The deliberate, malicious misuse of biosciences and technology, besides a motive, requires intention and material resources including technological infrastructure, access to information as well as necessary explicit and tacit knowledge (Vogel, 2006; Nixdorff, 2020). Since the turn of the millennium, there exists an apprehension that terrorists or other state- or non-state actors might circumvent existing biosecurity regulatory systems by acquiring new tacit knowledge, expertise, or vulnerabilities to develop biological weapons (Riedel, 2005; Cole and Bergman, 2010; Mondange et al., 2022). Explicit knowledge includes standard operation procedures for producing and processing biological agents and can be acquired through open-access scientific literature. In fact, a vast amount of knowledge and information from many decades of anthrax research is available and publicly accessible (Morris and Boyack, 2005; Savcı, 2019). Undeniably, globalization and the internet have significantly diminished the barrier to acquiring explicit knowledge. Tacit knowledge on the other hand is not only acquirable through hands-on encounters but it remains a hindrance to weapon efficiency and effectiveness (Tennenbaum et al., 2021). However, the required tacit knowledge to produce risky biological products is constantly decreasing due to the combination of SynBio with AI and automation.

Principally, forums such as DIY biology classes and Journal of Visualized Experiments (JoVE) videos can transfer the necessary skill and knowledge needed to use otherwise highly sophisticated techniques such as CRISPR during the development of an anthrax BW. Moreover, available kits can help to reduce knowledge and skill requirements during such CRISPR experiments given that the actor can select the appropriate kit and troubleshoot as needed (Paris, 2023). However, it is important to keep in mind that while some terrorist groups may want to genetically engineer B. anthracis; others may be satisfied with the most simple way to produce spores.

There are multiple ways to acquire the anthrax bioagent. Here, we more closely examine three, the isolation from natural sources, the illegal procurement from authorized laboratories, and the modification of related organisms to an anthrax bioagent. Furthermore, we examine the possibilities to include antibiotic resistances.

From the mid-1980s until 2003, the genome of B. anthracis was successfully sequenced (Read et al., 2003) and the two main megaplasmids carrying the main virulence factors pXO1 for the toxins factors edema factor (EF), protective antigen (PA), and lethal factor (LF) (110 MDa, 181 kb) (Green et al., 1985) as well as pXO2 for the capsule (60 MDa, 95 kb) encoding the three genes capB, capC, and capA (Makino et al., 1989; Okinaka et al., 1999) protecting from phagocytosis (Makino et al., 1989) were identified. Strains lacking either plasmid are either avirulent or significantly attenuated (Okinaka et al., 1999; Pilcher, 2003). In the upcoming years, knowledge about other pathogenicity factor genes increased, and “at the dawn of the 21st century, the scientific field of anthrax was perceived as a dead end” (Mondange et al., 2022).

However, as with the progress achieved with recombinant DNA in the 1970s and the rise of synthetic biology in the 2000s, the emergence of genome editing technologies, such as CRISPR in 2012, raised fears about that novel engineered strains of B. anthracis could become available for bioterrorism. Knowing the decoded B. anthracis genome with its more than 5,000 genes (Read et al., 2002; Pilcher, 2003), CRISPR made more precise editing of multiple genes simultaneously possible. In addition, genetic engineering for a fraction of the cost of predecessor technologies became feasible. Genetic modifications were now possible that used to be too demanding, laborious, or expensive in the past (Wang et al., 2019).

A more elaborate approach to obtain an anthrax-causing agent would be to modify a related microorganism, e.g., B. cereus, and convey all the characteristics of B. anthracis. One candidate for such a method could be B. cereus G9241, causing anthrax-like symptoms (Marston et al., 2016; Baldwin, 2020). As of 2022, this particular strain was available for purchase in limited amounts. Working with B. cereus requires only BSL-2 conditions, whereas with B. anthracis BSL-3 conditions have to be applied for intended aerosol production (Baldwin, 2020). Access restrictions become stricter with higher biosafety levels. Therefore, it is conceivable that such an organism may be misused by an actor aiming to “reproduce” anthrax by exploiting advances in biotechnology. Based on the information on the virulence factors given above, up to 14 genes may require editing depending on the original organism to be engineered. The chromosomal engineering could be conducted using the CRISPR/Cas kits which are readily available. Wang and coworkers (Wang et al., 2019) successfully edited the genomic DNA of B. cereus and B. anthracis using CRISPR/Cas9 and showed its efficacy for genome editing in the B. cereus group.

