Phage hunt so far…

As if I were mining for gold, I had been digging and scraping soil samples from different locations within Auckland. With each sample I had collected, I sensed hope; that I would perhaps find gold. Bacteriophages (phages) are like specs of gold found in dirt, they hold potential powers that could be the answer to many bacterial diseases.

gold

Image 1. Both gold and bacteriophages show similarities in its origins in the soil and value.

Who knew that phages could be found almost everywhere on Earth? They are found ranging from soil, sewage, the depths of oceans to the food we eat (3). Phages are viruses that are abundant in the biosphere with an estimation of 10^32 phages (7). It is often the case that when something becomes abundant, the value drops. But that is not the case here. Several university education programs such as the ‘Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science’ (SEA-PHAGES) are located across the globe. Students participating in these programs such as myself cannot get enough phages. There is a lot we do not know about phages. Currently, 13,074 phages have been found worldwide and counting according to (5), which is comparable to a grain of sand within a desert. The more phages found, the better as we can add to the existing databases of sequenced phage DNA and increase our understanding.

Phages are in fact an old discovery. They were first found in 1915 by Edward Twort and later 1917 by Felix d’Herelle (10). Phages, a strange looking organism as pictured in (Image 2) that resembles a spider. They are specific in that they only target specific strains of bacteria, whilst being ineffective towards mammalian cells (7). Phages infect bacteria by inserting DNA or RNA that incorporates into the bacterium and undergoes either lysogenic cycle or lytic cycle (3). The lysogenic cycle is when genetic material is integrated within the bacterial DNA, and remains dormant, allowing bacteria to replicate normally until it is triggered by an environmental factor, switching it to a lytic cycle. The lytic cycle is when the phage genetic material is replicated by mechanism in bacteria, synthesizing new phages, that build up inside the cell walls of a bacteria until it bursts open (lyses), killing the bacteria (3). 

phage

Image 2. A bacteriophage resembling the shape of a spider.

By the 1940s, phages were outshone by chemical antibiotics which emerged. Antibiotics have same superpowers as phages by destructing bacteria, however, they work differently. Instead of lysing bacteria, they can destroy the cell wall or inhibit bacteria to synthesize proteins for growth (6).

Antibiotics, like an ageing old man, grew weaker and weaker. Even a walking stick cannot aid this old man. He could not handle the younger and more active bacteria that adapted faster and increased their resistance. Antibiotic resistance rose rapidly, especially with misuse and over-prescription of antibiotics (1). 

We have come to a point where modern medicine is no longer effective. This problem worsened as fewer pharmaceutical companies are manufacturing new antibiotics. Luckily, an alternative exists called phage therapy, one of the strategies implemented the Department of Health (2). Once again, phages were rediscovered.

This comes back to the reason to why I and several other students in 246.202, a class in Massey University may have been out collecting soil. Be it at the crack of dawn or in the thick of the night. At random locations, such as the urban forests, parks, or sidewalk where other passersby would probably question if collecting soil was a weird hobby of mine. It has been a difficult journey so far, with many failed attempts to find a phage, totaling up to 20 soil samples and many other repetitive procedures in the laboratory. Currently, I am up to the stage of characterizing my phage, all derived from a singular pinprick plaque (a clearing on an agar plate due to bacterial lysis) I had found in my 19th soil sample. 

The future of phages looked promising to me. Phage therapy so far has been proven successful, having already treated a few diseases before antibiotics (3). They are natural and not chemically harmful such as antibiotics. They are safe towards humans as they specificity targeted bacterial cells. Due to the specificity, they also did not target the good bacteria in your body, unlike antibiotics (6).

Until I heard a question in class; could bacteria could become resistant to phages? I started to question all our efforts of phage hunting. What was the point of finding phages, if the same outcome of resistance were to occur? I wonder if these minuscule things found in soil really is the answer? I have primed myself into seeing phages as gold from the beginning, thinking that resistance would not be possible. However, phage resistance is a possibility. It arises differently from antibiotic resistance, from the incapability of detecting the bacterial antigen (a molecule found on the surface of the bacteria). A mutation on a bacterial antigen will mean that phages cannot attach onto and infect its host (4). Another way a phage becomes resistance is the bacteria’s natural mechanism to cut foreign DNA with enzymes (4). This causes a phage to be resistant to that bacteria it used to be able to infect. While converting to another phage that targets the same bacteria, or a cocktail of phages (use of multiple phages) could be hopeful. This could also fail yet again if no phages or if one phage in the phage cocktail is effective (4). If it were hopeful, new phages would need to be continuously found to make these cocktails, and additionally have ideal characteristics such as fast infection and lysis producing numerous phages (4). It seems like a cycle of needing more phages. It comes down to an ongoing search to isolate more phages that infect the same bacteria in case of phage resistance, which for now is at low risk in comparison to antibiotics.

For now, digging for more soil may be the way to go. Alchemy has not reversed its magic. Gold is still gold. However, not pure gold as it seems.

 


References

(1) Antibiotic resistance. (2015). Retrieved from http://www.who.int/en/news-room/fact-sheets/detail/antibiotic-resistance

(2) Henein, A. (2013). What are the limitations on the wider therapeutic use of phage? Bacteriophage, 3(2), e24872. https://doi.org/10.4161/bact.24872

(3) Kutter, E., Guram, G., Alavidze, Z., & Brewster, E. (2013). Chapter 8: Phage therapy.In Grassberger, M., Sherman, R.A., Gileva, O.S., Kim, C.M.H., & Mumcuoglu, K.Y (Eds.). Biotherapy – history, principles and practice: a practical guide to the diagnosis and treatment of disease using living organisms (pp. 191-231). Dordrecht, Netherlands: Springer Netherlands.

