Showing papers on "Bacillus anthracis published in 2021"

Discrimination of Bacillus cereus Group Members by MALDI-TOF Mass Spectrometry.

Abstract: Matrix-Assisted Laser Desorption/Ionization Time Of Flight Mass Spectrometry (MALDI-TOF MS) technology is currently increasingly used in diagnostic laboratories as a cost effective, rapid and reliable routine technique for the identification and typing of microorganisms. In this study, we used MALDI-TOF MS to analyze a collection of 160 strains belonging to the Bacillus cereus group (57 B. anthracis, 49 B. cereus, 1 B. mycoides, 18 B. wiedmannii, 27 B. thuringiensis, 7 B. toyonensis and 1 B. weihenstephanensis) and to detect specific biomarkers which would allow an unequivocal identification. The Main Spectra Profiles (MSPs) were added to an in-house reference library, expanding the current commercial library which does not include B. toyonensis and B. wiedmannii mass spectra. The obtained mass spectra were statistically compared by Principal Component Analysis (PCA) that revealed seven different clusters. Moreover, for the identification purpose, were generated dedicate algorithms for a rapid and automatic detection of characteristic ion peaks after the mass spectra acquisition. The presence of specific biomarkers can be used to differentiate strains within the B. cereus group and to make a reliable identification of Bacillus anthracis, etiologic agent of anthrax, which is the most pathogenic and feared bacterium of the group. This could offer a critical time advantage for the diagnosis and for the clinical management of human anthrax even in case of bioterror attacks.

A CRISPR/Cas12a-based DNAzyme visualization system for rapid, non-electrically dependent detection of Bacillus anthracis.

Abstract: As next-generation pathogen detection methods, CRISPR-Cas-based detection methods can perform single-nucleotide polymorphism (SNP) level detection with high sensitivity and good specificity. They do not require any particular equipment, which opens up new possibilities for the accurate detection and identification of Bacillus anthracis. In this study, we developed a complete detection system for B. anthracis based on Cas12a. We used two chromosomally located SNP targets and two plasmid targets to identify B. anthracis with high accuracy. The CR5 target is completely new. The entire detection process can be completed within 90 min without electrical power and with single-copy level sensitivity. We also developed an unaided-eye visualization system based on G4-DNAzyme for use with our CRISPR-Cas12a detection system. This visualization system has good prospects for deployment in field-based point-of-care detection. We used the antisense nucleic acid CatG4R as the detection probe, which showed stronger resistance to interference from components of the solution. CatG4R can also be designed as an RNA molecule for adaptation to Cas13a detection, thereby broadening the scope of the detection system.

A whole genome-based gene-by-gene typing system for standardised high resolution strain typing of Bacillus anthracis

Abstract: Whole-genome sequencing (WGS) has been established for bacterial subtyping and is regularly used to study pathogen transmission, to investigate outbreaks, and to perform routine surveillance. Core-genome multilocus sequence typing (cgMLST) is a bacterial subtyping method that uses WGS data to provide a high-resolution strain characterization. This study aimed at developing a novel cgMLST scheme for Bacillus anthracis, a notorious pathogen that causes anthrax in livestock and humans worldwide. The scheme comprises 3,803 genes that were conserved in 57 B. anthracis genomes spanning the whole phylogeny. The scheme has been evaluated and applied to 584 genomes from 50 countries. On average, 99.5% of the cgMLST targets were detected. The cgMLST results confirmed the classical canonical single-nucleotide-polymorphism (SNP) grouping of B. anthracis into major clades and subclades. Genetic distances calculated based on cgMLST were comparable to distances from whole-genome-based SNP analysis with similar phylogenetic topology and comparable discriminatory power. Additionally, the application of the cgMLST scheme to anthrax outbreaks from Germany and Italy led to a definition of a cutoff threshold of five allele differences to trace epidemiologically linked strains for cluster typing and transmission analysis. Finally, the association of two clusters of B. anthracis with human cases of injectional anthrax in four European countries was confirmed using cgMLST. In summary, this study presents a novel cgMLST scheme that provides high-resolution strain genotyping for B. anthracis. This scheme can be used in parallel with SNP typing methods to facilitate rapid and harmonized interlaboratory comparisons, essential for global surveillance and outbreak analysis. The scheme is publicly available for application by users, including those with little bioinformatics knowledge.

Characterization of the UK anthrax vaccine and human immunogenicity.

Abstract: The manufacture of the UK Anthrax vaccine (AVP) focuses on the production of Protective Antigen (PA) from the Bacillus anthracis Sterne strain. Although used for decades, several of AVP’s fundament...

Two approaches for the stabilization of Bacillus anthracis recombinant protective antigen

Abstract: Anthrax is a zoonotic disease caused by the gram-positive spore-forming bacteria Bacillus anthracis. There is a need for safe, highly effective, long-term storage vaccine formulations for mass vacc...

