Detection of differentially culturable tubercle bacteria in sputum using mycobacterial culture filtrates

Abstract

Rapid detection of tuberculosis (TB) infection is paramount to curb further transmission. The gold standard for this remains mycobacterial culture, however emerging evidence confirms the presence of differentially culturable tubercle bacteria (DCTB) in clinical specimens. These bacteria do not grow under standard culture conditions and require the presence of culture filtrate (CF), from axenic cultures of Mycobacterium tuberculosis (Mtb), to emerge. It has been hypothesized that molecules such as resuscitation promoting factors (Rpfs), fatty acids and cyclic-AMP (cAMP) present in CF are responsible for the growth stimulatory activity. Herein, we tested the ability of CF from the non-pathogenic bacterium Mycobacterium smegmatis (Msm) to stimulate the growth of DCTB, as this organism provides a more tractable source of CF. We also interrogated the role of Mtb Rpfs in stimulation of DCTB by creating recombinant strains of Msm that express Mtb rpf genes in various combinations. CF derived from this panel of strains was tested on sputum from individuals with drug susceptible TB prior to treatment. CF from wild type Msm did not enable detection of DCTB in a manner akin to Mtb CF preparations and whilst the addition of RpfABMtb and RpfABCDEMtb to an Msm mutant devoid of its native rpfs did improve detection of DCTB compared to the no CF control, it was not statistically different to the empty vector control. To further investigate the role of Rpfs, we compared the growth stimulatory activity of CF from Mtb, with and without Rpfs and found these to be equivalent. Next, we tested chemically diverse fatty acids and cAMP for growth stimulation and whilst some selective stimulatory effect was observed, this was not significantly higher than the media control and not comparable to CF. Together, these data indicate that the growth stimulatory effect observed with Mtb CF is most likely the result of a combination of factors. Future work aimed at identifying the nature of these growth stimulatory molecules may facilitate improvement of culture-based diagnostics for TB.

Culture-Free Enumeration of Mycobacterium tuberculosis in Mouse Tissues Using the Molecular Bacterial Load Assay for Preclinical Drug Development

“…When the MPN method was included in the analysis, it gave further support to the MBL assay detecting a population of bacteria that were still viable rather than residual rRNA from non-viable organisms. In vitro studies have begun to characterise these populations [34][35][36]. Our data further support the existence of these populations in mouse lungs.…”

Characterization of Differentially Detectable Mycobacterium tuberculosis in the Sputum of Subjects with Drug-Sensitive or Drug-Resistant Tuberculosis before and after Two Months of Therapy

“…The latter effect was alleviated in sputa from the cohort with DS TB after initiation of drug treatment, suggesting that the prior drug exposure experienced by subjects with DR TB may have contributed to this difference. What components of CF exert these effects and by what mechanisms remain unknown ( 12 ), making it difficult to draw biologic inferences. As a practical matter, it is advisable to perform MPN-LD assays both with and without CF to maximize detection of DD- Mtb .…”

“…This approach is often supplemented or replaced by assessing time to positivity (TTP) of sputum samples in an automated liquid culture system that measures the rate of depletion of oxygen from culture medium as a proxy for M. tuberculosis growth and quantity. These techniques may miss subpopulations of M. tuberculosis that are incapable of growing on solid medium or in liquid medium if the suspension has not been extensively diluted ( 3 – 12 ). These bacteria have been called by the interchangeable terms “viable but nonculturable” (VBNC- Mtb ) ( 13 ), “differentially culturable” (DCTB) ( 10 ), and “differentially detectable” M. tuberculosis (DD- Mtb ) ( 8 ).…”

Small RNA F6 Provides Mycobacterium smegmatis Entry into Dormancy

“…The relationship between biofilm formation and Rpfs in M. smegmatis has been recently demonstrated by Ealand et al [ 35 ]; they observed that simultaneous deletion of rpf genes hampered the development of biofilms and reduced drug tolerance, and that these effects were accompanied by a decrease in muropeptide production and altered peptidoglycan cross-linking. Recently, the same group has examined the role of M. tuberculosis Rpfs in reactivation processes by expressing them in M. smegmatis [ 36 ]. Their results indicate that the growth stimulatory effect observed with the culture filtrate is most likely the result of a combination of Rpfs with other factors [ 36 ].…”

“…Recently, the same group has examined the role of M. tuberculosis Rpfs in reactivation processes by expressing them in M. smegmatis [ 36 ]. Their results indicate that the growth stimulatory effect observed with the culture filtrate is most likely the result of a combination of Rpfs with other factors [ 36 ]. In our study, the changes in RpfE2 expression had a dramatic effect on M. smegmatis entering into the ‘non-culturable’ state but did not affect its reactivation from dormancy.…”