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ABSTRACTBioluminescence imaging (BLI) enables real-time, noninvasive tracking of infection in vivoand longitudinal infection studies. In this study, a bioluminescent Francisella tularensisstrain, SCHU S4-lux, was used to develop an inhalational infection model in BALB/c mice. Mice were infected intranasally, and the progression of infection was monitored in real time using BLI. A bioluminescent signal was detectable from 3 days postinfection (3 dpi), initially in the spleen and then in the liver and lymph nodes, before finally becoming systemic. The level of bioluminescent signal correlated with bacterial numbers in vivo, enabling noninvasive quantification of bacterial burdens in tissues. Treatment with levofloxacin (commencing at 4 dpi) significantly reduced the BLI signal. Furthermore, BLI was able to distinguish noninvasively between different levofloxacin treatment regimens and to identify sites of relapse following treatment cessation. These data demonstrate that BLI and SCHU S4-luxare suitable for the study of F. tularensispathogenesis and the evaluation of therapeutics for tularemia.

Whole body imaging technologies, coupled with the generation of stable light emitting strains of infectious agents, make it possible to monitor the location and kinetics of agent growth inside an animal by noninvasive nonterminal methodologies throughout the course of the infection. However, imaging of viable hazardous pathogens, such as Yersinia pestis, requires specialized containment facilities to prevent contamination of both personnel and the wider environment. Here we describe the successful establishment of an in vivobioimaging capability specifically addressing the requirement for safe and secure work with hazard group 3 pathogens and demonstrate its utility for imaging mice infected with a bioluminescence producing strain of Y. pestis.

Whole body imaging technologies, coupled with the generation of stable light emitting strains of infectious agents, make it possible to monitor the location and kinetics of agent growth inside an animal by noninvasive nonterminal methodologies throughout the course of the infection. However, imaging of viable hazardous pathogens, such as Yersinia pestis, requires specialized containment facilities to prevent contamination of both personnel and the wider environment. Here we describe the successful establishment of an in vivobioimaging capability specifically addressing the requirement for safe and secure work with hazard group 3 pathogens and demonstrate its utility for imaging mice infected with a bioluminescence producing strain of Y. pestis.

ABSTRACTBurkholderia pseudomallei, the causative agent of melioidosis, has complex and poorly understood extracellular and intracellular lifestyles. We used transposon-directed insertion site sequencing (TraDIS) to retrospectively analyze a transposon library that had previously been screened through a BALB/c mouse model to identify genes important for growth and survival in vivo. This allowed us to identify the insertion sites and phenotypes of negatively selected mutants that were previously overlooked due to technical constraints. All 23 unique genes identified in the original screen were confirmed by TraDIS, and an additional 105 mutants with various degrees of attenuation in vivowere identified. Five of the newly identified genes were chosen for further characterization, and clean, unmarked bpsl2248, tex, rpiR, bpsl1728, and bpss1528deletion mutants were constructed from the wild-type strain K96243. Each of these mutants was tested in vitroand in vivoto confirm their attenuated phenotypes and investigate the nature of the attenuation. Our results confirm that we have identified new genes important to in vivovirulence with roles in different stages of B. pseudomalleipathogenesis, including extracellular and intracellular survival. Of particular interest, deletion of the transcription accessory protein Tex was shown to be highly attenuating, and the texmutant was capable of providing protective immunity against challenge with wild-type B. pseudomallei, suggesting that the genes identified in our TraDIS screen have the potential to be investigated as live vaccine candidates.

ABSTRACTLiposome-encapsulated ciprofloxacin for inhalation (CFI) was investigated as a putative postexposure therapeutic for two strains of Francisella tularensis. The efficacies of oral ciprofloxacin and intranasally instilled CFI could not be distinguished in a mouse model of infection with the F. tularensislive vaccine strain (LVS), where a single dose of either formulation offered full protection against a lethal challenge. However, mouse studies with the more virulent Schu S4 strain of F. tularensisdemonstrated that a higher level of protection against a lethal aerosol infection is provided by CFI than by oral ciprofloxacin. In addition, using this infection model, it was possible to discriminate the efficacy of intranasally instilled CFI from that of aerosolized CFI, with aerosolized CFI providing full protection after just a single dose. The improved efficacy of CFI compared to oral ciprofloxacin is likely due to the high sustained concentrations of ciprofloxacin in the lung. In summary, CFI may be a promising therapy, perhaps enabling the prophylactic regimen to be shortened, for use in the event of a deliberate release of F. tularensis. The prophylactic efficacy of CFI against other biological warfare (BW) threat agents also warrants investigation.