Biosensors for the Detection of Bacillus anthracis

Abstract: ConspectusBacillus anthracis, present in two forms of vegetative cells and spores, is a pathogen that infects humans through contact with infected animals or contaminated animal products and is also maliciously used in terrorist acts. Therefore, a rapid and sensitive test for B. anthracis is necessary but challenging. The challenge comes from the following aspects: an accurate distinction of B. anthracis from other Bacillus species due to their high genomic similarity and the horizontal gene transfer between Bacillus members; direct detection of the B. anthracis spores without damaging them for component extraction to avoid the risk of spore atomization; and the rapid detections of B. anthracis in complex samples, such as soil and suspicious powders, without sample pretreatments and expensive large-scale equipment. Although culturing B. anthracis from samples is the conventional method for the detection of B. anthracis, it is time-consuming and the detection results would not be easy to interpret because many Bacillus species share similar phenotypic features such as a lack of motility and hemolysis, resistance to gamma phages, and so on. Intensive and extensive effort has been expended to develop reliable detection technologies, among which biosensors exhibit comprehensive advantages in terms of sensitivity, specificity, and portability. Here, we briefly review the research progress, providing highlights of the latest achievements and our own practice and experience. The contents can be summarized in three aspects: the discovery of detection targets, including genes, toxins, and other components; the creation of molecular recognition elements, such as monoclonal antibodies, single-chain antibody fragments, specific peptides, and aptamers; and the design and construction of biosensing systems by the integration of appropriate molecular recognition elements and transducer devices. These sensor devices have their own characteristics and different principles. For example, the surface plasmon resonance biosensor and quartz crystal microbalance biosensor are very sensitive, while the multiplex PCR-on-a-chip can detect multitargets. Biosensors for direct spore detection are highly recommended because they are not only fast but also avoid contamination from aerosol-containing spores. The introduction of nanotechnology has significantly improved the performance of biosensors. Superparamagnetic nanoparticles and phage-displayed gold nanoparticle ligand peptides have made the results of spore detection visible to the naked eye. Because of space constraints, many advanced biosensors for B. anthracis are not described in detail but are cited as references. Although biosensors provide a variety of options for various application scenarios, the challenges have not been fully addressed, which leaves room for the development of more advanced and practical B. anthracis detection means.

Ultrasensitive Detection of Bacillus anthracis by Real-Time PCR Targeting a Polymorphism in Multi-Copy 16S rRNA Genes and Their Transcripts.

Abstract: The anthrax pathogen Bacillus anthracis poses a significant threat to human health. Identification of B. anthracis is challenging because of the bacterium’s close genetic relationship to other Bacillus cereus group species. Thus, molecular detection is founded on species-specific PCR targeting single-copy genes. Here, we validated a previously recognized multi-copy target, a species-specific single nucleotide polymorphism (SNP) present in 2–5 copies in every B. anthracis genome analyzed. For this, a hydrolysis probe-based real-time PCR assay was developed and rigorously tested. The assay was specific as only B. anthracis DNA yielded positive results, was linear over 9 log10 units, and was sensitive with a limit of detection (LoD) of 2.9 copies/reaction. Though not exhibiting a lower LoD than established single-copy PCR targets (dhp61 or PL3), the higher copy number of the B. anthracis–specific 16S rRNA gene alleles afforded ≤2 unit lower threshold (Ct) values. To push the detection limit even further, the assay was adapted for reverse transcription PCR on 16S rRNA transcripts. This RT-PCR assay was also linear over 9 log10 units and was sensitive with an LoD of 6.3 copies/reaction. In a dilution series of experiments, the 16S RT-PCR assay achieved a thousand-fold higher sensitivity than the DNA-targeting assays. For molecular diagnostics, we recommend a real-time RT-PCR assay variant in which both DNA and RNA serve as templates (thus, no requirement for DNase treatment). This can at least provide results equaling the DNA-based implementation if no RNA is present but is superior even at the lowest residual rRNA concentrations.

AtxA-Controlled Small RNAs of Bacillus anthracis Virulence Plasmid pXO1 Regulate Gene Expression in trans.

