Worms and honey, the unlikely heroes

Liz Harry: Welcome everyone to UTS Science in Focus: Worms and Honey, the Unlikely Heroes, with Associate Professor Sheila Donnelly and Nural Cokcetin. I’m Liz Harry, Professor of Biology and the Director of the ithree institute, which stands for infection, immunity and innovation, at UTS. Before we officially start tonight’s very fascinating talks, I would like to acknowledge the Gadigal people of the Eora nation upon whose ancestral lands our campus now stands. I would also like to pay respect to the elders, both past and present, acknowledging them as the traditional custodians of knowledge for this land. I have some housekeeping reminders: the toilets are pretty much across the hall that way, and the fire exits are the same direction. Please turn off your mobile phones, or better still, put them onto silent and tweet to your heart’s content about tonight. Note that we’re filming this event, and the video will be made available online to watch within the next couple of weeks. We also have a photographer taking photos, so put your best smile on. Tonight, at the end of the two talks, we’ll invite you to ask your questions to our speakers, so have a think about what you’d really like to ask. I’m absolutely thrilled to be emcee tonight to introduce two outstanding scientists from UTS. They’re passionate about communicating this science, so not only are they good at doing research, but they love to tell people about it, and they love to tell other scientists, but more importantly I think they like to talk to the public. And they like to use their research, or they have done, use their research to discover new things that will help, that will have real impact in human health. Associate Professor Sheila Donnelly. She’s in the School of Life Sciences in the Faculty of Science at UTS. She studies parasitic worms call helminths, which you’ll hear about more. Her research has two major streams: understanding how these parasites successfully manipulate their host’s immune system, and using this knowledge to identify novel therapeutics for the treatment of autoimmune disease. She’s been leading a research team that investigates how these worms may be used to stop the progression of multiple sclerosis. Sheila joined the UTS team in 2004. She obtained her PhD in virology at Trinity College Dublin, in Ireland. She was postdoctoral fellow at the National University of Island Maynooth. In 2001 she moved to Dublin City University where she started her work on the immune modulating protein secreted by these parasites. At UTS she’s continued to investigate interactions between these parasitic worms and these human hosts. She’s published widely in high impact scientific journals, and has been awarded several research grants for this work. More recently, her team’s novel discoveries have revealed a potential solution to treating autoimmune diseases, and a few years ago they began the long journey of translating these findings to the clinic. She’s a fantastic program director for our Bachelor of Biotechnology degree, and an associate member of the ithree institute. Please join me in welcoming Associate Professor Sheila Donnelly.

[Applause]

Sheila Donnelly: Thank you Liz for a very generous invitation, and thanks to all of you for giving up your time this evening to come and hear about the science we do here at UTS. As Liz said, I’m a parasitologist, and the title of my talk is A Worm a Day dot dot dot. It’s yet unfinished, so this really is representing an ongoing research project we’re doing here. We haven’t quite got to the end of it; I don’t know if we ever will, as scientists. But much like the proverb, ‘An apple a day keeps the doctor away’, I’m hoping by the end of this talk I’ll convince you that perhaps infectious pathogenic worms taken every day may have health benefits. So, the focus of my research is primarily around, as Liz said, the host immune response, and this is a tool that we evolved as a species to protect us; to help us survive, fight against, infection, be it bacteria, viruses, fungi. Fight against those and survive so we can procreate and survive as a species. And that’s a tool that works very well for us. We can see that every day in society. Those people, for example, who have a deficient immune response, maybe through infection such as HIV, chemotherapy as cancer treatment, they’ve lost their immune response and they succumb to very simple infections. So the immune response works very, very well – it’s evolved over man, many years to fight against these infections. There are two main areas of the immune response. It’s a bit of a complex slide, but don’t worry too much. There is an early or innate immune response, and then there’s an adaptive, or learnt, immune response, and through this we create a memory of previous infections, we fight them and survive. This also leads to inflammation, which most of you will see if you get a cut on your hand – you get that red, raised skin, a bit of pus. That’s the immune response working really well to protect you against an infection and heal that wound. And the immune response is a very regulated thing, so you get inflammation, you beat the infection, the immune response is regulated, and you’re back to normal baseline. So