An Accidental Breakthrough: Bacteriophage Discovery

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

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

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

 

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

 

He wrote in his memoirs;

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

 

Felix D'Herelle

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

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

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

 

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

 

IMG_1109

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

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

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

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

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

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

 

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

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

 

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

 

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

 

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

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

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

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

 

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

 

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

 

References:

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

Figure 2: Photograph taken by Leani Oosthuizen

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

 

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

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

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What is Life?

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

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

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

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

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

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

 

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Antibiotics and Microbiomes

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

Microbiomes

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

 

Good Bacteria

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

 

What antibiotics do to our microbiomes

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

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

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

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

Alternatives

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

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

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

Kombucha_Mature.jpg

Kombucha including the culture (Mgarten, 2007)

 

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

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

 

References:

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

 

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Phage Hunt: The Phinal Phrontier

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

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

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

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

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

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

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

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A Phage of a Different Colour

Throughout the year, our team of phage hunters has been focused on bacteriophages (phages) which specialise in infecting Mycobacterium smegmatis bacteria.  Mycobacterium smegmatis replicates quickly and is non-pathogenic (Smith, 2003), making it an appropriate model organism to safely study other mycobacterial species.  M. smegmatis shares a unique cell wall structure and partial genetic homology with Mycobacterium tuberculosis (Wikipedia, 2017), and our work with M. smegmatis is contributing to an international effort to develop new methods to combat Tuberculosis disease (TB).

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Chest X-ray showing Mycobacterium tuberculosis infection (STD.GOV Blog, 2017).

 

But it’s not only humans that could benefit from a greater investment into phage therapy and bacteriophage research.  The honey bee (Apis spp.) plays an integral role in insect pollination of flowering plants and food crops (Michigan State University, n.d.), however honey bee populations worldwide are threatened by the contagious bacterial disease American foulbrood (BeeAware, n.d.).

American foulbrood disease affects the larval and pupal stages of juvenile honey bees, and is caused by the bacterium Paenibacillus larvae (Alippi, Lo´pez, & Aguilar, 2002).  P. larvae spores are ingested by honey bee larvae and begin to reproduce in the midgut, before progressing into tissues and causing death of the individual (Djukic et al., 2014).  The disease can be identified in the field by a progressive discolouration of the larvae to brown and black (Alippi et al., 2002) before it dies and is reduced to a viscous material within its cell.

foulbrood1

Brood cells showing infestation of American foulbrood (Bee Informed Partnership, 2013).

 

Characterisation of P. larvae bacteriophages could lead to an alternative treatment for the colonies of commercial beekeepers.  Destruction of an entire hive by burning is often resorted to in order to prevent the spread of American foulbrood; other methods can be costly, and antibiotic treatment has been disallowed in many countries due to residual product being detected in the resulting honey (Beims et al., 2015).  Research into these phages is relatively recent, with full genome sequences of six P. larvae bacteriophages being published in 2014 (Merrill, Grose, Breakwell, & Burnett), allowing for genomic analysis, comparison between the individuals sequenced, and identification of important genes.  The University of Minho took a more specific approach to this research by exploring the potential of hydrolytic enzymes used in bacteriophage replication to control P. larvae (Oliveira et al., 2015).

Brigham Young University and the University of Nevada are investigating bacteriophage treatment of Paenibaccilus larvae in beehives.  Yost, Tsourkas and Amy (2016) experimented with a cocktail of several different P. larvae phages, and were able to observe an increase in Apis mellifera larvae survival rates using post-infection and especially preventative treatments.  Brigham Young University’s ‘Phage Hunters’ class has inspired undergraduates to research P. larvae phages and ways that they can be used to treat American foulbrood (Hollingshead, 2014).  After isolating different phages, the host range of each was tested on 59 strains of Paenibaccilus larvae, and a cocktail was used to demonstrate complete protection, with 0% of treated hives developing American foulbrood, compared to an infection rate of 80% in untreated hives used in control experiments (Brady et al., 2017).  Treatment using a phage cocktail was found by both studies to have no adverse impact on bee mortality rates.

