Anthracis Infection Research Articles

The Impact of drinking water source on disease transmission among pastoralists: The Case study of the Ngorongoro Conservation Area, Tanzania

The study examined the impact of drinking water source on disease transmission among pastoralists, using a case study of the Ngorongoro Conservation Area (NCA) in Tanzania. A total of four villages located in the NCA were sampled and studied, namely: Endulen, Nainokanoka, Kayapus and Meshili. This was a cross-sectional study which deployed questionnaires, direct observation, document reviews and focus group discussions. A total of 396 households participated in the survey. The collected data were analyzed using qualitative data analysis techniques and descriptive statistics such as frequencies. The study findings revealed that unfiltered waterholes shared among humans, livestock and wildlife accounted for 82.1%; indicating that it was the primary source of disease transmission. Other sources of disease transmissions were: the use of untreated tap water, rivers, spring water and rainwater. The factors influencing the use of unfiltered waterholes were limited access to tap water, scarcity of drinking water sources, drought, population increase and tourists. The co-usage of water coupled with lack of latrines suggest fecal contamination of drinking water sources. Women were more susceptible to waterborne diseases as they were more directly engaging in all domestic activities including fetching for water as compared to men. The waterborne diseases affecting pastoralist communities were diarrhea, cholera, skin infection, dysentery, worms and typhoid fever. The study concluded that there is a nexus between shared drinking water sources and the prevalence of high anthrax (Bacillus anthracis) infection rates. It is recommended that the adverse impacts of disease transmission on drinking water sources can be mitigated through increasing supply of tap water, using treated water and encouraging community use of pit latrines.

Identification and characterization of two Bacillus anthracis bacteriophages

Anthrax is an acute infectious zoonotic disease caused by Bacillus anthracis, a bacterium that is considered a potential biological warfare agent. Bacillus bacteriophages shape the composition and evolution of bacterial communities in nature and therefore have important roles in the ecosystem community. B. anthracis phages are not only used in etiological diagnostics but also have promising prospects in clinical therapeutics or for disinfection in anthrax outbreaks. In this study, two temperate B. anthracis phages, vB_BanS_A16R1 (A16R1) and vB_BanS_A16R4 (A16R4), were isolated and showed siphovirus-like morphological characteristics. Genome sequencing showed that the genomes of phages A16R1 and A16R4 are 36,569 bp and 40,059 bp in length, respectively. A16R1 belongs to the genus Wbetavirus, while A16R4 belongs to the genus Hubeivirus and is the first phage of that genus found to lyse B. anthracis. Because these two phages can comparatively specifically lyse B. anthracis, they could be used as alternative diagnostic tools for identification of B. anthracis infections.

Doxycycline, levofloxacin, and moxifloxacin are superior to ciprofloxacin in treating anthrax meningitis in rabbits and NHP.

Efficient treatment of anthrax-related meningitis in patients poses a significant therapeutic challenge. Previously, we demonstrated in our anthrax meningitis rabbit model that ciprofloxacin treatment is ineffective with most of the treated animals succumbing to the infection. Herein we tested the efficacy of doxycycline in our rabbit model and found it highly effective. Since all of our findings are based on a rabbit model, we test the efficacy of ciprofloxacin or doxycycline in a specific central nervous system (CNS) model developed in non-human primates (NHPs). Similar to rabbits, ciprofloxacin treatment was ineffective, while doxycycline protected the infected rhesus macaques (n = 2) from the lethal CNS Bacillus anthracis infection. To test whether the low efficacy of Ciprofloxacin is an example of low efficacy of all fluoroquinolones or only this substance, we treated rabbits that were inoculated intracisterna magna (ICM) with levofloxacin or moxifloxacin. We found that in contrast to ciprofloxacin, levofloxacin and moxifloxacin were highly efficacious in treating lethal anthrax-related meningitis in rabbits and NHP (levofloxacin). We demonstrated (in naïve rabbits) that this difference probably results from variances in blood-brain-barrier penetration of the different fluoroquinolones. The combined treatment of doxycycline and any one of the tested fluoroquinolones was highly effective in the rabbit CNS infection model. The combined treatment of doxycycline and levofloxacin was effective in an inhalation rabbit model, as good as the doxycycline mono-therapy. These findings imply that while ciprofloxacin is highly effective as a post-exposure prophylactic drug, using this drug to treat symptomatic patients should be reconsidered.