it’s a highly evolved, regulated machinery. However, sometimes it goes horribly wrong. As I’ve said, the immune response evolved to fight against infection. We know some of these bacteria are dangerous, they’re pathogenic; they cause disease and horrible clinical symptoms. However, in some cases, the immune response fights against your own tissue, and this leads to what we know as immune-mediated or autoimmune disease. And I’m sure everyone in this audience knows someone, if someone in the audience doesn’t have one, or knows someone who has an autoimmune disease. Some of these are type 1 diabetes, shown on the left. In the middle, we have multiple sclerosis and rheumatoid arthritis. There’s currently a list of 100 plus known diseases caused by the immune response, and this is the tool that we evolved to help us survive. Somehow it’s turning on our own tissue and destroying that tissue. We know it’s the immune response – if we can stop that immune response against tissue, we can certainly stop the progression of some of the symptoms. So a lot of the leading clinical treatments for autoimmune disease are anti-inflammatory, or immune suppressant. They do work to some degree, but they have horrible cytotoxic side effects. So they’re not particularly effective, and largely a clinically unmet need. What’s interesting, I find, about these diseases, is that they have a very specific geographical distribution. So this map shows the current presence of type 1 diabetes in societies across the world. And what you notice on this map is that type 1 diabetes is highly prevalent in developed societies, like Australia, Europe, Northern America. In other parts of the world, like Latin America, Africa, less developed, very little if none of these autoimmune diseases. So some people look at this map and say, ‘Oh, must be genetic. So these populations are inbred, they’ve been evolving together over many years; there must be a genetic element to this disease – a genetic susceptibility.’ That’s interesting, and certainly we’re looking for that; however, the rate at which these diseases are increasing in society is too fast for it to be a genetically modified disease, or a genetic disease. If it’s a genetic disease, typically it takes a few generations before it manifests within a population. Every year we’re seeing increases of about 3-5 per cent of these diseases, which is far too fast for it to be a genetic disease. So what we think is there’s probably environmental factors at play. So either something has been added to the environment to induce these autoimmune diseases, or taken away from the environment to cause the induction of these diseases. As a parisitologist, I will have to say straight up, the next set of slides are possibly quite biased. However, I believe parasitic worms are that environmental factor. Is the fact that in a developed western society, where we clean up all the time, we use toilets very well, we wash our hands incessantly – my mother’s adage is if you have a spill in the kitchen nowadays, you renovate. Everything is really, really clean. So we’re not exposed to these parasitic worms at all – they’re no longer endemic in our population. And that’s only really since the 1960s, 1950s, 1960s. So the removal of these parasitic worms from our population is quite a recent occurrence, and if you look at the maps, you can see – oops! Excuse me – it’s a direct inverse relationship. So there’s the worms currently present in the world today; there’s the autoimmune disease. As I said, as a parasitologist, I like to think we’re missing parasitic worms and somehow they can influence development of these autoimmune diseases. Who are these worms? They come in lots of shapes and sizes; they infect us lots of ways. They get into the body through various means; end up in lots of different tissues. These three worms are typical human – or the leading human infectious disease. We have the hookworm on the right, apparently the inspiration for the alien in the movie. That worm migrates into the body through the skin, burrows through lots of tissues, ending up in the intestine where it latches onto the intestinal wall, feeding on blood, and it can stay in the intestine for 5-10 years, so they’re a long living pathogen. And remember that: they’re pathogens, or infectious organisms. The middle is my favourite – I’m going to talk about this later in the talk, but the liver fluke. As the name suggests, it migrates through the liver, ends up in the bile duct. And finally on the end [inaudible]. So these are all common human parasites found around the world, and in parts of the world where they’re endemic, many populations harbour more than just one of these, and hundreds in number, so there’s quite a large amount of these parasitic worms in the human. As I said, some of them can be quite long lasting, so they can last up to 20 years, some of them, within a human host. You have to ask the question at the very start: Sheila, you told us we had an immune system that has involved over millions of years to these infectious organisms, to fend of pathogens. How is it that we have a three-centimetre worm living in my intestine for 20 years? Well, the worms have evolved fantastic strategies to modulate or change our immune response. So