Clindamycin Protects Nonhuman Primates Against Inhalational Anthrax But Does Not Enhance Reduction of Circulating Toxin Levels When Combined With Ciprofloxacin.

BACKGROUND: Inhalational anthrax is rare and clinical experience limited. Expert guidelines recommend treatment with combination antibiotics including protein synthesis-inhibitors to decrease toxin production and increase survival, although evidence is lacking. METHODS: Rhesus macaques exposed to an aerosol of Bacillus anthracis spores were treated with ciprofloxacin, clindamycin, or ciprofloxacin + clindamycin after becoming bacteremic. Circulating anthrax lethal factor and protective antigen were quantitated pretreatment and 1.5 and 12 hours after beginning antibiotics. RESULTS: In the clindamycin group, 8 of 11 (73%) survived demonstrating its efficacy for the first time in inhalational anthrax, compared to 9 of 9 (100%) with ciprofloxacin, and 8 of 11 (73%) with ciprofloxacin + clindamycin. These differences were not statistically significant. There were no significant differences between groups in lethal factor or protective antigen levels from pretreatment to 12 hours after starting antibiotics. Animals that died after clindamycin had a greater incidence of meningitis compared to those given ciprofloxacin or ciprofloxacin + clindamycin, but numbers of animals were very low and no definitive conclusion could be reached. CONCLUSION: Treatment of inhalational anthrax with clindamycin was as effective as ciprofloxacin in the nonhuman primate. Addition of clindamycin to ciprofloxacin did not enhance reduction of circulating toxin levels.

Progress towards the Development of a NEAT Vaccine for Anthrax II: Immunogen Specificity and Alum Effectiveness in an Inhalational Model.

Bacillus anthracis is the causative agent of anthrax disease, presents with high mortality, and has been at the center of bioweapon efforts. The only currently U.S. FDA-approved vaccine to prevent anthrax in humans is anthrax vaccine adsorbed (AVA), which is protective in several animal models and induces neutralizing antibodies against protective antigen (PA), the cell-binding component of anthrax toxin. However, AVA requires a five-course regimen to induce immunity, along with an annual booster, and is composed of undefined culture supernatants from a PA-secreting strain. In addition, it appears to be ineffective against strains that lack anthrax toxin. Here, we investigated a vaccine formulation consisting of recombinant proteins from a surface-localized heme transport system containing near-iron transporter (NEAT) domains and its efficacy as a vaccine for anthrax disease. The cocktail of five NEAT domains was protective against a lethal challenge of inhaled bacillus spores at 3 and 28 weeks after vaccination. The reduction of the formulation to three NEATs (IsdX1, IsdX2, and Bslk) was as effective as a five-NEAT domain cocktail. The adjuvant alum, approved for use in humans, was as protective as Freund's Adjuvant, and protective vaccination correlated with increased anti-NEAT antibody reactivity and reduced bacterial levels in organs. Finally, the passive transfer of anti-NEAT antisera reduced mortality and disease severity, suggesting the protective component is comprised of antibodies. Collectively, these results provide evidence that a vaccine based upon recombinant NEAT proteins should be considered in the development of a next-generation anthrax vaccine.

Evaluation of liposomal ciprofloxacin formulations in a murine model of anthrax.

The in vivo efficacy of liposomal encapsulated ciprofloxacin in two formulations, lipoquin and apulmiq, were evaluated against the causative agent of anthrax, Bacillus anthracis. Liposomal encapsulated ciprofloxacin is attractive as a therapy since it allows for once daily dosing and achieves higher concentrations of the antibiotic at the site of initial mucosal entry but lower systemic drug concentrations. The in vivo efficacy of lipoquin and apulmiq delivered by intranasal instillation was studied at different doses and schedules in both a post exposure prophylaxis (PEP) therapy model and in a delayed treatment model of murine inhalational anthrax. In the mouse model of infection, the survival curves for all treatment cohorts differed significantly from the vehicle control. Ciprofloxacin, lipoquin and apulmiq provided a high level of protection (87-90%) after 7 days of therapy when administered within 24 hours of exposure. Reducing therapy to only three days still provided protection of 60-87%, if therapy was provided within 24 hours of exposure. If treatment was initiated 48 hours after exposure the survival rate was reduced to 46-65%. These studies suggest that lipoquin and apulmiq may be attractive therapies as PEP and as part of a treatment cocktail for B. anthracis.

The Fluorocycline TP-271 Is Efficacious in Models of Aerosolized Bacillus anthracis Infection in BALB/c Mice and Cynomolgus Macaques.