Abstract: Small regulatory RNAs (sRNAs) are short transcripts that base-pair to mRNA targets or interact with regulatory proteins. sRNA function has been studied extensively in Gram-negative bacteria; comparatively less is known about sRNAs in Firmicutes. Here we investigate two sRNAs encoded by virulence plasmid pXO1 of Bacillus anthracis, the causative agent of anthrax. The sRNAs, named "XrrA and XrrB" (for pXO1-encoded regulatory RNA) are abundant and highly stable primary transcripts, whose expression is dependent upon AtxA, the master virulence regulator of B. anthracis. sRNA levels are highest during culture conditions that promote AtxA expression and activity, and sRNA levels are unaltered in Hfq RNA chaperone null-mutants. Comparison of the transcriptome of a virulent Ames-derived strain to the transcriptome of isogenic sRNA-null mutants revealed multiple 4.0- to >100-fold differences in gene expression. Most regulatory effects were associated with XrrA, although regulation of some transcripts suggests functional overlap between the XrrA and XrrB. Many sRNA-regulated targets were chromosome genes associated with branched-chain amino acid metabolism, proteolysis, and transmembrane transport. Finally, in a mouse model for systemic anthrax, the lungs and livers of animals infected with xrrA-null mutants had a small reduction in bacterial burden, suggesting a role for XrrA in B. anthracis pathogenesis.

Spore-Associated Proteins Involved in c-di-GMP Synthesis and Degradation of Bacillus anthracis .

Abstract: Bis-(3'-5')-cyclic-dimeric GMP (c-di-GMP) is an important bacterial regulatory signaling molecule affecting biofilm formation, toxin production, motility, and virulence. The genome of Bacillus anthracis, the causative agent of anthrax, is predicted to encode ten putative GGDEF/EAL/HD-GYP-domain containing proteins. Heterologous expression in Bacillus subtilis hosts indicated that there are five active GGDEF domain-containing proteins and four active EAL or HD-GYP domain-containing proteins. Using an mCherry gene fusion-Western blotting approach, the expression of the c-di-GMP-associated proteins was observed throughout the in vitro life cycle. Of the six c-di-GMP-associated proteins found to be present in sporulating cells, four (CdgA, CdgB, CdgD, and CdgG) contain active GGDEF domains. The six proteins expressed in sporulating cells are retained in spores in a CotE-independent manner and thus are not likely to be localized to the exosporium layer of the spores. Individual deletion mutations involving the nine GGDEF/EAL protein-encoding genes and one HD-GYP protein-encoding gene did not affect sporulation efficiency, the attachment of the exosporium glycoprotein BclA, or biofilm production. Notably, expression of anthrax toxin was not affected by deletion of any of the cdg determinants. Three determinants encoding proteins with active GGDEF domains were found to affect germination kinetics. This study reveals a spore association of cyclic-di-GMP regulatory proteins and a likely role for these proteins in the biology of the B. anthracis spore. IMPORTANCE The genus Bacillus is composed of Gram-positive, rod shaped, soil-dwelling bacteria. As a mechanism for survival in the harsh conditions in soil, the organisms undergo sporulation, and the resulting spores permit the organisms to survive harsh environmental conditions. Although most species are saprophytes, Bacillus cereus and Bacillus anthracis are human pathogens and Bacillus thuringiensis is an insect pathogen. The bacterial c-di-GMP regulatory system is an important control system affecting motility, biofilm formation, and toxin production. The role of c-di-GMP has been studied in the spore-forming bacilli Bacillus subtilis, Bacillus amyloliquefaciens, B. cereus, and B. thuringiensis. However, this regulatory system has not heretofore been examined in the high-consequence zoonotic pathogen of this genus, B. anthracis.

An Estimate of Global Anthrax Prevalence in Livestock: A Meta-analysis.

Abstract: Background and aim Anthrax, caused by the soil-borne spore-forming bacteria called Bacillus anthracis, is a zoonotic disease that persists worldwide in livestock and wildlife and infects humans. It is a great hazard to livestock; henceforth, evaluating the global concerns about the disease occurrence in livestock is essential. This study was conducted to estimate the global prevalence of anthrax and predict high-risk regions, which could be an input to veterinarians to take necessary steps to control and avoid the disease. Materials and methods A literature review was performed using online databases, namely, PubMed, Google Scholar, Scopus, Biomed Central, and Science Direct, to extract relevant publications worldwide between 1992 and 2020. Initially, 174 articles were selected, and after scrutinizing, 24 articles reporting the prevalence of anthrax were found to be adequate for the final meta-analysis. The statistical study was accompanied by employing fixed effects and random effects models using R. Results The pooled prevalence of anthrax globally was 28% (95% confidence interval, 26-30%) from 2452 samples through the fixed effects model. Continent-wise subgroup analysis through the random effects model revealed that the pooled prevalence of anthrax was highest in Africa (29%) and least in North America (21%). Conclusion In these publications, anthrax causes economic loss to farmers and, thus, to the world. Hence, controlling anthrax infections in high-risk regions are essential by implementing appropriate control measures to decrease the effect of the disease, thereby reducing economic loss.

