Studies on the phenotypes of Mycobacterium tuberculosis in sputum

Transcriptional, cytological and culture-based analyses of Mycobacterium tuberculosis (Mtb) in sputum have revealed multiple traits indicating the presence of a persister-like or dormant mycobacterial population. Between patients, variable proportions of bacilli in sputum appear to be slow or non-growing, contain lipid bodies (LBs) and depend on exogenous Resuscitation promoting factors for growth. More recently by using Auramine O/Nile-red staining the presence of non-acid-fast (NAF) Mtb-like bacilli containing abundant LBs has been noted. Based on these findings, Mtb in sputum may present in multiple populations and express distinctive transmission adapted phenotypes. Identifying these phenotypes and replicating them in in vitro settings may lead to important new understanding of Mtb in vivo. To study the suspected NAF Mtb cells in sputum, immunofluorescence (IF), peptide nucleic acid (PNA) probe and fluorescence Kinyoun methods were developed and studied. The IF and PNA methods detected only minor components of sputum and variable proportions of in vitro grown cells. Various conditions such as freeze thawing, growth phase and biofilm cultures were shown to alter Auramine NAF proportions. In contrast the fluorescence Kinyoun method labelled the majority of Mtb cells in the preparations studied and provides a promising method for future studies when combined with a suitable LB stain. The capacity of biofilm cultures to replicate the Mtb bacillary populations in sputum was studied. Three phases of biofilm cultures (pellicle, planktonic and attached layers) were studied for gene expression, cytological, growth, antibiotic tolerance and [superscript 3]H-uracil labelling properties comparable to the Mtb phenotypes seen in sputum. The three layers replicated to differing degrees the sputum phenotypes including LB and NAF content, and modest Rpf-dependancy. Attached and planktonic cells gave well correlated transcriptional patterns. Overall, it appears plausible that biofilm grown cells in patient’s lungs could contribute to the populations seen in sputum.