(4) Nilsson, A. S. (2014). Phage therapy-constraints and possibilities. Upsala Journal of Medical Sciences, 119(2), 192-198. https://doi.org/10.3109/03009734.2014.902878

(5) The actinobacteriophage database. (n.d). Retrieved from
http://phagesdb.org/

(6) Trimarchi, M. (n.d.). How do antibiotics work? Retrieved from https://health.howstuffworks.com/medicine/medication/question88.htm

(7) Wittebole, X., Roock, S. D., & Opal, S. M. (2014). A historical overview of bacteriophage therapy as an alternative to antibiotics for the treatment of bacterial pathogens. Virulence, 5(1), 226-235. https://doi.org/10.4161/viru.25991

(Images 1) Smarter hobby. (n.d.). Gold nuggests [Photograph]. Retrieved from
https://www.smarterhobby.com/metal-detecting/

(Images 2) Usagi no Nedoko Kyoto. (n.d.). Enterobacteria phage T4 [Photograph].  Retrieved from https://usaginonedoko.online/products/enterobacteria-phage-t4

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A Killing Machine, a Chicken, a Group of Heartless Monsters & You

The concept of a lifeless machine made of DNA and protein hijacking the innards of a living being to then use these innards to replicate itself is frightening. For this machine to replicate in such numbers that the unfortunate host bursts at the seams as it fatally-births the offspring of its assailant is objectively terrifying.

For not just one but for a group of six individuals to intentionally subject countless beings of lesser complexity to this fate is cruelty of the highest degree. For these six individuals to willingly and knowingly cultivate, perpetuate and propagate this process, well that’s just what we do in phage-lab.

Perhaps we’re heartless monsters but if it’s of any consolation, I know that I, for one, have my reasons for being so and although I doubt I could sell M. smegmatis on them, I could sure get Robyn’s approval and I think I’ll get yours too.

To kick things off, let’s talk a little background.

What’s M. smegmatis? Put simply, M. smegmatis is a bacterium in the phylum Actinobacteria and the genus Mycobacterium. Along with this M. smegmatis is a genus relative of the deadly M. tuberculosis and is the host, the ‘being of lesser complexity’ mentioned above.

Okay, well what was the grizzly process described above and is it really that nasty?

Yeah it is, although the host isn’t sentient, and we are therefore unable to say much to how M. smegmatis feels about the whole ordeal the results are as described above. The ‘lifeless machine made of DNA and protein’ is a phage (a form of virus) and the description is a summary of the lytic cycle and the culmination of the lysogenic cycle, two of the ways that phages make baby phages.

More details – phages and making babies.

Viruses can replicate only via host cells. Unlike all living species on this earth, viruses lack the ability to reproduce, which in most circumstances would be seen as a factor that would limit their potential to exist. Phages though have found a way around this. A phage attaches to a specific host cell, infects it with their own genetic material and uses some of the organelles (the cellular-machinery that makes up the innards of a cell) to make clones of itself. In the lytic cycle, this leads directly to the whole seam-rupturing-death-thing where in the lysogenic cycle there are a few ‘dormant’ stages, during which the host cell makes more host cells that are also infected with the phage-DNA before all of the infected cells transition to the seam-rupturing-death-thing too.

Okay, so who’s Robyn?

Originally, Robyn was a chicken (pictured below and at her very sassy best).

Screen Shot 2018-05-03 at 11.40.13 PM

Unfortunately, after a long and truly happy life, this incarnation of Robyn passed away earlier this year, which poses the question, who’s Robyn now? Well, Robyn is the name I’ve lovingly given (in tribute to the chicken) to my phage. My very own virulent-phage, more specifically a bacteriophage (a phage that targets only bacterial cells). I isolated Robyn (the phage not the chicken) from a compost-based environmental sample and have since studied and kept ‘her’ happily fed and sustained with a steady supply of our bacterial friend M. smegmatis.In summary, Robyn is a genetically unique bacteriophage that predates M. smegmatis and whose discovery I’m hoping will be entirely new to science.

In the picture below Robyn-phage is far too tiny to see, but the apparent holes (called plaques) visible in the agar-gel are areas where Robyn and or/other phages have killed all the bacteria growing in the plate.

IMG_1606[1]

Time to wrap things up.

Why, oh why study bacteriophages?

For me the reasons are myriad, although primarily it falls to my love of science and my favourite aspect of science. Science is about discovery and exploration. To uncover something entirely new to science has been a goal of mine since childhood and studying bacteriophages gives me a chance to do just this. Unless you’re my biased mum, that doesn’t inherently benefit you though, and I said before I’d get your approval, so why should you care?

How’d you like to die of a papercut? It could be a real possibility for the human race in the near future. Antibiotic resistance amongst bacteria has been termed as a crisis (Neu, 1992). The issue here is that bacteria are not only capable of becoming resistant to antibiotics but via natural selection, research indicates the more that we use antibiotics the more likely it is that antibiotic-resistant strains of bacteria will emerge (Gregory, Saunders, & Saunders, 2010), potentially taking our biggest guns for keeping bacterial infections at bay out of commission. This is where the phages and their incredible bacteria-killing-powers come in.

I mentioned earlier that M. smegmatis (which is relatively benign) is related to M. tuberculosis (which is very much the opposite of benign) and fortunately for us, there are phages out there that are adapted to kill each. Phages are highly specialised to their host cells which means that, although it is extremely unlikely that Robyn as phage of M. smegmatisis also a phage of M. tuberculosis, to study Robyn and other bacteriophages is to better understand how bacteriophages operate. To better understand how bacteriophages operate could just be the silver bullet that humanity needs to prevent a bacterial driven apocalypse.

This field of research in its early stages, but we’re doing our bit, we’re being monsters for you and I hope that makes you smile.

I’ll keep you posted.

Bibliography

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Trust the Process

It’s fair to say that the phage journey so far has taken me by surprise. For someone who is more passionate about chemistry, physics and mathematics I did not expect to enjoy my time in the laboratory as much as I have. Although it has been far from smooth sailing, the hurdles have been positive stepping stones for becoming a better overall scientist.

A bacteriophage or in short, a phage is a virus which infects and kills bacteria. Phage’s can vary significantly in their genetic material with some of them being useful towards killing unwanted bacteria(1). In my phage hunt class this year we are specifically targeting phage’s which infect the bacteria mycobacterium smegmatis, which is closely related to Mycobacterium Tuberculosis. Mycobacterium Tuberculosis is a bacteria responsible for one of the oldest known human diseases(2). The research we are conducting will hopefully yield results which could be used to help combat this disease, improving our ability to control it.