The fluorocycline TP-271 was evaluated in mouse and nonhuman primate (NHP) models of inhalational anthrax. BALB/c mice were exposed by nose-only aerosol to Bacillus anthracis Ames spores at a level of 18 to 88 lethal doses sufficient to kill 50% of exposed individuals (LD50). When 21 days of once-daily dosing was initiated at 24 h postchallenge (the postexposure prophylaxis [PEP] study), the rates of survival for the groups treated with TP-271 at 3, 6, 12, and 18 mg/kg of body weight were 90%, 95%, 95%, and 84%, respectively. When 21 days of dosing was initiated at 48 h postchallenge (the treatment [Tx] study), the rates of survival for the groups treated with TP-271 at 6, 12, and 18 mg/kg TP-271 were 100%, 91%, and 81%, respectively. No deaths of TP-271-treated mice occurred during the 39-day posttreatment observation period. In the NHP model, cynomolgus macaques received an average dose of 197 LD50 of B. anthracis Ames spore equivalents using a head-only inhalation exposure chamber, and once-daily treatment of 1 mg/kg TP-271 lasting for 14 or 21 days was initiated within 3 h of detection of protective antigen (PA) in the blood. No (0/8) animals in the vehicle control-treated group survived, whereas all 8 infected macaques treated for 21 days and 4 of 6 macaques in the 14-day treatment group survived to the end of the study (56 days postchallenge). All survivors developed toxin-neutralizing and anti-PA IgG antibodies, indicating an immunologic response. On the basis of the results obtained with the mouse and NHP models, TP-271 shows promise as a countermeasure for the treatment of inhalational anthrax.

Green tea and epigallocatechin-3-gallate are bactericidal against Bacillus anthracis.

Bacillus anthracis, the etiological agent of anthrax, is listed as a category A biothreat agent by the United States Centers for Disease Control and Prevention. The virulence of the organism is due to expression of two exotoxins and capsule, which interfere with host cellular signaling, alter host water homeostasis and inhibit phagocytosis of the pathogen, respectively. Concerns regarding the past and possible future use of B. anthracis as a bioterrorism agent have resulted in an impetus to develop more effective protective measures and therapeutics. In this study, green tea was found to inhibit the in vitro growth of B. anthracis. Epigallocatechin-3-gallate (EGCG), a compound found abundantly in green tea, was shown to be responsible for this activity. EGCG was bactericidal against both the attenuated B. anthracis ANR and the virulent encapsulated B. anthracis Ames strain. This study highlights the antimicrobial activity of green tea and EGCG against anthrax and suggests the need for further investigation of EGCG as a therapeutic candidate against B. anthracis.

In Vitro and In Vivo Activity of Omadacycline against Two Biothreat Pathogens, Bacillus anthracis and Yersinia pestis.

The in vitro activity and in vivo efficacy of omadacycline (OMC) were evaluated against the causative pathogens of anthrax and plague, Bacillus anthracis and Yersinia pestis, respectively. MICs of OMC were determined by broth microdilution according to CLSI guidelines for 30 isolates each of Y. pestis and B. anthracis The in vivo efficacy of omadacycline was studied at a range of dosages in both a postexposure prophylaxis (PEP) murine model of anthrax and plague as well as in a delayed treatment model of inhalational anthrax. Omadacycline was active in vitro against Y. pestis (MIC90 of 1 µg/ml) and B. anthracis (MIC90 of 0.06 µg/ml). Omadacycline was less active in vitro than ciprofloxacin (CIP) against Y. pestis (CIP MIC90 of 0.03 µg/ml) but was more potent in vitro against B. anthracis (CIP MIC90 of 0.12 µg/ml). In the mouse model of infection, the survival curves for all treatment cohorts differed significantly from the vehicle control (P = 0.004). The median survival for the vehicle-treated controls was 6 days postchallenge, while all antibiotic-treated mice survived the entire study. Omadacycline treatment with 5, 10, or 20 mg/kg of body weight twice daily for 14 days had significant efficacy over the vehicle control in the treatment of aerosolized B. anthracis Additionally, for postexposure prophylaxis treatment of mice infected with Y. pestis, the survival curves for omadacycline (40 mg/kg twice daily), ciprofloxacin, and doxycycline cohorts differed significantly from the vehicle control (P < 0.0001). Omadacycline is potent and demonstrates efficacy against both B. anthracis and Y. pestis The well-characterized oral and intravenous pharmacokinetics, safety, and tolerability warrant further assessment of the potential utility of omadacycline in combating these serious biothreat organisms.

