Evolutionary Trade Off: A solution to anti-biotic resistance?

We all have heard of a saying, “That which doesn’t kill us makes us stronger” by Friedrich Nietzsche. This is especially true in case of bacteria, organisms that are found everywhere ranging from “on us” to “inside us”. these microscopic organisms are responsible for many of the most deadliest diseases to humankind. Advent of antibiotics provided relief as they were considered to be the best solution to the diseases caused by bacteria. however, on a longer run it was established that these organisms indeed develop resistance to the antibiotics making them invulnerable to the effects of antibiotics and making them more stronger in the process.

Antibiotic Resistance. Image Source: PHARMAC, NZ

This antibiotic resistance is the result of mutations that takes place in a bacterial population exposed to a specific drug leading to a evolved population which becomes impenetrable by the drug. The infections that are caused by these newly evolved strains of bacteria become very challenging to cure emanating longer stays in hospitals. This very evolution however, has opened channels to explore for solutions to the issue of antibiotic resistance as the phenomena of evolution which causes to build resistance against one type of drug might also result in developing hypersensitivity to others, thereby preventing multi-drug resistance. This strategy is termed as collateral sensitivity, which is a two way trade-off amounting to increased resistance to one drug and causing increased sensitivity to the other drug (Szybalski & Bryson, 1952).

Barbosa, Roemhild et al. attempted to exploit this aspect to decipher that evolving collateral sensitivity will ultimately slowdown the evolution of resistance by combination and therapies that are sequenced (Rodriguez De Evgrafov et al., 2015). Their biggest challenge was to determine the stability of their system such that at one point the bacterial population goes extinct or at least render them invulnerable to develop multi-drug resistance. In order to test their system they incorporated the bacterium Pseudomonas aeruginosa as it is believed to develop collateral sensitivity to to different drug treatments

The Experiment:

They subjected Pseudomonas aeruginosa to a two-step evolution, they utilized already evolved extremely resistant populations of P. aeruginosa, procured by serial passage experiments having increased concentrations of bactericidal antibiotics which were clinically relevant. They tested collateral sensitivity which would reciprocate i.e. the first target drug would create resistance while the second drug would show impend hypersensitivity on in first set-up and vice-versa. They also treated the bacteria by switching the antibiotics to collateral sensitivity but the administration of first drug remained continued along with the administration of the second drug hence they confirmed a constraint environment. Altogether, they laid down a total of four conditions which were running parallelly i.e. minimal or maximal increase of second drug with and without the presence of first drug. Simultaneously run control experiments i.e. without any antibiotic ensured treatment success. incorporation of quantification of extinct population frequency, absorbance measurements and characterization of changes amounting to antibiotic resistance of the evolving bacteria in comparison to what was previously observed in Pseudomonas aeruginosa further ensured the proofing of the experiment.

Authors conducted experiment to test stability in the evolution of reciprocal/reverse collateral sensitivity by exposing the clones from previous resistant populations with new set of antibiotics at high concentrations which resulted hypersensitivity in resistant populations. they performed a series of evolution experiments for 12 days following serial transfer protocol with beginning population size of approximately 106 CFU/ml. They evaluated each population in eight replicates and 5 treatment groups i.e.:

  1. Controls (without anti-biotic)
  2. Low level of increasing concentration of antibiotic (Unconstrained Evolution)
  3. High level of increasing concentration of antibiotic (Unconstrained Evolution)
  4. Low level of increasing concentration of antibiotic (Constrained Evolution)
  5. High level of increasing concentration of antibiotic (Constrained Evolution)

The authors also validated their findings by conducting repetitive evolution experiments by incorporating resistant population as the initial material. they used approximately 107 cells in contrast to a single clone, but reduced the treatments. In total they utilised 38 resistant populations.

Experimental design for testing collateral sensitivity
Image Source: (Barbosa et al., 2019)

Drug Combination:

  1. (PIT) Piperacillin/tazobactam and (STR) streptomycin
  2. (CAR) Carbenicillin and (GEN) Gentamicin

The authors in their experiment found that it is highly circumstantial that collateral sensitivity could be exploited for sequence based treatments as it validity is dependent on the combination of drugs used and also the order in which they are used, they also found that epistatic genetic interactions also play a role in their selection. The increased extinction rates determines that bacterial adaptation was constrained in treatments when a switch to ß-lactam was made. This effect was maximised when the second drug was administered in constraint environment. Their findings opened doors to the possibilities of further dwelling into deeper research of an unexplored strategy of treatment i.e. using single drug therapy after being treated with combination treatment. in this manner, the evolutionary trade-off of drug sensitivity could be maximised.

To be specific, the drug pair of CAR/GEN after deep analysis showed high extinction in comparison to growth improvements when given strong dose administration over mild dose. It is interesting to see how rate of extinction are often ignored and left unreported as a part of evolutionary outcome in corresponding studies (Yen & Papin, 2017). The authors are convinced upon the fact that, since antibiotic therapy is aimed towards attempt to eliminate bacterial pathogens, the extinction frequencies from dated experiments on evolution have shown variance depending upon different types of treatment and hence these deliberations could serve in order to refine the understanding of efficacy of the treatment.

In their experiment on CAR/GEN pair at clonal level demonstrated stability in collateral sensitivity as a result of slow adaptation and efficiency of re-sensitization. however, it was the negative epistasis of drug-specificity that re-sensitization was seen during switch from aminoglycoside to a ß-lactam. On the contrary the reciprocal collateral sensitivity test showed less stability as evident from low levels of extinction and absence of re-sensitisation. Few outcomes of their findings were unexplained with their current data set and the high instability could not be interpreted. Their work has drawn certainly drawn attention towards the need of careful evaluation of novel options of treatment.

Evolution is unique and inevitable, and so is the development of new methodologies to tackle the issue of drug-resistance. With many factors to play a role in achieving success to slow down the rate of drug-resistance or to completely eliminate the pathogenic bacteria, evolution amounting to collateral sensitivity has drawn attention towards designing a sustainable and stable approach of drug therapy to control infection in coalescence with traditional and other newly developed techniques.

Barbosa, C., Romhild, R., Rosenstiel, P., & Schulenburg, H. (2019, Oct 29). Evolutionary stability of collateral sensitivity to antibiotics in the model pathogen Pseudomonas aeruginosa. Elife, 8. https://doi.org/10.7554/eLife.51481

Rodriguez De Evgrafov, M., Gumpert, H., Munck, C., Thomsen, T. T., & Sommer, M. O. A. (2015). Collateral Resistance and Sensitivity Modulate Evolution of High-Level Resistance to Drug Combination Treatment in Staphylococcus aureus. Molecular Biology and Evolution, 32(5), 1175-1185. https://doi.org/10.1093/molbev/msv006

Szybalski, W., & Bryson, V. (1952). Genetic studies on microbial cross resistance to toxic agents. I. Cross resistance of Escherichia coli to fifteen antibiotics. Journal of bacteriology, 64(4), 489-499. https://doi.org/10.1128/JB.64.4.489-499.1952

Yen, P., & Papin, J. A. (2017). History of antibiotic adaptation influences microbial evolutionary dynamics during subsequent treatment. PLOS Biology, 15(8), e2001586. https://doi.org/10.1371/journal.pbio.2001586

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Why make it when you can take it?

I don’t know about you, but when I think of evolution I think of gaining the ability to do something. Just look at humans who live at high altitudes; they make a massive amount of red blood cells (among other things) so that they can breathe when the air is thinner. Sure the ability to breathe may not necessarily seem like a whole lot, we can all do it after all, but put a person who has adapted this ability at an altitude where the air is “normal” and step back and watch the magic. They don’t fatigue like the rest of us. Any aerobic athlete would KILL for this ability – look at Lance Armstrong, he ruined his reputation and cost himself 10 million dollars for the chance to exchange oxygen as efficiently as a Tibetan native. But what if adapting to a certain environment involves the loss of an ability? Not necessarily because the adaption is harmful, but because it is simply unnecessary. Athletes who train at high altitudes for just two weeks begin to adapt to life at low oxygen levels very quickly. Among these adaptions, the elusive increase in red blood cell count. Take these freshly adapted humans, put them back in life at sea level and there you have it, a super human ability for oxygen transport. But there’s a catch: these changes aren’t permanent. Want to maintain this wonderful ability to breathe super well? Back up the mountain you go! 

If you’re anything like me, you’re probably wondering why this happens. It seems to me that this specific adaption is really, really helpful. So why would we lose the ability to keep an abnormally high red blood cell count when it so clearly increases our fitness (both literally and evolutionarily). For this I have one simple answer: it’s just not an ability we need to have. The average human doesn’t have an evolutionary need to run a fast 10K, we just need to be able to be fit to survive long enough to reproduce. And the effort it takes our body’s to make these extra cells isn’t worth the benefit that we get from it. Evolution isn’t about being exceptionally good at living – it’s just about being able to survive. 

Personally, this came as a bit of a surprise to me. We are so used to associating evolution with natural selection and then in turn associating natural selection with the idea of the “survival of the fittest”. So what exactly is natural selection? How does it work and why have we not ended up with a super human population? 