My phage journey began with an unexpected positive start. Having minimal existing experience in biology labs, I was unfamiliar with certain techniques which were vital for carrying out successful experiments. However, to my surprise this didn’t seem to be an issue initially with the early discovery of plaques. These are small transparent clearings on the plates which are good indications that the host bacteria have been infected by phage(s). Below is an example of some of the positive results I received when I diluted my samples to attempt to isolate phage’s. The photos from left to right represent 10-3ul, 10-4ul, 10-5ul and 10-6ul (ul=10-6L) dilutions of the original neat sample. The small clearings are they plaques formed as a result of the presence of phage(s).

plate 1plate 2plate 3plate 4

Diluted plate 10-3ul  Diluted plate 10-4ul  Diluted plate 10-5ul  Diluted plate 10-6ul

I had spoken to students the year above me about their phage experience and had been given the impression that if it does go to plan, it doesn’t last for long. I was fortunate enough to have the first couple of weeks go reasonably well. However, things didn’t continue this way for very long.

It all started with the second round of serial dilutions. The first round went well, I recorded all my results and left the lab feeling positive. The next lab session I had to carry out serial dilutions once again but this time using a well isolated plaque from one of the diluted plates. This was successful to begin

plate 5

Above is an example of a contaminated plate.

with but when I returned the following week every single plate had been contaminated as shown to the right. This threw me a bit as it pushed me back and made me rethink my techniques. From here I had to use my neat samples from last time to carry out dilutions once again. Returning to the next lab after my previous contamination, I found no plaques on any of the plates which is when I realised I may have lost track of which tubes I was meant to be using. By this time, I realised I may have made a crucial mistake by not labelling my tubes properly. All the work leading up to this point could have been for nothing.journey

However, there was a silver lining. After my fourth round of serial dilutions my persistence was rewarded as I finally managed to regain some plaques. I had managed to find my original neat samples so was able to revive my phage, which I thought had been lost. This was very satisfying and helped me realise every day in phage is going to be different as well as a challenge, which will be both difficult and rewarding. I have now been able to gain more of an insight into what life in the scientific community will be like after my studies. In some ways it seems daunting, but in others I see it as a number of challenges which can only make me better.

What I’ve found interesting from the experience so far is that nothing will ever go according to plan no matter how well you think you’ve followed instructions. It is important to anticipate minor hiccups along the way so that when they do occur you aren’t confused as to where to go next. Although this was an inconvenience, I am thankful for this experience as it has made me focus more on the process rather than the end result. Too often it becomes easy to get caught up in striving to reach completion without actually understanding and trusting the process. I feel as though I am guilty of this and so it seems appropriate that this has acted as a wakeup call.

 

challenge

 

  1. Brussow, H., & Hendrix, R. W. (2002). Phage Genomics: Small is beautiful. Cell, 108(1), 13-16.
  2. Smith, I. (2003). Mycobacterium tuberculosisPathogenesis and Molecular Determinants of Virulence. Clinical Microbiology Reviews, 16(3), 463-496.
  3. Image retrieved from https://www.datasciencecentral.com/profiles/blogs/big-data-science-expectation-vs-reality
  4. Image retrieved from http://thetrigirlchronicles.com/challenge-your-limits/

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My Phage Hunt Experience

After thinking about what to write for my blog post, I decided to write about my experience so far in 246.202. However rather than be proactive, and finish my blog post early. I was…unproductive and left it to sit. Now coming back to it later (and close to its due date), I looked at what I had written, and saw that I was talking about all my struggles at the time, and what I had done wrong. And now, still see what I did wrong. But now just see more where I wasn’t right, and what I should’ve done differently. The following are those thoughts:

I have finally found a phage, HOORAY! (imaginary confetti cannons explode). And am feeling great as I have not just found one morphology of phages but….two. Yes I have done it, I have officially completed one of my learning objectives, and am looking forward to the road ahead of nurturing and growing my phages as I study them more indepthly. But now as I hold my two phages in my hands and proceed forward with them I look back at my other 30 or so samples, and reflect on what I did differently, what I did wrong and where I was just unlucky.

Firstly I look back at all the other environmental samples I took. From the soil and dirt, to the pond, stream and puddle water, and finally, to the compost, compost and compost. Compost from the beginning of the paper had been identified as an ideal environment to acquire a sample from, with it being the most successful in years prior. However, I did not find initial success with compost, and ended up collecting 38 samples before  finding my phages. And guess what medium the sample was from….it was compost. Samples 22 and 23 both contained bacteriophages, and were both located in the compost bins from my Grandfather’s garden. Now I know what you are thinking ‘why am I complaining about finding a phage in compost, when I was told that compost was the best environment to obtain phages from’ or ‘why did it then take me so long to figure this out’ or ‘why was compost not even my first pick’ or ‘why did I not take all my samples from compost’ or ‘why’….Ok, ok, ok, I get it. Maybe I should have had more compost samples, but at the end of the day, that still did not ensure that I would find a bacteriophage.

A big part of finding a bacteriophage is just luck. Some people get lucky and find a phage on their first try, and others (me)…don’t. I mean there is countless bacteriophages in the biosphere, with them making up a large group of organisms in the global population. With estimates claiming up to 10^31 bacteriophage particles on earth (1). On top off this the global phage population is both old and dynamic, but yet the knowledge on bacteriophages is incredibly shallow, as a large proportion of bacteriophages are still undiscovered and unsequenced. So surely I would have been able to find a bacteriophage, I mean I can’t be that unlucky…can I?

So if it wasn’t that my samples were bad, and that I’m not that unlucky, then what could the reason be that it took me so long to find phages, hmmmm….(jaws theme starts playing)…dunnn…hmm what could it be….dunn….no surely not that….dunn dunn…no…dunn dunn…..CONTAMINATION! Yes maybe the reason that I could not find any phages was that I contaminated all my samples during my direct phage isolation protocol. Maybe the whole time it wasn’t bad luck, or choosing the wrong sites for my samples. It was just me, entirely…my…fault.

