Bacillus anthracis(Anthrax)

Authors: Arthur M. Friedlander, M.D.,Henry Heine, Ph.D., Mary E. Wright, M.D., MPH

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Microbiology

Bacillus anthracisis an aerobic, non-motile, sporulating, large non-hemolytic Gram-positive rod that grows well on blood agar. Individual colonies are gray-white, rough, and sticky when teased with a bacteriologic loop. On bicarbonate agar in the presence of 5-20% CO2the colonies are mucoid and the organism has a prominent capsule. Susceptibility to a gamma bacteriophage, immunological detection of capsule and cell wall polysaccharide and PCR for the presence of toxin and capsule genes confirms the identity of the organism. All virulent strains are pathogenic for mice.

Epidemiology

B. anthracisspores may persist in a dormant state in the soil for long periods of time, probably at least decades. Infection of mammals results in massive amplification of the vegetative bacillus and subsequent seeding of the soil with spore formation after death of the animal. Anthrax is largely a disease of herbivores, and humans become accidentally infected through contact with infected animals or their contaminated products (18). Naturally occurring human anthrax in the United States is now a rare disease, with less than one cutaneous case per year reported to the CDC over the last 30 years, although it continues to be a significant disease in less developed countries. Until the recent bioterrorist-related outbreak, inhalational anthrax had been a disease mainly of historic interest. Since then, cases of naturally-occurring infections have occurred with most from exposure to drums made from contaminated animal hides (10, 63, 11, 2, 57). Systemic anthrax has also been reported in Europe among intravenous drug users who injected heroin contaminated by anthrax transported in affected animal skins (1, 5, 26). Epidemics of gastrointestinal anthrax continue to be sporadically reported from developing countries.

Clinical Manifestations

The clinical manifestations of human anthrax are quite striking. Cutaneous anthrax, the most common form of naturally occurring disease, begins as a small, painless, pruritic papule that within 2 days enlarges, develops vesicles, and ulcerates to form an eschar. It is associated with local edema which can be significant. In the absence of recent antimicrobial therapy cultures of the vesicular fluid are usually positive. During the 2001 letter attacks when post-exposure prophylaxis with antibiotics was used, immunohistochemical identification of B. anthracis antigen was needed to establish the diagnosis for some cases (56). Prompt antibiotic treatment, while not stopping the progression of the local infection to eschar formation, is associated with low mortality rates (<1 %) in individuals without septicemia.
Inhalational anthrax follows inhalation of infectious doses of anthrax spores. During the first several days, symptoms are non-specific with myalgia, fatigue that may be profound, fever and chills, sweats, minimal non-productive cough, nausea or vomiting and some chest discomfort. Symptoms of an upper respiratory infection are characteristically absent. Patients then develop the rapid onset of severe dyspnea, cyanosis, increased chest pain, and eventually shock. Chest x-rays typically show a widened mediastinum secondary to hemorrhagic necrotic mediastinal lymphadenitis and mediastinitis. Large pleural effusions, which may be hemorrhagic, are common. In the 2001 outbreak, widened mediastinum was not recognized in every case but all went on to develop pleural effusions with increased survival noted in those who underwent pleural drainage (36). High levels of B. anthracis lethal factor and cell wall antigen have been found in pleural fluid (57, 34). About half of all patients develop secondary anthrax meningitis, and virtually all cases are fatal if untreated.

Oropharyngeal and gastrointestinal anthrax follow ingestion of grossly contaminated and undercooked meat. Pharyngeal ulcers, lymphadenitis, and brawny edema of the neck result from local oropharyngeal multiplication of anthrax bacilli. Gastrointestinal anthrax is associated with ulcers in the terminal ileum, cecum, or stomach with mesenteric lymphadenitis, ascites, diarrhea, and septicemia. One unusual United States case resulted from exposure to anthrax spores at an event using drums made from contaminated animal hides (11). Mortality rates from oropharyngeal and particularly gastrointestinal anthrax are high (approximately 40%).

Anthrax due to direct injection of contaminated heroin into the skin or vein leads to presentation with fatigue and severe bruising and edema at the index injection site. Despite early surgical debridement and aggressive antimicrobial therapy, distant separate sites of severe edema can develop. The case fatality rate for patients with injectional anthrax is approximately 37% (5).

Laboratory Diagnosis

The organism grows readily on routine microbiological media. Definitive diagnosis is established by isolation of the organism from tissues. A non-motile, spore-forming, large, non-hemolytic Gram-positive rod that produces a capsule in the presence of 5-20% CO2 should be presumptively consideredB.anthracis. Confirmation is established by sensitivity to gamma bacteriophage, identification of cell wall antigen and capsule by immunofluorescence, and PCR for toxin and capsule genes. Immunohistological stains of tissue for cell wall polysaccharide and capsule are available. Rapid diagnostic tests are now available based on detection of toxin proteins and capsule (6) along with PCR on serum and other bodily fluids. These tests may be of particular use in samples obtained after institution of antibiotics as cultures may be negative. Serologic diagnosis is possible with use of a sensitive and specific ELISA test but is only of value retrospectively.

