I don’t Zinc it worked

Bacteriophages (phages) are all around us, they are soil dwellers, water dwellers and possibly even air dwellers. The time has arrived for the 2019 Phage Hunt as six classmates set off to attempt successfully isolate phages. The first step of extracting a phage from their environment is to isolate them. The sample is processed through filtration and then progressing to plating collected solution onto a bacterial lawn of base agar dosed with the target host bacterial strain.

This year, there seemed to be an inconsistency regarding the typical frequency of successfully finding a phage using host strain Mycobacterium smegmatis mc2155. Hence a protocol was incorporated into the direct isolation stage (before protocol 5.3) in attempt to increase the chances of phage isolation- phage precipitation by zinc chloride (referred to here as ZnCl2 precipitation).

sourced from:




sourced from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4691450/

ZnCl2 precipitation, as proposed by Santos, was more commonly used for rapid extraction and purification of phage genomic DNA (Santos, 1991). However, the scope of ZnCl2 precipitation is being redefined, as it is now also being adapted for use in the isolation phage particles from environmental samples (Czajkowski, Ozymko, & Lojkowska, 2016).

As phage particles are mostly comprised of a protein coat that protects genetic material( i.e. found in head and tail proteins), ZnCl2 precipitation works alongside the process of “salting out” proteins in assays for protein precipitation(Arakawa & Timasheff, 1984). Likewise, when phage particles are in a solution containing high salt content, precipitation occurs due to electrolyte-nonelectrolyte interaction (Czajkowski et al., 2016). This causes the nonelectrolyte phage particles to be less soluble at high ZnCl2  concentrations (Czajkowski et al., 2016). Experiments done by Czajkowski et al. suggest the sensitivity of phage detection, which were seen as plaque-like formations on the top agar, using this precipitation method was 10-100 times higher (Czajkowski et al., 2016).

The effects of ZnCl2  precipitation method was experienced first-hand as analysis of plates resulting from samples processed using this method had significantly more plaque counts compared to plates of which the samples were not processed in this way. However, although the sensitivity of phage detection increased, the actual chances of finding phage had not risen. Many plaque-like formations were picked and spot tested however none had given a positive result of a presence of a phage.

Using this method had given many of our classmates false hope, but we had learnt that research is not all about positive findings. A deeper understanding of phage structure and how components of their structure are able to interact with the environment in vivo.

sourced from: https://blog.red-badger.com/2017/3/15/are-you-learning-to-fail-or-failing-to-learn

References

Arakawa, T., & Timasheff, S. N. (1984). Mechanism of protein salting in and salting out by divalent cation salts: balance between hydration and salt binding. Biochemistry, 23(25), 5912-5923. doi:10.1021/bi00320a004

Czajkowski, R., Ozymko, Z., & Lojkowska, E. (2016). Application of zinc chloride precipitation method for rapid isolation and concentration of infectious Pectobacterium spp. and Dickeya spp. lytic bacteriophages from surface water and plant and soil extracts. Folia microbiologica, 61(1), 29-33. doi:10.1007/s12223-015-0411-1

Santos, M. A. (1991). An improved method for the small scale preparation of bacteriophage DNA based on phage precipitation by zinc chloride. Nucleic Acids Res, 19(19), 5442. doi:10.1093/nar/19.19.5442

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Phage: not your usual therapist

Antibiotic drugs have never been more accessible to the general public as it is now. The discovery of antibiotics has been one of the most significant advances for humans, yet now in the present world, its proving to be a double edged sword.  

There seems to be a trend amongst the general population where taking antibiotics is the most convenient and efficient way of battling common colds and fevers. Access to these drugs is becoming a major problem in various countries on a global scale (Carlet & Pittet, 2013). Unregulated “over the counter” prescriptions, inappropriate use/mis-use are all actions contributing to the rise in antimicrobial resistance (AMR) (Carlet & Pittet, 2013).

sourced from: https://www.who.int/en/

The presence of antibiotics in the body exerts immense selection pressure onto the bacterial strains (Schmidt, 2019). This selects for certain bacterial cells that have certain mutations in their genome that allow them to survive and hence produce more colonies with the anti-biotic resistance.

Currently, AMR causes 700,000 deaths per year and is estimated to rise to 10 million deaths to occur yearly after 2050 (Tagliabue & Rappuoli, 2018). It is now an arms race between research advances and bacterial evolution as AMR threatens.

One of the methods to battle AMR is phage therapy. Phage therapy involves bacteriophages (phage), natural viruses that target specific bacterial strains that causes them to lyse (Iftikhar, 2019).

The scope of phage therapy is currently mainly restricted in  food industry to inhibit bacteria growth in food that may cause food poisoning (Iftikhar, 2019). This is due to little research being done to investigate the effects of applying phage therapy onto humans, save for a few rare cases.