Now I don’t know whether I contaminated all my previous samples, or whether I even had bacteriophages in those samples. But now, after having archived my bacteriophage and waiting to look at it under an electron microscope I look back at what I have done to get to this point, and how different I see it all now.

I now look back at my choice of samples, and question some of them. Not because they weren’t all compost, but because I did not really think before choosing them. Bacteriophages inhabit environments that mirror the bacteria that it infects. We used the bacteria Mycobacterium smegmatis. If I had thought more carefully about my samples, by simply digging deeper for my soil and compost samples, I may very well have had more luck at finding phages.

Speaking of luck, whether I was lucky or not, and found or did not find phages is negligible. As finding a phage was not a race, and neither is scientific discovery…okay, okay. A race by definition is “a situation in which individuals or groups compete to be first to achieve a particular objective.” And scientific discovery is kind of a race, but not in a sense of the first one wins, and that is the end of it. There are many different paths that can lead to the same objective in science, and all can be ‘winners’.

‘Winning’ in science is not defined by being the first person to the end goal, but the person with the best and most sound theory. This can mean that you are not ‘lucky’ and discover something right away. It can also mean that you do contaminate your results, but as long as you gain information and knowledge from it that is fine. As doing well in scientific discovery is putting your best information forward, and that includes your mistakes.

So, in conclusion I have learnt a lot from 246.202, as it has been much more of practical paper that has really simulated what an actual laboratory would be like. It has taught me to think more scientifically about my investigatory choices. To accept that discovery and progression both take time in science, and that there is no time limit for either of them. And finally, that doing something wrong is not always bad, as you still gain information and experience from it.

References:

  1. Hendrickson, H. (2018). 246.202 Bacteriophage Discovery and Genomics, Course and Assessment Guide. Auckland, New Zealand: Massey University
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Bacteria Wars Episode 5. The Bubonic Strikes Back!

Just a mere 150 years ago, a small cut could easily seal your fate. Just a small cut, enough to form what was one of the greatest killers in human history. Infection! An infection can be a multitude of things, being a broad term to cover many different ailments all deriving from a bacteria breeding and living inside your flesh. Potentially leading to life threatening septicaemia – poisoning of the blood – this leads to bacteria entering the heart, brain and other vital organs. Potentially leading to complete shutdown of vital systems. However, we have the greatest discovery of the 20th century to thank! ANTIBIOTICS!

Discovered by near accident in 1928 by Scottish scientist Alexander Flemming. When he noticed a small culture of Staphylococci had been left open, now contaminated with a blue-green mould. Upon closer inspection Flemming noticed that the bacterial growth had been inhibited or completely stopped by the presence of this fungus. Known as Penicillium. This would lead to the eventual creation of the worlds first true antibiotic. Penicillin. (1)

However, this was not a discovery without issue. Flemming himself, and other scientists knew that bacteria would develop resistance to these antibiotics through horizontal gene transfer and evolution through the selection pressure we were now applying. However, this warning was not heeded. In the decades since antibiotics have been overused, and abused by the medical industry, households, and most of all, the food industry. With saturation of antibiotics into animal feed we have advanced the rate at which bacteria are exposed to these antibiotics, increasing the speed at which they can develop resistance. (2)

Now then, how can we stop, prevent or mitigate these effects? well the answer is simple and may sound like your nutty aunt. WE USE NATURE! There are organisms called BACTERIOPHAGE which are a type of virus that targets and use bacteria to reproduce. How they do this is through injecting their own genetic material into the bacteria. This then integrates its self into the hosts DNA forcing it to produce more of the Bacteriophage. This stops the bacteria from reproducing.  In the same token, it further produces more of the Bacteria fighting Phage.

But, I hear you ask “aren’t viruses potentially dangerous to us too?” technically yes. BUT, in this case it is a Bacteriophage and these types of viruses target a specific strain of bacteria. This is of no danger to the human body and tests are already being done on human subjects as to its potential use and effects. However this poses other issues, with the high specificity of Bacteriophages a specific treatment will have to be developed for each individual off of a cocktail of different phages.

Bacteriophage were first reported in 1915 by Frederick Twort and in 1917 by Felix d’Hérelle. Both noticing when the phage was present in in stool samples of patients sick with dysentery they would soon begin to recover. This was an imediate recognition in the medical field as a new way forward for eliminating bacterialpathogens. However, the equipment necessary to isolate, locate and purify specific Phages had not yet been developed. Since 1923 the Eliava institute in Tbilisi, Georgia has carried on this research and is the first noted institute to use Phage therapy as a legitimate medicine and viable treatment option. (3)

Screen Shot 2018-05-02 at 7.33.36 PM.png

(F.1)          Frederick Twort

However, currently a major issue with Phage therapy is that it has not yet been legislated as safe in western hospitals. With the exception of “experimental” or “supplemental use” in some US states. However change is on the horizon, and it has to be with the rising cases of “super bugs” in hospitals across the world. In 2013 there was the 20th biennial evergreen international phage meeting. Showing promise for international cooperation in this new field of medical science.

Phage therapy is a proven form of treatment for terminal bacterial diseases. As in the Russian federation they report a 50% efficacy of their treatment. Meaning out of every 100 patients with a terminal illness 50 are healed and survive the condition that would normally kill them. Although this is not a desirable percentage. It must be remembered that this treatment is still in its infancy and it is at times very difficult to determine the cause of the infection to create a valid treatment.(4)

Now, the question lies in the hand of the next generation of scientists and the public. As more research needs to be done in the field. While public knowledge and opinion needs to change on the topic. This is potentially the medical discovery of the century. However, when you are at the cutting edge of science, it is very easy to slip.

 

References:

 

Figure 1:Obituary Notices of Fellows of the Royal Society, Vol. 7, No. 20.

1  Haven, Kendall F. (1994). Marvels of Science : 50 Fascinating 5-Minute Reads. Littleton, CO: Libraries Unlimited

2  Bud, Robert (2009). Penicillin: Triumph and Tragedy. Oxford University Press. 

Twort, F. W. (1915). “An investigation on the nature of ultra-microscopic viruses”

4 В.Н.Крылов (5 April 2007) (translated)

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When life gives you mould, make penicillin.