Essentially the theory of natural selection is as follows:

  1. Traits, or characteristics are heritable. This means we get things from our parents. Eye colour, nose size, beak shape, short temper – all thanks to mum and dad.
  2. In any population (human, elephant, bacterial) certain individuals will inherit traits that make them better at reproducing and surviving than the rest of the population. These guys go on to have the most offspring.
  3. Because these good traits are heritable, they will pass them onto their children and because these traits allow them to have the most children, eventually these traits will become more common.
  4. Over generations, the original population will become adapted to its environment.

So basically natural selection doesn’t give us the fittest possible population available, it gives us the fittest population FOR THAT ENVIRONMENT. Rather than survival of the fittest we get survival of the least likely to die.

Let’s look at an example:

So if natural selection works to make sure that beneficial traits are passed on, we can assume that every trait we inherit from our parents is beneficial, right? Not quite. Natural selection isn’t the only evolutionary force in action, in fact some evolution happens purely by random chance. Now this may be a bit confusing seeing as I’ve just been going on about evolution ensuring that good traits get passed on and bad ones don’t. But there’s one little thing I didn’t mention about natural selection – it’s only really effective when we’re talking about large populations. Occasionally, a population can suddenly decrease in size. This can happen for a number of reasons: maybe a handful of individuals decide to move away from the rest to make their own new population, a predator might come in and eat all but a few or perhaps a natural disaster strikes. Either way, populations can suddenly go from huge to small, and when this sudden size decrease happens the individuals that remain are there purely by chance. This chance survival can mean that we end with a lot of individuals within this left over population that have traits we don’t necessarily want. And this random change in the prevalence of a certain trait in the population, due to “random population sampling” (i.e. taking only a small portion of the population) is called genetic drift.

If we refer back to the beetle colour example, natural selection would ensure that the population ends up being mostly green because the green beetles are the ones who are most likely to survive. But suppose one day massive rain fall caused a flood that killed 75% of the beetle population, with the remaining 25% surviving by chance and chance alone. The random survival of these individuals could mean that we end up with most of these surviving individuals being black rather than green, even though green is more advantageous. Then when these individuals reproduce, they will pass their colour trait down to their offspring and the population will remain mostly black in colour. So here we’ve ended up with a population that is mostly black, despite the fact that being black is actually a disadvantage. 

So how can we tell if a trait is present because it’s beneficial or because of random chance? This is where a little something called experimental evolution comes in. Experimental evolution allows scientist to use lab experiments or controlled field environments to explore the dynamics of evolution. This particular question was asked by a couple of scientists ( Glen D’Souza and Christian Kost) who noticed that bacteria sometimes lose the ability to make some nutrients and/or molecules that they need to survive. Bacteria are known for their ever-changing genome (think of a genome as a genetic blue print), so the loss and gain of genetic information is not a new thing for bacteria. But it does seem odd that they would lose the ability to carry out a task as important as making a molecule essential for their survival. So the question was asked: is the loss of this ability favoured by natural selection or does it occur as a result of random genetic drift?

D’Souza and Kost set out to answer this question by performing experimental evolution on a type of bacteria calledE.coli. Personally I think E.coli are underappreciate and misunderstood. Yea sure if the wrong strain populates your insides in the wrong place then they could kill you. But they’re not all out to get you, in fact we all contain a population ofE. coli in our gut that helps us break down and digest the food we eat. If that isn’t enough to change your mind then go and look at a video of them growing under a microscope – adorable. Plus, on top of all of that, their tiny genomes, super-fast reproductive rate and the fact that they’re super easy to manipulate make them the perfect model organism to perform evolutionary experiments on. 

D’Souza and Kost’s particular question of interest was to see whether or not E. coli would evolve to stop making amino acids (think of amino acids as the building blocks used to make proteins, and thus VERY essential) when they were provided by the environment. Firstly they looked at whether or not bacteria grow faster in an environment that lacks amino acids, compared to an environment that contains amino acids. They found that E.coli grown in the presence of amino acids reached a much greater cell density, or population size, than those that weren’t (figure 1A.). Remember how natural selection needs big populations to work? A larger population means it’s more likely that natural selection is actually happening and that any adaptions and mutations that happen are likely to be beneficial. 

So then the next question was – do these populations grow larger because of the increase in available nutrients? To determine this they subtracted the growth rate of populations in the presence of environmental amino acids from the growth rate of populations not in the presence of environmental amino acids and calculate the difference. If this number was a negative value it showed better growth in the presence of amino acids but if this number was positive it showed better growth in the absence of amino acids. If you look at the figure 1B you can see how these values over time. Initially the presence of amino acids was not the cause of increased growth rate, but over time that reversed. So by the time 2000 generations had passed, the E. coli grew far better when amino acids were available to them from the environment. So what does that mean? It means that over time the bacteria in this population have become dependent on the environmental amino acids for growth. 

A possible answer for this dependence is that the bacteria evolved amino acid auxotrophies. This means that they lost the ability to make these amino acids themselves and now rely on the environment to provide them. To confirm this, the genomes of 1000 bacterial colonies were analysed. The results were converted into a heat map (figure 2.) which shows the percentage of the population that had developed an amino acid auxotrophy along with how many generations it took them to obtain that auxotrophic mutation. Any square that is black shows no auxotrophies, any square that is pink or darker indicates auxotrophies. If you look at figure 2, you can see A and B – A shows populations grown in the presence of amino acids, B shows populations grown in the absence of amino acids. Both of these heat maps show pink squares – indicating auxotrophies evolve when grown in either environment (although more when amino acids were provided). Coupled with the large population sizes, these results suggest that the development of auxotrophs are in fact evolutionarily advantageous. 

So how can we confirm that having these auxotrophic mutations are advantageous? Well we can compare the fitness of the cells that don’t have them with the cells that do have them, which is exactly what D’Souza and Kost did. This figure below may look a little bit confusing, but essentially it shows the fitness of one strain compared to another in either of the two possible environments. The red boxes are auxotrophs, the bacteria that can no longer make certain amino acids, and the green boxes are bacteria that can make all of the amino acids themselves (these guys are called prototrophs). If the box sits higher up on the graph it has a greater fitness. So now looking at this figure we can see that when grown in the presence of amino acids, the auxotrophs have a greater fitness than the prototrophs, but when grown in the absence of amino acids this is reversed. The fitness of the phototrophs doesn’t really change at all. And this pattern was seen regardless of whether the strains evolved in the presence (A) or absence (B) of amino acids. 

It seems odd that these bacteria would evolve to lose the ability to make amino acids, even when there are no amino acids available from the environment right? Absolutely! But the thing is, the environment doesn’t just mean the physical place in which the bacteria grow. The fact that there was the development of auxotrophs when amino acids weren’t provided in the growth liquid shows that these bacteria were able to get the amino acids they needed from somewhere else. But where? Well not all the bacteria in these populations evolved into auxotrophs, some still retained the ability to make all the amino acids they needed on their own (prototrophs). So even when there are no amino acids obviously available in the growth liquid, the prototrophic cells that evolved with them provide a source of amino acids to the cells that need them. This is evident when looking at the how the fitness of the auxotrophs changes when they are assessed with the prototrophs.

So now we know that:

  1. Bacterial populations grow faster and when grown in a liquid media that contains extra nutrients (like amino acids)
  2. Over time bacteria evolve to become reliant on the nutrients provided by the environment
  3. This reliance is due to a loss in the ability to make the nutrients on their own (auxotrophs).
  4. When auxotrophs are grown in an environment that provides the nutrients they have a higher fitness levels than the cells that make all of these themselves.
  5. Auxotrophs can get the nutrients they need not only from the liquid their grown in but from the cells that can still make all the nutrients required.

There is one more important take away from this paper, and it involves something called negative frequency dependent selection. Sounds fancy and complicated right? It’s actually pretty simple. Basically it means that a certain trait is maintained at a low frequency because the more common it becomes in a population, the more its fitness decreases. So basically the trait is only beneficial when it’s not very common. It was determined that this is the case with these auxotrophic bacteria found in the population – they only had an increased fitness when they were present in low amounts. Why? Because the more auxotrophic bacteria there are in the population, the more the whole population relies on the environment to provide what they need. Think of the environment like a grocery store – the more people that need to buy groceries, the less there are available for everyone.  But if 75% of the population can grow their own vegetables at home then only 25% of the population needs to buy them. The less people that buy, the longer the stock lasts.

So there you have it, sometimes losing the ability to do something can actually increase the fitness of an individual. Yea sure making an essential nutrient is a good thing, but why make it when you can just take it?  

This blog was based on the following paper:

D’Souza, G., & Kost, C. (2016). Experimental evolution of metabolic dependency in bacteria. PLoS genetics12(11), e1006364.

Find it here: https://journals.plos.org/plosgenetics/articleid=10.1371/journal.pgen.1006364

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An experimental evolution-inspired step towards saving our planet

Do you every look up into the night sky of a big city in the hope to see stars, only to be disappointed by bleak smoggy darkness? Fossil fuel emissions are not pretty, and they certainly are not good for our environment. Annual Carbon Dioxide (CO2) emissions from fossil fuels have increased dramatically in the past decade and continue to rise rapidly. This has obvious negative health and environmental impacts such as accelerating global warming and deforestation.

