c Liposome-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. tularensis live 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. tularensis demonstrated 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.

Burkholderia pseudomallei is 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 vitro analysis 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 vivo studies 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. pseudomallei under acidic conditions in vitro and 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.

Deposition of Burkholderia pseudomallei within either the lungs or nasal passages of the Balb/c murine model resulted in different infection kinetics. The infection resulting from the inhalation of B. pseudomallei within a 12 μm particle aerosol was prolonged compared to a 1 μm particle aerosol with a mean time-to-death (MTD) of 174.7 ± 14.9 h and 73.8 ± 11.3 h, respectively. Inhalation of B. pseudomallei within 1 μm or 12 μm particle aerosols resulted in a median lethal dose (MLD) of 4 and 12 cfu, respectively. The 12 μm particle inhalational infection was characterized by a marked involvement of the nasal mucosa and extension of bacterial colonization and inflammatory lesions from the olfactory epithelium through the olfactory nerves (or tracts) to the olfactory bulb (100%), culminating in abscessation of the brain (33%). Initial involvement of the upper respiratory tract lymphoid tissues (nasal-associated lymphoid tissue (NALT) and cervical lymph nodes) was observed in both the 1 and 12 μm particle inhalational infections (80–85%). Necrotising alveolitis and bronchiolitis were evident in both inhalational infections, however, lung pathology was greater after inhalation of the 1 μm particle aerosol with pronounced involvement of the mediastinal lymph node (50%). Terminal disease was characterized by bacteraemia in both inhalational infections with dissemination to the spleen, liver, kidneys, and thymus. Treatment with co-trimoxazole was more effective than treatment with doxycycline irrespective of the size of the particles inhaled. Doxycycline was more effective against the 12 μm particle inhalational infection as evidenced by increased time to death. However, both treatment regimes exhibited significant relapse when therapy was discontinued with massive enlargement and abscessation of the lungs, spleen, and cervical lymph nodes observed.

No abstract

Burkholderia pseudomallei is the causative agent of the tropical disease melioidosis. Its genome encodes an arsenal of virulence factors that allow it, when required, to switch from a soil dwelling bacterium to a deadly intracellular pathogen. With a high intrinsic resistance to antibiotics and the ability to overcome challenges from the host immune system, there is an increasing requirement for new antibiotics and a greater understanding into the molecular mechanisms of B . pseudomallei virulence and dormancy. The peptidoglycan remodeling enzymes, lytic transglycosylases (Ltgs) are potential targets for such new antibiotics. Ltgs cleave the glycosidic bonds within bacterial peptidoglycan allowing for the insertion of peptidoglycan precursors during cell growth and division, and cell membrane spanning structures such as flagella and secretion systems. Using bioinformatic analysis we have identified 8 putative Ltgs in B . pseudomallei K96243. We aimed to investigate one of these Ltgs, LtgG (BPSL3046) through the generation of deletion mutants and biochemical analysis. We have shown that LtgG is a key contributor to cellular morphology, division, motility and virulence in BALB/c mice. We have determined the crystal structure of LtgG and have identified various amino acids likely to be important in peptidoglycan binding and catalytic activity. Recombinant protein assays and complementation studies using LtgG containing a site directed mutation in aspartate 343, confirmed the essentiality of this amino acid in the function of LtgG.