Genome-wide identification of bHLH gene family and screening of candidate gene in response to salt stress in kiwifruit

As the second largest family of transcription factors in plants, bHLH is widely involved in the growth and development of organisms and their response to stress. In the past, there was no systematic division of this gene family in kiwifruit. In this study, we identified 206 AcbHLH genes, divided into 22 subfamilies, and analyzed chromosomal localization, physicochemical properties, conserved domains, and intron/exon composition. In addition, the repeat events of the AcbHLH gene in kiwifruit and the evolutionary relationship with Arabidopsis thaliana and Oryza sativa were further analyzed. We analyzed the expression levels of the AcbHLH genes under biotic and abiotic stresses (heat, low temperature, drought, and Bacillus anthracis infection). In addition, we also investigated the biological function of AcbHLH146 under salt stress using a VIGS-mediated gene silencing method. In the AcbHLH146-VIGS line, the content of H2O2 and O2- was lower and the damage was less. SOD, POD, and CAT enzyme activities are higher. These results suggest that AcbHLH146 may play a negative moderating role in the response of kiwifruit to salt stress. Our findings provide new insights into the potential role of AcbHLH146 under abiotic stress, and provide a theoretical basis for the molecular mechanism of Kiwifruit adaptation to salt stress.

Season of death, pathogen persistence and wildlife behaviour alter number of anthrax secondary infections from environmental reservoirs.

An important part of infectious disease management is predicting factors that influence disease outbreaks, such as R, the number of secondary infections arising from an infected individual. Estimating R is particularly challenging for environmentally transmitted pathogens given time lags between cases and subsequent infections. Here, we calculated R for Bacillus anthracis infections arising from anthrax carcass sites in Etosha National Park, Namibia. Combining host behavioural data, pathogen concentrations and simulation models, we show that R is spatially and temporally variable, driven by spore concentrations at death, host visitation rates and early preference for foraging at infectious sites. While spores were detected up to a decade after death, most secondary infections occurred within 2 years. Transmission simulations under scenarios combining site infectiousness and host exposure risk under different environmental conditions led to dramatically different outbreak dynamics, from pathogen extinction (R < 1) to explosive outbreaks (R > 10). These transmission heterogeneities may explain variation in anthrax outbreak dynamics observed globally, and more generally, the critical importance of environmental variation underlying host-pathogen interactions. Notably, our approach allowed us to estimate the lethal dose of a highly virulent pathogen non-invasively from observational studies and epidemiological data, useful when experiments on wildlife are undesirable or impractical.

Anthrax disease burden: Impact on animal and human health

Anthrax is a zoonotic infectious disease caused by Bacillus anthracis. According to current knowledge, the disease originates in sub-Saharan Africa, especially Egypt and Mesopotamia. Laboratory tests involving direct staining or culture of samples taken from malignant pustules, sputum, blood, or patient discharge must be performed to establish a diagnosis. B. anthracis infection can enter the body through the skin, mouth, or nose. Human infection is usually caused by contact with infected animals or animal products. Anthrax causes a reduction in resource efficiency and decreases livestock productivity. B. anthracis spores are resistant to extreme temperatures, pressure, pH, drying, solvents, and ultraviolet light. The biological weapon of this disease may be fatal if it is designed to spread B. anthracis spores by aerosols. In the past, the treatment of human anthrax with penicillin at a high dose was the preferred method. The public can take several measures to prevent anthrax infection, such as purchasing and consuming meat that has been legally certified to have been slaughtered in a slaughterhouse, consuming healthy and properly cooked animal meat, and washing hands with antiseptic soap after handling, processing, and cooking animal products. This review aimed to describe the etiology, pathogenesis, mechanism of infection, epidemiology, diagnosis, clinical symptoms, transmission, risk factors, public health importance, economic impact, potential as a bio-warfare agent, treatment, and control of anthrax. Keywords: animal health, anthrax, human health, infectious disease.