Image URL: https://www.cbsnews.com/pictures/top-10-smoggiest-cities-in-us/

Therefore, we need to find alternative, more sustainable sources of energy so that we can reduce our carbon footprint for future generations. Of such, bioethanol is a renewable biofuel energy source that could meet our requirements as well as being environmentally sustainable [4]. Bioethanol is currently the most highly produced biofuel, meaning demand is high [4]. The process of bioethanol generation, called second generation bioethanol production, involves utilising cellulose and hemicellulose (plant biomass); the structural components of plants, into a renewable energy source called bioethanol [1].

So, you may be wondering why bioethanol, derived from plants, is so much better than fossil fuels? Well, in addition to being renewable and highly abundant, bioethanol has much lower CO2 emissions in comparison to fossil fuels. Also, the little amount of carbon dioxide that is released into the air by bioethanol use can be absorbed and utilised by the plants themselves, so that we can grow more plants and generate more biofuel. Hence the word “renewable”, It is an endless cycle of give and take, which is better for both us, our planet, and our environment.

The general process of second-generation bioethanol production involves pre-treating the plant biomass to weaken their strong structure provided by cellulose. This is followed by hydrolysis (breaking apart) of these cellulose structures into individual sugars [2]. Next, these sugars are fermented into bioethanol, which is assisted by a fermentable agent or microbe such as the commonly used yeast (S. cerevisiae) [2]. However, a problem arises when the hydrolysis step exceedingly releases acetic acid as a by-product. Yeast do not like acetic acid, it stops them from growing and being able to ferment ☹ [1]. In the big picture, this is bad because it means that bioethanol production yield will be reduced!

Expanding on this problem, experimental evolution has been an attractive approach for researchers to improve the efficacy of second-generation bioethanol production. Experimental evolution means that scientists can manipulate an environment experimentally and explore how this affects the evolution of a certain species [3]. In saying this, what if we could utilise experimental evolution to improve the efficacy of second-generation bioethanol production? A study that I will discuss in this blog post did just this, and the results were impressive!

González‑Ramos et al. (2016) used an experimental evolution approach to modify yeast to evolve a lasting tolerance to acetic acid, meaning these yeasts could thrive and survive in higher concentrations of acetic acid. This would allow the fermentation step of bioethanol production to be much more efficient and also improve the outcome of bioethanol production.

As a starting point, the researchers first tested whether prior exposure to acetic acid helps yeast to acquire tolerance. They did this by growing yeast either in the presence or absence of acetic acid (of a tolerable concentration), and then testing how the pre-adapted yeast cells grow, in comparison to non-adapted yeast cells, in acetic acid. To quantify these results, the researchers measured Final OD600 as an indicator of yeast biomass yield and were also able to determine specific growth rates (see Figure 1). So, as expected, both pre-adapted and non-adapted yeast cells were not very tolerant at all, as shown in Figure 1. There was a significant negative correlation between acetic acid concentration and growth of both pre-adapted and non-adapted yeasts. In saying this, the “pre-adapted” yeasts were slightly more resistant in concentrations greater than 10g/L, where they sustained slightly more survival (see Figure 1). Also, this tolerance of pre-adapted yeasts was lost after one exposure to non-stressed (low acetic acid concentration) conditions. Considering that the concentration of acetic acid can be more than 10g/L during bioethanol production, we want them to thrive in higher concentrations than this, and in a lasting manner, so this yeast has a fair way to go yet!

Figure 1: Growth and survival of either pre-adapted (grey lines) or non-adapted (black line) yeasts in gradually increasing acetic acid concentrations.

So here is where experimental evolution comes in…the researchers wanted to find a way to make these yeast able to thrive in and tolerate higher concentrations of acetic acid. To combat this, yeast cells were UV mutagenized (irradiating the cells with UV light to introduce mutations) and grown being transferred between conditions alternating from the absence and presence of acetic acid (of gradually increasing concentration); a technique called serial microaerobic transfer. Just like we train at the gym to build fitness, the yeast cells were training in the lab to build acetic acid tolerance. Over time, and approximately 50 transfers back and forth, these yeast cells were finally able to thrive in the higher acetic acid concentrations! They could even survive in 18g/L acetic acid, nearly twice the initial concentration that most yeasts could not survive in (Figure 2). Check out Figure 2 to see an example of the progress of one of these experimentally evolved yeasts with improved tolerance to acetic acid.

Figure 2: Acquired acetic acid tolerance of experimentally evolved yeast over serial microaerobic batch transfer. Acetic acid concentration is indicated by the grey line. Black dots are final OD600 measurements in the absence of acetic acid, while grey squares are final OD600 measurements in the presence of acetic acid.

 So, they experimentally evolved this yeast to be able to survive and grow in higher acetic acid concentrations, which is pretty cool (for a science nerd like me, anyways…), but the questions remain; what is going on? How is this happening? To approach this, researchers turned to genetics in the hopes to answer their questions. The research group used fancy scientific techniques allowing them to get a big archive of all the genes in these tolerant yeasts and compared these to the original non-tolerant yeast. This helped them to pinpoint a few culprits, mutated genes, for this acquired tolerance to acetic acid. They observed that many of these tolerant yeasts contained similar specific mutated genes (or, in ‘sciency’ terms; single nucleotide polymorphisms).

But what if this was just a crazy coincidence, and these mutated genes actually had nothing at all to do with this acquired acetic acid tolerance? To prove that this was not just a coincidence, and that these specific mutated genes were causal of this tolerance to acetic acid, the researchers carried out more fancy scientific techniques. They introduced each of these special mutated genes, individually, back into the non-tolerant yeast and… voila, these non-tolerant yeasts became tolerant to higher acetic acid concentrations! What is also cool is that the more of these mutated genes that were introduced back into the yeast, the more tolerant they became to high acetic acid concentrations (see Figure 3).

Figure 3: Growth of yeast with different variations of the identified mutant genes for acquired acetic acid tolerance.

In summary, using an experimental evolution approach, González‑Ramos et al. (2016) was able to genetically engineer yeast cells there were able to constitutively survive higher concentrations of acetic acid, which normally impairs yeast productivity and stops yeast from growing. This has beneficial implications in improving the efficacy of second-generation bioethanol production.

I found this study interesting because of the potential applications that it has. This study struck out to me as it shed some light on the clever way that we can apply scientific techniques like experimental evolution to biotechnology for the benefit of our environment and our future. I hope that you found this study as interesting as I did. Hopefully with more applicable studies like this, our cities will look less like *first picture* and more like *bottom picture*.

Image URL: https://www.azocleantech.com/article.aspx?ArticleID=618

Thanks for reading,

Bianca

Here are the papers I referred to:

  1. González-Ramos, D., Gorter de Vries, A. R., Grijseels, S. S., van Berkum, M. C., Swinnen, S., van den Broek, M., . . . van Maris, A. J. A. (2016). A new laboratory evolution approach to select for constitutive acetic acid tolerance in Saccharomyces cerevisiae and identification of causal mutations. Biotechnology for Biofuels, 9(1), 173. 10.1186/s13068-016-0583-1
  2. Rastogi, M., Shrivastava, S. J. R., & Reviews, S. E. (2017). Recent advances in second generation bioethanol production: an insight to pretreatment, saccharification and fermentation processes. 80, 330-340.
  3. Kawecki, T. J., Lenski, R. E., Ebert, D., Hollis, B., Olivieri, I., Whitlock, M. C. J. T. i. e., & evolution. (2012). Experimental evolution. 27(10), 547-560.
  4. Branco, R. H., Serafim, L. S., & Xavier, A. M. J. F. (2019). Second generation bioethanol production: on the use of pulp and paper industry wastes as feedstock. 5(1), 4.
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Fight or flight, bacterial edition: adapting your defense to suit your foe

Imagine you were surrounded by enemies, and had a choice of two defenses. You could send out a targeted laser to kill each bad guy, or instead, put in a bit more effort to form a clever disguise so that none of them could find you. How many baddies would there have to be to make the disguise worth the extra effort?

I recently read an article; The effect of phage genetic diversity on bacterial resistance evolution[1]. The authors deliberately evolved viruses that infect bacteria, to see if exposure to a more diverse group of these viruses would result in a change to the bacteria’s defense strategies.

Pseudomonas aeruginosa (pronounced “Soo-da-moan-us orig-in-osa”) is a bacteria that causes disease in both plants and animals, including humans[2]. It’s important for us to know more about it because it can have a negative impact on agricultural industries, and it’s able to form multi-cellular structures called biofilms to develop chronic infections on medical devices like catheters[3]. It’s an opportunistic pathogen, and one of the most common species associated with infections of immune-compromised or vulnerable patients such as those with cystic fibrosis[4].

Pseudomonas aeruginosa. Image credit: bioMérieux.

