The Armed Forces Radiobiology Research Institute (AFRRI) in the U. S. Department of Defense conducts biomedical research on the effects of ionizing radiation. It has the largest radiobiology program in the United States and is a national resource in the response to nuclear and radiation accidents. Bacterial spores are potential biological weapons because they can be prepared and distributed by aerosol, they endure harsh environmental conditions, and they are infectious. Decontamination procedures for large concentrations of spores must be effective and practical. We determined the dose response of bacterial spores to three qualities, or types, of ionizing radiation. Inactivation of dry and hydrated bacterial spores with gamma radiation has been more thoroughly studied than spore inactivation with neutron radiation. Decimalreduction curves were produced at AFRRI for Bacillus atrophaeus (B. subtilis var. niger, B. globigii, " BG "), B. pumilus, B. thuringiensis, and B. anthracis Sterne spores, both wet and dry, using doses of 0.3 to 7.2 kGy neutrons delivered at a dose rate of 44 to 49 Gy/min (D-n/D-T = 0.95) in the AFRRI training, research, isotope-producing General Atomic (TRIGA) Mark-F nuclear reactor, and doses of 0.6 to 24.0 kGy gamma rays delivered at dose rates of 112 to 120 Gy/min in the AFRRI cobalt-60 ((CO)-C-60) gamma-photon irradiation facility. Decimal-reduction curves were constructed by plotting the spore survival fraction in terms of colony-forming units vs. radiation dose. All four species showed greater sensitivity to neutron radiation than to gamma radiation, regardless of the state of hydration. Dry spores of all four species were more sensitive to gamma radiation than were hydrated spores. In contrast, the state of hydration, whether dry or hydrated, of spores of B. subtilis and B. pumilus, which were embedded in filter paper strips, did not affect their sensitivity to neutron radiation. Wet B. thuringiensis spores were only slightly more sensitive to neutrons than were B. thuringiensis spores in dry powdered form. Furthermore, the species most resistant to neutron and gamma radiations was a concentrated B. anthracis Sterne spore suspension. When the starting spore concentration, the Bacillus species used, the radiation quality (neutron or gamma), and the state of hydration are known, the radiation decimal-reduction curves generated in these studies can be used to predict bacterial spore survival. Electron-beam radiation (e-beam) has been used to inactivate microorganisms in spices, fresh food, medical components, and hazardous waste. AFRRI assessed the efficacy of using an e-beam for decontamination of bulk biological agents and of byproducts of the decontamination procedures such as wipes and aqueous runoff. Biological agent surrogates were tested under controlled conditions to determine the effectiveness of e-beam for decontamination. Using the AFRRI linear accelerator (LINAC) to deliver doses of 2 to 20 kGy at a dose rate of 1 kGy/min, radiation Decimal-reduction curves were constructed for Bacillus atrophaeus spores in a dry powder and B. anthracis Sterne spores in a slurry. Doses of 0.25 to 1.0 kGy were delivered to vegetative Gram-negative bacterial cells of Serratia marcescens. The LINAC produced 13-MeV electrons at 30 pulses/sec with a 4-mu sec pulse width generated through a water scatterer. Spore samples were irradiated in an array of three screw-capped polystyrene tubes. Dosimetry was performed at the beginning of each experimental run with LiF:Ti,Mg thermoluminescent dosimeters (TLDs), product type TLD-100 (Bicrong (R)). TLDs were processed with the Bicron (R)/Harshaw Model 5500 Automatic TLD Reader. The inactivation data for dry B. atraphaeus spores, B. anthracis Sterne spores, and S. marcescens were fitted to a mathematical ton-rula. The e-beam decimal-reduction curves for the bacterial spores were similar to our previously generated gamma-photon radiation curves. The vegetative bacterial cells of S. marcescens were more susceptible to high-speed electrons than were the bacterial spores. These experimental findings support the concept of using a truck-mounted transportable LINAC in the field for decontaminating bulk materials that are contaminated with pathogenic bacteria.

The terrorist attack in September 2001, with the dissemination of Bacillus anthracis spores in letters sent through the U.S. Postal Service, brought home the reality of bioterrorism. These attacks have heightened concerns about future largescale aerosol attacks with powders of B. anthracis spores and other pathogens that cause smallpox, pneumonic plague, tularemia, and viral hemorrhagic fevers, as well as toxins such as botulinum toxin, ricin or Staphylococcus enterotoxin B. Means to prevent the use of these agents, and to manage the consequences of their use, have become a high priority. One of the tools that can play a role in disease prevention and consequence management is nonionizing radiation in the form of germicidal short-wavelength ultraviolet (UV) light. This article presents background information on the pathogen Bacillus anthracis, the causative agent for anthrax, and its susceptibility to killing by germicidal UV. The results of two experimental studies are also presented that examine UV inactivation of B. anthracis vegetative cells and spores, and spores of closely related Bacillus species, in suspension, dried on surfaces, and as free-flowing powders.

