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Bacillus cereus biovar anthracis (Bcbva) causes anthrax-like disease in animals, particularly in the non-human primates and great apes of West and Central Africa. Genomic analyses revealed Bcbva as a member of the B. cereus species that carries two plasmids, pBCXO1 and pBCXO2, which have high sequence homology to the B. anthracis toxin and polyglutamate capsule encoding plasmids pXO1 and pXO2, respectively. To date, only a few studies have investigated the effect of variations in Bcbva sporulation, toxin, and capsule synthesis on animal and macrophage pathogenicity compared to B. anthracis, therefore more research is needed to gain a better understanding of the pathogenesis of this emerging infection. Here, we report that Bcbva can multiply and vegetatively survive on nutrient-rich media for a minimum of six days while generating spores. Sporulation of Bcbva occurred faster and more extensively than B. anthracis Ames. Bcbva tended to secrete less protective antigen (PA) than B. anthracis Ames when cultured in growth medium. We found Bcbva produced a substantially higher amount of attached poly-ƴ-D-glutamic acid (PDGA) capsule than B. anthracis Ames when grown in medium supplemented with human serum and CO2. In a phagocytosis assay, Bcbva spores showed reduced internalization by mouse macrophages compared to B. anthracis Ames. Our research demonstrated that Bcbva is more virulent than B. anthracis Ames using two in vivo models, Galleria mellonella larvae and guinea pigs. Following that, the efficacy of the veterinary vaccine Sterne strain 34F2 against anthrax-like disease was assessed in guinea pigs. Sterne vaccinated guinea pigs had significantly increased anti-PA titers compared to the unvaccinated control group. Toxin neutralizing antibody titers in vaccinated guinea pigs correlated with anti-PA titers. This indicates the Sterne vaccine provides adequate protection against Bcbva infection in laboratory animals., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Environmental and climatic factors, as well as host demographics and behaviour, significantly influence the exposure of herbivorous mammalian hosts to pathogens such as Bacillus anthracis, the causative agent of anthrax. Until the early 1990s in Kruger National Park (KNP), kudu (Tragelaphus strepsiceros) was the host species most affected by anthrax, with outbreaks occurring predominantly in the dry season, particularly during drought cycles. However, the most affected host species has shifted to impala (Aepyceros melampus), with more frequent anthrax outbreaks during the wet season. This study investigates the roles of environmental variation and other host species in this shift. Temporal trends in environmental variables such as precipitation, soil moisture, temperature, and normalised difference vegetation index (NDVI) were analyzed in relation to anthrax occurrence (presence/ absence and counts). Additionally, correlations between host species' densities and anthrax mortalities over time were examined. Anthrax cases in 1990 were concentrated in the central and northern regions of KNP(excluding Pafuri), primarily affected kudus; while subsequent mortalities affected mostly impala and were restricted to the far north, in Pafuri. Significant correlations were found between kudu anthrax mortality and a decrease in NDVI, average temperature, SPI-6 and SPI-12 (Standardised Precipitation Index in various time intervals. Conversely, anthrax occurrence in impalas was associated with a decline in SPI-3, and temperature rise, with increased mortality during the rainy season. Elephant density correlated negatively with kudu mortality, but a positive correlation with both impala mortality and impala density. The study concludes that environmental variables and species' densities may alter the diversity and frequency of hosts exposed to B. anthracis. Climate extremes and alterations therein may exacerbate anthrax severity by modifying species susceptibility and their probability of exposure over time., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

The formation of endospores has been considered the unique survival and transmission mode of sporulating Firmicutes due to the exceptional resistance and persistence of this bacterial form. However, nonsporulated bacteria (Spo - ) were reported at the early stages following the death of a host infected with Bacillus thuringiensis, an entomopathogenic sporulating bacterium. Here, we investigated the characteristics of the bacterial population in the late stages of an infection in the B. thuringiensis/Galleria mellonella infection model. Using fluorescent reporters and molecular markers coupled to flow cytometry, we demonstrated that the Spo - cells persist and constitute about half of the population 2 weeks post-infection (p.i.). Protein synthesis and growth recovery assays indicated that they are in a metabolically slowed-down state. These bacteria were extremely resistant to the insect cadaver environment, which did not support growth of in vitro -grown vegetative cells and spores. A transcriptomic analysis of this subpopulation at 7 days p.i. revealed a signature profile of this state, and the expression analysis of individual genes at the cell level showed that more bacteria mount an oxidative stress response as their survival time increases, in agreement with the increase of the free radical level in the host cadaver and in the number of reactive oxygen species (ROS)-producing bacteria. Altogether, these data show for the first time that nonsporulated bacteria are able to survive for a prolonged period of time in the context of an infection and indicate that they engage in a profound adaptation process that leads to their persistence in the host cadaver. IMPORTANCE Bacillus thuringiensis is an entomopathogenic bacterium widely used as a biopesticide. It belongs to the Bacillus cereus group, comprising the foodborne pathogen B. cereus sensu stricto and the anthrax agent Bacillus anthracis. Like other Firmicutes when they encounter harsh conditions, these Gram-positive bacteria can form dormant cells called spores. Due to its highly resistant nature, the spore was considered the unique mode of long-term survival, eclipsing any other form of persistence. Breaking this paradigm, we observed that B. thuringiensis was able to persist in its host cadaver in a nonsporulated form for at least 14 days. Our results show that these bacteria survived in the cadaver environment, which proved hostile for actively growing bacteria by engaging in a profound adaptation process. Studying this facet of the life cycle of a sporulating bacterium provides new fundamental knowledge and might lead to the development of strategies to combat sporulating pathogenic species., Competing Interests: The authors declare no conflict of interest.

Using data collected from previous (n = 86) and prospective (n = 132) anthrax outbreaks, we enhanced prior ecological niche models (ENM) and added kernel density estimation (KDE) approaches to identify anthrax hotspots in Kenya. Local indicators of spatial autocorrelation (LISA) identified clusters of administrative wards with a relatively high or low anthrax reporting rate to determine areas of greatest outbreak intensity. Subsequently, we modeled the impact of vaccinating livestock in the identified hotspots as a national control measure. Anthrax suitable areas included high agriculture zones concentrated in the western, southwestern and central highland regions, consisting of 1043 of 1450 administrative wards, covering 18.5% country landmass, and hosting 30% of the approximately 13 million cattle population in the country. Of these, 79 wards covering 5.5% landmass and hosting 9% of the cattle population fell in identified anthrax hotspots. The rest of the 407 administrative wards covering 81.5% of the country landmass, were classified as low anthrax risk areas and consisted of the expansive low agricultural arid and semi-arid regions of the country that hosted 70% of the cattle population, reared under the nomadic pastoralism. Modelling targeted annual vaccination of 90% cattle population in hotspot administrative wards reduced > 23,000 human exposures. These findings support an economically viable first phase of anthrax control program in low-income countries where the disease is endemic, that is focused on enhanced animal and human surveillance in burden hotspots, followed by rapid response to outbreaks anchored on public education, detection and treatment of infected humans, and ring vaccination of livestock. Subsequently, the global anthrax elimination program focused on sustained vaccination and surveillance in livestock in the remaining few hotspots for a prolonged period (> 10 years) may be implemented., (© 2022. The Author(s).)

Large area sampling approaches have been developed and implemented by the US Environmental Protection Agency (EPA) to increase sample sizes, and potentially representativeness, in outdoor urban environments (e.g., concrete, asphalt, grass/landscaping). These sampling approaches could be implemented in response to an outdoor biological contamination incident or bioterrorism attack to determine the extent of contamination and for clearance following remediation. However, sample collection over large areas often contains an extensive amount of co-collected debris and native background microorganisms that interfere with the detection of biological threat agents. Sample processing methods that utilize basic laboratory equipment amenable to field deployment were selected and applied to turbid aqueous samples (TAS) to reduce particulates and native environmental organisms prior to culture and rapid viability-polymerase chain reaction (RV-PCR) analytical methods. Bacillus anthracis Sterne (BaS) spores were spiked into TAS collected by soil grab, wet vacuum collection from an outdoor concrete surface, or storm water runoff from an urban parking lot. The implementation of a sample processing method improved the sensitivity of culture and RV-PCR analytical methods for BaS spore detection in soil and wet vacuum TAS samples compared to baseline (minimal to no field processing methods applied). For soil, when the processing method was applied, samples with 15 colony forming units (CFU)/ml (60 CFU/g) and 1.5 CFU/mL (6 CFU/g) BaS spore load were detected using culture and RV-PCR, respectively. Most notably, the processing methods greatly improved the sensitivity of the RV-PCR analytical method for the wet vacuum TAS from no detection at the 1500 CFU/mL BaS spore load level to as low as 1.5 CFU/mL BaS spore load., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Reversible protein phosphorylation at serine/threonine residues is one of the most common protein modifications, widely observed in all kingdoms of life. The catalysts controlling this modification are specific serine/threonine kinases and phosphatases that modulate various cellular pathways ranging from growth to cellular death. Genome sequencing and various omics studies have led to the identification of numerous serine/threonine kinases and cognate phosphatases, yet the physiological relevance of many of these proteins remain enigmatic. In Bacillus anthracis, only one ser/thr phosphatase, PrpC, has been functionally characterized; it was reported to be non-essential for bacterial growth and survival. In the present study, we characterized another ser/thr phosphatase (PrpN) of B. anthracis by various structural and functional approaches. To examine its physiological relevance in B. anthracis, a null mutant strain of prpN was generated and shown to have defects in sporulation and reduced synthesis of toxins (PA and LF) and the toxin activator protein AtxA. We also identified CodY, a global transcriptional regulator, as a target of PrpN and ser/thr kinase PrkC. CodY phosphorylation strongly controlled its binding to the promoter region of atxA, as shown using phosphomimetic and phosphoablative mutants. In nutshell, the present study reports phosphorylation-mediated regulation of CodY activity in the context of anthrax toxin synthesis in B. anthracis by a previously uncharacterized ser/thr protein phosphatase-PrpN., Competing Interests: The authors have declared that no competing interests exist.

