INFECTION AND IMMUNITY, Nov. 2002, p. 6231–6241 0019-9567/02/$04.00⫹0 DOI: 10.1128/IAI.70.11.6231–6241.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 70, No. 11
Postexposure Prophylaxis against Anthrax: Evaluation of Various Treatment Regimens in Intranasally Infected Guinea Pigs Zeev Altboum,1* Yehoshua Gozes,1 Ada Barnea,2 Avi Pass,2 Moshe White,2 and David Kobiler1 Departments of Infectious Diseases1 and Biotechnology,2 Israel Institute for Biological Research, Ness-Ziona 74100, Israel Received 3 April 2002/Returned for modification 13 June 2002/Accepted 14 August 2002
The efficiency of postexposure prophylaxis against Bacillus anthracis infection was tested in guinea pigs infected intranasally with either Vollum or strain ATCC 6605 spores (75 times the 50% lethal dose [LD50]and 87 times LD50, respectively). Starting 24 h postinfection, animals were treated three times per day for 14 days with ciprofloxacin, tetracycline, erythromycin, cefazolin, and trimethoprim-sulfamethoxazole (TMP-SMX). Administration of cefazolin and TMP-SMX failed to protect the animals, while ciprofloxacin, tetracycline, and erythromycin prevented death. Upon cessation of treatment all erythromycin-treated animals died; of the tetracycline-treated animals, two of eight infected with Vollum and one of nine infected with ATCC 6605 survived; and of the ciprofloxacin group injected with either 10 or 20 mg/kg of body weight, five of nine and five of five animals, respectively, survived. To test the added value of extending the treatment period, Volluminfected (46 times the LD50) animals were treated for 30 days with ciprofloxacin or tetracycline, resulting in protection of eight of nine and nine of nine animals, respectively. Once treatment was discontinued, only four of eight and five of nine animals, respectively, survived. Following rechallenge (intramuscularly) of the survivors with 30 times the LD50 of Vollum spores, all ciprofloxacin-treated animals were protected while none of the tetracycline-treated animals survived. In an attempt to confer protective immunity lasting beyond the termination of antibiotic administration, Vollum-infected animals were immunized with a protective antigen (PA)-based vaccine concurrently with treatment with either ciprofloxacin or tetracycline. The combined treatment protected eight of eight and nine of nine animals. Following cessation of antibiotic administration seven of eight and eight of eight animals survived, of which six of seven and eight of eight resisted rechallenge. These results indicate that a combined treatment of antibiotics together with a PA-based vaccine could provide long-term protection to prevent reoccurrence of anthrax disease. Sverdlovsk, Russia, in 1979, which caused fatal disease in at least 66 exposed individuals (17, 30). In October and November 2001, a bioterrorism-related attack launched by spreading highly sophisticated powdered B. anthracis spores through the U.S. mail caused infection in 22 people; 11 cases were diagnosed as inhalational anthrax and 11 (7 confirmed and 4 suspected) were diagnosed as cutaneous anthrax (5, 11). Patients were treated with multidrug antibiotics and supportive care (7, 8, 10, 24, 33). Six patients who received the antibiotic treatment at the initial phase of illness survived. Patients treated at the severe phase of the illness died. Given the rapid course of respiratory anthrax, early antibiotic administration is of crucial importance. Delaying treatment of infected patients, even by hours, may substantially reduce the chances for survival (1, 28). In 1944, Murphy et al. (31) reported the first successful use of penicillin in treating anthrax in humans. Since that time penicillin has been considered the first choice for antibiotic treatment of anthrax, although occasional reports have described the isolation of penicillin-resistant strains (4, 14, 27a). The World Health ORganization-recommended treatment for severe forms of anthrax is penicillin G as an initial treatment followed by procaine penicillin either alone or synergically with streptomycin. In the event of allergy to penicillin, effective alternatives include tetracycline, erythromycin, gentamicin, and chloramphenicol (37). Currently, the recommended antibiotics for postexposure prophylaxis are ciprofloxacin, doxycycline, and procaine penicillin G (9, 21).
Anthrax is primarily a disease of herbivorous animals caused by Bacillus anthracis, a gram-positive, nonmotile, spore-forming rod (18). In humans, three types of anthrax have been recorded according to the route of infection: cutaneous, gastrointestinal, and respiratory (13). Respiratory anthrax is a rare disease usually associated with either industrial exposure to spores (2) or bioterrorism (24). This form of the disease is difficult to diagnose and virtually always fatal, even with vigorous antibacterial therapy (3, 24). In animal models following pulmonary deposition, the spores are phagocytized by alveolar macrophages and transferred through the lymphatic channels to the tracheobronchial lymph nodes (after 4 h), where the spores germinate and grow as vegetative cells (18 h). These cells then enter the bloodstream, causing systemic disease (35). Within the alveolar macrophages, the germinating spores synthesize an antiphagocytic capsule and secrete lethal and edema toxins (36). In humans the disease begins with nonspecific symptoms that resemble cough-like diseases, which last 2 to 3 days, and then suddenly change to severe respiratory distress. Death occurs within 24 to 36 h from respiratory failure, sepsis, and shock. The severity of inhalation anthrax was described in detail following an accidental burst of B. anthracis spores over * Corresponding author. Mailing address: Department of Infectious Diseases, Israel Institute for Biological Research, P.O. Box 19, NessZiona 74100, Israel. Phone: 972-8-9381414. Fax: 972-8-9381639. Email:
[email protected]. 6231
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Prophylactic experiments for treatment of respiratory anthrax have been conducted in monkeys (15, 16, 20, 28) guinea pigs (25, 38), mice (26, 28a), and sheep (16). Treatment of infected rhesus monkeys with penicillin for 20 or 30 days protected the animals, but upon cessation of treatment only approximately 70% of the animals survived (15, 20). Treatment with doxycyline and ciprofloxacin for 30 days provided protection during treatment, and after termination of antibiotic injections approximately 90% of the animals survived (15). The surviving animals did not develop protective immune responses, and following rechallenge, 85 to 100% of the animals developed fatal disease. Full protection was achieved by combination of treatment with penicillin or doxycycline together with active immunization with protective antigen (PA)-based vaccines, which include components of the lethal and edema toxins (15, 20). Lincoln et al. (28) described a successful treatment that protected 84% of septicemic monkeys by administration of antibiotics together with anti-Sterne antisera and the PA-based vaccine. Successful treatment of sheep that were infected via inhalation with Vollum spores was achieved by 5 days of injections of penicillin or tetracycline with and without coadministration of the PA-based vaccine. Experiments with infected guinea pigs were performed with penicillin (38) and doxycycline and ciprofloxacin (25). In the latter experiment, the antibiotic treatment was begun 2 days prior to infection. During treatment the infected animals survived. However, after cessation of treatment, 14% of the penicillin-treated animals survived, compared to 92.5 and 90% of the animals treated with doxycycline and ciprofloxacin, respectively. Similar experiments with mice indicated that penicillin and streptomycin could provide either partial (28a) or full (26) protection. Chloramphenicol failed to protect the animals (28a). Another prophylactic approach to anthrax that was tested in guinea pigs was passive protection using anti-PA antibodies (27, 29; S. F. Little, B. E. Ivins, P. F. Fellows, and A. M. Friedlander, Abstr. 