You’re lying in a bath with water over 50 degrees. There isn’t much time; veins under your skin are pulsating in distress. In seconds heatstroke paralyses your body. Your vessels expand, dragging the blood to the surface. Sweat emanates, pooling in the crevices of your skin. Every cell in your body is beginning to die.

The most powerful of humans are incapable of surviving such extreme temperatures. No other organism could, right?

Wrong.

Where humans can only survive temperatures of 45 degrees, select bacteria thrive upwards of double this. Some are able to survive in extreme salt, others prefer uninhabitable cold. Extremophiles are organisms that can survive in historically unlivable environments. Discovered in a boiling hot spring in the 1970’s, these tiny remarkables could rid humanity of some of the most dangerous diseases.
In the same decade, the success of vaccinations had eradicated smallpox worldwide. The measles, mumps and rubella preventatives had massive success and were combined into one life-saving vaccine (MMR). While most of the world was waiting in hope and anticipation, some were impatient and suspicious. The Diphtheria, Tetanus and Pertussis vaccine had only been created twenty years prior, but the 1970’s saw the world’s first international controversy over its safety. 25 years later, this small rumble sparked an avalanche. The vaccine under scrutiny? MMR. The allegation?

The MMR vaccine may have direct links to autism.

In Britain, a doctor named Andrew Wakefield wrote a paper accusing medical professionals of unsafely releasing the vaccine. Fear seized the public instantly, taking hold even stronger than in the 1970’s. Through further investigation, it was found that this “successful” doctor had been bribed by a law firm. The firm wanted evidence in support of parents who alleged MMR had harmed their children. It was only in 2010 Wakefield’s initial paper was retracted and he was barred from practicing medicine. All further studies have found no link to autism, but some still support Wakefield’s work.

All the while, the increasing use of antibiotics was wreaking silent havoc on our world. Since Sir Alexander Fleming first discovered the antibiotic penicillin, resistance has been known and feared. In the acceptation of his Nobel Prize, Fleming stated, “It is not difficult to make microbes resistant” in a warning over the future of antibiotics. To this day, almost all antibiotics have undergone some form of resistance. According to ‘The Review on Antimicrobial Resistance’, these mutant bacteria will kill more people than cancer by 2050. Antibiotic resistance is deadly and holds immense dangers for the future of humanity.

You may be thinking that at this point it’s time to throw in the towel. How could we possibly get control of microscopic killers when our global culture can’t decide if pineapple goes on pizza? Simple: we find something else to take control of the bacteria. Bacteriophages are viruses that specifically attack bacteria. They inject their DNA into the bacteria and force reproduction of countless bacteriophages. These offspring go on to inject more bacteria, producing an army of viruses.
If we were to imagine a bacterial disease such as Tuberculosis, the manipulation of bacteriophages could be revolutionary. Our ability to engineer a bacteriophage to override the deadly Tuberculosis DNA may be far more successful than the mildly-successful vaccine that already exists. Instead of fighting bacteria with more bacteria, we can use a medium that has no detriment to the future of humanity.  It seems a fool-proof way to destroy bacterial diseases without the side-effects of current antibiotics.
It is not, however, that easy. Researchers are stuck in that most bacteriophages struggle to survive harsh environments. Our body undergoes extremes, especially when sick with a bacterial infection or cancer. To harness the power of bacteriophages, we must manipulate rare strains’ abilities to survive extremes. A perfect example is of geophiles, extremophiles that thrive in soil. It was recently discovered that these extremophiles prevent gastric cancer caused by bacterial infections. Overall, we have the process’ of bacteriophage reproduction and the survival success of extremophiles. Bacteriophage extremophiles may save humanity.

Fast forward to 2012 and Samuel Dubosie is awarded a grant that merges together these powerful elements of biology. The ‘Bill and Melinda Gates Foundation’ works to “combat infectious diseases that particularly affect the poorest”. They award the grant with developing countries in mind and create products that are easily available and cheap to source. In awarding Dubosie, there is vital funding for creating a new wave of vaccines. One that doesn’t overuse antibiotics or encourage their evolution into resistance.
In hideously scientific terms, this grant will aid in “developing a vaccine platform that uses bacteriophages that are structurally stable in one of Earth’s most extreme environments”. This extreme environment is the toxic soda lakes in Kenya, Africa. It’s a lake of extreme salt and high pH, deadly to almost all organisms on Earth. Certain bacteriophage extremophiles thrive in these lakes, injecting their DNA into other extremophiles and reproducing at immense rates. In harnessing these bacteriophages, Dubosie can engineer them to fight the diseases that kill millions worldwide. Extremes of disease within the body can be directly targeted, opening the world to a whole new form of medicine.

The work that Dubosie is attempting is representative of how science needs to evolve. Our current practices with antibiotics will lead us nowhere but to extinction. This is our future unless we harness new ways of thinking. Manipulating the successes of nature for our own survival is something only humanity can do. Instead of plaguing the future, we must protect the future.

P.S. Pineapple does go on pizza

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An Accidental Breakthrough: Bacteriophage Discovery

Based largely on The Forgotten Cure (2012) by Anna Kuchment .

Bacteriophages are incredibly tiny bacteria-killing viruses that most people don’t even know about, let alone think about often. You may however have noticed a bit of a theme in the many blog posts here, we sure do love to talk about these invisible assassins! The way we get so excited about these viruses must make them seem like a revolutionary new discovery in microbiology. While it is true that great progress is being made when it comes to learning more about phages, and they seem to hold great promise to solve many issues we face today (see my previous post, or some of the other posts by my fellow phage hunters), the discovery of bacteriophages actually occurred almost exactly 100 years ago.

The discovery, though very exciting at the time came to be overshadowed by the rise of antibiotics, and it is only recently that the western world has begun to pay attention to bacteriophages once more. Therefore I thought it might be interesting to take a look at just how bacteriophages came to be known to science in order to see just how far things have changed.

