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.

Why both slow and fast dividing subpopulations of bacteria are missed during routine culture?

During routine culture, the number of bacteria on solid agar medium that can form distinct colonies, separated from each other are usually around 100. Among 100 colonies, we may not notice slow or fast dividing subpopulations.

It is easy to explain why we miss slow dividing bacteria during routine culture- their number is very small and they divide slowly. By the time these subpopulation of bacteria start to grow and form colonies, the normal bacterial population would have already grown and covered the agar. Hence, to isolate those subpopulations of small colony variats (SCV), the normally dividing population needs to be eliminated, which can be done with the help of aminoglycosides (see the blogpost on Oct.5).

On the other hand, one would expect to notice fast dividing subpopulation more frequently. Even if their initial number is low, one would expect them to gradually dominate the population since their growth rate is higher than the normal. However, this may not happen if this fast dividing subpopulation undergoes senescence. As they divide, the growth rate of mother cell gradually reduces and becomes comparable to the normal population whereas the daughter cell may be a rejuvenated offspring. It may be due to the mother cell undergoing senescence that the fast dividing subpopulation does not dominate the culture. However, the fast dividing subpopulation can be selected by removing the normal population by repeatedly growing the culture in early exponential phase (as described in the previous blog on Sep.19).

Thus, bacterial senescence can explain why the fast dividing, hypervirulent subpopulation of bacteria that can be isolated by repeatedly growing the culture in early exponential phase does not dominate the whole population even if they have the growth advantage over the normal population.

Next- Shifting bacterial population distribution to the right or left

Experiment that could indicate that SCVs are senescent bacteria

One of the markers of senescence is the accumulation of oxidative carbonylated proteins. Cells of an E. coli population show asymmetry not only with respect to growth rate, but also with respect to protein oxidation levels (Desnues et al. 2003; Aguilaniu et al. 2003). An E. coli population consists of relatively low damaged daughter cells (low protein oxidation) that are reproductively competent and damaged mother cells with reduced reproductive ability (Desnues et al. 2003). In exponentially growing E. coli, the amount of protein aggregates increases over time and were found to be more prevalent in dead cells than in culturable cells (Maisonneuve et al. 2008a). Similarly, aggregated proteins accumulate in cells with older poles, which are associated with a decrease in reproductive ability (Lindner et al. 2008).

In the earlier blog, I had hypothesized that small colony variants of E. coli isolated using subinhibitory concentrations of aminoglycosides are senescent bacteria which are hypovirulent, slow dividing and form small colonies on solid medium. Measuring the levels of protein carbonylation can give an indication whether they are senescent bacteria. If they are senescent bacteria, one can expect the protein carbonylation levels to be high. As far as I know, level of protein carbonylation in small colony variants has never been measured (a search of “small colony variants” and protein carbonylation returned only two results in Google scholar).

However, increased protein carbonylation may not be conclusive evidence that SCVs are replicative senescent bacteria. Increased carbonylation can be a feature of both conditional and replicative senescence. Hence further research may be required to differentiate between these two. In fact, there is possibility that mutants that form SCVs (like hemin, menadione or thiamine mutants) may also show increased carbonylation which may be due to conditional senescence and not replicative senescence. Hence, I assume that both mutant SCVs and non-mutant SCVs may show increased protein carbonylation, the former due to conditional senescence and the latter due to replicative senescence.

If the yeast cells isolated by repetitively growing in early exponential phase have low number of bud scars (described earlier) and SCVs isolated using aminoglycosides have increased protein carbonylation levels, I can say with increased confidence that my model of bacterial aging is different from that proposed by Stewart et al. (2005) and that SCVs are senescent bacteria

Next- Why both slow and fast dividing subpopulation of bacteria are missed during routine culture?

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

Aguilaniu et al. (2003). Asymmetric inheritance of oxidatively damaged proteins during cytokinesis. Science 299(5613), 1751-3.

Desnues et al. (2003). Differential oxidative damage and expression of stress defence regulons in culturable and non-culturable Escherichia coli cells. EMBO Rep 4(4), 400-4.

Maisonneuve et al. (2008). Protein aggregates: an aging factor involved in cell death. J Bacteriol 190(18), 6070-5.