94th Gen. Meet. Am. Soc. Microbiol. 1994, abstr. E-64, p.154, 1994). In this work, we tested the efficacy of recommended antibiotics and antibiotics not tested previously as postexposure prophylactic agents against anthrax. The effectiveness of ciprofloxacin, tetracycline, erythromycin, cefazolin, and trimethoprim-sulfamethoxazole were tested. The effectiveness of ciprofloxacin and tetracycline for postexposure prophylaxis was demonstrated in a rhesus monkey model of experimental inhalation anthrax. The effectiveness of erythromycin has not been tested in an experimental model even though it is recommended as an antibiotic for treatment, particularly in children and pregnant women that are sensitive to other antibiotics. An antibiotic sensitivity test indicated that B. anthracis strains are sensitive to cefazolin (narrow-spectrum of cephalosporins); because this antibiotic is widely distributed in hospitals and can be administrated via intramuscular injections, it was included in this study. Another antibiotic that is commonly used in hospitals is trimethoprim-sulfamethoxazole, which is well absorbed following oral administration. In vitro assays indicated that B. anthracis strains have intermediate resistance to this antibiotic; however this antibiotic was included in this study to test its in vivo effect against anthrax. We chose the guinea pig as a model animal because these animals are highly sensitive to infection with B. anthracis
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spores, the disease is well characterized, and protection could be provided by different vaccine preparations. In contrast to the human disease, which is characterized as a two-phase disease, anthrax in guinea pigs develops rapidly and death occurs within 2 to 4 days postinfection. There are no physically specific symptoms that might indicate that the animals are ill, except that shortly before death the animals start breathing with difficulty. Guinea pigs are sensitive to certain antibiotics, such as penicillin, a fact which may narrow the feasibility of testing a wide range of antibiotics. However, working with small animals has the advantage of allowing simultaneous comparison of the effectiveness of various anti-B. anthracis agents. In this study the animals were infected through the respiratory system by intranasal instillation of spores from two different virulent strains, and treatment was begun 24 h postinfection. We analyzed the effectiveness of protection provided by the antibiotics included in this study. To study the influence of the duration of antibiotic administration, we analyzed the persistence of spores in the lungs either by monitoring survival after cessation of antibiotic therapy or by counting the number of bacteria in the lungs. We monitored the immunity status of the surviving animals either by determination of antibody titers to various bacterial components or by their resistance to rechallenge by a lethal dose of spores. We also tested the added value of a PA-based vaccine in providing reliable long-term protection. MATERIALS AND METHODS B. anthracis strains. The virulent ATCC 14578 (Vollum) (a Tox⫹ Cap⫹) and ATCC 6605 (a Tox⫹ Cap⫹) strains were used in this study. The 50% lethal doses (LD50s) by the intranasal route for both strains in guinea pigs are 4 ⫻ 104 and 8 ⫻ 104, respectively (calculated according to the method of Reed and Muench [33a]). The LD50 of the Vollum strain in guinea pigs by intramuscular administration is 50 spores (12, 34). The ATCC 14185 strain (Tox⫹ Cap⫺) was used for purification of PA for vaccine preparations (34). Antibiotics. The following antibiotics were used in this study: ciprofloxacin (a gift from Bayer), tetracycline hydrochloride (Sigma catalog no. T-3383), erythromycin lactobionate (Abbott Laboratories), cefazolin (Totacef) (Bristol catalog no. 7339-21) or cefamezin (cefazolin; Teva), and Septrin, trimethoprim-sulfamethoxazole, and co-trimoxazole (Wellcome). For disk sensitivity tests, the following disks were used: CIP5 (Oxoid) for ciprofloxacin; TE30 (BBL) for tetracycline; E15 (Oxoid) for erythromycin; CZ30 (Difco) for cefazolin; and SXT (BBL) for trimethoprim-sulfamethoxazole. For determination of the MIC by the E-test (a predefined gradient that is used to determine the MIC of individual antimicrobial agents for a microorganism), the following strips (all from AB Biodisk, Solna, Sweden) were used: CI for ciprofloxacin, EM for erythromycin, CE for cephalothin, TS for trimethoprim-sulfamethoxazole, and DC for doxycycline. PA-based vaccine. Purified PA isolated from strain ATCC 14185 was absorbed to Alhydrogel (Superfos Biosector) as previously described (34). Vaccination was done by subcutaneous injection of 0.5 ml of vaccine, by two injections at 2-week intervals. This quantity of PA vaccine is equivalent to the amount used to evaluate the efficiency of PA-based vaccines in guinea pigs with either MDPH-PA (the anthrax vaccine licensed for human use in the United States) (22, 23) or our vaccine (34). Sporulation. A bacterial culture (cultured for 16 h) in Bacto Tryptose broth was diluted into sporulation medium G (19) in an Erlenmeyer flask (well aerated) that was shaken (250 rpm) at 34°C for 48 h. Spores were centrifuged at 8,000 ⫻ g for 30 min, washed several times with cold distilled water, and stored frozen at ⫺20°C. Heat-shocked spores (70°C for 20 min) were used for inoculation of the guinea pigs. Animal studies. Female Hartley guinea pigs (250 to 300 g) obtained from Charles River Laboratories (Motgate, Kent, United Kingdom) were used. Guinea pigs were anesthetized by subcutaneous injection of a mixture of ketamine HCl (40 mg/kg of body weight) and xylazine (5 mg/kg). The animals were then inoculated intranasally by unilateral instillation of Vollum or ATCC 6605 spores (100 l). At 24 h postinfection, six groups of eight animals per group
