Investigating the Influence of ANTXR2 Gene Mutations on Protective Antigen Binding for Heightened Anthrax Resistance.

Bacillus anthracis is the bacterium responsible for causing the zoonotic disease called anthrax. The disease presents itself in different forms like gastrointestinal, inhalation, and cutaneous. Bacterial spores are tremendously adaptable, can persist for extended periods and occasionally endanger human health. The Anthrax Toxin Receptor-2 (ANTXR2) gene acts as membrane receptor and facilitates the entry of the anthrax toxin into host cells. Additionally, mutations in the ANTXR2 gene have been linked to various autoimmune diseases, including Hyaline Fibromatosis Syndrome (HFS), Ankylosing Spondylitis (AS), Juvenile Hyaline Fibromatosis (JHF), and Infantile Systemic Hyalinosis (ISH). This study delves into the genetic landscape of ANTXR2, aiming to comprehend its associations with diverse disorders, elucidate the impacts of its mutations, and pinpoint minimal non-pathogenic mutations capable of reducing the binding affinity of the ANTXR2 gene with the protective antigen. Recognizing the pivotal role of single-nucleotide polymorphisms (SNPs) in shaping genetic diversity, we conducted computational analyses to discern highly deleterious and tolerated non-synonymous SNPs (nsSNPs) in the ANTXR2 gene. The Mutpred2 server determined that the Arg465Trp alteration in the ANTXR2 gene leads to altered DNA binding (p = 0.22) with a probability of a deleterious mutation of 0.808; notably, among the identified deleterious SNPs, rs368288611 (Arg465Trp) stands out due to its significant impact on altering the DNA-binding ability of ANTXR2. We propose these SNPs as potential candidates for hypertension linked to the ANTXR2 gene, which is implicated in blood pressure regulation. Noteworthy among the tolerated substitutions is rs200536829 (Ala33Ser), recognized as less pathogenic; this highlights its potential as a valuable biomarker, potentially reducing side effects on the host while also reducing binding with the protective antigen protein. Investigating these SNPs holds the potential to correlate with several autoimmune disorders and mitigate the impact of anthrax disease in humans.

'We dry contaminated meat to make it safe': An assessment of knowledge, attitude and practices on anthrax during an outbreak, Kisumu, Kenya, 2019.

INTRODUCTION: Anthrax is the highest-ranked priority zoonotic disease in Kenya with about ten human cases annually. Anthrax outbreak was reported in Kisumu East Sub County after some villagers slaughtered and ate beef from a cow suspected to have died of anthrax. We aimed at establishing the magnitude of the outbreak, described associated factors, and assessed community knowledge, attitude, and practices on anthrax. METHODS: We reviewed human and animal records, conducted case search and contact tracing using standard case definitions in the period from July 1through to July 28, 2019. A cross-sectional study was conducted to assess community knowledge, attitude, and practices towards anthrax. The household selection was done using multistage sampling. We cleaned and analyzed data in Ms. Excel and Epi Info. Descriptive statistics were carried out for continuous and categorical variables while analytical statistics for the association between dependent and independent variables were calculated. RESULTS: Out of 53 persons exposed through consumption or contact with suspicious beef, 23 cases (confirmed: 1, probable: 4, suspected: 18) were reviewed. The proportion of females was 52.17% (12/23), median age 13.5 years and range 45 years. The attack rate was 43.4% (23/53) and the case fatality rate was 4.35% (1/23). Knowledge level, determined by dividing those considered to be 'having good knowledge' on anthrax (numerator) by the total number of respondents (denominator) in the population regarding cause, transmission, symptoms and prevention was 51% for human anthrax and 52% for animal anthrax. Having good knowledge on anthrax was associated with rural residence [OR = 5.5 (95% CI 2.1-14.4; p<0.001)], having seen a case of anthrax [OR = 6.2 (95% CI 2.8-14.2; p<0.001)] and among those who present cattle for vaccination [OR = 2.6 (95% CI 1.2-5.6; p = 0.02)]. About 23.2% (26/112) would slaughter and sell beef to neighbors while 63.4% (71/112) would bury or burn the carcass. Nearly 93.8% (105/112) believed vaccination prevents anthrax. However, 5.4% (62/112) present livestock for vaccination. CONCLUSION: Most anthrax exposures were through meat consumption. Poor knowledge of the disease might hamper prevention and control efforts.