ABSTRACTThere is a need to identify vaccines that can protect against Brucella, a potential bioterrorism agent. We have developed mouse models of infection with aerosolized Brucella melitensisand Brucella suisand demonstrated their utility for the evaluation of vaccines using the model live B. melitensisvaccine strain Rev.1.

ABSTRACTMelioidosis is an emerging disease of humans in Southeast Asia and tropical Australia. The bacterium causing this disease, Burkholderia pseudomallei, is also considered a bioterrorism agent, and as yet there is no licensed vaccine for preventing B. pseudomalleiinfection. In this study, we evaluated selected proteins (LolC, PotF, and OppA) of the ATP-binding cassette systems of B. pseudomalleias candidate vaccine antigens. Nonmembrane regions of the B. pseudomalleiproteins were expressed and purified from Escherichia coliand then evaluated as vaccine candidates in an established mouse model of B. pseudomalleiinfection. When delivered with the monophosphoryl lipid A-trehalose dicorynomycolate adjuvant, the proteins stimulated antigen-specific humoral and cellular immune responses. Immunization with LolC or PotF protein domains afforded significant protection against a subsequent challenge with B. pseudomallei. The most promising vaccine candidate, LolC, provided a greater level of protection when it was administered with immune-stimulating complexes complexed with CpG oligodeoxynucleotide 10103. Immunization with LolC also protected against a subsequent challenge with a heterologous strain of B. pseudomallei, demonstrating the potential utility of this protein as a vaccine antigen for melioidosis.

Melioidosis is an emerging disease of humans in Southeast Asia and tropical Australia. The bacterium causing this disease, Burkholderia pseudomallei, is also considered a bioterrorism agent, and as yet there is no licensed vaccine for preventing B. pseudomallei infection. In this study, we evaluated selected proteins (LolC, PotF, and OppA) of the ATP-binding cassette systems of B. pseudomallei as candidate vaccine antigens. Nonmembrane regions of the B. pseudomallei proteins were expressed and purified from Escherichia coli and then evaluated as vaccine candidates in an established mouse model of B. pseudomallei infection. When delivered with the monophosphoryl lipid A-trehalose dicorynomycolate adjuvant, the proteins stimulated antigen-specific humoral and cellular immune responses. Immunization with LolC or PotF protein domains afforded significant protection against a subsequent challenge with B. pseudomallei. The most promising vaccine candidate, LolC, provided a greater level of protection when it was administered with immune-stimulating complexes complexed with CpG oligodeoxynucleotide 10103. Immunization with LolC also protected against a subsequent challenge with a heterologous strain of B. pseudomallei, demonstrating the potential utility of this protein as a vaccine antigen for melioidosis.

Bacillus anthracis is the causative agent of anthrax, a disease that affects wildlife, livestock, and humans. Protection against anthrax is primarily afforded by immunity to the B. anthracis protective antigen (PA), particularly PA domains 4 and 1. To further the development of an orally delivered human vaccine for mass vaccination against anthrax, we produced Salmonella enterica serovar Typhimurium expressing full-length PA, PA domains 1 and 4, or PA domain 4 using codon-optimized PA DNA fused to the S. enterica serovar Typhi ClyA and under the control of the ompC promoter. Oral immunization of A/J mice with Salmonella expressing full-length PA protected five of six mice against a challenge with 105CFU of aerosolized B. anthracis STI spores, whereas Salmonella expressing PA domains 1 and 4 provided only 25% protection (two of eight mice), and Salmonella expressing PA domain 4 or a Salmonella-only control afforded no measurable protection. However, a purified recombinant fusion protein of domains 1 and 4 provided 100% protection, and purified recombinant 4 provided protection in three of eight immunized mice. Thus, we demonstrate for the first time the efficacy of an oral S. enterica-based vaccine against aerosolized B. anthracis spores.

ABSTRACTBacillus anthracisis the causative agent of anthrax, a disease that affects wildlife, livestock, and humans. Protection against anthrax is primarily afforded by immunity to the B. anthracisprotective antigen (PA), particularly PA domains 4 and 1. To further the development of an orally delivered human vaccine for mass vaccination against anthrax, we produced Salmonella entericaserovar Typhimurium expressing full-length PA, PA domains 1 and 4, or PA domain 4 using codon-optimized PA DNA fused to the S. entericaserovar Typhi ClyA and under the control of the ompCpromoter. Oral immunization of A/J mice with Salmonellaexpressing full-length PA protected five of six mice against a challenge with 105CFU of aerosolized B. anthracisSTI spores, whereas Salmonellaexpressing PA domains 1 and 4 provided only 25% protection (two of eight mice), and Salmonellaexpressing PA domain 4 or a Salmonella-only control afforded no measurable protection. However, a purified recombinant fusion protein of domains 1 and 4 provided 100% protection, and purified recombinant 4 provided protection in three of eight immunized mice. Thus, we demonstrate for the first time the efficacy of an oral S. enterica-based vaccine against aerosolized B. anthracisspores.