Impact of traditional cutaneous scarification on anthrax lesions: A series of cases from Cubal, Angola

ObjectivesBacillus anthracis infection is a worldwide zoonosis that affects the most vulnerable population and has a high mortality rate without treatment, especially in non-cutaneous presentations. Cutaneous scarification is still common in some regions of the world for the treatment of certain diseases as part of traditional medicine. We describe a series of cutaneus anthrax from a rural setting in Angola where cutaneus scarification is common. Case presentationThis is a retrospective observational study describing a series of cutaneous anthrax cases from Cubal (Angola), many of whom were treated with skin scarification before admission. A total of 26 cases were diagnosed from January 2010 to December 2018. None of the cases were confirmed and eight (30.8%) were probable cases according to the Centers for the Disease Control and Prevention anthrax case definition. The median age was 11 (4.7-30.5) years, 17 (65.4%) had lesions on the head, face, or neck and 15 (57.7%) were treated with cutaneous scarification. Nine (34.6%) patients died. Traditional cutaneous scarification was significantly associated with cutaneous superinfection, respiratory, systemic involvement, and death. ConclusionOur case series points to increased complications and worse outcome of cutaneous anthrax disease if treated with skin scarification.

Bacillus anthracis infection in humans and animals

Bacillus anthracis infection in humans and animals

Anthrax lethal toxin and tumor necrosis factor-α synergize on intestinal epithelia to induce mouse death.

Bacillus anthracis lethal toxin (LT) is a determinant of lethal anthrax. Its function in myeloid cells is required for bacterial dissemination, and LT itself can directly trigger dysfunction of the cardiovascular system. The interplay between LT and the host responses is important in the pathogenesis, but our knowledge on this interplay remains limited. Tumor necrosis factor-α (TNF-α) is a pleiotropic pro-inflammatory cytokine induced by bacterial infections. Since LT accumulates and cytokines, predominantly TNF, amass during B. anthracis infection, co-treatment of TNF + LT in mice was used to mimic in vivo conditions for LT to function in inflamed hosts. Bone marrow transplantation and genetically engineered mice showed unexpectedly that the death of intestinal epithelial cells (IECs) rather than that of hematopoietic cells led to LT + TNF-induced lethality. Inhibition of p38α mitogen-activated protein kinase (MAPK) signaling by LT in IECs promoted TNF-induced apoptosis and necroptosis of IECs, leading to intestinal damage and mouse death. Consistently, p38α inhibition by LT enhanced TNF-mediated cell death in human colon epithelial HT-29 cells. As intestinal damage is one of the leading causes of lethality in anthrax patients, the IEC damage caused by LT + TNF would most likely be a mechanism underneath this clinical manifestation and could be a target for interventions.

Postexposure Prophylaxis and Treatment of Bacillus anthracis Infections: A Systematic Review and Meta-analyses of Animal Models, 1947-2019.