Silkworm model for Bacillus anthracis infection and virulence determination.

Bacillus anthracis is an obligate pathogen and a causative agent of anthrax. Its major virulence factors are plasmid-coded; however, recent studies have revealed chromosome-encoded virulence factors, indicating that the current understanding of its virulence mechanism is elusive and needs further investigation. In this study, we established a silkworm (Bombyx mori) infection model of B. anthracis. We showed that silkworms were killed by B. anthracis Sterne and cured of the infection when administered with antibiotics. We quantitatively determined the lethal dose of the bacteria that kills 50% larvae and effective doses of antibiotics that cure 50% infected larvae. Furthermore, we demonstrated that B. anthracis mutants with disruption in virulence genes such as pagA, lef, and atxA had attenuated silkworm-killing ability and reduced colonization in silkworm hemolymph. The silkworm infection model established in this study can be utilized in large-scale infection experiments to identify novel virulence determinants and develop novel therapeutic options against B. anthracis infections.

A Stochastic Intracellular Model of Anthrax Infection With Spore Germination Heterogeneity.

We present a stochastic mathematical model of the intracellular infection dynamics of Bacillus anthracis in macrophages. Following inhalation of B. anthracis spores, these are ingested by alveolar phagocytes. Ingested spores then begin to germinate and divide intracellularly. This can lead to the eventual death of the host cell and the extracellular release of bacterial progeny. Some macrophages successfully eliminate the intracellular bacteria and will recover. Here, a stochastic birth-and-death process with catastrophe is proposed, which includes the mechanism of spore germination and maturation of B. anthracis. The resulting model is used to explore the potential for heterogeneity in the spore germination rate, with the consideration of two extreme cases for the rate distribution: continuous Gaussian and discrete Bernoulli. We make use of approximate Bayesian computation to calibrate our model using experimental measurements from in vitro infection of murine peritoneal macrophages with spores of the Sterne 34F2 strain of B. anthracis. The calibrated stochastic model allows us to compute the probability of rupture, mean time to rupture, and rupture size distribution, of a macrophage that has been infected with one spore. We also obtain the mean spore and bacterial loads over time for a population of cells, each assumed to be initially infected with a single spore. Our results support the existence of significant heterogeneity in the germination rate, with a subset of spores expected to germinate much later than the majority. Furthermore, in agreement with experimental evidence, our results suggest that most of the spores taken up by macrophages are likely to be eliminated by the host cell, but a few germinated spores may survive phagocytosis and lead to the death of the infected cell. Finally, we discuss how this stochastic modelling approach, together with dose-response data, allows us to quantify and predict individual infection risk following exposure.

One Health approach for elimination of human anthrax in a tribal district of Odisha: Study protocol.

BACKGROUND: Anthrax is a major but neglected zoonotic disease of public health concern in India with Odisha contributing a major share to the disease burden. Bacillus anthracis spores can be found naturally in soil and commonly affect both animals and humans around the world. Domestic and wild animals such as cattle, sheep, goats, and deer can become infected when they inhale or ingest spores from contaminated soil, plants, or water. Anthrax can be fatal if patients are not treated promptly with antibiotics. This protocol aims to describe the implementation and evaluation of the 'One Health' intervention model based on the principles of Theory of Change (ToC) to eliminate human anthrax from a tribal district in Odisha, India. METHODS: This study would test the effectiveness of a complex public health intervention package developed using the ToC framework for the elimination of human anthrax in Koraput district by a comparative analysis of baseline and end-line data. We plan to enroll 2640 adults across 14 geographically divided blocks in Koraput district of Odisha for baseline and end-line surveys. After baseline, we would provide capacity building training to stakeholders from the department of health, veterinary, forest, academic and allied health institutions followed by workshops on sensitization and awareness through IEC (Information Education Communication)/BCC (Behavior Change Communication) activities in the community. We would establish a state-level laboratory facility as a robust system for timely diagnosis and management of human anthrax cases. Surveillance network will be strengthened to track the cases in early stage and risk zoning will be done for focused surveillance in endemic areas. Advocacy with district level administration will be done for maximizing the coverage of livestock vaccination in the entire district. Interdepartmental coordination would be established for the effective implementation of the intervention package. CONCLUSION: This would be a first study applying One Health concept for the elimination of human anthrax in India. The findings from this study will offer important insights for policy-making and further replication in other endemic regions of the state and country. TRIAL REGISTRATION: The authors confirm that all ongoing and related trials for this intervention are prospectively registered with the Clinical Trials Registry of India [CTRI/2020/05/025325] on 22 May 2020.