Anthrax is a serious infectious disease caused by Bacillus anthracis, rod-shaped gram-positive bacteria. The disease infects both humans and animals and causes severe illness.  Many vaccines have been developed for anthrax, but the vaccine with very high efficacy is yet to be developed. To overcome the problems of efficacy posed by the existing vaccines, a recombinant chimeric fusion protein containing domain 1 of lethal factor (LFD1) and domain 4 of Bacillus anthracis protective antigen (PA4) was used as antigen in copolymeric nanocapsules (NCs). Accordingly, the solvent evaporation double emulsion method was used to produce NCs containing recombinant chimeric fusion protein (LFD1-PA4). Zeta sizer and potential of nanoparticles, nanoparticle loading efficiency, release pattern of recombinant protein, and the possible effect of polylactic acid-polyethylene glycol (PLA-PEG) nanoparticle production method were investigated. Mice were used to test and evaluate the immune response. The mean titer of antibody produced against loaded LFD1-PA4 compared to free form showed a significant difference. The difference in antibody titer between the groups of once injected, twice injected, and free antigen was significant, and the highest antibody titer was found in the mice twice injected. In addition, a single-time loaded injection showed significantly higher antibodies than the free form injection indicating that loaded LFD1-PA4 into PLA-PEG nanoparticles elicits a stronger immune response. This study showed that LFD1-PA4 fusion protein from Bacillus anthracis served as an active antigen in mice. Also, the nanocarrier (PLA-PEG) containing the antigen can stimulate the immune system in the mice, owing to their controlled release property.

Bacillus anthracis is the causative agent of anthrax. This Gram-positive bacterium poses a substantial risk to human health due to high mortality rates and the potential for malicious use as a bioterror weapon. To survive within the vertebrate host, B. anthracis relies on two-component system (TCS) signaling to sense host-induced stresses and respond to alterations in the environment through changes in target gene expression. HitRS and HssRS are cross-regulating TCSs in B. anthracis that respond to cell envelope disruptions and high heme levels, respectively. In this study, an unbiased and targeted genetic selection was designed to identify gene products that are involved in HitRS and HssRS signaling. This selection led to the identification of inactivating mutations within dnaJ and clpX that disrupt HitRS- and HssRS-dependent gene expression. DnaJ and ClpX are the substrate-binding subunits of the DnaJK protein chaperone and ClpXP protease, respectively. DnaJ regulates the levels of HitR and HitS to facilitate signal transduction, while ClpX specifically regulates HitS levels. Together, these results reveal that the protein homeostasis regulators, DnaJ and ClpX, function to maintain B. anthracis signal transduction activities through TCS regulation.

Anthrax is considered one of the most dangerous bioweapon agents, and concern about multidrug-resistant strains has led to the development of alternative therapeutic approaches that target the antiphagocytic capsule, an essential virulence determinant of Bacillus anthracis , the causative agent. Capsule depolymerase is a γ-glutamyltransferase that anchors the capsule to the cell wall of B. anthracis . Encapsulated strains of B. anthracis can be treated with recombinant capsule depolymerase to enzymatically remove the capsule and promote phagocytosis and killing by human neutrophils. Here, we show that pegylation improved the pharmacokinetic and therapeutic properties of a previously described variant of capsule depolymerase, CapD-CP, when delivered 24 hours after exposure every 8 hours for 2 days for the treatment of mice infected with B. anthracis. Mice infected with 382 LD 50 of B. anthracis spores from a nontoxigenic encapsulated strain were completely protected (10 of 10) after treatment with the pegylated PEG-CapD-CP S334C , whereas 10% of control mice (1 of 10) survived with control treatment using bovine serum albumin ( P < 0.0001, log-rank analysis). Treatment of mice infected with five LD 50 of a fully virulent toxigenic, encapsulated B. anthracis strain with PEG-CapD-CP S334C protected 80% (8 of 10) of the animals, whereas 20% of controls (2 of 10) survived ( P = 0.0125, log-rank analysis). This strategy renders B. anthracis susceptible to innate immune responses and does not rely on antibiotics. These findings suggest that enzyme-catalyzed removal of the capsule may be a potential therapeutic strategy for the treatment of multidrug- or vaccine-resistant anthrax and other bacterial infections.

Background: Inhalational anthrax is rare and clinical experience limited. Expert guidelines recommend treatment with combination antibiotics including protein synthesis-inhibitors to decrease toxin production and increase survival, although evidence is lacking., Methods: Rhesus macaques exposed to an aerosol of Bacillus anthracis spores were treated with ciprofloxacin, clindamycin, or ciprofloxacin + clindamycin after becoming bacteremic. Circulating anthrax lethal factor and protective antigen were quantitated pretreatment and 1.5 and 12 hours after beginning antibiotics., Results: In the clindamycin group, 8 of 11 (73%) survived demonstrating its efficacy for the first time in inhalational anthrax, compared to 9 of 9 (100%) with ciprofloxacin, and 8 of 11 (73%) with ciprofloxacin + clindamycin. These differences were not statistically significant. There were no significant differences between groups in lethal factor or protective antigen levels from pretreatment to 12 hours after starting antibiotics. Animals that died after clindamycin had a greater incidence of meningitis compared to those given ciprofloxacin or ciprofloxacin + clindamycin, but numbers of animals were very low and no definitive conclusion could be reached., Conclusion: Treatment of inhalational anthrax with clindamycin was as effective as ciprofloxacin in the nonhuman primate. Addition of clindamycin to ciprofloxacin did not enhance reduction of circulating toxin levels., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Soils contaminated with heavy metals such as Chromium (Cr) and Cadmium (Cd) severely impede plant growth. Several rhizospheric microorganisms support plant growth under heavy metal stress. In this study, Cr and Cd stress was applied to in vitro germinating seedlings of a Legume plant species, Sesbania sesban, and investigated the plant growth potential in presence and absence of Bacillus anthracis PM21 bacterial strain under heavy metal stress. The seedlings were exposed to different concentrations of Cr (25-75 mg/L) and Cd (100-200 mg/L) in Petri plates. Growth curve analysis of B. anthracis PM21 revealed its potential to adapt Cr and Cd stress. The bacteria supported plant growth by exhibiting ACC-deaminase activity (1.57-1.75 μM of α-ketobutyrate/h/mg protein), producing Indole-3-acetic acid (99-119 μM/mL) and exopolysaccharides (2.74-2.98 mg/mL), under heavy metal stress condition. Analysis of variance revealed significant differences in growth parameters between the seedlings with and without bacterial inoculation in metal stress condition. The combined Cr+Cd stress (75 + 200 mg/L) significantly reduced root length (70%), shoot length (24%), dry weight (54%) and fresh weight (57%) as compared to control. Conversely, B. anthracis PM21 inoculation to seedlings significantly increased (p ≤ 0.05) seed germination percentage (5%), root length (31%), shoot length (23%) and photosynthetic pigments (Chlorophyll a: 20%; Chlorophyll b: 16% and total chlorophyll: 18%), as compared to control seedlings without B. anthracis PM21 inoculation. The B. anthracis PM21 inoculation also enhanced activities of antioxidant enzymes, including superoxide dismutase (52%), peroxidase (66%), and catalase (21%), and decreased proline content (56%), electrolyte leakage (50%), and malondialdehyde concentration (46%) in seedlings. The B. anthracis PM21 inoculated seedlings of S. sesban exhibited significantly high (p ≤ 0.05) tissue deposition of Cr (17%) and Cd (16%) as compared to their control counterparts. Findings of the study suggested that B. anthracis PM21 endured metal stress through homeostasis of antioxidant activities, and positively impacted S. sesban growth and biomass. Further experiments in controlled conditions are necessary for investigating phytoremediation potential of S. sesban in metal-contaminated soils in presence of B. anthracis PM21 bacterial strain., (Copyright © 2020. Published by Elsevier Inc.)