Pathogenesis

B.anthracisis an invasive bacterium that grows to high concentrations in the blood and tissues. It produces three well-established virulence factors: an antiphagocytic capsule, and the lethal and edema toxins. The toxins interfere with neutrophil, macrophage as well as T and B lymphocyte function (60) and likely contribute to the impaired inflammatory response and edema observed in tissues. The lethal toxin is lytic for macrophages and may release proinflammatory cytokines and other mediators that contribute to the sepsis syndrome, toxemia, and shock. Evidence in experimental animals also suggests that in late stages of infection the high levels of toxins may directly impair cardiac and hepatic function (41). In inhalational anthrax, mediastinitis is associated with lymphatic and vascular obstruction, pleural effusions and pulmonary hemorrhage and edema, all of which contribute to death. Pathologically, vasculitis involving large and small vessels is prominent (25).

Susceptibility in vitro and in vivo

There have only been a few studies of antimicrobial susceptibility testing inB.anthracis. The methods of testing have varied and until the most recent studies, there has not been any attempt to standardize the testing nor were there guidelines for interpretation of results. The Clinical Laboratory Standards Institute (CLSI) formally the National Committee for Clinical Laboratory Standards (NCCLS) has now addressed and published standards and interpretive criteria forB.anthracis (14). Where definitive interpretive criteria are unavailable or incomplete, staphylococci antibiotic breakpoints are being used as a guide. Lightfoot et al. (39) evaluated nine antimicrobial agents with 33 epidemiologically distinct isolates by the agar dilution method. The criteria for setting susceptibility breakpoints was not specified, but 90% of the strains were determined to be sensitive to penicillin, amoxicillin, gentamicin, streptomycin, erythromycin, tetracycline, chloramphenicol and ciprofloxacin (Table 1). Most of the strains tested were resistant to cefuroxime. Doganay and Aydin (17) also used the agar dilution method for testing thirty antibiotics with 22B.anthracisstrains. They identified additional beta-lactams, aminoglycosides, clindamycin, vancomycin, ofloxacin and co-trimoxazole as antibiotics having activity (Table 1). A second study also showed susceptibility to azithromycin, clarithromycin, erythromycin and roxithromycin (58). In another study using a disk diffusion assay, 44 isolates were tested and susceptibilities determined by comparing inhibition zones to aStaphylococcus aureustype strain with generally similar results (49). All 44 isolates in this study were reported as resistant to sulfamethoxazole and trimethoprim. Co-trimoxazole was not tested. It should be noted that in all of these studies penicillin resistance and beta-lactamase activity was observed for only one isolate tested (39).

Studies from the Centers for Disease Control (CDC) and the United States Army Medical Research Institute for Infectious Diseases (USAMRIID) (48, 30, 31) utilized the broth microdilution assay system and followed CLSI standards (15). Both studies were in general agreement of susceptibilities with the exception of penicillin and chloramphenicol (Table 1). The CDC study looked at 9 antibiotics with 65 isolates. Included in those 65 isolates were 15 clinical isolates from the 2001 bio-terrorism release in the United States. Using Staphylococcal breakpoints they established that greater than 90% of the strains tested were sensitive to chloramphenicol, ciprofloxacin, clindamycin, penicillin, rifampin, tetracycline and vancomycin. The majority of the strains were of intermediate activity for erythromycin and ceftriaxone. All 15 recent B. anthracis isolates from the United States were susceptible to penicillin, doxycycline, and ciprofloxacin (48). The USAMRIID studies looked at 20B.anthracisstrains and 39 antibiotics including many recently approved by the FDA and others in phase 3 trials (30, 31). Data from this study indicate susceptibilities to additional fluoroquinolones, sparfloxacin and levofloxacin. In addition, three new classes of antibiotics showin vitroactivity: the streptogramins, quinupristin/dalfopristin; the oxazolidinones, linezolid; and the carbapenems, imipenem, meropenem (Table 1). Several recently approved or IND antibiotics also havein vitroactivity. These are oritavancin,dalbavancin, tigecycline, daptomycin and faropenem (27, 29, 32, 22).

Anotable discrepancy between the USAMRIID and CDC studies is the penicillin data that are likely explained by differences in the inoculum preparation used in the two testing methods. The USAMRIID study adjusted log-phase broth cultures while the CDC study resuspended colonies from 18-hour plates. Both methodologies are accepted CLSI methods for inoculum preparation (15). Induction of beta-lactamase has been observed bothin vitro(39, 47, 48) and as the result of suboptimal treatment in a nonhuman primate model (23). Older studies have previously shown thatB.anthracishas the ability to increase expression of a "penicillinase" as a function of inoculum size (4) and this may well explain the differences between the results of the two studies. In the CDC study observation of beta-lactamase production was variable, while the USAMRIID study consistently observed activity. There was overlap in the strains tested in these two studies and it may be that in addition to inoculum size, growth phase and other factors effect beta-lactamase induction. It should be emphasized that the previous data of Lightfoot et al. (39) and Doganay (17) showed all 55 strains except one to be highly susceptible to penicillin. Nevertheless, the possibility of an inducible beta-lactamase activity should be taken into consideration under clinical conditions where high numbers of organisms are to be expected.

Animal efficacy-Experimental therapeutics

Development of several animal models and in-vitro hollow fiber systems has led to an improved understanding of both efficacy and optimal dosing of new therapeutics (28, 64, 16, 42). Application of doses derived from hollow fiber and or pharmacodynamics determination have been applied to a murine aerosol model of infection showing efficacy for faropenem, daptomycin, oritavancin, dalbivancin, doxycycline, ciprofloxacin, and levofloxacin (22, 28, 27, 29, 32, 16). Ciprofloxacin has been shown to be efficacious in a rhesus monkey model for treatment of inhalational anthrax (62). Other animal studies in guinea pigs and rabbits have demonstrated efficacy of ciprofloxacin, levofloxacin and doxycycline (50, 64).