Convergent evolution of diverse Bacillus anthracis outbreak strains toward altered surface oligosaccharides that modulate anthrax pathogenesis.

Bacillus anthracis, a spore-forming gram-positive bacterium, causes anthrax. The external surface of the exosporium is coated with glycosylated proteins. The sugar additions are capped with the unique monosaccharide anthrose. The West African Group (WAG) B. anthracis have mutations rendering them anthrose deficient. Through genome sequencing, we identified 2 different large chromosomal deletions within the anthrose biosynthetic operon of B. anthracis strains from Chile and Poland. In silico analysis identified an anthrose-deficient strain in the anthrax outbreak among European heroin users. Anthrose-deficient strains are no longer restricted to West Africa so the role of anthrose in physiology and pathogenesis was investigated in B. anthracis Sterne. Loss of anthrose delayed spore germination and enhanced sporulation. Spores without anthrose were phagocytized at higher rates than spores with anthrose, indicating that anthrose may serve an antiphagocytic function on the spore surface. The anthrose mutant had half the LD50 and decreased time to death (TTD) of wild type and complement B. anthracis Sterne in the A/J mouse model. Following infection, anthrose mutant bacteria were more abundant in the spleen, indicating enhanced dissemination of Sterne anthrose mutant. At low sample sizes in the A/J mouse model, the mortality of ΔantC-infected mice challenged by intranasal or subcutaneous routes was 20% greater than wild type. Competitive index (CI) studies indicated that spores without anthrose disseminated to organs more extensively than a complemented mutant. Death process modeling using mouse mortality dynamics suggested that larger sample sizes would lead to significantly higher deaths in anthrose-negative infected animals. The model was tested by infecting Galleria mellonella with spores and confirmed the anthrose mutant was significantly more lethal. Vaccination studies in the A/J mouse model showed that the human vaccine protected against high-dose challenges of the nonencapsulated Sterne-based anthrose mutant. This work begins to identify the physiologic and pathogenic consequences of convergent anthrose mutations in B. anthracis.

Detection of Bacillus anthracis and Bacillus anthracis-like spores in soil from state of Rio de Janeiro, Brazil.

BACKGROUND: Bacillus anthracis is the aetiologic agent of anthrax, a re-emerging, septicaemic, haemorrhagic and lethal disease that affects humans, domestic ruminants and wildlife. Plasmids pXO1 and pXO2 are attributes that confer pathogenicity to B. anthracis strains. This bacterium was used as biological weapon in the World Wars and in the biological attack in the United States of America at 2001. B. anthracis is classified as a Tier 1 bioterrorism agent by the Centers for Diseases Control and Prevention. Anthrax is recognised as a re-emerging disease. Several studies concerning the dynamics of B. anthracis cycle in soil revealed that nonpathogenic B. anthracis strains due to lack of pXO2 plasmid are commonly found in some types of soil. OBJECTIVES: This study aimed isolation and identification of B. anthracis spores in soil samples of the state of Rio de Janeiro, Brazil. METHODS: Phenotypic and genotypic approaches were used to identify isolates including MALDI-TOF/MS, motility test, susceptibility to gamma phage and penicillin, survey for pag and cap genes as surrogates of pXO1 and pXO2 plasmids, respectively, and sequencing of 16SrRNA-encoding gene. Physicochemical analysis of the soil samples were carried out to describe soil characteristics. FINDINGS: We observed the presence of one B. anthracis pXO1+ and pXO2- isolated from clay loam soil; one B. anthracis-like strain pXO1+ and pXO2-isolated from loamy sand; and 10 Bacillus spp. strains sensitive to phage-gamma that need better characterisation to define which their species were recovered from loamy sand. MAIN CONCLUSIONS: This work showed promising results and it was the first study to report results from an active surveillance for B. anthracis in Brazil.

