Dual excitation channel ratiometric fluorescent probes for visual and fluorescent detection of anthrax spore biomarker and tetracycline hydrochloride.

Long-term and excessive use of tetracycline hydrochloride (TC) can lead to its accumulation in the environment, which can cause water contamination, bacterial resistance, and food safety problems. 2,6-Pyridine dicarboxylic acid (DPA) is a major biomarker of Bacillus anthracis spores, and its rapid and sensitive detection is of great significance for disease prevention and counter-terrorism. A bifunctional ratiometric fluorescent nanoprobe has been fabricated to detect DPA and TC. 3,5-dicarboxyphenylboronic acid (BOP) was intercalated into layered europium hydroxide (LEuH) by the ion-exchange method and exfoliated into nanosheets as a fluorescent nanoprobe (PNP). DPA and TC could significantly enhance the red fluorescence of Eu3+ through the antenna effect under different excitation wavelengths, while the fluorescence of BOP can be used as a reference based on the constant emission intensity, realizing ratiometric detection. A low limit of detection (LOD) for the target (DPA: 9.7 nM, TC: 21.9 nM) can be achieved. In addition, visual detection of DPA and TC was realized using color recognition software based on the obvious color changes. This is the first ratiometric fluorescent nanoprobe based on layered rare-earth hydroxide (LRH) for the detection of DPA and TC simultaneously, which opens new ideas in the design of multifunctional probes.

Backtracking: Improved methods for identifying the source of a deliberate release of Bacillus anthracis from the temporal and spatial distribution of cases.

Reverse epidemiology is a mathematical modelling tool used to ascertain information about the source of a pathogen, given the spatial and temporal distribution of cases, hospitalisations and deaths. In the context of a deliberately released pathogen, such as Bacillus anthracis (the disease-causing organism of anthrax), this can allow responders to quickly identify the location and timing of the release, as well as other factors such as the strength of the release, and the realized wind speed and direction at release. These estimates can then be used to parameterise a predictive mechanistic model, allowing for estimation of the potential scale of the release, and to optimise the distribution of prophylaxis. In this paper we present two novel approaches to reverse epidemiology, and demonstrate their utility in responding to a simulated deliberate release of B. anthracis in ten locations in the UK and compare these to the standard grid-search approach. The two methods-a modified MCMC and a Recurrent Convolutional Neural Network-are able to identify the source location and timing of the release with significantly better accuracy compared to the grid-search approach. Further, the neural network method is able to do inference on new data significantly quicker than either the grid-search or novel MCMC methods, allowing for rapid deployment in time-sensitive outbreaks.

Risk perceptions and preventive preparedness toward anthrax re-emergence within the lens of one health in Northcentral Nigeria.

Anthrax has re-emerged in domestic ruminants in Nigeria with public health concerns. This study assessed stakeholders' perceptions and preventive preparedness toward further resurgence and spread at the human-animal-environment interface. A questionnaire-based cross-sectional study was conducted in Nigeria. Descriptive and analytical statistical analyses were performed at 95% confidence levels. All the 384 recruited stakeholders responded. The majority (96.2%) of animal health practitioners (AHPs) and 56.7% of pastoralists were aware of the recent anthrax re-emergence in Nigeria (p < 0.001). Noteworthy, 88.5% of the AHPs and 32.2% of pastoralists mentioned that anthrax has an environmental component in its transmission to humans and animals. From the environmental perspective, 87.7% of AHPs and 24.0% of pastoralists significantly perceived that soil and aerosol contamination with anthrax spores are highly plausible explanation routes for its re-emergence. Extreme weather events (high rainfall, flooding, winds, and drought) (p = 0.001); grazing of livestock on pastures grown on contaminated soil (p < 0.001), transboundary movement and trade of animals (p = 0.001); introduction of new animals into the herds without quarantine (p = 0.001); and bioterrorism (p < 0.001) were more likely to influence the re-emergence and spread of anthrax. To tackle gaps in knowledge and risk perceptions, and address the socio-economic and anthropogenic drivers, cooperation and collaborations through the lens of the One Health approach are needed. The partnership will promote an integrated disease surveillance system from planning to implementation for the realization of elimination or reduction of the burden of anthrax and other zoonoses in Nigeria and contribute to achieving food safety, food security, and public and ecosystem health.

Pan-genome analysis reveals novel chromosomal markers for multiplex PCR-based specific detection of Bacillus anthracis.