Combination Drugs

There are no published studies that have evaluatedin vitrosusceptibility ofB.anthracisto antibiotic combinations. Limited animal data suggest that streptomycin and presumably other aminoglycosides may enhance the effectiveness of penicillin (40). Inaddition, there are no published data on possible antagonism between combinations of antibiotics. A review of human cases of inhalational anthrax suggests that combination antimicrobial treatment results in greater survival than treatment with single drugs (35). For systemic anthrax infections, combination antimicrobial treatment is recommended including one drug that inhibits protein synthesis for the theoretical advantage of more rapidly reducing toxin production (34). There is some evidence supporting this for in vitro grown B. anthracis (43) but it has not been definitively shown to be of benefit in animal models or human infections.

Adjunctive Therapy

Antitoxins have been used in the treatment of anthrax in the pre-antibiotic era with suggestive evidence for efficacy but no carefully controlled studies were reported (19). Two antitoxin preparations, one a humanized monoclonal antibody (raxibacumab, GlaxoSmithKline, London, UK) against the protective antigen component of the anthrax toxins and the other, a human polyclonal antiserum (AIGIV, Cangene Corporation, Winnipeg, Canada) produced from plasma of individuals vaccinated with Anthrax Vaccine Adsorbed (BioThrax) are available from the CDC Strategic National Stockpile. Studies show increased survival in rabbits and monkeys given raxibacumab alone compared to no treatment (46). However, while raxibacumab together with levofloxacin gave improved survival compared to animals treated with levofloxacin alone the difference was not statistically significant (34). Similar results have been obtained with the polyclonal AIGIV (34, 37).

Antimicrobial Therapy

Cutaneous Anthrax

Penicillin remains the drug of choice for the treatment of cutaneous anthrax. Organisms are rapidly cleared from skin lesions; 25 patients with cutaneous anthrax and positive initial cultures of vesicular fluid were given 2 million units of penicillin G intravenously and the fluid was cultured hourly (53); 5 h after the initiation of therapy, all cultures were negative. The duration of therapy with penicillin is not well established, and in the absence of controlled observations, therapy with penicillin for 7 to 10 days is recommended. Parenteral therapy should be used for cases with systemic symptoms while in mild cases, oral drugs can be used.
Very rarely naturally occurring strains resistant to penicillin have been reported. (8, 13, 54). As noted above induction of penicillin resistance due to a beta-lactamase can occurin vitro (4, 39, 48) and has been reported in one case of an inadequately treated non-human primate (23). It has never been reported in any human case of anthrax treated with penicillin. The inducible penicillinase may be clinically significant when a large number of organisms are present as would be expected in a patient with established systemic infection such as in inhalational anthrax.

Tetracyclines,chloramphenicol, anderythromycinhave also been highly effective in treating cutaneous anthrax and are alternative drugs for penicillin-sensitive patients (24).

Single-dose oral therapy withdoxycyclinehas also been evaluated clinically (54). Thirty-three patients with cutaneous anthrax were treated with a single oral dose of doxycycline and observed in a hospital setting for 3 days. There was dramatic clinical improvement, and all patients were bacteriologically negative by the fourth day. The authors propose that cutaneous anthrax in adults can be safely treated with a single 500-mg oral dose of doxycycline, while children and adolescents could be treated with single doses of 100 to 300 mg orally. Nevertheless, conservative practice suggests that treatment should be given for a minimum of 7 to 10 days.

Case fatality rates as high as 20% have been reported for untreated cutaneous anthrax, but with appropriate antibiotic treatment, fatalities are now very unusual. However, cutaneous lesions, even if promptly treated with antibiotics, continue to progress through the eschar phase.In the context of a possible bioweapon exposure, initial therapy should be started with a fluoroquinolone or doxycycline unless the strain is shown to be sensitive to penicillin (34).

Inhalational Anthrax

The life-threatening nature of inhalational anthrax requires intravenous therapy with two or more antibiotics at the very first consideration of this disease. This is all the more important because of the possibility of an antibiotic resistant organism being used in a biological attack. Resistance to multiple antibiotics (58) including the fluoroquinolones has been described in vitro (12, 52). Thus, initial therapy should include a minimum of two drugs including a fluoroquinoloneand a drug that inhibits protein synthesis, either clindamycin or linezolid. Alternative drugs include meropenem, imipenem, doripenem or vancomycin as recently recommended by the CDC Expert Panel on Prevention and Treatment of Anthrax (34). Because of the possibility of an inducible beta-lactamase, a penicillin should not be given by itself. The rationale for these recommendations is based primarily onin vitro sensitivities, as well as the limited data from treatment of experimental inhalational anthrax in animal models (21), the experience treating cutaneous anthrax in humans and the small number of recent human cases of inhalational anthrax. As soon as antibiotic sensitivities are determined, patients should be treated with the most sensitive, least toxic, available antibiotics.Special consideration should be given to patients who may have meningitis by using a minimum of three antibiotics active against B. anthracis as recommended by the recent CDC Expert Panel report (e.g. a fluoroquinolone, meropenem or penicillin if susceptible, and linezolid or clindamycin or rifampin or chloramphenicol) that are likely to achieve therapeutic concentrations in the cerebrospinal fluid (34).The use of corticosteroids in adult meningitis remains controversial and there are no controlled studies supporting their use in anthrax.