Structural Integrity of the Alveolar-Capillary Barrier in Cynomolgus Monkeys Challenged with Fully Virulent and Toxin-Deficient Strains of Bacillus anthracis.

Inhalational anthrax, a disease caused by inhaling Bacillus anthracis spores, leads to respiratory distress, vascular leakage, high-level bacteremia, and often death within days. Anthrax lethal toxin and edema toxin, which are composed of protective antigen (PA) plus either lethal factor (LF) or edema factor (EF), respectively, play an important yet incompletely defined role in the pulmonary pathophysiology. To better understand their contribution, we examined the structural integrity of the alveolar-capillary barrier in archival formalin-fixed lungs of cynomolgus monkeys challenged with the fully virulent B. anthracis Ames wild-type strain or the isogenic toxin-deficient mutants ΔEF, ΔLF, and ΔPA. Pulmonary spore challenge with the wild-type strain caused high mortality, intra-alveolar hemorrhages, extensive alveolar septal sequestration of bacteria and neutrophils, diffuse destabilization of epithelial and endothelial junctions, increased markers of coagulation and complement activation (including tissue factor and C5a), and multifocal intra-alveolar fibrin deposition. ΔEF challenge was lethal and showed similar alveolar-capillary alterations; however, intra-alveolar hemorrhages, bacterial deposition, and markers of coagulation or complement were absent or markedly lower. In contrast, ΔLF or ΔPA challenges were nonlethal and showed no signs of alveolar bacterial deposition or alveolar-capillary changes. These findings provide evidence that lethal toxin plays a determinative role in bacterial dissemination and alveolar-capillary barrier dysfunction, and edema toxin may significantly exacerbate pulmonary pathologies in a systemic infection.

Frontline Science: Anthrax lethal toxin-induced, NLRP1-mediated IL-1ß release is a neutrophil and PAD4-dependent event.

Anthrax lethal toxin (LT) is a protease that activates the NLRP1b inflammasome sensor in certain rodent strains. Unlike better-studied sensors, relatively little is known about the priming requirements for NLRP1b. In this study, we investigate the rapid and striking priming-independent LT-induced release of IL-1ß in mice within hours of toxin challenge. We find IL-1ß release to be a NLRP1b- and caspase-1-dependent, NLRP3 and caspase-11-independent event that requires both neutrophils and peptidyl arginine deiminiase-4 (PAD4) activity. The simultaneous LT-induced IL-18 response is neutrophil-independent. Bone marrow reconstitution experiments in mice show toxin-induced IL-1ß originates from hematopoietic cells. LT treatment of neutrophils in vitro did not induce IL-1ß, neutrophil extracellular traps (NETs), or pyroptosis. Although platelets interact closely with neutrophils and are also a potential source of IL-1ß, they were unable to bind or endocytose LT and did not secrete IL-1ß in response to the toxin. LT-treated mice had higher levels of cell-free DNA and HMGB1 in circulation than PBS-treated controls, and treatment of mice with recombinant DNase reduced the neutrophil- and NLRP1-dependent IL-1ß release. DNA sensor AIM2 deficiency, however, did not impact IL-1ß release. These data, in combination with the findings on PAD4, suggest a possible role for in vivo NETs or cell-free DNA in cytokine induction in response to LT challenge. Our findings suggest a complex interaction of events and/or mediators in LT-treated mice with the neutrophil as a central player in induction of a profound and rapid inflammatory response to toxin.

