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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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The definition of two model variations describing the generation and resuscitation of NC M. tuberculosis bacilli in potassium-limiting conditions. The high MPN/low CFU NC model variant (red circle) is plotted alongside an alternative high MPN/high CFU NC variant (green triangle). A 10-day-old inoculum was used to generate the high MPN/low CFU NC phenotype, whereas a 15-day-old inoculum resulted in a high MPN/high CFU NC phenotype. Measurements are detailed in CFU ml−1 over 50 days of incubation; corresponding MPN measurements for NC bacilli are marked. Experiments were repeated five times with similar results; a representative experiment is shown. Bars represent s.d. variation.
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RSOB140106F6: The definition of two model variations describing the generation and resuscitation of NC M. tuberculosis bacilli in potassium-limiting conditions. The high MPN/low CFU NC model variant (red circle) is plotted alongside an alternative high MPN/high CFU NC variant (green triangle). A 10-day-old inoculum was used to generate the high MPN/low CFU NC phenotype, whereas a 15-day-old inoculum resulted in a high MPN/high CFU NC phenotype. Measurements are detailed in CFU ml−1 over 50 days of incubation; corresponding MPN measurements for NC bacilli are marked. Experiments were repeated five times with similar results; a representative experiment is shown. Bars represent s.d. variation.

Mentions: The number and MPN/CFU ratio of NC bacilli resulting from this K+-deficient model could be adjusted by altering the age of inoculum used to generate the NC phenotype (figure 6). We have described above NC bacilli with a high MPN/low CFU phenotype (7.0 × 106 ml−1 and 1.1 × 103 ml−1, respectively), where 99.7% of cells were NC. A second NC model variant (high MPN/high CFU phenotype) with a higher relative proportion of culturable bacilli (5.5 × 107 MPN ml−1 and 7.0 × 106 CFU ml−1, where 80% of cells were NC) was used to recover greater biomass from the K+-deficient NC model, thus enabling a comparator transcriptional dataset to be generated without the requirement for RNA amplification that was used to verify NC mRNA signatures. Transcriptional adaptations to the NC state (in comparison to log-phase bacilli) were broadly mirrored in both NC phenotypes, verifying the metabolic signatures associated with the NC state. Experiments to ensure the representation of RNA amplification and transcriptional analysis showed good correlation between model variants (hp 9.63 × 10−71; electronic supplementary material, table S5), thus validating the experimental approach to profile low numbers of NC bacilli. Furthermore, comparing transcriptome patterns between cultures with varying MPN/CFU ratio enabled mapping of the phenotypic state of NC bacilli without recourse to defining changes relative to log phase or over time.Figure 6.


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)

The definition of two model variations describing the generation and resuscitation of NC M. tuberculosis bacilli in potassium-limiting conditions. The high MPN/low CFU NC model variant (red circle) is plotted alongside an alternative high MPN/high CFU NC variant (green triangle). A 10-day-old inoculum was used to generate the high MPN/low CFU NC phenotype, whereas a 15-day-old inoculum resulted in a high MPN/high CFU NC phenotype. Measurements are detailed in CFU ml−1 over 50 days of incubation; corresponding MPN measurements for NC bacilli are marked. Experiments were repeated five times with similar results; a representative experiment is shown. Bars represent s.d. variation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB140106F6: The definition of two model variations describing the generation and resuscitation of NC M. tuberculosis bacilli in potassium-limiting conditions. The high MPN/low CFU NC model variant (red circle) is plotted alongside an alternative high MPN/high CFU NC variant (green triangle). A 10-day-old inoculum was used to generate the high MPN/low CFU NC phenotype, whereas a 15-day-old inoculum resulted in a high MPN/high CFU NC phenotype. Measurements are detailed in CFU ml−1 over 50 days of incubation; corresponding MPN measurements for NC bacilli are marked. Experiments were repeated five times with similar results; a representative experiment is shown. Bars represent s.d. variation.
Mentions: The number and MPN/CFU ratio of NC bacilli resulting from this K+-deficient model could be adjusted by altering the age of inoculum used to generate the NC phenotype (figure 6). We have described above NC bacilli with a high MPN/low CFU phenotype (7.0 × 106 ml−1 and 1.1 × 103 ml−1, respectively), where 99.7% of cells were NC. A second NC model variant (high MPN/high CFU phenotype) with a higher relative proportion of culturable bacilli (5.5 × 107 MPN ml−1 and 7.0 × 106 CFU ml−1, where 80% of cells were NC) was used to recover greater biomass from the K+-deficient NC model, thus enabling a comparator transcriptional dataset to be generated without the requirement for RNA amplification that was used to verify NC mRNA signatures. Transcriptional adaptations to the NC state (in comparison to log-phase bacilli) were broadly mirrored in both NC phenotypes, verifying the metabolic signatures associated with the NC state. Experiments to ensure the representation of RNA amplification and transcriptional analysis showed good correlation between model variants (hp 9.63 × 10−71; electronic supplementary material, table S5), thus validating the experimental approach to profile low numbers of NC bacilli. Furthermore, comparing transcriptome patterns between cultures with varying MPN/CFU ratio enabled mapping of the phenotypic state of NC bacilli without recourse to defining changes relative to log phase or over time.Figure 6.

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