Review | Open | Open Peer Review | Published:
Alternative pre-approved and novel therapies for the treatment of anthrax
BMC Infectious Diseasesvolume 16, Article number: 621 (2016)
Bacillus anthracis, the causative agent of anthrax, is a spore forming and toxin producing rod-shaped bacterium that is classified as a category A bioterror agent. This pathogenic microbe can be transmitted to both animals and humans. Clinical presentation depends on the route of entry (direct contact, ingestion, injection or aerosolization) with symptoms ranging from isolated skin infections to more severe manifestations such as cardiac or pulmonary shock, meningitis, and death. To date, anthrax is treatable if antibiotics are administered promptly and continued for 60 days. However, if treatment is delayed or administered improperly, the patient’s chances of survival are decreased drastically. In addition, antibiotics are ineffective against the harmful anthrax toxins and spores. Therefore, alternative therapeutics are essential. In this review article, we explore and discuss advances that have been made in anthrax therapy with a primary focus on alternative pre-approved and novel antibiotics as well as anti-toxin therapies.
A literature search was conducted using the University of Manitoba search engine. Using this search engine allowed access to a greater variety of journals/articles that would have otherwise been restricted for general use. In order to be considered for discussion for this review, all articles must have been published later than 2009.
The alternative pre-approved antibiotics demonstrated high efficacy against B. anthracis both in vitro and in vivo. In addition, the safety profile and clinical pharmacology of these drugs were already known. Compounds that targeted underexploited bacterial processes (DNA replication, RNA synthesis, and cell division) were also very effective in combatting B. anthracis. In addition, these novel compounds prevented bacterial resistance. Targeting B. anthracis virulence, more specifically the anthrax toxins, increased the length of which treatment could be administered.
Several novel and pre-existing antibiotics, as well as toxin inhibitors, have shown increasing promise. A combination treatment that targets both bacterial growth and toxin production would be ideal and probably necessary for effectively combatting this armed bacterium.
Bacillus anthracis, the etiological agent of anthrax, is a Gram-positive, sporulating and toxin-producing, rod-shaped bacterium [1, 2]. It is readily found in soil and is responsible for causing disease in livestock including cows, sheep, and goats and wild animals (bison, buffalo) . This pathogen can be transmitted to humans via direct contact, ingestion, aerosolization or injection of vegetative cells or spores resulting in cutaneous, gastrointestinal, inhalational or injectional anthrax, respectively . Cutaneous anthrax (CA), the least severe, albeit the most common form of anthrax, represents approximately 95 % of all reported cases [5, 6]. Clinical presentation of CA often manifests as isolated infections on the face, neck, and arms and is characterized by a black necrotic skin eschar [5, 6]. This form is rarely fatal and can be effectively treated with antibiotics . Gastrointestinal anthrax (GA) is more severe although rare, with no cases having ever been reported in the United States (USA) . Symptoms of GA are considered non-specific (nausea, vomiting, fever, bloody diarrhea and malaise) often resulting in misdiagnosis, leading to treatment delays and high mortality rates of over 50 % [3, 7, 8]. Inhalational anthrax (IA) is the most severe manifestation of anthrax with a mortality rate of up to 90 % if left untreated [9–11]. Similar to GA, this respiratory infection is often misdiagnosed due to non-specific symptoms (fever, cough, fatigue and chest or abdominal pain) [9, 10]. IA rapidly progresses to a fulminant stage of infection resulting in cardiac and pulmonary shock. It can also commonly spread to the brain resulting in meningitis, which is quickly followed by death [9, 10]. The final and most recently identified clinical form of anthrax, known as injectional anthrax, has primarily been associated with heroin drug users in the United Kingdom (UK) and Europe . Since 2009, over 50 cases of injectional anthrax have been reported with a mortality rate of approximately 33 % [3, 12–15].
Over the last hundred years, there have been numerous documented anthrax outbreaks due to both natural and intentional causes [3, 6, 7, 11, 12, 14–18]. Anthrax is endemic in several developing countries in Africa, Latin America, Eastern Europe and Asia (see Fig. 1) [3, 6, 7, 19–21]. Turkey and Greece are particularly affected due to common practices of animal husbandry, lack of protective measures (such as animal vaccinations) and lack of knowledge about B. anthracis [22–24]. Contaminated heroin originating in Afghanistan likely contributed to the 2009 outbreak of injectional anthrax in Europe and the UK possibly due to casing the drug in skins of goats that died from anthrax . In 1979 in Ekaterinburg, Russia (formerly known as Sverdlosk), over 60 people were infected with anthrax due to the accidental release of B. anthracis spores from a military microbiology laboratory [18, 26]. Because of this air filter malfunction, 42 residents from the surrounding city perished from IA . In 1993, aerosolized spores were deliberately released by the Aum Shinrikyo cult over Kameido, Japan. However, since the attenuated B. anthracis Sterne 34 F2 strain was utilized, no infections were recorded . In 2001, B. anthracis Ames strain spores were sent through the USA post office to various news and government offices leading to the exposure of thousands of individuals to anthrax resulting in 22 reported cases (5 deaths) [2, 11, 17].
B. anthracis pathogenesis is mainly attributed to two large plasmids, pXO2 and pXO1, that are essential for full virulence (see Fig. 2) . pXO2, the smaller of the two plasmids, encodes 80 genes including the capBCAE operon responsible for the unique, negatively-charged, poly-γ-D-glutamate capsule that enables host immune evasion and macrophage intracellular survival [1, 27–29]. pXO1 encodes 140 genes including the tripartite exotoxin genes, pagA, lef and cya, which produce the protective antigen (PA), lethal factor (LF), and edema factor (EF), respectively . Once the toxin components are produced, they are secreted by the cell.
PA, the non-enzymatic portion of the toxin, is an 83 kilodalton (kDa) protein (PA83) that facilitates toxin entry into the host cell by binding endothelial cell surface receptors (i.e. tumor endothelial marker-8 [TEM-8] and capillary morphogenesis protein-2 [CMG-2]) throughout the body (see Fig. 3) [30, 31]. Once PA83 is bound to host receptors, it is cleaved by host proteases, namely furin, into two fragments referred to as PA20 (a 20 kDa PA) and PA63 (a 63 kDa PA) . Following the disassociation of PA20, the remaining cell-bound PA63 heptamerizes and binds up to 3 molecules of LF and/or EF to form the lethal toxin (LT) or edema toxin (ET), respectively [33–35]. The toxin complexes are subsequently translocated into the host cell via receptor-mediated endocytosis and delivered to the endosome. Here, the acidic pH induces a conformational change resulting in the release of LF and EF into the cell cytosol where they can exert their enzymatic properties [35, 36].
EF is an 89 kDa calcium and calmodulin (CaM)-dependant adenylate cyclase that catalyzes the reaction of adenosine triphosphate (ATP) to 3, 5′-cyclic adenosine monophosphate (cAMP) . The sudden increase of cAMP prevents apoptosis, cell motility, macrophage and neutrophil abilities as well as impairs the release of several inflammatory cytokines [38–40].
LF is a zinc-dependant metalloproteinase that cleaves and inactivates mitogen-activated protein kinase kinases (MAPKKs) resulting in the disruption of many signalling pathways such as macrophage activation, maturation, and chemotaxis, as well as induces cell death [40, 41]. In addition, in certain rodents, LT also targets NLRP1 (an inflammasome sensor) resulting in the activation of the caspase-1 dependent cell death signalling pathway [42, 43]. This pathway has been studied extensively and is the basis for many LT assays since LT is especially cytotoxic to rodents resulting in rapid macrophages lysis and cell death .
Through ET and LT, B. anthracis has evolved to target multiple cells throughout the host. In addition to targeting the innate immune system, ET interferes with the adaptive immune system by impairing lymphocyte function ensuring the establishment of the B. anthracis infection and future bacterial growth [43, 44]. Furthermore, it targets hepatocytes and intestinal epithelial cells leading to extensive tissue edema [43, 44]. LT primarily targets cardiomyocytes and smooth muscle cells [43–45]. Through liver and cardiovascular impairment, B. anthracis is able to impact two vital host systems resulting in lethal vascular collapse.
In order to effectively treat anthrax, prompt recognition and therapeutics are essential. Once the secreted toxins accumulate in the body, antibiotics are rendered ineffective and the patient’s chances of survival are decreased drastically [2, 11, 17]. Once B. anthracis is suspected, antibiotic administration should be commenced immediately and continued for 60 days . If clinical symptoms are absent, doxycycline or penicillin G should be administered orally or intravenously (i.v.) at 100 mg twice a day (BID) or 1,200,000 units every 12 h, respectively . Conversely, if clinical symptoms do manifest, an oral or i.v. formulation of 400 mg of ciprofloxacin BID is recommended . Despite this lengthy administration period, current antimicrobials are ineffective against the B. anthracis spore. In non-human primates (NHP), spores have been shown to germinate up to 100 days post infection resulting in death rates of up to 30 % [47–50]. Thus, the most promising way to eradicate B. anthracis might be to prevent the sporulation process altogether. Studies looking at the effect of the “gold standard” antibiotics on B. anthracis sporulation found that treatment with doxycycline (a bacteriostatic, protein synthesis inhibitor) resulted in a predominantly vegetative final population while treatment with the bactericidal ciprofloxacin led to a predominantly spore population [51, 52]. Hence, although doxycycline killed more slowly initially, overall it was considered more effective.
