Investigating the physiological adaptations of Burkholderia species to stress conditions

Burkholderia pseudomallei is an important tropical pathogen, biological threat, and the causative agent of melioidosis. It produces various dormant forms which may contribute to latent melioidosis infection. This project aimed to determine if Burkholderia species can undergo a transition to differential culturability, a physiological state where bacteria do not grow in standard media and do not produce colonies on agar. B. pseudomallei and Burkholderia thailandensis were incubated in phosphate buffered saline at either 21°C or 4°C for up to four weeks. During which the cultures were regularly sampled to determine viable counts and to allow microscopic analysis. Differentially culturable forms were generated during starvation of these bacteria, and the factors responsible for induction and resuscitation were characterised.

At 21°C Burkholderia produced short rods that remained culturable and restarted growth in nutrient rich conditions. Incubation at 4°C resulted in a 3-log reduction in colony forming unit counts, and caused an accumulation of cocci cells which were stained with SYTO 9 but not propidium iodide. These bacteria could be resuscitated with the addition of culture supernatant or Lysogeny Broth supplemented with Catalase 130U/mL and Tween 80 0.05% (v/v). Cell wall remodeling enzymes have previously been shown to contribute to the generation of rounded

cells in various Gram-negative bacteria. In accordance with these results a B. pseudomallei mutant with four deleted lytic transglycosylase genes, ΔltgGDFE produced less coccoid cells during starvation at 4°C and failed to resuscitate in the Catalase and Tween 80 supplemented media. The results reported in this thesis confirm that Burkholderia can produce differentially culturable forms (previously referred to as viable but non-culturable bacteria) and provide insights into the molecular mechanisms underlying the adaptation of Burkholderia to stress conditions.