Lindner et al. (2008). Asymmetric segregation of protein aggregates is associated with cellular aging and rejuvenation. Proc Natl Acad Sci U S A 105(8), 3076-81.

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.

Experimental proof that the fast dividing, hypervirulent bacteria are young bacterial population

In the last blog, I assumed that the fast dividing, hypervirulent bacteria isolated after repeated growth in early exponential phase are young bacterial population. This can be confirmed by doing the same experiment in Saccharomyces cerevisiae.

When yeast cells undergo replication by budding, a bud scar is left behind on mother cell’s surface. Bud scars remain permanently deposited on the surface and get accumulated as mother cells undergo more divisions. Thus, an old mother cell will have more number of bud scars whereas the number will be less in relatively young mother cells. Thus, the bud scars are used as a marker for the number of divisions a cell had undergone or the budding index. The budding index can be calculated by analyzing the cell wall for bud scars using confocal microscopy.

For the experiment, when a normally dividing yeast culture reaches an O.D. of 0.3-0.5, 100 ul of the culture should be withdrawn and added to 3 ml of fresh culture medium and incubated for growth. This process should be repeated 3-4 times. At the end of the fourth cycle, when yeast cells remain in early exponential phase itself, the budding index should be calculated. This can be compared with a yeast culture in one-time exponential phase and also with a culture in stationary phase. If the cells obtained after repeated culturing in early exponential phase are indeed young yeast cells, the number of bud scars will be lower than that in other two.

If they are found to be young yeast cells, the same can be true for bacteria also.

Next- Isolation of slow dividing, small colony forming, hypovirulent, senescent bacteria

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

Population distribution of an E. coli colony- comparison with Stewart et al. (2005) model

As per Stewart et al. (2005) (discussed in the last blog), the daughter cell formed from a mother cell is a rejuvenated offspring with full reproductive potential. So, if we designate x for virgin cells, x+1 for cells that have undergone 1 division, x+2 for those undergone 2 divisions and so on, then approximately 50% of a normally dividing  population in a colony of bacteria will be virgin cells (x), 25% will be x+1, 12.5% will be x+2 and so on. Hence if we plot the percentage of different populations on a graph, a half-bell curve distribution will be obtained. However, it may not be as perfect as one below as the growth rate of all cells are not the same (mother cells have reduced growth rate as it undergoes senescence).

However, my model of E. coli senescence is closer to that of S. pombe senescence (discussed on September 6 blogpost). In this model, the old cells do not give rise to rejuvenated offspring, but generate old cells itself. However, majority of the cells are relatively young cells with limited damaged or carbonylated proteins and this pool of young cells are large enough to prevent the extinction of the population. Another important feature in my model is the presence of a small population of virgin cells with no damaged or carbonylated proteins. Thus a major difference between Stewart et al. (2005) model and my model is that in the former, majority of the population are virgin cells (rejuvenated offspring) whereas in the latter, majority of the population are young bacteria with limited carbonylated proteins, along with a small population of virgin cells.

Thus, the population distribution in my model follows a normal distribution curve. However, it may not be a perfect bell shaped curve but can be skewed in favor of young cells. A bell-shaped curve indicates that there are small populations of fast-dividing young bacterial cells as well as slow-dividing senescent cells in any bacterial colony.

Is it possible to isolate any of these small populations? The answer is yes – both young cells and old cells can be isolated. In the next few sections, I will discus how to isolate these two populations separately.

Next- Isolation of fast dividing, hypervirulent, young bacteria 

Stewart et al. (2005) model of replicative senescence in E.coli

Perhaps, the earliest evidence of replicative senescence in bacteria was provided by Liu (1999). By tracking the bacterial growth in liquid media with high viscosity, Liu (1999) observed the unidirectional growth and reproduction of E. coli. He proposed that the bacterium has an intrinsic cell polarity with one end behaving as a mother compartment and the other end as the daughter compartment resulting in the formation of two bacteria of succeeding generations. His model defined bacterial age by its experienced chronological time. Based on this model, he predicted that, on bacterial division, the old strand of DNA remain with the mother bacterium whereas the new strand goes to the daughter bacterium and that this distribution of old and new strands of DNA between the mother and daughter cells is responsible for the intrinsic differences between the two.