The optimal duration of therapy in inhalational anthrax is unknown. The particular issue of persistence of ungerminated spores that dictates the prolonged use of antibiotics in the setting of prophylaxis after exposure but before the onset of clinical illness (see below) may not apply in the treatment of established disease and there is evidence in nonhuman primate models that prolonged treatment is not required (62). It is anticipated that sufficient antigenic mass would be present in a patient presenting with inhalational anthrax and bacteremia such that a robust immune response would occur. This has indeed been observed in the survivors of the recent outbreak of inhalational anthrax.Thus, the duration of treatment should be based upon clinical judgment but a minimum of 14 days is recommended.

Prior to the recent outbreak of inhalational anthrax, mortality was thought to approach 100%. However, this was based upon cases that were most often untreated or seen when moribund. Nine survivors out of approximately 75 cases were reported during the epidemic of inhalational anthrax in Sverdlovsk in 1979 (45). However, no definitive diagnosis was established for any of the nine purported survivors so it is impossible to interpret this data. The recent survival of 6 of the 11 recent cases of inhalational anthrax shows that with aggressive antibiotic and supportive modern therapy, survival can be anticipated particularly when patients present early in their course. In fact, of the patients that died, one had meningitis and several were moribund or in severe respiratory distress when treatment was begun (3, 36). This experience in humans is supported by experiments showing non-human primates can survive even when treatment is begun after the onset of mediastinitis or sustained bacteremia (23, 62).

Gastrointestinal Anthrax

Gastrointestinal and oropharyngeal anthrax are also associated with high rates of mortality and antibiotic recommendations are similar to those for inhalational anthrax.

Vaccines

Prevention of anthrax in animals has largely depended on the use of vaccines, since widespread decontamination of contaminated soil is impractical. The Sterne vaccine is composed of spores of a live, toxinogenic, unencapsulated attenuatedB.anthracisstrain and is used throughout the world as a veterinary vaccine. The main limitation to its use in humans is thought to be safety. A similar live spore vaccine has been used for humans in countries of the Former Soviet Union and China and is considered highly effective against cutaneous anthrax. A sterile protein-based anthrax vaccine was licensed for human use in the U.S. in 1970 and is currently produced by Emergent BioSolutions (Rockville, MD) (19). A less potent precursor of this vaccine has been field-tested and was found to be highly effective in preventing anthrax, predominantly cutaneous, in woolen mill workers (7). The current vaccine, Anthrax Vaccine Adsorbed (BioThrax), is an aluminum hydroxide-adsorbed cell-free culture filtrate from a toxinogenic unencapsulated strain ofB.anthraciscontaining the protective antigen host cell receptor binding component of the anthrax toxins.

Indications

Anthrax vaccine is recommended for persons whose occupations require frequent contact with imported animal products likely to be contaminated withB.anthracisspores and for laboratory workers who perform studies usingB.anthracis. Beginning in 1998, members of the armed forces at high risk have been vaccinated to protect against the use ofB.anthracisas a biowarfare weapon (20, 19, 9).

Doses and Schedules

The current FDA-approved dose schedule for anthrax vaccine consists of 0.5 mL given intramuscularly at 0 and 4 weeks and 6, 12, and 18 months followed by yearly boosters.

Adverse Effects

Extensive data on safety of the vaccine given by the previously recommended subcutaneous route indicated that mild local reactions occur in approximately 30% of individuals with moderate and severe local reactions occurring in 4% and <1% respectively and 1% reporting systemic symptoms (20, 51, 19). Reactions are self-limited and resolve without therapy.The change to the current FDA-approved intramuscular route in December 2008 resulted in fewer solicited local adverse events but no change in the incidence of systemic adverse events (44). More than 12 million doses of the vaccine have been given to more than 3 million individuals.

Prevention

General

B. anthracis spores are highly resistant to physical and chemical agents and they may persist in an inanimate environment. Parformaldehyde vapor and liquid disinfectants such as 5% hypochlorite or 5% phenol may be used for decontamination. Until recent times, prevention of anthrax ultimately depended on control of disease in animals. Effective vaccines for animals are available.

Postexposure Prophylaxis Of Inhalational Anthrax

The recent bioterrorist-related outbreak of 11 cases of inhalational anthrax in the U.S. has refocused interest on the question of postexposure prophylaxis (PEP) that was most recently addressed after the threat of anthrax during the 1990 Gulf War (18). This issue represents an unusual therapeutic situation that was appreciated in early animal trials of treatment, because inhaled spores may remain dormant and fail to germinate for prolonged periods of time (18). Earlier studies in experimental animals had shown that treatment with penicillin beginning 1 day after aerosol exposure to anthrax spores was protective during the 5 to 10 days of drug therapy, but animals died when the antibiotic was discontinued (33). In more recent studies, monkeys were challenged with aerosolized anthrax spores and beginning 1 day after exposure, groups of animals were given penicillin, ciprofloxacin or doxycycline for 30 days. A fourth group was immunized with anthrax vaccine after exposure and treated with doxycycline. All antibiotic regimens completely protected animals while they were on therapy and provided better long-term protection than the shorter 5- and 10-day treatment protocols. All animals that were immunized and treated with doxycycline survived. When these groups of monkeys were rechallenged with airborne anthrax spores, all succumbed except those who had been immunized (21). These data offer convincing proof that PEP is effective, although if given early after exposure it will prevent an immune response. Based upon these studies, recommendations have been made to provide prophylactic oral antibiotics with a fluoroquinolone or doxycycline for a 60-day period along with vaccination at 0, 2 and 4 weeks given subcutaneously to assure a rapid immune response (9). It is important to remember that PEP vaccination is given by the previously recommended subcutaneous route with three initial doses as this regimen provides a more rapid and greater immune response than the currently recommended pre-exposure prophylaxis schedule of two initial doses given intramuscularly (44). As for treatment of established disease, once sensitivities of isolated organisms are obtained, the most sensitive, least toxic, available antibiotic should be used. The addition of vaccination to a postexposure prophylactic antibiotic regimen may allow for reduction of the course of antibiotics to 30 to 45 days, although only limited animal data exist to support this recommendation (61).

