Final blog - a timeline of my findings

The reasons why I think that some of the concepts on bacterial growth and aging needs to change stems from the publications given below (in the order of time)

1. Persisters show heritable phenotype and generate bacterial heterogeneity and noise in protein expression (http://precedings.nature.com/documents/1411/version/2)

2. Senescent bacteria as potential live vaccine ( https://docs.google.com/viewer?a=v&pid=explorer&chrome=true&srcid=0B0-RiF-cW8KcNGJiOWMxNzUtMzdlYy00MzVlLThiOTUtOGY1Y2RmMWFiZTI4&hl=en_US)

3.  The systemic practice of misinterpretation of scientific data (http://www.universal-publishers.com/book.php?method=ISBN&book=1599428202)

4. The complete and unequivocal failure of climate hypothesis in cholera outbreaks: The case of Haiti and beyond  (https://docs.google.com/open?id=0B3eiZyPajPniMzdhMjAwZDEtYzY5Yi00YTRhLTkyYmItYzc1YjU3NGJlMjYy )

This blog is currently ending here....

The complete and unequivocal failure of climate hypothesis in the spread of cholera

Even though I am not a climate change skeptic, I have reservations on some of the research findings pertaining to the role of climate change in the spread of diseases. Some researchers argue that the global warming is one of the main reasons for the spreading of diseases like cholera or malaria. When cholera outbreak occurred in Haiti in October 2010, researchers were quick to suggest that extreme climatic patterns like La Nina was responsible for the outbreak. However, later research findings proved that climate or environment did not have any role in Haiti.

I feel that scientists and IPCC has to do more in their fight to reverse climate change. However, exaggerating the role of climate in cases like above will only question the validity of science and give plenty of room for skeptics to criticize research on climate change. My aim is to point out certain flaws in current research hypothesis so that necessary corrections can be made. My report can be read at

 https://docs.google.com/open?id=0B3eiZyPajPniMzdhMjAwZDEtYzY5Yi00YTRhLTkyYmItYzc1YjU3NGJlMjYy

Senescent bacteria as potential live vaccines

In 2008, I submitted a project to the Round 2 of Grand Challenges Exploration funded by Bill and Melinda Gates Foundation. This was one of the projects selected by the reviewers amongst 3000 application received by them. However, it failed to obtain funding after the due diligent process, the most probable reason being the lack of affiliation with any research institutes at the time.

In this project, I had made an assumption. i.e. the small colony variants obtained after incubating with sub-inhibitory concentrations of aminoglycosides are senescent bacteria (discussed previously).

The project can be read at https://docs.google.com/viewer?a=v&pid=explorer&chrome=true&srcid=0B0-RiF-cW8KcNGJiOWMxNzUtMzdlYy00MzVlLThiOTUtOGY1Y2RmMWFiZTI4&hl=en_US

Till now, the project has not been tested.

What is stationary phase of bacterial growth?

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

Senescent bacteria and persisters

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.

My model of bacterial senescence

The current model of bacterial senescence proposed by Stewart et al. (2005) suggests that as bacteria undergo aging, the growth rate of mother cell decreases with age whereas the daughter cell produced from the mother cell is a rejuvenated offspring with high growth rate which helps to maintain the bacterial lineage. In this case, mother cell retains the damaged proteins to itself whereas daughter cells are spared from accumulating damaged proteins.

In my model of bacterial senescence, different subpopulations of bacteria are considered. A bacterial colony comprises many subpopulations which may exhibit different growth rates. These subpopulations can be broadly divided into three - a small subpopulation of fast dividing young bacteria, a major population with intermediate growth rate and a small subpopulation of slow dividing bacteria that are towards the terminal stages of senescence.

The young bacterial population divides fast and is hypervirulent and may not contain any damaged proteins. After many divisions, the mother cell may gradually accumulate some damaged proteins whereas the daughter cells are rejuvenated offsprings. Thus a small population of young virgin bacteria is always maintained.

However, the majority of the population in a colony exhibit intermediate growth rate. Since they comprise the major population, the growth rate of the colony will be similar to the growth rate exhibited by this population. They may carry some amount of damaged proteins which are not segregated to mother cell alone, but are also transferred to daughter cells. However, with increasing amount of damaged proteins in mother cells, the daughter cells will also accumulate more damaged proteins.

Towards the terminal stages of senescence, mother cells may lose their ability to retain the damaged proteins and hence may be shared almost equally with daughter cells. At this stage, mother cell will also give rise to senescent daughter cells. Since both mother and daughter cells are old cells and divide slowly, a single senescent cell gives rise to small colony.

In a population, majority of the cells have only limited carbonylated proteins. This, along with a subpopulation of virgin cells without any damaged proteins, help to maintain a pool of young cells that are large enough to prevent the extinction of the population.

Thus, my model of bacterial aging is close to that of Schizosaccharomyces pombe, the fission yeast (discussed in the blogpost on Sep.6). The similarity may not be surprising given that both bacteria and S. pombe divide by binary fission.

Next- Senescent bacteria and persisters

Stewart et al. (2005). Aging and death in an organism that reproduces by morphologically symmetric division. PLoS Biol 3(2), e45.

Shifting bacterial population distribution curve to the right or left

As per my model, a bacterial population in a colony is heterogeneous with respect to growth rate and age. There are small subpopulations of both young and senescent bacterial population in addition to the major population which have an intermediate growth rate. The growth rate of the whole population will be close to the intermediate population as they comprise the major population.

However, the bacterial population distribution curve can be shifted either towards the left or the right. It can be shifted towards the left by growing the bacterial culture in early exponential phase. In this case, the growth rate of the culture gradually increases as more young bacteria are selected (as described in the previous blog on Sep.19). However, this increased growth rate of the culture may not be a permanent feature since the bacteria undergo senescence. Thus, if the culture of young bacteria is allowed to grow and reach stationary phase, the growth rate may gradually decrease due to the gradual increase in senescent population. If 50 ul of this stationary phase culture is transferred to 3 ml of fresh medium and allowed to grow, the growth rate may further reduce. If this process is repeated, the growth rate may reach the initial rate.

On the other hand, the bacterial distribution curve can be shifted to right by incubating the bacterial culture with aminoglycosides which results in the selection of slow dividing senescent bacteria (see the blogpost on Oct.5). Whether they can be shifted back to normal depends upon the stage of senescence. Bacteria towards the terminal stage of senescence can not be reverted back and in this case it may not be possible to shift the growth rate to normal.

Next- My model of bacterial senescence.