Later, Stewart et al. (2005) studied the senescence in E. coli using automated time lapse microscopy by following repeated cycles of reproduction. They followed individual exponentially growing cells up to nine generations of growth and reproduction. Their findings were comparable to those reported previously by Liu (1999). The bacterium exhibits cell polarities which give rise to an old pole with a reduced growth rate and a new pole with higher growth rate. They found that the average growth rate of old pole cells was 2.2% slower than that of new pole cells and that the new pole cells were larger and divided sooner than the old pole cells. In addition, the old pole cells were also more likely to die than the new pole cells. They concluded that the two apparently identical cells are functionally asymmetrical, with the old pole cell behaving as the aging mother cell and the new pole cell as the rejuvenated offspring.

Thus, according to these models, bacteria undergo aging and that 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.

Next- Population distribution of an E. coli colony- a comparison with Stewart et al. (2005) model

Liu, S. V. (1999). Tracking bacterial growth in liquid media and a new bacterial life model. Science in China 42, 644-654.

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

Replicative senescence in Caulobacter crescentus

Replicative senescence has been reported in Caulobacter crescentus, a bacterium in which cytokinesis is intrinsically asymmetrical. The bacterium can freely swim in water and this free swimming cell represents the swarmer cell. The swarmer cell is non-reproductive, but after a period of free swimming, it gets differentiated to a sessile reproductive stalked cell. The stalked cell remains attached to the substrate but produces progeny swarmer cells which then separates from the stalked cells and begin free swimming.

Ackerman et al. (2003) studied the replicative senescence in Caulobacter crescentus by using microscopy flow chambers in which the stalked cells were attached to the chamber while the swarmer cells produced from the stalked cells were removed by the medium flowing through the chamber. They noticed that, as the stalked cells gave rise to more progenies, their rate of division slowed and finally stopped completely. However, the progenies produced, i.e. the swarmer cells, were rejuvenated offspring. Moreover, those progenies produced towards the end were indistinguishable from any of the young cells produced earlier in the experiment in terms of cell division time. These experiments proved that the stalked cells undergo senescence and that their reproductive output decreases with increasing aging. However, the major difference was that, unlike S. cerevisiae, S. pombe, or D. melanogaster, the progenies from both the older and younger mother cells were rejuvenated offspring and indistinguishable from each other. i.e. older bacterium produces only young bacterium.

Next- Stewart et al. (2005) model of replicative senescence in E.coli

Ackermann et al. (2003). Senescence in a bacterium with asymmetric division. Science 300(5627), 1920.

Replicative senescence in Schizosaccharomyces pombe

Unlike Saccharomyces cerevisiae which divides by budding, Schizosaccharomyces pombe (S. pombe) divides by binary fission and hence named as fission yeast. They grow by elongation at their ends and divide by medial fission to produce two daughter cells and in this sense, it undergo morphologically symmetric division (however, the division is not symmetric in all aspects).

Minois et al. (2006) studied the replicative senescence of S. pombe by measuring the occurrence and intensity of protein carbonylation (a marker of senescence) in single and symmetrically dividing cells of S. pombe. In S. cerevisiae, cabonylated proteins are segregated unevenly between mother and daughter cells during cytokinesis, with most of the carbonylated proteins retained by the mother cell. However, in S. pombe, proteins are not segregated to one half of the cell but are shared between the two cells. They found that carbonylated proteins are never segregated to mother cell alone but are shared between the two. However, with increase in carbonylation, they are shared less equally. This pattern of sharing is opposite to that of S. cerevisiae. This indicates that in S. pombe, damaged cells give rise to damaged cells itself. In a population, most of the cells have only low levels of carbonylation whereas a few of the cells show high levels of carbonylation which is consistent with senescence model of S. cerevisiae in which a gradual accumulation of carbonylated proteins occurs as the cells undergo aging. Cells with high levels of carbonylation exhibit lower fitness and are less likely to divide. However, since majority of the cells have only limited carbonylated proteins, this indicate that the pool of young cells are large enough to prevent the extinction of the population.

