Mycobacterium smegmatis – Why are we hunting for phages that infect this species of bacteria?

If you’ve read any of the other posts on this Phage Hunt NZ blog, you may already know a little about the struggles us hunters go through to find phages. The ultimate aim of our hunt is to discover brand new types of bacteriophages (viruses that infect and usually kill bacteria), in an effort to not only contribute to the world’s collective knowledge of these genetic parasites but also in an attempt to understand our local ecosystems. As microscopically tiny as viruses are, they play a huge role in the microecology of our world. The epic unseen struggle between phages and the bacteria they parasitise, drives a fierce cycle of coevolution which can have big impacts on whole ecologies. This is a very interesting field of research, and despite being discovered more than 100 years ago, with new advances in genetic technology there is now renewed interest in phages.

 

Phages are one of the most abundant organisms on the planet so would think this phage hunt business would be pretty straightforward, right? [1] Not quite. When we search for evidence of phages in our environmental samples, we use a method that filters out bacteria and other large organisms and the leftover solution consisting of phage (hopefully) and added nutrients necessary for phage replication is poured over a lawn of bacteria growing on agar – consisting of one strain of bacteria. In the case of the Phage Hunt New Zealand group the bacterial host we used was Mycobacterium smegmatis, a fairly common soil bacteria. If phages are present and manage to infect the bacteria then small clearances where phages have killed the bacteria, called plaques will form.

 

IMG_1109.JPG

Plaques of my phage Daegal

This first step is where we encounter problems. The issue is that phages are very specific in what bacteria they can infect, with many having host ranges of only a few strains of bacteria. Though there will very likely be many thousands, millions or even billions of phage particles in one small sample, this method will only screen for those that infect the specific bacteria you used to make the bacterial lawn. Furthermore, the conditions which these new strains of phage need to survive may be very different from what is expected, and even if there are many phages present that infect your strain of bacteria the process of extracting them from your samples may be enough to destroy them.

Why not introduce your samples to other types of bacteria? If M. smegmatis phages are so hard to find, why bother sticking with it? The answer is pretty complex and I could probably spend forever discussing it but for now I’ll just give a brief introduction to M. smegmatis and give some reasons as to why we use it.

 

M. smegmatis is a fast growing growing species and this, combined with its comparative non-pathogenicity makes it a very useful substitute for studying the earlier mentioned pathogenic bacteria that it shares many similarities with. [2] Why should this matter to us?

 

Tuberculosis is a serious and potentially deadly disease caused by M. tuberculosis bacteria infecting humans. It spreads through inhalation of the bacteria and can easily spread through populations. It is still a huge issue in many developing countries, and even in New Zealand there are approximately 300 cases of TB diagnosed each year. Though healthy adults infected with the bacteria rarely experience any adverse effects or even show symptoms, in those with compromised or vulnerable immune systems like babies, the elderly or those with AIDS the disease can cause serious illness and if left untreated often results in death. [3]

Luckily an intensive course of antibiotics is usually very effective in treating TB and mortality due to the disease has been reduced significantly. [3] The disease that once claimed entire families is all but nonexistent in the minds of many New Zealanders.

 

Screen-Shot-2014-10-22-at-9.29.23-AM-e1413984813213

Graph showing Tuberculosis mortality among HIV-positive people. (Source: https://www.undispatch.com/map-day-people-die-tuberculosis/)

 

In recent times, the issue of antibiotic resistance has become a real concern. Overuse of antibiotics and a lack of progress in finding new antibiotics has meant that strains of bacteria have evolved that are immune to treatment. These strains threaten to plunge us back into the pre-antibiotic era. 480, 000 people globally are infected with multi-drug resistant TB every year and this number will likely increase. [4]

Antibiotic resistance is causing many to turn to phage therapy, an alternative to antibiotics that involves exposing patients to phages that are specific to the bacteria causing the infection. The phages only infect and kill these target bacteria, leaving the rest of the helpful bacteria in the patient’s bacteria unharmed. [1]

The hope is that along the way in finding new strains of M. smegmatis bacteriophage, we could find phages that infect M. tuberculosis as well and these could potentially be used in phage therapy to treat cases of antibiotic resistant tuberculosis. The development of new medical treatments is a long and arduous process and though not every phage has the chops to be the downfall of tuberculosis, by discovering and studying new that infect M. smegmatis phages we can know that not only are we contributing to scientific progression but possibly to a happier and healthier planet.

PHAGE

References:

 

  1. Bacteriophage therapy treats patient near death with MDR Acinetobacter baumannii. (2017, April 25). Outbreak News Today. Retrieved from http://outbreaknewstoday.com/bacteriophage-therapy-treats-patient-near-death-mdr-acinetobacter-baumannii-45488/

 

  1. Mycobacterium smegmatis. (Last edited 2011, April 22). Microbewiki. https://microbewiki.kenyon.edu/index.php/Mycobacterium_smegmatis

 

  1. Tuberculosis disease. (Last updated 2016, September 9). Ministry of Health. http://www.health.govt.nz/your-health/conditions-and-treatments/diseases-and-illnesses/tuberculosis-disease

 

  1. Antimicrobial Resistance. (Last updated 2016, September). World Health Organisation. http://www.who.int/mediacentre/factsheets/fs194/en/

 

 

Advertisements
Posted in Uncategorized | Leave a comment

The end of Illness?