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infected with Vollum spores and six groups of nine animals each infected with ATCC 6605 spores were injected subcutaneously three times per day with one of five antibiotics for 14 days. One untreated group from each infection served as the control for monitoring the development of fatal anthrax disease. The following antibiotics and doses were used: ciprofloxacin, 7.5 to 20 mg/kg per injection; tetracycline hydrochloride, 20 mg/kg per injection; erythromycin lactobionate, 40 mg/kg; cefazolin, 50 mg/kg per injection; and Septrin (trimethoprim-sulfamethoxazole; co-trimoxazole), 16 mg of trimethoprim/ml and 80 mg of sulfamethoxazole/ml, given as 7 mg of trimethoprim/kg per injection. In longer antibiotic treatment experiments (for 30 days), five groups with nine animals in each group were infected with Vollum spores and treated with tetracycline or ciprofloxacin and with the same antibiotic combined with active immunization with a PA-based vaccine on days 8 and 22 postinfection. The fifth untreated group served as a control group. In all the experiments, the animals were observed for survivors for at least 30 days after termination of treatment. To evaluate the anti-PA antibody titers provided by the combined treatment with antibiotics and vaccination, an additional two groups each of nine naive animals were immunized with PA vaccine while being treated for 30 days with either tetracycline or ciprofloxacin. To evaluate the acquired immunity that the surviving animals had developed, the animals were tested for resistance to rechallenge by intramuscular injection with 1.5 ⫻ 103 (30 times the LD50) of Vollum spores. This spore dose was used for the evaluation of the effectiveness of anti-B. anthracis vaccine preparations in guinea pigs (12, 22, 34). For determination of the number of bacteria in various organs, animals were sacrificed. Blood samples were drawn from the hearts, the isolated spleens were minced in saline, and the lungs were cut into small fragments and homogenized in an Omni mixer homogenizer. Serial dilutions of each sample were plated on tryptose agar plates (Difco). For determination of residual spores in the lungs, the samples were plated before and after incubation at 70°C for 20 min. Each animal that died during the experiments was tested for the presence of B. anthracis bacteria in the blood, spleen, kidney, and liver, and tissue sections taken from all organs, including the brain, were examined by histopathological staining. These tests enabled us to distinguish death from anthrax and nonanthrax death. In all experiments, the animals were cared for according to the National Institutes of Health guidelines for the care and use of laboratory animals (32). The Israel Institute for Biological Research animal use committee approved all experimental protocols. Antibiotic sensitivity tests. Antibiotic sensitivity tests were performed according to National Committee for Clinical Laboratory Standards protocols, using Mueller-Hinton II broth and agar plates (Becton Dickinson, Sparks, Md.). For determination of the MIC by broth macrodilution tests, antibiotics were diluted twofold into 2 ml of Mueller-Hinton II broth, and 0.02 ml of a bacterial culture at the logarithmic phase of growth with turbidity equivalent to 0.5 McFarland was plated and incubated for 20 to 24 h at 37°C. E-tests were performed by spreading bacterial cultures of an optical density at 540 nm of 1.0 by using impregnated swabs on Mueller-Hinton II plates, on which E-test strips were layered. For determination of the antibiotic concentration in the serum, animals were injected seven times (for 2 days plus the first injection on the third day) with each antibiotic at 8-h intervals; and thereafter, serum samples were collected from three animals at each time point up to 24 h after the last injection. The serum inhibition concentration (SIC) for each antibiotic was determined essentially as broth MIC tests by plating the bacterial strains into 2 ml of twofolddiluted serum in Mueller-Hinton II broth. The highest serum dilution that inhibited the growth of both B. anthracis strains was recorded. ELISA. For the enzyme-linked immunosorbent assay (ELISA), purified PA was diluted to 1 g/ml with 0.05 M carbonate buffer (pH 9.6), and 0.1 ml/well was added to 96-well microtiter plates. For antispore and anti-vegetative cell antibodies, approximately 106 formalin-killed spores or bacterial cells were plated into each well in carbonate buffer (pH 9.6). The plates were incubated overnight at 4°C, washed with phosphate-buffered saline–0.05% Tween 20 (PT), incubated with 0.2 ml of PT–2% bovine serum albumin per well for 60 min at 37°C, and washed again with PT. Serial dilutions of the tested sera (0.1 ml/well) were added, and the plates were incubated for 60 min at 37°C. The plates were washed with PT and incubated with 0.1 ml of alkaline phosphatase conjugated to rabbit anti-guinea pig antibodies (Sigma)/well for 30 min at 37°C. The plates were washed with PT incubated with 0.1 ml of alkaline phosphatase substrate/well for 60 min at 37°C according to the manufacturer’s instructions. Plates were read on a micro-ELISA reader at a wavelength of 405 nm. Readings higher than three times that of the controls were scored as positive reactions. Statistical analyses. Fisher’s exact test was performed to compare results between treatment groups, and statistical significance was established at a P value of ⬍0.05.
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TABLE 1. Virulence of Vollum and ATCC 6605 spores to guinea pigs via intranasal infectiona Spore dose (CFU)
Strain
2 ⫻ 107 2 ⫻ 106 2 ⫻ 105 2 ⫻ 104 2 ⫻ 103 2 ⫻ 102
Vollum
ATCC 6605 3 ⫻ 106 3 ⫻ 105 3 ⫻ 104 3 ⫻ 103 3 ⫻ 102 3 ⫻ 10
No. of deaths/no. infected
% Death
MTTD (days)
7/3 6/3, 1/4 3/3, 5/4, 1/5, 1/7 2/3, 2/4, 1/7, 1/12 None None
7/7 7/8 10/12 6/12 0/8 0/4
100 87.5 83.3 50 0 0
3 3.1 4.1 5.5
6/3, 3/4, 1/5 3/3, 2/4, 1/5, 1/7, 1/14 1/4, 1/9, 1/14 None 1/3 None
10/10 8/10 3/10 0/10 1/6 0/6
100 80 30 0 16.6 0
3.5 5.3 9.0
No. of deaths/day
a The LD50 of the Vollum strain for intranasal administration is 4 ⫻ 104 CFU and that of ATCC 6605 strain is 8 ⫻ 104 CFU, calculated according to the method of Reed and Muench (33a).
RESULTS Infectivity of intranasally administered B. anthracis spores to guinea pigs. The virulence of B. anthracis Vollum and ATCC 6605 strains via intranasal instillation in guinea pigs is described in Table 1). Results indicated that the LD50s of the Vollum and ATCC 6605 strains are 4 ⫻ 104 and 8 ⫻ 104, respectively. The rate of bacterial infiltration from the lungs to the spleen and bloodstream was examined by sacrificing animals at various times postinfection and determining the numbers of B. anthracis CFU in the spleen and blood. The results indicated that at 24 h postinfection no B. anthracis bacteria were detected in either the spleen or the blood. Bacteremia could be detected beginning at about 36 h postinfection, and bacteria were detected in the spleen and/or the blood in two of three animals tested. At 48 h all three animals tested were bacteremic (Table 2). Statistical analysis of the results indicated that the P value was 0.14, suggesting that the results are not statistically different. Since bacteremia was not detected at 24 h postinfection, antibiotic treatment was begun at 24 h postinfection, at the prebacteremic stage.