OBJECTIVES Sublethal ionizing doses of radiation increase the susceptibility of mice to Bacillus anthracis Sterne infection. In this study, we investigated the efficacy of clindamycin in 60Co-gamma-photon-irradiated and sham-irradiated mice after intratracheal challenge with B. anthracis Sterne spores. Clindamycin has in vitro activity against B. anthracis and inhibits the production of toxin from other species, although no direct evidence exists that production of B. anthracis toxin is inhibited. METHODS Ten-week-old B6D2F1/J female mice were either sham-irradiated or given a sublethal 7 Gy dose of 60Co-gamma-photon radiation 4 days prior to an intratracheal challenge with toxigenic B. anthracis Sterne spores. Mice were treated twice daily with 200 mg/kg clindamycin (subcutaneous or oral), 100 mg/kg moxifloxacin (oral), 50 mg/kg ciprofloxacin (subcutaneous) or a combination therapy (clindamycin + ciprofloxacin). Bacteria were isolated and identified from lung, liver and heart blood at five timed intervals after irradiation. Survival was recorded twice daily following intratracheal challenge. RESULTS The use of clindamycin increased survival in gamma-irradiated and sham-irradiated animals challenged with B. anthracis Sterne in comparison with control mice (P < 0.001). Ciprofloxacin-treated animals had higher survival compared with clindamycin-treated animals in two experiments, and less survival in a third experiment, although differences were not statistically significant. Moxifloxacin was just as effective as clindamycin. Combination therapy did not improve survival of sham-irradiated animals and significantly decreased survival among gamma-irradiated animals (P = 0.01) in comparison with clindamycin-treated animals. B. anthracis Sterne was isolated from lung, liver and heart blood, irrespective of the antimicrobial treatment. CONCLUSIONS Treatment with clindamycin, ciprofloxacin or moxifloxacin increased survival in sham-irradiated and gamma-irradiated animals challenged intratracheally with B. anthracis Sterne spores. However, the combination of clindamycin and ciprofloxacin increased mortality associated with B. anthracis Sterne infection, particularly in gamma-irradiated animals.

Biological agents and ionizing radiation lead to more severe clinical outcomes than either insult alone. This study investigated the survival of non-irradiated and (60)Co-gamma-irradiated mice given therapy for inhalation anthrax with ciprofloxacin (CIP) or a clinically relevant mixture of clarithromycin (CLR) and its major human microbiologically important metabolite 14-hydroxy clarithromycin (14-OH CLR). All B6D2F1/J 10-week-old female mice were inoculated intratracheally with 3 x 10(8) c.f.u. of Bacillus anthracis Sterne spores 4 days after the non-lethal 7 Gy dose of (60)Co gamma radiation. Twenty-one days of treatment with CLR/14-OH CLR, 150 mg kg(-1) twice daily, or CIP, 16.5 mg kg(-1) twice daily, began 24 h after inoculation. Pharmacokinetics indicate that the area under the curve (AUC) for 14-OH CLR on the concentration-versus-time graph was slightly higher in gamma-irradiated than non-irradiated animals. Neither drug was able to increase survival in gamma-irradiated animals. CIP and CLR/14-OH CLR therapies in non-irradiated animals increased survival from 49 % (17/35 mice) in buffer-treated animals to 94 % (33/35) and 100 %, respectively (P < 0.001). B. anthracis Sterne only was isolated from 25-50 % of treated mice with or without irradiation. Mixed infections with B. anthracis Sterne were present in 50-71 % of gamma-irradiated mice but only in 5-10 % of mice without irradiation.

Klebsiella pneumoniae is a common cause of nosocomially acquired pneumonia in immunocompromised patients. Previously, we established a pneumonia model using Klebsiella pneumoniae in B6D2F1/J mice sublethally irradiated with 7-Gy 60Co gamma-radiation and inoculated intratracheally. In the study reported here, we investigated survival of mice following 10 days of antimicrobial therapy with ceftriaxone, gentamicin, gatifloxacin, and a ceftriaxone-gentamicin combination given once daily. Survival was significantly prolonged in response to all therapies. However, survival of mice was 95% when treated with the ceftriaxone-gentamicin combination followed by ceftriaxone alone (75%), and gatifloxacin (80%), whereas survival for controls was 0%. In addition, resistance to any of the treatments did not develop during the study. We conclude that an immunocompromised status does not alter the Infectious Disease Society of America's primary recommendation for treating community-acquired K. pneumoniae pneumonia using a third-generation cephalosporin, with or without an aminoglycoside.