UCSD professor of psychiatry, Tom Patterson, marks the breakthrough success of applying phage therapy to human patients. Patterson had contracted a multiple drug resistant (MDR)  Acinetobactor baumannii infection while in Egypt(Schmidt, 2019). The infection progressed at an alarming speed and Patterson fell into a coma following organ failure (Schmidt, 2019). A viral cocktail of multiple phages were finally administered intravenously as a last resort and the improvement was drastic.

sourced from https://www.utoronto.ca/news/u-t-alumna-champions-century-old-therapy-treat-superbug-and-save-husband-s-life

This clinical experience proves that phage therapy is a valuable addition to the tool box of methods in combating AMR diseases. It does not make us choose between antibiotics or phage therapy, but rather gives us hope in making a positive change in medical history of thinking antibiotics and phage therapy. With increasing amounts of research being conducted on phage therapy, there is hope in discovering many possible treatments for life-threatening diseases.

References

Carlet, J., & Pittet, D. (2013). Access to antibiotics: a safety and equity challenge for the next decade. Antimicrobial resistance and infection control, 2(1), 1-1. doi:10.1186/2047-2994-2-1

Iftikhar, N. (2019). What is Phage Therapy? Retrieved from https://www.healthline.com/health/phage-therapy

Schmidt, C. (2019). Phage therapy’s latest makeover. Nat Biotechnol, 37(6), 581-586. doi:10.1038/s41587-019-0133-z

Tagliabue, A., & Rappuoli, R. (2018). Changing Priorities in Vaccinology: Antibiotic Resistance Moving to the Top. Front Immunol, 9, 1068. doi:10.3389/fimmu.2018.01068

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A taste of parenting

Parenting. From what I have been told and read about on the subject, parenting can be good, bad, and wonderful. Although I personally have not experienced what it is like to be a parent to a human baby and probably won’t for another 10 years, I believe that I experienced partially what it is like to be one to my phage, Bazzle. Yes, you may be thinking, ‘Bailey, how in the world would being an actual parent compare to one of a bacteriophage?’ According to Noba (1), there are six stages of parenthood: the image-making stage; the nurturing stage; the authority stage; the interpretive stage; the interdependent stage; and the departure stage. Now don’t get me wrong, some of these stages and what occurs during them may not relate to my personal experience, but most do in some way or form. 

I entered the first stage of parenthood when I was still isolating environmental samples. Dreaming about what it would feel like to finally see a plaque on my plates with my own eyes. Wondering what the morphology would be, what it would actually look like under the electron microscopy. 

I personally believe I entered the second stage, the nurturing stage, when I got to see my phage under the electron microscope. The day I had been waiting for since I had discovered my phage. I was beyond excited and had been counting down the days till I got to see what my baby would look like. When the day had finally arrived, it was just my luck that I was the last one in the group to see mine. It was nerve-wracking having to sit there and watch expecting parents see their phages and the excitement they possessed. The time had eventually arrived for my turn. The gif below sums up my reaction to seeing my phage for the first time. It is one long boy. With an average tail length of 273.7 nm and a head length of 60.1 nm it definitely was different to any other phages in the group.  It might sound completely crazy, but when I saw Bazzle, I felt more connected to it knowing that what I was seeing was mine.

Electron microscope image of phage Bazzle

I believe that I personally did not experience the interpretive stage or the interdependent stage with my phage.  According to Noba the interpretive stage occurs when the children are increasingly exposed to the world and the parents decide what experiences to provide for them. Clearly this cannot relate to the parenting of a phage. The Interdependent stage occurs when the children are now in their teenage years and the parents must ‘redefine their authority’.  I am so glad that phages do not go through a ‘rebellious’ teenage stage with drinking, arguing etc. Although it would be quite amusing to watch it occur. 

However, I did experience the final stage, the departure stage. This occurred on the last day in the lab. The day when we were packing up all our samples, sorting out which was going where and making sure we had enough of the DNA to send off to America. At this point we didn’t know which phage was going to be sequenced, so it felt like I was saying goodbye to Bazzle for the last time.  

Now that the semester is over, and we have no more wet labs left to complete, I am so thankful of the time I spent in the lab for this class. The skills and experiences I learned along the way I would not trade for anything in the world, especially being a parent to Bazzle. 

References:

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The Mystery Bacteria

Sometimes in science, we discover things that are not at all what we set out to find. Through the process of phage hunting, I found a mystery bacteria! The initial discovery was described in my first blog, so feel free to catch up here: https://wordpress.com/read/blogs/67175144/posts/1974. To sum it up, my soil sample came back with a bacteria that was exhibiting phage-like behaviour, but over time it became clear that this was not a phage at all. This leads to some seriously interesting theories. The one I originally came up with is that I had discovered a predatory bacterium! My support for this theory lay in the fact that predatory bacteria behave in a way similar to phages (1). Without sequencing the DNA of my potential bacteria, I took a guess it could be something similar to Bdellovibrio bacteriovorus. This is an example that seemed to be similar in behaviour to mine, but I wouldn’t expect it to be the same, as Bdellovibrio kills gram-negative bacteria (1) and M. smegmatis is gram-positive. There has not been much research done on bacteria that attack gram-positive bacteria, but there are a huge number of similarities in behaviour between Bdellovibrio and my sample, so for now, the research I did sat well with some ideas I came up with.

