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Acid-Fast Positive and Acid-Fast Negative : The Koch Paradox

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  • Authors: Catherine Vilchèze1, Laurent Kremer2
  • Editors: William R. Jacobs Jr.3, Helen McShane4, Valerie Mizrahi5, Ian M. Orme6
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461; 2: IRIM (ex-CPBS) UMR 9004, Infectious Disease Research Institute of Montpellier (IDRIM), Université de Montpellier, CNRS, 34293 Montpellier, France; 3: Howard Hughes Medical Institute, Albert Einstein School of Medicine, Bronx, NY 10461; 4: University of Oxford, Oxford OX3 7DQ, United Kingdom; 5: University of Cape Town, Rondebosch 7701, South Africa; 6: Colorado State University, Fort Collins, CO 80523
    • Source: microbiolspec March 2017 vol. 5 no. 2 doi:10.1128/microbiolspec.TBTB2-0003-2015
  • Received 02 November 2015 Accepted 02 February 2017 Published 24 March 2017
  • Catherine Vilchèze, [email protected]; Laurent Kremer, [email protected]
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  • Abstract:

    Acid-fast (AF) staining, also known as Ziehl-Neelsen stain microscopic detection, developed over a century ago, is even today the most widely used diagnostic method for tuberculosis. Herein we present a short historical review of the evolution of AF staining methods and discuss Koch’s paradox, in which non-AF tubercle bacilli can be detected in tuberculosis patients or in experimentally infected animals. The conversion of from an actively growing, AF-positive form to a nonreplicating, AF-negative form during the course of infection is now well documented. The mechanisms of loss of acid-fastness are not fully understood but involve important metabolic processes, such as the accumulation of triacylglycerol-containing intracellular inclusions and changes in the composition and spatial architecture of the cell wall. Although the precise component(s) responsible for the AF staining method remains largely unknown, analysis of a series of genetically defined mutants, which are attenuated in mice, pointed to the primary role of mycolic acids and other cell wall-associated (glyco)lipids as molecular markers responsible for the AF property of mycobacteria. Further studies are now required to better describe the cell wall reorganization that occurs during dormancy and to develop new staining procedures that are not affected by such cell wall alterations and that are capable of detecting AF-negative cells.

  • Citation: Vilchèze C, Kremer L. 2017. Acid-Fast Positive and Acid-Fast Negative : The Koch Paradox. Microbiol Spectrum 5(2):TBTB2-0003-2015. doi:10.1128/microbiolspec.TBTB2-0003-2015.

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/content/journal/microbiolspec/10.1128/microbiolspec.TBTB2-0003-2015
2017-03-24
2021-04-14

Abstract:

Acid-fast (AF) staining, also known as Ziehl-Neelsen stain microscopic detection, developed over a century ago, is even today the most widely used diagnostic method for tuberculosis. Herein we present a short historical review of the evolution of AF staining methods and discuss Koch’s paradox, in which non-AF tubercle bacilli can be detected in tuberculosis patients or in experimentally infected animals. The conversion of from an actively growing, AF-positive form to a nonreplicating, AF-negative form during the course of infection is now well documented. The mechanisms of loss of acid-fastness are not fully understood but involve important metabolic processes, such as the accumulation of triacylglycerol-containing intracellular inclusions and changes in the composition and spatial architecture of the cell wall. Although the precise component(s) responsible for the AF staining method remains largely unknown, analysis of a series of genetically defined mutants, which are attenuated in mice, pointed to the primary role of mycolic acids and other cell wall-associated (glyco)lipids as molecular markers responsible for the AF property of mycobacteria. Further studies are now required to better describe the cell wall reorganization that occurs during dormancy and to develop new staining procedures that are not affected by such cell wall alterations and that are capable of detecting AF-negative cells.