Thus, Minois et al. (2006) have shown that cells of S. pombe also undergo aging. However, unlike S. cerevisiae, the carbonylated proteins are shared between the mother and the daughter cells and hence the old cells of S. pombe generate old cells itself.

My model of E.coli senescence is almost similar to that of S. pombe. This will be discussed later.

Next- Replicative senescence in Caulobacter crescentus

Minois et al. (2006). Symmetrically dividing cells of the fission yeast Schizosaccharomyces pombe do age. Biogerontology 7:261-267.

Replicative senescence in Saccharomyces cerevisiae

Kennedy et al. (1994) studied the replicative aging of the budding yeast S. cerevisiae by microscopically following the mother cells through a number of cell divisions. They noticed that the mother cell underwent a finite number of divisions and that the size of both the mother cell and the daughter cell increased with age. They also noticed that older mother cells underwent a more symmetrical division in which the daughter cell that budded from the mother cell was almost the same size as the mother cell at division. Similarly, the daughter cells of older mother cells had a shorter lifespan than those of younger mother cells. The daughter cells of older mother cells in the last 10% of their lifespan was found to undergo only 7.9 divisions, whereas those daughter cells budded from the mother cells in the first 70% of the lifespan, on an average, divided 26.5 times. Symmetrical division, therefore, does not give any advantage to the daughter cells as they undergo a smaller number of divisions than those derived from younger mother cell.

Aging is associated with the accumulation of damaged proteins, genetic materials or dysfunctional mitochondria. In S. cerevisiae, the levels of protein oxidation increase with the replicative age of mother cells (Aguilaniu et al. 2003). Oxidized proteins are unevenly distributed between the mother and daughter cells during cytokinesis, with mother cells retaining most of the oxidized proteins. However, with increasing age, mother cells lose their ability to retain the oxidized proteins (Aguilaniu et al. 2003). Thus, the amount of oxidized proteins gradually increases in daughter cells as the mother cell undergoes aging.

What happens to the progeny produced from symmetrical daughters? Even though the lifespan of the daughters arising from symmetric divisions is reduced, daughters formed from the daughters of old mother cells are restored back to normal (Kennedy et al. 1994). In other words, progeny from symmetrical daughters recover full life span potential following asymmetric division, indicating that the decrease in the lifespan in daughters of old mother cells is not heritable. Thus the damages accumulated in older mother cells may be diluted in subsequent generations.

Next- Replicative senescence in Schizosaccharomyces pombe

Aguilaniu et al. (2003). Asymmetric inheritance of oxidatively damaged proteins during cytokinesis. Science 299(5613), 1751-3.

Kennedy et al. (1994). Daughter cells of Saccharomyces cerevisiae from old mothers display a reduced life span. J Cell Biol 127(6 Pt 2), 1985-93.

Aging/senescence- introduction

Despite the obvious disadvantages of aging, it is not opposed by natural selection probably because it can be beneficial to the species by avoiding overcrowding and promoting further evolution, thus increasing the fitness of subsequent generations. There are many theories of aging which can be broadly divided into stochastic and non-stochastic theories of aging. The former views aging as a phenomenon resulting from random events leading to cellular damage whereas the latter considers aging a programmed or a predetermined phenomenon occurring in all organisms within a particular time-frame.

Senescence can be conditional or replicative. When a bacterial culture is exposed to stressful conditions such as starvation for prolonged periods, they gradually accumulate oxidative damages and their ability to recover from the damages may also be lost and the cells may finally undergo death. This conditional senescence (Nystrom 2003) is different from the replicative senescence in that the latter results from the sequential loss of fitness following multiple rounds of replication.

Whereas aging is visible and very evident in higher eukaryotic organisms, it is also observable in simple eukaryotes like the fruitfly Drosophila or yeast Saccharomyces. However, senescence in prokaryotes like bacteria was not known till recently. For more than a century, bacteria were considered to be functionally immortal organisms because of their symmetrical division. It was believed that a parent bacterium split symmetrically into two equal daughter cells and that both the cells receive damaged and new cellular constituents equally. Thus, bacteria were considered to be organisms immune to natural aging and death, even though they can be killed by many external agents like starvation or other stressors.