bdellovibrio pic

Bdellovibrio attacking a bacteria under a microscope (shown in false colours, but how cool??) (7)

Bdellovibrio are very small bacteria that attack and kill other bacteria (1). The miniscule size of predatory bacteria would explain their ability to get through our filters. Their victims are far less specific than that of a bacteriophage (5). They infect in two phases, “a free-living, non-replicative attack phase, and an intracellular growth phase” (1). In the attack phase, they search for their prey, attach, and enter – in much the same way as a lytic phage (2), but phage don’t have the ability to actively search for the bacteria they infect. In the growth phase, they replicate their chromosome and form more tail fibres, again, similar to a phage using the bacterial systems to replicate (3). However, new phages form in the hundreds, whereas Bdellovibrio usually forms only three to six new bacteria before the host cell’s resources are exhausted (1). The host cell then ruptures through lysis, another similarity to phage behaviour, and the new Bdellovibrio bacteriovorus’ are released to continue their mission.

Bdellovibrio attack

Bdellovibrio (yellow) attacking larger bacteria (blue), using the bacteria to replicate itself (6)

The two phases of predatory bacteria line up with the morphology of my plaques. The kill zone is in the middle, while the growth phase contributes to the halo effect.

JLR10-4

10^-4 dilution of my unknown bacteria exhibiting the halo effect

Not too much is known about predatory bacteria at a molecular level. There is still much work to be done to understand their behaviour and how we can utilize them. Even though I have adopted an actual phage from a friend to continue on with the Phage Hunt, we are saving my potential predatory bacterium because we find it too interesting to let go. Hopefully we will be able to sequence it and find out exactly what it is. I still have many questions floating around in my head. The main one comes back to the reason we are hunting phage in the first place. Our target bacterium, Microbacterium smegmatis, is a non-pathogenic cousin to Microbacterium tuberculosis (4). In isolating phages that affect M. smegmatis, we hope to begin the journey of using phage therapy to help those with the devastating TB disease. Taking the specificity of phage into consideration, this is a huge task. However, predatory bacteria are far less specific, and prey on a wide range of pathogens (5). Could we have stumbled onto something that could affect both M. smegmatis and M. tuberculosis? Would using a predatory bacteria be safe? Could we use it to cure TB, or would it merrily attack and kill every bacteria, good and bad, that it finds, much like antibiotics? Would it harm our basic human systems? Could we control it or would it get out of hand inside the human body? I have endless questions to be researched and answered. Questions I never would have considered, research I never would have done, if I hadn’t stumbled across one of the most interesting and exciting discoveries of my brand new scientific career.

 

References

  1. Makowski, Ł., et al., Initiation of Chromosomal Replication in Predatory Bacterium Bdellovibrio bacteriovorus. Frontiers in Microbiology, 2016. 7(1898).
  2. Porter, L.K.L., Bacteriophages. 2019, Treasure Island: StatPearls Publishing.
  3. Weigel, C., & Seitz, H. (2006). Bacteriophage replication modules. FEMS Microbiology Reviews, 30(3), 321-381. 10.1111/j.1574-6976.2006.00015.x
  4. Tyagi, J. S., & Sharma, D. (2002). Mycobacterium smegmatis and turberculosis. Trends in Microbiology, 10(2), 68-69.
  5. Kadouri, D. E., To, K., Shanks, R. M. Q., & Doi, Y. (2013). Predatory Bacteria: A Potential Ally against Multidrug-Resistant Gram-Negative Pathogens. PLOS ONE, 8(5), e63397. 10.1371/journal.pone.0063397
  6. https://www.sciencenews.org/article/live-antibiotics-use-bacteria-kill-bacteria
  7. https://www.npr.org/sections/health-shots/2018/09/06/643661823/predatory-bacteria-might-be-enlisted-in-defense-against-antibiotic-resistance

 

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Sensitive Phriends

Everyone has THAT friend, you know the one who cries watching romcoms or thinks that water is too spicy, lives off venti, half sweet, non-fat, caramel lattes and ignores you for 2 weeks when you roast them a little too much. Who would’ve thought that some bacteriophage, mini bacteria slayers of the world, have a soft and sensitive side too?

Mean girls gif accurately describing those sensitive phages (Retrieved from https://giphy.com/explore/mean-girls)

After tirelessly hunting through 50 environmental samples for a bacteriophage, (phage) I decided that adoption was a better option for me. I was lucky enough to adopt from my phage mate Danielle and could continue on with meeting the next milestone of amplification [1]. To my surprise, the plates that were meant to be webbed [2] in fact had fewer plaques [3] than my adopted isolation plates which raised concerns as to how phage could simply disappear. After some research, I found that it was possible my phage was highly sensitive and could be part of a different morphological family therefore, being better suited to different physiological conditions [4].

My phage Gavin producing small amount of plaques

Siphoviridae phage family are considered the most resistant to extreme environmental conditions, being functional even with large changes of temperature, pH and salinity. These were the predicted family of phage that slay Mycobacterium smegmatis [5] that I have been hunting and have quite the machinery to do so. They are equipped with long, non-contractible tails which allow them to punch through the cell wall of their prey and insert DNA. This DNA is linear and double stranded contained in a non-enveloped head, offering easy integration into the host whether it be hot or cold.