The ‘bad guys’ in this story are bacteriophages, or “phages” for short. Phages are viruses that specifically infect bacteria. They do this by attaching to the outside of the bacterial cell and injecting their DNA, before using the cell’s own machinery to replicate themselves. The newly formed phage then burst out of the cell to each infect their own targets, killing the host cell in the process[5]. Click here to see a fun video explaining how this happens & why studying phage is so important, as well as a sneaky peek at a P. aeruginosa chronic infection that was treated with phage therapy!

Transmission electron micrograph of Pseudomonas aeruginosa bacteriophage. Image credit: Watanabe et al., 2007.

A phage called DMS3vir was used in this study and is specific to P. aeruginosa, but bacteria can employ a variety of different strategies to protect themselves from infection.

One of these strategies is a general resistance mechanism called surface modification. Bacterial cells can make changes to the receptor proteins of their surface (which phages use to recognise the cell), to mask their identity as a target and evade infection[6]. There are also more targeted strategies where the cell actively damages the phage particle, such as the CRISPR-Cas adaptive immune system.

CRISPR-Cas (or CRISPR) is utilised when infecting phage are recognised by the bacteria; the cell uses an enzyme called Cas to cleave the viral DNA, and a short sequence is then incorporated into a special region of the bacterial cell’s genome (known as CRISPR). This short sequence is called a spacer, and bacteria can accumulate them over time. Spacers can be used to help the bacteria recognise and disable infecting phage sooner, by creating a guide to allow Cas enzymes to become targeted towards the phage which have that specific sequence[7]. This becomes a type of immune memory to allow for a more precise and prompt immune defense. You can learn more about CRISPR-Cas and how this targeted mechanism is being developed for gene editing here.

While CRISPR is an effective targeted strategy, the spacers acquired are specific to a particular phage’s DNA, and will only work against phage that also have that sequence. So bacteria have to accumulate multiple spacers to have a broader range of immunity against different phage, and the cost of using this defense increases with each spacer they accumulate. By contrast, surface modification has a fixed cost, as one change to the receptors on the cell surface will prevent recognition by a variety of phage. This could make it a very efficient system when there are a lot of different phage to defend against.

P. aeruginosa has been shown to readily evolve CRISPR-based immunity to phage DMS3vir in the lab, but the communities of bacteriophages we can isolate from the environment are likely to be far more diverse, which can impact the way that bacteria protect themselves. The authors of this article wanted to know whether phage diversity has an effect of the development of CRISPR-based immune defense by the host bacteria, and hypothesised that surface modification will be favoured in conditions when phage diversity is high.

They started by evolving the phage DMS3vir to be more diverse. To do this, they mixed the phage with a culture of host bacteria which lacked a DNA repair gene; they thought that if the bacteria was more likely to mutate, then the phage resulting from infections of that bacteria may be even more diverse than those evolved using a ‘normal’ or wild-type strain of P. aeruginosa.

Schematic diagram of the bacteriophage evolution experiment, conducted in 12 replicates over 17 days.

After letting the phage infect the bacteria for 24 hours, they killed and removed any remaining bacterial cells before using the new collection of phage to infect a fresh bacterial culture. Each time they did this, the resulting population of phage was able to accumulate mutations and become more diverse. They repeated this for 17 days using 12 separate replicate experiments. At the end of the evolution experiment, they also took one purified phage (referred to as clonal phage) from each diverse population for use as a control to show that it was the diversity of phage bringing about any change in the bacterial response, not the evolved state of the phage alone.

They then obtained DNA sequences for each of these 12 diverse populations and clonal phage, as well as the ‘ancestral’ phage that they started with, to determine how many single-nucleotide polymorphisms (changes), or SNPs, had occurred as a result of the phages evolving. As shown in Figure 1A, the diverse phage populations had a much higher frequency of SNPs than the clonal phage, indicating that each type of phage within the diverse populations is likely to have different SNPs to each other.

Figure 1, Broniewski et al., 2020.

They then tested each of these phage against bacteria that had some CRISPR-based immunity already. For the bacteria, they used lab strains that each had a unique spacer against phage DMS3vir (1-12 in Figure 1B), as well as 6 strains that each had 2 spacers & would be harder for the phage to infect (combined, ‘2sp BIM’), and one surface-modified mutant (‘SM’). While none of the phage could infect the 2-spacer strains or the SM mutant, the diverse phage had a greater infection capability than its paired clonal isolate against each of the single-spacer strains.

The authors wanted to see how phage diversity affects which defense strategy the bacteria will develop. For this, they used wild-type bacteria with no existing CRISPR spacers for the DMS3vir phage. After exposing this bacteria to infecting phage for 3 days to allow bacterial defenses to come into play, they then added them to both a wild-type phage and a modified phage that had a gene to disable CRISPR-based immunity. If the bacteria was resistant to both of these phages, the authors assumed it had developed a surface modification, whereas resistance to the wild-type but not the anti-CRISPR modified phage was assessed to be due to a CRISPR-based immunity. Figure 2B shows that bacteria challenged by a diverse phage population utilised surface modification (SM) more often than when challenged by clonal phage, whereas bacteria challenged by a clonal phage population almost always developed a CRISPR-based immunity.

Figure 2B, adapted from Broniewski et al., 2020.

It was also observed that when bacteria challenged by a diverse population did develop CRISPR-based immunity, they were more likely to acquire multiple rather than single spacers. As shown in Figure 3A, the majority of bacteria challenged by one type of phage (clonal phage) only acquired a single spacer, as that was all that was required, whereas exposure to a more diverse population of phages more often resulted in multiple spacers being accumulated by P. aeruginosa.

Figure 3, Broniewski et al., 2020.

Overall, this study showed that an increased phage diversity promoted surface modification as a form of generalised resistance to the phage. It was also demonstrated that when CRISPR-based immunity was developed, a diverse phage population increased the likelihood of multiple spacers being acquired in the bacterial host.

While I found the research interesting, I found the ‘scoring’ for type of bacterial resistance to be based on a huge assumption that other strategies were not coming into play. Bacteria have many different ways to protect themselves against phage, and the authors don’t really mention how they eliminated other mechanisms as possible explanations for what they observed.

I also don’t feel that a lab-evolved population of bacteriophages is ever going to be an accurate representation of the diversity in an environmental sample; while this team had a readily available stock of phage DMS3vir in the lab, I would have been interested to hear their justification for not isolating P. aeruginosa bacteriophages from the environment to use in testing the effect of phage diversity either alongside or instead of their lab-evolved phage.

What do you think?

References

1.      Broniewski, J.M., et al., The effect of phage genetic diversity on bacterial resistance evolution. The ISME Journal, 2020. 14(3): p. 828-836.

2. Wu, W., et al., Chapter 41 – Pseudomonas aeruginosa, in Molecular Medical Microbiology (Second Edition), Y.-W. Tang, et al., Editors. 2015, Academic Press: Boston. p. 753-767.

3.      Khatoon, Z., et al., Bacterial biofilm formation on implantable devices and approaches to its treatment and prevention. Heliyon, 2018. 4(12): p. e01067.

4.      Bassetti, M., et al., How to manage Pseudomonas aeruginosa infections. Drugs in context, 2018. 7: p. 212527-212527.

5.      White, H.E. and E.V. Orlova, Bacteriophages: Their Structural Organisation and Function, in Bacteriophages – Perspectives and Future, R. Savva, Editor. 2019.

6.      Westra, Edze R., et al., Parasite Exposure Drives Selective Evolution of Constitutive versus Inducible Defense. Current Biology, 2015. 25(8): p. 1043-1049.

7.      Westra, E.R., et al., Evolution and Ecology of CRISPR. Annual Review of Ecology, Evolution, and Systematics, 2016. 47(1): p. 307-331.

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Phage Hunt NZ Guest Post: A First Hand Experience with Phage Therapy, Post 2

The following is the second of two guest posts by SamD* from Auckland New Zealand who describes his first hand account of his unsuccessful Phage Therapy in Georgia. Sam had a complicated infection treated by antibiotics for many years. Post 1 is his tale and Post 2 is his thoughts about phage therapy considering his experiences.

Sam is very interested in thoughts or suggestions regarding his case and can be contacted at nicois@gmail.com *This is a pseudomonym.