References

1. Abbara A, Brooks T, Taylor GP, Nolan M, Donaldson H, Manikon M, Holmes A. Lessons for control of heroin-associated anthrax in Europe from 2009-2010 outbreak case studies, London, UK. Emerg Infect Dis. 2014;20:1115-22

2. Anaraki S, Addiman S, Nixon G, Krahé D, Ghosh R, Brooks T, Lloyd G, Spencer R, Walsh A, McCloskey B, Lightfoot N. Investigations and control measures following a case of inhalation anthrax in East London in a drum maker and drummer, October 2008. Euro Surveill. 2008;18:13.

3. Barakat LA, Quentzel HL, Jernigan JA, Kirschke DL, Griffith K, Spear SM, Kelley K, Barden D, Mayo D, Stephens DS, Popovic T, Marston C, Zaki SR, Guarner J, Shieh W-J, Carver HW II, Meyer RF, Swerdlow DL, Mast EE, Hadler JL. 2002. Fatal inhalational anthrax in a 94-year-old Connecticut Woman. JAMA 2002;287:863-8.

4. Barnes JM. Penicillin and B. anthracis. J. Pathol Bacteriol 1947;59:113-25.

5. Berger T, Kassirer M, Aran AA. Injectional anthrax - new presentation of an old disease. Euro Surveill. 2014;19. pii: 20877.

6. Boyer AE, Quinn CP, Hoffmaster AR, Kozel TR, Saile E, Marston CK, Percival A, Plikaytis BD, Woolfitt AR, Gallegos M, Sabourin P, McWilliams LG, Pirkle JL, Barr JR. Kinetics of lethal factor and poly-D-glutamic acid antigenemia during inhalation anthrax in rhesus macaques. Infect Immun 2009;77:3432-41.

7. Brachman PS, Gold H, Plotkin SA, Fekety FR, Werrin M, Ingraham NR. Field evaluation of a human anthrax vaccine. Am J Public Health 1962;52:632-45.

8. Bradaric N, Punda-Polic V. Cutaneous anthrax due to penicillin-resistant Bacillus anthracis transmitted by an insect bite. Lancet 1992;340:306-7.

9. Centers for Disease Control and Prevention. Use of anthrax vaccine in the United States. Recommendations of the Advisory Committee on Immunization Practices (ACIP), 2009. MMWR 2010;59(RR-6):1–30. www. cdc.gov/mmwr/PDF/rr/rr5906.pdf.

10. Centers for Disease Control and Prevention (CDC). Cutaneous anthrax associated with drum making using goat hides from West Africa--Connecticut, 2007. MMWR Morb Mortal Wkly Rep. 2008;57:628-31.

11. Centers for Disease Control and Prevention (CDC). Gastrointestinal anthrax after an animal-hide drumming event - New Hampshire and Massachusetts, 2009. MMWR Morb Mortal Wkly Rep. 2010;59:872-7.

12. Choe C, Bouhaouala S, Brook I, Elliot TB, Knudson GB. In vitro development of resistance to ofloxacin and doxycycline in Bacillus anthracis Sterne. Antimicrob Agents Chemother 2000;44:1766

13. Christie AB. Infectious Diseases: epidemiology and clinical practice. Churchill Livingstone, New York 1987;2:997:983-1003.

14. Clinical and Laboratory Standards Institute. 2010. Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria, 2nd ed. Vol. 30, no 18 Approved Guideline M45-A2. CLSI, Wayne, PA.

15. Clinical and Laboratory Standards Institute. 2012. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 9th ed., vol. 32, no. 2. Approved standard M7-A9. CLSI, Wayne, PA.

16. Deziel, MR, Heine, H, Louie, A, Kao, M, Byrne, WR, Bassett, J, Miller, L, Bush, K, Kelly, M, Drusano, GL. Effective antimicrobial regimens for use in humans for therapy of Bacillus anthracis Infections and postexposure prophylaxis. Antimicrob Agents Chemother. 40:5099-5106 (2005)

17. Doganay M, Aydin N. Antimicrobial susceptibility of Bacillus anthracis. Scand J Infect Dis 1991;23:333-5.

18. Friedlander AM. Anthrax-Clinical Features, Pathogenesis, and Potential Biological Warfare Threat. In Current Clinical Topics in Infectious Diseases 2000;20:335-49 (Remington JS, Swartz MN, eds.)