 

So before I start let me set the scene. It’s the late 1880s and a young gentleman by the name of Felix D’Herelle has just graduated from high-school. Idealistic, self-confident and somewhat cocky, the young man is on a boat returning from Rio de Janeiro to his home city of Paris. As a graduation gift from his mother, D’Herelle had just finished an exciting 3 month trip around South America when yellow fever, a deadly mosquito-borne disease broke out among the ship’s occupants. Passengers and crew alike perished from the disease, and despite the horrors D’Herelle witnessed it was here that it became clear to our intrepid young adventurer that his life calling was study infectious diseases such as yellow fever.

 

He wrote in his memoirs;

“It is probable that I have, by birth, the first required quality needed to make a good microbe hunter. Most of the passengers were in anguish: I was perfectly calm, I thought I was invincible.”

 

Felix D'Herelle

Figure 1: Felix D’Herelle. Look at this twirly mustachioed rascal. Of course he’d end up in a life or death situation and immediately think about career prospects.

In 1894 D’Herelle moved back to his city of birth, Montreal, and set up his own home laboratory. Despite no formal scientific training, through family connections D’Herelle landed a government position studying fermentation. His lack of formal qualifications didn’t stop D’Herelle from accepting position after position studying fermentation and pest control – including becoming involved in innovative research in locust control using bacteria.

A few years later in 1911 D’Herelle moved back to Paris for an unpaid assistant position at the newly formed Pasteur Institute. With a wealth of knowledge and experts in the quickly blossoming field of microbiology at his disposal, D’Herelle was quickly at work pursuing his own research.

 

Now, before I go on to describe the actual discovery of bacteriophages is important to mention that our intrepid protagonist was not actually the first scientist to publish an academic work describing the phenomenon of bacteriophages.Two years prior to D’Herelle’s discovery a British scientist Fredrick William Twort who described small clearings in his bacterial colonies that we would describe as plaques (See Fig. 2 for an example), indicating the presence of phage. Twort however, attributed these to be a transparent product of the bacteria and thus since D’Herelle was the first to propose that the phenomena was caused by a different organism entirely, the credit for the discovery is shared between the two scientists.

 

IMG_1109

Figure 2: An agar plate containing a lawn of Mycobacterium smegmatis (the white, more opaque part), and showing characteristic plaques (the more transparent circular clearances) which are formed by phage infecting and killing bacterial cells in those areas.

As for D’Herelle’s discovery of bacteriophages, like Twort his attention was brought to phages because of their infection of bacterial cultures he was cultivating as part of another project in which he was studying locust control. When small, transparent clearances appeared on his bacterial lawn D’Herelle was puzzled. Unable to replicate the results of these infected cultures and unable to observe the phages under the light microscopes that were available at the time, D’Herelle assumed that the plaques must have been somehow related to the disease of the locusts.

It was only years later in 1916, during World War I when D’Herelle was studying stool samples from soldiers infected with dysentery on the battlefront that he noticed the plaque phenomenon once more. This was a game-changer as it demonstrated to D’Herelle that this was not an event exclusive to the coccobacilli bacteria he studied in the locusts, but could occur in multiple kinds of bacteria from multiple hosts.

events-first-world-war-wwi-western-front-german-soldiers-in-a-stormed-B45EK0.jpg

Figure 3: Photo showing German soldiers in a stormed french trench position on the Western Front during World War I. In the abysmal conditions of the trenches, it’s easy to see why disease killed so many soldiers during WWI. Virulent bacteria and by extension their bacteriophage adversaries would thrive in these close quarters.

Perhaps the most important aspect of the discovery was that D’Herelle noticed that plaques only seemed to appear in the bacterial samples from patients that seemed to be recovering from dysentery. The idea that whatever was causing the plaques could help fight disease was something that D’Herrelle immediately ran with.

 

D’Herrelle dived headfirst into his new discovery, conducting a test where he created a bacterial culture from a dysentery sufferers’ stool and filtered the bacterial culture along with an early form of the phage broth we used in the lab through a ceramic filter. He then added this filtrate to a liquid sample of the patient’s dysentery-causing bacteria and compared it to a control tube containing only bacterial sample and his early phage broth. For a few days the affected patient showed no sign of recovery and both test tubes were cloudy from bacterial growth. The next morning the tube with the filtrate was completely clear while the control tube was still cloudy from bacteria. Even more remarkably the patient was quite miraculously feeling a lot better.

D’Herelle was now certain that whatever was in the filtrate had the ability to kill the disease-causing bacteria, and thus he gave bacteriophages their name which means “bacteria eater.”

 

The method D’Herelle used was in principle the same method we used in our phage identification, but instead of using liquid cultures and judging phage presence based on the amount of bacteria present, we poured our filtrate onto a lawn bacteria and used plagues as indication of phage.

 

What followed was a paper published by D’Herelle in 1917 outlining his new discovery, and multiple trials by him and other researchers using isolated phage to treat illness. Some of these medical trials showed extremely promising results, with one study by physicians at the Baylor University College of Medicine in Dallas reporting 90% survival rate in a group of children suffering from dysentery that were treated with phage, as opposed to a 60% survival rate in the untreated control group. [1] Numerous reports of great success meant that phage therapy only became more and more popular as time went on, before the discovery of antibiotics of course.

 

D’Herelle’s discovery did not go unchallenged however. In his first 1917 report the scientist made some very bold guesses as to the nature of bacteriophages, some of which were flat out wrong and contradicted much of the emerging discoveries of the time.

By conducting tests that showed that a small dose of bacteriophage was just as effective as a large dose, D’Herelle correctly asserted that bacteriophages were living organisms as they must be capable of reproduction.

One of his more dubious suggestions was that bacteriophages were actually the “true microbe of immunity” which helped fight off disease in humans.This was in direct opposition to the work of Jules Bordet, his colleague at the Pasteur Institute who won a Nobel Prize for his work with antibodies in the blood and their involvement in the human immune system.

Furthermore while D’Herelle noticed that bacteriophage were specific to certain strains of bacteria, he suggested that phages could adapt to new strains by a process of acclimatization (exposure to other bacteria). We know now that phage can only adapt to infect new bacterial hosts due to mutations, but often at the cost of their ability to infect their previous host as bacteria posses quite specific and complex defense mechanisms against bacteriophages.