Siphoviridae electron microscope image. Retrieved from https://www.sciencedirect.com/science/article/pii/B9780123846846000045

Bacteria should also beware of Myoviridae, phage that are highly resistant to dry conditions. Just looking at them you know they mean business, kitted out with a helical tail consisting of a contractile sheath and a central tube, add a bowler hat and they’ll be off to Meet the Robinsons. They also have linear DNA and a non-enveloped head showing these same traits of Siphoviradae, present in more hard-wearing phage families.

From L to R: Myoviridae electron microscope image retrieved from http://www.mbio.ncsu.edu/esm/phage/phage.html) , Doris resembling Myoviridae from Meet the Robinsons retrieved from https://disney.fandom.com/wiki/DOR-15) .

Corticoviridae is a family very sensitive to pH changes and function best between 6-8 on the scale. If pH drops below 5, they lose their ability to attack therefore rely on basic substances such as Sodium Chloride and Calcium Chloride to maintain alkalinity. These little softies do not have tails instead have a non-enveloped icosohedral head containing supercoiled, double stranded, circular DNA. Their lack of protection makes them vulnerable to changes in their environment, therefore less stable and more difficult to maintain.

Labelled diagram and electron microscope image of Corticoviridae retrieved from https://talk.ictvonline.org/ictv-reports/ictv_9th_report/dsdna-viruses-2011/w/dsdna_viruses/106/corticoviridae-figures

To identify exactly how sensitive my phage was I needed to amplify it to gain a high enough concentration of particles to be viewed under an electron microscope. To do this I again amplified two samples, one with the normal phage buffer and another with a glycerol-based buffer to accommodate to a picky phage. When receiving my plates after incubation it was discovered that my phage magically returned to creating a large amount of plaques on the normal buffer as well as the glycerol buffer. Evidence of the electron microscope supported this and revealed my phage was bluffing and is actually a cheeky Siphovirdae as originally predicted, clearly shown by its effective tail.

From L to R: My phage Gavin as an electron microscope image and a Siphoviridae phage electron microscope image from https://www.sciencedirect.com/science/article/pii/B9780123846846000045 both showing similarities in morphology.

As phage hunting is quite the rollercoaster, I’m expecting many more phage antics to come. Stay tuned for my next blog which will hopefully see an improvement in my misbehaving, drama queen phage.

Bibliography

1. Hatfull. G., J.-S.D., Pope. W., Poxleitner. M., & Sivanathan. V, The Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science Discovery Guide, 2018. Retrieved from https://seaphagesphagediscoveryguide.helpdocsonline.com/7-0-overview

2. Hatfull. G., J.-S.D., Pope. W., Poxleitner. M., & Sivanathan. V, The Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science Discovery Guide, 2018. Retrieved from https://seaphagesphagediscoveryguide.helpdocsonline.com/7-1-protocol

3. Hatfull. G., J.-S.D., Pope. W., Poxleitner. M., & Sivanathan. V, The Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science Discovery Guide, 2018. Retrieved from https://seaphagesphagediscoveryguide.helpdocsonline.com/5-0-toc

4. Jonczyk, E. et al., The influence of external factors on bacteriophages-review, 2011, pp. 191-200.

5. Hatfull. G., J.-S.D., Pope. W., Poxleitner. M., & Sivanathan. V, The Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science Discovery Guide, 2018. Retrieved from https://seaphagesphagediscoveryguide.helpdocsonline.com/4-1-msmegmatis



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Uncovering phage StrongArm: Part 1…

It all started out too smoothly, like a dream of some sorts which I couldn’t have imagined myself. I found my phage StrongArm from my first lot of samples. Sample 10 to be exact, where I found two plaque (1) regions (see image below), suggesting there may be two bacteriophages (2) (phages) present (Sample 10.1 & 10.2). Whilst everyone else as struggling away finding new samples and undergoing all these different types of isolation techniques (3) there was me just going about my own business wondering why it is taking so long for everyone else to find one. It was actually quite lonely. I wanted to do all these steps together with my fellow phage hunting mates, but fate just didn’t seem to want it that way. Weeks went by with still no one showing any sign of finding a phage. While here I was carrying on, going through rounds of serial dilutions (4) and collecting titers of my phage lysate (5) for both my possible phage samples.

My first plaques that grew from my original sample to show that a phage may be present (Sample 10.1 & 10.2).
Drain outside my house where I found sample 10, which showed signs of being a phage due to plaque growth on agar plates.

There are two types of phage I could possibly end up with. There are phages that undergo a lytic (2) cycle or phage that undergo a lysogenic (2) cycle. I ended up possibly having a mutant of my 10.1 sample as I had some lytic (clear) looking plaques while the rest were more lysogenic and turbid (cloudy) in appearance (see image below). I also had my 10.2 sample which looked clearer and more lytic compared to my 10.1 sample. After very hard and considerate thinking I decided the best choice for my two other possible phage was to adopt them out to two loving families who are fellow Phage Hunters, as I just couldn’t look after them all. It was the hardest decision I’ve had to face so far so I didn’t take it lightly. However, this dream run didn’t end up being so perfect as just a week later I found out the worst news of my life… Hold onto your seats here as this one is a biggy and it rocked my world. This is where the smooth sailing ended. I just knew it was too good to be true…

My small turbid (cloudy) looking plaques after 3 rounds of serial dilutions (Sample 10.1).

