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Potassium availability triggers Mycobacterium tuberculosis transition to, and resuscitation from, non-culturable (dormant) states.

Salina EG, Waddell SJ, Hoffmann N, Rosenkrands I, Butcher PD, Kaprelyants AS - Open Biol (2014)

Bottom Line: In particular, latent tuberculosis may result from the maintenance of Mycobacterium tuberculosis bacilli in non-replicating states in infected individuals.Uniquely, growth of M. tuberculosis in aerobic conditions in potassium-deficient media resulted in the generation of bacilli that were non-culturable (NC) on solid media but detectable in liquid media.Transcriptomic and proteomic profiling through adaptation to, and resuscitation from, this NC state revealed a switch to anaerobic respiration and a shift to lipid and amino acid metabolism.

View Article: PubMed Central - PubMed

Affiliation: Institution of the Russian Academy of Sciences A.N. Bach Institute of Biochemistry RAS, Moscow, Russia elenasalina@yandex.ru.

ABSTRACT
Dormancy in non-sporulating bacteria is an interesting and underexplored phenomenon with significant medical implications. In particular, latent tuberculosis may result from the maintenance of Mycobacterium tuberculosis bacilli in non-replicating states in infected individuals. Uniquely, growth of M. tuberculosis in aerobic conditions in potassium-deficient media resulted in the generation of bacilli that were non-culturable (NC) on solid media but detectable in liquid media. These bacilli were morphologically distinct and tolerant to cell-wall-targeting antimicrobials. Bacterial counts on solid media quickly recovered after washing and incubating bacilli in fresh resuscitation media containing potassium. This resuscitation of growth occurred too quickly to be attributed to M. tuberculosis replication. Transcriptomic and proteomic profiling through adaptation to, and resuscitation from, this NC state revealed a switch to anaerobic respiration and a shift to lipid and amino acid metabolism. High concordance with mRNA signatures derived from M. tuberculosis infection models suggests that analogous NC mycobacterial phenotypes may exist during disease and may represent unrecognized populations in vivo. Resuscitation of NC bacilli in potassium-sufficient media was characterized by time-dependent activation of metabolic pathways in a programmed series of processes that probably transit bacilli through challenging microenvironments during infection.

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Antibiotic-mediated killing in actively growing and NC M. tuberculosis. Mycobacteria were exposed to 5 µg ml−1 RIF, 1 μg ml−1 INH or 0.2 μg ml−1 BTZ for 7 days. Viability was estimated by MPN assay and compared to drug-free controls. Open bars correspond to log-phase cells derived from K+-rich media, grey bars correspond to NC cells. The standard deviations of three independent experiments are marked.
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RSOB140106F3: Antibiotic-mediated killing in actively growing and NC M. tuberculosis. Mycobacteria were exposed to 5 µg ml−1 RIF, 1 μg ml−1 INH or 0.2 μg ml−1 BTZ for 7 days. Viability was estimated by MPN assay and compared to drug-free controls. Open bars correspond to log-phase cells derived from K+-rich media, grey bars correspond to NC cells. The standard deviations of three independent experiments are marked.

Mentions: We tested the activity of two first-line anti-tuberculosis drugs, isoniazid (INH) and rifampicin (RIF), and the newly developed compound benzothiazinone-043 (BTZ) [36] on NC and log-phase bacilli. The effectiveness of these antimicrobials was estimated by measuring the ability of NC cells to resuscitate after 7-day drug exposure by MPN assay. In contrast with plating on solid media, this approach enables the maximal number of potentially viable cells to be detected after antibiotic exposure. This allows non-culturability on solid media to be distinguished from drug killing, providing a suitable measure of the effect of these drugs on NC bacilli. A log-phase culture grown in +K+ Sauton medium at a similar cell density (estimated by MPN assay) was used as a reference. Seven-day exposure to RIF or INH caused approximately 2.5 log decrease in viable cells in log-phase bacilli compared with drug-free culture (figure 3). BTZ reduced the viable count of +K+ log-phase bacilli by two logs as measured by MPN. The same drugs were tested for their ability to kill NC cells derived from K+-deficient media. Both BTZ-043 and INH had little effect on NC cell viability: less than 1 log kill after 7-day exposure (figure 3). However, RIF efficacy remained similar to that observed for log-phase bacilli (approx. 2.5 log kill).Figure 3.