 

Brigham Young University has also released an informative video summarizing their research in addressing American foulbrood, which gives an excellent educational overview without getting too technical (Brigham Young University, 2014):

 

This summer school I am undertaking a research project within Biosciences as part of my undergraduate degree.  Pending permissions to work with Paenibaccilus larvae, I hope to initiate the first New Zealand-based contribution towards both a preventative and post-infection treatment for American foulbrood.  In addition to being integral to the success and diversity of our national flora, apiculture (beekeeping) represents an important sector of our nation’s economy, and I am excited for the opportunity to support the growing health of this industry.

 

 

References:

Alippi, A. M., Lo´pez, A. C., & Aguilar, O. M. (2002). Differentiation of Paenibacillus larvae  subsp. larvae, the Cause of American Foulbrood of Honeybees, by Using PCR and                Restriction Fragment Analysis of Genes Encoding 16S rRNA. Applied and      Environmental Microbiology, 68(7), 3655-3660.

Bee Informed Partnership. (2013). American Foulbrood (AFB). Retrieved from https://beeinformed.org/2013/10/21/american-foulbrood-afb/

BeeAware. (n.d.). American foulbrood.   Retrieved from http://beeaware.org.au/archive-pest/american-foulbrood/#ad-image-0

Beims, H., Wittmann, J., Bunk, B., Spröer, C., Rohde, C., Günther, G., . . . Steinert, M. (2015). Paenibacillus larvae-Directed Bacteriophage HB10c2 and Its Application in American Foulbrood-Affected Honey Bee Larvae. Applied and Environmental Microbiology, 81(16), 5411-5419.

Brady, T. S., Merrill, B. D., Hilton, J. A., Payne, A. M., Stephenson, M. B., & Hope, S. (2017). Bacteriophages as an alternative to conventional antibiotic use for the prevention or treatment of Paenibacillus larvae in honeybee hives. Journal of Invertebrate Pathology, 150, 94-100.

Brigham Young University. (2014). Bee Killers: Using Phages Against Deadly Honeybee Diseases.   Retrieved from https://www.youtube.com/watch?v=rj9_QGBJN0w

Djukic, M., Brzuszkiewicz, E., Fünfhaus, A., Voss, J., Gollnow, K., Poppinga, L., . . . Daniel, R. (2014). How to Kill the Honey Bee Larva: Genomic Potential and Virulence Mechanisms of Paenibacillus larvae. PLoS ONE, 9(3).

Hollingshead, T. (2014). Using microscopic bugs to save the bees. BYU News.

Merrill, B. D., Grose, J. H., Breakwell, D. P., & Burnett, S. H. (2014). Characterization of Paenibacillus larvae bacteriophages and their genomic relationships to firmicute bacteriophages. BMC Genomics, 15(745).

Michigan State University. (n.d.). Pollination.   Retrieved from http://www.canr.msu.edu/nativeplants/pollination/

Oliveira, A., Leite, M., Kluskens, L. D., Santos, S. B., Melo, L. D. R., & Azeredo, J. (2015). The First Paenibacillus larvae Bacteriophage Endolysin (PlyPl23) with High Potential to Control American Foulbrood. PLoS ONE, 10(7).

Smith, I. (2003). Mycobacterium tuberculosis Pathogenesis and Molecular Determinants of Virulence. Clinical Microbiology Reviews, 16(3), 463-496.

STD.GOV Blog. (2017). Bacterial Diseases.   Retrieved from https://www.std-gov.org/blog/bacterial-diseases/#4-tuberculosis

Wikipedia. (2017). Mycobacterium smegmatis.   Retrieved from https://en.wikipedia.org/wiki/Mycobacterium_smegmatis

Yost, D. G., Tsourkas, P., & Amy, P. S. (2016). Experimental bacteriophage treatment of honeybees (Apis mellifera) infected with Paenibacillus larvae, the causative agent of American Foulbrood Disease. Bacteriophage, 6(1).