TABLE 2. Infiltration of Vollum bacteria into the spleen and blood following intranasal spore infection Time postinfection (h)a
Animal no.b
CFU/spleen
CFU/ml of blood
24
1 2 3
0 0 0
0 0 0
36
4 5 6
0 0 6 ⫻ 104
0 6 ⫻ 10 4 ⫻ 102
48
7 8 9
3 ⫻ 10 6 ⫻ 103 5 ⫻ 108
4 ⫻ 10 6 ⫻ 102 2 ⫻ 106
a
Intranasal infection with 30 LD50s of Vollum spores. The experiment contained nine animals; three were sacrificed at each indicated time. Fisher’s exact test indicated that P was ⬎0.05. b
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INFECT. IMMUN. TABLE 3. MIC and SIC of antibiotics tested against B. anthracis strains MIC (g/ml) for strain
Antibiotic
Ciprofloxacin Tetracycline Doxycycline Erythromycin Cefazolin Cephalothin Trimethoprim-sulfamethoxazole
Vollum
ATCC 6605
Macrodilution
E-test
Macrodilution
0.15 0.007
0.094
0.15 0.015
0.4 0.15
0.047 1.0 0.5 ⬎32
0.4 0.45
Injected dose (mg/kg)
SICa
10 20
40 80
40 50
20 320
7
⬍2
E-test
0.094 0.032 1.0 0.75 ⬎32
a
The SIC was measured 60 min after antibiotic injection, and the results are presented as the maximum serum dilution that inhibited the growth of the tested B. anthracis strains.
Determination of the MIC and SIC. The sensitivities of B. anthracis strains to ciprofloxacin, tetracycline, erythromycin, cefazolin, and trimethoprim-sulfamethoxazole were tested by disk sensitivity tests and by determination of the MIC of each antibiotic by either broth sensitivity test or E-test (Table 3). The results indicated that both strains exhibited similar sensitivities to the antibiotics. Pharmacokinetic studies of the administered antibiotics revealed that the peak concentration in serum was achieved at 60 min postinjection. Administration of ciprofloxacin (10 mg/kg), tetracycline, erythromycin, and cefazolin resulted in SICs of 40, 80, 20, and 320 times the MICs, respectively, for both B. anthracis strains. SICs were detected up to 4 h for ciprofloxacin, 5 h for tetracycline, 3 h for cefazolin, and 2 h for erythromycin. By disk sensitivity tests with trimethoprim-sulfamethoxazole, the results suggest intermediate resistance. However, E-tests indicated a MIC of 32 g/ml; no SICs were detected in the sera of guinea pigs. The response of naive guinea pigs to treatment with antibiotics. In order to test the effect of the antibiotic dose on uninfected animals, guinea pigs (five per group) were injected with antibiotics at the doses indicated in Materials and Methods three times per day for a period of 14 days. Animals were observed for changes in body weight and other symptoms. Following injection of saline as a control, a body weight gain of 16% ⫾ 2% was observed. Injection of tetracycline, cefazolin, and trimethoprim-sulfamethoxazole resulted in body weight changes of 10% ⫾ 3%, 14% ⫾ 5%, and 16% ⫾ 2%, respectively. Injection of erythromycin resulted in a body weight change of only 3%, and injection of ciprofloxacin (20 mg/kg) caused the death of one animal during antibiotic administration and resulted in a body weight change of ⫺5% ⫾ 2%. The following symptoms were observed following antibiotic injections: tetracycline caused necrotic wounds in the skin, and ciprofloxacin and erythromycin caused hair loss and stiffness of the skin, which indicated loss of fluids. Animals injected with cefazolin and trimethoprim-sulfamethoxazole displayed no ab-
normal effects. Following cessation of antibiotic administration, all animals treated except the ciprofloxacin-treated group gained 31 to 42% of body weight in 14 days, while the ciprofloxacin-treated animals gained only 4% of their body weight. Effectiveness of post-B. anthracis exposure prophylaxis with 14 days of treatment with various antibiotics in guinea pigs. Guinea pigs were infected intranasally with either 75 times the LD50 of strain Vollum or 87 times the LD50 of strain ATCC 6605. At 24 h postinfection, the animals were treated with the following antibiotics: ciprofloxacin, tetracycline, erythromycin, cefazolin, and trimethoprim-sulfamethoxazole three times a day for a period of 14 days. Survival rates of the animals during the antibiotic treatment and 2 weeks thereafter are presented in Fig. 1. The control (untreated) guinea pigs infected with one of the two B. anthracis strains died, with a mean time to death (MTTD) of 2.8 days. Treatment with tetracycline, ciprofloxacin, and erythromycin prevented death of infected animals during treatment. Upon termination of antibiotic administration, none of the erythromycin-treated animals survived, (MTTD of 6.0 and 3.8 days after cessation of treatment of the Vollum- and ATCC 6605 strain-infected animals, respectively). Following the tetracycline treatment, only two of eight (25%)Vollum-infected animals and one of nine (11%) ATCC 6605 strain-infected animals survived (MTTD of 6.6 and 6.8 days after cessation of treatment of animals, respectively). Animals that were treated with 20 mg of ciprofloxacin/kg (Fig. 1B) were sensitive to the antibiotic, and three of eight animals died from non-anthrax-related causes. Bacteria were not cultivated from the different tissues, except the lungs, and were not detected by histopathological examination. The remaining five animals were protected and survived upon cessation of antibiotic administration. Treatment with ciprofloxacin at 10 mg/kg (Fig. 1G) prevented the deaths of the animals during antibiotic treatment; however, once treatment was discontinued, four of nine animals died on day 4, resulting in 55% survival. Treatment with cefazolin and with trimethoprim-sulfamethoxazole
FIG. 1. Protection of B. anthracis-infected guinea pigs treated for 14 days with various antibiotics. Survival rates of Vollum-infected animals (A to E) and 6605-infected animals (F to J) following treatment with tetracycline (A and F), ciprofloxacin (B and G), erythromycin (C and H), cefazolin (D and I), and trimethoprim-sulfamethoxazole (E and J). Guinea pigs were intranasally infected with the B. anthracis spores, and antibiotic administration was started 24 h postinfection three times a day for 14 days. The animals were observed for at least an additional 14 days after cessation of treatment. Statistical analysis that compared the results presented in panels A to E indicate a P value of ⬍0.0001 on days 14 and 30. Comparison of the results that are presented in panels F to J indicate a P value of ⬍0.0001 on day 14 and a P value of ⬍0.05 on day 30.
