Analysis of the genome of Francisella tularensis has revealed few regulatory systems, and how the organism adapts to conditions in different niches is poorly understood. The stringent response is a global stress response mediated by (p)ppGpp. The enzyme RelA has been shown to be involved in generation of this signal molecule in a range of bacterial species. We investigated the effect of inactivation of the relA gene in Francisella by generating a mutant in Francisella novicida. Under amino acid starvation conditions, the relA mutant was defective for (p)ppGpp production. Characterization showed the mutant to grow similarly to the wild-type, except that it entered stationary phase later than wild-type cultures, resulting in higher cell yields. The relA mutant showed increased biofilm formation, which may be linked to the delay in entering stationary phase, which in turn would result in higher cell numbers present in the biofilm and reduced resistance to in vitro stress. The mutant was attenuated in the J774A macrophage cell line and was shown to be attenuated in the mouse model of tularaemia, but was able to induce a protective immune response. Therefore, (p)ppGpp appears to be an important intracellular signal, integral to the pathogenesis of F. novicida.

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.