Sex-Specific Elk Resource Selection during the Anthrax Risk Period

Abstract: Anthrax, caused by the spore-forming bacterium Bacillus anthracis, is a zoonosis affecting animals and humans globally. In the United States, anthrax outbreaks occur in wildlife and livestock, with frequent outbreaks in native and exotic wildlife species in Texas, livestock outbreaks in the Dakotas, and sporadic mixed outbreaks in Montana. Understanding where pathogen and host habitat selection overlap is essential for anthrax management. Resource selection and habitat use of ungulates may be sex-specific and lead to differential anthrax exposure risks across the landscape for males and females. We evaluated female elk (Cervus canadensis) resource selection in the same study areas as male elk in a previous anthrax risk study to identify risk of anthrax transmission to females and compare transmission risk between females and males. We developed a generalized linear mixed-effect model to estimate resource selection for female elk in southwest Montana during the June to August anthrax transmission risk period. We then predicted habitat selection of female and male elk across the study area and compared selection with the distribution of anthrax risk to identify spatial distributions of potential anthrax exposure for the male and female elk. Female and male elk selected different resources during the anthrax risk period, which resulted in different anthrax exposure areas for females and males. The sex-specific resource selection and habitat use could infer different areas of risk for anthrax transmission, which can improve anthrax and wildlife management and have important public health and economic implications.

Enzyme-Linked Phage Receptor Binding Protein Assays (ELPRA) Enable Identification of Bacillus anthracis Colonies.

Abstract: Bacteriophage receptor binding proteins (RBPs) are employed by viruses to recognize specific surface structures on bacterial host cells. Recombinant RBPs have been utilized for detection of several pathogens, typically as fusions with reporter enzymes or fluorescent proteins. Identification of Bacillus anthracis, the etiological agent of anthrax, can be difficult because of the bacterium's close relationship with other species of the Bacillus cereussensu lato group. Here, we facilitated the identification of B. anthracis using two implementations of enzyme-linked phage receptor binding protein assays (ELPRA). We developed a single-tube centrifugation assay simplifying the rapid analysis of suspect colonies. A second assay enables identification of suspect colonies from mixed overgrown solid (agar) media derived from the complex matrix soil. Thus, these tests identified vegetative cells of B. anthracis with little processing time and may support or confirm pathogen detection by molecular methods such as polymerase chain reaction.

Effectiveness of calcium hypochlorite, quaternary ammonium compounds, and sodium hypochlorite in eliminating vegetative cells and spores of Bacillus anthracis surrogate

Abstract: BACKGROUND The spore-forming bacterium Bacillus anthracis causes anthrax, an often-fatal infection in animals. Therefore, a rapid and reliable strategy to decontaminate areas, humans, and livestock from B. anthracis is very critical. OBJECTIVES The aim of this study was performed to evaluate the efficacy of sodium hypochlorite, calcium hypochlorite, and quaternary ammonium compound (QAC) sanitizers, which are commonly used in the food industry, to inhibit spores and vegetative cells of B. anthracis surrogate. METHODS We evaluated the efficacy of sodium hypochlorite, calcium hypochlorite, and a QAC in inhibiting vegetative cells and spores of a B. anthracis surrogate. We treated a 0.1-mL vegetative cell culture or spore solution with 10 mL sanitizer. The samples were serially diluted and cultured. RESULTS We found that 50 ppm sodium hypochlorite (pH 7), 1 ppm calcium hypochlorite, and 1 ppm QAC completely eliminated the cells in vegetative state. Exposure to 3,000 ppm sodium hypochlorite (pH 7) and 300 ppm calcium hypochlorite significantly eliminated the bacterial spores; however, 50,000 ppm QAC could not eliminate all spores. CONCLUSIONS Calcium hypochlorite and QAC showed better performance than sodium hypochlorite in completely eliminating vegetative cells of B. anthracis surrogate. QAC was ineffective against spores of the B. anthracis surrogate. Among the three commercial disinfectants tested, calcium hypochlorite most effectively eliminated both B. anthracis vegetative cells and spores.

Further Insights into the Toxicity of Bacillus cytotoxicus Based on Toxin Gene Profiling and Vero Cell Cytotoxicity Assays.

Abstract: Bacillus cytotoxicus belongs to the Bacillus cereus group that also comprises the foodborne pathogen Bacillus cereus sensu stricto, Bacillus anthracis causing anthrax, as well as the biopesticide Bacillus thuringiensis. The first B. cytotoxicus was isolated in the context of a severe food poisoning outbreak leading to fatal cases of diarrheal disease. Subsequent characterization of the outbreak strain led to the conclusion that this Bacillus strain was highly cytotoxic and eventually resulted in the description of a novel species, whose name reflects the observed toxicity: B. cytotoxicus. However, only a few isolates of this species have been characterized with regard to their cytotoxic potential and the role of B. cytotoxicus as a causative agent of food poisoning remains largely unclear. Hence, the aim of this study was to gain further insights into the toxicity of B. cytotoxicus. To this end, 19 isolates were obtained from mashed potato powders and characterized by toxin gene profiling and Vero cell cytotoxicity assays. All isolates harbored the cytK1 (cytotoxin K1) gene and species-specific variants of the nhe (non-hemolytic enterotoxin) gene. The isolates exhibited low or no toxicity towards Vero cells. Thus, this study indicates that the cytotoxic potential of B. cytotoxicus may be potentially lower than initially assumed.