Before moving into bacterial senescence, I will discuss the aging model in simple eukaryotes.

Next- Replicative senescence in Saccharomyces cerevisiae

Nystrom, T. (2003). Conditional senescence in bacteria: death of the immortals. Mol Microbiol 48(1), 17-23

Small colony variants- concluding remarks

Small colony variants (SCVs) can be isolated in vitro and in vivo under a variety of conditions, including antibiotic pressure. Various reports indicate that they are electron-deficient mutants. However, many SCVs may not revert in the presence of hemin, menadione, thiamine or thymidine and thus may not be auxotrophic mutants. It is also not known whether SCVs can revert to normal colony forms in vivo. Even though they can be isolated from many chronic infections, their role in causing these infections has not been proven conclusively. In most of the reported cases, SCVs were co-cultured with normal colony forms. Since there are no indications that the large colony forms are the revertants of SCVs, chronic infections could be due to the normal colony forms only that have persisted under those conditions. SCVs may not be responsible for fatal infections; reports associating SCVs with fatal infections are highly questionable. SCVs can be part of the normal life cycle of bacteria and may not have much clinical significance. However, the isolation of SCVs in vivo following antibiotic therapy may be indicative of the failure of antibiotics to reach throughout the site of infection at optimal concentrations, which may result in the survival of some normal bacteria and the selection of SCVs. The surviving normal bacteria may re-grow once the antibiotic pressure is removed, whereas SCVs may not have much role and may remain without causing infection since they are less virulent.

My blogs on SCVs are ending here. Next, I will start with senescent bacteria and bacterial aging model

Intracellular persistence of small colony variants

It has been suggested that small colony variants (SCVs) can persist intracellularly and are protected against antibiotics and host innate defense system which may contribute to chronic infections (von Eiff et al. 2001). They may produce low levels of alpha-toxins which may help in the intracellular persistence by preventing cell lysis or apoptosis.

Tuchscherr et al. (2010) investigated the infection of endothelial cells with highly virulent wild type isolates and isogenic SCVs of S. aureus. They found that wild type bacteria upregulated the expression of a number of endothelial genes and proteins whereas SCVs upregulated only a few of them. Similarly, the levels of chemokine release after 3 days of infection was much higher with wild type cells when compared to SCVs. The data from the above article indicates that SCVs are less virulent when compared to wild type. They may be better in avoiding the host innate defense system and hence may persist inside the cells. Since they divide slowly, they may produce fewer products that activate host cell responses.

However, whether SCVs are adapted phenotypes that can cause chronic infections is unproven. In fact, previous experiments with animal models have also shown that SCVs are less virulent than their wild type counterparts. But whether they can occasionally give rise to normal wild type bacteria through in vivo reversion has not been demonstrated even though such reversion has been shown in vitro.

von Eiff et al. (2001). Intracellular persistence of Staphylococcus aureus small-colony variants within keratinocytes: a cause for antibiotic treatment failure in a patient with darier's disease. Clin Infect Dis 32(11), 1643-7.

Tuchscherr et al. (2010). Staphylococcus aureus small colony variants are adapted phenotypes for intracellular persistence. Journal of Infectious Diseases 202(7): 1031-1040

Are small colony variants responsible for fatal infections?

There are a few reports suggesting that small colony variants (SCVs) may be responsible for fatal infections. Some are given below.

1. Haussler et al. (2003). Fatal outcome of lung transplantation in cystic fibrosis patients due to small-colony variants of the Burkholderia cepacia complex. Eur J Clin Microbiol Infect Dis 22(4), 249-53.

2. Seifert et al. (1999). Fatal case due to methicillin-resistant Staphylococcus aureus small colony variants in an AIDS patient. Emerg Infect Dis 5(3), 450-3.

3. Adler et al. (2003). Emergence of a teicoplanin-resistant small colony variant of Staphylococcus epidermidis during vancomycin therapy. Eur J Clin Microbiol Infect Dis 22(12), 746-8.