Ethnopharmacological Relevance: Gastrointestinal anthrax, a disease caused by Bacillus anthracis, remains an important but relatively neglected endemic disease of animals and humans in remote areas of the Indian subcontinent and some parts of Africa. Its initial symptoms include diarrhea and stomachache. In the current study, several common plants indicated for diarrhea, dysentery, stomachache or as stomachic as per traditional knowledge in the Indian subcontinent, i.e., Aegle marmelos (L.) Correa (Bael), Allium cepa L. (Onion), Allium sativum L. (Garlic), Azadirachta indica A. Juss. (Neem), Berberis asiatica Roxb. ex DC. (Daruharidra), Coriandrum sativum L. (Coriander), Curcuma longa L. (Turmeric), Cynodon dactylon (L.) Pers. (Bermuda grass), Mangifera indica L. (Mango), Morus indica L. (Black mulberry), Ocimum tenuiflorum L. (Ocimum sanctum L., Holy Basil), Ocimum gratissimum L. (Ram Tulsi), Psidium guajava L. (Guava), Zingiber officinale Roscoe (Ginger), were evaluated for their anti-Bacillus anthracis property. The usage of Azadirachta indica A. Juss. and Curcuma longa L. by Santals (India), and Allium sp. by biblical people to alleviate anthrax-like symptoms is well documented, but the usage of other plants is traditionally only indicated for different gastrointestinal disturbances/conditions., Aim of the Study: Evaluate the above listed commonly available edible plants from the Indian subcontinent that are used in the traditional medicine to treat gastrointestinal diseases including those also indicated for anthrax-like symptoms for the presence of potent anti-B. anthracis activity in a form amenable to use by the general population in the endemic areas., Materials and Methods: Aqueous extracts made from fourteen plants indicated above were screened for their anti-B. anthracis activity using agar-well diffusion assay (AWDA) and broth microdilution methods. The Aqueous Garlic Extract (AGE) that displayed most potent anti-B. anthracis activity was assessed for its thermostability, stability under pH extremes encountered in the gastrointestinal tract, and potential antagonistic interaction with bile salts as well as the FDA-approved antibiotics used for anthrax control. The bioactive fractions from the AGE were isolated by TLC coupled bioautography followed by their characterization using GC-MS., Results: Garlic (Allium sativum L.) extract was identified as the most promising candidate with bactericidal activity against B. anthracis. It consistently inhibited the growth of B. anthracis in AWDA and decreased the viable colony-forming unit counts in liquid-broth cultures by 6-logs within 6-12 h. The AGE displayed acceptable thermostability (>80% anti-B. anthracis activity retained on incubation at 50 °C for 12 h) and stability in gastric pH range (2-8). It did not antagonize the activity of FDA-approved antibiotics used for anthrax control. GC-MS analysis of the TLC separated bioactive fractions of AGE indicated the presence of previously unreported constituents such as phthalic acid derivatives, acid esters, phenyl group-containing compounds, steroids etc. CONCLUSION: The Aqueous Garlic Extract (AGE) displayed potent anti-B. anthracis activity. It was better than that displayed by Azadirachta indica A. Juss. (Neem) and Mangifera indica L., while Curcuma longa L. (Turmeric) did not show any activity under the assay conditions used. Further work should be undertaken to explore the possible application of AGE in preventing anthrax incidences in endemic areas., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Two component systems (TCSs) are a primary mechanism of signal sensing and response in bacteria. Systematic characterization of an entire TCS could provide a mechanistic understanding of these important signal transduction systems. Here, genetic selections were employed to dissect the molecular basis of signal transduction by the HitRS system that detects cell envelope stress in the pathogen Bacillus anthracis. Numerous point mutations were isolated within HitRS, 17 of which were in a 50-residue HAMP domain. Mutational analysis revealed the importance of hydrophobic interactions within the HAMP domain and highlighted its essentiality in TCS signaling. In addition, these data defined residues critical for activities intrinsic to HitRS, uncovered specific interactions among individual domains and between the two signaling proteins, and revealed that phosphotransfer is the rate-limiting step for signal transduction. Furthermore, this study establishes the use of unbiased genetic selections to study TCS signaling and provides a comprehensive mechanistic understanding of an entire TCS., Competing Interests: No authors have competing interests.

Dipicolinic acid (DPA) is employed as a significant biomarker to detect Bacillus anthracis, which can do serious damages to the health of human beings. Hence, it is crucial to develop a fast and highly efficient strategy for DPA monitoring. In this work, based on silicon nanoparticles (Si NPs) and terbium metal-organic frameworks (Tb-MOFs), a hybrid structure (Si NPs/Tb-MOFs) as a novel dual-emitting fluorescence probe was fabricated for ratiometric detection of DPA, where blue light-emitting Si NPs (Ex: 280 nm; Em: 422 nm) are encapsulated into green light-emitting Tb-MOFs (Ex: 280 nm; Em: 547 nm). The optical properties and chemical composition of the as-obtained Si NPs/Tb-MOFs were characterized in detail. The Si NPs/Tb-MOFs probe not merely possesses the merits of a facile synthesis method but also is an excellent fluorescence probe. The response time towards DPA is less than 30 s, revealing that the process of detecting DPA can be completed in such a short time. The limit of detection for DPA is 5.3 nM, which is four orders of magnitude lower than an infectious dosage of anthrax spores for human beings (60 μM). This dual-emitting Si NPs/Tb-MOFs probe with interference-free and self-calibrating properties may be a potential candidate for further development in medical diagnosis. Graphical abstract.

The efficient biological treatment of saline wastewater has been limited by the low activities of microorganisms under saline conditions. High salinity poses unbalance osmotic stress across the cell wall and even leads to cell plasmolysis. In this work, we aim to isolate salt-tolerant bacterial strains from activated sludge, and apply them for degrading chemical oxygen demand (COD) of saline organic wastewater. Two salt-tolerant strains were screened and isolated from activated sludge, which was domesticated with salty water for over 300 days. The two strains were identified as Bacillus cereus (strain A) and Bacillus anthracis (strain B) through 16S rRNA sequencing. The degradation characteristics of strain A were explored. The results showed the relative membrane permeability of strain A remained stable under high salt stress, which glycine and proline play an important role to maintain cell osmotic. The protein and soluble sugar amounts of strain were increased by higher salt concentrations. In simulating saline wastewater, the optimum culture temperature, pH, salinity, influent COD concentration and inoculation amount of strain A were 35 °C, 9, 4%, 8000 mg L -1 , 6%, respectively. Optimal conditions could provide guidance for the treatment of practical saline wastewater. The linear regression model of each impact factor built based on the result PB experiment revealed that cross-linking time has the most significant influence on COD removal for salt-tolerant strains. It will provide theoretical basis for biological treatment of saline organic wastewater.

A recent outbreak of anthrax disease, severely affecting reindeer herds in Siberia, has been reportedly associated to the presence of infected carcasses or spores released from the active layer over permafrost, which is thawing and thickening at increasing rates, thus underlying the re-emerging nature of this pathogen in the Arctic region because of warming temperatures. Anthrax is a global zoonotic and epizootic disease, with a high case-fatality ratio in infected animals. Its transmission is mediated by environmental contamination through highly resistant spores which can persist in the soil for several decades. Here we develop and analyze a new epidemiological model for anthrax transmission that is specifically tailored to the Arctic environmental conditions. The model describes transmission dynamics including also herding practices (e.g. seasonal grazing) and the role of the active layer over permafrost acting as a long-term storage of spores that could be viable for disease transmission during thawing periods. Model dynamics are investigated through linear stability analysis, Floquet theory for periodically forced systems, and a series of simulations with realistic forcings. Results show how the temporal variability of grazing and active layer thawing may influence the dynamics of anthrax disease and, specifically, favor sustained pathogen transmission. Particularly warm years, favoring deep active layers, are shown to be associated with an increase risk of anthrax outbreaks, and may also foster infections in the following years. Our results enable preliminary insights into measures (e.g. changes in herding practice) that may be adopted to decrease the risk of infection and lay the basis to possibly establish optimal procedures for preventing transmission; furthermore, they elicit the need of further investigations and observation campaigns focused on anthrax dynamics in the Arctic environment.