Ionizing radiation depresses host defenses and enhances susceptibility to local and systemic infection due to endogenous or exogenous microorganisms. Exposure of mice to a lethal dose of ionizing 60Co-gamma radiation induces a dose-related reduction in the number of both aerobic and anaerobic bacteria from 10(10-12) to 10(4-6) per gram of stool within 4 days. The number of anaerobic bacteria stays low, but the number of Enterobacteriaceae per gram of stool increases significantly up to 10(9) by the 12th day after irradiation. This increase is associated with bacterial translocation of these organisms and fatal bacteremia. The use of quinolones in the irradiated animals was effective in controlling systemic endogenous Gram-negative infection after irradiation. Supplementation with penicillin prevented treatment failures due to Streptococcus spp. and increased survival. Quinolones given for 21 days also were effective in management of systemic exogenous infections due to orally ingested Klebsiella pneumoniae and Pseudomonas aeruginosa. Effectiveness of quinolones may be attributed to inhibition of exogenous organism growth within the gut lumen while preserving the anaerobic gut flora as well as their systemic antibacterial activity. Based on these findings, antimicrobial agents recommended for therapy of infection after exposure to irradiation are: ciprofloxacin, levofloxacin, ceftriaxone, cefepime, gentamicin +/- amoxicillin, or vancomycin.

Bacteria such as Klebsiella pneumoniae can invade and colonize an immunocompromised host and complicate clinical recovery. In the study reported here, an experimental model of induced pneumonia was developed in 60Co gamma-photon-irradiated mice for the purpose of evaluating efficacy of therapeutic agents. The model was characterized by use of probit analysis of bacterial dose, and microbiologic, and histopathologic results. Bacterial colony-forming-unit (CFU) values producing 50% mortality within 30 days (LD50/30) and their 95% confidence intervals were 4.0 x 10(4) [1.7 x 10(4) - 8.9 x 10(4)] for 0-Gray (Gy)-irradiated mice, 1.9 x 10(4) [7.0 x 10(3) - 4.8 x 10(4)] for 5-Gy-irradiated mice, and 1.0 x 10(3) [2.8 x 10(2) - 3.3 x 10(3)] for 7-Gy-irradiated mice. Probit regression line fits calculated by use of an iterative, weighted least-squares fit, were used to assess a dose-modifying factor (DMF). The DMFs for mortality, compared with that for the 0-Gy dose, with their 95% confidence intervals, were 2.2 [0.63 - 7.7] for the 5-Gy and 38.9 [9.6 -165.0] for 7-Gy doses. The 5-Gy probit line did not significantly differ (P = 0.21) from the 0-Gy probit line (dose ratios did not significantly differ from 1), whereas the 7-Gy probit line differed significantly from the 0-Gy probit line (P < 0.001). These results demonstrate that 7-Gy 60Co gamma-photon radiation in combination with intratracheal K. pneumoniae challenge induces a valid pulmonary infection model in immunocompromised female B6D2F1/J mice.

ABSTRACTChallenge with both nonlethal ionizing radiation and toxigenicBacillus anthracisspores increases the rate of mortality from a mixed bacterial infection. If biological weapons, such asB. anthracisspores, and nuclear weapons were used together, casualties could be more severe than they would be from the use of either weapon alone. We previously discovered that a polymicrobial infection developed in B6D2F1/J mice after nonlethal (7-Gy)60Co γ irradiation and intratracheal challenge withB. anthracisSterne spores 4 days after irradiation. In this present study, we investigated the survival of mice and the response of the polymicrobial infection during the course of antimicrobial therapy with penicillin G procaine, ofloxacin, trovafloxacin, or gatifloxacin. Survival was prolonged, but not ensured, when the mice were treated with either broad-spectrum ofloxacin or narrow-spectrum penicillin G for 7 days beginning 6 or 24 h after challenge. Survival was not prolonged when therapy was delayed more than 24 h after challenge. When these two antimicrobial agents were given for 21 days, the survival rate was increased from 0% for the controls to 38 to 63% after therapy. Therapy with trovafloxacin or gatifloxacin reduced the incidence of mixed infection and improved the rate of survival to 95% (trovafloxacin) or 79% (gatifloxacin), whereas the rate of survival for the controls was 5%. We conclude that the mixed infection induced byB. anthracisin irradiated mice complicates effective therapy with a single antimicrobial agent. To limit mortality following nonlethal irradiation and challenge withB. anthracisspores, antimicrobial therapy needs to be initiated within a few hours after challenge and continued for up to 21 days.