Bacillus anthracis chain length, a virulence determinant, is regulated by membrane localized serine/threonine protein kinase PrkC

Abstract: Anthrax is a zoonotic disease caused by Bacillus anthracis, a spore-forming pathogen that displays a chaining phenotype. It has been reported that the chaining phenotype acts as a virulence factor in B. anthracis In this study, we identify a serine/threonine protein kinase of B. anthracis, PrkC, the only kinase localized at the bacteria-host interface, as a determinant of B. anthracis chain length. In vitro, prkC disruption strain (BAS ΔprkC) grew as shorter chains throughout the bacterial growth cycle. A comparative analysis between the parent strain and BAS ΔprkC indicated that the levels of proteins, BslO and Sap, associated with the regulation of the bacterial chain length, were upregulated in BAS ΔprkC BslO is a septal murein hydrolase that catalyzes daughter cell separation and Sap is an S-layer structural protein required for the septal localization of BslO. PrkC disruption also has a significant effect on bacterial growth, cell wall thickness, and septa formation. Upregulation of ftsZ in BAS ΔprkC was also observed. Altogether, our results indicate that PrkC is required for maintaining optimum growth, cell wall homeostasis and most importantly - for the maintenance of the chaining phenotype.IMPORTANCEChaining phenotype acts as a virulence factor in Bacillus anthracis This is the first study that identifies a 'signal transduction protein' with an ability to regulate the chaining phenotype in Bacillus anthracis We show that the disruption of the lone surface-localized serine/threonine protein kinase, PrkC, leads to the shortening of the bacterial chains. We report upregulation of the de-chaining proteins in the PrkC disruption strain. Apart from this, we also report for the first time that PrkC disruption results in an attenuated cell growth, a decrease in the cell wall thickness and aberrant cell septa formation during the logarithmic phase of growth - a growth phase where PrkC is expressed maximally.

Antibiotic resistance modifying ability of phytoextracts in anthrax biological agent Bacillus anthracis and emerging superbugs: a review of synergistic mechanisms

Abstract: BACKGROUND AND OBJECTIVES The chemotherapeutic management of infections has become challenging due to the global emergence of antibiotic resistant pathogenic bacteria. The recent expansion of studies on plant-derived natural products has lead to the discovery of a plethora of phytochemicals with the potential to combat bacterial drug resistance via various mechanisms of action. This review paper summarizes the primary antibiotic resistance mechanisms of bacteria and also discusses the antibiotic-potentiating ability of phytoextracts and various classes of isolated phytochemicals in reversing antibiotic resistance in anthrax agent Bacillus anthracis and emerging superbug bacteria. METHODS Growth inhibitory indices and fractional inhibitory concentration index were applied to evaluate the in vitro synergistic activity of phytoextract-antibiotic combinations in general. FINDINGS A number of studies have indicated that plant-derived natural compounds are capable of significantly reducing the minimum inhibitory concentration of standard antibiotics by altering drug-resistance mechanisms of B. anthracis and other superbug infection causing bacteria. Phytochemical compounds allicin, oleanolic acid, epigallocatechin gallate and curcumin and Jatropha curcas extracts were exceptional synergistic potentiators of various standard antibiotics. CONCLUSION Considering these facts, phytochemicals represents a valuable and novel source of bioactive compounds with potent antibiotic synergism to modulate bacterial drug-resistance.

Construction of a bivalent vaccine against anthrax and smallpox using the attenuated vaccinia virus KVAC103

Abstract: Anthrax and smallpox are high-risk infectious diseases, and considered as potential agents for bioterrorism. To develop an effective countermeasure for these diseases, we constructed a bivalent vaccine against both anthrax and smallpox by integrating a gene encoding protective antigen (PA) of Bacillus anthracis to the genome of the attenuated vaccinia virus strain, KVAC103. Immunization with this bivalent vaccine induced antibodies against both PA and vaccinia virus in a mouse model. We also observed that the efficacy of this vaccine can be enhanced by combined immunization with immunoadjuvant-expressing KVAC103. Mouse groups co-immunized with PA-expressing KVAC103 and either interleukin-15 (IL-15) or cholera toxin subunit A (CTA1)-expressing KVAC103 showed increased anti-PA IgG titer and survival rate against B. anthracis spore challenge compared to the group immunized with PA-expressing KVAC103 alone. We demonstrated that the attenuated smallpox vaccine KVAC103 is an available platform for a multivalent vaccine and co-immunization of immunoadjuvants can improve vaccine performance.