Aside from its inefficiency against spores, the lengthy treatment regimen can also result in a decrease in patient compliance (as seen in 2001 when compliance rates were only 40 %) [53, 54]. Furthermore, the possibility of selection of antibiotic resistant mutants due to high antibiotic usage is a risk since B. anthracis resistance has been shown to occur gradually over time [55, 56]. In fact, studies have shown that after 21 subcultures, B. anthracis ciprofloxacin sensitivity was decreased from 0.1 to 1.6 mg/l . Likewise, after repeated subculturing with doxcycline, the minimum inhibitory concentration (MIC) was 600 times its initial MIC . Although natural resistance is considered to be low in B. anthracis, with no naturally occurring ciprofloxacin or doxycycline resistant strains documented to date, lactamase genes (that cleave lactam drugs i.e. penicillin) have been discovered, albeit in order to be functional, they need to be induced . Induction of these genes results in clinical isolates with MICs as high as 128 g/ml and surveillance has found penicillin resistance in approximately 15 % of reported cases [58, 59]. Additionally, reports have been published showing that penicillin, ciprofloxacin and doxycycline resistant strains can be easily bioengineered [55, 60]. Therefore, in the event of a bioterrorist attack, it may be possible that the microorganism being used might be resistant to first-line antibiotic therapy (i.e. penicillin, doxycycline, or ciprofloxacin).
In addition to antibiotics, under certain specific circumstances (i.e. not generally across the population) vaccination is also recommended to aid in the development of an active immune response and to prevent infection with B. anthracis. There are two anthrax vaccines that are currently licensed for use: Anthrax Vaccine Precipitated (AVP) which is licensed in the UK and Anthrax Vaccine Absorbed (AVA) which is licensed in the USA. AVP, first licensed in 1979, is a PA-based vaccine made from an avirulent B. anthracis Sterne 34 F2 strain and requires 4 intramuscular doses over a period of 32 weeks . AVA or Biothrax (Emergent BioDefense Operations Lansing LLC), a cell-free PA-based vaccine made from the V770-NP1-R B. anthracis strain, contains an aluminum adjuvant and is part of the USA strategic national stockpile [7, 62]. A subcutaneous (SC) dose of 0.5 mL of AVA must be administered as five doses at 0 and 4 weeks and at 6, 8 and 12 months to be effective as pre-exposure prophylaxis [63, 64]. Although it is primarily used for pre-exposure prophylaxis in high-risk populations such as military personnel, researchers or veterinarians, AVA has also been shown to be effective in post-exposure situations [62, 65, 66].
Aside from requiring multiple doses, there are many other points to be considered with the current anthrax vaccine. Specifically, AVA has varying amounts of PA per batch, and a limited shelf life (stocks must be replaced every 4 years) [67–69]. Although it is considered safe, adult patients have reported some side effects (lymphadenopathy, immune system disorders, tremor, ulnar nerve neuropathy, as well as musculoskeletal, connective tissue and bone disorders) . Also, the safety of AVA in children less than 18 years of age is currently unknown. In addition, in a 2008 observational study it was found that pregnant USA military women that had been vaccinated with AVA during their first trimester had slightly elevated rates of birth defects compared to non-vaccinated pregnant women. Therefore, AVA is not recommended for pregnant women or people under 18 years of age unless all other options have been exhausted .
Furthermore, although PA can elicit a humoral immune response, it is limited in its ability to promote long-lasting immunity (due to a declining anti-PA response over time) [64, 71]. Storage of both the vaccine and antibiotics in such large quantities along with the requirement for constant patient monitoring make these strategies less practical. Consequently, treatment following a scenario of a biological anthrax attack where a large number of individuals are exposed would not be feasible using the current treatment regimens.
In this review article, we explore and discuss advances that have been made in anthrax therapeutics with a primary focus on alternative pre-approved and novel antibiotics as well as anti-toxin therapies that could be useful in the event of a bioterror attack.
Qualifications for new drugs for the treatment of B. Anthracis infections
Since B. anthracis is primarily an agent of bioterrorism, naturally occurring human infections are infrequent. Moreover, due to the high morbidity and mortality associated with anthrax, it would be unethical to perform clinical studies in humans. In the case of bioterrorism agents, the Food and Drug Administration (FDA) may approve a drug based on the Animal Efficacy Rule which states that a drug may receive approval for the treatment of IA if its efficacy has been demonstrated in more than one animal model as long as the models serve as a reasonable substitute for humans . Furthermore, pharmacokinetic studies must be conducted in animals and humans allowing proper dose selection and the mechanisms of drug toxicity must be relatively well understood. Finally, it is expected that the drug will have a favorable endpoint and will improve host survival .
Antibiotics currently approved for the treatment of other bacterial diseases
It is often recommended that the medication being evaluated already be approved for the treatment of other illnesses since their safety profile and clinical pharmacology will already be known. Doxycycline and penicillin G, two antibiotics that have recently been approved for their use in the treatment of IA, had been on the market for upwards of 50 years and had been used to treat over 100 million patients in the USA prior to their approval for anthrax therapy .
Second and third generation fluoroquinolones, such as ofloxacin, levofloxacin, and moxifloxacin, have already been approved by the USA FDA under the respective names of Floxin (Ortho-McNeil Pharmaceutical), Levaquin (Janssen Incorporated), and Avelox (Bayer HealthCare Pharmaceuticals) for the treatment of various respiratory and skin infections [74–76]. These fluoroquinolones are bactericidal against a broad spectrum of microorganisms and function by inhibiting the bacterial DNA gyrase and topoisomerase IV (Fig. 3) [75, 77, 78]. Fluoroquinolone resistance has been assessed and although spontaneous in vitro resistance is rare (10−9–10−10), bioengineering of resistant strains in a laboratory setting is possible and should be taken into consideration . Athamna et al. investigated the ability of the B. anthracis ST-1 and Sterne strains to develop resistance to ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin and garenoxacin . Within 10 passages, resistance was recorded for all quinolones and after 18 passages, all of the MIC increased from 0.03 to 8 mg/L with the exception of garenoxacin which increased from 0.015 to 0.5 mg/L for the ST-1 strain. Cross-resistance among the fluoroquinolones was also demonstrated; however, being resistant to one fluoroquinolone did not necessarily indicate resistance to all others .
Levofloxacin, an isomer of ofloxacin, has demonstrated improved in vitro potency and reduced toxicity compared to its second generation precursor . In 2008, levofloxacin, or more specifically Levaquin (Janssen Incorporation), received approval by the FDA as an alternative therapeutic for the treatment of IA . A 60-day regimen of Levaquin should be administered to both adult and pediatric patients. However, adults and children weighing > 50 kg should be given 500 mg/kg every day (QD) while children weighing < 50 kg should be administered 8 mg/kg BID [75, 79]. Side effects of Levaquin have only been assessed in adult populations and are similar to those of other fluoroquinolones (tendon rupture and tendinopathy, peripheral neuropathy, arthralgia, myalgia, dermatologic reactions, thrombocytopenia, and interstitial nephritis) . In addition, in some juvenile animal studies quinolones have been associated with osteochondrosis [74, 80]. Although the safety profile of using levofloxacin long term (for example in a 60-day regimen) is still currently unknown, due to the gravity of IA and to the current lack of approved alternative antibiotics, the benefits of using a fluoroquinolone such as levofloxacin greatly out-weigh the risks.
Moxifloxacin, another third generation quinolone, has also been assayed for its effectiveness against B. anthracis. In a recent study, a hollow-fiber pharmacodynamic model (IPDM) was used to compare the efficacies of moxifloxacin, linezolid and meropenem to the currently prescribed antibiotics in killing the spore forming Sterne and non-spore forming CR4 B. anthracis strains . Against the CR4 strain, meropenem killed the fastest followed by the fluoroquinolones (moxifloxacin and ciprofloxacin), with doxycycline and linezolid exhibiting the slowest kill rate. Heat-shock studies demonstrated that the bacterial populations exposed to bactericidal antibiotics (fluoroquinolones and meropenem) consisted primarily of spores while populations treated with doxycycline and linezolid consisted primarily of replicating bacteria. A possible reason for the latter could be because linezolid and doxycycline are protein synthesis inhibitors and may have prevented the population from converting into the spore form [73, 81]. Although these bacteriostatic antibiotics kill at a slower rate, their potential ability to increase the window of antibiotic exposure due to the prevention of spore formation may result in better overall rate of clearance of the total population. These findings were corroborated by another group who compared the efficacy of linezolid to ciprofloxacin in treating B. anthracis Sterne strain infections in an IPDM . Their study demonstrated that a dose of 600 mg of linezolid was sufficient to prevent vegetative B. anthracis from converting to spores while a 500 mg dose of ciprofloxacin BID was not .
