Monday, November 21, 2011
When bacteria are added to fresh growth medium, they undergo four growth phases. The lag phase, exponential phase, stationary phase and the death phase. Initial lag in growth may result from adaptation to the new environment. Once adapted, they undergo exponential growth phase. However, after many divisions, they enter the stationary phase wherein the number of bacteria remains a constant resulting from equilibrium between the rate of cell growth and cell death.
Stationary phase is thought to result from a combination of factors including lack of nutrients, lack of space and the accumulation of toxic products. However, whether the above factors are responsible for stationary phase is questionable. This is because cell-free supernatant from a stationary phase E. coli culture can still support bacterial growth (Carbonell et al. 2002). This result was confirmed in my experiments also. In my experiments (unpublished findings), an overnight grown E.coli culture at stationary phase was centrifuged and the supernatant was collected and incubated again. Few residual cells remaining after centrifugation have the ability for further division and growth (even though the turbidity will be less than the parent culture at stationary phase). In fact, in my experiments, bacterial growth (though, to less extent) was supported by the medium for two more rounds of incubation. This indicates that nutrient limitation or accumulation of toxic products is not the major responsible factor in reaching stationary phase.
Similarly, by repeatedly growing the bacterial culture in early exponential phase, fast growing, hypervirulent bacteria (which I assume as young bacteria) can be isolated which when incubated results in higher number of bacteria/ml at stationary phase (discussed earlier on the blogpost on September 19). This also indicates that the above factors may not be the major responsible factors in reaching the stationary phase. If they were the factors responsible for reaching stationary phase, a higher number of bacteria/ml would not have occurred.
The number of bacteria at stationary phase depends on the initial growth stage or the age of bacteria. When we add a small amount of overnight incubated culture into fresh medium and further incubate overnight (as in most of the experiments), a specific OD and number of bacteria/ml will be noticed at stationary phase. However, if we start with fast growing hypervirulent bacteria, the OD and the number of bacteria/ml will be higher. On the other hand, if we start with senescent bacteria (which are slow dividing, hypovirulent bacteria), the OD and the number of bacteria/ml will be much less (the medium will not turn to turbidity even after reaching stationary phase) (Jacob 2007) . Thus, the major factor that determines the stationary phase is the growth stage or the age of bacteria and not the nutrient or space limitation or accumulation of toxic products (they may be minor factors only).
Whether quorum sensing molecules have any role in stationary phase is an open question. However, considering the increased number of cells at stationary phase with hypervirulent bacteria (one would expect higher concentration of quorum sensing molecule) and their decreased number with hypovirulent bacteria (the concentration may be too low to induce a stationary phase), their role is also under question. It is tempting to state that the growth stage or the age is the only important factor in determining the stationary phase (others may have only a minor role).
Carbonell et al. (2002). Control of Escherichia coli growth rate through cell density. Microbiol. Res. (2002) 157, 257–265
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
Thursday, November 3, 2011
Persisters are small subpopulation of bacteria that are neither killed nor grown in the presence of antibiotics. Features of persister bacteria have already been discussed previously.
Integrating the features of persistence and bacterial senescence, Klapper et al. (2007) proposed that persisters are senescent bacteria. Their proposal is based on the model of bacterial senescence put forward by Stewart et al. (2005). As per the model, mother cells undergo gradual aging and have a reduced growth rate and finally stop dividing, whereas the daughter cell produced from a mother cell is a rejuvenated offspring capable of faster growth. Klapper et al. (2007) also made an assumption that the older cells are more tolerant to antibiotics than the younger cells due to their slow growth rate.
Thus, as per Klapper et al. (2007), when a bacterial culture is treated with bactericidal antibiotics, the younger cells are killed due to their fast growth rate whereas the older mother cells (persisters) survive. However, upon removal of antibiotics, the rejuvenated offspring produced from the mother cells quickly repopulate the culture. Since, during the exponential phase, the number of older cells is low, there may not be many survivors when antibiotics are used against exponential phase bacteria. Thus, Kappler et al. (2005) argued that senescence can explain all the features of persister cells. Their argument would have been correct if the current model of bacterial aging were true. However, the current model of bacterial aging may not be complete as I discussed previously.
One drawback with Stewart et al. (2005) model of aging is that it cannot explain WHY the presence of mother cells in a bacterial culture is advantageous to the whole population. As per this model, the older mother cells also give rise to rejuvenated offspring. If their model is correct, senescence is disadvantageous only to the individual mother cell but is advantageous to the population because old mother cells not only can give rise to rejuvenated offspring but also are resistant to antibiotics.
Stewart et al. (2005). Aging and death in an organism that reproduces by morphologically symmetric division. PLoS Biol 3(2), e45.
Klapper et al. (2007). Senescence can explain microbial persistence. Microbiology 153(Pt 11), 3623-30.