Application of CRISPR/Cas9 System for Plasmid Elimination and Bacterial Killing of Bacillus cereus Group Strains

Abstract: The CRISPR-Cas system has been widely applied in prokaryotic genome editing with its high efficiency and easy operation. We constructed some "scissors plasmids" via using the temperature-sensitive pJOE8999 shuttle plasmid, which carry the different 20nt (N20) guiding the Cas9 nuclease as a scissors to break the target DNA. We successfully used scissors plasmids to eliminate native plasmids from Bacillus anthracis and Bacillus cereus, and specifically killed B. anthracis. When curing pXO1 and pXO2 virulence plasmids from B. anthracis A16PI2 and A16Q1, respectively, we found that the plasmid elimination percentage was slightly higher when the sgRNA targeted the replication initiation region (96-100%), rather than the non-replication initiation region (88-92%). We also tried using a mixture of two scissors plasmids to simultaneously eliminate pXO1 and pXO2 plasmids from B. anthracis, and the single and double plasmid-cured rates were 29 and 14%, respectively. To our surprise, when we used the scissor plasmid containing two tandem sgRNAs to cure the target plasmids pXO1 and pXO2 from wild strain B. anthracis A16 simultaneously, only the second sgRNA could guide Cas9 to cleave the target plasmid with high efficiency, while the first sgRNA didn't work in all the experiments we designed. When we used the CRISPR/cas9 system to eliminate the pCE1 mega-virulence plasmid from B. cereus BC307 by simply changing the sgRNA, we also obtained a plasmid-cured isogenic strain at a very high elimination rate (69%). The sterilization efficiency of B. anthracis was about 93%, which is similar to the efficiency of plasmid curing, and there was no significant difference in the efficiency of among the scissors plasmids containing single sgRNA, targeting multi-sites, or single-site targeting and the two tandem sgRNA. This simple and effective curing method, which is applicable to B. cereus group strains, provides a new way to study these bacteria and their virulence profiles.

Complement C5 inhibition protects against hemolytic anemia and acute kidney injury in anthrax peptidoglycan-induced sepsis in baboons.

Abstract: Late-stage anthrax infections are characterized by dysregulated immune responses and hematogenous spread of Bacillus anthracis, leading to extreme bacteremia, sepsis, multiple organ failure, and, ultimately, death. Despite the bacterium being nonhemolytic, some fulminant anthrax patients develop a secondary atypical hemolytic uremic syndrome (aHUS) through unknown mechanisms. We recapitulated the pathology in baboons challenged with cell wall peptidoglycan (PGN), a polymeric, pathogen-associated molecular pattern responsible for the hemostatic dysregulation in anthrax sepsis. Similar to aHUS anthrax patients, PGN induces an initial hematocrit elevation followed by progressive hemolytic anemia and associated renal failure. Etiologically, PGN induces erythrolysis through direct excessive activation of all three complement pathways. Blunting terminal complement activation with a C5 neutralizing peptide prevented the progressive deposition of membrane attack complexes on red blood cells (RBC) and subsequent intravascular hemolysis, heme cytotoxicity, and acute kidney injury. Importantly, C5 neutralization did not prevent immune recognition of PGN and shifted the systemic inflammatory responses, consistent with improved survival in sepsis. Whereas PGN-induced hemostatic dysregulation was unchanged, C5 inhibition augmented fibrinolysis and improved the thromboischemic resolution. Overall, our study identifies PGN-driven complement activation as the pathologic mechanism underlying hemolytic anemia in anthrax and likely other gram-positive infections in which PGN is abundantly represented. Neutralization of terminal complement reactions reduces the hemolytic uremic pathology induced by PGN and could alleviate heme cytotoxicity and its associated kidney failure in gram-positive infections.

Loss of the ClpXP Protease Leads to Decreased Resistance to Cell-Envelope Targeting Antimicrobials in Bacillus anthracis Sterne.