My phage stopped working! No plaques were produced on my agar plates when I was testing out my high titer lysate (5). I know it’s quite upsetting and incomprehensible but don’t say I didn’t warn you about it. It was how I’d imagine losing a child, as I’d cared for it and brought it up to become who it was.


An image of me in the lab. Photo credit to Heather Hendrickson.

The true cause for my phage StrongArm to stop responding was unknown at first. Many specialised tests were done to distinguish the true cause (as shown through images below). It came to light that the original Mycobacterium smegmatis (6) (bacterial) culture that the class was using was thought to be contaminated so it no longer resembled M. smegmatis. This contaminated solution of bacteria is what my phage StrongArm infected, so when the original culture ran out and a new culture of bacteria was made my phage StrongArm showed no signs of life. This is because it was most likely trying to infect a different bacterium to what it originally infected, so my phage was now useless to my experiment. This was hard to comprehend as my teeny tiny little lovable phage was unusable to me. I had to say goodbye and move on with my life and find a new phage.

So here I am, phageless and heartbroken back at square one. What am I going to do? I am running out of time and have just wasted weeks purifying (7) and amplifying (8) what I thought was going to be “the one”. Now what?

To see what happens next in my chapter, look out for part 2 coming soon…

References:

1. Griffiths. A., Miller. J., Suzuki. D., Lewontin. R., & Gelbart. W. (2000). Bacteriophage Genetics. An Introduction to Genetic Analysis. 7th edition. Retrieved from https://www.ncbi.nlm.nih.gov/books/NBK21824/

2. Kahn Academy. (n.d.). Bacteriophages. Retrieved from https://www.khanacademy.org/science/biology/biology-of-viruses/virus-biology/a/bacteriophages

3. Hatfull. G., Jacobs-Sera. D., Pope. W., Poxleitner. M., & Sivanathan. V. (2018). Chapter 5: Direct Isolation. The Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science. Supported by Howard Hughes Medical Institute. Retrieved from https://seaphagesphagediscoveryguide.helpdocsonline.com/5-0-overview

4. Hatfull. G., Jacobs-Sera. D., Pope. W., Poxleitner. M., & Sivanathan. V. (2018). Protocol 6.2: Serial Dilutions. The Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science. Supported by Howard Hughes Medical Institute. Retrieved from https://seaphagesphagediscoveryguide.helpdocsonline.com/6-2-protocol

5. Hatfull. G., Jacobs-Sera. D., Pope. W., Poxleitner. M., & Sivanathan. V. (2018). Protocol 6.3: Collecting Plate Lysates. The Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science. Supported by Howard Hughes Medical Institute. Retrieved from https://seaphagesphagediscoveryguide.helpdocsonline.com/6-3-protocol

6. Hatfull. G., Jacobs-Sera. D., Pope. W., Poxleitner. M., & Sivanathan. V. (2018). An Introduction to the Host Bacteria, Mycobacterium smegmatis. The Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science. Supported by Howard Hughes Medical Institute. Retrieved from https://seaphagesphagediscoveryguide.helpdocsonline.com/4-1-msmegmatis

7. Hatfull. G., Jacobs-Sera. D., Pope. W., Poxleitner. M., & Sivanathan. V. (2018). Chapter 6: Picking a Plaque. The Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science. Supported by Howard Hughes Medical Institute. Retrieved from https://seaphagesphagediscoveryguide.helpdocsonline.com/6-0-overview

8. Hatfull. G., Jacobs-Sera. D., Pope. W., Poxleitner. M., & Sivanathan. V. (2018). Chapter 7: Phage Amplification. The Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science. Supported by Howard Hughes Medical Institute. Retrieved from https://seaphagesphagediscoveryguide.helpdocsonline.com/7-0-overview

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The strange things discovered in a phage hunt…

Finding a phage! The excitement is indescribable! To see those clearings of death on the bacterial plates is truly a thing of beauty. Here at Massey University, we have an entire paper dedicated to discovering these tiny viruses – aptly named Phage Hunt. As a passionate genetics major, this paper has quickly become one of my favourites. It began with environmental samples taken from anywhere I thought our target bacteria, Mycobacterium smegmatis, would grow. I took a huge range of samples, excitedly helped by my 5 year old daughter, my partner, and my dog.

Kailee Phage Hunter

A young phage hunter photographed in the foggy wilderness (of our back yard)

My friends started referring to me as “dirt girl”, and that made me bizarrely happy. After all the fun of collecting samples and processing them in the lab to see if I had found a phage, I finally came into the lab one fateful Wednesday and had a plaque! A plaque is a clearing on the plated bacterial lawn, which indicates phage have killed the bacteria in that area.