BACKGROUND: Bacillus anthracis is a highly pathogenic bacterium that can cause lethal infection in animals and humans, making it a significant concern as a pathogen and biological agent. Consequently, accurate diagnosis of B. anthracis is critically important for public health. However, the identification of specific marker genes encoded in the B. anthracis chromosome is challenging due to the genetic similarity it shares with B. cereus and B. thuringiensis. METHODS: The complete genomes of B. anthracis, B. cereus, B. thuringiensis, and B. weihenstephanensis were de novo annotated with Prokka, and these annotations were used by Roary to produce the pan-genome. B. anthracis exclusive genes were identified by Perl script, and their specificity was examined by nucleotide BLAST search. A local BLAST alignment was performed to confirm the presence of the identified genes across various B. anthracis strains. Multiplex polymerase chain reactions (PCR) were established based on the identified genes. RESULT: The distribution of genes among 151 whole-genome sequences exhibited three distinct major patterns, depending on the bacterial species and strains. Further comparative analysis between the three groups uncovered thirty chromosome-encoded genes exclusively present in B. anthracis strains. Of these, twenty were found in known lambda prophage regions, and ten were in previously undefined region of the chromosome. We established three distinct multiplex PCRs for the specific detection of B. anthracis by utilizing three of the identified genes, BA1698, BA5354, and BA5361. CONCLUSION: The study identified thirty chromosome-encoded genes specific to B. anthracis, encompassing previously described genes in known lambda prophage regions and nine newly discovered genes from an undefined gene region to the best of our knowledge. Three multiplex PCR assays offer an accurate and reliable alternative method for detecting B. anthracis. Furthermore, these genetic markers have value in anthrax vaccine development, and understanding the pathogenicity of B. anthracis.

Omadacycline is active in vitro and in vivo against ciprofloxacin-resistant Bacillus anthracis.

Bacillus anthracis, the causative agent of anthrax, is among the most likely bacterial pathogens to be used in a biological attack. Inhalation anthrax is a serious, life-threatening form of infection, and the mortality from acute inhaled anthrax can approach 100% if not treated early and aggressively. Food and Drug Administration-approved antibiotics indicated for post-exposure prophylaxis (PEP) or treatment of anthrax are limited. This study assessed the in vitro activity and in vivo efficacy of omadacycline and comparators against clinical isolates of B. anthracis, including a ciprofloxacin-resistant isolate. Minimum inhibitory concentrations (MICs) of omadacycline, ciprofloxacin, and doxycycline were determined against animal and human clinical isolates of B. anthracis, including the ciprofloxacin-resistant Ames strain BACr4-2. Mice were challenged with aerosolized BACr4-2 spores, and survival was monitored for 28 days post-challenge. Treatment was initiated 24 h after aerosol challenge and administered for 14 days. Omadacycline demonstrated in vitro activity against 53 B. anthracis isolates with an MIC range of ≤0.008-0.25 µg/mL, and an MIC50/MIC90 of 0.015/0.03 µg/mL. Consistent with this, omadacycline demonstrated in vivo efficacy in a PEP mouse model of inhalation anthrax caused by the Ames BACr4-2 ciprofloxacin-resistant B. anthracis isolate. Omadacycline treatment significantly increased survival compared with the vehicle control group and the ciprofloxacin treatment group. As antibiotic resistance rates continue to rise worldwide, omadacycline may offer an alternative PEP or treatment option against inhalation anthrax, including anthrax caused by antibiotic-resistant B. anthracis.

Development of an amperometric biosensor for the detection of Bacillus anthracis spores.

INTRODUCTION: Due to most likely use of Bacillus anthracis in biological terrorism agents, the rapid and sensitive detection of its spores is crucial in both taking prophylactic measures and proper treatment. This study aimed to develop an amperometric electrochemical immunosensor for the detection of B. anthracis spores. METHODS: A new amperometric biosensor was designed using a combination of magnetic beads and multiplex screen-printed electrodes. This method measures changes in current intensity resulting from oxidation and reduction in the working electrode directly to spore concentrations. RESULTS: A standard curve was formed to test the number of live spores between 2 × 102-2 × 104 spores/ml concentrations. LOD and LOQ values were found to be 92 and 272 spores/ml, respectively. No cross-reactions were seen for Bacillus subtilis, Bacillus cereus and Bacillus thuringiencis spores. CONCLUSIONS: It is shown that the designed Anthrax immunosensor has high sensitivity and selectivity with rapid detection results.