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-1β in mice within hours of toxin challenge. We find IL-1β 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-1β originates from hematopoietic cells. LT treatment of neutrophils in vitro did not induce IL-1β, neutrophil extracellular traps (NETs), or pyroptosis. Although platelets interact closely with neutrophils and are also a potential source of IL-1β, they were unable to bind or endocytose LT and did not secrete IL-1β 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-1β release. DNA sensor AIM2 deficiency, however, did not impact IL-1β 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., (©2020 Society for Leukocyte Biology.)

Anthrax toxin is the major virulence factor secreted by Bacillus anthracis, causing high mortality in humans and other mammals. It consists of a membrane translocase, known as protective antigen (PA), that catalyzes the unfolding of its cytotoxic substrates lethal factor (LF) and edema factor (EF), followed by translocation into the host cell. Substrate recruitment to the heptameric PA pre-pore and subsequent translocation, however, are not well understood. Here, we report three high-resolution cryo-EM structures of the fully-loaded anthrax lethal toxin in its heptameric pre-pore state, which differ in the position and conformation of LFs. The structures reveal that three LFs interact with the heptameric PA and upon binding change their conformation to form a continuous chain of head-to-tail interactions. As a result of the underlying symmetry mismatch, one LF binding site in PA remains unoccupied. Whereas one LF directly interacts with a part of PA called α-clamp, the others do not interact with this region, indicating an intermediate state between toxin assembly and translocation. Interestingly, the interaction of the N-terminal domain with the α-clamp correlates with a higher flexibility in the C-terminal domain of the protein. Based on our data, we propose a model for toxin assembly, in which the relative position of the N-terminal α-helices in the three LFs determines which factor is translocated first., Competing Interests: The authors have declared that no competing interests exist.

Bacillus anthracis, the bacteria that causes anthrax, a disease that primarily affects herbivorous animals, is a soil borne endospore-forming microbe. Environmental distribution of viable spores determines risky landscapes for herbivore exposure and subsequent anthrax outbreaks. Spore survival and longevity depends on suitable conditions in its environment. Anthrax is endemic in Queen Elizabeth Protected Area in western Uganda. Periodic historical outbreaks with significant wildlife losses date to 1950s, but B. anthracis ecological niche in the ecosystem is poorly understood. This study used the Maximum Entropy modeling algorithm method to predict suitable niche and environmental conditions that may support anthrax distribution and spore survival. Model inputs comprised 471 presence-only anthrax occurrence data from park management records of 1956-2010, and 11 predictor variables derived from the World Climatic and Africa Soil Grids online resources, selected considering the ecology of anthrax. The findings revealed predicted suitable niche favoring survival and distribution of anthrax spores as a narrow-restricted corridor within the study area, defined by hot-dry climatic conditions with alkaline soils rich in potassium and calcium. A mean test AUC of 0.94 and predicted probability of 0.93 for anthrax presence were registered. The five most important predictor variables that accounted for 93.8% of model variability were annual precipitation (70.1%), exchangeable potassium (12.6%), annual mean temperature (4.3%), soil pH (3.7%) and calcium (3.1%). The predicted suitable soil properties likely originate from existing sedimentary calcareous gypsum rocks. This has implications for long-term presence of B. anthracis spores and might explain the long history of anthrax experienced in the area. However, occurrence of suitable niche as a restricted hot zone offers opportunities for targeted anthrax surveillance, response and establishment of monitoring strategies in QEPA., Competing Interests: The authors declare having no competing interests.

Aims: To determine the mechanism of killing of spores of Bacillus thuringiensis Al Hakam, a Bacillus anthracis spore surrogate, in a blast environment with or without HIO 3 and whether the spores are truly dead., Methods and Results: Spores exposed to an aluminium-based blast environment with or without HIO 3 with dynamic peak gas phase temperatures near 1000°C persisting for 10's of ms, were killed 97 and 99·99% without and with HIO 3 respectively and the spores were truly dead. The survivors of the detonations did not acquire mutations, did not become wet heat sensitive, became sensitive to elevated NaCl but not lack of glucose in recovery media, and many dead spores remained phase bright and retained their Ca-dipicolinic acid. A large fraction of the dead spores could germinate, but most of these germinated spores were dead., Conclusions: Most spores exposed to a blast environment are truly dead, and HIO 3 increases spore death. The likely mechanism of spore killing in these blast environments is damage to some essential spore protein, although spore inner membrane damage could contribute., Significance and Impact of the Study: This work shows that spores of a surrogate for B. anthracis spores are killed in a blast environment without or with HIO 3 present, this approach could inactivate up to 99·99% of dry B. anthracis spores, and the spores are likely killed by damage to some essential spore protein., (© 2020 The Society for Applied Microbiology.)

Inflammasomes act as sensors of infection or damage to initiate immune responses. While extensively studied in rodents, understanding of livestock inflammasomes is limited. The NLRP1 inflammasome sensor in rodents is activated by Toxoplasma gondii , Bacillus anthracis lethal toxin (LT), and potentially other zoonotic pathogens. LT activates NLRP1 by N-terminal proteolysis, inducing macrophage pyroptosis and a pro-inflammatory cytokine response. In contrast, NLRP1 in macrophages from humans and certain rodent strains is resistant to LT cleavage, and pyroptosis is not induced. Evolution of NLRP1 sequences towards those leading to pyroptosis is of interest in understanding innate immune responses in different hosts. We characterized NLRP1 in cattle ( Bos taurus ) and American bison ( Bison bison ). Bovine NLRP1 is not cleaved by LT, and cattle and bison macrophages do not undergo toxin-induced pyroptosis. Additionally, we found a predicted Nlrp1 splicing isoform in cattle macrophages lacking the N-terminal domain. Resistance to LT in bovine and human NLRP1 correlates with evolutionary sequence similarity to rodents. Consistent with LT-resistant rodents, bovine macrophages undergo a slower non-pyroptotic death in the presence of LPS and LT. Overall, our findings support the model that NLRP1 activation by LT requires N-terminal cleavage, and provide novel information on mechanisms underlying immune response diversity.

Bacillus anthracis is a spore-forming bacterium that causes devastating infections and has been used as a bioterror agent. This pathogen can survive hostile environments through the signaling activity of two-component systems, which couple environmental sensing with transcriptional activation to initiate a coordinated response to stress. In this work, we describe the identification of a two-component system, EdsRS, which mediates the B. anthracis response to the antimicrobial compound targocil. Targocil is a cell envelope-targeting compound that is toxic to B. anthracis at high concentrations. Exposure to targocil causes damage to the cellular barrier and activates EdsRS to induce expression of a previously uncharacterized cardiolipin synthase, which we have named ClsT. Both EdsRS and ClsT are required for protection against targocil-dependent damage. Induction of clsT by EdsRS during targocil treatment results in an increase in cardiolipin levels, which protects B. anthracis from envelope damage. Together, these results reveal that a two-component system signaling response to an envelope-targeting antimicrobial induces production of a phospholipid associated with stabilization of the membrane. Cardiolipin is then used to repair envelope damage and promote B. anthracis viability. IMPORTANCE Compromising the integrity of the bacterial cell barrier is a common action of antimicrobials. Targocil is an antimicrobial that is active against the bacterial envelope. We hypothesized that Bacillus anthracis , a potential weapon of bioterror, senses and responds to targocil to alleviate targocil-dependent cell damage. Here, we show that targocil treatment increases the permeability of the cellular envelope and is particularly toxic to B. anthracis spores during outgrowth. In vegetative cells, two-component system signaling through EdsRS is activated by targocil. This results in an increase in the production of cardiolipin via a cardiolipin synthase, ClsT, which restores the loss of barrier function, thereby reducing the effectiveness of targocil. By elucidating the B. anthracis response to targocil, we have uncovered an intrinsic mechanism that this pathogen employs to resist toxicity and have revealed therapeutic targets that are important for bacterial defense against structural damage., (Copyright © 2020 Laut et al.)

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., (Copyright © 2020 Sharma, Bhatnagar and Gaur.)