Abstract: The ClpX ATPase is critical for resistance to cell envelope targeting antibiotics in Bacillus anthracis, however, it is unclear whether this is due to its function as an independent chaperone or as part of the ClpXP protease. In this study, we demonstrate that antibiotic resistance is due to formation of the ClpXP protease through construction of a ClpX complementation plasmid that is unable to interact with ClpP. Additionally, we genetically disrupted both clpP genes, clpP1 and clpP2, found in B. anthracis Sterne and find that the loss of either increases susceptibility to cell envelope targeting antimicrobials, although neither has as strong of a phenotype as loss of clpX and neither clpP gene is essential for virulence in a G. mellonella model of infection. Lastly, we looked at changes to cell envelope morphology that could contribute to increased antibiotic sensitivity. We find no difference in cell charge or cell lysis, although we do see increased hydrophobicity in the ΔclpX strain, decreased cellular density and slightly thinner cells walls. We also see significant cell division defects in ΔclpX, although only when cells are grown in the mammalian cell culture medium, RPMI. We conclude that the intrinsic resistance of B. anthracis to cell wall active antimicrobials is dependent on formation of the ClpXP protease and that this could be due, at least in part, to the role of ClpX in regulating cell envelope morphology.

Direct Regulons of AtxA, the Master Virulence Regulator of Bacillus anthracis.

Abstract: AtxA, the master virulence regulator of Bacillus anthracis, regulates the expression of three toxins and genes for capsule formation that are required for the pathogenicity of B. anthracis. Recent transcriptome analyses showed that AtxA affects a large number of genes on the chromosome and plasmids, suggesting a role as a global regulator. However, information on genes directly regulated by AtxA is scarce. In this work, we conducted genome-wide analyses and cataloged the binding sites of AtxA in vivo and transcription start sites on the B. anthracis genome. By integrating these results, we detected eight genes as direct regulons of AtxA. These consisted of five protein-coding genes, including two of the three toxin genes, and three genes encoding the small RNAs XrrA and XrrB and a newly discovered 95-nucleotide small RNA, XrrC. Transcriptomes from single-knockout mutants of these small RNAs revealed changes in the transcription levels of genes related to the aerobic electron transport chain, heme biosynthesis, and amino acid metabolism, suggesting their function for the control of cell physiology. These results reveal the first layer of the gene regulatory network for the pathogenicity of B. anthracis and provide a data set for the further study of the genomics and genetics of B. anthracis. IMPORTANCE Bacillus anthracis is the Gram-positive bacterial species that causes anthrax. Anthrax is still prevalent in countries mainly in Asia and Africa, where it causes economic damage and remains a public health issue. The mechanism of pathogenicity is mainly explained by the three toxin proteins expressed from the pXO1 plasmid and by proteins involved in capsule formation expressed from the pXO2 plasmid. AtxA is a protein expressed from the pXO1 plasmid that is known to upregulate genes involved in toxin production and capsule formation and is thus considered the master virulence regulator of B. anthracis. Therefore, understanding the detailed mechanism of gene regulation is important for the control of anthrax. The significance of this work lies in the identification of genes that are directly regulated by AtxA via genome-wide analyses. The results reveal the first layer of the gene regulatory network for the pathogenicity of B. anthracis and provide useful resources for a further understanding of B. anthracis.

A serological survey of Bacillus anthracis reveals widespread exposure to the pathogen in free-range and captive lions in Zimbabwe.

Abstract: Numerous unknown factors influence anthrax epidemiology in multi-host systems, especially at wildlife/livestock/human interfaces. Serology tests for anti-anthrax antibodies in carnivores are useful tools in identifying the presence or absence of Bacillus anthracis in a range. These were employed to ascertain whether the disease pattern followed the recognized high- and low-risk anthrax zonation in Zimbabwe and also to establish whether anthrax was absent from Hwange National Park in which there have been no reported outbreaks. African lions (Panthera leo) (n = 114) drawn from free-range protected areas and captive game parks located in recognized high- and low-risk zones across Zimbabwe were tested for antibodies to anthrax PA antigen using the ELISA immunoassay. A random selection of 27 lion sera samples comprising 17 seropositive and 10 seronegative sera was further tested in the species- independent toxin neutralization assay (TNA) in order to validate the former as a surveillance tool for anthrax in African lions. Using the ELISA-PA immunoassay, 21.9% (25/114) of the lions tested positive for antibodies to anthrax. Seropositivity was recorded in all study areas, and there was no significant difference (p = .852) in seropositivity between lions in high- and low-risk anthrax zones. Also, there was no significant difference (McNemar's chi-square test = 0.9, p = .343) in the proportion of lions testing positive to anti-PA anthrax antibodies on ELISA-PA immunoassay compared with the TNA, with fair agreement between the two tests [kappa (K) statistic = 0.30; 0.08 < K<0.613]. Results of this study indicate that anthrax could be more widespread than 42 currently realized in Zimbabwe, and present in recognized high- and low-risk zones, including 43 where it has not been reported in over 20 years such as Hwange National Park. This is also the 44 first report documenting the presence of anthrax lethal toxin-neutralizing antibodies in naturally 45 infected carnivores, further confirming exposure to B. anthracis. The research results point to a 46 need for revisiting the currently recognized anthrax risk zones in Zimbabwe. This should be based 47 on improved surveillance of the disease in both wild and domestic animals for better understanding and control of the disease.

