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.

Nuclear, biological, and chemical training in the U.S. Army Reserves: mitigating psychological consequences of weapons of mass destruction.

Weapons of mass destruction (WMD) and their associated delivery systems pose a major threat to the national security of the United States. The Department of Defense is pursuing a number of activities to counter paramilitary and terrorist threats from nuclear, biological, and chemical (NBC) agents. These efforts include supporting, training, and equipping the U.S. Army Reserves (USAR) for the medical management of physical injuries and psychological trauma resulting from the use of NBC weapons both in the United States and overseas. The USAR will play an important role in responding to a WMD incident because most of the Army's support assets are in the USAR. The USAR is training to perform its mission in an NBC-contaminated environment by engaging in realistic WMD exercises using state-of-the-art protective equipment and medical support. Realistic training builds confidence in medical defenses and in NBC protective equipment. This translates into accomplishing the mission while minimizing the psychological and physical casualties in an NBC-contaminated battlefield or in support of a WMD terrorist incident.

Performance decrement after combined exposure to ionizing radiation and Shigella sonnei.

Ionizing radiation could increase morbidity from common bacterial infections in military personnel on the modern battlefield. The combined effects of a sublethal dose of ionizing radiation and the bacterial diarrheal agent Shigella sonnei on body weight and forelimb grip strength in mice were assessed over a 30-day period. Individually housed B6D2F1 female mice were divided into four groups: control, sham irradiation + gavage with saline vehicle; 3 Gy 60Co gamma radiation at 0.4 Gy/min radiation + saline gavage; sham irradiation + 1.3 x 10(8) colony-forming units (CFUs) S. sonnei via gavage, administered 4 days postirradiation; and the combination of 3 Gy 60Co gamma radiation + 1.3 x 10(8) CFUs S. sonnei. Behavioral tests were conducted 3 days preirradiation and on days 9, 14, and 22 postirradiation. Body weight was significantly reduced in the radiation + Shigella group on days 5 to 10 postirradiation. Forelimb grip strength was reduced for mice in the radiation + Shigella group on days 9 and 14 postirradiation. These data demonstrate that an exposure to gamma radiation in combination with the bacterial agent S. sonnei can lead to a synergistic loss of body weight and degradation in performance.

Combined effects of Venezuelan equine encephalitis IIIA virus and gamma irradiation in mice.

The combined effects of injury from exposure to ionizing radiation and the potential biological warfare agent Venezuelan equine encephalitis (VEE) virus remain largely unknown. To study these effects, 4- to 5-week-old B6D2F1/J female mice were given a sublethal whole-body 7 Gy dose of 60Co gamma-photon radiation followed 48 hours later by aerosol or intraperitoneal challenge with enzootic VEE IIIA virus. Survival was observed for 30 days. A single sublethal 7 Gy dose of gamma radiation reduced the LD50/30 of VEE IIIA virus, in intraperitoneal challenged mice by a factor of 10(4) from 1.1 x 10(6) plaque-forming units (pfu) to 1 x 10(2) pfu, and in aerosol challenged mice, by a factor of 5 from 70 pfu to 14 pfu. These findings further confirm there is a combined effect of exposure to ionizing radiation and biological warfare agents, which could be devastating to unprotected populations and thus should be investigated further.

Identification of antigens of pathogenic free-living amoebae by protein immunoblotting with rabbit immune and human sera.

Prominent antigens of pathogenic and nonpathogenic free-living amoebae were identified by using polyclonal rabbit immune sera in immunoblot assays. The intent was to determine if prominent epitopes identified with rabbit immune sera could also be recognized by human sera. With rabbit sera, the development of immunoreactive bands was restricted to molecular masses of greater than 18.5 kDa for Naegleria, Hartmannella, and Vahlkampfia antigens. Two or more broad bands of less than 18.5 kDa were prominent features in three different Acanthamoeba species. Few cross-reactive antibodies could be detected between representative species of the three different subgroups of Acanthamoeba. Naegleria antigen was likewise serologically distinct, as were Hartmannella and Vahlkampfia antigens. The relative lack of cross-reacting antibodies between the pathogenic amoebae suggested that i would be desirable to use a panel of amoebic antigens to represent the range of serologically distinct antigens for assessing reactive antibodies in human sera. In pooled human sera (10 serum specimens per pool), the appearance of minimally reactive bands ranging from 32.5 to 106 kDa was a common feature of all six antigens. A prominent band of less than 18.5 kDa was identified in the Acanthamoeba culbertsoni antigen lane in 2 of the 10 human serum specimen pools. When sera from each of the two groups were tested individually by immunoblotting, the reaction with A. culbertsoni antigen could be associated with one individual. By using a panel of amoebic antigens, this method could prove useful in recognizing undiagnosed amoebic infections by revealing specific reactive antibodies.

