Enhanced Methodologies for Detecting Phenotypic Resistance in Mycobacteria
Lipid droplets found in algae and other microscopic organisms have become of interest to many researchers partially because they carry the capacity to produce bio-oil for the mass market. They are of importance in biology and clinical practice because their presence can be a phenotypic marker of an altered metabolism, including reversible resistance to antibiotics, prompting intense research.
A useful stain for detecting lipid bodies in the lab is Nile red. It is a dye that exhibits solvatochromism; its absorption band varies in spectral position, shape and intensity with the nature of its solvent environment, it will fluoresce intensely red in polar environment and blue shift with the changing polarity of its solvent. This makes it ideal for the detection of lipid bodies within Mycobacterium spp. This is because mycobacterial lipid bodies’ primary constituents are nonpolar lipids such as triacylglycerols but bacterial cell membranes are primarily polar lipid species. In this chapter we describe an optimal method for using Nile red to distinguish lipid containing (Lipid rich or LR cells) from those without lipid bodies (Lipid Poor or LP). As part of the process we have optimized a method for separating LP and LR cells that does not require the use of an ultracentrifuge or complex separation media. We believe that these methods will facilitate further research in these enigmatic, transient and important cell states.
Key wordsTuberculosis Dormancy Phenotypic resistance Lipid body
- 6.Deb C, Lee CM, Dubey VS, Daniel J, Abomoelak B, Sirakova TD, Pawar S, Rogers L, Kolattukudy PE (2009) A novel in vitro multiple-stress dormancy model for Mycobacterium tuberculosis generates a lipid-loaded, drug-tolerant, dormant pathogen. PLoS One 4(6):e6077. https://doi.org/10.1371/journal.pone.0006077 CrossRefPubMedPubMedCentralGoogle Scholar
- 8.Hammond RJH, Baron VO, Oravcova K, Lipworth S, Gillespie SH (2015) Phenotypic resistance in mycobacteria: is it because I am old or fat that I resist you? J Antimicrob Chemother. https://doi.org/10.1093/jac/dkv178
- 9.Schmidt WA (1919) Process and apparatus for separating finely-divided materials. Google PatentsGoogle Scholar
- 13.Boogaerts MA, Vercelotti G, Roelant C, Malbrain S, Verwilghen RL, Jacob HS (1986) Platelets augment granulocyte aggregation and cytotoxicity: uncovering of their effects by improved cell separation techniques using Percoll gradients. Scand J Haematol 37(3):229–236. https://doi.org/10.1111/j.1600-0609.1986.tb02302.x CrossRefPubMedGoogle Scholar
- 14.Kurnick JT, Östberg L, Stegagno M, Kimura AK, Örn A, Sjöberg O (1979) A rapid method for the separation of functional lymphoid cell populations of human and animal origin on PVP-silica (Percoll) density gradients. Scand J Immunol 10(6):563–573. https://doi.org/10.1111/j.1365-3083.1979.tb01391.x CrossRefPubMedGoogle Scholar
- 16.Raposo G, Nijman HW, Stoorvogel W et al (1996) B lymphocytes secrete antigen-presenting vesicles. J Exp Med 183(3). https://doi.org/10.1084/jem.183.3.1161
- 18.Daniel J, Maamar H, Deb C, Sirakova TD, Kolattukudy PE (2011) Mycobacterium tuberculosis uses host triacylglycerol to accumulate lipid droplets and acquires a dormancy-like phenotype in lipid-loaded macrophages. PLoS Pathog 7(6):e1002093. https://doi.org/10.1371/journal.ppat.1002093 CrossRefPubMedPubMedCentralGoogle Scholar
- 19.Martin NJ, Macdonald RM (2015) Separation of non-filamentous micro-organisms from soil by density gradient centrifugation in Percoll. J Appl Bacteriol 51(2):243–251. https://doi.org/10.1111/j.1365-2672.1981.tb01238.x CrossRefGoogle Scholar
- 23.Ishidate K, Creeger E, Zrike J, Deb S, Glauner B, MacAlister T, Rothfield L (1986) Isolation of differentiated membrane domains from Escherichia coli and Salmonella typhimurium, including a fraction containing attachment sites between the inner and outer membranes and the murein skeleton of the cell envelope. J Biol Chem 261(1):428–443PubMedGoogle Scholar
- 25.Auty MAE, Gardiner GE, McBrearty SJ, O’Sullivan EO, Mulvihill DM, Collins JK, Fitzgerald GF, Stanton C, Ross RP (2001) Direct in situ viability assessment of bacteria in probiotic dairy products using viability staining in conjunction with confocal scanning laser microscopy. Appl Environ Microbiol. https://doi.org/10.1128/AEM.67.1.420-425.2001