There are indeed certain genetic similarities between B. anthracis and B. cereus G9241, both possess two plasmids in the bacterial cytoplasm. One of the B. cereus plasmids, pBCX01, has a 99.63% homology with the pXO1 plasmid of B. anthracis, Ames strain. However, B. cereus G9241 lacks the pXO2 plasmid responsible for the formation of the polyglutamic acid capsule of B. anthracis (Hoffmaster et al., 2006). This physiological trait allows B. anthracis to evade immune response-mediated phagocytosis. The pXO2 plasmid encoding the polyglutamic acid capsule can in principle be synthesized de novo using oligonucleotides with overlapping sequences, an approach also used for the de novo synthesis of the polio virus (Cello et al., 2002). Oligos can be combined using assembly PCR. Afterward, the plasmid can be transferred into B. cereus G9421 by applying electroporation or other established methods (Ehling-Schulz et al., 2019).

In the context of the genetic engineering of B. anthracis, the incident around the Aum Shinrikyo cult might be of some interest. The characterization of a B. anthracis strain associated with the cult’s activities revealed no evidence of genetic modification (Keim et al., 2001). According to the results of the molecular genetic typing, the strain cultivated by Aum represented the Sterne vaccine strain, known to lack the pXO2 plasmid. On the other hand, Danzig and coworkers (Danzig et al., 2012) formulated a hypothesis that at some stage during their biological weapons program, one of the members of the Aum Shinrikyo cult attempted to transfer the genetic information for the capsule formation into the Sterne strain.

This would parallel the hypothetical scenario of B. cereus transformation discussed above. Both, B. cereus G9241 and B. anthracis Sterne do not possess the important virulence factor, the pXO2 plasmid for capsule formation. In principle, the plasmid could be transferred into the respective microorganism using the established tools of molecular biology. Without discussing the plausibility and likelihood of such an experiment performed by the Aum Shinrikyo cult, it can be insightful to compare the state of knowledge and technological advancement at that time and today.

Already 1988, at the time, when supposedly the biological weapons program of the Aum Shinrikyo cult was in progress, Makino and coworkers demonstrated the possibility of cloning the genetic region required for the encapsulation (Cap region) into Escherichia coli and B. anthracis (Cap-), which resulted in the encapsulation of both species in the presence of CO₂ (Makino et al., 1989). A year later Stepanov and coworkers performed the transduction of pXO2 plasmid into different strains of B. anthracis (STI-1, Sterne, KM33, KM35) and reported that a “dramatic increase of virulence for white mice has been registered for B. anthracis strains having acquired the pXO2 plasmid replicon” (Stepanov et al., 1996). These experiments, among others, show that genetic manipulation of non-pathogenic B. anthracis or other microorganisms to convey the particular characteristics of lethal wild type anthrax was already feasible at the end of the 20th century.

The de novo synthesis of the respective genetic material for the encapsulation and the subsequent bacterial transformation would spare the necessity of acquiring such a regulated strain in the first place. In principle, a de novo synthesis of a Cap region could also be performed using the solid-state phosphoramidite method developed by Caruthers (Caruthers et al., 1987). The sequence of the 3.2 kbp long Cap region was published by Makino et al., in 1989 (Makino et al., 1989). By 1995, the longest DNA segment synthesized chemically and assembled from a large number of oligonucleotides was about 2.7 kbp (Stemmer et al., 1995).

No doubt, the possibilities for misuse of B. anthracis by terrorists have been expanded by the advances in science and technology and the huge amount of knowledge that has accumulated around B. anthracis. The (mis-)use of emerging technologies to genetically modify a harmless microorganism to produce anthrax toxins has been well documented in prokaryotes as well as eukaryotes. One of the candidates is E. coli, for which the expression of LF, EF, and PA of B. anthracis and their subsequent purification from this Gram-negative bacterium have been reported (Robertson and Leppla, 1986; Sharma et al., 1996; Kumar et al., 2001). Additionally, a Gram-positive spore-forming bacterium, B. subtilis, was used in one of the studies to produce recombinant LF (Gholami et al., 2021). Since E. coli and B. subtilis are broadly used in biochemistry and molecular, biology the barrier for an actor with malicious intent is rather low. In addition, there are other alternative systems for the expression of anthrax toxins. For instance, the yeast species Pichia pastoris was used for the expression of the de novo synthesized toxin of Bacillus thuringensis (Gurkan and Ellar, 2005). The advantage of using a eukaryotic organism is the post-translational modification of the toxins produced, which is lacking in prokaryotes.