William Stewart the surgeon general of the United States of America has said “The time has come to close the book on infectious diseases. We have basically wiped out infection in America”. This sounds like pretty good news for a change doesn’t it? Except of course that he said it in 1967 and thus we all know that he was wrong.

Somehow I don’t imagine many people with medical degrees would be stupid enough to say something like that today. The fact is that with smallpox the only significant exception all the diseases that have troubled us in the past are still with humans today. (Yes that includes leprosy and the plague) In fact many of them due to antibiotic resistance have become much more difficult to treat than ever before. The first lecture I heard this year was by Dr Heather Hendrickson on a post antibiotic era we are entering when even trivial infections would often be fatal. Sounds pretty scary right. How did this happen?  Well there are a number of ways we have misused antibiotics.  It is estimated that about 70% of antibiotics used in the developed world are given to farm animals. This is not really a problem by itself but the farmers often don’t bother trying to find out which animals are sick instead they just put the antibiotics in stock feed for all of the animals. The more the bacteria are exposed though the more chances they get to become immune to antibiotics.

That is not to say those used by humans are necessarily put to good use either. Did you ask for antibiotics to treat the last cold you had? Hopefully not because the cold is caused by a virus and the antibiotics will have no effect. But how the antibiotics are used is not the only problem there are also not enough being developed not one entirely new antibiotic was found between the 1970s and 2003. Another issue is that if a drug company could develop a drug that people have to take every day for a month or a drug they have to take every day for the rest of their lives they make a little bit more money if they pick the later option.

is fortunate then that bacteria can themselves get sick. A virus is a non living* pathogen consisting of a piece of DNA in a protein capsid that can reprogram a cell to produce more viruses. While some viruses target cells of animals or plants others target bacteria. These bacteriophages can be used in the fight against bacteria in fact they already have been and they present a number of advantages over antibiotics. 1. They target specific bacteria while antibiotics usually affect any bacteria in the vicinity including those that help us. One scientist working with bacteriophages compared antibiotics and bacteriophages to a “bomb blast and a sneaky ninja” respectively. 2. Bacteriophages can evolve to counter bacterial resistance. Instead of having to find new ones every time bacteria become resistant we can use the same bacteriophages to counter bacteria again and again. 3. They multiply at the site of infection so only a tiny quantity is needed. But we need to discover a bacteriophage before we can use it so university science classes such as mine search for and study bacteriophages so that they may be used to treat illnesses and get practical experience in our study.IMG_1236

This all sounds very promising but bacteriophages are still relatively little known and it is too early to tell whether they are really the silver bullet doctors are hoping for. Then there are still those viruses that target us many of which we have no idea how to treat. So to the honourable surgeon general sorry Billy we got a long way to go mate.

Posted in Uncategorized | Leave a comment

When lab safety goes wrong.

 

Aseptic technique in the microbiology lab is really important with the main aim being to avoid contamination.  Even in the lab there is risk of contamination everywhere, especially when working with highly contagious strains of bacteria. This contamination can be incredibly irritating, expensive and even disastrous.  Contamination risks are everywhere. They are present in the air as airborne microorganisms, on our bodies as dust and other particles and even on lab equipment and surfaces.[1] Proper aseptic technique should reduce the chances of contamination of your experiment and most importantly keep your lab technician happy. This is because it means that resources aren’t wasted and you don’t have to repeat experiments for trivial reasons. It will also maintain the purity of stock samples.

Proper aseptic technique is important for safety in the lab in order to prevent infection and contamination of the environment and people in the lab. In our lab we are working with bacteriophages. Bacteriophages are viruses that infects and kills specific bacteria.

I now understand the importance of this technique after what I believed to be was my beloved first found phage actually turned out to be a form of contamination. In our experience in the lab, we have begun to understand and appreciate the importance of aseptic technique. This technique was new to me and took some getting used to. I often dropped lids and put my hand or sleeve to close to the Bunsen burner flame. Aseptic technique  was especially important in our lab as when working with unique phages, it is important to make sure there is no cross contaminations between individuals work.  We were lucky that there was no cross contamination between individuals and no one ended up with the same phage due to contamination. Thankfully our aseptic technique was up to scratch.

Phages were in fact first discovered by contamination by Frederick William Twort. In 1915 he discovered plaques on his agar plates.[2] Contamination not only leaves opportunities for many new discoveries, it also is the cause of many issues. Only a month ago, the CDC centre for disease control made headlines after 84 laboratory workers were exposed to a potentially deadly strain of anthrax. An investigation into the incident found that the lab was using expired disinfectant and were storing samples in unlocked freezes in unrestricted areas. Fortunately, this outbreak was contained.[3] In other cases, people weren’t so lucky. In 1977 there was an outbreak of influenza in China which spread globally but luckily the virus only caused which caused moderate symptoms such as a light fever.  In another instance, the famous foot and mouth outbreak, which began in Britain from a biosafety lab and caused billions of dollars in damage. This particular disease is spread by cloven-hoofed animals and in the end required over 1500 animals to be culled.[4]

In our lab we work with Mycobacterium Smegmatis. We use this as a bacteria host as it has a similar make up to Mycobacterium Tuberculosis, also know as TB. There are obvious reasons as to why we do not use TB in our labs. TB is highly contagious and is a dangerous bacteria that causes around 1.8 million deaths world wide.[5] This is one of the main reasons we don’t use it and as newbie lab scientists, our aseptic technique would not be sufficient to quantify the risk. This is why we hope to find a phase that can infect Mycobacterium Smegmatis and therefore might also be able to infect and cure TB patients.