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did not confer total protection, and the animals died during the antibiotic treatment. During the injections of cefazolin to guinea pigs infected with Vollum and ATCC 6605 spores, only four of eight and five of nine infected animals, respectively, were protected. After cessation of treatment, only one of eight and two of nine infected animals survived. The results with cefazolin were disappointing since both B. anthracis strains exhibited high sensitivity to the antibiotic in vitro, and its administration to guinea pigs resulted in high concentrations in blood (peak level of 320 times the MIC) and in the lungs (9 times the MIC), kidneys (69 times the MIC) and liver (5 times the MIC) (data not presented). Administrations of trimethoprim-sulfamethoxazole to guinea pigs infected with either Vollum or ATCC 6605 spores protected five of eight and five of nine infected animals, respectively; and following termination of injections five of eight and none of nine infected animals survived. Statistical analysis to compare the effectiveness of protection provided by the different antibiotics during 14 days of treatment and on day 30 postinfection resulted in a P value of ⬍0.05 for both time points. This finding further indicated that there is a significant difference in the effectiveness of protection provided by the different antibiotics as postexposure treatment. Comparison of the number of guinea pigs infected with spores of both bacterial strains surviving on day 30 postinfection indicates that ciprofloxacin protected 71.4% (10 of 14 animals), trimethoprim-sulfamethoxazole protected 29.4% (5 of 17 animals), tetracycline and cefazolin each protected 17.6% (3 of 17) of the animals, and erythromycin protected 0.0% (0 of 17 animals). These results indicate that ciprofloxacin is the most effective antibiotic for postexposure treatment. The surviving animals infected with ATCC 6605 spores and treated with various antibiotics were tested for the development of specific antibodies against B. anthracis. Antibodies against PA could be detected beginning on day 45 postinfection. Three animals from the ciprofloxacin-treated group developed anti-PA antibodies, with titers of 200, 400, and 400; antispore antibodies, with titers of 200, 200, and 400; and anticapsule antibodies, with titers of 50, 100, and 100. Two survivors from the tetracycline- and cefazolin-treated groups developed an anti-PA titer of 400; antispore titers of 100 and 200, respectively; and an anticapsule titer of 50. No antivegetative antibodies were detected (titer of ⬍50). To examine residual deposition of spores in the lungs, the survivors from infection with strain ATCC 6605 that were treated with various antibiotics were sacrificed on days 45 to 56 postinfection. Two animals from the ciprofloxacin-treated group contained 4.1 ⫻ 103 and 3.8 ⫻ 104 spores in the lungs, respectively; survivors from cefazolin-treated animals contained 2.4 ⫻ 102 and 1.2 ⫻ 104 spores, respectively; and a survivor from the tetracycline-treated group contained 1.2 ⫻ 102 spores in its lungs. These results indicate that antibiotic administration did not eliminate all the spores from the lungs. To test whether the survivors had acquired protective immunity that could prevent reestablishment of a fatal disease, survivors of the Vollum-infected animals (five animals from the ciprofloxacin-treated group, two from the tetracyclinetreated group, and five from the trimethoprim-sulfamethoxazole-treated group) were rechallenged on day 30 after cessation of antibiotic administration by an intramuscular injection
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of 30 times the LD50 of strain Vollum. None of the animals survived, and the animals died with a MTTD of 2.6 days, similar to that of the four infected naive animals. Effectiveness of postexposure prophylaxis with 30 days of treatment with ciprofloxacin or tetracycline in Vollum-infected guinea pigs. Since 14 days of antibiotic treatment resulted finally in the death of most infected animals, we tested whether prolongation of administration of tetracycline or ciprofloxacin could eradicate the spores from the lungs of infected animals with enhanced effectiveness, so that upon cessation of antibiotic treatment reestablishment of a fatal disease would be prevented. We extended the antibiotic treatment of Volluminfected animals with 46 times the LD50 to 30 days. The results are presented in Fig. 2A and C for tetracycline and ciprofloxacin, respectively. Treatment with tetracycline (Fig. 2A) protected all nine animals. However, upon termination of antibiotic administration, only five (55%) of the animals survived, compared to 25% (two of eight) following 14 days of treatment. None of the survivors developed protective immunity against intramuscular rechallenge with 30 times the LD50 of strain Vollum. Ciprofloxacin (Fig. 2C) was injected at a dose of 15 mg/kg for 10 days followed by 7.5 mg/kg for additional 20 days. A dose of 20 mg of ciprofloxacin/kg was found to be toxic to guinea pigs. However, upon termination of treatment the animals survived. A dose of 10 mg of ciprofloxacin/kg was well tolerated by the guinea pigs, but after termination of antibiotic, four of nine animals died from anthrax. To try to improve the effectiveness of the antibiotic treatment so that after discontinuation of the antibiotic administration all the infected animals would survive, we chose to treat the animals with a dose of 15 mg of ciprofloxacin/kg. However, on day 10 after the initiation of treatment, a control (uninfected) animal that was treated with the same dose of antibiotic died. For that reason we continued to treat the animals with a dose of 7.5 mg of ciprofloxacin/kg. Treatment of Vollum-infected animals with ciprofloxacin (Fig. 2C) protected eight (89%) of nine animals. One animal died from anthrax on day 25 after initiation of treatment. Upon cessation of treatment, four (50%) of the eight remaining animals developed fatal anthrax disease. The remaining four animals were rechallenged by intramuscular injection of 30 times the LD50 of strain Vollum, and all four animals survived, indicating that the animals had developed protective immunity. Sera were not collected in these experiments for determination of antibody titers, in order to avoid a potential spreading of the retained spores from the guinea pig lungs to the heart or other tissues. Statistical analysis comparing the effectiveness of 30 days of treatment with ciprofloxacin or tetracycline resulted in a P value of 1.0, which indicated that the effectiveness of protection provided by both antibiotics is similar. Similarly, no difference was detected in the effectiveness of the treatment with both antibiotics after 60 days postinfection (P ⫽ 1.0). However, the statistical analysis that compared the number of survivors on day 80 postinfection and after the rechallenge test indicated that the P was ⬍0.05, emphasizing that there is a significant difference in the protection provided by both antibiotics. The results (Fig. 2A and C) show that while treatment with cipro-