As soon as I found the plaques on my plate, I knew there was something different about them. They had a distinct area of bacterial death, but they exhibited an unusual halo around the plaque. The plaque was not entirely clear, and the halo even less so. In the undiluted plates, an unusual dimpling effect had taken place, almost as if the “phage” had been killing off the top agar, but not the firmer agar underneath. There were also small specks of what looked like dirt.

As the dilution increased, the morphology of the plaques changed. They became clearer, more circular, more like typical phage plaques, but still were different from anything my colleagues and lecturer had ever seen when working with M. smegmatis. My plates were also much more difficult to photograph than expected, as the plaque clearings did not go all the way through the agar, so light was needed behind the plate in order to see them, and the halos barely show at all.

jlr105e-1.jpg

10^-1 dilution

JLR10^-2

10^-2 dilution

JLR10-4

10-4 dilution, side on angle. Note the central plaque and the halo around it. Very unusual to have this sort of raised effect!!

In my naivety, I thought I had discovered a completely new phage. Looking back, I don’t think this is unreasonable. Phages show an astonishing amount of diversity in the way they behave, and due to the fact that there are estimated to be 1031 phages (1), it is not hard to conclude that us scientists will be seeing things previously never even considered (which is why working with phages is so exciting)!!

I went home and did some research, finding phages that had similar morphologies. These phages were not ones that infected M. smegmatis, but I found that the halos of these online examples were created due to the release of an enzyme that goes ahead of the phage and begins to break down the capsule of the bacteria (2)(see photo below). These enzymes can move more easily through a medium such as a bacterial lawn, as they are smaller than the phage itself. It would be interesting if my “phage” was exhibiting this sort of behaviour, as there is little evidence of this happening with Mycobacterium strains. I thought I had been the one to discover the first M. smegmatis phage that was using enzymes to help their deadly attack! I was full of the excitement of the unknown, and neglected all of my other subjects to read about phages deep into the night.

crater1

Actual phage plaques exhibiting halos from enzymatic activity (2)

Coming into the lab on Friday, I excitedly tied up my hair, pulled on my lab coat and gloves, and raced to the front to grab my plates. My…. empty plates. There was nothing on them. Many things can cause a phage to be “lost” while you are working with it. I had possibly contaminated my plates, or not created my lysate properly. That was okay. I still had my original samples. I would re-plate and see what happened. I was feeling disheartened, but I also accept these things happen as a student trying many things for the first time. I had a gut feeling something was up with my beloved, unusual phage. And I was right. Coming back to my plates after re-plating my original sample, I saw the strangest thing. My plate was growing in a similar way to before… but the growth was yellow! They weren’t behaving at all like a phage anymore. Instead, they showed bacterial characteristics. This stumped everyone in the lab. How could bacteria have gotten through the extremely small filters we use for phage? How could it have created clear death zones in my original M. smegmatis?

I decided to adopt a friends phage to carry on with the work for the paper (meet it here: https://phagesdb.org/phages/Squee/), but I also chose to streak my potential bacteria to see its behaviour, and as I expected, it created colonies on plain plates. A phage would never show this kind of growth. As disappointed as I was to realise that my exciting first discovery wasn’t a phage, my heart soon lifted at the fact that this was something new! What could have been in my innocent looking soil sample? What on earth was it? Keep an eye out for my second blog…. The Mystery Bacteria!

References

  • Welkin H Pope, C. A. B., Daniel A Russell, Deborah Jacobs-Sera, David J Asai, Steven G Cresawn, William R Jacobs Jr, Roger W Hendrix, Jeffrey G Lawrence, Graham F Hatfull Is a corresponding author , Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science, Phage Hunters Integrating Research and Education, Mycobacterial Genetics Course (2015). Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity. eLIFE 
  • Eklund, C., & Wyss, O. (1962). ENZYME ASSOCIATED WITH BACTERIOPHAGE INFECTION. Journal of Bacteriology, 84(6), 1209-1215
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The resilience behind phage hunting

The putrid aroma wafted through the air till it reached my senses. Not in my wildest dreams did I think that when I signed up for genetics, I would be frolicking around, elbows in decomposing matter. With my head held high in determination, I dove my gloved covered hands into the filth that was before me.  This was not my first compost diving adventure but boy oh boy was I hoping it was going to be my last. As the weeks flew by and the negative results and failed samples racked up, my determination to success was being tested. But I never gave up. 

As a millennial, I have been told through the media time and time again that my generation shows no resilience (1). My collection of environmental samples proved that I, as a millennial can show resilience.  I grew up in a household which supported and pushed me to show resilience in everyday situations. I personally believe that good things happen when we as individuals learn to become more resilient in our everyday lives. Being resilient isn’t just doing one thing, it’s about bouncing back with optimism, being flexible about how we see things, taking a little risk and doing something about it. It’s about the emotional strength you hold inside (2). The more resilient you become, the more you adapt in situations and see the positive side of the negatives. The most resilient approach of all is reaching out, asking for help and recognizing that we need support (3).   

