B. anthracis Ames. Ninety-three DNA sequences that were present in B. anthracis but absent from the non-anthrax-causing Bacillus genomes were isolated. Furthermore, 28 of these sequences were not found in a collection of 10 non-anthrax-causing Bacillus species but were present in all members of a representative collection of B. anthracis strains. These sequences map to distinct loci on the B. anthracis genome and can be assayed simultaneously in multiplex PCR assays for rapid and highly specific DNA-based detection of B. anthracis.     

Genetic distribution of 295 Bacillus cereus group members based on adk-screening in combination with MLST (Multilocus Sequence Typing) used for validating a primer targeting a chromosomal locus in B. anthracis

The genetic distribution of 295 Bacillus cereus group members has been investigated by using a modified Multilocus Sequence Typing method (MLST). By comparing the nucleic acid sequence of the adk gene fragment, isolates of B. cereus group members most related to B. anthracis may be easily identified. The genetic distribution, with focus on the B. anthracis close neighbours, was used to evaluate a new primer set for specific identification of B. anthracis. This primer set, BA5510-1/2, targeted the putative B. anthracis specific gene BA5510. Real-time PCR using BA5510-1/2 amplified the target fragment from all B. anthracis strains tested and only two (of 289) non-B. anthracis strains analysed. This is one of the most thoroughly validated chromosomal B. anthracis markers for real-time PCR identification, in which the screened collection contained several very closely related B. anthracis strains.     

Sampling and analytical strategies for biomarker discovery using mass spectrometry

There is an often unspoken truth behind the course of scientific investigation that involves not what is necessarily academically worthy of study, but rather what is scientifically worthy in the eyes of funding agencies. The perception of worthy research is, as cost is driven in the simplest sense in economics, often driven by demand. Presently, the demand for novel diagnostic and therapeutic protein biomarkers that possess high sensitivity and specificity is placing major impact on the field of proteomics. The focal discovery technology that is being relied on is mass spectrometry (MS), whereas the challenge of biomarker discovery often lies not in the application of MS but in the underlying proteome sampling and bioinformatic processing strategies. Although biomarker discovery research has been historically technology-driven, it is clear from the meager success in generating validated biomarkers that increasing attention must be placed at the pre-analytic stage, such as sample retrieval and preparation. As diseases vary, so do the combinations of sampling and sample analyses necessary to discover novel biomarkers. In this review, we highlight different strategies used toward biomarker discovery and discuss them in terms of their reliance on technology and methodology.