The use of ozone gas for the inactivation of Bacillus anthracis and Bacillus subtilis spores on building materials.

A study was conducted to assess the efficacy of ozone gas in inactivating spores of both Bacillus anthracis and Bacillus subtilis inoculated onto six building materials (glass, wood, carpet, laminate, galvanized metal, and wallboard paper). Testing conditions consisted of ozone gas concentrations ranging from 7,000-12,000 parts per million (ppm), contact times from 4 to 12 h, and two relative humidity (RH) levels of 75 and 85%. Results showed that increasing the ozone concentration, contact time, and RH generally increased decontamination efficacy. The materials in which the highest decontamination efficacy was achieved for B. anthracis spores were wallboard paper, carpet, and wood with ≥ 6 log10 reduction (LR) occurring with 9,800 ppm ozone, 85% RH, for 6 h. The laminate and galvanized metal materials were generally more difficult to decontaminate, requiring 12,000 ppm ozone, 85% RH, and 9-12 h contact time to achieve ≥6 LR of B. anthracis. Lastly, overall, there were no significant differences in decontamination efficacy between the two species.

Progress towards the Development of a NEAT Vaccine for Anthrax II: Immunogen Specificity and Alum Effectiveness in an Inhalational Model.

Bacillus anthracis is the causative agent of anthrax disease, presents with high mortality, and has been at the center of bioweapon efforts. The only currently U.S. FDA-approved vaccine to prevent anthrax in humans is anthrax vaccine adsorbed (AVA), which is protective in several animal models and induces neutralizing antibodies against protective antigen (PA), the cell-binding component of anthrax toxin. However, AVA requires a five-course regimen to induce immunity, along with an annual booster, and is composed of undefined culture supernatants from a PA-secreting strain. In addition, it appears to be ineffective against strains that lack anthrax toxin. Here, we investigated a vaccine formulation consisting of recombinant proteins from a surface-localized heme transport system containing near-iron transporter (NEAT) domains and its efficacy as a vaccine for anthrax disease. The cocktail of five NEAT domains was protective against a lethal challenge of inhaled bacillus spores at 3 and 28 weeks after vaccination. The reduction of the formulation to three NEATs (IsdX1, IsdX2, and Bslk) was as effective as a five-NEAT domain cocktail. The adjuvant alum, approved for use in humans, was as protective as Freund's Adjuvant, and protective vaccination correlated with increased anti-NEAT antibody reactivity and reduced bacterial levels in organs. Finally, the passive transfer of anti-NEAT antisera reduced mortality and disease severity, suggesting the protective component is comprised of antibodies. Collectively, these results provide evidence that a vaccine based upon recombinant NEAT proteins should be considered in the development of a next-generation anthrax vaccine.

Bacillus anthracis Poly-γ-D-Glutamate Capsule Inhibits Opsonic Phagocytosis by Impeding Complement Activation.

