Experimental demonstration of persisters

In most of the experiments demonstrating persisters, bacterial cultures were incubated in the presence of antibiotics for 3-6 h. At the end of the incubation period, the majority of the bacteria were found to be dead, except for a small subpopulation of persisters. During the incubation period, the persisters were either growing slowly or not at all, but once the antibiotic was removed, they were able to grow normally.

Researchers had incubated bacterial cultures using a single concentration and dose of the antibiotic against a specific inoculum size for a very short incubation time and then assumed that the bacteria that survived the antibiotic treatment are persisters that have the capability to undergo a phenotypic shift. However, a different result would have been obtained had the researchers used  

1. different inoculum sizes (inoculum effect)

2. different concentrations of antibiotic

3. different total time of incubaton and

4. multiple doses of antibiotics

The definition of persisters as a small subpopulation of bacteria (less than 1% of the total populaton) that are not killed in the presence of bacteria is based on a particular experimental condition only. This definition can change once the conditions are also changed. For example, had the researchers incubated the bacterial culture with antibiotic for 24-48 h, rather than 3-6 h (at the same antibiotic concentration and inoculum size used in their experiments), they would have noticed one of three conditions.

1. Complete sterilization of the culture without any persisters;

2. Re-growth of the remaining survivors even in the presence of antibiotics;

3. Or, less frequently, the surviving bacteria remaining in the dormant stage itself without growth.

Similarly, had the researchers tested different antibiotic concentrations and inoculum sizes followed by incubation for 24-48 h, they would have noticed the optimal conditions for complete sterilization. The perceived phenotypic shift demonstrated in many experiments can be the result of the failure of antibiotics to kill all bacteria owing to the suboptimal conditions of killing provided in the experiments.

Next- Where 94% is a small subpopulation…