Potassium availability triggers Mycobacterium tuberculosis transition to, and resuscitation from, non-culturable (dormant) states.

Salina EG, Waddell SJ, Hoffmann N, Rosenkrands I, Butcher PD, Kaprelyants AS - Open Biol (2014)

Antibiotic-mediated killing in actively growing and NC M. tuberculosis. Mycobacteria were exposed to 5 µg ml−1 RIF, 1 μg ml−1 INH or 0.2 μg ml−1 BTZ for 7 days. Viability was estimated by MPN assay and compared to drug-free controls. Open bars correspond to log-phase cells derived from K+-rich media, grey bars correspond to NC cells. The standard deviations of three independent experiments are marked.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4221891&req=5

RSOB140106F3: Antibiotic-mediated killing in actively growing and NC M. tuberculosis. Mycobacteria were exposed to 5 µg ml−1 RIF, 1 μg ml−1 INH or 0.2 μg ml−1 BTZ for 7 days. Viability was estimated by MPN assay and compared to drug-free controls. Open bars correspond to log-phase cells derived from K+-rich media, grey bars correspond to NC cells. The standard deviations of three independent experiments are marked.
Mentions: We tested the activity of two first-line anti-tuberculosis drugs, isoniazid (INH) and rifampicin (RIF), and the newly developed compound benzothiazinone-043 (BTZ) [36] on NC and log-phase bacilli. The effectiveness of these antimicrobials was estimated by measuring the ability of NC cells to resuscitate after 7-day drug exposure by MPN assay. In contrast with plating on solid media, this approach enables the maximal number of potentially viable cells to be detected after antibiotic exposure. This allows non-culturability on solid media to be distinguished from drug killing, providing a suitable measure of the effect of these drugs on NC bacilli. A log-phase culture grown in +K+ Sauton medium at a similar cell density (estimated by MPN assay) was used as a reference. Seven-day exposure to RIF or INH caused approximately 2.5 log decrease in viable cells in log-phase bacilli compared with drug-free culture (figure 3). BTZ reduced the viable count of +K+ log-phase bacilli by two logs as measured by MPN. The same drugs were tested for their ability to kill NC cells derived from K+-deficient media. Both BTZ-043 and INH had little effect on NC cell viability: less than 1 log kill after 7-day exposure (figure 3). However, RIF efficacy remained similar to that observed for log-phase bacilli (approx. 2.5 log kill).Figure 3.

Bottom Line: In particular, latent tuberculosis may result from the maintenance of Mycobacterium tuberculosis bacilli in non-replicating states in infected individuals.Uniquely, growth of M. tuberculosis in aerobic conditions in potassium-deficient media resulted in the generation of bacilli that were non-culturable (NC) on solid media but detectable in liquid media.Transcriptomic and proteomic profiling through adaptation to, and resuscitation from, this NC state revealed a switch to anaerobic respiration and a shift to lipid and amino acid metabolism.

View Article: PubMed Central - PubMed

Affiliation: Institution of the Russian Academy of Sciences A.N. Bach Institute of Biochemistry RAS, Moscow, Russia elenasalina@yandex.ru.

ABSTRACT
Dormancy in non-sporulating bacteria is an interesting and underexplored phenomenon with significant medical implications. In particular, latent tuberculosis may result from the maintenance of Mycobacterium tuberculosis bacilli in non-replicating states in infected individuals. Uniquely, growth of M. tuberculosis in aerobic conditions in potassium-deficient media resulted in the generation of bacilli that were non-culturable (NC) on solid media but detectable in liquid media. These bacilli were morphologically distinct and tolerant to cell-wall-targeting antimicrobials. Bacterial counts on solid media quickly recovered after washing and incubating bacilli in fresh resuscitation media containing potassium. This resuscitation of growth occurred too quickly to be attributed to M. tuberculosis replication. Transcriptomic and proteomic profiling through adaptation to, and resuscitation from, this NC state revealed a switch to anaerobic respiration and a shift to lipid and amino acid metabolism. High concordance with mRNA signatures derived from M. tuberculosis infection models suggests that analogous NC mycobacterial phenotypes may exist during disease and may represent unrecognized populations in vivo. Resuscitation of NC bacilli in potassium-sufficient media was characterized by time-dependent activation of metabolic pathways in a programmed series of processes that probably transit bacilli through challenging microenvironments during infection.

Show MeSH
Related in: MedlinePlus