Bacillus anthracis poly-γ-D-glutamic acid (PGA) capsule is an essential virulent factor that helps the bacterial pathogen to escape host immunity. Like other encapsulated bacterial species, the B. anthracis capsule may also inhibit complement-mediated clearance and ensure bacterial survival in the host. Previous reports suggest that B. anthracis spore proteins inhibit complement activation. However, the mechanism through which the B. anthracis capsule imparts a survival advantage to the active bacteria has not been demonstrated till date. Thus, to evaluate the role of the PGA capsule in evading host immunity, we have undertaken the present head-to-head comparative study of the phagocytosis and complement activation of non-encapsulated and encapsulated B. anthracis strains. The encapsulated virulent strain exhibited resistance toward complement-dependent and complement-independent bacterial phagocytosis by human macrophages. The non-encapsulated Sterne strain was highly susceptible to phagocytosis by THP-1 macrophages, after incubation with normal human serum (NHS), heat-inactivated serum, and serum-free media, thus indicating that the capsule inhibited both complement-dependent and complement-independent opsonic phagocytosis. An increased binding of C3b and its subsequent activation to C3c and C3dg, which functionally act as potent opsonins, were observed with the non-encapsulated Sterne strain compared with the encapsulated strain. Other known mediators of complement fixation, IgG, C-reactive protein (CRP), and serum amyloid P component (SAP), also bound more prominently with the non-encapsulated Sterne strain. Studies with complement pathway-specific, component-deficient serum demonstrated that the classical pathway was primarily involved in mediating C3b binding on the non-encapsulated bacteria. Both strains equally bound the complement regulatory proteins C4BP and factor H. Importantly, we demonstrated that the negative charge of the PGA capsule was responsible for the differential binding of the complement proteins between the non-encapsulated and encapsulated strains. At lower pH closer to the isoelectric point of PGA, the neutralization of the negative charge was associated with an increased binding of C3b and IgG with the encapsulated B. anthracis strain. Overall, our data have demonstrated that the B. anthracis capsule inhibits complement fixation and opsonization resulting in reduced phagocytosis by macrophages, thus allowing the bacterial pathogen to evade host immunity.

Increased Excess Intracellular Cyclic di-AMP Levels Impair Growth and Virulence of Bacillus anthracis.

Cyclic di-AMP (c-di-AMP) is a recently identified bacterial second messenger that regulates biological processes. In this study, we found that inactivation of two c-di-AMP phosphodiesterases (PDEs), GdpP and PgpH, resulted in accumulation of 3.8-fold higher c-di-AMP levels than in the parental strain Sterne in Bacillus anthracis and inhibited bacterial growth. Moreover, excess c-di-AMP accumulation decreased bacterial toxin expression, increased sensitivity to osmotic stress and detergent, and attenuated virulence in both C57BL/6J and A/J mice. Complementation of the PDE mutant with a plasmid carrying gdpP or pgpH in trans from a Pspac promoter restored bacterial growth, virulence factor expression, and resistance to detergent. Our results indicate that c-di-AMP is a pleiotropic signaling molecule in B. anthracis that is important for host-pathogen interaction.IMPORTANCE Anthrax is an ancient and deadly disease caused by the spore-forming bacterial pathogen Bacillus anthracis Vegetative cells of this species produce anthrax toxin proteins and S-layer components during infection of mammalian hosts. So far, how the expression of these virulence factors is regulated remains largely unknown. Our results suggest that excess elevated c-di-AMP levels inhibit bacterial growth and reduce expression of S-layer components and anthracis toxins as well as reduce virulence in a mouse model of disease. These results indicate that c-di-AMP signaling plays crucial roles in B. anthracis biology and disease.

Using old antibiotics to treat ancient bacterium-ß-lactams for Bacillus anthracis meningitis.

As Bacillus anthracis spores pose a proven bio-terror risk, the treatment focus has shifted from exposed populations to anthrax patients and the need for effective antibiotic treatment protocols increases. The CDC recommends carbapenems and Linezolid (oxazolidinone), for the treatment of anthrax, particularly for the late, meningeal stages of the disease. Previously we demonstrated that treatment with Meropenem or Linezolid, either as a single treatment or in combination with Ciprofloxacin, fails to protect rabbits from anthrax-meningitis. In addition, we showed that the failure of Meropenem was due to slow BBB penetration rather than low antibacterial activity. Herein, we tested the effect of increasing the dose of the antibiotic on treatment efficacy. We found that for full protection (88% cure rate) the dose should be increased four-fold from 40 mg/kg to 150 mg/kg. In addition, B. anthracis is a genetically stable bacterium and naturally occurring multidrug resistant B. anthracis strains have not been reported. In this manuscript, we report the efficacy of classical ß-lactams as a single treatment or in combination with ß-lactamase inhibitors in treating anthrax meningitis. We demonstrate that Ampicillin based treatment of anthrax meningitis is largely efficient (66%). The high efficacy (88-100%) of Augmentin (Amoxicillin and Clavulonic acid) and Unasyn (Ampicillin and Sulbactam) makes them a favorable choice due to reports of ß-lactam resistant B. anthracis strains. Tazocin (Piperacillin and Tazobactam) proved inefficient compared to the highly efficient Augmentin and Unasyn.