The Lessons I learned regarding Phages

  • Patients needing treatment of multi-pathogenic environments with a history of extensive antibiotic treatment require a treatment facility who are experienced, who are transparent about potential treatment outcomes, rigorously establish sensitivities to antibiotics and use them unfailingly concurrently, and persevere with the concurrent treatment until all pathogenic responses are resolved.
  • A phage treatment plan’s schedule must differentiate between physiologically complex, possibly multi-pathogenic environments like urogenital and airway systems, versus muscle-tissue infections like the classic lower limb examples often associated with phages, and allocate time and money accordingly, the first as much as a magnitude of 6 over the second.
  • That 6 over 1 assessment: Much of the time allotted to be spent at a treatment provider is spent waiting for the production of phages, if problems arise as a result of the actual use of the phage, there will very likely be no time to deal with the consequences unless significant extra funds are spent. Arrival >> tests taking up to 7 days for results >> Phage preparation time (if a match is available) 2 weeks to 3+ months >> Virulent reaction to phage >> the long process starts all over again.. and.. etc.
  • Phage in-Clinic treatment v. long-distance both have pros and cons that at first glance confers no distinct advantage either way. An in-Clinic presence should offer more prompt treatment and better communication but the enormous consideration of waiting time and charges should negate these advantages, until the virulent reactions are considered. In many countries, rigorous testing and the availability of a wide range of antibiotics for best sensitivity choice may be absent, so the distance patient upon taking the new bacteriophage** and antibiotic (only targeted to the last infection, not the next) would be in a difficult position medically if another infection appeared as a result.
  • The application of the first bacteriophage didn’t only bring about a new dominant infection, it meant the phage treatment of the original target had to be immediately discontinued and on retesting, the original bacteriophage was no longer highly-specific to the original infection and had to be re-produced. As well, antibiotic sensitivities of the original infection changed markedly. (Note: in an uncomplicated treatment schedule that phage would not be discontinued, and one would expect to remove the pathogen, albeit with months of phage-taking, alternating 3 weeks on, 3 weeks off, to avoid immune system responses).

phage

What happens next?

  • I had read the NZ Herald Massey Phages Project article in the closing days in Tbilisi and contacted Dr Hendrickson soon after my return, hence this post. She showed great interest and has been extremely helpful in analysing the elements of phage treatment and encouraging the treatment experience to be recorded on the Phage Hunt NZ blog.
  • I next wrote to the second “International” Bacteriophage Clinic in Tbilisi giving them the phage treatment/repeat infection details with no country/clinic details to see what they would say; a paraphrased summary of several emails is “With multi-pathogenic environments like the urogenital system, we must always administer antibiotics concurrent with a phage. It is common for new, ascendant infections to occur. Nevertheless, we continue producing phage solutions and administering concurrent antibiotics for each occurrence, without stopping until no more appear. We usually find the ascendant infections are variants of the original infection being treated, as against your second different bacteria infection.” (The current infection has similar antibiotic sensitivities to the last Staphylcoccus haemolyticus report which suggests it is now a variant of the last infection).
  • Being back in New Zealand with an unknown infection also means a return to a medical system which offers no answers or treatment ability for complex urogenital infections. As before, the infection became chronic in the two weeks that passed in returning to New Zealand from its first occurrence. My system seems to have seen so many antibiotic courses and repeat infections that an infection quickly moves to a state where symptoms are virulent and debilitating, but sub-acute, and the local simplistic detection method of urinalysis fails the WBC level entry requirement for culture. No positive culture, no treatment of any kind. I have failed over a long time to find anyone in New Zealand in public medical practice who understands the significant differences in physiological status, and detection methods required, between acute and chronic pathogenic urogenital conditions. Microscopy, EPS tests, a consideration of the role of bacterial slime and biofilms have all proved to be beyond the scope of locally available treatment. Post-phage I am back to depending on an antibiotic to poorly maintain a condition more virulent and with a wider tissue spread than at any time in the past.

HK97 from Life in our phage world

HK97 from “Life in Our Phage World”, available in full (for free) here: http://2015phage.org/art.php

Bacteriophage Therapy Protocol Summary

  • It would be an understatement to say I know how a phage guinea pig might feel after a failed experiment; but more recently received information confirms for me that although my experience cannot be easily learned from available internet-based information, the phage treatment Clinic kept the realities of treatment complications close to the chest, only revealing them when they had to and were quick to deny further treatment when the going required persistence and resilience.
    The second commercial Tbilisi phage Clinic has now offered to pick up where their peers left off, and rigorously progress treatment to a conclusion, however the cost involved so far has been fairly substantial and the money has run out.
  • A Bacteriophage protocol in this area appears to need a lot more work; ideally an in-vitro test of an environment sample with the newly produced bacteriophage before application, if such a thing were possible, and a phage cocktail of a much wider spectrum for any one bacterial species. Otherwise the current treatment method as seen in Georgia presents an unaffordable monetary cost for many prospective patients.

If anyone has questions or observations you’d like a reply to, you can write to me here: nicois@gmail.com

**Where Bacteriophage is written in BOLD, Sam had initially used the word Autophage, a term that he came across in Georgia and which appears to be the way clinicians there refer to Bacteriophages used therapeutically.

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Phage Hunt NZ Guest Post: A First Hand Experience with Phage Therapy, Post 1

The following is the first of two guest posts by SamD* from Auckland New Zealand who describes his first hand account of his recent experiences with unsuccessful Phage Therapy in Georgia. Sam has a complicated infection that was treated with antibiotics for many years. Post 1 will be his tale and Post 2 will be his thoughts about phage therapy considering his experiences.

Sam is very interested in thoughts or suggestions regarding his case and can be contacted at nicois@gmail.com *This is a pseudomonym.

Phage Therapy in Georgia: A Clinical Perspective

In this post I detail my recent experience with Bacteriophage treatment in Georgia, the challenges the treatment presented, the lessons learned, a “Where to from Here” and my thoughts about how Bacteriophage therapy needs to be developed for my situation.

Phage therapy has increasingly gripped the Western world lately with media headlines of great wonder and promise. After a longtime on the receiving end of increasingly severe, resistant, urogenital infections, the potential promise of bacteriophages was not new to me, it had long been my plan “Y”; when and if absolutely all else failed, phages would be my salvation.

Phage Treatment in Georgia
Plan “Y” came early in March 2015 in New Zealand, after 29 years of antibiotic-dependency, including 15 years of continuous antibiotics, subsequent complications from long-term antibiotic side-effects and finally, unyielding nosocomial infections, a casualty of prior successful medical treatment. Preparations were made to go to a Tbilisi phage Clinic with the only initial reservation being that my phage research had always lacked one vital factor; reported, verifiable (male) urogenital-related clinical outcomes were virtually non-existent. I had found most phage clinical material in general very dated with narrow repetitive treatment topics. A new perplexing factor crept in at the 11th hour; the treating Clinic mentioned they sometimes administered antibiotics concurrently with phages. Apart from a few very specific PAS studies, this conflicted with my idea of the principles of the specificity of bacteriophages v. broad-spectrum medications and got me thinking about a pathogen acquiring resistance in mid-course treatment with the two technologies. I attached no more importance to this apart from it remaining as a mild irritant in the back of my mind. But, I was to find it was probably to be one of two specific things crucial to my medical treatment success.

An image of Félix d’Herelle.

On arrival in Tbilisi, the high apartment window view, coincidentally, was very “phage”;  there was a leafy view of Félix d’Herelle’s residence, totally obscured at street level, having been a KGB and now State Security facility, walled up like Fort Knox and bristling with cameras, as well as an aerial view of the actual Eliava Institute building and more distantly, the Zoo which was soon to be destroyed in disastrous flooding.

The Tbilisi Phage Clinic took samples and using a third-party laboratory, identified a “strong” infection of Enterococcus faecalis and “non-pathogenic” Staphylococcus haemolyticus (both the infections a casualty of prior treatment referred to above) with detailed report results including a wide range of graduated drug sensitivities, antibody counts and other non-translated information. These test results confirmed results obtained two weeks earlier in China en route to Georgia. Frustratingly, in the preceding 18 months in New Zealand, all similar tests were pronounced negative, in spite of debilitating, ongoing symptoms.

My too-Simplistic Perception of Phages becomes Apparent
The Eliava Institute, as another third-party entity, successfully located a match for the Enterococcus; enhancing and producing an bacteriophage took 4 weeks and the three application techniques produced a near-miraculous result. Over three days, all the prior 22 months of urogenital pain and urinary urgency disappeared and a sense of normal life started to rapidly return; but it was short-lived, on the 4th day an acute bladder-urethral infection ensued and a sample was sent to pathology. A return flight home was due in 3 days, too late to change the departure date.Medical tourism is essentially uninsurable.Remaining in Tbilisi likely meant the time and cost would again be spent unable to do anything, waiting for production of another bacteriophage. The treating Clinic promised that another bacteriophage would be produced and sent by courier, and so a very uncomfortable 33 hour trip back to New Zealand followed.

The new infection was identified as the “non-pathogenic” Staphylococcus haemolyticus shortly after leaving. Once the immediate-area Enterococcus had been removed by its bacteriophage, it appeared the new environment enabled the Staphylococcus to become virulently ascendant. This new infection not only quickly and aggressively occupied the same areas as the previous Enterococcus, but spread outside the urogenital system. The laboratory report included a list of 23 antibiotic sensitivities, with just one of the 4 that showed good sensitivity being available in New Zealand, but proving ineffective as single therapy as its drug notes indicated.phage

Seven long weeks followed, with no information from the Clinic on the E.T.A. of the new bacteriophage. It had become obvious that a “long-distance” patient had either gone to the bottom of the treatment priorities, or that other forces were at play. So, a return flight ensued, and upon arrival the Clinic announced that the Institute (apparently the only bacteriophage producer in Tbilisi) had a phage match and it would take the standard 2 weeks to produce. It was very tempting to think that the coming phage would be the total solution after a bad start but the former thoughts of “will phages work for me” had well and truly changed to “what’s going to happen with the next phage” and the coming days passed slowly.