To date, linezolid has not been approved by the FDA for the treatment of B. anthracis infections. However, it has been approved for the treatment of a variety of other Gram-positive microorganisms including methicillin-resistant Staphylococcus aureus (MRSA) and multidrug resistant Streptococcus pneumonia under the name Zyvox (Pfizer Canada Inc) . Linezolid is a bacteriostatic oxazolidinone that inhibits bacterial protein synthesis by preventing the formation of the 70S ribosomal complex . Since linezolid has such a unique mode of action, cross resistance with other antibiotic classes is unlikely and has not yet been observed. Thus, linezolid is an attractive therapeutic for penicillin or fluoroquinolone resistant bioterrorism agents. In vitro linezolid resistance occurs at a frequency of approximately 10−10 and has been associated with point mutations in the bacterial 23S rRNA gene . Studies have shown that linezolid can reduce the production of the S. aureus toxin, toxic shock syndrome toxin-1 . Since linezolid is a protein synthesis inhibitor that can decrease toxin production in other Gram-positive organisms it is reasonable to consider that it may also prevent B. anthracis toxin production. Louie et al. looked at the effect of ciprofloxacin and linezolid on B. anthracis PA production at various times throughout a 10-day experiment . They detected PA in the control after 3 h and in levofloxacin from 3 to 8 h. However, no PA was observed with linezolid treatment . In a similar manner, linezolid has also been found to prevent spore production while non-protein synthesis inhibitors, such as moxifloxacin and meropenem, could not . These findings support the hypothesis that a protein synthesis inhibitor (i.e. linezolid) may be a practical way to prevent the deleterious effects of the anthrax toxins while preventing sporulation. Studies looking at resistance to, and the pharmacodynamics of, linezolid have found that a pharmacodynamically optimized regimen of 700 mg QD did not lead to resistance and was just as effective at killing as the clinically prescribed linezolid (600 mg BID) and gold standard ciprofloxacin (500 mg BID) . Not only does this dosing regimen decrease the total dosage patients would be exposed to, but it would also aid in patient compliance by decreasing the dose frequency from BID to QD making it more feasible and more cost effective.
Aside from being a protein synthesis inhibitor, linezolid also has excellent bioavailability and is available in both oral and i.v. formulations [52, 82, 85, 86]. Conversely, one drawback to linezolid is that it is quite costly. The average price of medication for one patient for one day costs approximately $140 Canadian dollars (CAD) which is much higher than levofloxacin ($2 CAD), ciprofloxacin ($2.50 CAD), and doxycycline ($3 CAD) combined making linezolid undesirable if a 60-day regimen is required [87–90]. In addition, although linezolid is considered relatively safe when used for a short term (<2 weeks), side effects such as peripheral neuropathy, thrombocytopenia, and neutropenia have been associated with long term use (>28 days) [86, 91–94]. This toxicity may, however, be reduced by modifying the currently acceptable linezolid regimen to the suggested pharmacodynamic dosage schedule suggested by Louie et al. . By the same token, linezolid could be used initially in the event of a bioterror attack and once susceptibilities of the agent have been determined patients could switch to alternative antibiotics such as ciprofloxacin, penicillin or doxycycline.
A newer class of antibiotic, the cyclic lipopeptides, and more specifically daptomycin, has recently gained more interest as a possible surrogate for anthrax therapy. This agent binds to the bacterial membrane depolarizing the membrane potential resulting in DNA, RNA and protein synthesis inhibition ultimately leading to cell death [95–97]. This unique mechanism of action sets daptomycin apart from other antibiotic classes and to date, no resistant isolates have been documented. Currently, cyclic lipopeptides are only approved for the treatment of antimicrobial resistant Gram-positive microorganisms like those of the staphylococcus, streptococcus and enterococcus genera . However, studies have demonstrated that this class is also efficacious against B. anthracis in vitro [95, 96]. One study in particular found that a 21-day treatment of daptomycin (SC injection of 50 mg/kg BID) was as effective as a 21-day treatment of ciprofloxacin (intraperitoneal [IP] injection of 30 mg/kg of body weight BID) in treating B. anthracis Ames strain infected mice with both treatments yielding survival rates of 90 % . Moreover, no bacteria (vegetative or spores) were cultivable from the lung, spleen and mediastinum samples collected from all of the surviving mice . This mouse inhalational study suggests daptomycin may be a potential candidate as an alternative therapeutic and further in vivo B. anthracis studies are warranted.
Several studies have evaluated the effect of combination therapy in order to further increase the rate of bacterial killing and to circumvent bacterial resistance [98, 99]. The combination of rifampin and clindamycin and the combination of telithromycin and amoxicillin have been shown to work synergistically against the B. anthracis Sterne strain . Other therapies containing a combination of either ciprofloxacin or tetracycline with clindamycin, rifampin, or linezolid have demonstrated indifference while combinations containing penicillin were found to be antagonistic . Similarly, when combined, linezolid and levofloxacin have been shown to behave antagonistically and resulted in decreased B. anthracis killing . Due to the alarming increase in antibiotic resistance and the resilience of B. anthracis, further research determining effective combination therapies is merited and will be invaluable.
Exploiting novel targets for antibiotic development
With an increase in antimicrobial resistance, there is a greater need for the development of newer classes of antimicrobials. Studying essential bacterial processes such as DNA replication, RNA synthesis, and cell division that are currently underexploited would potentially broaden our arsenal against this microbe by leading to new and innovative antibiotics for which there is no pre-existing bacterial resistance mechanism.
Targeting bacterial DNA replication has been successful in the past; however, specifically targeting the bacterial DNA helicase or primase are relatively new ideas [2, 101–103]. Through the use of high-throughput screening, several coumarin-based helicase inhibitors were identified for their efficacy against B. anthracis and S. aureus . Chemical optimization of these inhibitors led to the discovery of two potent biphenyl coumarin compounds (20 and 22) with half maximal inhibitory concentration (IC50) values of 3 and 1 μM (against both microorganisms) that worked non-competitively when inhibiting DNA helicase . In contrast, benzobisthiazole helicase inhibitors and their derivatives work in a competitive manner with DNA and ATP substrates by binding to the helicase active site . Compound 59, the most potent helicase inhibitor identified to date, has a high selectivity index (greater than 500), no observable cytotoxicity, and inhibits B. anthracis with an IC50 of 0.2 μM .
In a recently reported dose–response assay, doxorubicin (an interferon inhibitor), and tilorone (an interferon inducer) demonstrated low micromolar inhibitory activities towards the B. anthracis 34 F2 Sterne strain primase DnaG . One challenge for making DnaG inhibitors is that in order to be effective these compounds must be able to access the bacterial cytoplasm. Doxorubicin was able to traverse the bacterial envelope and exert its bacteriostatic effects on the 34 F2 Sterne strain with a MIC of 6.6 μM; however, tilorone could not . Since DnaG is an essential enzyme for chromosomal DNA replication, is moderately conserved among bacterial species (30 % sequence identity between Bacillus and Mycobacterium), and shares very low sequence similarity to the eukaryotic topoisomerase II, targeting this enzyme could lead to a novel inhibitor with high species specificity [107–109].
Anthracimycin, another inhibitor that targets the DNA replication process, is a structurally unique compound composed of a rare combination of 14-and 6-member rings and is produced by a marine-derived actinomycete . This tricarbocyclic metabolite has demonstrated good potency against the B. anthracis UM23C1-1 strain with a MIC of 0.031 μg/mL as well as inhibitory activities against other Gram-positive microorganisms (staphylococci, enterococci, streptococci) . Chlorination of anthracimycin leads to improved bioactivity against a broader spectrum of microbes (including Gram-negatives) while retaining significant potency against B. anthracis (MIC of 0.0625 μg/mL) . Although the exact mechanism of action is yet to be elucidated, it is thought to be a DNA and RNA synthesis inhibitor. Furthermore, it has also been suggested that anthracimycin works synergistically with the cathelicidin, LL-37, making microorganisms more sensitive to cathelicidin-mediated killing [111, 112]. LL-37 plays an important part in the host immune response especially in the recruitment of neutrophils, monocytes, and T cells and has been shown to play a role in containing the B. anthracis infection [113–115]. Having an antibiotic that works synergistically with a host cationic peptide may increase the initial immune response when exposed to B. anthracis. Although in vivo studies looking at the effect of anthracimycin of B. anthracis have yet to be carried out, one study looking at the effect of this novel compound on MRSA-infected CD1 mice found that anthracimycin retained high potency in vivo and was well tolerated . Due to its broad spectrum abilities, high potency, and possible host synergy, anthracimycin and its derivatives represent a potential new and unique class of antibiotics. Further studies are required to determine the exact mechanism of action by which this compound exerts its effects as well as safety, resistance and appropriate dosing.
Aside from hindering bacterial DNA replication, targeting cell-to-cell cross-talk or other important cell processes (nutrient acquisition or cell division) have also proven to be invaluable [116–118]. The compound 3Z1, which is a FtsZ-targeting oligoclorophen, was recently explored as a clinical agent and was found to have a MIC of 320 nM against the B. anthracis Sterne strain (comparable to tetracycline and penicillin G) . Aside from being a potent antimicrobial, developing bacterial resistance to 3Z1 may also be difficult since FtsZ is a conserved protein essential for bacterial cell division [120–122]. Indeed, when studies looked at resistance they found that the B. anthracis Sterne 7702 strain developed resistance at a much lower rate to the FtsZ inhibitor, 3Z1, compared to rifampin (4.34x1010 and 2.65x109 per generation, respectively) .
Baulamycins, produced by Streptomyces tempisquensis, target bacterial siderophore synthesis genes necessary for iron sequestration important for growth and survival in iron deficient environments . B. anthracis produces an iron scavenger named petrobactin, which is synthesized by a nonribosomal peptide synthetase independent siderophore synthetase, AsbA [118, 123]. Baulamycins A and B inhibited AsbA with an in vitro IC50 of 180 μM and 200 μM, respectively, and demonstrated good cell solubility [118, 124–126]. Moreover, these compounds exhibited broad spectrum activities against MRSA, Escherichia coli, and Shigella flexneri. Further studies are necessary to improve potency and target selectivity; however, baulamycins show great potential.