Personally, I believe that I showed all of these traits during my time in the labs. With every negative sample, I kept my chin up high. I used different methods of isolation to see if they achieved different results. Most importantly, I asked for help when I knew that I needed it in order to improve. 

Over the last 12 weeks I have spent almost 72 hours in the lab, failing and succeeding. 42 of those hours I spent using various methods to isolate potential phages from my samples. When my peers in my lab were discovering phages of their own, I was beginning to lose hope, not only in myself but also in my ability as a phage hunter.  I gave my blood, sweat, and tears into discovering a phage and still all I was having were negative samples.

Figure 1: Picture of myself in the lab directly isolating my environmental samples.
Photo credit to Heather Hendrickson.

On the last day that I was allowed to isolate my latest my sample before I had to adopt, I finally had a plaque on one of my own plates. Not only that but it was also sample 100. This was a huge weight off my chest. This milestone of reaching 100 samples before finding a phage shows the resilience I displayed in order to overcome the many negative results. From just the short amount of time I’ve spent inside the labs, I’ve learned an awful lot about myself and the strength I store inside. Truth be told, I have never felt true resilience until I became a phage hunter. 

Figure 2: Picture of sample 100 showing the plaque found using enrichment isolation.

References:

  1. Mccolgan, C. (2018). Millennials: The resilient generation. Retrieved from https://businessnewswales.com/millennials-resilient-generation/
  2. Alessandra, T. (2014). What does resilience mean to you?Retrieved from https://www.speakersoffice.com/what-does-resilience-mean-to-you/
  3. American Psychological Association. (n.d.). The road to resilience.Retrieved from https://www.apa.org/helpcenter/road-resilience
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The side of scientific research that they don’t tell you about . . .

When you think of scientific research and discoveries, you often think of those Aha! moments in popular media like when Doc Martin comes up with the idea for the flux capacitor or when Doctor Frankenstein created his monster by harnessing the power of lightning! “It’s alive!”. However, like most things in movies, that’s just Hollywood exaggerating and glamorizing reality for our entertainment. What Hollywood and scientists don’t show or tell you about is the hours and hours of blood, sweat and, tears that goes on behind the scenes of those big Aha! moments which is something that this phage hunter is slowly learning.
This blog however is not going to be all doom and gloom, I thought I’d share the adventure that Phage hunting has taken me on thus far and give my perspective on why failure is a good thing.

So, here’s some stuff about me to give this blog (and future blogs) a bit more context, I am an undergraduate student at Massey University completing a Bachelor of Science, double majoring in genetics and psychology. I am really interested in research from both disciplines and for obvious reasons I really enjoy experimental psychology and human genetics. One day I would love to be able to do research into neurological degenerative disorders such as Parkinson’s disease or looking at specific learning disorders. Basically, I want to be able to stick electrodes to people’s heads and look at their genome and hopefully find a way to help improve their lives.

My introduction to scientific research has been an interesting one, up until the 5th of March 2019, I had had nothing but disappointment (yes, I thought this would be easy like in the movies). I went through a grand total of 92 samples in order to find my phage (stay tuned for details on Phage Golddigger to come in a future blog).
I had collected everything from multiple different compost samples, to river bank samples, to homemade kombucha, to rabbit poo, to fresh spring water out of a drinking well (Picture one is of me in the well collecting my sample), and everything in between.

Picture One: Picture of me climbing out of the water well after collecting one of my samples. The well was on private land (thank you to the land owner for suggesting it) near Wainamu Lake, Auckland (DMMW 10 on phage hunting map, link in next paragraph).
Photo credit to my Dad, Graeme Williams.

As you can see in picture One, my extensive phage hunting has taken me to some areas of New Zealand that I ordinarily wouldn’t prioritize time for during semester (Here is a link to a map of where some of my samples were found https://drive.google.com/open?id=17CSRYFl1B39D-lElJ2cR3x2m14ygn3CW&usp=sharing ) and I enjoyed the adventure that came with looking for different samples, even if it became a bit tedious and frustrating after the 40th odd sample…
Phage hunting has also allowed me to spend time with and teach a lot of my friends and family about bacteriophage and other things that I am learning about in my other papers. This has helped them to understand why I am always studying, working or doing assignments and hardly ever have time to reply to texts and spend time with them. Recruiting the people I care about to be my collection assistants has also helped to keep my stress levels down and ground me a bit, because students easily get caught up and consumed by deadlines and everything that we have to do and complete and spending an hour digging through compost and collecting samples with my mum is way cheaper then therapy.

Peltzer and Pengpid (2015) (https://www-tandfonline-com.ezproxy.massey.ac.nz/doi/abs/10.3109/13651501.2015.1082598) discovered in a questionnaire based study conducted with 20222 undergraduate university students, that 24% had moderate depression symptoms and 12.8% had severe depression symptoms. The researchers identified that stress and regression of social variables and support networks were major factors in depression symptoms and risk-taking behaviour (such as smoking, excessive alcohol consumption and lack of self-care) among undergraduate students.