 

All and all despite D’Herelle’s lack of formal training and some dubious hypothesis along the way, his determination and ingenuity led to a discovery that would be built upon by countless scientists to come and be used for a variety of tasks. From phage therapy treating bacterial infections to transgenic organisms made using bacteriophage vectors, we owe a lot of what we know and can do today at least in part to Felix D’Herelle. What must have seemed like an annoying blight on his bacterial cultures turned out to be such a massive stroke of luck!

 

If you enjoyed this story you might enjoy The Forgotten Cure (2012) by Anna Kuchment [2], upon which I based a lot of this blog on. It’s a fairly light and compact recounting of the history of bacteriophage and goes into more detail about the rise and decline of phage therapy and what occured after D’Herelle’s disovery.

 

References:

Figure 1: Ryzhikov, B.A., N. Devdariani, & Various at Pasteur Museum. (14 Mar 2017). Under the Sign of Bacteriophage. Science First Hand, 46. https://scfh.ru/en/papers/under-the-sign-of-bacteriophage-paris-tbilisi/

Figure 2: Photograph taken by Leani Oosthuizen

Figure 3: INTERFOTO / Alamy Stock Photo. http://www.alamy.com/stock-photo-events-first-world-war-wwi-western-front-german-soldiers-in-a-stormed-19790260.html

 

  1.  MacNeal, W. J. and Frisbee, Frances C.: Bacteriophage as a Therapeutic Agent in Staphylococcus Bacteremia, Journal of the American Medical Association 99: 1150–1155 (Oct. 1) 1932.

2. Kuchment, A. (2012). The Forgotten Cure: The Past and Future of Phage Therapy. New York, NY, USA: Copernicus Books.

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What is Life?

For a change I think I will start this blog by talking about one of my other courses rather than the phage hunt course I am supposed to talk about. Instead I will discuss astronomy. One of the most interesting parts of astronomy is speculating about the possibility of finding extra-terrestrial life. Of course there would be many challenges first the sheer scale of the universe, along with the background radiation that means we can only search for signals at few frequencies, the fact that life could be microscopic and even that life might not be too happy about being found… However one thing that science fiction seldom includes but which was brought up by Dr Ian Bond is that even if we did find life we might not even realise it because it would be so “alien”.

So I started to think about what extra-terrestrial life might be like if it could be so different to life on earth that we don’t recognise it. Then it occurred to me that we already right here on earth have things that we cant be completely sure of their status as life. This brings me to the course I was supposed to talk about. Viruses are in essence a bundle of genetic material (DNA or RNA) surrounded by a protein coat. They therefore have the same material that allows us to live and they certainly reproduce and evolve by natural selection but does that make them alive?

It does not help that we don’t think much about what makes something alive we usually know life when we see it but if we cant easily define it in an objective way. There have been attempts to define it but these are not necessarily ideal definitions. I learned in year 11 of the acronym MRS GREN that includes the 7 signs of life movement, respiration, sensitivity, growth, reproduction, excretion and nutrition. This seemed very easy at the time so a bee is alive but a river is not alive and fire is almost alive but it is not sensitive. Using this definition a virus is not alive as it does not excrete and cannot move independently.

Later though I found out that it used to be MRS C GREN the C being circulation. The C was removed after it was recognised that bacteria are do not have circulatory systems. This problem here was that the definition was changed to suit what we already consider to be life rather than being used to discern what is or isn’t life. Why then couldn’t you drop other letters and say that a virus is alive. Admittedly life is not the only concept that is difficult to define (science, culture, consciousness…) but these are not the focus of this blog as I don’t want to spend my whole summer holidays writing it even if I was allowed to.

However it seems important to know what life could be. Even if it is not useful to know if a virus is alive maybe so we could know if we find alien life or whether scientists could really produce artificial life. So does the fact that virus can reproduce and evolve by natural selection make it alive? It may well not fire can make copies of itself and natural selection in fact works on anything that can make non identical copies of itself. The historian/scientist Jared Diamond has hypothesised that societies can evolve in a similar way and evolution is fairly easily repeated on a computer. The fact that viruses contain genetic material is also not relevant as any organism when killed still contains genetic material.

It has been hypothesised that viruses may have evolved from cells that when parasitising other cells became progressively simpler which would seem to suggest that they must be alive. However unless one subscribes to the idea of creationism you would have to consider that life itself must have arisen from non living things so the reverse happening does not seem implausible (in any case this is merely a hypothesis). What seems even more convincing (at least to me) though is that some viruses can in fact infect other viruses which makes it impossible to say that they simply something that infects life but is not itself alive.  The most logical solution is probably that there is no sharp divide between life and non living things. That is that there is a set of characteristics we group together and call life but which can exist in varying combinations and we can choose any of them as being essential or unnecessary.  Viruses have been described in one article as biological replicators noting the important thing about viruses is that they interact with life. (The article also notes that everything that we have observed natural selection affecting has some biological origin such as a computer program that is man made). So in nature there entities that replicate, move react to their environment and many other things and entities that don’t it is up to each individual to decide whether those things are alive.

 

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Antibiotics and Microbiomes

As I mentioned in my previous blog “Antibiotic resistance and what we can do about it”, antibiotic resistance is big problem we are currently facing. Every usage of antibiotics contributes to the resistance problem and therefore we need to reduce our use of antibiotics and find alternatives, such as phage therapy. What I have since learnt is that there are more reasons to find alternatives to antibiotics than just antibiotic resistance. Antibiotics can have an impact on our microbiome and that is what I am going to talk about today.

Microbiomes

We have a lot of microorganisms in our bodies. The microorganisms I am discussing are mainly bacteria but there are also other microorganisms such as from the Domain Archaea. It has been estimated that there are around 10 times the bacterial cells in our bodies than human cells [1]. More bacteria than you! Microorganisms are found all over and in our bodies. Different areas contain different communities with different organisms. The mouth and gut communities of one person can be more different then the microbes in a reef and prairie [1]. For more information on this watch this video.