19. Friedlander AM, Grabenstein JD, Brachman PS, Anthrax vaccines. 2013. In Vaccines. 6th Edition. (Plotkin SA, Orenstein WA, Offit PA, eds.) pp 127-40. WB Saunders, Elsevier, Philadelphia.

20. Friedlander AM, Pittman PR, Parker GW. Anthrax vaccine: evidence for safety and efficacy against inhalational anthrax. JAMA 1999;282:2104-6.

21. Friedlander AM, Welkos SL, Pitt ML, Ezzell JW, Worsham PL, Rose KJ, Ivins BE, Lowe JR, Howe GB, Mikesell P, Lawrence WB. Postexposure prophylaxis against experimental inhalation anthrax. J Infect Dis 1993;167:1239-42.

22. Gill SC, Rubino CM, Bassett J, Miller J, Ambrose PG, Bhavnani SM, Beaudry A, Li J, Clawson-Stone K, Critchley I, Janjic N, Heine HS. 2010. Pharmacokinetic-pharmacodynamic assessment of faropenem in a lethal murine Bacillus anthracis inhalation postexposure prophylaxis model. Antimicrob Agents Chemother. 2010;54:1678-83.

23. Gochenour WS Jr., Sawyer WD, Henderson JE, Gleiser CA, Kuehne RW, Tigertt WD. On the recognition and therapy of Simian woolsorter’s disease. J Hyg 1963;61:317-22.

24. Gold H. Anthrax: a report of one hundred seventeen cases. AMA Arch Intern Med 1955;96:387-96.

25. Grinberg LM, Abramova FA, Yampolskaya OV, Walker DH, Smith JH. Quantitative pathology of inhalational anthrax I: quantitative microscopic findings. Mod Pathol 2001;14:482-95.

26. Hanczaruk M, Reischl U, Holzmann T, Frangoulidis D, Wagner DM, Keim PS, Antwerpen MH, Meyer H, Grass G. Injectional anthrax in heroin users, Europe, 2000-2012. Emerg Infect Dis. 2014;20:322-3.

27. Heine HS, Bassett J, Miller L, Bassett A, Ivins BE, Lehoux D, Arhin FF, Parr TR, Moeck G. Efficacy of oritavancin in a murine model of Bacillus anthracis spore inhalation anthrax. Antimicrob Agents Chemother. 2008;52:3350-7.

28. Heine HS, Bassett J, Miller L, Hartings JM, Ivins BE, Pitt ML, Fritz D, Norris SL, Byrne WR. Determination of antibiotic efficacy against Bacillus anthracis in a mouse aerosol challenge model. Antimicrob Agents Chemother. 2007;51: 1373-9.

29. Heine 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: 4471-3.

30. Heine HS, Dicks R, Andrews G. In vitro activity of oratavancin (LY33328), levofloxacin, meropenem, GAR936 and linezolid against strains of Bacillus anthracis. 41st Interscience Conference on Antimicrobial Agents and Chemotherapy. Chicago, Ill, Dec. 16-19, 2001; Abst E-534, p173.

31. Heine HS, Dicks R, Byrne WR. In vitro activity of daptomycin, sparfloxacin, quinupristin-dalfopristin and other antibiotics against Bacillus anthracis. 40st Interscience Conference on Antimicrobial Agents and Chemotherapy. Toronto Can, Sept 17-20, 2000; Abst 517, p167.

32. Heine HS, Purcell BK, Bassett J, Miller L, Goldstein BP. Activity of dalbavancin against Bacillus anthracis in vitro and in a mouse inhalation anthrax model. Antimicrob. Agents Chemother. 2010;54:991-6.

33. Henderson DW, Peacock S, Belton FC. Observations on the prophylaxis of experimental pulmonary anthrax in the monkey. J Hyg 1956;54:28-36.

34. Hendricks KA, Wright ME, Shadomy SV, Bradley JS, Morrow MG, Pavia AT, Rubinstein E, Holty JE, Messonnier NE, Smith TL, Pesik N, Treadwell TA, Bower WA; Workgroup on Anthrax Clinical Guidelines.Centers for Disease Control and Prevention expert panel meetings on prevention and treatment of anthrax in adults. Emerg Infect Dis [Internet]. 2014 Feb [date cited]. http://dx.doi.org/10.3201/eid2002.130687

35. Holty JE, Bravata DM, Liu H, Olshen RA, McDonald KM, Owens DK. Systematic review: a century of inhalational anthrax cases from 1900 to 2005. Ann Intern Med. 2006;144:270-80.

36. Jernigan JA, Stephens DS, Ashford DA, Omenaca C, Topiel MS, Galbraith M, Tapper M, Fisk TL, Zaki S, Popovic T, Meyer RF, Quinn CP, Harper SA, Fridkin SK, Sejvar JJ, Shepard CW, McConnell M, Guarner J, Shieh WJ, Malecki JM, Gerberding JL, Hughes JM, Perkins BA. Bioterrorism-related inhalational anthrax: the first 10 cases reported in the United States. Emerg Infect Dis 2001;7:933-44.

37. Kammanadiminti S, Patnaikuni RK, Comer J, Meister G, Sinclair C, Kodihalli S. Combination therapy with antibiotics and anthrax immune globulin intravenous (AIGIV) is potentially more effective than antibiotics alone in rabbit model of inhalational anthrax. PLOS One 2014;9:1-10.