The new bacteriophage was delivered and a repeat of the first bacteriophage reaction followed, except this time, the next ascendant infection appeared even more acutely in just 24 hours, so much so that antibiotics were used immediately to control it and a two week course halted any other progress. By now the summer break holidays were approaching and soon, behind closed doors, one of the Doctors for the first time broached the subject that resistant multi-pathogenic environments can be a problem to treat with phages, that the environment is easily unbalanced by the high specificity of a phage. Apparently, the “sometimes” use of antibiotics at the same time as the bacteriophage was started was specifically for this reason. I wasn’t convinced the increasingly virulent ascendant pathogens were unavoidable, I was getting a distinct impression that each phage was conferring some kind of virulence on other bacteria. Further, it appeared that the high phage specificity, single phage dosing, a variable concurrent antibiotic policy and a lack of awareness of the microbiology of the targeted area required a higher-level phage treatment protocol than the Clinic was offering. They were appearing at this point as little more than a matching agency for 3rd-party laboratory and phage production services.

This very competent Doctor formed a plan, the second bacteriophage** should be resumed, it would surely cause a repeat of the last infection and so all-important urinalysis would be done, and we would continue like before.. another bacteriophage. Airtickets were extended for an extra 2 weeks, giving nearly 4 in all, to accommodate this, with my distinct unease that phage treatment may never bring the resolution I sought.

And this is how the plan went; the organising Doctor went off for the Summer holidays, the Clinic’s Director (Managing Doctor) took over, a sample from a very virulent ensuing infection was sent off for pathology, however after waiting 5 days the Director declared it “an unusable sample”, and by now 4 days of injected and oral antibiotics getting the forced new infection back under control made getting another sample of no use. Further, the Director refused over the next few days to arrange an EPS which may have revealed a non-antibiotic-affected culprit, holding out until I had to meet my airline departure deadline. Now came the long trip back to New Zealand, uncomfortable in the knowledge that the clinic didn’t have the medical bacteriophage resilience required to engage with my condition. Further, in comparison to when I first went to Georgia, I now had a far more virulent unidentified infection and was antibiotic-dependant again, a return to a past I had hoped I had left for good.

If anyone has questions or observations you’d like a reply to, you can write to me here: nicois@gmail.com
**Where Bacteriophage is written in BOLD, Sam had initially used the word Autophage, a term that he came across in Georgia and which appears to be the way clinicians there refer to Bacteriophages used therapeutically.

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To be or not to be? The question of virulence as posed by polio

In a previous post we discussed the identity of polio.
If you haven’t yet read it and aren’t well-versed in who or what polio is, might I recommend a brief glance? If you’re already up to date on cVDPVs, let’s proceed, deep into the tangled web of RNA that is… poliovirus.

(Previous article: https://thismicrobiallife.wordpress.com/2019/09/12/youve-heard-about-measles-youve-probably-even-heard-about-smallpox-but-what-do-you-know-about-polio/ )

Step One – Find the Trouble Maker

Of the three types of attenuated poliovirus contained within the oral vaccine, it appears that Type II is by far the most likely to regain virulence. With this in mind, many researchers have focused on Type II, trying to pin down precisely what is occurring, at the genetic level.
Note that poliovirus is an RNA virus rather than a DNA virus, which means it is more prone to mutate, as during replication many errors are made and not corrected, unlike with DNA.

Some background on viruses (differences in DNA vs. RNA viruses), as well as an explanation of live vaccines, can be found here: https://sciencing.com/rna-mutation-vs-dna-mutation-3260.html

Step Two – What Makes a Trouble-Maker Troublesome

For the rest of this blog, most of the material discussed is referenced from an article titled: The Evolutionary Pathway to Virulence of an RNA Virus.
Link: https://www.cell.com/fulltext/S0092-8674(17)30292-1

Combining genetic sequencing and experimental evolution (E.E.) – a handy method of studying evolutionary processes under experimental conditions – authors attempted to identify whether or not the mutations found in cVDPVs were liable to reoccur. By doing this they were able to help answer an important question – was there a parallelism between independent cVDPVs?

(In short, could there be a common element in the many cases of attenuated poliovirus reverting to virulence?)

Looking first at cVDPV sequences from Belarus, China, Egypt, Madagascar, and Nigeria, a series of nine common mutations were seen across the countries. This indicated that even without any interaction, avirulent vaccine strains were undergoing similar evolutionary changes to revert to virulence.
To delve deeper, researchers set up a model where human cells (a cell culture, not a human trial) were infected with the attenuated Type II. After a set time these cells were taken and the virus extracted. Some of the viral particles were then reintroduced into new cells, and some of them were sequenced. This cycle was repeated several times, with both a 33°C and 39.5°C model.

Retrieved from: Stern et al. “The Evolutionary Pathway to Virulence of an RNA Virus“, 2016.
Fig 1. A diagram showing the path of mutation from attenuated vaccine strain to virulence.

(These two apparently arbitrary temperatures might cause a raised eyebrow, but the rational is that vaccines are ordinarily produced at 33°C , and a human body under an immune response often reaches a 39.5°C febrile state.)

Whilst little synonymous mutation was seen at 33°C (there’s as always a base rate but no particular mutation was steadily seen significantly more than others), the 39.5°C model followed a similar trajectory to that seen in the cVDPVs analysed. Of the nine noted mutations seen in cVDPVs, four were observed to occur at heightened levels in cell culture, as shown below.

Retrieved from: Stern et al. “The Evolutionary Pathway to Virulence of an RNA Virus“, 2016.
Fig 2. The four mutations seen at heightened levels at 39.5°C (as indicated by the bold, coloured line). The baseline (in grey) shows the expected frequency, were the mutation not being actively selected. Dashes indicate levels at 33°C .

Three of these four were dubbed “gateway” mutations (seen as red lines in the above image) and were found to occur much more frequently than by random chance – indicating they were being selected.
The term “gateway” was used by the authors to clarify that in order of evolution, these mutations tended to precede further mutations. It appears that they are acting as an opening passage, where once they occur a series of further mutations can then occur, leading to reversion. To clarify whether or not these gatekeeper mutations were indeed likely to be “leading the charge”, samples were taken from vaccinated individuals, 14 days after vaccination. The sequences of these samples were compared to the initial attenuated Type II sequence used in vaccine production, and it was found that not only were these gatekeeper mutations tending to precede further mutations, A481G was usually seen prior to the other two gatekeeper mutations.

Taming the Shrew – Ahem, Trouble

All of this suggests a very delicate evolutionary pattern is taking place, and lends hope that with understanding, prevention can occur.
Now that particular key sites have been identified, one option would be to proactively remove an additional portion of the attenuated Type II strain, adding in another step it must take in order to regain virulence. A good target for this would be the gatekeeper mutation A481G, as it has been shown to be a key player through cVDPV analysis, cell culture E.E. and screening of vaccinated individuals.

In expanding this work to other vaccines, it is likely that similar patterns are detectable and could be prevented, before events progress to the point where polio currently is. This might include running a short E.E. experiment to see if there’s a sudden reversion when the vaccine of interest is taken from it’s 33°C production environment into a ~39.5°C environment. Any rapid reversion to a sequence resembling the initial virulent virus would be a red flag indicating a need to alter the vaccine.

It can be concerning to hear of a vaccine causing illness, but as is the case here, there is often much more to the picture. The number of cases of polio has dropped drastically world-wide since vaccinations began and were there a more effective vaccination system in place, cVDPVs wouldn’t have had the opportunity to develop. This, more than any other outcome, may be the most important finding, and something that needs to be amended for further eradication efforts.

Thank you for taking the time to read this (and the previous) post, I hope it was informative and left you wanting to do more of your own research in the future.

Posted in Experimental Evolution | Tagged , , , | 7 Comments

Experimental Evolution OF Evolution.

Evolution is something the vast majority of the 21st century agrees on.
TV shows like the big bang theory have created a cosy little bandwagon where after an 8 hour shift, Joe blow can switch off to the terminology and just go with the flow of the episode.

Knowledge of the basics and by extension their appreciation, is a luxury afforded to only those who sought it out in the first place. Without this, the ‘flow of the episode’ takes us where it wants us to go.

Going against the grain, and fighting against the flow of the episode is something that sets people apart, and this is not a concept lost on evolution itself.

We both know Joe blow isn’t going to be making any power moves in his life time, but what about his children?
If they are anything like their father, probably not. But if for some reason, their genes are expressed differently, they might just have the opportunity to ‘blow’ up the family name.

The world they grow up in will of course be different to their dad, but what if it wasn’t?
Would they still have the ability to change?
By how much would they change?

The study I chose to write this blog about asks that same question but in the context of Drosophila melanogaster fruit flies.

How does the expression of genes change when the environment is kept constant?
How does the expression of genes change when the environment itself changes regularly?

The Evolution of evolution guys yes.

Inception etc.