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 2.38×10 4 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bacillus anthracis , the etiological agent of anthrax, is a well-established model organism. For B. anthracis and most other infectious diseases, knowledge regarding transmission and infection parameters in natural systems, in large part, comprises data gathered from closely controlled laboratory experiments. Fatal, natural anthrax infections transmit the bacterium through new host-pathogen contacts at carcass sites, which can occur years after death of the previous host. For the period between contact and death, all of our knowledge is based upon experimental data from domestic livestock and laboratory animals. Here we use a noninvasive method to explore the dynamics of anthrax infections, by evaluating the terminal diversity of B. anthracis in anthrax carcasses. We present an application of population genetics theory, specifically, coalescence modeling, to intrainfection populations of B. anthracis to derive estimates for the duration of the acute phase of the infection and effective population size converted to the number of colony-forming units establishing infection in wild plains zebra ( Equus quagga ). Founding populations are small, a few colony-forming units, and infections are rapid, lasting roughly between 1 d and 3 d in the wild. Our results closely reflect experimental data, showing that small founding populations progress acutely, killing the host within days. We believe this method is amendable to other bacterial diseases from wild, domestic, and human systems., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

To understand the epidemiological and genetic background of anthrax cases occurring in Vietnam from 2011 to 2015, we surveilled and genetically analyzed Bacillus anthracis isolated in the north of the country. Epidemiological surveillance showed that most human cutaneous anthrax cases occurred in association with animal dissection. Whole-genome sequences were obtained from six B. anthracis strains from human patients with cutaneous anthrax in the endemic area. Comparative genomic analysis showed that the genetic homogeneity among Vietnamese B. anthracis strains was very high. All Vietnamese B. anthracis strains belonged to the canSNP lineage of A.Br.011/009, which mostly consists of strains of the trans-Eurasian (TEA) group, including the most closely related strain, Carbosap. To clarify the genetic diversity of Vietnamese strains and strains belonging to A.Br.011/009 and A.Br.008/011 canSNP lineages, we applied a reference genome-based single-nucleotide polymorphism (SNP) and gene-by-gene genomic analysis (whole-genome MLST) strategy. The phylogeny from core genome SNPs revealed that the Vietnamese strains were positioned close to each other; moreover, several SNPs specific to Vietnamese B. anthracis were identified. Whole-genome MLST analysis revealed the differences in the number of SNPs between Vietnamese strains, which could enable discrimination at the strain level., Competing Interests: The authors have declared that no competing interests exist.

Bacillus anthracis is the causative agent of anthrax in animals and humans. The organism lies in a dormant state in the soil until introduced into an animal via, ingestion, cutaneous inoculation or inhalation. Once in the host, spores germinate into rapidly growing vegetative cells elaborating toxins. When animals die of anthrax, vegetative bacteria sporulate upon nutrient limitation in the carcass or soil while in the presence of air. After release into the soil environment, spores form a localized infectious zone (LIZ) at and around the carcass. Laboratory strains of B. anthracis produce fewer proteins associated with growth and sporulation compared to wild strains isolated from recent zoonotic disease events. We verified wild strains grow more rapidly than lab strains demonstrating a greater responsiveness to nutrient availability. Sporulation was significantly more rapid in these wild strains compared to lab strains, indicating wild strains are able to sporulate faster due to nutrient limitation while laboratory strains have a decrease in the speed at which they utilize nutrients and an increase in time to sporulation. These findings have implications for disease control at the LIZ as well as on the infectious cycle of this dangerous zoonotic pathogen., Competing Interests: The authors have declared that no competing interests exist.

Small organisms, like the nematode C. elegans, are emerging as insightful models in which to study host/pathogen interactions and the evolving interplay between host defenses and microbial offenses. In C. elegans the innate immune response has been shown to be connected to the DAF-2 insulin/insulin-like growth factor 1 (IGF-1) signal pathway, a critical transduction pathway that mediates stress response in the worms via the DAF-16 FOXO/forkhead transcription factor. Our studies of the C. elegans' phenotypes that are associated with behavioral innate immune response (avoidance behavior) and IGF-1 signaling perturbations (lifespan effects) led us to question the cause of the avoidance behavior observed when C. elegans are challenged with B. anthracis. While worms indeed avoid B. anthracis, and this behavior seems to be partly tied to IGF-1 signaling, the bacteria have neither nematocidal nor visible pathogenic effects on the worms. In fact, worms fed B. anthracis alone exhibit extended lifespans. We demonstrate that the extended lifespan phenotype seen in worms fed B. anthracis is likely the result of calorie restriction, and that worms do not eat B. anthracis even when avoidance behaviors have been suppressed. We further demonstrate a large time lag between the onset of avoidance behavior (which occurs upon contact with B. anthracis), and the induction of IGF-1 signaling (which occurs much later) in worms fed B. anthracis. Taken together, our data demonstrate behavioral avoidance that does not appear to be linked to a measurable immune response. We propose that, in some situations, avoidance behaviors categorized as immunological might be more accurately described as broad foraging behaviors induced in worms presented with a non-preferred food choice, or with a food choice that is either difficult or impossible for the worms to ingest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Aims: To add a spore germination step in order to reduce decontamination temperature and time requirements compared to the current hot, humid air decontamination parameters, which are 75-80°C, ≥72 h, 70-90% RH, down to ≤60°C and ≤24 h total decontamination time., Methods and Results: Bacillus anthracis spore germination with l-alanine+inosine+calcium dipicolinate (CaDPA) was quantified at 0-40°C, several time points and spore concentrations of 5-9 log 10 per ml. Germination efficiency at 0-40°C was >99% at <8 log 10 spores per ml. The temperature optimum was 20°C. Germination efficiency was significantly higher but slower at 0°C compared to ≥30°C at ≥8 log 10 spores per ml. A single germinant application followed by 60°C, 1-h treatment consistently inactivated >2 log 10 (>99%) of spores. However, a repeat application of germinant was needed to achieve the objective of ≥6 log 10 spore inactivation out of a 7 log 10 challenge (≥99·9999%) for ≤24 h total decontamination time for nylon and aircraft performance coating., Conclusions: l-alanine+inosine+CaDPA stimulated germination across wide temperature and spore concentration ranges., Significance and Impact of the Study: Germination expands the scope of spore decontamination to include materials from any industry sector that can be sprayed with an aqueous germinant solution., (Published 2019. This article is a U.S. Government work and is in the public domain in the USA.)

Bacillus anthracis is a spore-forming, Gram-positive bacterium responsible for anthrax, an acute infection that most significantly affects grazing livestock and wild ungulates, but also poses a threat to human health. The geographic extent of B. anthracis is poorly understood, despite multi-decade research on anthrax epizootic and epidemic dynamics; many countries have limited or inadequate surveillance systems, even within known endemic regions. Here, we compile a global occurrence dataset of human, livestock and wildlife anthrax outbreaks. With these records, we use boosted regression trees to produce a map of the global distribution of B. anthracis as a proxy for anthrax risk. We estimate that 1.83 billion people (95% credible interval (CI): 0.59-4.16 billion) live within regions of anthrax risk, but most of that population faces little occupational exposure. More informatively, a global total of 63.8 million poor livestock keepers (95% CI: 17.5-168.6 million) and 1.1 billion livestock (95% CI: 0.4-2.3 billion) live within vulnerable regions. Human and livestock vulnerability are both concentrated in rural rainfed systems throughout arid and temperate land across Eurasia, Africa and North America. We conclude by mapping where anthrax risk could disrupt sensitive conservation efforts for wild ungulates that coincide with anthrax-prone landscapes.

Anthrax, a lethal, weaponizable disease caused by Bacillus anthracis, acts through exotoxins that are primary mediators of systemic toxicity and also targets for neutralization by passive immunotherapy. The ease of engineering B. anthracis strains resistant to established therapy and the historic use of the microbe in bioterrorism present a compelling test case for platforms that permit the rapid and modular development of neutralizing agents. In vitro antigen-binding fragment (Fab) selection offers the advantages of speed, sequence level molecular control, and engineering flexibility compared to traditional monoclonal antibody pipelines. By screening an unbiased, chemically synthetic phage Fab library and characterizing hits in cell-based assays, we identified two high-affinity neutralizing Fabs, A4 and B7, against anthrax edema factor (EF), a key mediator of anthrax pathogenesis. Engineered homodimers of these Fabs exhibited potency comparable to that of the best reported neutralizing monoclonal antibody against EF at preventing EF-induced cyclic AMP production. Using internalization assays in COS cells, B7 was found to block steps prior to EF internalization. This work demonstrates the efficacy of synthetic alternatives to traditional antibody therapeutics against anthrax while also demonstrating a broadly generalizable, rapid, and modular screening pipeline for neutralizing antibody generation.

Inhalation of Bacillus anthracis spores can lead to an anthrax infection that can be fatal. Previously published mathematical models have extrapolated kinetic rates associated with bacterial growth in New Zealand White (NZW) rabbits to humans, but to date, actual measurements of the underlying processes associated with anthrax virulence between species have not been conducted. To address this knowledge gap, we have quantified species-specific rate constants associated with germination, proliferation, and immune cell inactivation of B. anthracis Sterne using an in vitro test platform that includes primary lung epithelial and immune cells. The generated data was then used to develop a physiologically based biokinetic model (PBBK) which quantitatively compares bacterial growth and mean time to death under lethal conditions in rabbits and humans. Simulations based upon our in vitro data and previously published in vivo data from rabbits indicate that disease progression is likely to be faster in humans than in NZW rabbits under comparable total deposited dose conditions. With the computational framework established, PBBK parameters can now be refined using experimental data for lethal B. anthracis strains (e.g. Ames) under identical conditions in future studies. The PBBK model can also be linked to existing aerosol dosimetry models that account for species-specific differences in aerosol deposition patterns to further improve the human health risk assessment of inhalation anthrax., Competing Interests: The authors have declared that no competing interests exist.