38. Li F, Nandy P, Chien S, Noel GJ, Tornoe CW. Pharmacometrics-based dose selection of levofloxacin as a treatment for postexposure inhalational anthrax in children. Antimicorb Agents Chemother. 2010;54(1):375-9.

39. Lightfoot NF, Scott RJ, Turnbull PC. Antimicrobial susceptibility of Bacillus anthracis. Salisbury Med Bull Suppl 1990;68:95-8.

40. Lincoln RE, Walker JS, Klein F, Haines BW. Anthrax. Advances Vet Sciences 1964;9:327-68.

41. Liu S, Moayeri M, Leppla SH. Anthrax lethal and edema toxins in anthrax pathogenesis. Trends in Microbiology 2014;22:317-25.

42. Louie A, VanScoy BD, Brown DL, Kulawy RW, Heine HS, Drusano 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.

43. Louie A, VanScoy BD, Heine HS, Liu W, Abshire T, Holman K, Kulawy R, Brown DL, Drusano GL. Differential effects of linezolid and ciprofloxacin on toxin production by Bacillus anthracis in an in vitro pharmacodynamic system. Antimicrob Agents Chemother. 2012;56:513-7.

44. Marano N, Plikaytis BD, Martin SW, Rose C, Semenova VA, Martin SK, Freeman AE, Li H, Mulligan MJ, Parker SD, Babcock J, Keitel W, El Sahly H, Poland GA, Jacobson RM, Keyserling HL, Soroka SD, Fox SP, Stamper JL, McNeil MM, Perkins BA, Messonnier N, Quinn CP; Anthrax Vaccine Research Program Working Group. Effects of a reduced dose schedule and intramuscular administration of anthrax vaccine adsorbed on immunogenicity and safety at 7 months: a randomized trial. JAMA 2008;300:1532–43. Erratum in: JAMA 2008;300:2252.

45. Meselson M, Guillemin J, Hugh-Jones M, Langmuir A, Popova I, Shelokov A, Yampolskaya O. The Sverdlovsk anthrax outbreak of 1979. Science 1994;266:1202-8.

46. Migone TS, Subramanian M, Zhong J, Healey LM, Corey A, Devalaraja M Lo L, Ullrich S, Zimmerman J, Chen A, Lewis M, Meister G, Gillum K, Sanford D, Mott J, Bolmer SD. Raxibacumab for the treatment of inhalational anthrax. N Engl J Med. 2009;361:135-44.

47. Mikesell P, Hillanbrand DA, Friedlander AM, Bush K. Antibiotic susceptibility profiles and beta-lactamase activity in strains of Bacillus anthracis. Amer Soc Micro Annual Meeting 1992;Abstract A-122.

48. Mohammed MJ, Marston CK, Popovic T, Weyant RS, Tenover FC. Antimicrobial susceptibility testing of Bacillus anthracis: comparison of results obtained by using the National Committee for Clinical Laboratory Standards broth microdilution reference and Etest agar gradient diffusion methods. J Clin Microbiol. 2002;40:1902-7.

49. Odendaal MW, Pieterson PM, de Vos V, Botha AD. The antibiotic sensitivity patterns of Bacillus anthracis isolated from the Kruger National Park. Onderstepoort J Vet Res 1991;58:17-9.

50. Peterson JW, Moen ST, Healy D, Pawlik JE, Taormina J, Hardcastle J, Thomas JM, Lawrence WS, Ponce C, Chatuev BM, Gnade BT, Foltz SM, Agar SL, Sha J, Klimpel GR, Kirtley ML, Eaves-pyles T, Chopra AK. Protection afforded by fluoroquinolones in animal models of respiratory infections with Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Open Microbiol J. 2010;4:34-46.

51. Pittman PR, Gibbs PH, Cannon TL, Friedlander AM. Anthrax vaccine: short-term safety experience in humans. Vaccine 2001;20:972-8.

52. 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.

53. Ronaghy HA, Azadeh B, Kohout E, Dutz W. Penicillin therapy of human cutaneous anthrax. Curr Ther Res 1972;14:721-5.

54. Saggar SN, Joseph MM, Bell WJ. Treatment of cutaneous anthrax with a single oral dose of doxycycline. East Afr Med J 1974;51:889-94.

55. Severn M. A fatal case of pulmonary anthrax. Brit Med J 1976;1:748.

56. Shieh WJ, Guarner J, Paddock C, Greer P, Tatti K, Fischer M, Layton M, Philips M, Bresnitz E, Quinn CP, Popovic T, Perkins BA, Zaki SR; Anthrax Bioterrorism Investigation Team. The critical role of pathology in the investigation of bioterrorism-related cutaneous anthrax. Am J Pathol. 2003;163:1901-10.

57. Sprenkle MD, Griffith J, Marinelli W, Boyer AE, Quinn CP, Pesik NT, Hoffmaster A, Keenan J, Juni BA, Blaney DD. Lethal factor and anti-protective antigen IgG levels associated with inhalation anthrax, Minnesota, USA. Emerg Infect Dis. 2014;20:310-4.

58. Stepanov AV, Marinin LI, Pomerantsev AP, Staritsin NA. Development of novel vaccines against anthrax in man. J Biotechnol 1996;44:155-60.

59. Sumerkan B, Aygen B, Doganay M, Sehmen E. Antimicrobial susceptibility of Bacillus anthracis against macrolides. Salisbury Med Bull Supplement 1996;87:138.