Operation Desert Shield: medical aspects of weapons of mass destruction.

Our concern over possible use of weapons of mass destruction against U.S. forces in the Middle East has increased because Iraq has violated the Geneva Protocol of 1925 and the 1972 Biological Weapons Convention, attempted to acquire nuclear capability and delivery systems, and is reported to be developing biological weapons. The Army Medical Department has had no experience, since World War I, in the management and treatment of mass casualties contaminated by chemical agents, and has never treated casualties resulting from the use of nuclear or biological weapons used against our soldiers. Management and diagnosis of casualties will be complicated by their possible exposure to a mixture of chemical warfare and biological warfare agents. Triage is an essential aspect in the management of mass casualties since the number of injured patients will exceed the maximum medical capability to treat each patient on arrival. All levels of medical support must be prepared to protect themselves, their equipment and supplies, and their patients from contamination. In contaminated operations on the integrated battlefield, it will be of utmost importance to incorporate flexibility and innovation to match the medical and tactical situation.

Immunization against anthrax with aromatic compound-dependent (Aro-) mutants of Bacillus anthracis and with recombinant strains of Bacillus subtilis that produce anthrax protective antigen.

The safety and efficacy of five prototype, live anthrax vaccines were studied in Hartley guinea pigs and CBA/J and A/J mice. Two of the strains, Bacillus anthracis FD111 and FD112, are Aro- mutants derived by Tn916 mutagenesis of B. anthracis UM23-1. Bacillus subtilis PA1 and PA2 contain a recombinant plasmid, pPA101 or pPA102, respectively, that carries the gene from B. anthracis encoding synthesis of protective antigen (PA). The final strain, B. subtilis PA7, was isolated in this study from B. subtilis DB104 transformed with pPA101. All five strains were less virulent in guinea pigs and A/J and CBA/J mice than the toxinogenic, nonencapsulated B. anthracis veterinary vaccine Sterne strain. A/J and CBA/J inbred mice represent strains that are innately susceptible and resistant, respectively, to the Sterne strain. These differences in susceptibility are due to differences in ability to produce complement component 5. In guinea pigs, immunization with PA1 or PA2 vegetative cells or PA7 spores protected greater than or equal to 95% from an intramuscular spore challenge with the virulent, "vaccine-resistant" B. anthracis Ames strain. Strain PA2 vegetative cells and strain PA7 spores were as effective as the Sterne strain in Sterne-resistant CBA/J mice, protecting 70% of the mice from Ames strain spore challenge. Immunization with FD111 or FD112 vegetative cells fully protected guinea pigs from challenge. Immunization with FD111 cells protected up to 100% of CBA/J mice and up to 70% of A/J mice.

Transposon Tn916 mutagenesis in Bacillus anthracis.

Mutagenesis of Bacillus anthracis by the streptococcal tetracycline resistance transposon Tn916 is described. Tn916 was transferred from Streptococcus faecalis DS16C1 to B. anthracis VNR-1 by conjugation in a standard filter mating procedure. Tetracycline-resistant (Tcr) transconjugants were obtained at a frequency of 1.6 X 10(-8) per donor CFU. When donor and recipient cells were treated with nafcillin before conjugation, the frequency was increased nearly 10-fold. Nafcillin pretreatment of donor and recipient strains was used in all subsequent conjugation experiments. S.faecalis CG110, containing multiple chromosomal insertions of Tn916, transferred the transposon to B. anthracis VNR-1 at a frequency of 9.3 x 10(-5). A Tcr B. anthracis transconjugant, strain VNR-1-tet-1, transferred Tn916 to B. anthracis UM23-1 and Bacillus subtilis BST1 at frequencies of 2.1 x 10(-4) and 5.8 X 10(-6), respectively. The transfer of Tn916 occurred only on membrane filters, since no Tcr transconjugants were obtained when strains VNR-1-tet-1 and UM23-1 were mixed and incubated in broth culture. The presence of the Tn916-associated tetM gene in Tcr B. anthracis and B. subtilis transconjugants was confirmed in hybridization experiments by using a 5-kilobase-pair DNA fragment containing the tetM gene as a probe. Of 3,000 B. anthracis UM23-1 Tcr transconjugants tested, 21 were phenylalanine auxotrophs and 2 were auxotrophic for phenylalanine, tyrosine, and tryptophan.