Anthrax is endemic to many countries, including the United States. The causative agent, Bacillus anthracis, poses a global bioterrorism threat. Without effective antimicrobial postexposure prophylaxis (PEPAbx) and treatment, the mortality of systemic anthrax is high. To inform clinical guidelines for PEPAbx and treatment of B. anthracis infections in humans, we systematically evaluated animal anthrax treatment model studies. We searched for survival outcome data in 9 scientific search engines for articles describing antimicrobial PEPAbx or treatment of anthrax in animals in any language through February 2019. We performed meta-analyses of efficacy of antimicrobial PEPAbx and treatment for each drug or drug combination using random-effects models. Pharmacokinetic/pharmacodynamic relationships were developed for 5 antimicrobials with available pharmacokinetic data. Monte Carlo simulations were used to predict unbound drug exposures in humans. We synthesized data from 34 peer-reviewed studies with 3262 animals. For PEPAbx and treatment of infection by susceptible B. anthracis, effective monotherapy can be accomplished with fluoroquinolones, tetracyclines, β-lactams (including penicillin, amoxicillin-clavulanate, and imipenem-cilastatin), and lipopeptides or glycopeptides. For naturally occurring strains, unbound drug exposures in humans were predicted to adequately cover the minimal inhibitory concentrations (MICs; those required to inhibit the growth of 50% or 90% of organisms [MIC50 or MIC90]) for ciprofloxacin, levofloxacin, and doxycycline for both the PEPAbx and treatment targets. Dalbavancin covered its MIC50 for PEPAbx. These animal studies show many reviewed antimicrobials are good choices for PEPAbx or treatment of susceptible B. anthracis strains, and some are also promising options for combating resistant strains. Monte Carlo simulations suggest that oral ciprofloxacin, levofloxacin, and doxycycline are particularly robust choices for PEPAbx or treatment.

Structural and Functional Analysis of Toxin and Small RNA Gene Promoter Regions in Bacillus anthracis.

It was previously demonstrated that anthrax toxin activator (AtxA) binds directly to the σA-like promoter region of pagA (encoding protective antigen, PA) immediately upstream of the RNA polymerase binding site. In this study, using electrophoretic mobility shift assays and in vivo analyses, we identified AtxA-binding sites in the promoter regions of the lef and cya genes (encoding lethal and edema factors, respectively) and of two Bacillus anthracis small RNAs (XrrA and XrrB). Activities of all four newly studied promoters were enhanced in the presence of CO2/bicarbonate and AtxA, as previously seen for the pagA promoter. Notably, the cya promoter was less activated by AtxA and CO2/bicarbonate conditions. The putative promoter of a recently described third small RNA, XrrC, showed a negligible response to AtxA and CO2/bicarbonate. RNA polymerase binding sites of the newly studied promoters show no consensus and differ from the σA-like promoter region of pagA. In silico analysis of the probable AtxA binding sites in the studied promoters revealed several palindromes. All the analyzed palindromes showed very little overlap with the σA-like pagA promoter. It remains unclear as to how AtxA and DNA-dependent RNA-polymerase identify such diverse DNA-sequences and differentially regulate promoter activation of the studied genes. IMPORTANCE Anthrax toxin activator (AtxA) is the major virulence regulator of Bacillus anthracis, the causative agent of anthrax. Understanding AtxA's mechanism of regulation could facilitate the development of therapeutics for B. anthracis infection. We provide evidence that AtxA binds to the promoters of the cya, lef, xrrA, and xrrB genes. In vivo assays confirmed the activities of all four promoters were enhanced in the presence of AtxA and CO2/bicarbonate, as previously seen for the pagA promoter. The cya and lef genes encode important toxin components. The xrrA and xrrB genes encode sRNAs with a suggested function as cell physiology regulators. Our data provides further evidence for the direct regulatory role of AtxA that was previously shown with the pagA promoter.

A Novel Toll-Like Receptor 2 Agonist Protects Mice in a Prophylactic Treatment Model Against Challenge With Bacillus anthracis.

