Gregory B. Knudson

In vitro resistance of Bacillus anthracis Sterne to doxycycline, macrolides and quinolones.

Bacillus anthracis is a potential biological warfare agent. Its ability to develop resistance to antimicrobial agents currently recommended for the treatment of anthrax infection is a major concern. B. anthracis Sterne was grown from a live veterinary vaccine and used it to test for the development of resistance after 21 sequential subcultures in sub-inhibitory concentrations of doxycycline and three quinolones (ciprofloxacin, alatrofloxacin and gatifloxacin) and 15 sequential subcultures in sub-inhibitory concentrations of three macrolides (erythromycin, azithromycin and clarithromycin). After 21 subcultures the minimal inhibitory concentrations (MICs) increased from 0.1 to 1.6 mg/l for ciprofloxacin, from 1.6 to 12.5 mg/l for alatrofloxacin, from 0.025 to 1.6 mg/l for gatifloxacin and from 0.025 to 0.1 mg/l for doxycycline. After 15 passages of sequential subculturing with macrolides, the MICs increased from 12.5 to 12.5 or 50.0 mg/l for azithromycin, from 0.2 to 1.6 or 0.4 mg/l for clarithromycin and from 6.25 to 6.25 or 50 mg/l for erythromycin. After sequential passages with a single quinolone or doxycycline, each isolate was cross-tested for resistance using the other drugs. All isolates selected for resistance to one quinolone were also resistant to the other two quinolones, but not to doxycycline. The doxycycline-resistant isolate was not resistant to any quinolone.

In vitro development of resistance to ofloxacin and doxycycline in Bacillus anthracis Sterne.

Anthrax is an infection caused by Bacillus anthracis . It occurs endemically and could also be used for biological warfare with devastating effects ([3][1]). The worldwide increase in the development of drug resistance in bacteria is a major concern. This phenomenon can develop after in vitro

Management of Postirradiation Infection: Lessons Learned from Animal Models

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.

High-Dose Ultraviolet C Light Inactivates Spores of Bacillus Atrophaeus and Bacillus Anthracis Sterne on Nonreflective Surfaces

Ultraviolet light (UV) is frequently used in limited clinical settings to reduce the risk of nosocomial infections. The World Health Organization Global Solar UV Index (UVI) divides UV into A (315-400 nm), B (280-315 nm), and C (100-280 nm) bands. Light in the C band (UV-C) is listed in the UVI as the most harmful to living organisms because of its propensity to damage DNA and RNA. It is also the least relevant in the natural setting since it is completely filtered by the atmosphere and does not reach Earth’s surface in levels measurable with commercially available equipment. Often small surface areas or airflow in high-risk areas are treated with UV-C to decrease infectious microorganism populations. Other means of decontamination are sometimes employed when a large area has been contaminated. Gaseous disinfection with ethylene oxide, chlorine dioxide, or formaldehyde is costly, hazardous to workers and the environment, and requires prolonged evacuation of the treatment area (Rehork et al., 1990). Liquid disinfectants must be manually applied and removed and may damage exposed materials such as electrical devices. Ionizing radiation will kill in adequate doses but is hazardous to workers, difficult to contain, and not practical for general working space disinfection (Rehork et al., 1990). The possibility of using UV-C (254 nanometer range) to decontaminate or sterilize work areas and to avoid the problems listed above led to the development by two of the authors (JLD, DRD) of the Ultraviolet Area Sterilizer (UVAS). The device is unique in that it generates intense levels of UV-C and then utilizes measured UV-C intensities reflected from the walls, ceilings, floors, or other treated areas to calculate the operation time to deliver the programmed lethal dose for infectious microorganisms. UV-C has been found to be highly effective against a wide spectrum of microorganisms (Banrud & Moan, 1999; Druce et al., 1995; Inamoto et al., 1979; Knudson, 1985). The development of a method to deliver a lethal and predictable UV-C dose can greatly increase the potential uses for UV-C in decontamination. Since the biological activity of UV-C is not limited to microorganisms, the UVAS has multiple safety features including remote controls, motion sensors, and audible voice warnings. These safety features were active during the course of this study. The ability of the UVAS device to deliver lethal doses of UV-C to bacterial spores on nonreflective surfaces was evaluated by comparing the susceptibilities of Bacillus atrophaeus (formerly named B. subtilis var. niger, and B. globigii) and Bacillus anthracis Sterne spores to incremental UV-C doses. Additionally, the susceptibility of Bacillus atrophaeus spores in the presMarie U. Owens1, David R. Deal2, Michael O. Shoemaker3, Gregory B. Knudson3, Janet E. Meszaros4, and Jeffery L. Deal2

Susceptibility of irradiated mice to Bacillus anthracis sterne by the intratracheal route of infection.

The susceptibility of sublethally irradiated mice to pulmonary infection with Bacillus anthracis was investigated in a mouse model. Female B6D2F1/J mice were challenged intratracheally with 4.3 x 10(6), 3.7 x 10(7) and 4.4 x 10(8) cfu of B. anthracis Sterne spores 4 days after 60Co gamma-radiation at a dose of 0, 1, 2, 3, 4, 5, 6 or 7 Gy. Bacterial cultures were obtained from lung, spleen homogenates and heart blood. A biphasic mode of mortality was observed, with a constant response of up to 3 or 4 Gy (up to 18% mortality), after which a sharp increase in mortality occurred (up to 100%). When irradiation was delayed beyond 15 days after inoculation, the susceptibility to B. anthracis infection and subsequent mortality disappeared. B. anthracis was recovered from the organs and blood of up to 89% of the animals. However, organisms of enteric origin were also isolated mixed with B. anthracis from up to 36% of the animals exposed to 3, 5 or 7 Gy. Inoculation of B. anthracis delta-Sterne-1 that lacks lethal toxin and oedema toxin also induced infection with B. anthracis, but not translocation of enteric micro-organisms. The synergic adverse effect of exposure to gamma-radiation followed by intratracheal challenge with B. anthracis was observed above 4 Gy. The lethal toxin of B. anthracis may enhance the emergence of polymicrobial infection with B. anthracis and enteric micro-organisms.

Antimicrobial Therapy for Bacillus anthracis-Induced Polymicrobial Infection in 60Co γ-Irradiated Mice

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