Survival and infectivity of mycobacterium tuberculosis after aerosolization

<p dir="ltr">Tuberculosis (TB) remains the leading cause of death by a single infectious organism with over 1.3 million people succumbing to the disease every year. Despite efficient antibiotic treatment the disease continues to spread with transmission being the major contributor. TB is caused by the bacterium Mycobacterium tuberculosis (Mtb), an intracellular pathogen the transmits through the air. Importantly, airborne transmission is what makes this disease difficult to study, detect and control. Survival to aerosol transport is critical for the mycobacteria to be able to successfully transmit and infect new individuals. However, little is known on how Mtb survives aerosol transport and its impact on the subsequent ability to establish an infection.</p><p dir="ltr">The aim of this PhD project was to study Mycobacterium tuberculosis adaptations to aerosol transport and determine how this knowledge can be employed in transmission studies.</p><p dir="ltr">With this in mind, we sought to create a method by which aerosols carrying Mtb could be studied. In Paper I, we tested the feasibility of using a Coriolis® u air sampler, a wet-walled cyclone sampler designed for collection of viable airborne microorganisms, for the study of Mtb aeromicrobiology. In Paper II, we employed this air sampler together with the Anderssen cascade impactor (ACI), a sampler that impacts aerosols onto agar plates, to investigate the influence of temperature and humidity on the survival of aerosolized Mtb, as well as the changes to its gene expression in response to stress and its recovery capacity after aerosolization. Finaly, in Paper III, we developed a protocol to capture and detect Mtb aerosols using an electrostatic air sampler in combination with the widely used GeneXpert® platform and employed it to collect cough generated aerosols from TB patients.</p><p dir="ltr">In Paper I, we investigated the sampling efficiency of the Coriolis for aeromicrobiology studies. The sampling protocol suggested by the manufacturer of 1 h continuous sampling with a single collector cone was evaluated. We found this protocol to be detrimental to the collection capacity after 30 min of sampling. From this time point, no further material seemed to be collected and even a decrease in the total material collected observed after 1 h. We then developed a cumulative sampling protocol, where a new collector cone was replaced every 10 min, which improved collection capacity. Furthermore, we detected a loss of material from the collector cone during sampling periods longer than 10 min, meaning that collected material was being re-aerosolized. Indeed, we were able to measure contamination of all the tubing parts of the air sampler following a collection run. With this in mind, we adapted an HEPA filter to the device to eliminate contamination between collection runs and environments. The device was then tested in a real-life scenario during a biological spill simulation in a laboratory setting. Following a spill of one billion microspheres in 500 ml of water, virtually no particles were detected with the Coriolis at bench height. Taken together, the results of this study provide a stable and effective method for collecting airborne particles without cross contamination or losses. Additionally, our simulation experiment suggests that there is no scientific basis for evacuating a laboratory following a spill, on account of aerosol generation. Instead, it may be better to inactivate and clean the spill as soon as possible.</p><p dir="ltr">In Paper II, we studied how Mtb is able to survive the process of aerosolization and its impact on the bacteria. For this, we aerosolized the H37Rv lab strain of Mtb inside an aerosol-tight, hygrothermally-controlled aerosol chamber in a biosafety level 3 laboratory. Our results demonstrated that Mtb suffers a loss of viability when aerosolized in conditions of lower humidity independent of the temperatures tested. These effects were exacerbated by the time that the bacteria were allowed to dwell in the air, with almost no recovery of culturable Mtb observed after 30 min. We then sought to determine if virulence affected Mtb ability to survive aerosol transport under intermediate and high humidity conditions. The avirulent H37Ra laboratory strain of Mtb exhibited a lower survival compared to its virulent counterpart H37Rv independent of humidity. Aerosol transport also induced the expression of reactive oxygen species (ROS) detoxifying genes. Under intermediate humidity conditions this expression also increased the longer the bacteria remained in aerosol. Mtb also induced the expression of the hspX gene after aerosolization. This gene is known to block Mtb replication. Indeed, when aerosolized Mtb were returned to liquid media, its replication was arrested after 24 h, partly recovering after 48 hours. Altogether these results provide novel insights into how Mtb reacts to aerosolization, suggesting humidity and partly virulence dependent survival, as well as active adaptations in response to ROS damage suffered during the aerosol transport.</p><p dir="ltr">In Paper III, we set to determine the feasibility of detecting Mtb in respiratory aerosols and its clinical relevance for diagnostics. We used an electrostatic air sampler previously developed in the group to collect aerosols from TB patients. The detection limit of the device was tested by aerosolizing the TB vaccine M. bovis BCG, collecting it on the air sampler and then analyzing detection of bacterial DNA using the Xpert MTB/RIF Ultra cartridge. Our results showed a detection limit of 0.5 genomes/Lair. We deemed that this sensitivity was conductive to the device being tested in a clinical setting. We then proceeded to use the device to detect Mtb DNA in cough aerosols of TB patients in a clinic in South Africa. Overall, the sensitivity of this method was 46%, with sensitivity increasing to 60% in patients with higher Mtb burden in their sputum. We were also able to detect Mtb DNA in the coughs of sputum-negative patients, although the clinical implications of these results remain unclear. Detection of Mtb DNA was associated with male sex, high sputum burden and reported fever. These findings show that detection of Mtb DNA in patients cough aerosol is feasible in primary care. Moreover, the results also support the possibility of transmission by sputum negative patients.</p><h3 dir="ltr">List of scientific papers</h3><p dir="ltr">I. Operative and Technical Modifications to the Coriolis® μ Air Sampler That Improve Sample Recovery and Biosafety During Microbiological Air Sampling. Nuno Rufino de Sousa, Lei Shen, David Silcott, Charles J Call, Antonio Gigliotti Rothfuchs. Annals of Work Exposures and Health. 2020, 64(8): 852-865. <a href="https://doi.org/10.1093/annweh/wxaa053" target="_blank">https://doi.org/10.1093/annweh/wxaa053</a></p><p dir="ltr">II. Mycobacterium tuberculosis survival and replication is impaired after short-term aerosolization. Nuno Rufino de Sousa, Grisna Isabel Prensa, Veronika Krmeská, Laura Steponaviciute, Samantha L. Sampson, Grant Theron, Antonio Gigliotti Rothfuchs. [Manuscript]</p><p dir="ltr">III. Detection of Aerosolized Mycobacterium tuberculosis DNA From Adults Being Investigated for Pulmonary Tuberculosis via an Electrostatic Sampler in a South African Primary Care Setting. Jay Achar, Rouxjeane Venter, Jamie van Schalkwyk, Zandile Booi, Zama Mahlobo, Zaida Palmer, Nuno Rufino de Sousa, Knut Lönnroth, James A Seddon, Antonio Gigliotti Rothfuchs, Grant Theron. Open Forum Infectious Diseases. 2025; 12(10):ofaf593. <a href="https://doi.org/10.1093/ofid/ofaf593" target="_blank">https://doi.org/10.1093/ofid/ofaf593</a></p>