Targeting the bacterial quorum sensing mechanism which co-ordinates many behaviors (colonization, persistence and often virulence) has been shown to attenuate other pathogenic bacteria . Many Bacillus, including B. anthracis, synthesize small autoinducers that allow the co-ordination of the toxin genes and cell growth. A study by Jones et al. looked at the effect of (5Z)-4-bromo-5-(bromo-methylene)-3-butyl-2(5H)-furanone and several furanone derivatives on the B. anthracis autoinducer, Al-2 . Not only did the furanones inhibit log-phase growth on multiple B. anthracis strains when added 3 h post-inoculation, but they also significantly reduced toxin gene expression. Moreover, these naturally synthesized furanones have been shown to be stable under storage conditions . Although quorum sensing systems have the potential to lead to promising new therapeutics, little work has been collected on their toxicity in animals; therefore further analysis is still required.
Currently, the main priority for treating many illnesses is to eliminate the replicating bacteria. However, in the case of anthrax, preventing the effects of the toxins is equally important when combatting late stage disease. Throughout the last decade, extensive research has been conducted looking at B. anthracis anti-toxins in order to find cheaper, more stable and immunogenic molecules, albeit very few anti-toxins are currently approved for anthrax treatment. Toxin inhibitors can target several steps in the toxin entry process, which include: (i) PA83 binding to host receptor, (ii) PA83 cleavage, (iii) PA83 heptamerization, (iv) LF or EF binding, (v) LF proteolysis, (vi) LF inflammasome activation, and (vii) EF adenylyl cyclase activity (Fig. 3). Since PA is essential for toxin entry and is a component of both ET and LT, many studies have focused on targeting this component (for a full review see Chen, Moayeri, and Purcell ). Raxibacumab (Abthrax; GlaxoSmithKline), a human IgG1 monoclonal antibody (mAb) against PA, received FDA approval in 2012 for treating anthrax based on the Animal Efficacy Rule [128–130]. It binds PA with an affinity of 2.78 nM and prevents PA-receptor binding . It is recommended for treating adult and pediatric patients with IA and should be administered in combination with antibacterial drugs. Indeed, when combined with ciprofloxacin, raxibacumab demonstrated potency and did not affect ciprofloxacin function . In addition, it is recommended for IA prophylaxis if alternative therapies are not unavailable . In adults, a 40 mg/kg dose of Raxibacumab should be diluted in 0.9 % Sodium Chloride, USP to a final volume of 250 mL then administered as a single i.v. dosage over 2 h and 15 min. [128, 130] To reduce the risk of reaction, 25–50 mg of diphenhydramine should be administered within 1 h of Raxibacumab. Common adverse reactions include rash, pain in extremity, pruritus, and somnolence . To date, Raxibacumab is the best anti-PA option available; however, there is still opportunity for improvement. This anti-PA mAb cannot cross the blood brain barrier and is not antibacterial; therefore it cannot prevent or treat meningitis (often a consequence of late stage anthrax).  In addition, for storage, this medication must be kept refrigerated (2 to 8 °C) and should not be exposed to light. Furthermore, modifying the current route of administration from i.v. to either SC or intramuscular would be more desirable in the event of a bioterror attack.
Anthim (Elusys Therapeutics), a humanized anti-PA mAb that also targets PA-receptor binding, binds PA with an affinity of 0.33 nM . As mentioned by Chen et al., this drug has received Fast-track and orphan drug status since it has demonstrated efficacy in both pre and post-exposure situations in various animal models . In studies where Anthim has been co-administered with levofloxacin, ciprofloxacin or doxycycline, IA-infected animals demonstrated higher survival outcomes compared to solo antibacterial therapy [127, 129]. In fact, in 2016, Anthim received FDA approval as an alternative treatment for adult and pediatric patients with IA and is recommended in combination with antibacterial drugs (ciprofloxacin) when alternatives are unavailable . Due to hypersensitivity reactions and anaphylaxis, Anthim is only recommended if its benefit outweighs the risk. Similar to Raxibacumab, patients must be pre-medicated with diphenhydramine and Anthim must be diluted in Sodium Chloride prior to use. Medication should be administered as a single i.v. dose of 16 mg/kg over 1 h and 30 min. In addition, Anthim does not have antibacterial activity, cannot cross the blood–brain barrier and requires storage in a dark, refrigerated (2 to 8 °C) area.
Targeting the next step in the toxin entry process, PA83 cleavage by Furin, has shown some merit in several studies [32, 132, 133]. Inter-α inhibitor protein (IαIp), a human serine protease, inhibits furin with good efficacy resulting in post-exposure protection in B. anthracis Sterne 34 F2-infected AJ mice when combined with moxifloxacin . In addition, treatment with this combination led to normal liver and spleen histopathology with no bacilli present. Since IαIp have been shown to be effective in murine models, the next step is to determine their efficacy in larger IA animal models. Some IαIp, like urinary trypsin inhibitors, have already demonstrated their safety in clinical trials and their potential for the treatment of anthrax disease seems promising [134, 135].
Another PA process that can be targeted is the formation of PA heptamers which enables LF and EF entry to the cell. Valortin (PharmAthene/Medarex) is a fully human anti-PA mAb generated from transgenic mice that interrupts PA heptamerization. This inhibitor has demonstrated prophylactic efficacy in rabbits and monkeys and has obtained Fast-Track, Orphan drug status .
As a result of more knowledge and understanding of both EF and LF structure and function, a variety of novel, small molecule inhibitors, antibodies and other drug-like, anti-toxins have been discovered. Curcumin, the active ingredient in turmeric spice (Curcuma longa), is often used in traditional medicine and has demonstrated beneficial activities in combating cancers, inflammatory diseases and anthrax [136–139]. Of late, curcumin has been shown to inhibit metalloproteinases (including LF) by binding to the zinc in their active site [138–141]. Furthermore, chemically modified curcumin has demonstrated improved solubility, stability, and bioavailability, with similar potency and less toxicity [138, 139]. Other lethal factor inhibitors (LFI) that contain a zinc chelating group are R9LF-1, R9LF-2 and modified peptidomimetic inhibitors (MPI) [142–145]. Both R9LF-1 and R9LF-2 are stable in solution and are efficient at inhibiting LF in kinetic assays. However, in a longer murine macrophage assay, the stability of R9LF-1 decreased drastically while R9LF-2 had better stability and macrophage protection. MPI containing portions of either BI-MFM3 (Cengent Therapeutics Incorporated) or L915 (Merck Research Laboratories) have demonstrated high binding abilities to both the LF substrate-binding groove and the catalytic zinc-binding site leading to good LF inhibition.
Aside from targeting LF’s proteolytic ability, studies have also looked at inhibiting LF’s ability to activate the NLRP1 inflammasome (i.e. caspase-mediated apoptosis) [146–149]. Both Auranofin (an organogold compound with anti-inflammatory abilities) and Idebenone (a benzoquinone that has previously been used in Alzheimer’s patients) are speculated to interfere with inflammasome activation. Specifically, Auranofin inhibits LT-mediated caspase-1 activation and catalytic activity while Idebenone inhibits the voltage-gated potassium channels. When combined, these two compounds work synergistically to strongly inhibit the LT activity of B. anthracis [146–149].
Although majority of studies have focused on PA and LF inhibition, significant research into EF inhibition has also been conducted (reviewed extensively in ). EF inhibition can occur through different ways including targeting the adenylyl cyclase, substrate binding, and allosteric sites. To date, the most promising anti-EF molecule is EF13D, a chimeric chimpanzee/human mAb that neutralizes EF with a very high affinity of 0.05–0.12 nM . Studies have shown that EF13D prevents edema formation as well as rescues ET-challenged mice. It functions by binding CaM and can also displace pre-bound CaM from EF.
Since CaM stimulates EF catalytic activity, certain studies have targeted CaM and the CaM-target interaction [37, 151–153]. Several well-known, potent CaM-inhibitors that have already been discovered and produced for their use in other illnesses, such as depression and psychosis, have demonstrated good efficacy against EF [151, 154–157]. Clomipramine (antidepressant), fluphenazine (antipsychotic), penfluridol (antipsychotic), and trifluoperazine (antipsychotic), were able to inhibit EF by 20 %, 30 %, 45 % and 40 %, respectively. In addition, Calmidazolium chloride (CDZ) was able to abolish EF activity all together . Interestingly, CDZ inhibits EF through an allosteric mechanism (while the other EF inhibitors directly target the CaM-EF binding region) . Unfortunately, CDZ often affects unintentional targets . In a like manner, P-site inhibitors (such as N-methyl anthraniloyl-nucleotides [(M)ANT-nucleotides] and adefovir), that function by targeting the adenylyl cyclase catalytic site, are non-selective between EF and mammalian adenylyl cyclases. Therefore, to date, have not been clinically useful.
An adenylyl cyclase inhibitor that has demonstrated good potential as an EF inhibitor is the fluorine-based compound, DC5 [159, 160]. This compound can inhibit EF with a more potent IC50 than prostaglandin E2-imidazole (a previously described front-runner EF inhibitor). In addition, it can prevent toxin-induced cAMP accumulation from both enterotoxinogenic E. coli and B. anthracis [159, 160]. Moreover, through modification of its aromatic group, DC5 derivatives have become more soluble and less toxic (but equally potent) compared to their parent compound.