Current therapies for anthrax include the use of antibiotics (i.e., doxycycline, and ciprofloxacin), an anthrax vaccine (BioThrax) and Bacillus anthracis-specific, monoclonal antibody (mAb) (i.e., Raxibacumab and obiltoxaximab). In this study, we investigated the activity of immunomodulators, which potentiate inflammatory responses through innate immune receptors. The rationale for the use of innate immune receptor agonists as adjunctive immunomodulators for infectious diseases is based on the concept that augmentation of host defense should promote the antimicrobial mechanism of the host. Our aim was to explore the anti-B. anthracis effector function of Toll-like receptor (TLR) agonists using a mouse model. Amongst the six TLR ligands tested, Pam3CSK4 (TLR1/2 ligand) was the best at protecting mice from lethal challenge of B. anthracis. We then evaluated the activity of a novel TLR2 ligand, DA-98-WW07. DA-98-WW07 demonstrated enhanced protection in B. anthracis infected mice. The surviving mice that received DA-98-WW07 when re-challenged with B. anthracis 20 days post the first infection showed increased survival rate. Moreover, ciprofloxacin, when treated in adjunct with a suboptimal concentration of DA-98-WW07 demonstrated augmented activity in protecting mice from B. anthracis infection. Taken together, we report the prophylactic treatment potential of DA-98-WW07 for anthrax and the utility of immunomodulators in combination with an antibiotic to treat infections caused by the B. anthracis bacterium.

Bacillus anthracis induces NLRP3 inflammasome activation and caspase-8–mediated apoptosis of macrophages to promote lethal anthrax

Lethal toxin (LeTx)-mediated killing of myeloid cells is essential for Bacillus anthracis, the causative agent of anthrax, to establish systemic infection and induce lethal anthrax. The "LeTx-sensitive" NLRP1b inflammasome of BALB/c and 129S macrophages swiftly responds to LeTx intoxication with pyroptosis and secretion of interleukin (IL)-1β. However, human NLRP1 is nonresponsive to LeTx, prompting us to investigate B. anthracis host-pathogen interactions in C57BL/6J (B6) macrophages and mice that also lack a LeTx-sensitive Nlrp1b allele. Unexpectedly, we found that LeTx intoxication and live B. anthracis infection of B6 macrophages elicited robust secretion of IL-1β, which critically relied on the NLRP3 inflammasome. TNF signaling through both TNF receptor 1 (TNF-R1) and TNF-R2 were required for B. anthracis-induced NLRP3 inflammasome activation, which was further controlled by RIPK1 kinase activity and LeTx-mediated proteolytic inactivation of MAP kinase signaling. In addition to activating the NLRP3 inflammasome, LeTx-induced MAPKK inactivation and TNF production sensitized B. anthracis-infected macrophages to robust RIPK1- and caspase-8-dependent apoptosis. In agreement, purified LeTx triggered RIPK1 kinase activity- and caspase-8-dependent apoptosis only in macrophages primed with TNF or following engagement of TRIF-dependent Toll-like receptors. Consistently, genetic and pharmacological inhibition of RIPK1 inhibited NLRP3 inflammasome activation and apoptosis of LeTx-intoxicated and B. anthracis-infected macrophages. Caspase-8/RIPK3-deficient mice were significantly protected from B. anthracis-induced lethality, demonstrating the invivo pathophysiological relevance of this cytotoxic mechanism. Collectively, these results establish TNF- and RIPK1 kinase activity-dependent NLRP3 inflammasome activation and macrophage apoptosis as key host-pathogen mechanisms in lethal anthrax.

Silkworm model for Bacillus anthracis infection and virulence determination

ABSTRACT 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.

Nociceptive Sensory Neurons Mediate Inflammation Induced by Bacillus Anthracis Edema Toxin.

Bacterial products are able to act on nociceptive neurons during pathogenic infection. Neurogenic inflammation is an active part of pain signaling and has recently been shown to impact host-pathogen defense. Bacillus anthracis Edema Toxin (ET) produces striking edema in peripheral tissues, but the cellular mechanisms involved in tissue swelling are not completely understood. Here, we find that nociceptive neurons play a role in ET-induced edema and inflammation in mice. Subcutaneous footpad infection of B. anthracis Sterne caused ET-dependent local mechanical allodynia, paw swelling and body weight gain. Subcutaneous administration of ET induced paw swelling and vascular leakage, the early phases of which were attenuated in the absence of Trpv1+ or Nav1.8+ nociceptive neurons. Nociceptive neurons express the anthrax toxin receptor ANTXR2, but this did not mediate ET-induced edema. ET induced local cytokine expression and neutrophil recruitment, which were dependent in part on Trpv1+ nociceptive neurons. Ablation of Trpv1+ or Nav1.8+ nociceptive neurons also attenuated early increases in paw swelling and body weight gain during live B. anthracis infection. Our findings indicate that nociceptive neurons play an active role in inflammation caused by B. anthracis and Edema Toxin to potentially influence bacterial pathogenesis.

