Bacillus anthracis: Molecular taxonomy, population genetics, phylogeny and patho-evolution

Bacillus anthracis, the etiological agent of anthrax, manifests a particular bimodal lifestyle. This bacterial species alternates between short replication phases of 20-40 generations that strictly require infection of the host, normally causing death, interrupted by relatively long, mostly dormant phases as spores in the environment. Hence, the B. anthracis genome is highly homogeneous. This feature and the fact that strains from nearly all parts of the world have been analysed for canonical single nucleotide polymorphisms (canSNPs) and variable number tandem repeats (VNTRs) has allowed the development of molecular epidemiological and molecular clock models to estimate the age of major diversifications in the evolution of B. anthracis and to trace the global spread of this pathogen, which was mostly promoted by movement of domestic cattle with settlers and by international trade of contaminated animal products. From a taxonomic and phylogenetic point of view, B. anthracis is a member of the Bacillus cereus group. The differentiation of B. anthracis from B. cereus sensu strict, solely based on chromosomal markers, is difficult. However, differences in pathogenicity clearly differentiate B. anthracis from B. cereus and are marked by the strict presence of virulence genes located on the two virulence plasmids pXO1 and pXO2, which both are required by the bacterium to cause anthrax. Conversely, anthrax-like symptoms can also be caused by organisms with chromosomal features that are more closely related to B. cereus, but which carry these virulence genes on two plasmids that largely resemble the B. anthracis virulence plasmids. (C) 2011 Elsevier B.V. All rights reserved.

Bovine Bacillus anthracis in Cameroon

Bovine Bacillus anthracis isolates from Cameroon were genetically characterized. They showed a strong homogeneity, and they belong, together with strains from Chad, to cluster A beta, which appears to be predominant in western Africa. However, one strain that belongs to a newly defined clade (D) and cluster (D1) is penicillin resistant and shows certain phenotypes typical of Bacillus cereus.

Identification of an African Bacillus anthracis Lineage That Lacks Expression of the Spore Surface-Associated Anthrose-Containing Oligosaccharide

The surfaces of Bacillus anthracis endospores expose a pentasaccharide containing the monosaccharide anthrose, which has been considered for use as a vaccine or target for specific detection of the spores. In this study B. anthracis strains isolated from cattle carcasses in African countries where anthrax is endemic were tested for their cross-reactivity with monoclonal antibodies (MAbs) specific for anthrose-containing oligosaccharides. Unexpectedly, none of the isolates collected in Chad, Cameroon, and Mali were recognized by the MAbs. Sequencing of the four-gene operon encoding anthrose biosynthetic enzymes revealed the presence of premature stop codons in the aminotransferase and glycosyltransferase genes in all isolates from Chad, Cameroon, and Mali. Both immunological and genetic findings suggest that the West African isolates are unable to produce anthrose. The anthrose-deficient strains from West Africa belong to a particular genetic lineage. Immunization of cattle in Chad with a locally produced vaccine based on anthrose-positive spores of the B. anthracis strain Sterne elicited an anti-carbohydrate IgG response specific for a synthetic anthrose-containing tetrasaccharide as demonstrated by glycan microarray analysis. Competition immunoblots with synthetic pentasaccharide derivatives suggested an immunodominant role of the anthrose-containing carbohydrate in cattle. In West Africa anthrax is highly endemic. Massive vaccination of livestock in this area has taken place over long periods of time using spores of the anthrose-positive vaccine strain Sterne. The spread of anthrose-deficient strains in this region may represent an escape strategy of B. anthracis.

Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples

A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.

Antibiotic susceptibility and molecular diversity of Bacillus anthracis strains in Chad: detection of a new phylogenetic subgroup

We genotyped 15 Bacillus anthracis isolates from Chad, Africa, using multiple-locus variable-number tandem repeat analysis and three additional direct-repeat markers. We identified two unique genotypes that represent a novel genetic lineage in the A cluster. Chadian isolates were susceptible to 11 antibiotics and free of 94 antibiotic resistance genes.