In all these cases, it can be noted that the patients had long and complicated clinical histories and had received antibiotic therapy for a long time. For example, in the first case, the patients had severe lung diseases and had undergone lung transplantation whereas in the second article, it was an AIDS patient who had a traffic accident. Similarly, in the third case, the patient was undergoing treatment for acute myeloid leukemia. Antibiotic therapy might have selected SCVs, and both large and small colony forms were cultured in all cases. However, there are no indications that SCVs are responsible for the fatal infections in any of the above cases. Just because SCVs were isolated from these patients, how can it be suggested that they are responsible for fatal infection especially considering their severe and complicated clinical histories?

Are small colony variants responsible for chronic infections?

Researchers propose that during unfavorable conditions, especially antibiotic therapy, small colony variants (SCVs) are selected due to their ability to tolerate antibiotics. This is considered to be a survival strategy of bacteria to overcome adverse conditions. Upon the removal of unfavorable conditions, the SCVs revert to normal growth and cause re-infection. Moreover, they have enhanced capacity to persist intracellularly which may protect them from antibodies and complements. In many chronic infections, SCVs are isolated in large numbers. Isolation of SCVs from osteomyelitis and cystic fibrosis supports a pathogenic role for SCVs in such chronic diseases. In these cases, both large colonies and SCVs are isolated and researchers have shown that both colony types are clonal indicating a common origin. Thus, it is proposed that the normal large colonies are the revertants of SCVs.

Even though both the colony types might have originated from the common ancestor, however, there is no indication that the large colonies are truly the revertants of SCVs. In fact, there are no clear data that have proven the reversion of SCVs to large colony types in vivo. The isolation of both large and small colony types from such infections only indicates that, antibiotics are unable to kill all bacteria. Since some of the normal bacteria also survive, they may result in re-infection later. Thus, researchers have given undue importance to SCVs assuming that normal colonies are reverted from SCVs.

The fundamental flaw here- the assumption that antibiotics are capable of killing all normal bacteria under such chronic infections. (It is well established that antibiotics may not be able to kill all bacteria in biofilms or in other pathological conditions resulting in cystic fibrosis or chronic osteomyelitis). However, the reversion of SCVs to normal types in vitro made researchers to assume that the normal colonies found in vivo are also the revertants of SCVs.

Next- Are SCVs responsible for fatal infections?

The switching mechanism between the wild type and small colony variants as proposed by Massey et al. (2001) is not convincing

Massey et al. (2001) proposed that small colony variants (SCVs) could emerge by switching from the wild type and vice versa. They found that SCVs of S. aureus could be isolated after just 30 min of exposure to gentamicin. Their number increased as the exposure time increased and reached a maximum by 14 h, but subsequently declined due to the emergence and overgrowth of gentamicin-resistant wild type bacteria. The authors hypothesized that the increase in the frequency of SCVs after gentamicin treatment for the first 14 h was either due to the very short generation time of SCVs or due to the switching from wild type to SCVs. 

To test which of the above hypotheses was responsible for the increase in SCV numbers, the mean generation time of SCVs and the wild type population was compared. The former hypothesis was ruled out since the actual mean generation time was found to be much higher than the wild type. Since the first hypothesis was wrong, they studied whether the increase in the frequency of SCVs was due to their presence in the inoculum. For this purpose, they reduced the initial inoculum size of bacteria by 100-fold and calculated the percentage of SCVs. They found that the proportion of SCVs at 24 h increased when the initial inoculum size was reduced. They calculated that, if the emergence of SCVs actually depended on the initial inoculum, their number should have reduced at 24 h at low inoculum. Since they found an increased percentage of SCVs, it was concluded that the emergence of SCVs was not dependent on its initial numbers, but was due to the switching from the wild type bacteria.

However, the increase in the number of SCVs at a low inoculum size may not be due to the switching of wild type bacteria. Until 14 h (in the above experiment), the increase in the population of SCVs could be due to the selective killing of normal bacteria along with the gradual multiplication of SCVs. But, at a high inoculum size, antibiotics may not kill all normal bacteria. Some bacteria may escape killing and may remain dormant for a short period of time but later may undergo adaptation and re-grow and overcome the SCV population. On the other hand, at a low inoculum size, most of the normal bacteria get killed and thus the percentage of SCVs may increase. The switching mechanism proposed by Massey et al. (2001) could have resulted from the selective and complete killing of normal bacteria at a lower inoculum size, resulting in the selective multiplication and the increase in the number of SCVs.