Ultrasensitive detection of Bacillus anthracis by real time PCR targeting a polymorphism in multi-copy 16S rRNA genes and their transcripts

Abstract: The anthrax pathogen Bacillus anthracis poses a significant threat to human health. Identification of B. anthracis is challenging because of the bacterium’s close genetic relationship to other Bacillus cereus group species. Thus, molecular detection is founded on species-specific PCR targeting single-copy genes. Here, we validated a previously recognized multi-copy target, a species-specific SNP present in 2-5 copies in every B. anthracis genome analyzed. For this, a hydrolysis probe-based real time PCR assay was developed and rigorously tested. The assay was specific as only B. anthracis DNA yielded positive results, was linear over 9 log10 units and was sensitive with a limit of detection (LoD) of 2.9 copies/reaction. Though not exhibiting a lower LoD than established single copy PCR targets (dhp61 or PL3), the higher copy number of the B. anthracis–specific 16S rRNA gene allele afforded ≤2 unit lower threshold (Ct) values. To push the detection limit even further, the assay was adapted for reverse transcription PCR on 16S rRNA transcripts. This RT-PCR assay was also linear over 9 log10 units and was sensitive with a LoD of 6.3 copies/reaction. In a dilution-series of experiments, the 16S RT-PCR assay achieved a thousand-fold higher sensitivity than the DNA-targeting assays. For molecular diagnostics, we recommend a real time RT-PCR assay variant in which both DNA and RNA serve as templates (thus, no requirement for DNase treatment). This will at least provide results equaling the DNA-based implementation if no RNA is present but will be superior even at the lowest residual rRNA concentrations.

A Suggested Diagnostic Approach for Sporadic Anthrax in Cattle to Protect Public Health

Abstract: The repeated occurrence of anthrax in grazing animals should be a reminder of a widespread presence of Bacillus anthracis spores in the environment. Its rapid diagnosis is critical to protect public health. Here, we report a case of anthrax in cattle that was investigated using conventional and molecular methods. In 2015, six cows suddenly died within three days and the number of dead animals increased to a total of 12 within two weeks. At necropsy, anthrax was suspected. Therefore, spleen tissue samples were collected (from 6/12 animals) and laboratory tests (microscopy, cultivation, and real-time PCR) performed. The results of tissue staining for microscopy and cultivation were in congruence, while B. anthracis real-time PCR outperformed both. Spleen tissues from all six animals were real-time PCR-positive, while B. anthracis was successfully cultivated and detected by microscopy from the spleen of only three animals. Additionally, the ear tissue from another (1/12) cow tested positive by real-time PCR, supporting the suitability of ear clippings for molecular confirmation of B. anthracis. Genotyping of the isolates using multiple-locus variable-number tandem repeat analysis (MLVA) revealed a common source of infection as all three typed isolates had an indistinguishable MLVA genotype, which has not been observed previously in Europe. The results indicate that molecular testing should be selected as the first-line tool for confirming anthrax outbreaks in animals to ensure timely protection of public health.

Poly-γ-Glutamic Acid Encapsulation of Bacillus anthracis Inhibits Human Dendritic Cell Responses

Abstract: The capsule of Bacillus anthracis is composed of a d isomer poly-γ-glutamic acid polymer, which is especially nonstimulatory to dendritic cells, even more so than similar mixed d, l isomer polymers from nonpathogenic Bacillus species. Capsule is an essential virulence factor for B. anthracis, protecting the bacilli from phagocytosis by innate immune cells. In this study, we demonstrate that encapsulation provides a further pathogenic advantage by shielding more inflammatory Ags on the bacillus surface, thereby reducing dendritic cell responses. We exposed human immature dendritic cells (DCs) to increasing multiplicities of infection (MOIs) of killed B. anthracis bacilli from the fully encapsulated wild-type Ames strain (WT) and an isogenic capsule-deficient strain (capA mutant). Both strains elicited robust cytokine responses, but IL-23, TNF-α, and IL-10 were significantly reduced in response to the encapsulated WT compared with capA mutant up to an MOI of 15. capA mutant bacilli could induce phenotypic maturation of immature DCs with upregulation of MHC classes I and II, CD83, and CCR7 at an MOI of 3.75, whereas encapsulated WT bacilli still did not induce significant upregulation of MHC classes I and II at an MOI of 15. DCs exposed to capA mutant bacilli (MOI 3.75) exhibited CCR7-dependent chemotaxis that was comparable to that of LPS-stimulated controls, whereas DCs exposed to encapsulated WT bacilli exhibited significantly less chemotaxis. We conclude that capsule shields more inflammatory surface Ags, delaying development of an adaptive immune response by reducing TNF-α, thereby inhibiting DC maturation.