A thirty-year time series analyses identifies coherence between oscillations in Anthrax outbreaks and El Niño in Karnataka, India.

Anthrax is an economically important zoonotic disease affecting both livestock and humans. The disease is caused by a spore forming bacterium, Bacillus anthracis, and is considered endemic to the state of Karnataka, India. It is critical to quantify the role of climatic factors in determining the temporal pattern of anthrax outbreaks, so that reliable forecasting models can be developed. These models will aid in establishing public health surveillance and guide strategic vaccination programs, which will reduce the economic loss to farmers, and prevent the spill-over of anthrax from livestock to humans. In this study, correlation and coherence between time series of anthrax outbreaks in livestock (1987-2016) and meteorological variables and Sea Surface Temperature anomalies (SST) were identified using a combination of cross-correlation analyses, spectral analyses (wavelets and empirical mode decomposition) and further quantified using a Bayesian time series regression model accounting for temporal autocorrelation. Monthly numbers of anthrax outbreaks were positively associated with a lagged effect of rainfall and wet day frequency. Long-term periodicity in anthrax outbreaks (approximately 6-8 years) was coherent with the periodicity in SST anomalies and outbreak numbers increased with decrease in SST anomalies. These findings will be useful in planning long-term anthrax prevention and control strategies in Karnataka state of India.

A genome-based investigation of the Priestia species isolated from anthrax endemic regions in Kruger National Park.

Priestia is a genus that was renamed from the genus Bacillus based on the conserved signature indels (CSIs) in protein sequences that separate Priestia species from Bacillus, with the latter only including species closely related to B. subtilis and B. cereus. Diagnosis of anthrax, a zoonotic disease, is implicated by tripartite anthrax virulence genes (lef, pagA, and cya) and poly-γ-D-glutamic acid capsular genes cap-ABCDE of Bacillus anthracis. Due to the amplification of anthrax virulence genes in Priestia isolates, the search for homologous anthrax virulence genes within the Priestia genomes (n = 9) isolated from animal blood smears was embarked upon through whole genome sequencing. In silico taxonomic identification of the isolates was conducted using genome taxonomy database (GTDB), average nucleotide identity (ANI), and multi-locus sequence typing (MLST), which identified the genomes as P. aryabhattai (n = 5), P. endophytica (n = 2) and P. megaterium (n = 2). A pan-genome analysis was further conducted on the Priestia genomes, including the screening of virulence, antibiotic resistance genes and mobile genetic elements on the sequenced genomes. The oligoribonuclease NrnB protein sequences showed that Priestia spp. possess a unique CSI that is absent in other Bacillus species. Furthermore, the CSI in P. endophytica is unique from other Priestia spp. Pan-genomic analysis indicates that P. endophytica clusters separately from P. aryabhattai and P. megaterium. In silico BLASTn genome analysis using the SYBR primers, Taqman probes and primers that target the chromosomal marker (Ba-1), protective antigen (pagA), and lethal factor (lef) on B. anthracis, showed partial binding to Priestia regions encoding for hypothetical proteins, pyridoxine biosynthesis, hydrolase, and inhibitory proteins. The antibiotic resistance genes (ARG) profile of Priestia spp. showed that the genomes contained no more than two ARGs. This included genes conferring resistance to rifamycin and fosfomycin on P. endophytica, as well as clindamycin on P. aryabhattai and P. megaterium. Priestia genomes lacked B. anthracis plasmids and consisted of plasmid replicon types with unknown functions. Furthermore, the amplification of Priestia strains may result in false positives when qPCR is used to detect the virulence genes of B. anthracis in soil, blood smears, and/or environmental samples.

[Multiple locus variable number tandem repeat analysis genotype polymorphism of Bacillus anthracis in China].

Objective: To analyze the multiple locus variable number tandem repeat analysis (MLVA) genotype polymorphism of Bacillus (B.) anthracis and establish a MLVA genotype database of B. anthracis in China. Methods: B. anthracis strains isolated from different sources in China since 1947 were collected. Genotype identification was carried out using the MLVA15 genotyping protocol based on 15 variable number tandem repeat loci. The genotypes were uniformly numbered and named. The distribution characteristics of the MLVA genotypes of strains were analyzed. Software Bionumerics was used to construct clustering diagrams to analyze the genetic relationships. Results: The MLVA15 clustering analysis subdivided the isolates into 4 major groups and 91 genotypes, 54 of which were unique to China. The genotypes from MLVA15-CHN1 to MLVA15-CHN6 were widely distributed throughout China and in all eras, while other genotypes were restricted to certain regions or eras. Conclusions: This study established a MLVA genotype database of B. anthracis, which provides basis for the understanding of MLVA genetic polymorphisms and the control and molecular source tracing of the anthrax outbreaks in China.