Bacillus anthracis is the causative agent of anthrax in humans, bovine, and other animals. B. anthracis pathogenesis requires differentiation of dormant spores into vegetative cells. The spores inherit cellular components as phenotypic memory from the parent cell, and this memory plays a critical role in facilitating the spores' revival. Because metabolism initiates at the beginning of spore germination, here we metabolically reprogrammed B. anthracis cells to understand the role of glycolytic enzymes in this process. We show that increased expression of enolase (Eno) in the sporulating mother cell decreases germination efficiency. Eno is phosphorylated by the conserved Ser/Thr protein kinase PrkC which decreases the catalytic activity of Eno. We found that phosphorylation also regulates Eno expression and localization, thereby controlling the overall spore germination process. Using MS analysis, we identified the sites of phosphorylation in Eno, and substitution(s) of selected phosphorylation sites helped establish the functional correlation between phosphorylation and Eno activity. We propose that PrkC-mediated regulation of Eno may help sporulating B. anthracis cells in adapting to nutrient deprivation. In summary, to the best of our knowledge, our study provides the first evidence that in sporulating B. anthracis , PrkC imprints phenotypic memory that facilitates the germination process.

Anthrax, caused by Bacillus anthracis , is a non-contagious infectious disease that affects a wide range of animal species (primarily ruminants) including humans. Due to the often-fatal outcome in humans, quick administration of definitely effective antimicrobials is crucial either as prophylaxis or as a clinical case therapy. In this study, 110 B. anthracis strains, temporally, geographically, and genetically different, isolated during anthrax outbreaks in Italy from 1984 to 2017, were screened using a broth microdilution method to determine their susceptibility to 16 clinically relevant antimicrobial agents. The strains were isolated from various matrices (human, animal, and environmental samples) and were representative of thirty distinct genotypes previously identified by 15-loci multiple-locus variable-number of tandem repeats analysis. The antimicrobials tested were gentamicin, ceftriaxone, streptomycin, penicillin G, clindamycin, chloramphenicol, vancomycin, linezolid, cefotaxime, tetracycline, erythromycin, rifampin, amoxicillin, ciprofloxacin, doxycycline, and trimethoprim. All isolates were susceptible to most of the tested antimicrobials, with the exception of trimethoprim for which all of them showed high minimal inhibitory concentration values. An intermediate level of susceptibility was recorded for ceftriaxone and cefotaxime. Although the Centers for Disease Control and Prevention recommend the use of doxycycline, ciprofloxacin, penicillin G, and amoxicillin for treatment of human cases and for post-exposure prophylaxis to anthrax spores, this study shows a high degree of in vitro susceptibility of B. anthracis to many other antimicrobials, suggesting the possibility of an alternative choice for prophylaxis and therapy.

In this study, an ELISA was developed for simultaneous detection of antibodies against both the important toxins of B. anthracis i.e. protective antigen (PA) and lethal factor (LF). A chimera of PA and LF was made by fusion and cloned and expressed in E. coli. The purified recombinant protein was used in plate ELISA for serodiagnosis of anthrax. The chimera could detect antibodies against both the toxins of Bacillus anthracis. The human serum samples (n = 98) collected from anthrax endemic and non-endemic areas were tested employing ELISA. The ELISA gave sensitivity of 100% (95% Confidence Interval [CI], 92.13 to 100) and specificity of 97.78% (95% Confidence Interval [CI], 88.23 to 99.94) with a J index of 0.97. The efficiency of ELISA was found to be 98.9% with the positive predictive value (PPV) and negative predictive value (NPV) of 97.8% and 100%, respectively. The chimera of PA and LF could be a better diagnostic antigen for serodiagnosis as the assay detects antibodies against both the toxins in early as well delayed infection cases of anthrax. Therefore, it can be a very useful tool for the surveillance as well as for confirmation of cutaneous anthrax cases., (Copyright © 2019 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

The spore forming pathogen Bacillus anthracis is the etiologic agent of anthrax in humans and animals. It cycles through infected hosts as vegetative cells and is eventually introduced into the environment where it generates an endospore resistant to many harsh conditions. The endospores are subsequently taken up by another host to begin the next cycle. Outbreaks of anthrax occur regularly worldwide in wildlife and livestock, and the potential for human infection exists whenever humans encounter infected animals. It is also possible to encounter intentional releases of anthrax spores, as was the case in October 2001. Consequently, it is important to be able to rapidly establish the provenance of infectious strains of B. anthracis. Here, we compare protein expression in seven low-passage wild isolates and four laboratory strains of B. anthracis grown under identical conditions using LC-MS/MS proteomic analysis. Of the 1,023 total identified proteins, 96 had significant abundance differences between wild and laboratory strains. Of those, 28 proteins directly related to sporulation were upregulated in wild isolates, with expression driven by Spo0A, CodY, and AbrB/ScoC. In addition, we observed evidence of changes in cell division and fatty acid biosynthesis between the two classes of strains, despite being grown under identical experimental conditions. These results suggest wild B. anthracis cells are more highly tuned to sporulate than their laboratory cousins, and this difference should be exploited as a method to differentiate between laboratory and low passage wild strains isolated during an anthrax outbreak. This knowledge should distinguish between intentional releases and exposure to strains in nature, providing a basis for the type of response by public health officials and investigators., Competing Interests: The authors have declared that no competing interests exist.

Nonencapsulated (∆pXO2) Bacillus anthracis strains are commonly used as vaccines and for anthrax research, mainly in the mouse model. Previously, we demonstrated that the infection of rabbits, intranasally or subcutaneously, with the spores of a fully virulent strain results in the systemic dissemination of the bacteria, meningitis, and death, whereas ∆pXO2 strains are fully attenuated in this animal model. We used the intravenous inoculation of rabbits to study the pathogenicity of the ∆pXO2 strain infection. Bacteremia, brain bacterial burden, and pathology were used as criteria to compare the Vollum∆pXO2 disease to the wild type Vollum infection. To test the role of adhesion in the virulence of Vollum∆pXO2, we deleted the major adhesion protein BslA and tested the virulence and immunogenicity of this mutant. We found that 50% of the rabbits succumb to Vollum∆pXO2 strain following i.v. infection, a death that was accompanied with significant neurological symptoms. Pathology revealed severe brain infection coupled with an atypical massive bacterial growth into the parenchyma. Contrary to the Vollum strain, deletion of the bslA gene fully attenuated the ∆pXO2 strain. Though the Vollum∆pXO2 cannot serve as a model for B. anthracis pathogenicity in rabbits, deletion of the bslA gene prevents central nervous system (CNS) infections, possibly leading to the generation of a safer vaccine.

Bacterial spores produced by the Bacillales are composed of concentric shells, each of which contributes to spore function. Spores from all species possess a cortex and coat, but spores from many species possess additional outer layers. The outermost layer of Bacillus anthracis spores, the exosporium, is separated from the coat by a gap known as the interspace. Exosporium and interspace assembly remains largely mysterious. As a result, we have a poor understanding of the overarching mechanisms driving the assembly of one of the most ubiquitous cell types in nature. To elucidate the mechanisms directing exosporium assembly, we generated strains bearing mutations in candidate exosporium-controlling genes and analyzed the effect on exosporium formation. Biochemical and cell biological analyses argue that CotE directs the assembly of CotO into the spore and that CotO might be located at or close to the interior side of the cap. Taken together with data showing that CotE and CotO interact directly in vitro , we propose a model in which CotE and CotO are important components of a protein interaction network that connects the exosporium to the forespore during cap formation and exosporium elongation. Our data also suggest that the cap interferes with coat assembly at one pole of the spore, altering the pattern of coat deposition compared to the model organism Bacillus subtilis We propose that the difference in coat assembly patterns between these two species is due to an inherent flexibility in coat assembly, which may facilitate the evolution of spore outer layer complexity. IMPORTANCE This work dramatically improves our understanding of the assembly of the outermost layer of the B. anthracis spore, the exosporium, a layer that encases spores from many bacterial species and likely plays important roles in the spore's interactions with the environment, including host tissues. Nonetheless, the mechanisms directing exosporium assembly into a shell surrounding the spore are still very poorly understood. In this study, we clarify these mechanisms by the identification of a novel protein interaction network that directs assembly to initiate at a specific subcellular location in the developing cell. Our results further suggest that the presence or absence of an exosporium has a major impact on the assembly of other more interior spore layers, thereby potentially explaining long-noted differences in spore assembly between B. anthracis and the model organism B. subtilis ., (Copyright © 2018 Boone et al.)