instead of responding like it’s a pathogen, the body thinks oh hey, a friend – let’s massage it, but let’s repair the tissue damage and let the worm stay there. All it’s going to do is latch on the intestine and feed blood. Not too bad, and we can repair those little pinholes in the intestine so we don’t get tissue damage. So what we find is the majority of parasitic infections are actually asymptomatic – you don’t know you have them. So unless you have hundreds and hundreds of worms, or because of their size you have physical discomfort, you actually don’t have an awful lot of clinical symptoms because of the immune response, because the worms have regulated that immune response. I’m going to switch that one and go to this one. The other important thing is not only have they modulated the immune response, they’ve been present in the human population as the immune response evolved. So they regulate the immune response, and they’ve been there all the time. The question is, is it possible that these worms are actually part of the human immune response? Is it possible that they’re actually the thing that regulates the human immune response? By taking that away, we now have the capacity to mount an immune response, but not to manage it or regulate it to turn it off when it goes wrong. Therefore, by taking the worms out of the immune response, you’ve tipped the balance of immunity, shown here on the left-hand side, towards an inflammation cascade which can’t be turned off. So by taking worms away, we’re not actually being able to regulate the immune response, and we get autoimmune disease. So that sounds great in theory, but do we have any evidence that this in fact is real? On the right-hand side is the first paper that’s published – a clinical trial where they recruited very happy volunteers that had Crohn’s disease, or ulcerative colitis, which are immune-mediated diseases. That is the immune system in the intestine has gone totally array and dysregulated. And this chap here, gastroenterologist, Joel Weinstock, recruited these and gave them 2500 worm eggs in a few mls of orange-flavoured Gatorade, which you can see he’s very proudly holding up. Apparently they taste fine – you can’t taste the worms, you can’t feel them in your body. And these were pig worms, so worms that had involved to infect pigs, not humans. He chose that approach because he didn’t want to initiate an infection in the body. He thought well, the worms might hatch from their larval stage, become juvenile worms and be there long enough to switch the immune response. And it seemed to work – he had very positive results. Seventy-five percent showed a reversion of their symptoms, and those people who had worms opted to keep the worms at the end of the clinical trial. Other trials are ongoing, so we have a few ongoing at the moment, but certainly there’s definite proof of concept that they work. On the other side is the delivery of hookworm, so that worm that was like the alien from the Alien movie, and they burrow through the skin, so you can see on the person’s arm there there’s a Band-Aid, and on that Band-Aid there’s 20 larvae worms. And they just burrow through the skin, ending up in the intestine, and you harbour those worms in the intestine. Again, there’s clinical trials going on using hookworms in certain diseases. Excuse me. And again, anybody who has these say they can’t really feel any discomfort – there’s no obvious signs they are infected. Importantly, remember the worms don’t replicate in the body, so they’re not like bacteria and viruses. If you’re infected with 20 worms, you’ll just have 20 worms; you’ll never have more than that. They produce eggs, and the eggs get passed out of the body for the life cycle to continue. So in that sense, it’s perhaps a manageable dose. However – I hope no one’s had tea recently – this is colonoscopy of a person who is infected with one hookworm. I’m hoping you can see the hookworm wriggling around there in the corner. So this is one worm, and you can see it’s attaching to the intestine. You can see it feeding on the blood, producing its waste and so on, so it’s not very pleasant. It is a live worm, it is a pathogen, and it is destroying some tissue, so it’s having a physiological impact on the body. So yes, we can say it’s safe to a certain degree, but is it really wise to reintroduce pathogens into the world? And in terms of patient compliance and comfort, if we did a show of hands, I’m not sure how many would line up for live worm infections for treatment. What we’re asking here at UTS is: is there an alternative to live infection – is there some way we can replace the effect of the live worms with something synthetic or chemically made in the lab? And I’m here to tell you today that confidently that I’m very confident that yes there is. So what you can see here is the liver fluke – that’s the worm that we work on here at UTS. Quite a beautiful worm – it’s a flat worm, spread out along the surface, and you can see the tree-like branch structures throughout its body. They’re red in colour, because they’re full of haemoglobin. That parasite has fed on the blood of its host and its gut has completely fills up as it gets all the nutrients it needs from the blood. Once its finished digesting