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floxacin provided protective immunity to four (44.4%) of the nine infected animals, treatment with tetracycline did not. Effectiveness of postexposure prophylaxis with 30 days of treatment with tetracycline or ciprofloxacin combined with active immunization with PA-based vaccine in Vollum-infected guinea pigs. Another approach to increase the number of survivors at the post-antibiotic treatment periods was to actively immunize the infected animals with a PA-based vaccine along with treatment with antibiotics, so that upon cessation of antibiotic administration the acquired anti-PA antibodies will provide protection against disease reestablishment. In these experiments (Fig. 2B and D), the Vollum-infected animals (46 times the LD50) were treated with tetracycline or ciprofloxacin for 30 days, during which the animals were immunized by two injections of PA-based vaccine on days 8 and 22 postinfection. Cotreatment with tetracycline and PA vaccine (Fig. 2B) protected all nine infected animals from developing the disease during the 30 days of treatment; eight were protected for 30 days afer cessation of treatment (one animal died from a nonanthrax cause), and all resisted an intramuscular challenge with 30 LD50s Vollum spores. These results indicated that cotreatment with tetracycline and a PA-based vaccine, in comparison to treatment with tetracycline alone, significantly increased the number of survivors either during post-antibiotic treatment (survival, 100% [eight of eight] versus 55.5% [five of nine animals] and after rechallenge (survival, 100% [eight of eight] versus 0.0% [zero of nine animals]. Cotreatment with ciprofloxacin and PA-based vaccine protected eight of nine animals (one animal died from a cause other than anthrax) during the 30 days of antibiotic administration, after which seven (87.5%) of eight animals survived and six (85.7%) of seven animals resisted intramuscular challenge with 30 times the LD50 of strain Vollum. Comparison of the effectiveness of protection that was provided by the cotreatment with ciprofloxacin plus a PA-based vaccine to that of ciprofloxacin alone indicates that the number of survivors on day 30 postinfection is 100% (eight of eight) versus 88.8% (eight of nine), on day 60 postinfection is 87.5% (seven of eight) versus 44.4% (four of nine), and on day 81, after the rechallenge test, is 75% (six of eight) versus 44.4% (four of nine). Statistical analysis indicates that there is no significant difference between the combined treatment with either antibiotic (P ⫽ 1.0 for 60 days postinfection and P ⫽ 0.47 following rechallenge tests). These findings enable the comparison of the number of survivors for the cotreatment methods and that of antibiotics alone. On day 60 postinfection the number of survivors with the cotreatments versus antibiotic alone was 15 of 16 (93.7%) versus 9 of 18 (50%) animals, respectively, and on day 81 postinfection and after the rechallenge was 14 of 16 (87.5%) versus 4 of 18 (22.2%), respectively. These results strongly indicate the importance of immunization with a PAbased vaccine during the antibiotic treatment to ensure high survival rates after cessation of treatment. The immunity status of animals that were vaccinated during antibiotic treatment was tested on control animal groups. Uninfected animals (nine animals in each group) were injected with tetracycline or ciprofloxacin for 30 days during which the animals were immunized with PA vaccine on days 8 and 22 after the initiation of antibiotic administrations. One animal
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from the ciprofloxacin treated group died during the antibiotic administration. Serum samples were collected 5 days after the second vaccination, and ELISAs indicated anti-PA antibody titers with a geometric mean titer of 22,807 ⫾ 5,225 for animals injected with tetracycline and 16,127 ⫾ 8,506 for ciprofloxacintreated animals. Thirty-five days after the second immunization, the animals were challenged by intramuscular injection of 30 times the LD50 of strain Vollum. Among the PA-vaccinated tetracycline-treated animals, seven of nine (78%) survived, and among the PA-vaccinated ciprofloxacin-treated animals, eight of eight animals (100%) survived. Statistical analysis that compared the effectiveness of protection provided by the cotreatment of uninfected animals with both antibiotics and PA-based vaccine resulted in a P value of 0.47, which indicated that both treatments are effective and provide similar immunity. These results indicated that an effective protection as postexposure prophylaxis treatment against respiratory anthrax could be achieved by a combined treatment of tetracycline or ciprofloxacin together with active immunization with PA-based vaccines. DISCUSSION Respiratory anthrax is a severe disease, almost always fatal. We tested the effectiveness of different antibiotics as postexposure prophylaxis of guinea pigs infected intranasally with two different virulent B. anthracis strains, Vollum and ATCC 6605. We tested the effectiveness of treatment against two different strains to compare treatment efficiencies. Previously described postexposure prophylaxis experiments were performed with rhesus monkeys infected with Vollum spores (15, 20). Antibiotic prophylaxis in guinea pigs was tested in animals that were infected with either the Vollum or Ames strain (25). No major difference in the efficiency of the antibiotics to protect against a certain strain was observed in that work. In this study, groups of animals (eight and nine animals per group) were intranasally infected with (2.3 to 3) ⫻106 (46 to 75 times the LD50) Vollum spores and 7 ⫻ 106 (87 times the LD50) ATCC 6605 spores, in order to cause a fatal disease in all the animals. Lower spore doses did not result in death (Table 1). Antibiotic treatment was begun 24 h postinfection, in order to anticipate the bacteremia shown previously to begin beyond 24 h postinfection (Table 2). We followed the works of Henderson et al. (20) and Friedlander et al. (15), who started the antibiotic treatment postexposure, while Jones et al. (25) tested the efficacy of antibiotic treatment commencing 2 days prior to infection. Infected animals were treated with ciprofloxacin, tetracycline, erythromycin, cefazolin, and trimethoprim-sulfamethoxazole. The Federal Drug Administration has approved ciprofloxacin and tetracycline for treatment of anthrax disease (9, 21), and erythromycin was recommended by the World Health Organization as an alternative antibiotic (37). Previous experiments tested the effectiveness of postexposure treatment with ciprofloxacin and doxycycline (belongs to tetracycline antibiotics) (15, 25). Erythromycin has not been tested previously as a prophylactic antibiotic in experimental anthrax. Cefazolin and trimethoprim-sulfamethoxazole are commonly used in hospitals, and there was no information concerning their effectiveness in treating anthrax.