Trace detection of anthrax protective antigens via a competitive method based on surface-enhanced Raman scattering

Owing to the high mortality and infection rates of the Bacillus anthracis infection, commonly known as anthrax, the ability to detecting anthrax protective antigens (PA) is important in several domains, including food safety and bioterrorism. An ultrasensitive PA detection that employs a biosensor using a competitive method based on surface-enhanced Raman scattering (SERS) was developed herein. Au nanorod arrays (AuNRAs), conjugated with the PA-aptamer were prepared as substrates, whereas Au nanoparticles (AuNPs) were modified with 1,2-bis (4-pyridyl) ethylene (BPE) and PA as SERS tags. During detection, free PA and SERS tags bound competitively with active sites on the surface of AuNRAs. The Raman spectra of BPE were used to evaluate the limit of detection (LOD) for PA. Three enhanced Raman modes in this biosensor, namely AuNP self-enhancement, plasmonic resonance between single AuNP and AuNRAs, and nanogaps between two AuNP agglomerates and AuNRAs, were evaluated using the finite-difference time-domain (FDTD) method. Among AuNPs, AuNRAs and PA/BPE/AuNP/PA-apt/AuNRAs complex, the highest Raman signal of the Raman molecule BPE for the PA/BPE/AuNP/PA-apt/AuNRAs complex proved the existence of these three modes. Owing to the increased number of hotspots in biosensors, the ultra-sensitive detection of PA was achieved with an LOD of 1 pg/mL PA.

A Review of the Efficacy of FDA-Approved B. anthracis Anti-Toxin Agents When Combined with Antibiotic or Hemodynamic Support in Infection- or Toxin-Challenged Preclinical Models.

Anti-toxin agents for severe B. anthracis infection will only be effective if they add to the benefit of the two mainstays of septic shock management, antibiotic therapy and titrated hemodynamic support. Both of these standard therapies could negate benefits related to anti-toxin treatment. At present, three anthrax anti-toxin antibody preparations have received US Food and Drug Administration (FDA) approval: Raxibacumab, Anthrax Immune Globulin Intravenous (AIGIV) and ETI-204. Each agent is directed at the protective antigen component of lethal and edema toxin. All three agents were compared to placebo in antibiotic-treated animal models of live B. anthracis infection, and Raxibacumab and AIGIV were compared to placebo when combined with standard hemodynamic support in a 96 h canine model of anthrax toxin-associated shock. However, only AIG has actually been administered to a group of infected patients, and this experience was not controlled and offers little insight into the efficacy of the agents. To provide a broader view of the potential effectiveness of these agents, this review examines the controlled preclinical experience either in antibiotic-treated B. anthracis models or in titrated hemodynamic-supported toxin-challenged canines. The strength and weaknesses of these preclinical experiences are discussed.

Hydrocortisone decreases lethality and inflammatory cytokine and nitric oxide production in rats challenged with B. anthracis cell wall peptidoglycan