Since the EF catalytic site has demonstrated close similarity to other bacterial adenylyl cyclases, such as the heat-labile toxin of enterotoxinogenic E. coli and the cholera toxin, it may be possible to synthesize EF inhibitors with broad spectrum activity [159, 160]. However, since bacterial and mammalian adenylyl cyclase catalytic sites also share homology, constructing highly selective and potent EF inhibitors may be difficult . Therefore, research looking at targeting EF allosteric sites is also recommended, albeit caution is recommended since targeting allosteric sites, as seen with CDZ, can have off-target effects . Collectively, studies have demonstrated how problematic it has been to create a soluble, highly selective, and potent EF inhibitor.
It is known that combination therapy can have considerable benefits. Particularly, combining toxin inhibitors with antibiotics has proven to be a valuable way to combat several infections from Pseudomonas spp, klebsiella spp and B. anthracis [127, 129–131, 161–164]. Indeed, in a study by Karginov and colleagues, solo treatment with ciprofloxacin was only able to rescue 50 % of the Sterne-infected mice while the combination of ciprofloxacin and anti-PA antibodies was able to rescue more than 90 % . These studies, and others, reiterate the fact that combination therapy may be the most promising means for combatting B. anthracis.
Although B. anthracis has been a microorganism of high interest for many years, anthrax still remains a dangerous disease that is often untreatable. A great deal of progress has been made in anthrax therapies with many novel antibiotics and toxin inhibitors showing great potential. Utilizing antibiotics that have already been approved for the treatment of other bacterial infections may prove to be an asset in treating anthrax. Furthermore, targeting the anthrax toxins could increase the length of which treatment may be administered. A combination treatment that targets both bacterial growth and toxin production would be ideal and probably necessary for effectively combatting this armed bacterium.
Anthrax vaccine absorbed
Anthrax vaccine precipitated
Twice a day
5′-cyclic adenosine monophosphate
Colony forming units
Capillary morphogenesis protein-2
Food and drug administration
- IC50 :
Half maximal inhibitory concentration
In vitro hollow fiber pharmacodynamic model
Inter-α Inhibitor protein
Lethal factor inhibitors
Mitogen-activated protein kinase kinases
Minimum inhibitory concentration
Modified peptidomimetic inhibitors
Methicillin-resistant Staphylococcus aureus
20 kDa PA
63 kDa PA
83 kDa PA
Tumor endothelial marker 8
Kolsto AB, Tourasse NJ, Okstad OA. What sets Bacillus anthracis apart from other bacillus species. Annu Rev Microbiol. 2009;63:451–76.
Inglesby T, O`Toole T, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, et al. Anthrax as a biological weapon, 2002, updated recommendations for management. JAMA. 2002;287:2236–52.
Hicks CW, Sweeney DA, Cui X, Li Y, Eichacker PQ. An overview of anthrax infection including the recently identified form of disease in injection drug users. Intensive Care Med. 2012;38:1092–104.
Dugdale D, Vyas JM, Zieve D. Anthrax. A.D.A.M. Medical Encyclopedia. 2015. https://medlineplus.gov/ency/article/001325.htm. Accessed 24 Oct 2016.
Doganay M, Metan G, Alp E. A review of cutaneous anthrax and its outcome. J Infect Public Health. 2010;3:98–105.
Demirdag K, Ozden M, Saral Y, Kalkan A, Kilic SS, Ozdarendeli A. Cutaneous anthrax in adults: A review of 25 cases in the Eastern Anatolian Region of Turkey. Infection. 2003;31:327–30.
Kanafani ZA, Ghossain A, Sharara AI, Hatem JM, Kanj SS. Endemic gastrointestinal anthrax in 1960s Lebanon: Clinical manifestations and surgical findings. Emerg Infect Dis. 2006;9:520–5.
Onerci M, Ergin NT. Oropharyngealer Milzbrand (Oropharyngeal anthrax). Laryngorhinootologie. 1993;72:350–1.
Barakat LA, Quentzel HL, Jernigan JA, Kirschke DL, Griffith K, Spear SM, et al. Fatal inhalational anthrax in a 94-year-old connecticut woman. JAMA. 2002;287:863–8.
Jon E, Holty C, Bravata DM, Hau L, Olshen RA, McDonald KM, et al. Systematic Review: A century of inhalational anthrax cases from 1900 to 2005. Ann Intern Med. 2006;144:270–80.
Jernigan JA, Stephens DS, Ashford DA, Omenaca C, Topiel MS, Galbraith M, et al. Bioterrorism-related inhalational anthrax: the first 10 cases reported in the United States. Emerg Infect Dis. 2001;7:933–44.
Ringertz SH, Høiby EA, Jensenius M, Mæhlen I, Caugant DA, Myklebust A, et al. Injectional anthrax in a heroin skin-popper. Lancet. 2000;356:1574–5.
Ramsay CN, Stirling A, Smith J, Hawkins G, Brooks T, Hood J, et al. An outbreak of infection with Bacillus anthracis in injecting drug users in Scotland. Euro Surveill. 2010;15:19465.
Powell AG, Crozier JE, Hodgson H, Galloway DJ. A case of septicemic anthrax in an intravenous drug user. BMC Infect Dis. 2011. doi:10.1186/1471-2334-11-21.
Radun D, Bernard H, Altmann M, Schoneberg I, Bochat V, Van T, et al. Preliminary case report of fatal anthrax in an injecting drug user in North-Rhine-Westphalia, Germany, December 2009. Euro Surveill. 2010;15:19464.
Keim P, Smith KL, Keys C, Takahashi H, Kurata T, Kaufmann A. Molecular investigation of the Aum Shinrikyo anthrax release in Kameido, Japan. J Clin Microbiol. 2001;39:4566–7.
Jernigan DB, Raghunathan PL, Bell BP, Brechner R, Bresnitz EA, Butler JC, et al. Investigation of bioterrorism-related anthrax, United States, 2001: epidemiologic findings. Emerg Infect Dis. 2002;8:1019–28.
Abramova FA, Grinberg LM, Yampolskaya OV, Walker DH. Pathology of inhalational anthrax in 42 cases from the Sverdlovsk outbreak of 1979. Proc Natl Acad Sci U S A. 1993;90:2291–4.
Doganay M, Metan G. Human anthrax in Turkey from 1990 to 2007. Vector Borne Zoonotic Diseases. 2009;9:131–40.
Ozkurt Z, Parlak M, Tastan R, Dinler U, Saglam YS, Ozyurek SF. Anthrax in eastern Turkey, 1992—2004. Emerg Infect Dis. 2005;11:1939–41.
Louisiana State University School of Veterinary Medicine: World Anthrax Data Site. http://www.vetmed.lsu.edu/whocc/mp_world.html. 2003. Accessed 3 March 2014
Hugh-Jones M. 1996–97 global anthrax report. J Appl Microbiol. 1991;87:189–91.
Velimirovic B. Bacillus anthracis in Europe. Rev Sci Tech. 1984;3:527–59.
Chakraborty A, Khan SU, Hasnat MA, Parveen S, Islam SM, Mikolon A, et al. Anthrax Outbreaks in Bangladesh, 2009–2010. Am J Trop Mede Hyg. 2012;86:703–10.
Del Giudice P. Cutaneous complications of intravenous drug abuse. Br J Derm. 2004;150:1–10.
Meselson M, Guillemin J, Hugh-Jones M, Langmuir A, Popova I, Shelokov A, et al. The sverdlovsk anthrax outbreak of 1979. Science. 1994;266:1202–8.
Bourgogne A, Drysdale M, Hilsenbeck SG, Peterson SN, Koehler TM. Global Effects of Virulence Gene Regulators in a Bacillus anthracis Strain with both Virulence Plasmids. Infect Immun. 2003;71:2736–43.
Chitlaru T, Altboum Z, Reuveny S, Shafferman A. Progress and novel strategies in vaccine development and treatment of anthrax. Immunol Rev. 2010;239:221–36.
Park JM, Greten FR, Li ZW, Karin M. Macrophage Apoptosis by Anthrax Lethal Factor through p38 MAP Kinase Inhibition. Science. 2002;297:2048–51.
Carson-Walter EB, Watkins DN, Nanda A, Vogelstein B, Kinzler KW, St Croix B. Cell surface tumor endothelial markers are conserved in mice and humans. Cancer Res. 2001;61:6649–55.
Scobie HM, Rainey GJ, Bradley KA, Young JA. Human capillary morphogenesis protein 2 functions as an anthrax toxin receptor. Proc Natl Acad Sci U S A. 2003;100:5170–4.
Klimpel KR, Molloy SS, Thomas G, Leppla SH. Anthrax toxin protective antigen is activated by a cell surface protease with the sequence specificity and catalytic properties of furin. Proc Natl Acad Sci U S A. 1992;89:10277–81.
Cunningham K, Lacy DB, Mogridge J, Collier RJ. Mapping the lethal factor and edema factor binding sites on oligomeric anthrax protective antigen. Proc Natl Acad Sci U S A. 2002;99:7049–53.
Lacy DB, Mourez M, Fouassier A, Collier RJ. Mapping the anthrax protective antigen binding site on the lethal and edema factors. J Biol Chem. 2002;277:3006–10.
Miller CJ, Elliott JL, Collier RJ. Anthrax protective antigen: prepore-to-pore conversion. Biochem. 1999;38:10432–41.
Barth H, Aktories K, Popoff MR, Stiles BG. Binary bacterial toxins: Biochemistry, biology, and applications of common clostridium and bacillus proteins. Microbiol Mol Biol Rev. 2004;68:373–402.
Leppla SH. Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations in eukaryotic cells. Proc Natl Acad Sci U S A. 1982;79:3162–6.
