The emergence of differentially culturable tubercle bacteria in clinical drug sensitive mycobacterium tuberculosis strains
Date
2020
Authors
Padarath, Kiyasha
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Abstract
Mycobacterium tuberculosis (M. tuberculosis) is the causative agent of tuberculosis (TB)
infection and disease. Since its discovery, various diagnostic tools have been developed to
detect bacteria in sputum samples from TB-infected patients including bacterial culture,
auramine staining and nucleic acid amplification tests (NAATs). Recent studies have revealed
a distinct population of bacteria in sputum samples from patients with drug susceptible TB that
are undetected by standard culturing techniques. These bacteria, defined as differentially
culturable tubercle bacteria (DCTB), exist in a non-replicating state that is reactivated only in
liquid media, supplemented with growth factors, and not on solid media. Laboratory models
that induce this differentially culturable state are important for studying the physiology and
metabolism of these bacteria to ultimately develop new diagnostic tests for TB. In this study
we tested and optimised in vitro stress models in laboratory media using the laboratory strain
H37Rv, to determine the conditions that robustly generate DCTB. The quantity of DCTB was
assessed using the most probable number assay and colony forming units. In addition, the
phenotype of these cells was analysed using microscopy and flow cytometry, with metabolic
probes that target remodelling of the peptidoglycan (PG) component of the bacterial cell wall.
Our results indicated that application of the carbon starvation model to clinical M. tuberculosis
strains (Beijing and LAM), produced robust levels of DCTB as illustrated by limited growth
on agar plates and enhanced growth in liquid media supplemented with culture filtrate from an
axenic culture of M. tuberculosis. These DCTB cells were non-replicating and significantly
shorter compared to cells grown in normal media. Using probes that report on PG biosynthesis
representing metabolically active cells, confirmed a lack of de novo biosynthesis in DCTB,
providing evidence for the establishment of a non-replicating state. Microscopy revealed a lack
of metabolic probe uptake in a large proportion of the DCTB population, which was restored
when the bacteria were resuscitated. We also demonstrated that Beijing strains have greater
propensity to produce DCTB compared to LAM strains. Collectively, our observations allow
for the improvement of experimental systems that enable further investigation of DCTB and
how these bacteria contribute to treatment response. In addition, these efforts will further allow
for the development of a shorter TB regimen, with reduced daily pill burden and limited side
effects
Description
A dissertation submitted in fulfilment of the requirements for the degree of Master of Science in Medicine to the Faculty of Health Sciences, School of Pathology, University of the Witwatersrand, Johannesburg, 2020