Knowledge, attitude and practice related to anthrax among livestock farmers in West Kazakhstan.

BACKGROUND: Anthrax is the most prioritized zoonotic disease in Kazakhstan due to its threatening potential to the public health and agricultural sector. Sporadic anthrax outbreaks are being reported annually among human and livestock populations throughout the country, with the highest frequency occurring in West Kazakhstan. METHODS: A cross-sectional study was conducted using a survey-based face-to-face interview. From January to May 2022, 489 randomly selected participants were surveyed in 6 districts of the Baiterek province in West Kazakhstan oblast to evaluate the knowledge, attitude and practice (KAP) regarding anthrax among community members. This is the first KAP study conducted relating to outbreaks of anthrax in Kazakhstan. RESULTS: In this study, most participants (74%) surveyed were males, and 40% of respondents had a secondary level education. Overall, 91% of the community respondents were engaged in agriculture and livestock rearing. Among these community members, cattle rearing was the most common (67%) occupation compared to other livestock species. Additionally, over a 50% of the population studied had no knowledge about the zoonotic nature of the disease, and about 82% and 87% of respondents were unaware of any animal and human anthrax symptoms, respectively. About 70% of the respondents were interested in vaccinating their livestock against anthrax. Individuals aged 45-54 displayed notably higher animal vaccination rates (45%; 95% CI: 38.4-52.0; p < 0.025) compared to those aged 25-34 and 65-74. Respondents residing in the Beles district (20%; 95% CI: 17.1-24.7; p < 0.005) exhibited a significantly higher level of awareness concerning the fatality of anthrax in contrast to participants from Bolashak. Roughly 61% of respondents held the belief that anthrax is a lethal disease. An overwhelming majority of the survey participants (99%) affirmed their non-participation in the slaughter of infected animals. CONCLUSION: The findings of this study indicate that KAP among community members relating to anthrax is low and requires swift implementation of education programmes in building awareness of anthrax under the One Health approach, especially in anthrax prone regions.

Fabrication of a Terbium-Functionalized Cadmium Organic Framework with Proper Energy Levels as a Ratiometric Probe of an Anthrax Biomarker.

Anthrax bacillus is a very dangerous zoonotic pathogen that seriously endangers public health. Rapid and accurate qualitative and quantitative detection of its biomarkers, 2,6-dipicolinic acid (DPA), is crucial for the prevention and treatment of this pathogenic bacterium. In this work, a novel Cd-based MOF (TTCA-Cd) has been synthesized from a polycarboxylate ligand, [1,1':2',1″-terphenyl]-4,4',4″,5'-tetracarboxylic acid (H4TTCA), and further doped with Tb(III), forming a dual-emission lanthanide-functionalized MOF hybrid (TTCA-Cd@Tb). TTCA-Cd@Tb can be developed as a high-performance ratiometric fluorescent sensor toward DPA with a very low detection limit of 7.14 nM and high selectivity in a wide detection range of 0-200 µM, demonstrating a big advancement and providing a new option for the detection of DPA.

Bacillus anthracis in South Africa, 1975-2013: are some lineages vanishing?

The anthrax-causing bacterium Bacillus anthracis comprises the genetic clades A, B, and C. In the northernmost part (Pafuri) of Kruger National Park (KNP), South Africa, both the common A and rare B strains clades occur. The B clade strains were reported to be dominant in Pafuri before 1991, while A clade strains occurred towards the central parts of KNP. The prevalence of B clade strains is currently much lower as only A clade strains have been isolated from 1992 onwards in KNP. In this study 319 B. anthracis strains were characterized with 31-loci multiple-locus variable-number tandem repeat analysis (MLVA-31). B clade strains from soil (n = 9) and a Tragelaphus strepsiceros carcass (n = 1) were further characterised by whole genome sequencing and compared to publicly available genomes. The KNP strains clustered in the B clade before 1991 into two dominant genotypes. South African strains cluster into a dominant genotype A.Br.005/006 consisting of KNP as well as the other anthrax endemic region, Northern Cape Province (NCP), South Africa. A few A.Br.001/002 strains from both endemic areas were also identified. Subclade A.Br.101 belonging to the A.Br.Aust94 lineage was reported in the NCP. The B-clade strains seems to be vanishing, while outbreaks in South Africa are caused mainly by the A.Br.005/006 genotypes as well as a few minor clades such as A.Br.001/002 and A.Br.101 present in NCP. This work confirmed the existence of the rare and vanishing B-clade strains that group in B.Br.001 branch with KrugerB and A0991 KNP strains.