John AT, Collier J. Anthrax toxin: receptor binding, internalization, pore formation, and translocation. Annu Rev Biochem. 2007;76:243–65.
Molloy S, Bresnahan P, Leppla S, Klimpel KR, Thomas G. Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Argx-x-arg and efficiently cleaves anthrax toxin protective antigen. J Biol Chem. 2002;267:16396–402.
Tournuer JN, Paccani SR, Quesnel-Hellmann A, Baldari CT. Anthrax Toxins: A weapon to systematically dismantle the host immune defenses. Mol Aspects Med. 2009;30:456–66.
Moayeri M, Leppla SH. Cellular and systemic effects of anthrax lethal toxin and edema toxin. Mol Aspects Med. 2009;30:439–55.
Moayeri M, Leppla SH, Vrentas C, Pomerantsev AP, Liu S. Anthrax Pathogenesis. Annu Rev Microbiol. 2015;69:185–208.
Firoved AM, Miller GF, Moayeri M, Kakkar R, Shen Y, Wiggins JF, et al. Bacillus anthracis edema toxin causes extensive tissue lesions and rapid lethality in mice. Am J Pathol. 2005;167:1309–20.
Liu S, Zhang Y, Moayeri M, Liu J, Crown D, Fattah RJ, et al. Key tissue targets responsible for anthrax-toxin-induced lethality. Nature. 2013;501:63–8.
Suffredini DA, Sampath-Kumar H, Li Y, Ohanjanian L, Remy KE, Cui X, Eichacker PQ. Does Bacillus anthracis Lethal Toxin Directly Depress Myocardial Function? A Review of Clinical Cases and Preclinical Studies. Toxins. 2015;7:5417–34.
Department of Health and Human Services. Prescription drug products; doxycycline and penicillin G procaine administration for inhalational anthrax (post-exposure). Food and Drug Administration. 2001;66:55679–82.
U. S. Food and Drug Administration. Ciprofloxacin [Fact sheet]. 2013. http://www.accessdata.fda.gov/drugsatfda_docs/label/2013/019857s062lbl.pdf. Accessed 15 July 2014.
Henderson DW, Peacock S, Belton FC. Observations on the prophylaxis of experimental pulmonary anthrax in the monkey. J Hygiene. 1956;54:28–36.
Friedlander AM, Welkos SL, Pitt ML, Ezzell JW, Worsham PL, Rose KJ, et al. Postexposure prophylaxis against experimental inhalation anthrax. J Infect Dis. 1993;167:1239–43.
Kao LM, Bush K, Barnewall R, Estep J, Thalacker FW, Olson PH, et al. Pharmacokinetic considerations and efficacy of levofloxacin in an inhalational anthrax (postexposure) rhesus monkey model. Antimicrob Agents Chemother. 2006;50:3535–42.
Louie A, VanScoy BD, Brown DL, Kulawy RW, Heine HS, Drusanoa GL. Impact of spores on the comparative efficacies of five antibiotics for treatment of Bacillus anthracis in an in vitro hollow fiber pharmacodynamic model. Antimicrob Agents Chemother. 2012;56:1229–39.
Louie A, VanScoy BD, Heine III HS, Liu W, Abshire T, Holman K, et al. Differential effects of linezolid and ciprofloxacin on toxin production by Bacillus anthracis in an in vitro pharmacodynamic system. Antimicrob Agents Chemother. 2011;56:513–7.
Shepard CW, Soriano-Gabarro M, Zell ER, Hayslett J, Lukacs S, Goldstein S, et al. Antimicrobial postexposure prophylaxis for anthrax: Adverse events and adherence. Emerg Infect Dis. 2002;8:1124–32.
Centers for Disease Control and Prevention. Update: Investigation of bioterrorism-related anthrax and adverse events from antimicrobial prophylaxis. MMWR. 2001;50:973–6.
Athamna A, Athamna M, Abu-Rashed N, Medlej B, Bast DJ, Rubinstein E. Selection of Bacillus anthracis isolates resistant to antibiotics. J Antimicrob Chemother. 2004;54:424–8.
Brook I, Elliott TB, Pryor II HI, Sautter TE, Gnade BT, Thakar JH, et al. In vitro resistance of Bacillus anthracis Sterne to doxycycline, macrolides and quinolones. Int J Antimicrob Agents. 2001;18:559–62.
Pomerantsev AP, Shishkova NA, Marinin LI. Comparison of therapeutic effects of antibiotics of the tetracycline group in the treatment of anthrax caused by a strain inheriting tet-gene of plasmid pBC16. Antibiot Khimioter. 1992;37:31–4.
Chen Y, Tenover FC, Koehler TM. β-Lactamase Gene Expression in a Penicillin-Resistant Bacillus anthracis Strain. Antimicrob Agents Chemother. 2004;48:4873–7.
Lalitha MK, Thomas MK. Penicillin resistance in Bacillus anthracis. Lancet. 1997;349:1522–4.
Price LB, Vogler A, Pearson T, Busch JD, Schupp JM, Keim P. In vitro selection and characterization of Bacillus anthracis mutants with high-level resistance to ciprofloxacin. Antimicrob Agents Chemother. 2003;47:2362–5.
Baillie L, Hebdon R, Flick-Smith H, Williamson D. Characterisation of the immune response to the UK human anthrax vaccine. FEMS Immunol Med Microbiol. 2004;42:267–70.
Crowe SR, Garman L, Engler RJM, Farris AD, Ballard JD, Harley JB, et al. Anthrax vaccination induced anti-lethal factor IgG: Fine specificity and neutralizing capacity. Vaccine. 2011;29:3670–8.
Stern EJ, Uhde KB, Shadomy SV, Messonnier N. Conference report on public health and clinical guidelines for anthrax. Emerg Infect Dis. 2008. doi:10.3201/eid1404.070969.
Rynkiewicz D, Rathkopf M, Sim I, Waytes AT, Hopkins RJ, Girid L, et al. Marked enhancement of the immune response to BioThrax®(Anthrax Vaccine Adsorbed) by the TLR9 agonist CPG 7909 in healthy volunteers. Vaccine. 2011;29:6313–20.
Roy J, Kumar UC, Machiraju PK, Muttineni RK, Kumar S, Gundla R, et al. In silico studies on anthrax lethal factor inhibitors: Pharmacophore modeling and virtual screening approaches towards designing of novel inhibitors for a killer. J Mol Graph Model. 2010;29:256–65.
Ionin B, Hopkins RJ, Pleune B, Sivko GS, Reid FM, Clement KH, et al. Evaluation of immunogenicity and efficacy of anthrax vaccine adsorbed for postexposure prophylaxis. Clin Vaccine Immunol. 2013;20:1016–26.
Gorse GJ, Keitel W, Keyserling H, Taylor DN, Lock M, Alves K, et al. Immunogenicity and tolerance of ascending doses of a recombinant protective antigen (rPA102) anthrax vaccine: a randomized, double-blinded, controlled, multi-center trial. Vaccine. 2006;24:5950–9.
Campbell JD, Clement KH, Wasserman SS, Donegan S, Chrisley L, Kotloff KL. Safety, reactogenicity and immunogenicity of a recombinant protective antigen anthrax vaccine given to healthy adults. Hum Vacc Immunother. 2007;3:205–11.
Wasserman GM, Grabenstein JD, Pittman PR, Rubertone MV, Gibbs PP, Wang LZ, et al. Analysis of adverse events after anthrax immunization in US Army medical personnel. J Occup Environ Med. 2003;45:222–33.
Ryan MAK, Smith TC, Sevick CJ, Honner WK, Loach RA, Moore CA, et al. Birth defects among infants born to women who received anthrax vaccine in pregnancy. Am J Epidemiol. 2008;168:434–42.
Tross D, Klinma DM. Effect of CpG oligonucleotides on vaccine-induced B cell memory. J Immunol. 2008;181:5785–90.
U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research. Guidance for industry product development under the animal rule. 2014. http://www.fdanews.com/ext/resources/files/06/06-02-14-AnimalRule.pdf. Accessed 3 July 2014.
U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research. Guidance for industry inhalational anthrax (post-exposure)- developing antimicrobial drugs. 2002. http://ocw.jhsph.edu/courses/DrugDevelopment/PDFs/DD0FDAGuidance_InhalationalAnthrax.pdf. Accessed 3 July 2014.
U. S. Food and Drug Administration. Floxin tablets [Fact sheet]. 2008. http://www.accessdata.fda.gov/drugsatfda_docs/label/2008/019735s059lbl.pdf. Accessed 3 July 2014.
U. S. Food and Drug Administration. Levaquin [Fact sheet]. 2008. http://www.accessdata.fda.gov/drugsatfda_docs/label/2008/021721s020_020635s57_020634s52_lbl.pdf. Accessed 3 July 2014.
U.S. Food and Drug Administration. FDA- approved medication guide avelox. 2014. http://www.fda.gov/downloads/Drugs/DrugSafety/UCM231731.pdf. Accessed 3 July 2014.
Hooper DC. Mode of action of fluoroquinolones. Drugs. 1999;58:6–10.
Turnidge J. Pharmacokinetics and pharmacodynamics of fluoroquinolones. Drugs. 1999;58 Suppl 2:29–36.
Li F, Nandy P, Chien S, Noel GJ, Tornoe CW. Pharmacometrics-based dose selection of levofloxacin as a treatment for post-exposure inhalational anthrax in children. Antimicrob Agents Chemother. 2010;54:375–9.
Schadd UB. Use of quinolones in pediatrics. Eur J Clin Microbiol Infect Dis. 1991;10:355–60.