Evaluation of liposomal ciprofloxacin formulations in a murine model of anthrax.

The in vivo efficacy of liposomal encapsulated ciprofloxacin in two formulations, lipoquin and apulmiq, were evaluated against the causative agent of anthrax, Bacillus anthracis. Liposomal encapsulated ciprofloxacin is attractive as a therapy since it allows for once daily dosing and achieves higher concentrations of the antibiotic at the site of initial mucosal entry but lower systemic drug concentrations. The in vivo efficacy of lipoquin and apulmiq delivered by intranasal instillation was studied at different doses and schedules in both a post exposure prophylaxis (PEP) therapy model and in a delayed treatment model of murine inhalational anthrax. In the mouse model of infection, the survival curves for all treatment cohorts differed significantly from the vehicle control. Ciprofloxacin, lipoquin and apulmiq provided a high level of protection (87-90%) after 7 days of therapy when administered within 24 hours of exposure. Reducing therapy to only three days still provided protection of 60-87%, if therapy was provided within 24 hours of exposure. If treatment was initiated 48 hours after exposure the survival rate was reduced to 46-65%. These studies suggest that lipoquin and apulmiq may be attractive therapies as PEP and as part of a treatment cocktail for B. anthracis.

Genetic characterization of Bacillus anthracis strains circulating in Italy from 1972 to 2018.

In Italy anthrax is an endemic disease, with a few outbreaks occurring almost every year. We surveyed 234 B. anthracis strains from animals (n = 196), humans (n = 3) and the environment (n = 35) isolated during Italian outbreaks in the years 1972-2018. Despite the considerable genetic homogeneity of B. anthracis, the strains were effectively differentiated using canonical single nucleotide polymorphisms (CanSNPs) assay and multiple-locus variable-number tandem repeat analysis (MLVA). The phylogenetic identity was determined through the characterization of 14 CanSNPs. In addition, a subsequent 31-loci MLVA assay was also used to further discriminate B. anthracis genotypes into subgroups. The analysis of 14 CanSNPs allowed for the identification of four main lineages: A.Br.011/009, A.Br.008/011 (respectively belonging to A.Br.008/009 sublineage, also known Trans-Eurasian or TEA group), A.Br.005/006 and B.Br.CNEVA. A.Br.011/009, the most common subgroup of lineage A, is the major genotype of B. anthracis in Italy. The MLVA analysis revealed the presence of 55 different genotypes in Italy. Most of the genotypes are genetically very similar, supporting the hypothesis that all strains evolved from a local common ancestral strain, except for two genotypes representing the branch A.Br.005/006 and B.Br.CNEVA. The genotyping analysis applied in this study remains a very valuable tool for studying the diversity, evolution, and molecular epidemiology of B. anthracis.

Very Early Blood Diffusion of the Active Lethal and Edema Factors of Bacillus anthracis After Intranasal Infection.

BACKGROUND: Lethal and edema toxins are critical virulence factors of Bacillus anthracis. Few data are available on their presence in the early stage of intranasal infection. METHODS: To investigate the diffusion of edema factor (EF) and lethal factor (LF), we use sensitive quantitative methods to measure their enzymatic activities in mice intranasally challenged with a wild-type B anthracis strain or with an isogenic mutant deficient for the protective antigen. RESULTS: One hour after mouse challenge, although only 7% of mice presented bacteremia, LF and EF were detected in the blood of 100% and 42% of mice, respectively. Protective antigen facilitated the diffusion of LF and EF into the blood compartment. Toxins played a significant role in the systemic dissemination of B anthracis in the blood, spleen, and liver. A mouse model of intoxination further confirmed that LT and ET could diffuse rapidly in the circulation, independently of bacteria. CONCLUSIONS: In this inhalational model, toxins have disseminated rapidly in the blood, playing a significant and novel role in the early systemic diffusion of bacteria, demonstrating that they may represent a very early target for the diagnosis and the treatment of anthrax.