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Acid-Fast Positive and Acid-Fast Negative : The Koch Paradox
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Citation: Vilchèze C, Kremer L. 2017. Acid-fast positive and acid-fast negative : the koch paradox. microbiolspec 5(2): doi:10.1128/microbiolspec.TBTB2-0003-2015
/content/microbiolspec/10.1128/microbiolspec.TBTB2-0003-2015.fig1
FIGURE 1
Chemical structures of the major mycolic acids of . Cyclopropane rings and methyl branches are shown and annotated with the -adenosyl methionine-dependent methyl transferases responsible for their synthesis. , ; , .
microbiolspec/5/2/TBTB2-0003-2015-fig1_thmb.gif
microbiolspec/5/2/TBTB2-0003-2015-fig1.gif
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FIGURE 1

Chemical structures of the major mycolic acids of . Cyclopropane rings and methyl branches are shown and annotated with the -adenosyl methionine-dependent methyl transferases responsible for their synthesis. , ; , .

Source: microbiolspec March 2017 vol. 5 no. 2 doi:10.1128/microbiolspec.TBTB2-0003-2015
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FIGURE 2
Staining of using ZN (left) and auramine O (right). Magnification, ×100.
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FIGURE 2

Staining of using ZN (left) and auramine O (right). Magnification, ×100.

Source: microbiolspec March 2017 vol. 5 no. 2 doi:10.1128/microbiolspec.TBTB2-0003-2015
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FIGURE 3
Ser/Thr kinase-dependent signaling cascade resulting in phosphorylation of KasB and loss of acid-fastness. Modification of the cell wall composition in response to exogenous cues is central for adaptation to different environmental conditions. In response to an external signal, mycobacterial Ser/Thr kinases phosphorylate the different FAS-II components, including the β-ketoacyl ACP synthase KasB involved in the addition of the last carbon atoms during the mycolic acid elongation step. Phosphorylation on Thr334 and Thr336 decreases the condensation activity of KasB, resulting in the production of shorter mycolic acids, which probably affects the packing of the lipid layer and also results in the loss of the AF property and severe attenuation in mice.
microbiolspec/5/2/TBTB2-0003-2015-fig3_thmb.gif
microbiolspec/5/2/TBTB2-0003-2015-fig3.gif
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FIGURE 3

Ser/Thr kinase-dependent signaling cascade resulting in phosphorylation of KasB and loss of acid-fastness. Modification of the cell wall composition in response to exogenous cues is central for adaptation to different environmental conditions. In response to an external signal, mycobacterial Ser/Thr kinases phosphorylate the different FAS-II components, including the β-ketoacyl ACP synthase KasB involved in the addition of the last carbon atoms during the mycolic acid elongation step. Phosphorylation on Thr334 and Thr336 decreases the condensation activity of KasB, resulting in the production of shorter mycolic acids, which probably affects the packing of the lipid layer and also results in the loss of the AF property and severe attenuation in mice.

Source: microbiolspec March 2017 vol. 5 no. 2 doi:10.1128/microbiolspec.TBTB2-0003-2015
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FIGURE 4
Loss of AF staining coincides with the accumulation of TAG-containing intracellular lipid inclusions. Dual staining of grown under multiple stress conditions, using auramine O for AF-staining (green) and Nile red as a neutral lipid stain (red). Bacilli were observed by confocal laser scanning microscopy. Overlaid images of the dual-stained bacteria are shown. Bar = 4 μm. Quantification of the number of AF-positive and lipid-stain-positive bacilli grown as in (A). Auramine O-stained and Nile red-stained positive cells were counted from multiple scans. (Adapted from Deb et al. 4(6):e6077 with permission of the publisher.)
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FIGURE 4

Loss of AF staining coincides with the accumulation of TAG-containing intracellular lipid inclusions. Dual staining of grown under multiple stress conditions, using auramine O for AF-staining (green) and Nile red as a neutral lipid stain (red). Bacilli were observed by confocal laser scanning microscopy. Overlaid images of the dual-stained bacteria are shown. Bar = 4 μm. Quantification of the number of AF-positive and lipid-stain-positive bacilli grown as in (A). Auramine O-stained and Nile red-stained positive cells were counted from multiple scans. (Adapted from Deb et al. 4(6):e6077 with permission of the publisher.)

Source: microbiolspec March 2017 vol. 5 no. 2 doi:10.1128/microbiolspec.TBTB2-0003-2015
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