U.S. Food and Drug Administration. Zyvox [Fact sheet]. Pfizer Pharmacia and Upjohn Company. 2005. http://www.fda.gov/ohrms/dockets/ac/06/briefing/2006-4254b_02_05_KP%20LinezolidFDAlabel52005.pdf. Accessed 4 July 2014.
Diekema DJ, Jones RN. Oxazolidinone antibiotics. Lancet. 2001;385:1975–82.
Swaney SM, Aoki H, Ganoza MC, Shinabarger DL. The oxazolidinone linezolid inhibits initiation of protein synthesis in bacteria. Antimicrob Agents Chemother. 1998;42:3251–5.
Stevens DL, Ma Y, Salmi DB, McIndoo E, Wallace RJ, Bryant AE. Impact of antibiotics on expression of virulence-associated exotoxin genes in methicillin-sensitive and methicillin-resistant staphylococcus aureus. J Infect Dis. 2007;195:202–11.
Louie A, Heine HS, Kim K, Brown DL, VanScoy B, Liu W, et al. Use of in vitro pharmacodynamics model to derive a linezolid regimen that optimizes bacterial kill and prevents emergence of resistance in Bacillus anthracis. Antimicrob Agents Chemother. 2008;52:2486–96.
Physicians’ Desk Reference. Physician’s desk reference. 61st ed. Montvale: Thomson PDR; 2007. p. 2652–60.
Canada Drugs. Zyvox. 2013. https://www.canadadrugs.com/products/zyvox/600mg. Accessed 12 July 2013.
Canada Drugs. Ciprofloxacin. 2013. https://www.canadadrugs.com/products/cipro/500mg. Accessed 12 July 2013.
Canada Drugs. Vibramycin. 2013. https://www.canadadrugs.com/products/vibramycin-caps/100mg. Accessed 12 July 2013.
Canada Drugs. Levaquin. 2013. https://www.canadadrugs.com/products/levaquin/500mg. Accessed 12 July 2013.
Bishop E, Melvani S, Howden BP, Charles PGP, Grayson ML. Good clinical outcomes but high rates of adverse reactions during linezolid therapy for serious infections: a proposed protocol for monitoring therapy in complex patients. Antimicrob Agents Chemother. 2006;50:1599–602.
Stalker DJ, Wajszczuk CP, Batts DH. Linezolid safety, tolerance, and pharmacokinetics following oral dosing twice daily for 14.5 days [abstract A-115]. In: Program and abstracts of the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy (Toronto). Washington: American Society for Microbiology; 1997. p. 23.
Stalker DJ, Wajszczuk CP, Batts DH. Linezolid safety, tolerance, and pharmacokinetics after intravenous dosing twice daily for 7.5 days. In: 37th Interscience Conference on Antimicrobial Agents and Chemotherapy. Toronto, Canada: 1997. p. abstr A-116.
Rubinstein E, Isturiz R, Standiford C, Smith G, Oliphant TH, Cammarata S, et al. Worldwide assessment of linezolid’s clinical safety and tolerability: comparator-controlled phase III studies. Antimicrob Agents Chemother. 2003;47:1824–31.
U.S. Food and Drug Administration. Cubicin. Cubist Pharmaceuticals, Inc. 2011. http://www.accessdata.fda.gov/drugsatfda_docs/label/2011/021572s038lbl.pdf. Accessed 4 Feb 2015.
Hein HS, Bassett J, Miller L, Purcell BK, Byrne WR. Efficacy of Daptomycin against Bacillus anthracis in a Murine Model of Anthrax spore inhalation. Antimicrob Agents Chemother. 2010;54:4471.
Xing Y, Wang W, Dai S, Lio T, Tan J, Qu G, et al. Daptomycin exerts rapid bactericidal activity against Bacillus anthracis without disrupting membrane integrity. Acta Pharmacol Sin. 2014;35:211–8.
Athamna A, Athamna M, Nura A, Shlyakov E, Bast DJ, Farrell D, Rubinstein E. Is in vitro antibiotic combination more effective than single-drug therapy against Anthrax? Antimicrob Agents Chemother. 2005;49:1323–5.
Karginov VA, Robinson TM, Riemenschneider J, Golding B, Kennedy M, Shiloach J, Alibek K. Treatment of anthrax infection with combination of cipro£oxacin and antibodies to protective antigen of Bacillus anthracis. FEMS Immunol Med Microbiol. 2004;40:71–4.
Head BM, Alfa M, Sitar DS, Rubinstein E, Meyers FA. The in vitro evaluation of the effect of linezolid and levofloxacin on Bacillus anthracis toxin production, spore formation and cell growth. J Antimicrob Chemother. 2016. doi:10.1093/jac/dkw427.
McKay GA, Reddy R, Arhin F, Belley A, Lehoux D, Moeck G, et al. Triaminotriazine DNA helicase inhibitors with antibacterial activity. Bioorg Med Chem Lett. 2006;16:1286–90.
Zhang Y, Yang F, Kao YC, Kurilla MG, Pompliano DL, Dicker IB. Homogenous assays for Escherichia coli DnaB-stimulated DnaG primase and DnaB helicase and their use in screening for chemical inhibitors. Anal Biochem. 2002;304:174–9.
Earnshaw DL, Moore KJ, Greenwood CJ, Djaballah H, Jurewicz AJ, Murray KJ, et al. Time-resolved fluorescence energy transfer DNA helicase assays for high throughput screening. J Biomol Screen. 1999;4:239–48.
Aiello D, Barnes MH, Biswas EE, Biswas SB, Gu S, Williams JD, et al. Discovery, characterization and comparison of inhibitors of Bacillus anthracis and staphylococcus aureus replicative DNA helicases. Bioorg Med Chem Lett. 2009;17:4466–76.
Li B, Pai R, Di M, Aiello D, Barnes MH, Butler MM, et al. Coumarin-Based Inhibitors of Bacillus anthracis and Staphylococcus aureus replicative DNA helicase: chemical optimization, biological evaluation, and antibacterial activities. J Med Chem. 2012;55:10896–908.
Li B, Pai R, Aiello D, Di M, Barnes MH, Peet NP, et al. Optimization of a novel potent and selective bacterial DNA helicase inhibitor scaffold from a high throughput screening hit. Bioorg Med Chem Lett. 2013;23:3481–6.
Biswas T, Green KD, Garneau-Tsodikova S, Tsodikov OV. Discovery of Inhibitors of Bacillus anthracis Primase DnaG. Biochem. 2013;52:6905–10.
Kuchta RD, Stengel G. Mechanism and evolution of DNA primases. Biochim Biophys Acta. 1804;2010:1180–9.
Sanyal G, Doig P. Bacterial DNA replication enzymes as targets for antibacterial drug discovery. Expert Opin Drug Dis. 2012;7:327–39.
Jang KH, Nam SJ, Locke JB, Kauffman CA, Beatty DS, Paul LA, et al. Anthracimycin, a Potent Anthrax Antibiotic from a Marine-Derived Actinomycete. Angew Chem Int Ed. 2013;52:7822–4.
Hensler ME, Jang KH, Thienphrapa W, Vuong L, Tran DN, Soubih E, et al. Anthracimycin activity against contemporary methicillin-resistant staphylococcus aureus. J antibiotics. 2014;67:549–53.
Sakoulas G, Bayer AS, Pogliano J, Tsuji BT, Yang SJ, Mishra NN, et al. Ampicillin enhances daptomycin-and cationic host defense peptide-mediated killing of ampicillin-and vancomycin-resistant Enterococcus faecium. Antimicrob Agents Chemother. 2012;56:838–44.
Finlay BB, Hancock RE. Can innate immunity be enhanced to treat microbial infections. Nature Rev Microbiol. 2004;2:497–504.
Hancock RE, Diamond G. The role of cationic antimicrobial peptides in innate host defenses. Trends Microbiol. 2000;8:402–10.
Lisanby MW, Swiecki MK, Dizon BLP, Pflughoeft KJ, Koehler TM, Kearney JF. Cathelicidin administration protects mice from Bacillus anthracis spore challenge. J Immunol. 2008;181:4989–5000.
Jones MB, Jani R, Ren D, Wood TK, Blaser MJ. Inhibition of Bacillus anthracis growth and virulence-gene expression by inhibitors of quorum-sensing. J Infect Dis. 2005;191:1881–8.
Foss MH, Weibel DB. Oligochlorophens Are Potent Inhibitors of Bacillus anthracis. Antimicrob Agents Chemother. 2010;54:3988–90.
Tripathi A, Schofield MM, Chlipala GE, Schultz PJ, Yim I, Newmister SA, et al. Baulamycins A and B, Broad-Spectrum Antibiotics Identified as Inhibitors of Siderophore Biosynthesis in Staphylococcus aureus and Bacillus anthracis. J Am Chem Soc. 2014;136:1579–86.
Turnbull PC, Sirianni NM, LeBron CI, Samaan MN, Sutton FN, Reyes AE, et al. MICs of selected antibiotics for Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides from a range of clinical and environmental sources as determined by the Etest. J Clin Microbiol. 2004;42:3626–34.
Lock RL, Harry EJ. Cell-division inhibitors: new insights for future antibiotics. Nat Rev Drug Discov. 2008;7:324–38.
Michie KA, Lowe J. Dynamic filaments of the bacterial cytoskeleton. Annu Rev Biochem. 2006;75:467–92.
Shih YL, Rothfield L. The bacterial cytoskeleton. Microbiol Mol Biol Rev. 2006;70:729–54.