Screening, optimization and extraction of polyhydroxyalkanoates and peptidoglycan from Bacillus megaterium

Bioplastics are polymers (such as polyesters) produced from bacterial fermentations that are biodegradable and nonhazardous. They are produced by a wide variety of bacteria and are made only when stress conditions allow, such as when nutrient levels are low, more specifically levels of nitrogen and oxygen. These stress conditions cause certain bacteria to build up excess carbon deposits as energy reserves in the form of polyhydroxyalkanoates (PHAs). PHAs can be extracted and formed into actual plastic with the same strength of conventional, synthetic-based plastics without the need to rely on foreign petroleum. The overall goal of this project was to select for a bacteria that could grow on sugars found in the lignocellulosic biomass, and get the bacteria to produce PHAs and peptidoglycan. Once this was accomplished the goal was to extract PHAs and peptidoglycan in order to make a stronger more rigid plastic, by combing them into a co-polymer. The individual goals of this project were to: (1) Select and screen bacteria that are capable of producing PHAs by utilizing the carbon/energy sources found in lignocellulosic biomass; (2) Maximize the utilization of those sugars present in woody biomass in order to produce optimal levels of PHAs. (3) Use room temperature ionic liquids (RTILs) in order to separate the cell membrane and peptidoglycan, allowing for better extraction of PHAs and more intact peptidoglycan. B. megaterium a Gram-positive PHA-producing bacterium was selected for study in this project. It was grown on a variety of different substrates in order to maximize both its growth and production of PHAs. The optimal conditions were found to be 30°C, pH 6.0 and sugar concentration of either 30g/L glucose or xylose. After optimal growth was obtained, both RTILs and enzymatic treatments were used to break the cell wall, in order to extract the PHAs, and peptidoglycan. PHAs and peptidoglycan were successfully extracted from the cell, and will be used in the future to create a new stronger co-polymer. Peptidoglycan recovery yield was 16% of the cells’ dry weight.

Severe hepatotoxicity following ingestion of Herbalife nutritional supplements contaminated with Bacillus subtilis

BACKGROUND/AIMS: Nutritional supplements are widely used. Recently, liver injury after consumption of Herbalife preparations was reported but the underlying pathogenesis remained cryptic. METHODS: Two patients presented with cholestatic hepatitis and pruritus, and cirrhosis, respectively. Viral, alcoholic, metabolic, autoimmune, neoplastic, vascular liver diseases and synthetic drugs as the precipitating causes of liver injury were excluded. However, both patients reported long-term consumption of Herbalife products. All Herbalife products were tested for contamination with drugs, pesticides, heavy metals, and softeners, and examined for microbial contamination according to standard laboratory procedures. Bacteria isolated from the samples were identified as Bacillus subtilis by sequencing the 16S rRNA and gyrB genes. RESULTS: Causality between consumption of Herbalife products and disease according to CIOMS was scored "probable" in both cases. Histology showed cholestatic and lobular/portal hepatitis with cirrhosis in one patient, and biliary fibrosis with ductopenia in the other. No contamination with chemicals or heavy metals was detected, and immunological testing showed no drug hypersensitivity. However, samples of Herbalife products ingested by both patients showed growth of Bacillus subtilis of which culture supernatants showed dose- and time-dependent hepatotoxicity. CONCLUSIONS: Two novel incidents of severe hepatic injury following intake of Herbalife products contaminated with Bacillus subtilis emphasize its potential hepatotoxicity.

The prognostic value of cytology and fluorescence in situ hybridization in the follow-up of nonmuscle-invasive bladder cancer after intravesical Bacillus Calmette-Guérin therapy

Molecular markers reliably predicting failure or success of Bacillus Calmette-Guérin (BCG) in the treatment of nonmuscle-invasive urothelial bladder cancer (NMIBC) are lacking. The aim of our study was to evaluate the value of cytology and chromosomal aberrations detected by fluorescence in situ hybridization (FISH) in predicting failure to BCG therapy. Sixty-eight patients with NMIBC were prospectively recruited. Bladder washings collected before and after BCG instillation were analyzed by conventional cytology and by multitarget FISH assay (UroVysion, Abbott/Vysis, Des Plaines, IL) for aberrations of chromosomes 3, 7, 17 and 9p21. Persistent and recurrent bladder cancers were defined as positive events during follow-up. Twenty-six of 68 (38%) NMIBC failed to BCG. Both positive post-BCG cytology and positive post-BCG FISH were significantly associated with failure of BCG (hazard ratio (HR)= 5.1 and HR= 5.6, respectively; p < 0.001 each) when compared to those with negative results. In the subgroup of nondefinitive cytology (all except those with unequivocally positive cytology), FISH was superior to cytology as a marker of relapse (HR= 6.2 and 1.4, respectively). Cytology and FISH in post-BCG bladder washings are highly interrelated and a positive result predicts failure to BCG therapy in patients with NMIBC equally well. FISH is most useful in the diagnostically less certain cytology categories but does not provide additional information in clearly malignant cytology.