60. Tournier J-N, Paccani SR, Quesnel-Hellmann A, Baldari CT. Anthrax toxins: A weapon to systematically dismantle the host immune defenses. Molecular Aspects of Medicine 2009;30:456–66.

61. Vietri NJ, Purcell BK, Lawler JV, Leffel EK, Rico P, Gamble CS, Twenhafel NA, Ivins BE, Heine HS, Sheeler R, Wright ME, Friedlander AM. Short-course postexposure antibiotic prophylaxis combined with vaccination protects against experimental inhalational anthrax. Proc Natl Acad Sci U S A 2006;103:7813–6.

62. Vietri NJ, Purcell BK, Tobery SA, Rasmussen SL, Leffel EK, Twenhafel NA, Ivins BE, Kellogg MD, Webster WM, Wright ME, Friedlander AM. A short course of antibiotic treatment is effective in preventing death from experimental inhalational anthrax after discontinuing antibiotics. The Journal of Infectious Diseases 2009;199:336–41.

63. Walsh JJ, Pesik N, Quinn CP, Urdaneta V, Dykewicz CA, Boyer AE, Guarner J, Wilkins P, Norville KJ, Barr JR, Zaki SR, Patel JB, Reagan SP, Pirkle JL, Treadwell TA, Messonnier NR, Rotz LD, Meyer RF, Stephens DS. A case of naturally acquired inhalation anthrax: clinical care and analyses of anti-protective antigen immunoglobulin G and lethal factor. Clin Infect Dis. 2007;44:968-71.

64. Weiss S, Kobiler D, Levy H, Pass A, Ophir Y, Rothschild N, Tal A, Schlomovitz J, Altboum Z. Antibiotics cure anthrax in animal models. Antimicrob Agents Chemother. 2011;55:1533-42.

Table 1. Broth dilution minimum inhibitory concentrations (ug/ml) (Ref 39, 17, 48, 30, 31)

	MIC (ug/ml) (Lightfoot)			MIC (ug/ml) (Doganay)			MIC (ug/ml) (CDC)			MIC (ug/ml) (USAMRIID)
ANTIBIOTIC	Range	MIC50	MIC90	Range	MIC50	MIC90	Range	MIC50	MIC90	Range	MIC50	MIC90
Amikacin				0.03-0.06	0.03	0.06				1-4	2	2
Gentamicin	0.06-0.5	0.125	0.25	0.03-0.25	0.06	0.125				1-4	2	2
Netilmicin				0.015-0.125	0.06	0.125				2-8	2	4
Streptomycin	0.5-4	1	1	1-4	2	4				4-16	4	8
Tobramycin				0.25-1	0.25	1				1-16	2	4
Vancomycin				0.25-1	1	1	0.5-2	2	2	1-4	2	2
Oritavancin										<0.03-1	0.25	0.5
Erythromycin	0.25-1	0.5	1	0.25-1	0.5	1	0.5-1	1	1
Azithromycin				0.5-4	1	4				2-32	8	8
Clarithromycin				0.03-0.25	0.06	0.12				0.25-2	0.5	1
Clindamycin				0.5-1	1	1	<0.5-1	<0.5	1	0.12-1	0.25	0.5
Levofloxacin										0.06-1	0.25	0.5
Ofloxacin				0.03-0.06	0.06	0.06				0.25-2	1	2
Ciprofloxacin	0.03-0.06	0.06	0.06	0.03-0.06	0.03	0.06	0.03-0.12	0.06	0.06	0.06-2	0.25	1
Sparfloxacin										0.12-2	0.5	0.5
Novobiocin										1-4	2	2
Amox/clav (2:1)				0.015-0.015	0.015	0.015				0.5-16	1	2
Amoxicillin	0.03-64	0.06	0.125	0.015-0.03	0.015	0.015				8->64	64	>64
Ampicillin				0.03-0.125	0.03	0.03				4->64	64	>64
Penicillin G	0.015-64	0.06	0.125	0.015-0.03	0.015.	0.015	<0.06-128	<0.06	<0.06	2->64	64	>64
Piperacillin				0.125-0.5	0.25	0.5				16->64	64	>64
Imipenem										<0.03->64	<0.03	0.12
Meropenem										<0.03->64	0.06	0.12
Ceftazidime				128-256	128	256				>64	>64	>64
Cefotaxime				8-32	32	32				16->64	32	>64
Cefotetan										8-32	16	16
Cefuroxime	1-64	32	64	16-64	64	64				16->64	64	>64
Cefazolin				0.015-0.03	0.015	0.015				0.5-8	0.5	1
Ceftriaxone				16-32	16	32	14-32	16	32	16->64	16	64
Aztreonam				>128	>128	>128				>64	>64	>64
Sulfamethoxazole										2->64	>64	>64
Co-trimoxazole				8-16	16	16				2->64	>64	>64
Trimethoprim										>64	>64	>64
Tetracycline	0.06-1.0	0.125	0.125				0.03-0.06	0.03	0.06
Doxycycline										<0.03-0.12	0.06	0.06
Tigecycline										<0.03-0.5	0.12	0.5
Clofazamine										8-64	16	32
Rifampin							<0.25-0.5	<0.25	0.5	<0.03-1	0.5	0.5
Quinupristin-Dalfopristin										0.25-4	1	1
Chloramphenicol	2-4	4	4	1-2	2	2	2-8	4	4	8-64	16	16
Daptomycin										1-4	2	2
Linezolid										0.5-4	2	4