Development of a novel adjuvanted nasal vaccine: C48/80 associated with chitosan nanoparticles as a path to enhance mucosal immunity.

In a time in which mucosal vaccines development has been delayed by the lack of safe and effective mucosal adjuvants, the combination of adjuvants has started to be explored as a strategy to obtain potent vaccine formulations. This study describes a novel adjuvant combination as an effective approach for a nasal vaccine - the association of the mast cell activator compound 48/80 with chitosan based nanoparticles. It was hypothesized that mucoadhesive nanoparticles would promote the cellular uptake and prolong the antigen residence time on nasal cavity. Simultaneously, mast cell activation would promote a local microenvironment favorable to the development of an immune response. To test this hypothesis, two different C48/80 loaded nanoparticles (NPs) were prepared: Chitosan-C48/80 NP (Chi-C48/80 NP) and Chitosan/Alginate-C48/80 NP (Chi/Alg-C48/80 NP). The potential as a vaccine adjuvant of the two delivery systems was evaluated and directly compared. Both formulations had a mean size near 500nm and a positive charge; however, Chi-C48/80 NP was a more effective adjuvant delivery system when compared with Chi/Alg-C48/80 NP or C48/80 alone. Chi-C48/80 NP activated mast cells at a greater extent, were better internalized by antigen presenting cells than Chi/Alg-C48/80 NP and successfully enhanced the nasal residence time of a model antigen. Superiority of Chi-C48/80 NP as adjuvant was also observed in vivo. Therefore, nasal immunization of mice with Bacillus anthracis protective antigen (PA) adsorbed on Chi-C48/80 NP elicited high levels of serum anti-PA neutralizing antibodies and a more balanced Th1/Th2 profile than C48/80 in solution or Chi/Alg-C48/80 NP. The incorporation of C48/80 within Chi NP also promoted a mucosal immunity greater than all the other adjuvanted groups tested, showing that the combination of a mast cell activator and chitosan NP could be a promising strategy for nasal immunization.

Evaluation of mucoadhesive carrier adjuvant: toward an oral anthrax vaccine.

The aim of present study was to evaluate the potential of mucoadhesive alginate-coated chitosan microparticles (A-CHMp) for oral vaccine against anthrax. The zeta potential of A-CHMp was -29.7 mV, and alginate coating could prevent the burst release of antigen in simulated gastric fluid. The results indicated that A-CHMp was mucoadhesive in nature and transported it to the peyer's patch upon oral delivery. The immunization studies indicated that A-CHMp resulted in the induction of potent systemic and mucosal immune responses, whereas alum-adjuvanted rPA could induce only systemic immune response. Thus, A-CHMp represents a promising acid carrier adjuvant for oral immunization against anthrax.

Proteolytic assay-based screening identifies a potent inhibitor of anthrax lethal factor.

Anthrax lethal factor (LF), a Zn(2+)-dependent metalloprotease, is a key virulence component of anthrax toxin. Here, we used proteolytic assay-based screening to identify novel LF inhibitors from a naturally extracted chemical library. The screening identified four compounds that inhibited in vitro proteolytic activity of LF with an IC(50) of low micromolar range (11-20 µM). Three of these compounds were toxic to the mouse macrophage-like cell line, RAW 264.7. Compound 200 was non-toxic, however, and successfully protected Raw 264.7 cells from a lethal toxin challenge with an IC(50) of 39.2 µM. We also identified possible binding modes of compound 200 by molecular docking.

Post-exposure therapy of inhalational anthrax in the common marmoset.

The aim of this study was to compare the pharmacokinetics and efficacy of ciprofloxacin as post-exposure therapy against inhalational anthrax in the common marmoset (Callithrix jacchus) with other non-human primate models in order to determine whether the marmoset is a suitable model to test post-exposure therapies for anthrax. Pharmacokinetic (PK) and efficacy studies with ciprofloxacin were performed in the marmoset. Ciprofloxacin plasma pharmacokinetics were determined in six animals in separate single-dose and multiple-dose studies and were analysed by high-performance liquid chromatography (HPLC). A separate group of marmosets was exposed to ca. 100× the 50% lethal dose (LD(50)) of Bacillus anthracis Ames strain by the airborne route. On Day 5 of a twice-daily dosing regimen of 17.5 mg/kg, the ciprofloxacin half-life (t(1/2)), maximum drug concentration (C(max)) and area under the concentration-time curve (AUC) in marmoset plasma were 1.9 h, 2.1 µg/mL and 7.9 µg/mL/h, respectively. Naïve untreated control animals succumbed to infection by Day 9. All animals treated with ciprofloxacin, started on the day of exposure and continued for 10 days, remained healthy during the treatment period. Two antibiotic-treated animals (33%) died after withdrawal of antibiotic therapy, attributed to the germination of residual spores. In conclusion, in many respects the marmoset appears to respond to B. anthracis in a similar way to the macaque, suggesting that this small non-human primate is an acceptable, practical alternative model for the evaluation of medical countermeasures against respiratory anthrax infection.