I think it’s important to note here that risk taking behaviour can also be due to things like sudden freedom and reduction of authoritative figures (especially among first year students). However, on the other side of the coin increased depression and anxiety is often due to spoken and unspoken pressures put on the student. In todays society you’re expected to have some form of higher education (i.e. diploma, apprenticeship, degree, etc.) in order to get anything above an entry level retail or hospitality job and because this expectation is very isolated to our generation, a lot of students are the first in their family to go to University or even worse, are expected to follow in their older sibling’s, cousin’s or Parent’s footsteps and pursue a higher education which puts massive pressure on the student and creates a severe fear of failure and reinforces the idea that you have to do it yourself and that asking for help is failing. This is something that I struggled with (and still do to a degree), especially in my first year but if I could tell any students reading this one thing, failure is okay, just try again and that asking for help or further explanation is not failing and will actually help you in the long run.

So, what does all of this stress and failure teach me? Why is failure a good thing?
Well to put it bluntly, the stress and failure that I have experienced with phage hunting and the overwhelming pressure to complete my degree with decent grades has taught me a great deal about resilience and determination.
Dictonary.com defines resilience as the ability to recover readily from illness, depression or adversity (https://www.dictionary.com/browse/resilience). In other words, “just keep swimming” like Dory said to.
From personal experience, failure is a good thing because it gives you the opportunity to stand back for a second a assess your options and figure out your next steps; What are you going to do differently? Who can you talk to for advice? Where do you go next? And the most important one to remember, how do you climb a mountain?

So, to conclude, failure hurts but it teaches you how to be resilient and push through adversity in order to reach your goals and become better person and scientist .

References:

Peltzer, K., Pengpid, S. (2015) Depressive symptoms and social demographic, stress and health risk behaviour among university students in 26 low-, middle- and high-income countries. International journal of psychiatry in clinical practice, 19(4), 260-266. DOI 10.3109/13651501.2015.1082598
https://www-tandfonline-com.ezproxy.massey.ac.nz/doi/abs/10.3109/13651501.2015.1082598

https://www.dictionary.com/browse/resilience

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Phage’s fighting climate change

 

 

 

What a world we now live in. With a traffic jam lasting 10 days, beer not being classed as alcoholic in Russia until 2011 and the average American consuming 600 sodas a year, we certainly live during a time full of surprises(1). However, ultimately all of this has one thing in common and that is over consumption. Petrol, beer and soda make up the smallest fragment of the items we overconsume as a species. Overconsumption can be great! We all love indulging from time to time, but with over 7 billion people doing this from time to time it is our planet that ultimately pays the price in the form of a rapidly changing climate. However, our little friend’s bacteriophages are helping us feel better about our over indulgence by fighting back against climate change. Guess you might be able to have that extra soda after all…

 

The ocean is immense when you look at surface area of our planet alone, but when you look beneath the surface and find that the deepest part of the ocean is 10,902m, it is inevitable that the ocean is a lot more complex than it may initially seem(2). With bacteriophages being the most abundant viruses in the ocean, they are potentially an unsung hero. There exist bacteria called SAR11 which convert dissolved carbon molecules into carbon dioxide which gets released into the atmosphere(2). With carbon dioxide being one of the most abundant greenhouse gases this extra emission increases the amount of heat/energy that gets reflected back to earth, ultimately increasing global temperatures. However, our friend’s bacteriophages and specifically pelagiphages are fighting against these bacteria by destroying them and keeping their numbers under control(2). They are extremely helpful for reducing lots of carbon dioxide from entering the atmosphere and hence are helping us in the fight against climate change.

 

Our friends are also helping to reduce the rise in ocean temperatures which has a significant effect on species such as phytoplankton(3). These little guys are very important as they account for about 50% of the photosynthesis on Earth as well as removing loads of CO2 from the oceans. However, if the ocean temperatures continue to rise this could cause a shift in feeding patterns which could cause their food sources to be lost due to timing(3). With phage’s removing more and more carbon dioxide from the oceans the waters temperatures are kept under some control and these CO2 cycling machines known as phytoplankton can continue to play their part in climate change mitigation.

 

As our population continues to rise we continue to consume at an increased rate with our sinks such as the ocean having to work extra hard. As we continue to get stuck in traffic jams, consume ridiculous amounts of soda and get our heads around how much the Russians must love their beer, bacteriophages fight to neutralise the effects of our actions. We may not be able to see them or even physically see the good work they are doing but they act as the unsung hero’s of our planet.

 

 

 

 

1)    All That’s Interesting. (2018). 100 Interesting Facts About The World To Blow Your Mind. Retrieved from https://allthatsinteresting.com/interesting-facts-about-the-world#32

2)    Geology.com. (2018). Deepest Part of the Ocean – Deepest Ocean Trench. Retrieved from https://geology.com/records/deepest-part-of-the-ocean.shtml

3)    Danovaro, R., Corinaldesi, C., Dell’Anno, A., Fuhrman, J., Middelburg, J., Noble, R. and Suttle, C. (2011). Marine viruses and global climate change. FEMS Microbiology Reviews, 35(6), pp.993-1034.

4)  Keen, E. (2014). A century of phage research: Bacteriophages and the shaping of modern biology. BioEssays, 37(1), pp.6-9.

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