It can be argued that the respective technical challenge of genome synthesis in the laboratory is lower nowadays due to the possibility of obtaining the corresponding oligos from commercial suppliers. The cost of ordering such sequences has steadily decreased over the years (Hoose et al., 2023), making the technology more accessible for use in biolabs, but also for misuse for malicious purposes. The beginning of the synthetic biology era marks the possibility of ordering de novo synthesized DNA from a commercial provider at desired concentrations and 100% purity. The orders of synthetic DNA are not subjected to mandatory screening. However, a vast majority of the companies working in this field have voluntarily introduced screening procedures based on the guidelines by the United States Department of Health and Human Services (HHS), e.g., Screening Framework Guidance for Providers of Synthetic Double-Stranded DNA (U.S. Department of Health and Human Services, 2010). The ordered sequences are matched with databases regarding pathogen and toxin sequences. Nevertheless, it is possible to evade such control mechanisms. One novel way around this is re-coding, which can be done either, less promisingly, by changing the codons, resulting in the same amino acid sequence. Or with encryption, whereby ultimately codons code for other amino acids (iGem Team, 2017). This would be a much more promising way to circumvent such controls, although much costlier, as all the gene sequences within the agent would have to be encrypted in the same way to allow functionality. So far, large-scale “rewiring” is not feasible, but research in this field advances rapidly. These aspects show that current screening algorithms need to be redesigned according to the developments in biotechnology.

But also the Engineering Biology research Consortium (EBRC Engineering Biology Research Consortium, 2022) itself lists some gaps, for instance, sequences of 200 bp or smaller are usually not cross-checked, since the results of oligo screenings might be ambiguous and expensive, compared to the cost of the DNA synthesis itself. Such shorter sequences could be assembled into longer sequences, thus creating a backdoor for abuse. Furthermore, the guidance refers only and explicitly to double-stranded DNA (dsDNA), not to single-stranded DNA or RNA. Both can be converted to dsDNA in vitro. Furthermore, the working group assumes that about 80% of the world’s DNA synthesis capacity is combined under these provisions, which leaves out a significant 20%. This is a major loophole in biosecurity, which can result in the synthesis and shipment of sequences of concern such as toxins.

Alternatively, the synthesis of oligos and short dsDNA can nowadays be also performed fast and at relatively low costs directly in the lab using state-of-the-art benchtop synthesizers. For more details see Carter et al. (Carter et al., 2023). Some of the devices synthesizing nucleic acids greater than 1,500 bp in length are subjected to export controls under the AG (2021) (The Australian Department of Foreign Affairs and Trade, 1958), but this does neither apply to all commercially available instruments nor national trade.

Even with all regulations in place, there remains the risk that an order may evade screening. For example, the sequence in question may be camouflaged by benign genes. Such a construct has a high probability to circumvent the screening procedure. The camouflaging genes can thereafter be removed through methods such as CRISPR/Cas9, and the deletions repaired via homology-directed repair (HDR), leaving a sequence encoding for a dangerous toxin or a virulence factor. This scenario may sound technically elaborate. However, proof of concept has been conducted by Puzis et al. (Puzis et al., 2020). No respective obfuscated DNA encoding a toxic peptide was detected by the screening algorithm, and the order was moved to production.

It should be pointed out that B. anthracis is one of the most extensively studied microorganisms. Several mitigation measures and therapeutic strategies have been established over the years, which can be efficiently applied in the case of a potential outbreak (Supplementary Table S1). However, it is possible to introduce antibiotic resistance genes into the bacterial genome to circumvent these therapeutic strategies. Multidrug-resistant bacterial strains have been successfully engineered in the past (Dassanayake et al., 2021). In certain cases, multidrug resistance can lead to loss of virulence due to pleiotropic effects, as reported for the Francisella tularensis strain engineered to be resistant to multiple antibiotics as part of the USSR’s BW program (Leitenberg et al., 2012). There are also publications available, indicating that a B. anthracis strain (STI-1 vaccine strain) was engineered to resist several antibiotics (Stepanov et al., 1996). Therefore, the threat of a biological attack involving a multidrug-resistant lethal strain of B. anthracis cannot be understated.