The fundamental flaw with the above article is that they could find only two hypotheses for their results and assumed that if one of them is wrong, other should be correct. What, if there are more than two reasons or explanations for their results?

Massey, R. C., Buckling, A., and Peacock, S. J. (2001). Phenotypic switching of antibiotic resistance circumvents permanent costs in Staphylococcus aureus. Curr Biol 11(22), 1810-4.

Is SCV generation a survival strategy of bacteria?

Most of the SCVs are reported to be hemin, menadione, thymidine or thiamine auxotrophs. It is argued that SCV generation is a survival strategy of bacteria to resist adverse conditions, especially antibiotic therapy. Thus, in the presence of aminoglycoside antibiotics, only SCVs may survive whereas the normal wild type population gets killed by the antibiotic. However, once the antibiotic is removed, SCVs can revert to normal wild type and cause re-infection.

However, if the formation of SCV is a survival strategy, why do high frequencies of hemin-deficient mutants occur among the Enterobacteriacae family? It is documented that Enterobacteriacae lacks the ability to take up hemin (Sasarman et al. 1968). To revert to the normal wild type, it needs a second independent mutation that helps it take up hemin (Roggenkamp et al. 1998). However, the frequency of this second mutation is very low (Roggenkamp et al. 1998). This would mean that the hemin-deficient mutants of Enterobacteriacae will remain as SCVs even in the presence of hemin, thus offering them no growth advantages. If reversion is not possible, how it can be argued that SCVs are responsible for chronic infections?

Similarly, if the reversion to normal wild type is not possible, what is the fate of those Enterobacteriacae SCVs inside the body?

Next- A switching mechanism between the normal bacterial population and SCVs

Sasarman et al. (1968). Hemin-deficient mutants of Escherichia coli K-12. J Bacteriol 96(2), 570-2.

Roggenkamp et al. (1998). Chronic prosthetic hip infection caused by a small-colony variant of Escherichia coli. J Clin Microbiol 36(9), 2530-4.

Small colony variants- a survival strategy of bacteria: literature review

The formation of small colony variants (SCVs) is considered as a survival strategy of bacteria to evade antibiotic killing. In the presence of antibiotics, especially aminoglycosides, SCVs may survive due to reduced uptake of the antibiotic when most of the bacteria get killed. Thus, within a host, antibiotic pressure may select electron transport deficient mutants which may survive intracellularly due to low levels of free hemin and menadione within the host cell (McNamara and Proctor 2000). Since the intracellular milieu protects bacteria from antibodies, complements and many antibiotics, increased intracellular persistence of SCVs could be a survival strategy of S. aureus. However, once the antibiotic is removed, SCVs may revert to normal phenotype resulting in chronic infections.

In many clinical cases of chronic infections, both the SCVs and large colony types have been isolated. Researchers have shown that both colony types are clonal indicating a common origin. Isolation of SCVs from osteomyelitis and cystic fibrosis supports a pathogenic role for SCVs in such diseases (Proctor et al. 2006). SCVs have also been isolated from device-related infections, persistent wound infections and persistent bovine mastitis.

SCVs are implicated not only in chronic infections, but also in fatal infections. For example, a fatal infection due to SCVs of methicillin-resistant S. aureus in a patient with AIDS has been reported (Seifert et al. 1999). Similarly, SCVs of the Burkholderia cepacia complex has been reported to be responsible for the fatal outcome of lung transplantation in CF patients (Haussler et al. 2003).

Next- Is SCV generation a survival strategy of 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.

McNamara, P. J., and Proctor, R. A. (2000). Staphylococcus aureus small colony variants, electron transport and persistent infections. Int J Antimicrob Agents 14(2), 117-22.

Seifert et al. (1999). Fatal case due to methicillin-resistant Staphylococcus aureus small colony variants in an AIDS patient. Emerg Infect Dis 5(3), 450-3.

Haussler et al. (2003). Fatal outcome of lung transplantation in cystic fibrosis patients due to small-colony variants of the Burkholderia cepacia complex. Eur J Clin Microbiol Infect Dis 22(4), 249-53.