Inhalational anthrax, caused by the gram positive bacteria Bacillus anthracis, is a potentially fatal form of anthrax infection. It is initiated after inhaled spores are deposited in the lung, phagocytosed by immune cells, and subsequently transported to nearby lymph nodes. Intracellular spores that successfully germinate and become vegetative bacteria can lyse their host cell and contribute to bacterial outgrowth and toxin production. To better understand the early disease dynamics of the host-pathogen interaction, we develop a mathematical model of ordinary differential Equations and estimate parameters using available data. The model which consists of two subsystems is designed in accordance with an in vitro experimental protocol in which macrophages were challenged with varying doses of spores at spore-to-macrophage ratios of 1:1, 1:2, 1:10, 1:20. Initial modeling results suggested the need to consider two distinct subpopulations of anthrax bacteria: newly germinated bacteria which cannot replicate immediately and fully vegetative bacteria that can. Additional modeling results provide insights into possible reasons why macrophage-induced killing is more effective at the 1:20 ratio., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Environmentally transmitted diseases are comparatively poorly understood and managed, and their ecology is particularly understudied. Here we identify challenges of studying environmental transmission and persistence with a six-sided interdisciplinary review of the biology of anthrax (Bacillus anthracis). Anthrax is a zoonotic disease capable of maintaining infectious spore banks in soil for decades (or even potentially centuries), and the mechanisms of its environmental persistence have been the topic of significant research and controversy. Where anthrax is endemic, it plays an important ecological role, shaping the dynamics of entire herbivore communities. The complex eco-epidemiology of anthrax, and the mysterious biology of Bacillus anthracis during its environmental stage, have necessitated an interdisciplinary approach to pathogen research. Here, we illustrate different disciplinary perspectives through key advances made by researchers working in Etosha National Park, a long-term ecological research site in Namibia that has exemplified the complexities of the enzootic process of anthrax over decades of surveillance. In Etosha, the role of scavengers and alternative routes (waterborne transmission and flies) has proved unimportant relative to the long-term persistence of anthrax spores in soil and their infection of herbivore hosts. Carcass deposition facilitates green-ups of vegetation to attract herbivores, potentially facilitated by the role of anthrax spores in the rhizosphere. The underlying seasonal pattern of vegetation, and herbivores' immune and behavioural responses to anthrax risk, interact to produce regular 'anthrax seasons' that appear to be a stable feature of the Etosha ecosystem. Through the lens of microbiologists, geneticists, immunologists, ecologists, epidemiologists, and clinicians, we discuss how anthrax dynamics are shaped at the smallest scale by population genetics and interactions within the bacterial communities up to the broadest scales of ecosystem structure. We illustrate the benefits and challenges of this interdisciplinary approach to disease ecology, and suggest ways anthrax might offer insights into the biology of other important pathogens. Bacillus anthracis, and the more recently emerged Bacillus cereus biovar anthracis, share key features with other environmentally transmitted pathogens, including several zoonoses and panzootics of special interest for global health and conservation efforts. Understanding the dynamics of anthrax, and developing interdisciplinary research programs that explore environmental persistence, is a critical step forward for understanding these emerging threats., (© 2018 Cambridge Philosophical Society.)

Epidemiologic data indicate a global distribution of anthrax outbreaks associated with certain ecosystems that promote survival and viability of Bacillus anthracis spores. Here, we characterized three anthrax outbreaks involving humans, livestock, and wildlife that occurred in the same locality in Kenya between 2014 and 2017. Clinical and epidemiologic data on the outbreaks were collected using active case finding and review of human, livestock, and wildlife health records. Information on temporal and spatial distribution of prior outbreaks in the area was collected using participatory epidemiology. The 2014-2017 outbreaks in Nakuru West subcounty affected 15 of 71 people who had contact with infected cattle (attack rate = 21.1%), including seven with gastrointestinal, six with cutaneous, and two with oropharyngeal forms of the disease. Two (13.3%) gastrointestinal human anthrax cases died. No human cases were associated with infected wildlife. Of the 54 cattle owned in 11 households affected, 20 died (attack rate = 37%). The 2015 outbreak resulted in death of 10.5% of the affected herbivorous wildlife at Lake Nakuru National Park, including 745 of 4,500 African buffaloes (species-specific mortality rate = 17%) and three of 18 endangered white rhinos (species-specific mortality rate = 16%). The species mortality rate ranged from 1% to 5% for the other affected wildlife species. Participatory epidemiology identified prior outbreaks between 1973 and 2011 in the same area. The frequency and severity of outbreaks in this area suggests that it is an anthrax hotspot ideal for investigating risk factors associated with long-term survival of anthrax spores and outbreak occurrence.

Germination of Clostridium difficile spores is a crucial early requirement for colonization of the gastrointestinal tract. Likewise, C. difficile cannot cause disease pathologies unless its spores germinate into metabolically active, toxin-producing cells. Recent advances in our understanding of C. difficile spore germination mechanisms indicate that this process is both complex and unique. This review defines unique aspects of the germination pathways of C. difficile and compares them to those of two other well-studied organisms, Bacillus anthracis and Clostridium perfringens C. difficile germination is unique, as C. difficile does not contain any orthologs of the traditional GerA-type germinant receptor complexes and is the only known sporeformer to require bile salts in order to germinate. While recent advances describing C. difficile germination mechanisms have been made on several fronts, major gaps in our understanding of C. difficile germination signaling remain. This review provides an updated, in-depth summary of advances in understanding of C. difficile germination and potential avenues for the development of therapeutics, and discusses the major discrepancies between current models of germination and areas of ongoing investigation., (Copyright © 2018 American Society for Microbiology.)

In 2015, a laboratory of the United States Department of Defense (DoD) inadvertently shipped preparations of gamma-irradiated spores of Bacillus anthracis that contained live spores. In response, a systematic evidence-based method for preparing, concentrating, irradiating, and verifying the inactivation of spore materials was developed. We demonstrate the consistency of spore preparations across multiple biological replicates and show that two different DoD institutions independently obtained comparable dose-inactivation curves for a monodisperse suspension of B. anthracis spores containing 3 × 10 10 CFU. Spore preparations from three different institutions and three strain backgrounds yielded similar decimal reduction (D 10 ) values and irradiation doses required to ensure sterility (D SAL ) to the point at which the probability of detecting a viable spore is 10 -6 Furthermore, spores of a genetically tagged strain of B. anthracis strain Sterne were used to show that high densities of dead spores suppress the recovery of viable spores. Together, we present an integrated method for preparing, irradiating, and verifying the inactivation of spores of B. anthracis for use as standard reagents for testing and evaluating detection and diagnostic devices and techniques. IMPORTANCE The inadvertent shipment by a U.S. Department of Defense (DoD) laboratory of live Bacillus anthracis (anthrax) spores to U.S. and international destinations revealed the need to standardize inactivation methods for materials derived from biological select agents and toxins (BSAT) and for the development of evidence-based methods to prevent the recurrence of such an event. Following a retrospective analysis of the procedures previously employed to generate inactivated B. anthracis spores, a study was commissioned by the DoD to provide data required to support the production of inactivated spores for the biodefense community. The results of this work are presented in this publication, which details the method by which spores can be prepared, irradiated, and tested, such that the chance of finding residual living spores in any given preparation is 1/1,000,000. These irradiated spores are used to test equipment and methods for the detection of agents of biological warfare and bioterrorism.

The revelation in May 2015 of the shipment of γ irradiation-inactivated wild-type Bacillus anthracis spore preparations containing a small number of live spores raised concern about the safety and security of these materials. The finding also raised doubts about the validity of the protocols and procedures used to prepare them. Such inactivated reference materials were used as positive controls in assays to detect suspected B. anthracis in samples because live agent cannot be shipped for use in field settings, in improvement of currently deployed detection methods or development of new methods, or for quality assurance and training activities. Hence, risk-mitigated B. anthracis strains are needed to fulfill these requirements. We constructed a genetically inactivated or attenuated strain containing relevant molecular assay targets and tested to compare assay performance using this strain to the historical data obtained using irradiation-inactivated virulent spores.