No doubt, the emerging, converging and enabling technologies led to a decrease in the requirement for sophisticated equipment thereby expanding the realm of feasibility and hence the BW paradigm. In addition, tools traditionally siloed in academic and government labs are increasingly becoming accessible to a wider audience (Dunlap and Pauwels, 2017; Sanz et al., 2022). Moreover, during the process of B. anthracis weapon development, less dangerous and easily available surrogate microorganisms can be used. Historically, the Japanise, the United States, the UK, and Iraq used B. anthracis surrogates in biological warfare test studies (Balmer, 2001; United Nations, 2007; Greenberg et al., 2010). More recently, research data were generated that could be exploited for B. anthracis BW development with a surrogate microorganism. For example, B. thuringiensis was effectively used as an appropriate model for B. anthracis in aerosol and re-aerosolization testing allowing environmental release without pathogenicity concerns (Tufts et al., 2014). In addition, using B. thuringiensis as a surrogate test organism opens new possibilities even for alternative non-regulated cultivation technologies such as solid-state fermentation (SSF) application as a new production system (Lima-Pérez et al., 2019).

Furthermore, significant progress in frugal science, collectively describing the attempt to create cheap, easy-to-use low cost and low electricity-requiring scientific equipment alongside emerging technologies made for anyone, anywhere could potentially be exploited to develop biological weapons (Tennenbaum et al., 2021).

Novel developments and contemporary lab practices make the cultivation and scale-up of B. anthracis more feasible. For large-scale cultivation, B. anthracis could be grown in submerged high-cell-density fermenters, as shown for the comparable B. subtilis (Grossman and Losick, 1988; Riesenberg and Guthke, 1999). Further upscaling would usually require large liquid-state fermenters which are subject to export controls under the AG (The Australia Group, 2021). To circumvent this restriction, a novel, unrestricted alternative SSF with polyurethane foams could be performed. This method was developed for B. thuringensis but is in principle applicable to B. anthracis (Lima-Pérez et al., 2019). SSF would therefore represent an unrestricted alternative method. All these aspects must be considered when discussing the imminent threat of an anthrax attack.

There is also a large body of literature available on anthrax sporulation. It can be induced by a lack of nutrients in a freely available sporulation medium (Chen et al., 2020). Common histological stains (Moeller or Schaeffer-Fulton) are suitable to verify sporulation. Once obtained, the spores can be stored for extended timespans and disseminated by aerosolization. Not only can B. anthracis spores be stored for a long time, since they remain viable for decades, but they can also be easily transported in sealed containers and survive exposure to the Sun, air, rain, and violent dissemination methods. The spores are even so heat-resistant, they could be disseminated using explosives (Fetter, 1991).

Historical indices show that several state and non-state actors transformed cultivated B. anthracis spores into a powder form as a part of anthrax weaponization. The purpose of this step is to improve the dissemination and aerosolization of this bioagent. It was reported that the United States (Matsumoto, 2003) and USSR (Zilinskas, 2014) produced dried anthrax spores in the scope of their BW programs. Also, the Aum Shinrikyo cult attempted to obtain anthrax in powder form (Danzig et al., 2012), while Iraq experimented with lypholization (Mondange et al., 2022). The best-known case of deploying anthrax as a powder is the 2001 anthrax attack (v. s. Amerithrax), where the spores were dried to the concentration of 2.0 × 10¹² colony-forming units per Gram (USDOJ, 2010). Tufts and coworkers (Tufts et al., 2014) showed that B. thuringensis can be used as a surrogate to optimize the aerosolization of anthrax spores. Such a procedure requires sufficient technical expertise and special freeze- or spray-drying equipment. This category of dual-use equipment with a water evaporation capacity between 0.4 and 400 kg/h, and the ability to reach particle sizes below 10 µm or to sterilize or disinfect in situ is subjected to export controls under the EU regulations (European Union, 2021), implementing the AG-control lists (The Australia Group, 2021). Additional safety precautions such as a glovebox with negative pressure and protective clothing with an external oxygen supply are also highly recommended when working with aerosols containing pathogens. These items are also included in the AG control list of dual-use equipment and technologies. However, it can still be purchased within the country, second-hand, or crudely manufactured, albeit with technical challenges. Thus, the Aum Shinrikyo cult, although unsuccessfully employed a rudimentary self-made drier. Thus, aerosolization may be considered a bottleneck in the production of an anthrax BW. Probably the safest way to circumvent the regulations and controls in this regard would be to build the necessary equipment oneself, which would mean a significant increase in the necessary know-how in the field of engineering and infrastructure in the form of corresponding clandestine production facilities.