Immuno-detection of anthrose containing tetrasaccharide in the exosporium of Bacillus anthracis and Bacillus cereus strains

AIMS: Bacillus anthracis strains of various origins were analysed with the view to describe intrinsic and persistent structural components of the Bacillus collagen-like protein of anthracis glycoprotein associated anthrose containing tetrasaccharide in the exosporium. METHODS AND RESULTS: The tetrasaccharide consists of three rhamnose residues and an unique monosaccharide--anthrose. As anthrose was not found in spores of related strains of bacteria, we envisioned the detection of B. anthracis spores based on antibodies against anthrose-containing polysaccharides. Carbohydrate-protein conjugates containing the synthetic tetrasaccharide, an anthrose-rhamnose disaccharide or anthrose alone were employed to immunize mice. All three formulations were immunogenic and elicited IgG responses with different fine specificities. All sera and monoclonal antibodies derived from tetrasaccharide immunized mice cross-reacted not only with spore lysates of a panel of virulent B. anthracis strains, but also with some of the B. cereus strains tested. CONCLUSIONS: Our results demonstrate that antibodies to synthetic carbohydrates are useful tools for epitope analyses of complex carbohydrate antigens and for the detection of particular target structures in biological specimens. SIGNIFICANCE AND IMPACT OF THE STUDY: Although not strictly specific for B. anthracis spores, antibodies against the tetrasaccharide may have potential as immuno-capturing components for a highly sensitive spore detection system.

Bacillus Calmette-Guérin Failure in Patients with Non-Muscle-invasive Urothelial Carcinoma of the Bladder May Be Due to the Urologist's Failure to Detect Urothelial Carcinoma of the Upper Urinary Tract and Urethra

BACKGROUND: Various reasons exist for so-called bacillus Calmette-Guérin (BCG) failure in patients with non-muscle-invasive urothelial bladder carcinoma (NMIBC). OBJECTIVE: To explore whether urothelial carcinoma of the upper urinary tract (UUT) and/or prostatic urethra may be a cause for BCG failure. DESIGN, SETTING, AND PARTICIPANTS: Retrospective analysis of 110 patients with high-risk NMIBC repeatedly treated with intravesical BCG, diagnosed with disease recurrence, and followed for a median time of 9.1 yr. INTERVENTION: Two or more intravesical BCG induction courses without maintenance. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Primary outcome was pattern of disease recurrence (BCG failure) within the urinary tract categorised into UUT and/or urethral carcinoma (with or without intravesical recurrence), and intravesical recurrence alone. Secondary outcome was survival. Predictors of UUT and/or urethral carcinoma and the effect of pattern of disease recurrence on cancer-specific survival were assessed with multivariable Cox regression analysis adjusting for multiple clinical and tumour characteristics. RESULTS AND LIMITATIONS: Of the 110 patients, 57 (52%) had UUT and/or urethral carcinoma (with or without intravesical recurrence), and 53 (48%) had intravesical recurrence alone. In patients with UUT and/or urethral carcinoma, bladder carcinoma in situ (Tis) before the first and second BCG course was present in 42 of 57 (74%) and 47 of 57 (82%) patients, respectively. On multivariable analysis, bladder Tis before the first and/or second BCG course was the only independent predictor of UUT and/or urethral carcinoma. Of the 110 patients, 69 (63%) were alive at last follow-up visit, 18 (16%) had died due to metastatic urothelial carcinoma, and 23 (21%) had died of other causes. Pattern of disease recurrence within the urinary tract was not an independent predictor of cancer-specific survival. Main study limitations were retrospective design and limited power for survival analysis. CONCLUSIONS: In our patients with high-risk NMIBC failing after two or more courses of intravesical BCG, UUT and/or urethral carcinoma was detected in >50% of the cases during follow-up. The vast majority of these patients had bladder Tis before the first and/or second BCG course. In patients experiencing the so-called BCG failure, a diagnostic work-up of UUT and prostatic urethra should always be performed to exclude urothelial carcinoma before additional intravesical therapy or even a radical cystectomy is considered.