Mapping high probability area for the Bacillus anthracis occurrence in wildlife protected area, South Omo, Ethiopia.

Anthrax is a zoonotic disease caused by a spore-forming gram-positive bacterium, Bacillus anthracis. Increased anthropogenic factors inside wildlife-protected areas may worsen the spillover of the disease at the interface. Consequently, environmental suitability prediction for B. anthracis spore survival to locate a high-risk area is urgent. Here, we identified a potentially suitable habitat and a high-risk area for appropriate control measures. Our result revealed that a relatively largest segment of Omo National Park, about 23.7% (1,218 square kilometers) of the total area; 36.6% (711 square kilometers) of Mago National Park, and 29.4% (489 square kilometers) of Tama wildlife Reserve predicted as a high-risk area for the anthrax occurrence in the current situation. Therefore, the findings of this study provide the priority area to focus on and allocate resources for effective surveillance, prevention, and control of anthrax before it causes devastating effects on wildlife.

Lysins as a powerful alternative to combat Bacillus anthracis.

This review gathers all, to the best of our current knowledge, known lysins, mainly bacteriophage-derived, that have demonstrated activity against Bacillus anthracis strains. B. anthracis is a spore-forming, toxin-producing bacteria, naturally dwelling in soil. It is best known as a potential biowarfare threat, an etiological agent of anthrax, and a severe zoonotic disease. Anthrax can be treated with antibiotics (ciprofloxacin, penicillin, doxycycline); however, their administration may take up even to 60 days, and different factors can compromise their effectiveness. Bacterial viruses, bacteriophages (phages), are natural enemies of bacteria and use their lytic enzymes, endolysins (lysins), to specifically kill bacterial cells. Harnessing the potential of lysins to combat bacterial infections holds promise for diminishing antibiotic usage and, consequently, addressing the escalating antibiotic resistance in bacteria. In this context, we list the lysins with the activity against B. anthracis, providing a summary of their lytic properties in vitro and the outcomes observed in animal models. Bacillus cereus strain ATCC 4342/RSVF1, a surrogate for B. anthracis, was also included as a target bacteria. KEY POINTS: • More than a dozen different B. anthracis lysins have been identified and studied. • They fall into three blocks regarding their amino acid sequence similarity and most of them are amidases. • Lysins could be used in treating B. anthracis infections.

A participatory approach to move towards a One Health surveillance system for anthrax in Burkina Faso.

The One Health approach calls for collaboration across various sectors and different scales to improve understanding of complex health issues. Regarding epidemiological surveillance, this implies the development of integrated systems that link several surveillance components operating in different domains (human, domestic animals, environment) and involving several actor networks. However, surveillance continues to operate in a very compartmentalized way, with little interaction between sectoral institutions and with the community for the governance and operation of surveillance activities. This is partly explained by the insufficient consideration of the local context and the late involvement of national stakeholders when developing programmes that aimed at strengthening the integration of surveillance. In low- and middle-income countries in particular, there is a strong influence of external partners on the development of intersectoral programmes, including surveillance systems. In this context, we developed and implemented a participatory planning process to support stakeholders of the surveillance system of anthrax in Burkina Faso, in the definition of the One Health surveillance system they wish for and of the pathway to reach it. The workshop produced an action plan that reflects the views and perspectives of representatives of the different categories of stakeholders and beneficiaries of surveillance. In addition, the participation of stakeholders in this participatory co-construction process has also improved their knowledge and mutual understanding, fostering a climate of trust conducive to further collaboration for surveillance activities. However, the quality of the participation raises some questions over the results, and contextual factors may have influenced the process. This underlines the need to include a monitoring and evaluation plan in the process to assess its implementation and ability to produce One Health surveillance modalities that are appropriate, accepted and applied over the long term.

Rapid in vitro activity of telavancin against Bacillus anthracis and in vivo protection against inhalation anthrax infection in the rabbit model.