Efficacy of oritavancin in a murine model of Bacillus anthracis spore inhalation anthrax.

The inhaled form of Bacillus anthracis infection may be fatal to humans. The current standard of care for inhalational anthrax postexposure prophylaxis is ciprofloxacin therapy twice daily for 60 days. The potent in vitro activity of oritavancin, a semisynthetic lipoglycopeptide, against B. anthracis (MIC against Ames strain, 0.015 microg/ml) prompted us to test its efficacy in a mouse aerosol-anthrax model. In postexposure prophylaxis dose-ranging studies, a single intravenous (i.v.) dose of oritavancin of 5, 15, or 50 mg/kg 24 h after a challenge with 50 to 75 times the median lethal dose of Ames strain spores provided 40, 70, and 100% proportional survival, respectively, at 30 days postchallenge. Untreated animals died within 4 days of challenge, whereas 90% of control animals receiving ciprofloxacin at 30 mg/kg intraperitoneally twice daily for 14 days starting 24 h after challenge survived. Oritavancin demonstrated significant activity post symptom development; a single i.v. dose of 50 mg/kg administered 42 h after challenge provided 56% proportional survival at 30 days. In a preexposure prophylaxis study, a single i.v. oritavancin dose of 50 mg/kg administered 1, 7, 14, or 28 days before lethal challenge protected 90, 100, 100, and 20% of mice at 30 days; mice treated with ciprofloxacin 24 h or 24 and 12 h before challenge all died within 5 days. Efficacy in pre- and postexposure models of inhalation anthrax, together with a demonstrated low propensity to engender resistance, promotes further study of oritavancin pharmacokinetics and efficacy in nonhuman primate models.

Peptide inhibitors MAP the way towards fighting anthrax pathogenesis.

The pathogenesis of anthrax is such that unless antibiotic treatment is initiated at an early stage in the disease, it is ineffective against the bacteria-induced toxaemia that subverts the immune response, inflicts massive tissue damage and is ultimately the major factor contributing to death during anthrax infection. As current events have demonstrated the feasibility of the use of anthrax as a bioterrorism agent, and exemplified the difficulty of treating the ensuing infection, inhibition of anthrax toxin has become a major focus of research for the design of antitoxin therapeutics. In this issue of Biochemical Journal, Bracci and co-workers describe the discovery by competitive screening of a phage-display library of a peptide inhibitor of anthrax toxin assembly that shows great promise towards the treatment of anthrax.

Pharmacokinetic-pharmacodynamic analysis of fluoroquinolones against Bacillus anthracis.

Based on the pharmacokinetic-pharmacodynamic (PK-PD) parameters of ciprofloxacin in rhesus monkeys, the efficacies of levofloxacin, sparfloxacin, norfloxacin, and tosufloxacin against anthrax in humans were examined. The optimal PK-PD parameter for the prophylaxis or treatment of infection with Bacillus anthracis is not clearly defined. To evaluate the efficacy of fluoroquinolones against anthrax, PK-PD parameters and the protein-binding effect of fluoroquinolones are used. B. anthracis is very susceptible to fluoroquinolones in vitro, and levofloxacin, sparfloxacin, and tosufloxacin may be as effective against anthrax as ciprofloxacin by PK-PD analysis. However, additional studies of the in vivo model are necessary to define more clearly efficacy against anthrax and the pharmacodynamic relationship of fluoroquinolones.

Post-exposure prophylaxis of systemic anthrax in mice and treatment with fluoroquinolones.

