Wednesday, October 5, 2011
Isolation of slow dividing, small colony forming, hypovirulent, senescent bacteria
In a normally dividing population, a small subpopulation of slow dividing bacteria is present which can be isolated using aminoglycoside antibiotics. They are termed as small colony variants (SCV). SCVs had already been discussed before (please check the posts in the month of August). They constitute a naturally occurring, slow-growing subpopulation of bacteria that form small colonies (less than one-tenth of the size of parent colonies) on solid media (Proctor et al. 2006). Much has been published on the biochemical aspects and the significance of SCVs. However, there are two areas where I have difference of opinion from those in published articles.
1. SCVs are mutants that revert to normal growth in the presence of auxotrophic agents
2. SCVs are responsible for chronic infections
Whereas a number of mutants form SCVs and can be reverted to normal growth after adding hemin, menadione, thiamine or thymidine, all SCVs isolated in vitro after adding aminoglycosides may not be specific mutants. In fact all SCVs are not similar and may exhibit different protein profiles (Kriegeskorte et al. 2011). Similarly, the role of SCVs in chronic infections is questionable (please read the previous posts).
A pure culture of SCVs of E. coli DH-5alpha cells can be isolated after treating cells with subinhibitory concentration of aminoglycosides like kanamycin as explained in Jacob (2007). In short, 50 ul of stationary phase culture is added to 3 ml of fresh LB medium containing kanamycin at different concentrations and incubated for 2 days. Three factors are important to get a pure culture of SCVs- initial inoculum size, concentration of antibiotic and the total time of incubation. If the inoculum size is very low, SCVs may be missed, but if high, some normally dividing bacteria that have escaped killing may overgrow and mask SCVs. Since they are slow dividing bacteria, SCVs may take longer time to grow. With different concentrations of kanamycin, colonies of different sizes can be obtained.
The slow dividing SCVs have been shown to be hypovirulent also (Sifri et al. 2006). But, how can it be proved that they are senescent bacteria?
Next- Experiment that could indicate that SCVs are senescent bacteria
Proctor et al. (2006). Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections. Nat Rev Microbiol 4(4), 295-305.
Kriegeskorte et al. (2011). Small colony variants of Staphylococcus aureus reveal distinct protein profiles. PROTEOMICS, 11: 2476–2490.
Jacob, J (2007). Persisters show heritable phenotype and generate bacterial heterogeneity and noise in protein expression . Available from Nature Precedings http://hdl.handle.net/10101/npre.2007.1411.1
Sifri et al. (2006). Virulence of Staphylococcus aureus small colony variants in the Caenorhabditidis elegans infection model. Infection and Immunity, 74(2);1091-1096.