Inhalation anthrax is the most severe form of Bacillus anthracis infection, often progressing to fatal conditions if left untreated. While recommended antibiotics can effectively treat anthrax when promptly administered, strains engineered for antibiotic resistance could render these drugs ineffective. Telavancin, a semisynthetic lipoglycopeptide antibiotic, was evaluated in this study as a novel therapeutic against anthrax disease. Specifically, the aims were to (i) assess in vitro potency of telavancin against 17 B. anthracis isolates by minimum inhibitory concentration (MIC) testing and (ii) evaluate protective efficacy in rabbits infected with a lethal dose of aerosolized anthrax spores and treated with human-equivalent intravenous telavancin doses (30 mg/kg every 12 hours) for 5 days post-antigen detection versus a humanized dose of levofloxacin and vehicle control. Blood samples were collected at various times post-infection to assess the level of bacteremia and antibody production, and tissues were collected to determine bacterial load. The animals' body temperatures were also recorded. Telavancin demonstrated potent bactericidal activity against all strains tested (MICs 0.06-0.125 µg/mL). Further, telavancin conveyed 100% survival in this model and cleared B. anthracis from the bloodstream and organ tissues more effectively than a humanized dose of levofloxacin. Collectively, the low MICs against all strains tested and rapid bactericidal in vivo activity demonstrate that telavancin has the potential to be an effective alternative for the treatment or prophylaxis of anthrax infection.

European multi-centre study to establish MIC and zone diameter epidemiological cut-off values for Bacillusanthracis.

OBJECTIVES: Bacillus anthracis clinical breakpoints, representing a systematic approach to guide clinicians in selecting the most appropriate antimicrobial treatments, are not part of the guidance from the European Committee on Antimicrobial Susceptibility Testing (EUCAST). This is because defined distributions of MIC values and of epidemiological cut-off values (ECOFFs) have been lacking. In this study, a Europe-wide network of laboratories in collaboration with EUCAST, aimed at establishing standardized antimicrobial susceptibility testing methods, wild-type MIC distributions, and ECOFFs for ten therapeutically relevant antimicrobials. METHODS: About 335 B. anthracis isolates were tested by broth microdilution and disc diffusion methodologies. MIC and inhibition zone diameters were curated according to EUCAST SOP 10.2 and the results were submitted to EUCAST for ECOFFs and clinical breakpoint determination. RESULTS: Broth microdilution and disc diffusion data distributions revealed putative wild-type distributions for the tested agents. For each antimicrobial agent, ECOFFs were defined. Three highly resistant strains with MIC values of 32 mg/L benzylpenicillin were found. MIC values slightly above the defined ECOFFs were observed in a few isolates, indicating the presence of resistance mechanisms to doxycycline, tetracycline, and amoxicillin. DISCUSSION: B. anthracis antimicrobial susceptibility testing results were used by EUCAST to determine ECOFFs for ten antimicrobial agents. The MIC distributions were used in the process of determining clinical breakpoints. The ECOFFs can be used for the sensitive detection of isolates with resistance mechanisms, and for monitoring resistance development. Genetic changes causing phenotypic shifts in isolates displaying slightly elevated MICs remain to be investigated.

c-di-AMP accumulation impairs toxin expression of Bacillus anthracis by down-regulating potassium importers.

The Gram-positive bacterium Bacillus anthracis is the causative agent of anthrax and a bioterrorism threat worldwide. As a crucial second messenger in many bacterial species, cyclic di-AMP (c-di-AMP) modulates various key processes for bacterial homeostasis and pathogenesis. Overaccumulation of c-di-AMP alters cellular growth and reduces anthrax toxin expression as well as virulence in Bacillus anthracis by unresolved underlying mechanisms. In this report, we discovered that c-di-AMP binds to a series of receptors involved in potassium uptake in B. anthracis. By analyzing Kdp and Ktr mutants for osmotic stress, gene expression, and anthrax toxin expression, we also showed that c-di-AMP inhibits Kdp operon expression through binding to the KdpD and ydaO riboswitch; up-regulating intracellular potassium promotes anthrax toxin expression in c-di-AMP accumulated B. anthracis. Decreased anthrax toxin expression at high c-di-AMP occurs through the inhibition of potassium uptake. Understanding the molecular basis of how potassium uptake affects anthrax toxin has the potential to provide new insight into the control of B. anthracis.IMPORTANCEThe bacterial second messenger cyclic di-AMP (c-di-AMP) is a conserved global regulator of potassium homeostasis. How c-di-AMP regulates bacterial virulence is unknown. With this study, we provide a link between potassium uptake and anthrax toxin expression in Bacillus anthracis. c-di-AMP accumulation might inhibit anthrax toxin expression by suppressing potassium uptake.