OBJECTIVES: To compare the fluoroquinolones gatifloxacin and moxifloxacin with ciprofloxacin for post-exposure prophylaxis of systemic anthrax in a BALB/c mouse model. METHODS: Treated mice and controls were inoculated subcutaneously with 5 x 10(4) spores/mouse of Bacillus anthracis Ames strain and observed for 37 days after challenge. Treated mice were given 100 mg/kg of antibiotic orally twice daily for 14 days, starting at various times post-challenge. RESULTS: Treatment starting 6 h post-challenge resulted in survival rates of 90%, 15% and 40% for gatifloxacin, moxifloxacin and ciprofloxacin, respectively. Treatment commencing 24 h post-challenge resulted in survival rates of 65%, 10% and 5%, respectively. Treatment starting more than 24 h after exposure had little effect on survival. CONCLUSIONS: Gatifloxacin appeared to be more effective than moxifloxacin or ciprofloxacin, at similar doses, for early post-exposure treatment of murine systemic anthrax. However, these results might be due to differences in potency or pharmacokinetic properties.

PCR-based detection of Bacillus anthracis in formalin-fixed tissue from a patient receiving ciprofloxacin.

We demonstrate that Bacillus anthracis may be detected from a formalin-fixed, paraffin-embedded biopsy specimen, even after the patient has received antibiotic treatment. Although traditional PCR methods may not be sufficiently sensitive for anthrax detection in such patients, cycle numbers can be increased or PCR can be repeated by using an aliquot from a previous PCR as the template.

Antimicrobial therapy for bacillus anthracis-induced polymicrobial infection in (60)Co gamma-irradiated mice.

Challenge with both nonlethal ionizing radiation and toxigenic Bacillus anthracis spores increases the rate of mortality from a mixed bacterial infection. If biological weapons, such as B. anthracis spores, and nuclear weapons were used together, casualties could be more severe than they would be from the use of either weapon alone. We previously discovered that a polymicrobial infection developed in B6D2F(1)/J mice after nonlethal (7-Gy) (60)Co gamma irradiation and intratracheal challenge with B. anthracis Sterne spores 4 days after irradiation. In this present study, we investigated the survival of mice and the response of the polymicrobial infection during the course of antimicrobial therapy with penicillin G procaine, ofloxacin, trovafloxacin, or gatifloxacin. Survival was prolonged, but not ensured, when the mice were treated with either broad-spectrum ofloxacin or narrow-spectrum penicillin G for 7 days beginning 6 or 24 h after challenge. Survival was not prolonged when therapy was delayed more than 24 h after challenge. When these two antimicrobial agents were given for 21 days, the survival rate was increased from 0% for the controls to 38 to 63% after therapy. Therapy with trovafloxacin or gatifloxacin reduced the incidence of mixed infection and improved the rate of survival to 95% (trovafloxacin) or 79% (gatifloxacin), whereas the rate of survival for the controls was 5%. We conclude that the mixed infection induced by B. anthracis in irradiated mice complicates effective therapy with a single antimicrobial agent. To limit mortality following nonlethal irradiation and challenge with B. anthracis spores, antimicrobial therapy needs to be initiated within a few hours after challenge and continued for up to 21 days.

Bacillus anthracis and antibacterial agents.

Anthrax is one of the oldest threats to humankind, and remains endemic in animals in many parts of the world. Human cases are infrequent, and some result from biological warfare. This review summarizes the current knowledge on the antibacterial activity of available antibiotics. For potential use in the most severe cases of anthrax, antibacterials need to exhibit potent in vitro activity, intracellular bioactivity, and suitable locations in lymph nodes. In animal models, it has been shown that doxycycline and fluoroquinolones are the most active compounds. There is a lack of data for animal models for macrolides and ketolides, some of them exhibiting good in vitro activity. However, systemic anthrax (inhalation or gastrointestinal) is mainly due to anthrax toxin, and therapy directed against intoxication is needed as basic treatment.

Screening inhibitors of anthrax lethal factor.

The disease anthrax is caused by lethal factor, an enzyme component of the toxin produced by the spore-forming bacterium Bacillus anthracis. Here we describe substrate molecules for this factor that offer a means for high-throughput screening of potential inhibitors for use in anthrax treatment. Our assay should help to answer the urgent call for new and specific therapies to combat this pathogen after its recent emergence as a terrorist bioweapon.

Anthrax: ENT manifestations and current concepts.

In light of recent events, anthrax has once again taken center stage in the world of science and the world in which we live. Because patients with anthrax may initially present to otolaryngology clinics, it is important for the otolaryngologist to know how to diagnose and treat this entity. This article will present current information on epidemiology, microbiology, pathogenesis/clinical manifestations, diagnosis, and treatment with a particular interest in the head and neck region.