 

 

  1. Aseptic Technique and the Transfer of Microorganisms. 2016.
  2. jcturnbullnz, The Pioneers of Phage Virology. 2017.
  3. Newly disclosed CDC biolab failures ‘like a screenplay for a disaster movie. 2016.
  4. BENDER, J., Here Are 5 Times Infectious Diseases Escaped From Laboratory Containment. 2014.
  5. Tuberculosis. 2017.
Posted in Uncategorized | 1 Comment

Mushball’s Big Day Out

Happy life. Just me and my compost. I must admit, it does smell in here from all the food scraps. Such a waste, which makes me wonder why Homo Sapiens even eat? I can survive! All I need is my favourite bacterial host Mycobacterium smegmatis to inject my DNA into, and voilà! My apprentices do the rest.

Wait… I feel movement…there’s light. I’ve never seen light before. Now I’m all wet. I was perfectly happy in my dirt. What’s this substance anyway? Is that calcium chloride enrichment broth? Awesome! I love this stuff! I can reproduce so rapidly in this. Just gotta find some M.smeg to infect. We’re gonna have a party ladies and gentlemen! M.smeg is a bit far away from me, and Nature gave me no way of moving. But this shaking that’s going on is bringing it right to me! This is amazing. I wish my life was this perfect all the time.

Okay, party’s over phages. We are all going to get sucked out of the dirt and squeezed through the exit. The doorway is so small… M.smeg can’t fit through! But I need those hosts… How else can I replicate? I’m panicking now, and I don’t want to leave the party! 

All us phages just sitting here are getting bored, until all of a sudden, there’s so much M.smeg! Where did you guys all come from? I’m glad you’re all sacrificing your bodies for us to survive. How generous. Bathing in M.smeg seems a lot like Homo Sapiens bathing in wine. Such a delicacy.

It’s been a few days now of bathing in an ocean of calcium chloride, hot agar and M.smeg. It’s been so busy here, replicating me, “Mushball”, as much as I can, so I have an army of Mushballs to conquer the world. The other phages don’t have a chance. Every new bath I jump into, I see less and less foreigners and more and more Mushballs! We did it phages! We conquered the world! It’s just you, me, and M.smeg now. There’s so many of us. Billions… Maybe even trillions!

Me and my fellow Mushballs have infected most of the M.smeg bacteria, with heaps of cloned apprentice Mushballs bursting out of each one. I’m exhausted from all this infecting, so it’s good there’s not much more to go. We have to ration them out to last us the winter. 

The most weirdest thing is happening right now. The bacteria are being eaten by something. It’s like a plague dissolving everything so quickly. It’s seen us. It’s coming for the Mushballs. I think it wants to eat us too! It’s getting closer. My body can’t protect my DNA for much longer. Help!

Phew. That was close. The EDTA force has rescued us! We are saved! I just want to go home now, but there’s just one more thing I feel I need to do. I rally up some Mushballs and we clean ourselves off with resin to expose our luscious DNA locks, and rinse them with alcohol to make them shine.

I feel a strange sensation. I feel like I’m destined for something. We all are. Well, I’d hoped so since we don’t just shine up like this for any old picnic! A force is pulling me into position. I think my first photo is being taken. I’ve never had my picture taken before. It’s so exciting!

As the light shines onto me, my whole DNA radiates outwards, and a little voice whispers in my ear…

Say CHEESE! 

2017-5-26 tash

Gel electrophoresis photo for “Mushball” phage

Just a little adventure through the lab work working with a phage named “Mushball”. This image is a characterisation step with extracted DNA.

The Mushball population undergone enrichment, filtration, isolation, amplification, DNA extraction, restriction-enzyme digestion, and gel electrophoresis. 

 

Posted in Uncategorized | Leave a comment

A phage by any other name would smell as sweet – The trials and tribulations of naming your phage

If you’ve ever wandered into the clearance section of your local bookstore I’m sure you’ll have seen at least one book about baby names. Titles like “100,000 Baby Names”, “Cool Names for Babies” and the “The Baby Name Wizard” offer parents with seemingly endless ideas and options for choosing the perfect name for their little bundle of joy. Parents can spend months trying to settle on the right name for their child, and who can blame them? After all, a person’s name can affect how they are treated and perceived, impacting their interpersonal relationships, self-perception, and even their career. 

Naming can be quite a stressful affair.Though I have never had a child of my own, I found myself in a similar situation only a few weeks back when I was trying to find a name to call my phage.

 

Let me explain: For the past semester I have been involved in the SEAPHAGES Phage Hunt at Massey University. Bacteriophages (or phages) are viruses that infect bacteria, hijacking their cellular functions and forcing the bacteria to manufacture more and more virus particles until they lyse the bacteria and break out – ready to start the cycle all over. It’s like a tiny microscopic version of Ridley Scott’s Alien.

