Investigating Single Cell Growth Dynamics of Mycobacteria With Microfluidics.

As one of the world's most devastating diseases, tuberculosis is a global health crisis, with over two billion people infected. Despite extensive research into the disease spanning more than a century, it remains the number one killer due to a single infectious agent, making Mycobacterium tuberculosis, the causative agent, one of the most successful human pathogens. The bacterium is able to persist as a long-term infection, known as latent tuberculosis. Bacterial persistence is believed to be the root cause of latency. One characteristic of persister cells is that they are phenotypically tolerant to the action of antibiotics; a trait which has important implications in tuberculosis chemotherapy. Establishing in vitro models of persistence is an important element of the study of latent tuberculosis. To gain insight into persistence of tuberculosis, microfluidic devices were designed and manufactured in order to study single cell behaviour of the model organism Mycobacterium smegmatis. Microfluidic devices comprising polydimethylsiloxane and glass were designed and constructed using photolithography and soft lithography techniques. The growth of single cells of M. smegmatis expressing the green fluorescent protein was monitored using fluorescence time-lapse microscopy. The cells were exposed to an antibiotic, and subsequent cell death was directly observed. This technique allows cell genealogy to be determined and observation of rare events, such as persistence, can be made. Furthermore, a flow cytometry protocol was established in which nutrient starvation, and nutrient deprived stationary phase in M. smegmatis caused increased resistance to antibiotic stress, while maintaining viability in vitro. This method could ultimately allow high-throughput screenings for drugs against persistent M. tuberculosis