Another aspect in the context of anthrax weaponization is the encapsulation of the purified and dried spores. The encapsulation would impart additional stability and prevent aggregation (Matsumoto, 2003). It was suspected that the spores disseminated in the Amerithrax case were coated, based on the high silica level determined during the investigation. However, this hypothesis could not be verified experimentally. According to the results of the transmission electron microscopy, the silica was localized to the spore coat within the exosporium, and not on the surface. Therefore, it was concluded that silica was incorporated into the cells as a natural part of cell formation, rather than by a deliberate attempt to coat the spores. Despite this experimental evidence, the controversial debate on the spore coating in the Amerithrax case is still ongoing (Rosenberg, 2002; Bernstein, 2010; Epstein, 2010; National Research Council, 2011a).

From the perspective of this manuscript, it is of interest to evaluate the state of technology, which could in principle be applied to encapsulate the anthrax spores. As previously mentioned, B. subtilis can be used as a model organism for B. anthracis. Therefore, the encapsulation procedure described by Balkundi and coworkers (Balkundi et al., 2009) for B. subtilis has to be considered in the discussion on the advances in knowledge and technology, which might be misused for the weaponization of anthrax.

The method of BW deployment depends on the agent, its preparation, its stability, and the route of infection. In 1970, a World Health Organization (WHO) expert committee estimated that “an aircraft release of 50 kg of anthrax over an urban, developed population of 5 million would result in 250,000 casualties”. Medical resource limitation and capacity strain in such a scenario is enormous, ultimately requiring 13,000 hospital beds, 60-day of antibiotics for 125,000 patients leaving 95,000 dead. This would undoubtedly result in a quick and complete collapse of medical resources and civilian infrastructure. More recent estimations have confirmed the original WHO data (Congress, 1993). The CDC has developed an economic model that puts forward costs of $26.2 billion per 100,000 people exposed to an anthrax attack (Kaufmann et al., 1997). Fetter (Fetter, 1991) estimated that a missile armed with 30 kg of anthrax spores would affect an area of 6–80 square kilometers, delivering doses greater than 0.1 mg/min/m³ (the estimated ECt₅₀ for anthrax) depending on the weather conditions and kill an estimated 20,000–80,000 people if a large, sparsely populated city was attacked. Alternatively, bioterrorists may disperse B. anthracis spores through aerosols using knapsack sprayers or a crop-spraying light aircraft to disseminate the biological agent (Durrant, 2002; Haas, 2002; Aduojo et al., 2022). The intimidating scenario of an attack with a UAV, commonly known as a drone, on a vulnerable target delivering weaponized anthrax can be considered increasingly realistic. In the Sverdlovsk incident, 1 g of wind driven anthrax spores killed sheep at a distance of up to 50 km (Durrant, 2002). Despite international regulations such as well-established import-export control regimes, up-to-date drones offer terrorists the convenience of anonymity and bypass traditional security measures (Pethő-Kiss, 2022).

Shortly after the most recent bioterrorist Amerithrax attack and the complete genome sequencing of B. anthracis (Read et al., 2002), remarkable innovations and advances in the realm of anthrax detection and the newly initiated field of microbial forensics (Keim et al., 2001; Rasko et al., 2011) were made (Schmedes et al., 2019; Revill et al., 2022).