As a part of the Phage Hunt Program we collected soil samples to try find phages that infect Mycobacterium smegmatis, a close relative of the tuberculosis bacteria. The hope is that perhaps these bacteriophages could be used to kill these bacteria and treat diseases.

The process was a great learning experience where we got find phage, purify the tiny microscopic viruses, and even extract and analyze their DNA. And the cherry on top: we get to name our very own phage! This was the part I looked forward to the most, what an honor!

 

Created by Digital Micrograph, Gatan Inc.

This is my phage Daegal. Isn’t he just ADORABLE?

Now perhaps I was just naive but I didn’t expect the very first step of the process, finding a phage to be so hard. Despite searching in very many samples (including those from areas where others had found phage), I found not a single phage. Weeks went by as my classmates found phage after phage, and I grew more and more envious. By the time I finally adopted a phage from one of my friends I was determined to keep it alive and give it the best name I could possibly find.

 

The only thing is, naming is harder than you’d think. Especially when you have to think of name no one else has thought of. Unlike a human child, the phages registered as part of our hunt – on the PhagesDB database – could not have the same name as another. If there was already a phage named Brittany, that name is completely out of bounds for your phage. This makes sense, as every species should have a distinct name in order to avoid confusion. Still it is awfully disappointing when your phage, who is clearly a Marcus, has to be named something else. As a result, I scoured PhagesDB to see what names were still available, and I was surprised by what I saw.

ill-name-her-sarah-300x270

 

In the description of each phage, there is a section in which a hunter could explain why they named the phage what they did. As I curiously scrolled through the names, some of the explanations were surprisingly heartfelt. There was no shortage of phages named in honor of children, family, mentors and beloved pets. Names reflected the friendships forged between partner phage founders, honored significant events, and gave a curious insight into the phage hunters themselves. My favorite description was that of the phage “NoodleTree”: In the words of one of the phage discovers “I come to school to grow my noodle.” From a choice of name alone, you can get a snapshot of the friendship between phage hunters. You can read about this and many other great phages at PhagesDB.

 

This made me wonder, what makes a person name their phage what they do? And what does that say about them? In speaking with my classmates, they seemed to feel a sense of responsibility and privilege in choosing a name. My classmate Jo – who named her phage after a Grandmother who she never really got the chance to know as an adult – explained it in a way that seemed to resonate with how I felt. She said this might be our only ever chance to do something like this, name our very own species and leave our mark on the scientific world. We had worked so hard to get here, it would make sense that we would want to choose a name that was meaningful, carefully chosen, and would hopefully inspire in others the same interest in our phage that we had. Though I’m sure the phages couldn’t care less what we call them, what we call a phage often means a lot to us. And when you’re naming a creature you’ve never even seen with your bare eyes, it says a lot about you too.

 

 

Posted in Uncategorized | Leave a comment

The Viralcoaster: My highs and lows

Wow, we are at the end of a semester already!  I don’t know about the rest of my class, but I have been left wanting.  Despite all the time, effort and experience that has been packed into these last 14 weeks, all I want to do is keep coming into the lab and playing with my phage.

There’s something about this ‘choose your own adventure’ styled course that has gotten its hooks into me (or perhaps that’s just a wayward podovirus [1.]).  One of my absolute favourite parts of this journey has been designing or modifying my own experiments to characterise my phage, and I just don’t want to stop!

My hunt for phage began with a stubborn determination to shirk the easy route, the obvious choices.  For the first three weeks I was tramping through our beautiful (and muddy) native bush, picking my way across equine graveyards, and coming perilously close to falling into multiple lakes, all in the quest to conquer the challenge I set for myself – finding a phage somewhere other than a compost bin.  I should have paid more attention to the obvious facts: bacteria love compost, and phages love bacteria.

Eventually I caved, and I get a kind of sentimental warmth from knowing that my phage came from the location-that-shall-not-be-named of my darling mum, an avid gardener.  Not only did my search end somewhere symbolic of my mother, but the name I chose for my precious virus came from my father’s mother.  Both women are actually named Colleen, but my Nana was always known by her middle name, Dulcie.  We lost her when I was a teenager, and I think it’s nice that I’ve taken this opportunity to connect to her a little more in my adult life.  She wouldn’t have given a toss about bacteriophages, but she would have appreciated the thought.

nana photo

I know what you’re thinking: the resemblance is uncanny.

 

After finally hitting the jackpot with plaque assays that I tend to compare to swiss cheese, I proceeded to isolate and process my phage samples ready for the next stage of our adventure.  I started off isolating two phages, before abandoning one to focus my attention on Dulcie.

plate photo for blog

From discovery…                                  to isolation…                                      to amplification

 

Once I had isolated my phage and achieved a high-titer lysate to work with, I proceeded to extract dear Dulcie’s DNA and take a look at it using restriction enzyme digests and gel electrophoresis.  I was pretty stoked with the concentration of DNA in my fourth and fifth rounds of extraction, in which I used a spun-down sample of my lysate.  Those concentrated samples yielded an average of 250 µg of DNA per mL, six times more concentrated than previous extractions.  I had more DNA than I knew what to do with!