Influence of the phosphoenolpyruvate:carbohydrate phosphotransferase system on toxin gene expression and virulence in Bacillus anthracis.

AtxA, the master virulence gene regulator of Bacillus anthracis, is a PRD-Containing Virulence Regulator (PCVR) as indicated by the crystal structure, post-translational modifications and activity of the protein. PCVRs are transcriptional regulators, named for PTS Regulatory Domains (PRDs) subject to phosphorylation by the phosphoenolpyruvate phosphotransferase system (PEP-PTS) and for their impact on virulence gene expression. Here we present data from experiments employing physiological, genetic and biochemical approaches that support a model in which the PTS proteins HPr and Enzyme I (EI) are required for transcription of the atxA gene, rather than phosphorylation of AtxA. We show that atxA transcription is reduced 2.5-fold in a mutant lacking HPr and EI, and that this change is sufficient to affect anthrax toxin production. Mutants harboring HPr proteins altered for phosphotransfer activity were unable to restore atxA transcription to parent levels, suggesting that phosphotransfer activity of HPr and EI is important for regulation of atxA. In a mouse model for anthrax, a HPr- EI- mutant was attenuated for virulence. Virulence was restored by expressing atxA from an alternative, PTS-independent, promoter. Our data support a model in which HPr transfers a phosphate to an unidentified downstream transcriptional regulator to influence atxA gene transcription.

Determination of pathogenic bacteria-Bacillus anthrax spores in environmental samples by ratiometric fluorescence and test paper based on dual-emission fluorescent silicon nanoparticles.

Many lanthanide ions-based probes have been widely used for detecting anthrax spores biomarker-dipicolinic acid (DPA). However, little work has realized detection of bacillus anthrax spores in real environmental samples. In this work, a novel ratiometric fluorescent nanoprobe based on europium (Eu)-doped silicon nanoparticles (Eu@SiNPs) was fabricated for the first time by one-pot method without post-modification for determination of the DPA in bacillus subtilis spores (simulant bacillus anthrax spores). Based on Eu(III) in the Eu@SiNPs could be sensitized by DPA to emit intrinsic fluorescence and the fluorescence intensity of SiNPs in the Eu@SiNPs almost remained stable, a new ratiometric fluorescent method for determination of micro DPA in bacillus subtilis spores and bacillus subtilis spores in real environmental samples, such as Yellow river water, tap water and soil was established. Under the optimum conditions, the limit of detection (LOD) of the method toward bacillus subtilis spores was as low as 2.38×104 spore/mL. Simple, fast and visual DPA and bacillus subtilis spores determination was also achieved by the Eu@SiNPs-based test paper. Therefore, the newly established method was expected to be a powerful tool for efficiently determination of bacillus anthrax spores to avoid anthrax threats.

An insight into the complete biophysical and biochemical characterization of novel class A beta-lactamase (Bla1) from Bacillus anthracis.

Bacillus anthracis, a potent pathogen of anthrax is becoming resistant to many beta-lactam antibiotics because of the expression of two chromosomally encoded beta-lactamases Bla1 and Bla2. Bla1 is a class A beta-lactamase whereas Bla2 is a Metallo beta-lactamase. In the current study, we have attempted in-detailed characterization of Bla1 beta-lactamase by taking interdisciplinary approaches. Our study includes structure and sequence comparison of this enzyme with other members of the class, to know the conservation pattern that includes active site residues, secondary structure, conserved fold, evolutionary relationships, etc. Dynamic characterizations of the enzyme, unfolding kinetics were determined with the help of Molecular dynamics simulation. Detailed enzyme stability and catalytic activity towards various physical (Temperature and pH), and chemical parameters (Urea, GnHCl) were performed. Together, our study helps to develop a proper understanding of this beta-lactamase by characterizing its biochemical, biophysical, dynamic, kinetic and thermodynamic properties. This would help contribute towards a better understanding of beta-lactamase based AMR emergence.