As a side note like any other dream, some parts are more memorable in the morning when you wake up than others, and my rendition of this paper works out a bit like that. Certain things have been left out for the greater good of the take home message. The take home message here is that he was stuck to the floor . . .  never mind the holes he knows are at the bottom of everyone’s feet. It’s deeper than that. Moooooving along . . .  .

To recreate the idea of our hypothetical Joe Blow, researchers used a field collected Drosophila Melanogaster raised on a standard corneal food to establish two other large populations. One was given the time to adapt to a salt-enriched diet and the other was given the time to adapt to a cadmium-rich diet.
A cross was made between both of these to create 20 smaller populations.

These were split into four different environments,

A Cadmium-rich diet and a Salt-rich diet where the flies were given either of the two every generation as a food source.
A temporally variable environment where flies were reared in alternating generations of Salt-rich followed by Cadmium-rich food.
And a Spatially variable environment where in each generation, half of the flies were fed on one diet and the other half on the other diet, separated from one another up until the point of mating.

Before a gene can become what it needs to be, it must first be written down like a post it note. At any given point in time researchers are able to take a snapshot of all the post it notes that are around in a cell before whoever needs to do what’s on them finally gets them done.

After 130 generations researchers took all the post it notes of the drosophila populations and compared them to see if they prioritized having certain things done more often than others.

This is what they called ‘RNA-seq data.’

This would be an example of what the post it notes would like in an inflamed leg muscle that is under exercise

In the same way the culture of the world would change between Joe Blow and his kids, the times and the place affected these genes within the drosophila. Different post it notes become more and more prevalent amongst the evolved populations.

When they compared the populations that were given Cadmium-rich OR Salt-rich food every generation, they found 546 genes that showed what they called a selection history effect.
This is essentially an evolved difference in the amount of a specific post it note relative to the other diets. 
In a previous study by the same researchers they measured how often certain alleles showed up between an ancestral salt-rich and cadmium-rich fed populations. These alleles are simply different versions of the same gene (before they are written down in a post it note). A bit like how you would get skim milk, soy milk, rice milk, oat milk, goat milk, and finally cow milk. It’s all drinkable but a little bit different. 

Combing that data set with their results from the current evolution experiment, they were able separate genes based on whether the particular variation of that allele was located in coding regions, noncoding regions, or located in the DNA sections between genes (intergenic).
They found that the genes that were expressed more (or had more post it notes) also were the same genes they noticed previously had higher numbers of variations in their intergenic regions. Why would a mutation that appears between genes affect them?
Cis acting factors. Like little poltergeists, you might not see them initially, but after months and months of confusing results and occurrences you’re left thinking somethings toying with you .

These are regions that are near-by certain genes which can impose a regulatory function, giving them an idea on just how many post it notes are left in the booklet, and how many they can afford to take out to write on.
The reoccurrence of mutations in these intergenic regions, together with the overlap of an increase in gene expression, suggests that cis acting factors contribute significantly to the evolved difference in the amount of post it notes between populations.
They next chose to examine whether the genes that have more post it notes in cadmium exposure relative to salt exposure in the Ancestral Populations, were also upregulated in the 5 constant Cad populations.

Using the Grand Ancestor population as a point of reference (because it was naïve to BOTH diets), researchers identified 905 genes that showed a significant change in expression when the flies are reared on cadmium-rich food compared to salt.

They then again checked for overlap with the 546 genes that showed that selection history effect from before.
108 genes overlapped between the two gene sets, and further computer analysis showed that they had a reoccurring theme of being involved with the cell membrane.
In 91% of these genes, their response in terms of how many post it notes they had was opposite and contradictory to what appeared in the ancestral populations.

That is to say for example, A gene with more post it notes in the Ancestral populations will have evolved to have less post it notes in the populations that have been given the time to adapt. 
It is an example of what they called counter gradient evolution.
Where the genetic influences on a trait, oppose the environmental influences, creating far less of a change than what you would expect from the environment.

We expect that in a new environment, there will be far much more to do i.e. – more post it notes to deal with the changes, but what researchers found was the opposite of that.
There were less post it notes for the same genes even though the environments were different.
There are two common reasons behind the emergence of a counter gradient pattern.
If natural selection favours the same amount of post it notes across all the environments, but one environment induces a change, then opposing genetic changes are expected to evolve from what was normal.

The other reason is related to the stress that is experienced by a population exposed to a new environment. We expect that this stress would cause different post it notes to appear in higher amounts to cope with the changes. The result would be an abnormal display as above.

Abnormal displays like this build character, and after enough of them you might fight yourself changed for the better.
Someone who had already adapted however, would not warrant the same stress response as someone who wasn’t ‘built for it’.
The likely scenario is that after the 5 Cadmium populations had been given the time to adapt to the diet, they no longer needed to exhibit this stress specific response that was seen in the Naïve Ancestor.
This is the reason behind there being less post it notes for the same genes between the populations that are stressed and those who are not, who have already adapted.

The level of change that could appear in the amount of post it notes for a particular gene set is what these researchers termed its expression plasticity. We would expect a higher expression plasticity for the diets that vary spatially and temporally, compared to the two that were fed consistently every generation.

The researchers could not test this however just using the complete set of post it notes from a given population. They needed to first, identify specific genes where they expected either an increase or decrease before they were written down.

To do this, the screened for genes that could meet two criteria.
First, they required a significant difference in the amount of post it notes from the optimal that is seen in the Ancestral Salt/Cadmium populations.
Second, they needed to exclude genes that naturally have high levels of change in their number of post it notes between populations.

109 genes passed this screening test and for each gene in each population they calculated the change across diets in such a way that a positive value indicates the post it notes changed adaptively.

For each population they averaged the values across all 109 genes to obtain a single measure of adaptive plasticity that they could then compare between diets.
The mean score for both the spatially and temporally varying diets was significantly greater than 0.
This same mean score for the consistent diets was practically zero.

Their hypothesis that adaptive changes arise more readily and to a greater extent in these heterogeneous environments was confirmed once they assessed these results.
The change in expression that has been measures thus far is not the same as measuring how adaptive expression is in either diet.

To do this, the researchers figured out a mathematical formula that allowed them to represent how far the expression levels of genes are from their optimum.
As before the formula equates to a number that is a unique distance from zero, where zero represents optimal gene expression.

The Metric Φ in the Temporally and spatially variable diets was close to 0 for both, indicating them being close to optimal expression – i.e. the perfect amount of post it notes for what needs doing in that environment.
The same metric in a consistently fed population that has been transferred to either of the variable diets, was found to be significantly higher and much further away from optimal expression.

Obviously because they haven’t had the time to adapt.

The patterns of counter gradient variation that they see represent evolutionary responses that attempt to restore just the right amount of post it notes to handle the situation.

If the Goldilocks was built to handle the different types of porridge, then we wouldn’t have had our beloved nursery rhyme. It’s fair to say that when she sampled each of them, a bit of counter gradient variation occurred. She stalled too long making the post it notes that she needed to digest the meal, fell asleep, and the rest is history.

Ok so enough about Wolves, Joe blows, T-rexes and Leonardo DiCaprio, the bottom line really is as expected. Environments that have spatial or temporal variation elicit adaptive responses in the individuals who are naive. If an organism is already adapted to an environment then its response in terms of post it notes will not be adaptive in nature. It will know exactly what to do, how much to do, and at what times. No problem.

They mentioned some of the limitations they noticed in retrospect after having conducted the study.
They chose to count post it notes only from very young drosophila larvae, and this offers with it a problem when you attempt to extrapolate across other developmental stages.
The collections of genes they used were all reasonably highly expressed and that they could have been more liberal with their thresholds. This is a reoccurring problem in expression studies, where accepting a few more false positives would have given a higher resolution picture and idea of the post it notes involved in the specific response.

They also only took a single snapshot of the post it notes at a single time point in the populations and had they taken multiple, they would have noticed the inconsistencies that arise – as the tasks on the post it notes are being performed.

Moving forward, follow up studied perhaps using the same drosophila populations should have a focus on understanding (1) Why plasticity occurs

(2) What is the main cascade of events and who are the star players that underlie adaptation – i.e. where is the quarterback??

(3) How these events relate back to the change in post it notes that eventually cause different behaviour patterns and hopefully speciation away from the Joe Blow Lineage.

Thanks for reading !

Jesse.

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Want to make a new species? Put a little pressure on it…

A fascinating event that can occur in evolution is speciation; mutations bringing about new species that are significantly different to their ancestor. But how does this happen, what causes it, and how can we watch it happen?

Observing the processes of evolution can be a daunting task. After all, in an experiment you need to be able to measure the results, but evolution occurs over a long, long, LOOONNG time… doesn’t it?

evolution of man

A depiction of the evolution of man. Image credit: owutranscript.com 

Meyer et al. [1]  got around the challenge of waiting for organisms to evolve by studying those with a much shorter lifespan. Bacteriophage λ (lambda) is a virus that reproduces by infecting its bacterial host Escherichia coli.

You’ve probably heard of E. coli. – they live in our gut, often with us none the wiser, but some types can make us sick. E. coli. is readily available and easy to grow, and boy do they grow quickly! Each new generation of E. coli. can be ‘born’ in less than 20 minutes [2], and can have a different genetic fingerprint to their parent due to things like mutations and genetic material being switched around during reproduction. This short generation time allows scientists to observe THOUSANDS of generations worth of change within a few weeks.