The Immune Inhibitor A1 Protease of Bacillus anthracis

Bacillus anthracis, an organism ubiquitous in the soil and the causative agent of anthrax, utilizes multiple mechanisms to regulate secreted factors; one example is the activity of secreted proteases. One of the most abundant proteins in the culture supernates of B. anthracis is the Immune Inhibitor A1 (InhA1) protease. Here, I demonstrate that InhA1 modulates the abundance of approximately half of the proteins secreted into the culture supernates, including substrates that are known to contribute to the ability of the organism to cause virulence. For example, InhA1 cleaves the anthrax toxin proteins, PA, LF, and EF. InhA1 also targets a number of additional proteases, including Npr599, contributing to a complex proteolytic regulatory cascade with far-reaching affects on the secretome. Using an intra-tracheal mouse model of infection, I found that an inhA-null strain is attenuated in relation to the parent strain. The data indicate that reduced virulence of the inhA mutant strain may be the result of toxin protein deregulation, decreased association with macrophages, and/or the inability to degrade host antimicrobial peptides. Given the significant modulation of the secretome by InhA1, it is likely that expression of the protease is tightly regulated. To test this I examined inhA1 transcript and protein levels in the parent and various isogenic mutant strains and found that InhA1 expression is regulated by several mechanisms. First, the steady state levels of inhA1 transcript are controlled by the regulatory protein SinR, which inhibits inhA1 expression. Second, InhA1 abundance is inversely proportional to the SinR-regulated protease camelysin, indicating the post-transcriptional regulation of InhA1 by camelysin. Third, InhA1 activity is dependent on a conserved zinc binding motif, suggesting that zinc availability regulates InhA1 activity. The convergence of these regulatory mechanisms signifies the importance of tight regulation of InhA1 activity, activity that substantially affects how B. anthracis interacts with its environment.

Application of in vivo induced antigen technology (IVIAT) to Bacillus anthracis.

In vivo induced antigen technology (IVIAT) is an immuno-screening technique that identifies bacterial antigens expressed during infection and not during standard in vitro culturing conditions. We applied IVIAT to Bacillus anthracis and identified PagA, seven members of a N-acetylmuramoyl-L-alanine amidase autolysin family, three P60 family lipoproteins, two transporters, spore cortex lytic protein SleB, a penicillin binding protein, a putative prophage holin, respiratory nitrate reductase NarG, and three proteins of unknown function. Using quantitative real-time PCR comparing RNA isolated from in vitro cultured B. anthracis to RNA isolated from BALB/c mice infected with virulent Ames strain B. anthracis, we confirmed induced expression in vivo for a subset of B. anthracis genes identified by IVIAT, including L-alanine amidases BA3767, BA4073, and amiA (pXO2-42); the bacteriophage holin gene BA4074; and pagA (pXO1-110). The exogenous addition of two purified putative autolysins identified by IVIAT, N-acetylmuramoyl-L-alanine amidases BA0485 and BA2446, to vegetative B. anthracis cell suspensions induced a species-specific change in bacterial morphology and reduction in viable bacterial cells. Many of the proteins identified in our screen are predicted to affect peptidoglycan re-modeling, and our results support significant cell wall structural remodeling activity during B. anthracis infection. Identification of L-alanine amidases with B. anthracis specificity may suggest new potential therapeutic targets.

The role of the amino terminus of Bacillus subtilis sigmaK in transcription initiation

The sigma (σ) subunit of eubacterial RNA polymerase (RNAP) is required for specific recognition of promoter DNA sequences and transcription initiation. Regulation of bacterial gene expression can be achieved by modulating a factor activity. The Bacillus subtilis sporulation a σ factor, σ K, controls gene expression of the late sporulation regulon. σ K is synthesized as an inactive precursor protein, pro-σ K, with a 20 amino acid pro sequence. Proteolytic processing of the pro sequence produces the active form, σK, which is able to bind to the core subunits of RNAP to direct gene expression. Thus, the pro sequence renders σK inactive in vivo. After processing, the amino terminus of σK consists of region 1.2, which is conserved among various σ factors. To understand the role of the amino terminus of σK, namely the pro sequence and region 1.2, mutagenesis of both regions was pursued. NH 2-terminal truncations of pro-σK were constructed to address how the pro sequence silences σK activity. The work described here shows that the pro sequence inhibits the ability of σ K to associate with the core subunits and that a deletion of only six amino acids of the pro sequence is sufficient to activate pro-σ K for DNA binding and transcription initiation to levels similar to σ K. Additionally, site directed mutagenesis was used to obtain single amino acid substitutions in region 1.2 to address the role of region 1.2 in σ K transcriptional activity. Two mutations were isolated, converting a lysine (K) to an alanine (A) at position three, and an asparagine (N) to a tyrosine (Y) at position five, both of which alter the efficiency of transcription initiation by RNAP containing the mutant σKs. Surprisingly, σ KK3A increased transcript production when compared to wild type. This increase is due to improvement in DNA affinity and increased stability of RNAP-DNA promoter open complexes. σKN5Y showed a decrease in transcription activity that is related to defects in the ability of RNAP to make the transition from the closed to open RNAP-DNA complex. Results of both the pro sequence and region 1.2 analyses indicate that the amino terminus of σK is important for transcription activity and this work adds to the increasing body of evidence that the amino termini of many σ factors modulate transcription initiation by RNAP. ^