Detecting mycobacterial cell states using photonics

Abstract

Tuberculosis is an ancient disease with evidence of Mycobacterium tuberculosis bacilli being found in mummies from ancient Egypt. There are also contemporary cases of tuberculosis, worldwide, every day. Like a good parasite Mycobacterium tuberculosis can hide within its host undetected for long periods of time. This state of quiescence has numerous names but in this research we will be referring to mycobacterial dormancy. This study investigates the continuing problem of dormancy and associated antibiotic resistance in Mycobacterium tuberculosis (MTB). We focused on closely related research surrogates of MTB; M. smegmatis, M. fortuitum, M. marinum and M. bovis (BCG). Phenotypic resistance is defined as antibiotic resistance that arises as a function of an organism’s specific phenotype, rather than its genome and the genes it could express. Dormancy in MTB can arise when a culture of in vitro bacteria ages to the stationary phase or becomes otherwise stressed. In vivo the conditions surrounding dormancy are less well understood. Dormancy is an ill-defined state of being suggested for MTB characterised by a down shift in metabolic function and a resistance to chemotherapy. It has been noted that a similar phenotype is found in MTB cells that are expressing lipid bodies- lipid rich cells. We aimed to create a device that could differentiate between lipid rich and lipid poor cells rapidly using photonic technology. In so doing we created a rapid cell quantifying device, SLIC, which we have used and evaluated extensively with both mycobacteria and common nosocomial pathogens. As another approach we attempted to separate lipid rich from lipid poor cells. This was achieved using a novel buoyant density separation methodology in combination with an adapted lipophilic staining regimen. In combination these two techniques allowed us to generate discreet populations of ≥95% purity which we were then able to experiment on individually. Due to our novel separation methodology we were able to discover that lipid rich cells are much more resistant to the current anti-tuberculosis frontline treatment (≈40x more resistant). We showed that lipid rich cells down regulate certain nucleic acid markers associated with a quiescent cell state. We were also able to discover that lipid rich cells occur in young unstressed cultures indicating that the accumulation of lipid bodies is a natural part of the mycobacterial cell cycle. This hints at a possible reason for relapse in non-immunocompromised patients that maintain their drug regimen over the entire term. Given what we have achieved over the course of this work I believe that we are closer to understanding the effects of mycobacterial dormancy on the Mycobacterium tuberculosis bacilli in vivo. Combining this with the invention of SLIC and its capacity to rapidly detect bacteria at unprecedentedly low concentrations we are closer to being able to diagnose and treat patients faster and with less wasted antibiotics than was previously possible.