Investigation of factors affecting the aerosol transmission of Mycobacterium tuberculosis

Background: Mycobacterium tuberculosis (Mtb) is transmitted via aerosol droplets generated by infected persons. Transmission of Mtb is thought to be a selective process and that cell surface hydrophobicity (CSH) influences this selectivity. On expulsion into the environment, bacilli will be exposed to rapidly changing physical conditions (temperature, water activity, osmolarity and oxygen tension) as the droplet evaporates to a stable droplet nucleus. Adaptation of the bacilli to the changing environment will be a feature supporting their fitness for survival in the aerosol phase and will impact the phenotype of subsequent infection. In this project, the role of CSH on mycobacterial aerosolisation and techniques to measure CSH were investigated. Additionally, survival and changes in transcriptional profile of M. tuberculosis H37Rv and M. bovis BCG in aerosols over a period of 2 hours have been explored.

Methods: Lawn growth of either Mtb H37Rv or M. bovis BCG were scrapped and suspended in distilled water (DW) medium. Bacteral suspensions were nebulised using either Collison 3-Jet or ultrasonic Omron nebuliser for 5 min into a rotating drum. Aerosols samples were collected in DW or Guanidinium thiocyanate (GTC) at different time points over 2 hours for survival and transcriptional profile experiments, respectively. Survival of cells in aerosols was determined by colony-forming units (CFU), most probable number (MPN) and qPCR. For transcriptional changes, samples were subject to RNA-sequencing analysis by Illumina NextSeq-500. Differentially expressed genes were detected at q-value < 0.01.

Results: Mtb H37Rv and M. bovis BCG consistently showed > 50 % survival in aerosol for up to 2 hours. Differentially expressed genes of Mtb H37Rv were detected at different stages during aerosolisation and due to the nebulisation process. However, no obvious transcriptional pattern of Mtb cells under the tested conditions was obtained. Genes of Mtb universal stress regulon (DosR) were up- regulated at pre-nebulisation samples compared to the post nebulisation and aerosol samples.

Discussion: Investigation into the survival of Mtb and M .bovis BCG under different environmental conditions could provide better insight into the survival of these cells in aerosols. DosR regulon was induced prior nebulisation process indicating early stress conditions for Mtb cells during inoculum preparation.