 

Good Bacteria

When we hear about bacteria we may often think about all the ‘bad’ bacteria that cause infections and diseases. However, there is a lot of ‘good’ bacteria that we need to live. For example, some bacteria help us to break down plant fibres [2]. They are also primary sources for some of the nutrients we need. It is thought that bacteria help to ‘prime’ our immune systems while we are children to help prepare for pathogens in later life [2]. In addition, there are many bacteria found on our skin, so many that it may help to prevent other bacteria establishing [2].

 

What antibiotics do to our microbiomes

Antibiotics are drugs used to kill and treat bacterial infections. They are very commonly used and very important in medicine. The issue with antibiotics is that they are not specific in the type of bacteria that they kill and therefore when used will not just kill the type of bacteria that is being targeted. They will kill bacteria, both good and bad.

As we generally take antibiotics orally the gut microbiome is often affected. It can cause a change in around 30% of the bacteria and can have an impact on the function [3]. Once the antibiotic treatment has stopped the gut tends to revert to its original composition, but does not fully recover [3].

Antibiotic exposure in early life is thought to have the most effect on the microbiome. This is because the microbiome changes the most in early life. The first colonisation of microbes after birth is very important [4]. Interestingly the microbiome is effected by the mode of birth delivery. Caesarean births mean that the baby is not exposed to the vaginal microbes and the babies tend to begin with a gut microbiome like an adult skin microbiome. Whereas vaginally delivered birthed babies tend to have gut microbiomes like an adult vaginal microbiome [1]. This means caesarean births tend to give children with a more unstable microbiome, which may be associated with more allergies, asthma and obesity [4].

Then, in the first two to three years of a child’s life, their microbiome becomes more like an adult microbiome. During this time, they are receiving microbes from places such as food, breast milk and the environment [4]. Delays in the development of the microbiome may be caused by undernutrition or antibiotics [4]. Antibiotics can cause a huge change in the community and the earlier this happens the bigger the effect is likely to be.

Alternatives

So, what can we do to keep our microbiomes healthy?

This, along with antibiotic resistance may make it sound like antibiotics are evil and we should completely avoid them. However, we currently need antibiotics to treat bacterial infections. It is important that we use them only when necessary. As talked about in my previous blog, antibiotics are frequently misused and we need to change this.

To account for the loss in bacteria after using antibiotics probiotics can be used [4]. Probiotics, such as Kombucha, contain live ‘good’ bacteria for our gut and therefore help to replenish it.

Kombucha_Mature.jpg

Kombucha including the culture (Mgarten, 2007)

 

The most exciting alternative is using phages! Phages can be used as an alternative to antibiotics as mentioned in my previous blog. Unlike antibiotics phages are specific. This means that they only kill the specific type of bacteria they are targeting eliminating the problem of the microbiome and all the ‘good’ bacteria being killed.

By continuing to do research about phages we are helping to contribute to a future where phage therapy is a widespread alternative to antibiotics. It has been amazing to have had the opportunity to be part of this.

 

References:

  1. Knight, R., How our microbes make us who we are. 2014, TED Talks.
  2. Ashford, M. Could Humans Live Without Bacteria? 2010; Available from: https://www.livescience.com/32761-good-bacteria-boost-immune-system.html.
  3. Francino, M.P., Antibiotics and the Human Gut Microbiome: Dysbioses and Accumulation of Resistances. Front Microbiol, 2015. 6: p. 1543.
  4. Langdon, A., N. Crook, and G. Dantas, The effects of antibiotics on the microbiome throughout development and alternative approaches for therapeutic modulation. Genome Med, 2016. 8(1): p. 39.

 

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Phage Hunt: The Phinal Phrontier

To keep the continuation of these blogs going I thought I’d, again, talk about the adventures of lab work. In the last blog post, we had reached the stage of DNA sequencing as our samples had just been sent off to America. Low and behold, my phage was one of the chosen ones. I actually couldn’t believe this and thought it was very ironic as I had made my dislike of Phage Hunt apparent from the beginning. I’m pretty sure it was the universe telling me to suck it up and role with things! It was really exciting to know that I would be able to find out more about my bacteriophage and that the hard work and tears had paid off.

My phage, Beatrix, and Leani’s phage, Daegal, had been sequenced and those were the two phages that were being analysed by the class in this half of the semester.

We had all previously thought that the practical labs had been the hardest part of this double semester paper but we had yet to embark on the journey that is learning how to annotate genes. Annotating genes is a very confusing process and once you get the hang of it it’s just tedious but doable. Annotating genes consists of deciding on all of the important parts of the gene. If you don’t get it right then it’ll be engraved into the science guide book forever and you could be the reason why antibiotic resistance cannot be cured. It’s not as serious as that but you do want to make sure you’re as accurate as possible. For example, you need to have reasons and evidence as to why you’re calling the start codon at a specific place. For annotating genes, we have to label the start and stop codons, select the coding potential, Z score, gaps and all this other fancy stuff that I have just gotten used to.

We are lucky as we have resources and databases that help us to make the right calls when we’re annotating. DNA Master is one of these things. It is a beautiful software programme that has all of the genes of the phage listed and all of the information that is needed to make the calls about parts of the gene. We basically go through all the genes, adding notes and double checking all the information so it is as accurate and detailed as possible. Another good aspect of annotating is that we got to work in pairs and so the we we’re able to struggle through with at least some moral support.

Despite the theme of complaining about how hard everything is, Phage Hunt has taught me some valuable lessons about science and life in general. Phage Hunt is one of those papers that allows you to make your own discoveries and is very heavy on the self-directed learning which is really helpful as it teaches you that you can actually do things. It pushes you to do things for yourself and rely on yourself more which is what the real world expects. It also gives you a hand on experience in a field of science that no other paper does. It allows you to gain valuable skills and techniques in the laboratory which you can apply later on in life as we are all science majors. Any practical leaning experience is great to have. The work load is killer but it means you have to keep on top of everything and procrastination cannot exist in your schedule!

Phage Hunt also allowed me to discover more of the wonderful world of science memes which I greatly appreciate.

Phage Hunt has been a roller coaster of a ride but the skills and connections that have been made make it all worth it. Hopefully this has been a cute yet potentially cheesy insight into the life of a Phage Hunter and encourages others to have a go!

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