Anthrax is an era old deadly disease against which there are only two currently available licensed vaccines named anthrax vaccine adsorbed and precipitated (AVP). Though they can provide a protective immunity, their multiple side-effects owing to their ill-defined composition and presence of toxic proteins (LF and EF) of Bacillus anthracis , the causative organism of anthrax, in the vaccine formulation makes their widespread use objectionable. Hence, an anthrax vaccine that contains well-defined and controlled components would be highly desirable. In this context, we have evaluated the potential of various vaccine formulations comprising of protective antigen (PA) encapsulated trimethyl-chitosan nanoparticles (TMC-PA) in conjunction with either CpG-C ODN 2395 (CpG) or Poly I:C. Each formulation was administered via three different routes, viz., subcutaneous (SC), intramuscular (IM), and intraperitoneal in female BALB/c mice. Irrespective of the route of immunization, CpG or Poly I:C adjuvanted TMC-PA nanoparticles induced a significantly higher humoral response (total serum IgG and its isotypes viz., IgG1, IgG2a, and IgG2b), compared to their CpG or Poly I:C PA counterparts. This clearly demonstrates the synergistic behavior of CpG and Poly I:C with TMC nanoparticles. The adjuvant potential of TMC nanoparticles could be observed in all the three routes as the TMC-PA nanoparticles by themselves induced IgG titers (1-1.5 × 10 5 ) significantly higher than both CpG PA and Poly I:C PA groups (2-8 × 10 4 ). The effect of formulations on T-helper (T h ) cell development was assessed by quantifying the Th1-dependant (TNF-α, IFN-γ, and IL-2), Th2-dependant (IL-4, IL-6, and IL-10), and Th17-type (IL-17A) cytokines. Adjuvanation with CpG and Poly I:C, the TMC-PA nanoparticles triggered a Th1 skewed immune response, as suggested by an increase in the levels of total IgG2a along with IFN-γ cytokine production. Interestingly, the TMC-PA group showed a Th2-biased immune response. Upon challenge with the B. anthracis Ames strain, CpG and Poly I:C adjuvanted TMC-PA nanoparticles immunized via the SC and IM routes showed the highest protective efficacy of ~83%. Altogether, the results suggest that CpG or Poly I:C adjuvanted, PA-loaded TMC nanoparticles could be used as an effective, non-toxic, second generation subunit-vaccine candidate against anthrax.

Introduction: Cutaneous anthrax is a zoonotic disease caused by the spore-forming bacterium Bacillus anthracis, which typically presents with ulcers after contact with animals or animal products, and is rarely seen in high-income countries but is common in those with low- and middle-incomes. Objective. The aim of this study is to show the main clinical characteristics of cutaneous anthrax in endemic areas., Material and Methods: The study describes the main clinical characteristics of cutaneous anthrax in eight patients (six female and two male, age range 1 - 56 years) admitted to the rural General Hospital of Gambo, West Arsi Province of Ethiopia from 2010-2013., Results: In all cases, lesions began as an erythematous papule located on exposed sites (n=7 head; n=1 thigh) and subsequently became a necrotic black eschar surrounded by an edematous halo. Two patients presented with painful ipsilateral adenopathy near the black eschar. Four patients developed a malignant pustule on the suborbital region of the face. Patients responded positively to treatment, and the lesions resolved, leaving eschars. However, one patient suffered the loss of an eyeball, and another died 12 hours after starting treatment., Conclusions: Physicians working in rural areas of resource-poor settings should be trained in the clinical identification of cutaneous anthrax. Early antibiotic treatment is essential for decreasing morbidity and mortality.

The process of sporulation is vital for the stability and infectious cycle of Bacillus anthracis. The spore is the infectious form of the organism and therefore relevant to biodefense. While the morphological and molecular events occurring during sporulation have been well studied, the influence of growth medium and temperature on the proteins expressed in sporulated cultures is not well understood. Understanding the features of B. anthracis sporulation specific to natural vs. laboratory production will address an important question in microbial forensics. In an effort to bridge this knowledge gap, a system for sporulation on two types of agar-immobilized soils was used for comparison to cultures sporulated on two common types of solid laboratory media, and one liquid sporulation medium. The total number of proteins identified as well as their identity differed between samples generated in each medium and growth temperature, demonstrating that sporulation environment significantly impacts the protein content of the spore. In addition, a subset of proteins common in all of the soil-cultivated samples was distinct from the expression profiles in laboratory medium (and vice versa). These differences included proteins involved in thiamine and phosphate metabolism in the sporulated cultures produced on soils with a notable increase in expression of ATP binding cassette (ABC) transporters annotated to be for phosphate and antimicrobial peptides. A distinct set of ABC transporters for amino acids, sugars and oligopeptides were found in cultures produced on laboratory media as well as increases in carbon and amino acid metabolism-related proteins. These protein expression changes indicate that the sporulation environment impacts the protein profiles in specific ways that are reflected in the metabolic and membrane transporter proteins present in sporulated cultures.

Gastrointestinal (GI) anthrax is the most prevalent form of naturally acquired Bacillus anthracis infection, which is associated with exposure to vegetative bacteria in infected meat (carnivores) or to fermented rumen contents (herbivores). We assessed whether key host and pathogen factors modulate infectivity and progression of infection using a mouse model of GI infection. Gastric acid neutralization increases infectivity, but 30%-40% of mice succumb to infection without neutralization. Mice either fed or fasted before exposure showed similar infectivity rates. Finally, the pathogen's anthrax lethal factor is required to establish lethal infection, whereas its edema factor modulates progression and dissemination of infection., (Published by Oxford University Press for the Infectious Diseases Society of America 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Endospores of the genus Bacillus can be triggered to germinate by a limited number of chemicals. Mandelate had powerful additive effects on the levels and rates of germination produced in non-heat-shocked spores of Bacillus anthracis strain Sterne, Bacillus cereus , and Bacillus thuringiensis when combined with l-alanine and inosine. Mandelate had no germinant effect on its own but was active with these germinants in a dose-dependent manner at concentrations higher than 0.5 mM. The maximum rate and extent of germination were produced in B. anthracis by 100 mM l-alanine with 10 mM inosine; this was equaled by just 25% of these germinants when supplemented with 10 mM mandelate. Half the maximal germination rate was produced by 40% of the optimum germinant concentrations or 15% of them when supplemented with 0.8 mM mandelate. Germination rates in B. thuringiensis were highest around neutrality, but the potentiating effect of mandelate was maintained over a wider pH range than was germination with l-alanine and inosine alone. For all species, lactate also promoted germination in the presence of l-alanine and inosine; this was further increased by mandelate. Ammonium ions also enhanced l-alanine- and inosine-induced germination but only when mandelate was present. In spite of the structural similarities, mandelate did not compete with phenylalanine as a germinant. Mandelate appeared to bind to spores while enhancing germination. There was no effect when mandelate was used in conjunction with nonnutrient germinants. No effect was produced with spores of Bacillus subtilis , Clostridium sporogenes , or C. difficile IMPORTANCE The number of chemicals that can induce germination in the species related to Bacillus cereus has been defined for many years, and they conform to specific chemical types. Although not a germinant itself, mandelate has a structure that is different from these germination-active compounds, and its addition to this list represents a significant discovery in the fundamental biology of spore germination. This novel activity may also have important applied relevance given the impact of spores of B. cereus in foodborne disease and B. anthracis as a threat agent. The destruction of spores of B. anthracis , for example, particularly over large outdoor areas, poses significant scientific and logistical problems. The addition of mandelate and lactate to the established mixtures of l-alanine and inosine would decrease the amount of the established germinants required and increase the speed and level of germination achieved. The large-scale application of "germinate to decontaminate" strategy may thus become more practicable., (Copyright © 2017 American Society for Microbiology.)

Understanding the interaction between macrophage cells and Bacillus anthracis spores is of significant importance with respect to both anthrax disease progression, spore detection for biodefense, as well as understanding cell clearance in general. While most detection systems rely on specific molecules, such as nucleic acids or proteins and fluorescent labels to identify the target(s) of interest, label-free methods probe changes in intrinsic properties, such as size, refractive index, and morphology, for correlation with a particular biological event. Optical chromatography is a label free technique that uses the balance between optical and fluidic drag forces within a microfluidic channel to determine the optical force on cells or particles. Here we show an increase in the optical force experienced by RAW264.7 macrophage cells upon the uptake of both microparticles and B. anthracis Sterne 34F2 spores. In the case of spores, the exposure was detected in as little as 1 h without the use of antibodies or fluorescent labels of any kind. An increase in the optical force was also seen in macrophage cells treated with cytochalasin D, both with and without a subsequent exposure to spores, indicating that a portion of the increase in the optical force arises independent of phagocytosis. These results demonstrate the capability of optical chromatography to detect subtle biological differences in a rapid and sensitive manner and suggest future potential in a range of applications, including the detection of biological threat agents for biodefense and pathogens for the prevention of sepsis and other diseases.