But back to the star of the show (E. coli. is just the host after all): bacteriophage λ.

lambda phage

Artist’s interpretation of bacteriophage λ on an E. coli. cell. Image credit: theminione.com 

This virus (also known as ‘phage’) has the ability to attach itself to an E. coli. cell, inject its DNA, then use the host machinery to replicate itself. Once there are many copies of phage λ the cell bursts open, releasing new phage progeny into the environment. Phage λ does this by having binding proteins on its ‘feet’ (we call them tail fibers) that match up to receptor proteins on the cell’s outer membrane. Most λ phage can only match to one receptor called ‘LamB’, but some types of λ can use another receptor as well.

Teacup evolution

Meyer and his colleagues wanted to explore what would happen if they took a type of phage λ that could match with two different receptors (LamB and OmpF), and let it go through many cycles of reproduction with E. coli. cells that have the LamB receptor, OmpF receptor, or both. The type of E. coli. cells that were provided represent a selection pressure for each set of phage to change the way that it attaches to host cells – evolution towards being better able to utilise the resources available.

The phage that could use both receptors was grown with each of three different types of E. coli. (LamB only, OmpF only, or both receptors), with six replicates of each scenario. Every 8 hours the phage population was moved to a new culture of bacteria, to prevent the E. coli. progeny from evolving resistance to the phage and affecting its ability to adapt. They wanted to see how phage λ would respond to only having one receptor available, and what it would do when it had access to both.

With only one option available, phage λ almost always evolved to specialise to that receptor. All six of the phage populations grown with the LamB-only E. coli. lost their ability to use the OmpF receptor, and five out of six grown with the OmpF-only cells became unable to use LamB.

Pretty easy to make a decision when you only have one option! However, when both receptors were available, most λ populations developed weaker specialisations to either of the two receptors, and some retained the function to use both.

 

F1.large

Figure 1 [1]. In the ‘Allopatric’ group, red bars represent phage λ evolved exclusively with OmpF receptor while blue represents phage λ evolved with LamB. In the ‘Sympatric’ group, phage λ evolved with both OmpF and LamB were then isolated in equal proportions from cultures with one or the other receptor (red & blue bars respectively). The length of the bars shows the extent to which each group specialised, with -1 (OmpF) and 1 (LamB) indicating complete specialisation.

 

More LamB specialists evolved than OmpF, which the authors attributed to fewer mutations being required to become a LamB specialist, making it a simpler adaptation to achieve. This was determined by looking at the DNA sequence of J, a key gene in host recognition, from one phage progeny of each experimental group and comparing differences in the sequence that could bring about a change in function.

To test that these genetic sequence changes were responsible for the phage specialising on one receptor, Meyer et al. created phage with engineered genomes containing the mutations observed for each group. These modified phage were found to specialise or generalise in the same way as their evolved counterparts. A constructed hybrid with both sets of specialist mutations was not viable, indicating genetic incompatibility between the two specialists.

While this experiment didn’t go so far as to bring about the creation of a new species, it demonstrates how easily mutations can render two members of a species distinct from, and even incompatible with, each other. Genetic incompatibility is one of the criteria considered when defining speciation, with two members of different species being unable to produce offspring. While the phage λ progeny in this experiment were still far too genetically similar to qualify as distinct species, they are an example of how selective pressures can bring about adaptive mutations in a relatively short period of time, which has the potential to lead to speciation events. Let’s give them a few more weeks and see what happens…

 

References:

  1. Meyer, J.R., et al., Ecological speciation of bacteriophage lambda in allopatry and sympatry. Science, 2016. 354(6317): p. 1301-1304.
  2. Todar, K. The Growth of Bacterial Populations. Online Textbook of Bacteriology 2012; Available from: http://www.textbookofbacteriology.net.
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What in the heck is Convergent evolution? An explanation, and how scientists study it.

Growing up I was always obsessed with the universe and the life it birthed. Atoms forged and fired from dying stars at the outer reaches of the cosmos travelling eons to coalesce into a cloud of universal dust. Gravity taking hold; pressurizing atoms until rock is formed; then Earth. A single pale blue dot orbiting a 4.6 billion year old nuclear fusion reaction we call the “Sun”; It’s awesome. The baron rock we call Earth slowly morphing into the one we know today. Lush and full of life. But how did life get there? The answer is deceptively simple; Evolution, baby!

A Pikachu evolving into Raichu after encountering a thunderstone. Credit

Have you ever noticed unrelated animals share the same traits?

Did I say deceptively simple? What so deceptive about it? Animals have traits, and those traits are selected for by the environment and passed down to offspring. Not very deceptive at all you might say; until you think about Bats, Birds, and Butterflies. Mammals, aves, and insects respectfully. How is it that each of these three organisms all share a common trait so distinguishable as wings and yet be completely different in every other way? Surely long ago an ancient ancestor of each of these animals evolved wings right? What if told you that Birds, Bats, and butterflies each evolved wings independently of one another? This is what is known as convergent evolution and its something that researchers led by Pedro Simoes out of Portugal decided to elucidate through their published journal article.

What are these Scientists actually researching?

So lets look at this incredible image I have made to better understand what it is these researchers are looking for. The orange background is the environment, the circles are the generations of species A and species B; the colour changes represent new adaptations that have been selected for. So what do we see between species A and species B? Well they both stem from different coloured circles (which means both species have different traits to begin with) and as they evolve, they begin to differ more and more, until! Aha! In the top right corner, can you see it? Even though the starting circle In each box was a different colour, both boxes have ended with a circle that is yellow! This is convergent evolution in action! Two separate organisms developing the same trait independently of one another.

That’s great and all but how does this relate to the research by Pedro Simoes?

Well with the knowledge you now have on convergent evolution I’m sure you’ve already put two and two together. Since we can make an environment homogenous (make completely the same), could we then breed animals in that environment for 100s of generations, add new organisms, and then predict the traits the new organisms will develop based off the 100s of older generations? Well this is exactly what Pedro Simoes’ team set out to discover!

They took flies and recorded their fecundity traits (the number of eggs laid, the age of first reproduction, peak number of eggs laid) over the generations and compared that to the results of the older generations of flies that had been living in the lab for a much longer time. If the traits of the new flies became similar (converged) to the older flies, then that would mean we could theoretically predict how the flies would evolve when placed in the lab! How cool is that?

Here’s what they found!

Image source
Moving clockwise starting on the far left we have: The difference to the control, The different populations of flies, the traits they studied, and the number of generations. The 0 represents identical traits to the older generations of flies that have lived int he lab previously. The dotted lines represent the smaller number of generations, and the solid line represents the larger number of generations
It should also be mentioned that early fecundity is the number of eggs laid in the first week, and peak fecundity is the number of eggs laid after that week.

In order to get this data Simoes et al caught flies from separate regions in Portugal, brought them back to the lab, bred them, and after two generations began collecting data (this is to help standardize the data, which is done to decrease variables in the experiment). To collect data Simoes et al performed phenotypic assays of the flies in each generation, by transferring mated flies to fresh media every day, and counting the number of eggs laid in the first week of life (early fecundity), the number of eggs laid between days 8 and 12 (peak fecundity), and the number of days before the first eggs were laid (age of first reproduction).

How did they make these graphs?

In order to normalize the data and plot it on this graph some pretty heavy statistical methods were used.

Source: Giphy

In interest of transparency i couldn’t accurately explain it to you, so instead i will give you a very simple explanation that gives you the gist. The data was averaged, and then a linear regression of the data was performed, mix that in with some statistics that go way over my head and bingo bango, you’ve got this graph.

In the graph, we can see that as time goes on the traits begin to converge (as seen by the dotted lines, and solid lines moving towards the 0 mark) but then as more time passes (more generations are born) they begin to diverge again e.g., move away from the 0 line (as seen in the very late generations; specifically in the NARA and TW populations). Some traits don’t even converge at all! (looking at you; peak fecundity for the NARA population) The writers of this article have stated there is “an overall theme of convergence” which is true. Overall some convergence is occurring. Unfortunately, due to the transient divergence seen between the early, and late generations of the populations; there is no way you could accurately predict the fly’s evolutionary trajectory.

So all in all, as cool as it would be to be able to predict traits in animals, there is just so much variety when it comes to evolution that it leaves us struggling to predict the trajectory animals will take along their evolutionary path. Perhaps with time, and more generations, the traits of the new flies will converge in a manner more predictable. Until then we will just have to remain ignorant of our evolutionary trajectory. Despite the results. I hope you can walk away from your computer screen knowing you’ve learnt something about convergent evolution, and how experimental evolution can shed light on how this phenomena arises.

Source Material

Simoes, P., Fragata, I., Santos, J., Santos, M. A., Santos, M., Rose, M. R., Matos, M. (2019) How phenotypic convergence arises in experimental evolution. Evolution, 73(9), 1839-1849.  doi: 10.1111/evo.13806

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