Conventionally, samples are assessed via microbiological growth analysis, Gram-, spore-, and capsule staining, microscopic analysis, hemolysis tests and phage susceptibility (Zasada, 2020). These methods require highly trained laboratory personnel, BSL-3 facilities, and practices. Novel detection methods are based on diverse targets, from detection based on DNA (Pal and Alocilja, 2010; Hao et al., 2011; Kaittanis et al., 2011; Chen et al., 2020), chemical reactions (Boyer et al., 2007; Duriez et al., 2009; Čapek et al., 2010; Kuklenyik et al., 2011), antibodies (De et al., 2002; Biagini et al., 2006; Campbell and Mutharasan, 2006; McGovern et al., 2007; Hao et al., 2009; Mwilu et al., 2009; Tang et al., 2009; Zahavy et al., 2010; Wang, 2013; Atabakhshi-Kashi et al., 2020), phages (Schuch et al., 2002; Fujinami et al., 2007), peptides (Acharya et al., 2007; Park et al., 2009), aptamers (Alibek and Handelman, 1999; Huan et al., 2009; Cella et al., 2010; Oh et al., 2011; Kim et al., 2013) or even DNA-peptide chimeras (Zhang and Appella, 2007; Kim et al., 2015; Wang D-B. et al., 2021). To date, a variety of detection methods for environmental or clinical anthrax samples emerged, each with distinct advantages over conventional culture and PCR-based detection. Xu and coworkers (Xu et al., 2023) developed a rapid (<40 min), easy-to-implement and accurate DNA endonuclease targeted CRISPR trans reporter (DETECTR)-based detection and identification method as a novel screening and diagnostic user-friendly portable devices for pathogenic B. anthracis (Xu et al., 2023). Overall, the speed, sensitivity and accuracy of modern detection methods have increased, potentially saving uncounted lives in case of an anthrax BW attack. Early detection is the prerequisite for adequate treatment and mitigation. In order to make a difference, these detection methods, must be widely available.

In addition, the Amerithrax incident led to important investments in medical funding for biodefense. In the following decade, the United States, for example, spent 5.6 billion dollars on biodefense known as the Project BioShield Act 2004 (US Congress, 2004). While many wealthy countries followed the United States in an attempt to globally improve the capacity to face an emerging outbreak, although, with budgets that were and are orders of magnitudes lower (Mondange et al., 2022).

Currently, preparedness against the intentional use of B. anthracis relies on increased disease as well as environmental surveillance (US BioWatch program (National Research Council, 2011b)), laboratory capacity, information and system technology, education, and workforce training as well as clinical practice that integrates all accessible countermeasures such as new antimicrobials and advances in critical care (Blatny and Green, 2007; Scales and Horney, 2023). The armamentarium for PEP and treatment of anthrax involves numerous effective antimicrobials, including alternatives for resistant strains, antitoxins, and vaccines (Kaufer et al., 2020; Honein and Hoffmaster, 2022) that must be stockpiled in adequate quantities. Protocols to deal with anticipated B. anthracis scenarios are developed and tested in exercises.

Considering the preparedness towards anthrax attacks, there have been efforts to adopt strategies by various countries. Some examples are listed in the following: The US CDC (2015) published a clinical framework and medical countermeasure use during an anthrax mass-casualty incident. The focus was set on the allocation of scarce resources with different treatment plans depending on whether anthrax developed meningitis. The CDC recommends additional treatment with antitoxin in meningeal anthrax cases. The European Centre for Disease Prevention and Control (ECDC), however, only monitors cases in EU/European Economic Area countries and discusses them in their weekly Communicable Disease Threat Reports (ECDC. COMMUNICABLE, 2022). In their Annual Epidemiological Report for Anthrax, the ECDC also discusses the complementary administration of antitoxins, albeit additional benefits have been contested. The Department of Public Health of the Australian Government published a Public Health response plan for Anthrax (Australian Government Department of Health, 2012). Next to the description of clinical etiology and different treatment plans, this response plan entails measures for five different response codes (threat levels), for deliberate anthrax releases, defining the main actions and communication plans to be taken by the government and jurisdiction for each threat level and the key stakeholders.

Not surprisingly, the development of effective anthrax vaccines was spurred on by the potentially nefarious use of B. anthracis as a biological warfare agent. Already in 1953 and 1959 the USSR licensed their live spore vaccine for scarification and subcutaneous administration, respectively (Biselli et al., 2022). As new biochemistry methods in the 1950s and 1960s, paved the way for discovering and deciphering the capsule of B. anthracis (Smith et al., 1953; Thorne, 1960; Stanley et al., 1961), responsible for the toxin-mediated disease anthrax. In the 1970s, these breakthroughs and biodefense endeavors lead to the successful development and approval of a novel and enhanced cell-free human preparation of aluminum hydroxide gel adsorbed protective antigen, now known as anthrax vaccine adsorbed (AVA) formulation in the 1970s (Tournier et al., 2009; Tournier and Mohamadzadeh, 2010). At the same time, the WHO declares anthrax one of high-impact bioagents (World Health Organization, 1970).

