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...

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Autores principales:	Bhavna G. Gordhan, Julian S. Peters, Amanda McIvor, Edith E. Machowski, Christopher Ealand, Ziyaad Waja, Neil Martinson, Bavesh D. Kana
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/19ef0633fedb477db1d68450d3568b1c
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spelling	oai:doaj.org-article:19ef0633fedb477db1d68450d3568b1c2021-12-02T11:45:01ZDetection of differentially culturable tubercle bacteria in sputum using mycobacterial culture filtrates10.1038/s41598-021-86054-z2045-2322https://doaj.org/article/19ef0633fedb477db1d68450d3568b1c2021-03-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-86054-zhttps://doaj.org/toc/2045-2322Abstract 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 RpfAB Mtb and RpfABCDE Mtb 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.Bhavna G. GordhanJulian S. PetersAmanda McIvorEdith E. MachowskiChristopher EalandZiyaad WajaNeil MartinsonBavesh D. KanaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-11 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Bhavna G. Gordhan Julian S. Peters Amanda McIvor Edith E. Machowski Christopher Ealand Ziyaad Waja Neil Martinson Bavesh D. Kana Detection of differentially culturable tubercle bacteria in sputum using mycobacterial culture filtrates
description	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 RpfAB Mtb and RpfABCDE Mtb 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.
format	article
author	Bhavna G. Gordhan Julian S. Peters Amanda McIvor Edith E. Machowski Christopher Ealand Ziyaad Waja Neil Martinson Bavesh D. Kana
author_facet	Bhavna G. Gordhan Julian S. Peters Amanda McIvor Edith E. Machowski Christopher Ealand Ziyaad Waja Neil Martinson Bavesh D. Kana
author_sort	Bhavna G. Gordhan
title	Detection of differentially culturable tubercle bacteria in sputum using mycobacterial culture filtrates
title_short	Detection of differentially culturable tubercle bacteria in sputum using mycobacterial culture filtrates
title_full	Detection of differentially culturable tubercle bacteria in sputum using mycobacterial culture filtrates
title_fullStr	Detection of differentially culturable tubercle bacteria in sputum using mycobacterial culture filtrates
title_full_unstemmed	Detection of differentially culturable tubercle bacteria in sputum using mycobacterial culture filtrates
title_sort	detection of differentially culturable tubercle bacteria in sputum using mycobacterial culture filtrates
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/19ef0633fedb477db1d68450d3568b1c
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Detection of differentially culturable tubercle bacteria in sputum using mycobacterial culture filtrates

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