My first attempt at gel electrophoresis didn’t go so well, with some degradation of the DNA occurring due to over-enthusiastic nucleases [2].  Use of EDTA solved that smudgy little problem, and the next gel I ran had me dancing on the spot when I saw the photo:

copy for blog Dulcie gel electrophoresis labelled

Dulcie’s DNA following restriction enzyme digest and gel electrophoresis

 

From there, life in phage lab has been all about figuring out which experiments I wanted to run to distinguish Dulcie from other phages, and waiting for the much-anticipated trip to view ‘her’ using transmission electron microscopy.  Memories of that visit will have me smiling for the rest of the year; not solely due to the experience, but also the recollection of four grown-ass independent young women reduced to squeals, ‘ooh’s ‘aah’s and ‘whoa’s with each image that popped up on the monitor.

Created by Digital Micrograph, Gatan Inc.

Can you blame us?  Lots of little Dulcies is pretty phagin’ cool!

In the remaining weeks of semester, I have been putting Dulcie through her paces.  We’ve been testing whether she can switch to the lysogenic cycle (still not sure on that one, our tests were inconclusive), how hot she likes her spa pool (apparently 50ºC is just the ticket), and also just how viral a virus she is.

We are just about to send our DNA samples away to be sequenced under the SEA-PHAGES program, so the next exciting chapter will be finding out what her genome looks like.  Until then, I’ll be spending my time gazing wistfully out the window and daydreaming about that one time, in phage lab…

References:

[1]  Aksyuk, A. A., Bowman, V. D., Kaufmann, B., Fields, C., Klose, T., Holdaway, H. A., . . . Rossmann, M. G. (2012). Structural investigations of a Podoviridae streptococcus phage C1, implications for the mechanism of viral entry. Proceedings of the National Academy of Sciences, 109(35), 14001-14006.

[2]  Nishino, T., & Morikawa, K. (2002). Structure and function of nucleases in DNA repair: shape, grip and blade of the DNA scissors. Oncogene, 21(58), 9022-9032.

 

Posted in Uncategorized | Leave a comment

Super Bugs.

I was stumped as to what to write my blog about so I rang my sister who is studying medicine and asked her if she could give me any inspiration. We proceeded to chat about antibiotic resistance and discussed a news article from the weeks news. In this article, they talked about the bacterium MRSA which interested my sister as it is a bacterium in hospital that she regularly encounters. MRSA stands for Methicillin-resistant Staphylococcus aureus which is a strain of bacteria that is really difficult to treat in humans which she mentioned. The patients that are infected with this bacteria have to be screened and placed in a separate ward. The doctors and nurses working within these wards have to wear full safety gear to avoid contamination. To me, this seemed like extreme measures for a media exaggerated scare mongering superbug.

Following the talk with my sister I went on to do some research on common superbugs, namely MRSA. Antibiotic resistance is arguably the greatest threat to human health in the twenty first century. These bacteria have mutated and developed to become resistant to most or all antibiotics. Staphylococcus aureus the common original strain of the mrsa bacteria and is not always pathogenic.[1] It can be the cause of abscesses on the skin, skin infections, food poisoning and respiratory infections. This was first being treated in the 1940s with penicillin and 1950s it became more common. In 1961, we began using Methicillin to treat these resistant strains and within only a year of usage, resistant strains started to appear. These days MRSA is resistant to a large list of antibiotics including Vancomycin which is often considered a last line of defence. [2]

MRSA is carried by 30% of the population and is often found in areas such as under the armpits and around the groin.[2, 3] It only becomes a problem once it has penetrated into our skin. MRSA now contributes to more US deaths than HIV. 2] Resistant strains of bacteria is beginning to gain attention in the media.  The media label them superbugs and MRSA has appeared a number of times in the news this last month. There was a report that had findings from a study regarding hospitals screening for the bug.[4]

How does bacteria that causes a minor skin infection to start with become so scary. Media often refer to the superbugs as having developed or learnt to evade our bodies but infact it has evolved.[4] Bacteria can evolve quickly due to their short reproduction time and large population size. Despite the medias apparent scare mongering, antibiotic resistance is still a serious issue with around 700,000 dying worldwide due to this resistance. This number is only set to rise with an estimated 10 million fatalities annually by 2050. [5] Research and figures are scary but they highlight the importance of programs such as the one I’m involved. Phage hunt is a paper I take that aims to assist with the effort to find alternatives to antibiotics. Phage Hunt works to find bacteriophages that are able to destroy bacteria. Many strains of bacteria have become resistant to antibiotics and the bacteria that haven’t will soon become resistant with their rapid evolution.

In our lab we work with Mycobacterium Smegmatis which is similar to mycobacterium tuberculosis. I began to wonder if bacteriophages had had any success in the case of MRSA. I do not have to look far to find heaps of positive research that demonstrated using bacteriophage to successfully kill MRSA. One example was a student from Brigham Young University who had a keen interest in MRSA after his father had lost his leg due to the bacterial infection. The student was using a methods that was similar to the the ones we were using in our lab and  he was able to find six unique bacteriophages that could kill off MRSA cells and his research is continuing to find more.[2]

Other studies even incorporated phages with traditional antibiotics. [6] Continuing research in this field has exciting prospects with making a real difference in the fight against antibiotic resistance and bacterium superbugs.