The identification of Yersinia pestis as a potential bioterrorism agent and the emergence of antibiotic-resistant strains have highlighted the need for improved vaccines and treatments for plague. The aim of this study was to evaluate the potential for ATP-binding cassette (ABC) transporter proteins to be exploited as novel vaccines against plague. Western blotting of ABC transporter proteins using sera from rabbits immunized with killed whole Y. pestis cells or human convalescent-phase sera identified four immunologically reactive proteins: OppA, PstS, YrbD, and PiuA. Mice immunized with these proteins developed antibody to the immunogen. When the immunized mice were challenged with Y. pestis, the OppA-immunized mice showed an increased time to death compared to other groups, and protection appeared to correlate with the level of immunoglobulin G antibody to OppA.

ABSTRACTThe identification of Yersinia pestisas a potential bioterrorism agent and the emergence of antibiotic-resistant strains have highlighted the need for improved vaccines and treatments for plague. The aim of this study was to evaluate the potential for ATP-binding cassette (ABC) transporter proteins to be exploited as novel vaccines against plague. Western blotting of ABC transporter proteins using sera from rabbits immunized with killed whole Y. pestiscells or human convalescent-phase sera identified four immunologically reactive proteins: OppA, PstS, YrbD, and PiuA. Mice immunized with these proteins developed antibody to the immunogen. When the immunized mice were challenged with Y. pestis, the OppA-immunized mice showed an increased time to death compared to other groups, and protection appeared to correlate with the level of immunoglobulin G antibody to OppA.

Increasing resistance of bacteria to antibiotics is a serious global challenge and there is a need to unlock the potential of novel antibacterial targets. One such target is the essential prokaryotic endoribonuclease RNase E. Using a combination of in silicohigh-throughput screening and in vitrovalidation we have identified three novel small molecule inhibitors of RNase E that are active against RNase E from Escherichia coli, Francisella tularensisand Acinetobacter baumannii. Two of the inhibitors are non-natural small molecules that could be suitable as lead compounds for the development of broad-spectrum antibiotics targeting RNase E. The third small molecule inhibitor is glucosamine-6-phosphate, a precursor of bacterial cell envelope peptidoglycans and lipopolysaccharides, hinting at a novel metabolite-mediated mechanism of regulation of RNase E.

ABSTRACTBurkholderia pseudomalleiis the causative agent of melioidosis, a serious disease endemic in Southeast Asia and Northern Australia. Antibiotic treatment is lengthy and relapse often occurs. Finafloxacin is a novel fluoroquinolone with increased antibacterial activity in acidic conditions in contrast to other fluoroquinolones which demonstrate reduced activity at a lower pH. Therefore, finafloxacin may have improved efficacy against B. pseudomallei, which can survive within host cells where the local pH is acidic. In vitroanalysis was performed using MICs, minimal bactericidal concentrations (MBCs), time-kill assays, persister cell assays, and macrophage assays. Finafloxacin showed increased bactericidal activity at pH 5 in comparison to pH 7 and ciprofloxacin at pH 5. In vivostudies in BALB/c mice included pharmacokinetic studies to inform an appropriate dosing regimen. Finafloxacin efficacy was evaluated in an inhalational murine model of melioidosis where antibiotic treatment was initiated at 6 or 24 h postchallenge and continued for 14 days, and mice were observed for 63 days. The survival of infected mice following 14 days of treatment was 80%, 60% or 0% for treatments initiated at 6 h and 60%, 30% or 0% for treatments initiated at 24 h for finafloxacin, co-trimoxazole, or ciprofloxacin, respectively. In summary, finafloxacin has increased bactericidal activity for B. pseudomalleiunder acidic conditions in vitroand improves survival in a murine model of melioidosis compared with those for ciprofloxacin. Furthermore, finafloxacin improves bacteriological clearance compared with that of co-trimoxazole, suggesting it may offer an effective postexposure prophylaxis against B. pseudomallei.