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Our results indicated that treatment of B. anthracis-infected guinea pigs with either cefazolin or trimethoprim-sulfamethoxazole did not confer full protection, and the animals died during antibiotic administrations. Following cessation of cefazolin injections, only 3 of 17 (17.6%) animals infected with both B. anthracis strains survived. After the injections of trimethoprim-sulfamethoxazole were stopped, 5 of 17 (29.4%) infected animals survived. These results may indicate that cefazolin and trimethoprim-sulfamethoxazole should not be considered for treatment of anthrax. Antimicrobial susceptibility tests of B. anthracis isolates from the bioterrorism attack in the United States in September through November 2001 indicated that the strains are sensitive to ciprofloxacin, tetracycline, doxycycline, penicillin, amoxicillin, clarithromycin, clindamycin, vancomycin, and chloramphenicol; intermediately susceptible to erythromycin and azithromycin; and intermediately susceptible or resistant to ceftriaxone, which indicates the presence of cephalosporinase in the isolates. Based on these results, the Centers for Disease Control and Prevention do not recommend the use of cephalosporins for postexposure prophylaxis or treatment of B. anthracis infection (6). We used agar disk diffusion tests for determination of susceptibility of Vollum and ATCC 6605 strains to cephalosporins and found that both strains were sensitive to cephalothin (narrow-spectrum cephalosporins) and cefoxitin (expanded spectrum) but were resistant to cefuroxime (expanded spectrum) and cefotaxime, ceftriaxone, and ceftazidime (broad spectrum). Our results support the Centers for Disease Control and Prevention recommendation and prove that in spite of the fact that in vitro studies indicated susceptibility of both B. anthracis strains to cefazolin (narrowspectrum cephalosporins), in vivo studies indicated failure to protect B. anthracis-infected animals. Successful treatment of the infected guinea pigs was obtained with ciprofloxacin, tetracycline, and erythromycin, which prevented the development of fatal anthrax disease in the infected animals, during administration of antibiotics. However, when administration of these antibiotics was suspended, the spores that were retained in the lungs germinated, proliferated, and led to the death of the treated animals. We have shown that at 45 to 56 days postinfection, surviving animals still carry about 0.0017 to 0.58% of the initial infectious spore dose in their lungs. Henderson et al. (20) demonstrated that at 42 days postinfection, rhesus monkeys that were treated with penicillin and survived contained in their lungs 15 to 20% of the initially retained spores. Jones et al. (25) showed that most of the infected guinea pigs that were treated with ciprofloxacin and doxycyline retained spores in their lungs 17 and 25 days after cessation of treatment, respectively. This result emphasizes the ineffectiveness of the antibiotic administration to eradicate residual spores from the animal’s lungs. This fact was
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demonstrated in particular in animals that were treated with erythromycin. After cessation of erythromycin administrations, none of the animals survived, and the animals died with a MTTD of 3.8 and 6.0 days after cessation of treatment in Vollum- and ATCC 6605-infected animals, respectively. Since the control infected animals that were not treated with antibiotics died within 2 to 3 days, it is obvious that erythromycin failed to eradicate a significant number of spores from the lungs. Similar results were obtained with tetracycline that protected only 3 of 17 (17.6%) animals infected with both bacterial strains. Following termination of injections the animals died, with a MTTD of 6.6 and 6.8 days. These results indicate that 14 days of injection of either erythromycin or tetracycline are inefficient to cause a significant reduction in the number of retained spores in the infected lungs, and the animals died from reestablishment of fatal anthrax disease. Treatment of the infected guinea pigs for 14 days with ciprofloxacin at a high dose of 20 mg/kg was toxic to three of eight animals; however, the remaining five animals survived for at least 14 additional days but did not developed a protective immune response. Treatment with 10 mg of ciprofloxacin/kg was not toxic; all animals were protected during treatment, but upon cessation of treatment only five of nine (55%) animals survived. Ciprofloxacin is a bactericidal antibiotic that penetrates cells and can inhibit the growth of germinating spores in macrophages. Treatment with 20 versus 10 mg of ciprofloxacin/kg may result in higher drug concentrations within cells and macrophages, which could eliminate the bacteria more efficiently from various organs (including the brain) and thus provide better protection. In these experiments, treatment with ciprofloxacin was the most effective in protecting the infected animals, resulting in survival of 10 of 14 (71.4%) infected animals. Animals that survived 30 to 56 days postinfection developed a weak immune response to PA (ELISA titer in the range of 200 to 400), antispores (ELISA titer in the range of 100 to 400), and anti-bacterial capsule (ELISA titer in the range of 50 to 100). However, this immune response did not provide protection against Vollum spore challenge. Jones et al. (25) could not detect anti-PA antibodies in the surviving animals that were treated with either ciprofloxacin or doxycycline on days 38 and 46 postinfection. In addition, Friedlander et al. (15) did not detect anti-PA antibodies in the surviving rhesus monkeys that were treated with penicillin, ciprofloxacin, or doxycycline even on days 131 to 142 after exposure, and the animals did not develop significant protective immunity against rechallenge. It is possible that in our case, due to the route of infection (intranasal spore instillation) and the high infective dose (87 times the LD50), we could detect antibodies, albeit in very low titers, to different bacterial components. To further characterize the efficacy of antibiotic treatment,
FIG. 2. Protection of Vollum-infected guinea pigs with tetracycline and ciprofloxacin treatments for 30 days or by cotreatment of antibiotics with active immunization with a PA-based vaccine. Intranasally infected animals were treated with antibiotics for 30 days (A and C) or by a combined treatment with antibiotics and a PA-based vaccine (B and D). Animals were observed for additional 30 days after which they were intramuscularly challenge with Vollum spores. Statistical analysis that compared the results presented in panels A and C indicates that on day 30, P ⫽ 1.0; on day 60, P ⫽ 1.0; and on day 80, P ⬍ 0.05. Comparison of the results in panels B and D indicates that on day 30, P ⫽ 1.0; on day 60, P ⫽ 1.0; and on day 80, P ⫽ 0.47.