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Phage Hunt was my Phavorite

The hunt for bacteriophages has been my phavorite course this year.  Here is why:

1) Fun, supportive, enthusiastic and adventurous.  These are the qualities that our Phage Whānau (family) embodies.

2) Tauira (student) working along side tauira, encouraging one another in their endeavour to become independent scientists.

3) Real research and data that we can take ownership of and is useful.

Too often science undergraduates find them selves snowed under deadlines and content heavy courses, that they never have the oppurtunity to experience what it is like to be a scientist and produce relevant and useful results.

While the purpose of an undergraduate degree is to teach students a strong foundation, there should also be an oppurtunity for tauira to experience research.  If these oppurtunities were made avaiable as credited courses in the degree, more students would be likely to engage in these courses.  This may even lead to increased postgraduate enrollments as students get drawn in.

The SEAPHAGES program has proved that this is achievable.  The creation of a  bacteriophage database by international students and faculty, shows that course-based research can be successfully implemented on a large scale without compromising the authenticity or richness of scientific research.  Not only this but the spread of this course out of the U.S.A. into other countries like New Zealand has also shown how local culture can be incorporated into the course to further engage students.  The flexible and student-lead environment has allowed this to happen in our class, ultimately creating our Phage Whānau.  Therefore, this is also proving that you can incorporate Te Ao Maori into a science course with out compramising it’s authenticity.

Maori in Science

From 1994 to 2005 the number of Maori science undergradutes increased three fold, from 107 to 323 (1).  The popular areas of study included biological sciences and health and medical sciences.  Although this is a worthy cause for celebration, there is still so much more room for success.  Maori graduating with science degrees ranged from 8 – 10%, for non-Maori this was between 16.5 – 18.5% (1).  A study by Hook, Waaka and Raumati (2007) tried to identify some things that may help Maori tauira feel more engaged in their science courses.  You’ll find that our Phage Whanau already incorporates some of these values.

“Mentoring is a brain to pick, an ear to listen and a push in the right direction.” – John C. Crosby.

One of the tools that Hook, Waaka and Raumati talked about was the value of mentorship.  In the Phage Hunt the lecturers and faculty take a backseat role, becoming more like Phage Mentors, rather than scary, intimidating lecturers. Although they may not be Maori themselves, it’s their heart of inclusiveness that allows Maori students to thrive.

“The key to being a great mentor is to help people become more of who they already are – not to become more like you.” – Suze Orman

The following three values have the potential to address some of the cultural and racial issues associated with Māori students in science (and university in general)(2).

Whanaungatanga (family like relationship)

Te reo Māori (Māori language)

Rangatiratanga (leadership)

All members of our class commented on the welcoming environment of the class and so whanaungatanga is already established.  Te Reo has been welcomed and incorporated throughout the year as well.  Rangatiratanga is one that could be improved on.  This relates to the idea that more Māori role models and key figures are needed in science.  A barrier experienced by many tauira is that an absence of role models and key figures, prevents science from being relatable and achievable to Māori.

All three of these values related directly to the fact that almost half of Māori (41%) in tertiary education are the first in their families to attend university (3).  Feeling comfortable, well supported and guided is essential for Māori success in undergraduate, and hopefully postgraduate study.  I expect that as these issues continue to be recognised and these values incorporated, we will see a continued increase in tauira participation and achievement in science.

Phage hunt has been my phavorite because of the research opportunities that is has given me, but also because the potential the course has to positivley influence Māori student outcomes in the future.

Tēnā koutou, tēnā koutou, tēnā koutou katoa.

 

References
  1. Hook, G. R., Waaka, T., & Raumati, L. P. (2007). Mentoring Māori within a Pākehā framework. Mai Review, 3(1).
  2. Hook, G. R. (2008). Māori students in science: Hope for the future. MAI Review LW1(1), 11.
  3. Te Pōkai Tara Universities New Zealand (2016). New Zealand Universities Key Facts and Stats.
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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/

 

 

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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.

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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.
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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. 

 

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