Moir DT, Opperman TJ, Butler MM, Bowlin TL. New classes of antibiotics. Curr Opin Pharmacol. 2012;12:535–44.
Quadri LE. Strategic paradigm shifts in the antimicrobial drug discovery process of the 21st century. Infect Disord Drug Targets. 2007;7:230–7.
Miethke M, Marahiel MA. Sideophore-based iron acquisition and pathogen control. Microbiol Mol Biol Rev. 2007;71:413–51.
Ren D, Sims JJ, Wood TK. Inhibition of biofilm formation and swarming of Escherichia coli by(5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone. Environ Microbiol. 2001;3:731–6.
Chen Z, Moayeri M, Purcell R. Monoclonal Antibody Therapies against Anthrax. Toxins. 2011;3:1004–19.
Kummerfeldt CE. Raxibacumab: potential role in the treatment of inhalational anthrax. Infect Drug Resist. 2014;7:101–9.
Mazumdar S. Raxibacumab. MAbs. 2009;1:531–8.
U. S. Food and Drug Administration. Raxibacumab. Human Genome Sciences. 2012. http://www.accessdata.fda.gov/drugsatfda_docs/label/2012/125349s000lbl.pdf. Accessed 26 Aug 2016
U. S. Food and Drug Administration. ANTHIM® (obiltoxaximab) injection. Elusys Therapeutics, Inc. 2016. http://www.accessdata.fda.gov/drugsatfda_docs/label/2016/125509lbl.pdf. Accessed 26 Aug 2016.
Opal SM, Lim YP, Cristofaro P, Artenstein AW, Kessimian N, DelSesto D, et al. Inter-α inhibitor proteins: a novel therapeutics strategy for experimental anthrax infection. Shock. 2011;35:42–4.
Opal SM, Artenstein AW, Cristofaro PA, Jhung JW, Palardy JE, Parejo NA, et al. Inter-alpha-inhibitor proteins are endogenous furin inhibitors and provide protection against experimental anthrax intoxication. Infect Immun. 2005;73:5101–5.
Ying Z, Chen H, Li YM, Zheng SS, Chen YG, Li LJ, et al. Thymosin alpha1-and ulinastatin-based immunomodulatory strategy for sepsis arising from intra-abdominal infection due to carbapenem-resistant bacteria. J Infect Dis. 2008;198:723–30.
Yumin L, Hao C, Xun L, Wence Z, Minyan H, Chiriva-Internati M, et al. A new immunomodulatory therapy for severe sepsis: ulinastatin plus thymosin. J Intensive Care Med. 2009;24:47–53.
Ammon HP, Wahl MA. Pharmacology of Curcuma longa. Planta Med. 1991;57:1–7.
Kurup PNV. Handbook of Medicinal Plants. Lucknow. India: Central Council for Research in Ayurveda and Siddha;1979; p 78.
Zhang Y, Gu Y, Lee HM, Hambardjieva E, Vrankova K, Golub LM, et al. Design, synthesis and biological activity of new polyenolic inhibitors of matrix metalloproteinases: a focus on chemically-modified curcumins. Curr Med Chem. 2012;19:4348–58.
Antonelli AC, Zhang Y, Golub LM, Johnson F, Simon SR. Inhibition of anthrax lethal factor by curcumin and chemically modified curcumin derivatives. J Enzyme Inhib Med Chem. 2014;29:663–9.
Gupta SC, Prasad S, Kim JH, Patchva S, Webb LJ, Priyadarsini IK, et al. Multitargeting by curcumin as revealed by molecular interaction studies. Nat Prod Rep. 2011;28:1937–55.
Zhang Y, Golub LM, Johnson F, Wishnia A. pKa, zinc-and serum albumin-binding of curcumin and two novel biologically-active chemically-modified curcumins. Curr Med Chem. 2012;19:4367–75.
Calugi C, Trabocchi A, Lalli C, Guarna A. D-Proline-based peptidomimetic inhibitors of anthrax lethal factor. Eur J Med Chem. 2012;56:96–107.
Li F, Chvyrkova I, Terzyan S, Wakeham N, Turner R, Ghosh AK, et al. Inhibition of anthrax lethal factor, lability of hydroxamate as a chelating group. Appl Microbiol Biotechnol. 2012;94:1041–9.
Schepetkin IA, Khlebnikov AI, Kirpotina LN, Quinn MT. Novel small-molecule inhibitors of anthrax lethal factor identified by high-throughput screening. J Med Chem. 2006;49:5232–44.
Turk BE, Wong TY, Schwarzenbacher R, Jarrell ET, Leppla SH, Collier RJ, et al. The structural basis for substrate and inhibitor selectivity of the anthrax lethal factor. Nat Structural Mol Biol. 2004;11:60–6.
Newman ZL, Sirianni N, Mawhinney C, Lee MS, Leppla SH, Moayeri M, et al. Auranofin Protects against Anthrax Lethal Toxin-Induced Activation of the Nlrp1b Inflammasome. Antimicrob Agents Chemother. 2011;55:1028–35.
Kean WF, Hart L, Buchanan WW. Auranofin. Br J Rheumatol. 1997;36:560–72.
Bondeson J. The mechanisms of action of disease-modifying antirheumatic drugs: a review with emphasis on macrophage signal transduction and the induction of proinflammatory cytokines. Gen Pharmacol. 1997;29:127–50.
Lingetti M, Porfido FA, Ciarimboli M, Oliviero U, Cocozza M, Coto V, et al. Evaluation of the clinical efficacy of idebenone in patients affected by chronic cerebrovascular disorders. Arch Gerontol Geriat. 1992;15:225–37.
Seifert R, Dove S. Inhibitors of Bacillus anthracis edema factor. Pharmacol Therap. 2013;140:200–12.
Lübker C, Seifert R. Effects of 39 Compounds on Calmodulin-Regulated Adenylyl Cyclases AC1 and Bacillus anthracis Edema Factor. PLoS One. 2015;10:e0124017.
Wolff J, Cook GH, Goldhammer AR, Berkowitz SA. Calmodulin activates prokaryotic adenylate cyclase. Proc Natl Acad Sci U S A. 1980;77:3841–4.
Tang WJ, Krupinski J, Gilman AG. Expression and characterization of calmodulin-activated (type I) adenylylcyclase. J Biol Chem. 1991;266:8595–603.
Levin RM, Weiss B. Mechanism by which psychotropic drugs inhibit adenosine cyclic 3',5'-monophosphate phosphodiesterase of brain. Mol Pharmacol. 1976;12:581–9.
Weiss B, Prozialeck WC, Wallace TL. Interaction of drugs with calmodulin. Biochemical, pharmacological and clinical implications. Biochem Pharmacol. 1982;31:2217–26.
Douglass PM, Salins LL, Dikici E, Daunert S. Class-selective drug detection: Fluorescently-labeled calmodulin as the biorecognition element for phenothiazines and tricyclic antidepressants. Bioconjug Chem. 2002;13:1186–92.
Dikici E, Deo SK, Daunert S. A whole-cell assay for the high throughput screening of calmodulin antagonists. Anal Bioanal Chem. 2008;390:2073–9.
Gietzen K, Wuthrich A, Bader H. R 24571: A new powerful inhibitor of red blood cell Ca2 + −transport ATPase and of calmodulin-regulated functions. Biochem Biophys Res Commun. 1981;101:418–25.
Moen ST, Blumentritt CA, Slater TM, Patel SD, Tutt CB, Estrella-Jimenez ME, et al. Testing the efficacy and toxicity of adenylyl cyclase inhibitors against enteric pathogens using in vitro and in vivo models of infection. Infect Immun. 2010;78:1740–9.
Chen D, Martin ZS, Soto C, Schein CH. Computational selection of inhibitors of Abeta aggregation and neuronal toxicity. Bioorg Med Chem. 2009;17:5189–97.
Barekzi NA, Felts AG, Poelstra KA, Slunt JB, Grainger DW. Locally delivered polyclonal antibodies potentiate intravenous antibiotic efficacy against gram-negative infections. Pharm Res. 2002;19:1801–7.
Collins MS, Roby RE. Protective activity of an intravenous immune globulin (human) enriched in antibody against lipopolysaccharide antigens of Pseudomonas aeruginosa. Am J Med. 1984;76:168–74.
El-Zaim HS, Chopra AK, Peterson JW, Vasil ML, Heggers JP. Protection against exotoxin A (ETA) and Pseudomonas aeruginosa infection in mice with ETA-specific antipeptide antibodies. Infect Immun. 1998;66:5551–4.
Cryz SJ, Cross AS, Furer E, Chariatte N, Sado¡ JC, Germanier R. Activity of intravenous globulins against Klebsiella. J Lab Clin Med. 1986;108:182–9.
The authors of this paper would like to recognize Dr. Michelle Alfa and Dr. Dan Sitar of the University of Manitoba for their aid in reviewing this manuscript.
BH carried out the literature search and drafted the manuscript. ER aided in the outline of the manuscript and critically revised the intellectual content. AM aided in the design of the manuscript as well as critically revised the manuscript. All authors read and approved the final manuscript.
All authors have read BioMed Central’s guidance on competing interests and the authors declare that they have no competing interests (financial or otherwise) with other people or organizations that could inappropriately influence or bias the content of this paper.
Consent for publication
Consent to publish all figures in this manuscript has been obtained from all necessary authors and/or journals.
Ethics approval and consent to participate
Ethan Rubinstein Deceased