 

 

 

  1. Staphylococcus aureus.
  2. New method to treat antibiotic resistant MRSA: Bacteriophages. Science daily, 2015.
  3. ChB, D.A.S.M., Methicillin-resistant Staphylococcus aureus. 2015.
  4. Superbug, super-fast evolution. 2008.
  5. Rise of the Superbugs: How is Biotech Fighting Antibiotic Resistance? 2017.
  6. Sandeep Kaur, K.H., and Sanjay Chhibber, Methicillin-Resistant Staphylococcus aureus Phage Plaque Size

Enhancement Using Sublethal Concentrations of Antibiotics. American Society for Microbiology 2012. 78(23).

 

Posted in Uncategorized | Leave a comment

The Search for Mahuika

Tēnā koutou katoa.  Ngā mihi nui anō ki a koutou, ki ngā manuhiri o taku pae tukutuku.

Welcome everyone, again to my blog.


In this post I will be sharing with you some of the mahi – work, that I have been doing through Massey University, as well as intorudce you to my phage friend, Mahuika.

What did we learn last time we explored Te Ao o ngā Huakita me ngā Huaketo – The world of Bacteria and Viruses?

  1. Our health and enviornment can be affected by bacteria in good and bad ways
  2. Antibiotic resistance occurs, when BAD BUGS are immune to antibiotics
  3. Bacteriophages are viruses, that infect and kill bacteria

As we learned last time, bacteriophages are EVERYWHERE and they are very useful in treating bacterial infections.  This is an area that many scientists and medical professionals are reyling on, to provide an alternative to antibiotics.

To find an alternative, we must find or HUNT for new bacteriophages. They must possess the ability to infect and kill the types of bacteria that are infecting us.  This is why I joined the course at Massey University called The Phage Hunt.

In this course, a group of young, ambitious scientists joined forces in the lab; to discover, purify and sequence, undiscovered bacteriophages.

Photo on 6-2-17 at 12.09 PM #2 2 (1)

The Phage Whanau

How do you hunt for viruses that infect bacteria?

We look in places where there is a lot of bacteria!  For example taepu – soil.

My hunt began by collecting soil samples from my backyard, the beach and compost bins.  I  found a number of phages in the University compost bin!

How do you know that you have found a phage?

You add a phage sample to a plate (like the one pictured), that contains bacteria cells. Leave them over night and they form small clearings, called plaques.  These plaques are areas when the phage has infected the bacteria and killed them, leaving a clear spot on the plate.

UC1

Plaques on bacterial plate 

As seen in the picture, there are different sized plaques that have different morphologies (characteristics, i.e. cloudy or clear).  This means that there are different bacteriophages present.  From this plate I managed to purify three different phages.  Two of these phages were whangai – adopted out to other class mates, and I kept one to work on further.

The Phage Hunt course allows you use an Electron Microscope to get images of your phage and you get to name your phage.

So, I present to you, Mahuika.

Screen Shot 2017-06-02 at 9.38.04 PM

My phage, Mahuika

Mahuika is about 315 nanometers long.  That is 0.00035 of a millimeter.

He tino iti – very small!

The capsid contains the phage DNA.  The tail fibers allow the phage to recognize and attach to their host bacteria.  The tail allows the phage DNA to infect the host bacteria.  Mahuika’s sister phages that were adopted out are named Mooo and Naira.

Click here to have a look at the Phage Data Base

Why did I name my phage Mahuika?

Mahuika is the Māori Goddess of Fire.  She is the wife of Auahitūroa and the teina – younger sister of Hine-nui-o-te-pō.  Some of you will have heard about the Goddess of Fire from the pukapuka – story, about how Māui brought fire to the world; or maybe I should say tricked Mahuika and stole her fire!

Mahuika and Maui

Mahuika is not impressed by Māui (1)

*Tsk tsk tsk*

Here is a short version of the story.  Māui was curious about where fire came from.  So one night, he put out all the fires in the pa – village.  In the morning his mother Taranga, sent Māui to the ends of the earth to find Mahuika in the maunga – mountain of fire where she dwelled.

When Māui arrived he asked Mahuika for her fire to take back to the tāngata – people of the world.  She gave him one of her nails which contained the fire.  Māui left with the fire but thought to himself, what would happen if Mahuika didn’t have any fire left?  Where would she get more fire from?  So Māui threw the nail into a near by stream and  then returned to Mauhika’s maunga-mountain.

0dab71_9d375ef469bf4c94b6b85a1a89aa25e0~mv2_d_2932_4236_s_4_2

Mahuika giving her fire to Māui (2)

Maui then lied to Mahuika and said that he accidently dropped the first nail and needed another.  She gave him another one, but Māui also threw that one away.  Māui continued this nonsense, until Mahuika only had a few nails left.  When she realized what Māui was doing, she became very angry!  She threw one of her nails at Māui and a wild fire exploded around him!

