Isolation of fast dividing, hypervirulent, young bacteria- a simple experiment that can reveal many interesting properties

In one of my experiments aimed at eliminating persister bacteria (a small subset of slow growing bacterial population) from a normally dividing population, a culture of bacteria was repeatedly grown in early exponential phase (Jacob 2007). This experiment is simple and inexpensive (only an incubator and a spectrophotometer are needed) and quick (can be finished in a few days), but give some interesting results regarding bacterial growth kinetics.

In this experiment, when a culture of bacteria reached an O.D. of 0.3- 0.5 ( i.e. at a stage of light turbidity of the medium), 100 ul of the culture was transferred to 3 ml of fresh medium and incubated again. The same procedure was repeated 4 times. Repeated selection of bacteria at early exponential phase eliminated all slow growing bacteria. At the end of 4^th cycle, when the bacterial culture was allowed to continue its growth and reach a stationary phase, some interesting properties were noticed.

1. The growth rate of the selected bacteria was found to be much higher than a normally dividing culture

2. At stationary phase, the number of bacteria per ml of medium was higher

3. Persisters that were absent initially after repeatedly grown in early exponential phase reappeared at stationary phase

4. GFP expression of selected bacteria were higher

5. Activity of ampicillin was much reduced against the selected bacteria especially at higher initial inoculum size (in fact, bacteria grew as if antibiotic was not added; however, this lack of activity was not due to antibiotic resistance)

In the above article, only the third property was reported since the article was focused on the phenotypic shift of persisters. Based on the increased GFP expression and lack of activity of ampicillin (which I attribute to fast growth and increased production of enzymes that destroy the antibiotic, especially at higher inoculum size), I assumed that those bacteria were hypervirulent. Indeed, the hypervirulence of selected bacteria was later reported by Chapuis et al. (2011) after injection of Xenorhabdus nematophila into insects. My interpretation to the above result is that these fast dividing, hypervirulent bacteria are very young bacteria that are found in low in numbers in a normally dividing population (see the earlier blogpost).

Just because the selected bacteria are fast dividing and hypervirulent does not prove that they are young bacteria. However, it is possible to prove it with the help of another experiment.

Next- The experiment that can prove that the fast dividing, hypervirulent bacteria are young bacteria

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>

Chapuis et al. (2011). Virulence and pathogen multiplication: A serial passage experiment in the hypervirulent bacterial insect-pathogen Xenorhabdus nematophila. PLoS ONE 6(1): e15872. doi:10.1371/journal.pone.0015872