Mechanical transmission of Bacillus anthracis by stable flies (Stomoxys calcitrans) and mosquitoes (Aedes aegypti and Aedes taeniorhynchus).

We evaluated the potential of stable flies, Stomoxys calcitrans, and two species of mosquitoes, Aedes aegypti and Aedes taeniorhynchus, to transmit Bacillus anthracis Vollum 1B mechanically. After probing on Hartley guinea pigs with a bacteremia of ca. 10(8.6) CFU of B. anthracis per ml of blood, individual or pools of two to four stable flies or mosquitoes were allowed to continue feeding on either uninfected guinea pigs or A/J mice. All three insect species transmitted lethal anthrax infections to both guinea pigs and mice. Both stable flies and mosquitoes transmitted anthrax, even when they were held at room temperature for 4 h after exposure to the bacteremic guinea pig before being allowed to continue feeding on the susceptible animals. This study confirms that blood-feeding insects can mechanically transmit anthrax and supports recent anecdotal reports of fly-bite-associated cutaneous human anthrax. The potential for flies to mechanically transmit anthrax suggests that fly control should be considered as part of a program for control of epizootic anthrax.

Photoreactivation of ultraviolet-irradiated, plasmid-bearing, and plasmid-free strains of Bacillus anthracis.

The effects of toxin- and capsule-encoding plasmids on the kinetics of UV inactivation of various strains of Bacillus anthracis were investigated. Plasmids pXO1 and pXO2 had no effect on bacterial UV sensitivity or photoreactivation. Vegetative cells were capable of photoreactivation, but photo-induced repair of UV damage was absent in B. anthracis Sterne spores.

Comparative efficacy of Bacillus anthracis live spore vaccine and protective antigen vaccine against anthrax in the guinea pig.

Several strains of Bacillus anthracis have been reported previously to cause fatal infection in immunized guinea pigs. In this study, guinea pigs were immunized with either a protective antigen vaccine or a live Sterne strain spore vaccine, then challenged with virulent B. anthracis strains isolated from various host species from the United States and foreign sources. Confirmation of previously reported studies (which used only protective antigen vaccines) was made with the identification of 9 of the 27 challenge isolates as being vaccine resistant. However, guinea pigs immunized with the live Sterne strain spore vaccine were fully protected against these nine isolates. In experiments designed to determine the basis of vaccine resistance, guinea pigs which were immunized with individual toxin components and which demonstrated enzyme-linked immunosorbent assay antibody titers comparable to those induced by Sterne strain vaccine were not protected when challenged with a vaccine-resistant isolate. We concluded that antibodies to toxin components may not be sufficient to provide protection against all strains of B. anthracis and that other antigens may play a role in active immunity. As a practical matter, it follows that the efficacy of anthrax vaccines must be tested by using vaccine-resistant isolates if protection against all possible challenge strains is to be assured.

Photoreactivation of UV-irradiated Legionella pneumophila and other Legionella species.

Shortwave UV light was assessed as a feasible modality for the control of Legionnaires disease bacterium in water. The results of this study show that Legionella pneumophila and six other Legionella species are very sensitive to low doses of UV. However, all Legionella species tested effectively countered the germicidal effect of UV when subsequently exposed to photoreactiving light.

Isolation of plasmids in Legionella pneumophila and Legionella-like organisms.

Agarose gel electrophoresis was employed to screen nine strains of Legionella-like bacteria and one strain of Legionella pneumophila for the presence of extrachromosomal deoxyribonucleic acid. Cryptic plasmids with molecular weights ranging from 46.6 x 10(6) to 59.8 x 10(6) were found in three of the isolates examined.

A plasmid in Legionella pneumophila.

Sixteen strains from the six serogroups of Legionella pneumophila were examined for the presence of extrachromosomal genetic elements by a modified cleared lysate procedure, dye-buoyant centrifugation, and agarose gel electrophoresis. Two strains, Atlanta-1 and Atlanta-2 from serogroup II, each contained a plasmid of cryptic function with a molecular weight of ca. 30 megadaltons.