Māui fled from the mountain, into the forest.  The wild fire followed him and hit the Mahoe tree, the Tōtara, the Patete, the Pukatea, and the Kaikōmako trees.  Unlike Māui, the trees knew that Mahuika’s fire was a great gift, and so they grasp onto the fire.  When Māui returned to the pa – village, he brought with him dry wood from the trees to show the villagers how to start a fire by rubbing together the wood.

That is the pukapuka – story, of how Māui brought fire to the world.  This is one of my favorite stories of Māui’s adventures.

Unknown

PUKANA 

Although, this story doesn’t exactly relate to bacteria and viruses , I once read an account of an old koro (elderly man) exclaiming “E hika! Ko Mahuika koe!” (Oh my! You are Mahuika!) when he was shown a radio for the first time.  I do not think he meant that the radio was the Goddess of fire, or that it was going to burst into flames, rather it surprised and intrigued him.  This is how I felt about by phage!  Surprised and intrigued by the complexity of such a simple biological particle.

In addition, Māui’s curiosity is the same kind of curiosity that I feel when I think about Te Ao o ngā Huakita me ngā Huaketo.   Albert Einstein once said “I have no special talents; I am only passionately curious.” For me being a scientist simply means being curious enough and bold enough, to ask questions.

Māui’s curiosity lead him to Mahuika, just as my curiosity lead me to my phage.  This is why I named my phage after the Goddess of fire.  However, I do intend to treat my Mahuika better than Māui treated his.

Nō reira – therefore

Until next time whanau, think about what intrigues you?

Are you curious enough, like Māui or like me, to search for your Mahuika?

Ngā mihi ki a koutou – thank you all.

Ka kite anō,

Anezka Hoskin

 

References 

  1. Mahuika and Maui. Retrieved from https://sugarskulldragon.tumblr.com/post/156147644096/making-a-disney-reference-with-other-disney
  2. Amber Stotts (2007). Maui and Mahuika.  Retrieved from https://www.amberstottsart.com
Posted in Uncategorized | Leave a comment

Fern the Phage

As I mentioned in my last blog, at the beginning of this semester I found a phage that infects the bacterium Mycobacterium smegmatis. My journey with my phage, who I’ve named Fern, has been interesting and filled with many unexpected twists and turns.

Everything began back in March when we were collecting environmental samples. I collected a LOT of environmental samples, a grand total of… 40 samples. I was told compost bins are a good place to find them, so to compost bins I went… mine, my friend’s, my friend’s Grandma’s. Nothing. I continued trying in the garden, ponds and streams. Still my plates were free of plaques. Eventually, from a sample from the soil from on top of our old garage door that had been lying in the vegetable patch, I found a plaque, and not just one, I found 3 differently shaped plaques! The picture below shows this very plate with some plaques that have been circled. A plaque is a circular clearing that forms on plates due to a phage infecting the bacteria on the plate. If you’re interested in more information about how phages form plaques on plates click here [1].

IMG_1064

Photo of direct isolation plate

Unfortunately, a few labs later I was back to only one phage (the other two did not survive the phage buffer).

The plaques of the surviving phage seemed to have quite a range of different morphologies. Some were completely clear, while others had cloudy rings around the outside and some were small, some medium and some large, as you can see in the photo below. Plaques of one phage tend to have plaques with the same morphologies so I was cautious that I may have had multiple phages. However, multiple morphologies continued to be the case even after purification so I assumed that I had purified my phage.

10-3 serial dilutions 2 24:3

Photo from phage purification showing different plaque morphologies

A few weeks later we were extracting the DNA of our phage. I was very lucky and miraculously I only had to do it once. I was so surprised that something actually worked the first time as this was not my experience of phage lab so far. Some of this DNA will be sent off to the University of Pittsburgh to be sequenced!

One of my favourite parts about this journey was when I got to see my phage! Phages are way, way, way too small to see with the human eye. Fern has a head of about 62 nm and a tail of about 137nm. That’s 0.000137 mm! To see that small, we need an electron microscope. We went to the electron microscope at Auckland University and saw our phages. This is what Fern looks like…

Picture1.png

Fern the phage from the electron microscope

As you can see from these pictures the phage has a head and a tail. The head is covered by a capsid (a protein shell) and contains the DNA [2]. The tail is the part that attaches to the bacterium, pierces the bacterium then passes the phage DNA into the bacterium [3].

Near the end of the semester I was ready to explore more characteristics of my phage and I decided that burst size would be interesting. The burst size is the number of phages produced when a bacterium is infected by a phage. If you want to find out more about burst size click here [4]. I was modifying a protocol for a different type of bacteria using a lysate with a different concentration of phage so there was some guessing and trial and error involved. The first time I tried this experiment the concentration of my phage lysate was way too high and I couldn’t see anything. When I tried again I got the concentration right. However, I did not do the experiment for long enough.

IMG_1346.JPG

Plate from burst size experiment showing the two morphologies: small and large

Although I was unsuccessful in establishing the burst size I discovered something very interesting … I had two distinct plaque morphologies. Could there still be two phages? There were two distinct morphologies: small and large. So, did I ever purify my phage properly?

My first reaction was that there must be two phages. To try to figure this out I picked all the different plaques morphologies (large, small and cloudy or clear) and plated them separately. The new plates all showed small plaques, all with similar morphologies. This lead me to believe it was only one phage and why this was happening was still a mystery.