Currently, the two primarily used culture filtrate vaccines, the Europe- and US-licensed Anthrax Vaccine Adsorbed (AVA; trade name BioThrax) and the United Kingdom-licensed vaccine, AVP, contain PA and variable quantities of LF and EF. Guidelines “recommend vaccination for people at risk, such as veterinarians, abattoir workers, those working with animal hides or furs, laboratory workers and the armed forces in areas with a high risk of exposure. In addition to PrEP the anthrax vaccine is also recommended for PEP, along with antibiotics” (ECDC, 2013). For individuals 18–65 years of age, various Anthrax vaccines are licensed or in development for PEP (Wolfe et al., 2020).

In addition, the antibiotics ciprofloxacin, penicillin, and doxycycline were approved by the Food and Drug Administration (FDA) for the treatment of anthrax and may be also useful in combination with other antibiotics for the treatment of inhalation anthrax (Inglesby et al., 2002). In B. anthracis antibiotic resistance to, e.g., amoxicillin, penicillin G, and/or cotrimoxazole has been documented. Although drug resistance mechanisms of B. anthracis have not yet been fully exploited, beta-lactamases against β-lactam antibiotics and efflux-pump mediating cross-resistance to fluoroquinolone antibiotics like ciprofloxacin in B. anthracis have been reported. Genetic modification of B. anthracis (to induce resistance to vaccines or antimicrobial drugs) has not yet been achieved by terrorists. Yet, the illicit Soviet program was successful. Hence, the introduction of safer and more efficient chemotherapeutic options are required (Dassanayake et al., 2021).

Moreover, antibiotics are effective against bacteremia caused by antibiotic-susceptible strains of anthrax but not against the toxemia that drives pathogenesis. In fact, the quantities of secreted anthrax toxins in some cases lead to death despite efficient antibiotics administration. The discovery of the biochemical structure of LF and EF (Pannifer et al., 2001), of the cellular receptors of PA (Bradley et al., 2001), and description of the precise effects of LF and EF on the cell biology (Moayeri et al., 2015), therefore, were important scientific achievements in the toxin field. In 2009, the first monoclonal antibody targeting PA was finally authorized by the FDA (Migone et al., 2015). Nowadays, three anthrax antitoxins have been approved by the FDA and stockpiled by the United States: two monoclonal antibodies (raxibacumab and obiltoxaximab “Anthim”), and the human polyclonal purified IgG from vaccinated humans (intravenous anthrax immune globulin AIG-IV, also referred as Anthrasil) (Huang et al., 2015; Avril et al., 2022), regardless of uncertainties associated with the clinical effectiveness of antibodies. Hence, Anthim and Anthrasil can be administered solo or in combination with antibiotics for a more effective anthrax therapy. According to the CDC, the administration of both antibiotics and antibodies is recommended, regardless of recent studies doubting the efficiency of antibodies (Tournier et al., 2019; Avril et al., 2022).

Generally, remediation following a B. anthracis BW attack requires decontamination, confirmatory sampling, and testing. The decontamination strategy should include the decontamination of surfaces and affected areas (space), as well as the proper disposal of any decontamination wastewater (Urban-Sorensen, 2018). In the aftermath of the Amerithrax attack, both private and government facilities were affected, and their cleaning up was an unexpected challenge. The decontamination work was not only high-profile but also very time-consuming and expensive. A complete renovation of all facilities required over 3 years and cost about $320 million (Urban-Sorensen, 2018). Meanwhile, specific advances in nanotechnology and material sciences led to the improvement of decontamination and decontamination capabilities even against spore-forming bacilli. For decontamination applications against B. anthracis with up to 100% efficacy after 10–15 min, Ginghina (Ginghina et al., 2022) demonstrated the antimicrobial activity of organic solutions enriched with ZnO, TiO₂, and zeolite nanoparticles. Another effective strategy is to incorporate different semiconductors to enhance their bactericidal synergistic effects for water disinfection. A maximum antimicrobial activity against B. subtilis was shown by CuWO₄/CuS CuS nanopowder (Dong et al., 2022). Moreover, Nakonieczna (Nakonieczna et al., 2022) recently identified three new siphophages that can specifically infect and lyse siphophages that can specifically infect and lyse B. anthracis and have applications as decontaminants or disinfectants (of skin, surface, or clothes).