BackgroundLethal B. anthracis infection produces high proinflammatory peptidoglycan (PGN) burdens in hosts. We investigated whether the lethality and inflammation anthrax PGN can produce are related.MethodsAt 6 h before and the start of 24 h anthrax PGN infusions, rats (n = 198) were treated with diluent (controls) or one of three IV-doses of either hydrocortisone (125, 12.5 or 1.25 mg/kg) or TNF-soluble receptor (TNFsr; 2000, 1000 or 333 μg/kg), non-selective and selective anti-inflammatory agents, respectively.ResultsCompared to controls, hydrocortisone 125 and 12.5 mg/kg each decreased 7-day lethality (p ≤ 0.004). Hydrocortisone 125 mg/kg decreased IL-1β, IL-6, TNFα, MCP, MIP-1α, MIP-2, RANTES and nitric oxide (NO) blood levels at 4 and 24 h after starting PGN (except MCP at 24 h). Each decrease was significant at 4 h (except MIP-1α that was significant at 24 h) (p ≤ 0.05). Similarly, hydrocortisone 12.5 mg/kg decreased each measure at 4, 24 and 48 h (except TNFα at 24 h and MIP-1α at 24 and 48 h and NO at 48 h). Decreases were significant for IL-6 and NO at 4 h and RANTES at 48 h (p ≤ 0.05). Hydrocortisone 1.25 mg/kg had non-significant effects. Each TNFsr dose decreased lethality but non-significantly. However, when doses were analyzed together, TNFsr decreased lethality in a potential trend (p = 0.16) and IL-6 and NO significantly at 4 h (p = 0.05).ConclusionsPeptidoglycan-stimulated host inflammation may contribute to B. anthracis lethality.

Repurposing Clinically Approved Drugs for the Treatment of Bacillus cereus, a Surrogate for Bacillus anthracis.

Of the numerous infectious diseases afflicting humans, anthrax disease, caused by Bacillus anthracis, poses a major threat in its virulence and lack of effective treatment. The currently lacking standards of care, as well as the lengthy drug approval process, demonstrate the pressing demand for treatment for B. anthracis infections. The present study screened 1586 clinically approved drugs in an attempt to identify repurposable compounds against B. cereus, a relative strain that shares many physical and genetic characteristics with B. anthracis. Our study yielded five drugs that successfully inhibited B. cereus growth: dichlorophen, oxiconazole, suloctidil, bithionol, and hexestrol. These drugs exhibited varying levels of efficacy in broad-spectrum experiments against several Gram-positive and Gram-negative bacterial strains, with hexestrol showing the greatest inhibition across all tested strains. Through tests for the efficacy of each drug on B. cereus, bithionol was the single most potent compound on both solid and liquid media and exhibited even greater eradication of B. cereus in combination with suloctidil on solid agar. This multifaceted in vitro study of approved drugs demonstrates the potential to repurpose these drugs as treatments for anthrax disease in a time-efficient manner to address a global health need.

Structure of S-layer protein Sap reveals a mechanism for therapeutic intervention in anthrax.

Anthrax is an ancient and deadly disease caused by the spore-forming bacterial pathogen Bacillus anthracis. At present, anthrax mostly affects wildlife and livestock, although it remains a concern for human public health-primarily for people who handle contaminated animal products and as a bioterrorism threat due to the high resilience of spores, a high fatality rate of cases and the lack of a civilian vaccination programme1,2. The cell surface of B. anthracis is covered by a protective paracrystalline monolayer-known as surface layer or S-layer-that is composed of the S-layer proteins Sap or EA1. Here, we generate nanobodies to inhibit the self-assembly of Sap, determine the structure of the Sap S-layer assembly domain (SapAD) and show that the disintegration of the S-layer attenuates the growth of B. anthracis and the pathology of anthrax in vivo. SapAD comprises six β-sandwich domains that fold and support the formation of S-layers independently of calcium. Sap-inhibitory nanobodies prevented the assembly of Sap and depolymerized existing Sap S-layers in vitro. In vivo, nanobody-mediated disruption of the Sap S-layer resulted in severe morphological defects and attenuated bacterial growth. Subcutaneous delivery of Sap inhibitory nanobodies cleared B. anthracis infection and prevented lethality in a mouse model of anthrax disease. These findings highlight disruption of S-layer integrity as a mechanism that has therapeutic potential in S-layer-carrying pathogens.