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we chose to prolong the treatment with tetracycline and ciprofloxacin for 30 days. Increasing the duration of tetracycline treatment to 30 days resulted in a moderately higher survival rate, 5 of 9 (55.5%) versus 3 of 17 (17.6%), after cessation of antibiotic administration. Treatment with ciprofloxacin (15 mg/kg for 10 days, followed by 7.5 mg/kg for an additional 20 days) resulted in survivors in only four of eight (50%) infected animals. Interestingly, the survivors in the ciprofloxacintreated group developed protective immunity against spore challenge, whereas the survivors in the tetracycline treatment group did not. In contrast to ciprofloxacin, tetracycline is a bacteriostatic drug that affects only multiplying microorganisms. In this experiment the infected animals were initially treated with a high dose of ciprofloxacin, 15 mg/kg, which eliminates a high percentage of the spore dose, followed by 20 days of treatment with only 7.5 mg/kg. It is possible that the lower antibiotic dose enables some spores to germinate, grow as vegetative cells, and secrete the lethal and edema toxins, thus inducing the development of a protective immunity. In contrast, tetracycline treatment inhibited the growth of the germinated spores; no protective antibodies were developed so that the surviving animals could not resist challenge. We did not collect sera from the infected guinea pigs to examine their antibody responses because we did not want to contaminate the animals’ tissues with the spores retained in the lungs. Theoretically, anthrax can be cured by long periods of antibiotic treatment, until spores are cleared from the lungs. The Centers for Disease Control and Prevention recommendation for antibiotic treatment of inhalation anthrax is for at least for 60 days. This was based on the observation that in the accidental exposure in Sverdlovsk, Russia, patients developed a fatal disease up to 43 days after exposure. However, as long periods of antibiotic treatment are difficult to manage and patients may respond adversely to the antibiotics, it was suggested previously (15, 20, 28) that respiratory anthrax should be treated with antibiotics in combination with PA vaccines. In 1956, Henderson et al. (20) demonstrated that while treatment of infected monkeys with penicillin did not provide full protection, the addition of immunization with a PA vaccine during the antibiotic administration protected 10 of 10 animals infected by inhalation of spores. In 1993, Friedlander et al. (15) demonstrated full protection of rhesus monkeys infected by inhalation with a combination treatment with doxycycline and PA vaccine. In this study, cotreatment of the infected guinea pigs with tetracycline or ciprofloxacin together with active immunization with a PA-based vaccine led to full protection; 15 of 16 (93.7%) infected animals survived, indicating that antibiotic treatment did not interfere with the effectiveness of the vaccine and emphasizing the effectiveness of this treatment for inhalational anthrax. Control uninfected groups of animals that were vaccinated during antibiotic treatment developed high titers of anti-PA antibodies (ELISA titer [geometric mean titer] of 16,000 to 23,000). Two injections of a PA-based vaccine to naive guinea pigs on days 0 and 14 resulted within 4 weeks in anti-PA antibodies with a geometric mean titer of 36,921 (S. Reuveni, M. D. White, Y. Y. Adar, and Y. Kafri, personal communication). These results indicated that immunization during antibiotic treatment resulted in less than twofold reductions in antibody titers; however, since the assays
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were performed separately, the results might be in the range of a statistical error. The U.S. Department of Health and Human Services statement (www.hhs.gov/news) dated 18 December 2001 provided two additional options beyond the 60-day antibiotic course for those who were exposed to inhalational anthrax in the recent mail attacks in the United States: (i) an extended course of antibiotics for an additional 40 days and (ii) an additional 40 days of antibiotic treatment plus anthrax vaccine that will be given in three doses over a 4-week period, as an investigational postexposure treatment with anthrax vaccine (which is currently under an investigational new drug application). The findings reported here are in accordance with previous reports showing that tetracycline and ciprofloxacin treatment is very efficient during antibiotic administration (15, 25) if given early enough but may allow the flair-up of the disease upon cessation of administrations. The combined treatment of antibiotic and a PA-based vaccine is a reliable postexposure prophylaxis for treatment of respiratory anthrax. ACKNOWLEDGMENTS We thank Avigdor Shafferman and Baruch Velan for fruitful discussions. We thank Yaacov Chai, Moshe Redelman, Yoel Papir, Nili Rothschild, Jossef Shlomovich, and Pinchas Parnes for their excellent technical assistance. We thank Ziv Klausner for the detailed statistical analysis. REFERENCES 1. Barnes, J. M. 1947. Penicillin and B. anthracis. J. Pathol. Bacteriol. 194:113– 125. 2. Brachaman, P. S., A.F. Kaufman, and F. G. Dalldorf. 1966. Industrial inhalation anthrax. Bacteriol. Rev. 30:646–659. 3. Brachman, P. S. 1980. Inhalation anthrax. Ann. N. Y. Acad. Sci. 353:83–93. 4. Bradic, N., and V. Punda-Polic. 1992. Cutaneous anthrax due to penicillin resistant Bacillus anthracis transmitted by insect bite. Lancet 340:306–307. 5. Bush, L. M., B. H. Abrams, A. Beal, and C. C. Johnson. 8 November 2001, posting date. Index case of fatal inhalational anthrax due to bioterrorism in the United States. N. Engl. J. Med. 345:1607–1610. [Online.] http://www .nejm.org. 6. Centers for Disease Control and Prevention. 22 October 2001, posting date. CDC update: Antimicrobial susceptibility of Bacillus anthracis isolates associated with intentional distribution in Florida, New Jersey, New York, Pennsylvania, Virginia, and Washington D.C., September–October 2001. http: //www.bt.cdc.gov/DocumentsApp/Anthrax/10222001Advisory/10222001 advisory.asp. 7. Centers for Disease Control and Prevention. 2001. CDC update: investigation of bioterrorism-related anthrax and interim guidelines for clinical evaluation of persons with possible anthrax. Morb. Mortal. Wkly. Rep. 50:941– 948. 8. Centers for Disease Control and Prevention. 2001. CDC update: investigation of bioterrorism-related anthrax and adverse events from antimicrobial prophylaxis. Morb. Mortal. Wkly. Rep. 50:973–976. 9. Centers for Disease Control and Prevention. 2001. Interim recommendations for antimicrobial prophylaxis for children and breastfeeding mothers and treatment of children with anthrax. Morb. Mortal. Wkly. Rep. 50:1014– 1016. 10. Centers for Disease Control and Prevention. 2001. Adverse events associated with anthrax prophylaxis among postal employees—New Jersey, New York City, and the District of Columbia Metropolitan Area. Morb. Mortal. Wkly. Rep. 50:1051–1054. 11. Centers for Disease Control and Prevention. 2001. Investigation of bioterrorism-related anthrax – Connecticut. Morb. Mortal. Wkly. Rep. 50:1077– 1079. 12. Cohen, S., I. Mendelson, Z. Altboum, D. Kobiler, E. Elhanany, T. Bino, M. Leitner, I. Inbar, H. Rosenberg, Y. Gozes, R. Barak, M. Fisher, C. Korman, B. Valan and A. Shafferman. 2000. Attenuated nontoxinogenic and nonencapsulated recombinant Bacillus anthracis spore vaccines protect against anthrax. Infect. Immun. 68:4549–4558. 13. Dixon, T. C., M. Meselson, J. Guillemin, and P. C. Hanna. 1999. Anthrax. N. Engl. J. Med. 341:815–826. 14. Dognany, M., and N. Aydin. 1991. Antimicrobial susceptibility of Bacillus anthracis. Scand. J. Infect. Dis. 23:333–335. 15. Friedlander, A. M., S. L. Welkos, M. L. M. Pitt, J. W. Ezzell, P. L. Worsham,
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