Next I thought maybe it had something to do with the age of the bacteria culture as this had only started happening later when the bacteria were older. However, after plating with both old and new bacteria I saw no difference, so it wasn’t this. Further confusing me, this time there were no small plaques. There could be so many reasons why this is happening… rapid mutations, the time at which the phage infects the bacterium and I’m sure many more things of which I’m unaware, but currently it’s still a mystery.

This course has shown me firsthand how in the real world everything doesn’t fit into the categories and rules we often try to make in science. It’s a lot more complicated than that. Currently no one knows why my phage is making two different morphologies sometimes and that’s amazing. It really reminds me of how much we don’t know and how much there is to still discover.

References:

[1] Microbial Genetics (2002). Phage Plaques. Retrieved from: http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/phage/plaques.html

[2] Orlova, E. V. (2009). How viruses infect bacteria? The EMBO Journal28(7), 797–798. http://doi.org/10.1038/emboj.2009.71

[3] Ecole Polytechnique Fédérale de Lausanne. (2016). How viruses infect bacteria: A tale of a tail. ScienceDaily. Retrieved from http://www.sciencedaily.com/releases/2016/05/160518133816.htm

[4] S. T. Abedon. (n.d) Burst Size. Retrieved from: http://www.archaealviruses.org/terms/burst_size.html

Posted in Uncategorized | Tagged , , , | Leave a comment

The adventures of a budding scientist and her so-called bacteriophage.

Welcome back.

My relationship with phage lab is one of much uncertainty. One thing I am certain of is that it is testing my usual calm and positive attitude towards life. Science is very much like that and if I want to continue my work in the field of science I shall just try and get used to it. It can be frustrating and tedious and challenging.  Phages and success in phage lab seems to avoid me at me at all cost. If you have read my last blog, you will know that I haven’t had much luck with phage lab but after I had gathered a lysate from my phage I hoped my luck would change.

 

bacteriapresents-01

Bacteria Gifts

DNA extraction is the next step of phage lab and possibly the most tedious, closely rivaling direct isolation. DNA is a molecule that carries our genetic code and in order to find out more about the phage I had finally we had to extract the phage’s DNA. DNA provides all of the information you could ever want from an organism. A protocol similar to what we followed can be found here. [1]

Luck continued to evade me and for some unexplained reason I could not find my phages DNA to save my life. I had to repeat the DNA extraction protocol 6 times in order to find enough DNA to move on to the next step. The first reading I got was so low that there wasn’t even any point in keeping the sample. The process takes around an hour of continuous work and so it wasn’t the easiest protocol to follow but by the end of this stage I had all but memorised it. The next time I got another low reading and I was thinking ‘oh no, here we go again’. I thought I had gotten passed the unluckiness that plagued me but alas it had reared its ugly head again. I ended up having to spin my lysate (the liquid that contained my bacteriophages) so that the phages concentrated at the bottom in order to get a greater concentration of the bacteria. Thankfully the next time I did DNA extraction I got a much larger concentration of DNA but I still had to end up doing it three more times.

I had gotten to the stage where I had almost had enough DNA to take to the next step and then suddenly a whole tube of DNA appeared one day in lab. I had labelled the tube as being DNA but I had no record of it in my log book nor did I know where the heck it had come from. If this DNA sample was actually DNA then I would have more than enough DNA to take to the next step and wouldn’t have to go through another round of extraction which had become mind-numbing at this point. I ran the DNA on a gel which would tell me whether or not I had DNA. An in-depth explanation of this step can be found here.[2] Somehow my sample that had come out of nowhere was DNA and good DNA at that.

emily1234

This is “Mooo”.

We got to see our phages through an Electron Microscope which was awesome as its really hard to see any progress when working in lab because you can’t see your phage and so to see it in real life was rewarding. The Electron Microscope we used cost over half a million dollars and so I was almost too scared to touch it, knowing how clumsy I am.

I tend to be a person who doesn’t follow the recipe book exactly while cooking and so there may be too much of one thing and not enough of the over. This doesn’t really matter when it comes to cooking but when it comes to science it is a bit of a problem. Following a protocol exactly how it is lined out in the book is really important because accuracy is key to success in science. I struggled with this precision which lead to many mishaps and mistakes made. It would seem that maybe science wouldn’t be the thing for me due to my clumsy nature and inability to precisely follow a protocol. Things are not always as they seem and I’m here to show you that even clumsy people can eventually make fabulous scientists.

The next step of phage lab now is to sequence the DNA of our bacteriophage. I have named my phage “Mooo” which is fitting as its appearance is short and stubby, kind of like its founder! Science requires one to be persistent and to just keep going and that is exactly what we had to do. It challenges you in a way that can make you question your sanity and even though it may not seem like it, everything will turn out okay in the end. It’s a fickle business but getting through the lab work and being able to see what we achieved in the end makes it all worth it.

Things can only improve from here, surely.

See y’all in the next instalment of this scientific adventure.

 

References

  1. Gill, J.J., Gill: Phage Genomic DNA extraction 2015.
  2. Addgene, Agarose Gel Electrophoresis.

 

Posted in Uncategorized | Leave a comment