its meal, it will then secrete waste products along with a lot of enzymes out of its content, and we collect those secretions, because we know that the worm secretes these molecules the whole time its in the host, and therefore it must contain some capacity, perhaps, to modulate the host immune response. Can we actually look deeper into those? So it’s a big mishmash of molecules. You can see that on the right-hand side; this is a method we use in the lab to separate the molecules. Each of those bands represents a single molecule, and the thickness of the colour represents how much of it is there or how abundant it is. So I will say that just so that you know, that this slide actually summarises nine years of my time in the lab. This is all I produced. I did work very hard, but unlike the human situation where we understand the genome of the human and what genes produce what proteins and so on, we understand quite a lot about the human genome now. In parasitic worms, we actually know very little, so it’s been a very slow process to try and identify what all of these molecules are. But after all of that work, we did find one molecule from all of those secretions that we thought might work in the disease model. We these days can’t go straight into humans – we do have to take small pre-clinical steps to prove efficacy of a model or a treatment, and here we use some animal models of disease, and I’m going to show you the work today around the MS. And this is work we’ve done in collaboration with the group at the University of Queensland with Judith Greer. And this is an animal model that recapitulates the human relapsing remitting condition of multiple sclerosis. And you can see each individual colour in this graph represents an individual mouse that we track over 70 days, recording the disease. The scale on the left, going from zero to five, is the clinical score. It’s not linear, in that zero is obviously no clinical symptom, one is a light paralysis of the tail of the mouse, whereas five is complete paralysis. So we record the level of paralysis induced in these mice, and you can see at the start, for example, the blue and the green lines, they were terminated because they got so sick, but then you can see the waves of remission and relapse across the 70 days, which recapitulates the human disease. So these are our mice with MS that we’re trying to treat, and this graph is the outcome of 3-4 years’ work where we treated mice in this animal model with the parasite protein. So this is a protein that we’ve identified that’s secreted by the liver fluke parasite. We make in the lab synthetically and then inject it into mice. We only give a very short-term injection, so we’re not giving it very day, making it a more effective and better treatment than what’s there already. We don’t see any side effects, because the worm, you remember, lives in the human host and has really done all the work for us. It’s evolved this protein to have a very specific effect with very little cytotoxicity, and what you can see is the dark black line represents those mice that were untreated, and you can see again they have that relapse remission profile of disease, but the grey line on the bottom, which is almost flat, are those mice that were given the parasite protein. And in those mice, 20 per cent got no disease, 50 per cent of the mice only had one attack – and you can see when the attacks occur, they only reach a level of one. So the parasite protein clearly in this animal model of disease, which is an accepted pre-clinical step, is showing efficacy. Wow. So what’s it doing? I’m not going to go into all of the scientific details, but as I said, it’s quite specific. So the worm has spent millennia, if you like, evolving these molecules to have very specific effects, and we luckily have identified one of those proteins. So what I’m just showing you here is we know that it binds to one of those cells within the immune response, so it’s directly targeting a cell of the immune response. On the top you can see a macrophage, which is one of the cells of the immune system. The membrane are out, the surface is stained red and the nuclei are stained blue. And on the bottom in green, we can see the parasite protein attaching to that cell. So we can identify the cell it attached to, and basically if you look at the graph on the left-hand side, that’s how activated the cell is, and with the presence of the parasite protein, that graph is going right down to zero, showing that it’s preventing the activation of those cells, which in turn drive the autoimmune response. This means – I’m hoping you can see this – that on the left-hand side, this is the histology from the brain of those mice, and on the top, hopefully you can see there’s blue stain, which represents the myelin, and that’s the insulation sheath around the nerve, which is destroyed. You can see there’s lots of holes and tears in it, and on the bottom are those that were treated with the parasite protein. It’s a continuous blue line, except where we have the blood vessels. So treating with this parasite has meant the immune response is dampened and all the cells stay within the blood vessels, meaning we don’t get any destruction in the brain itself. So can we see a future? I’m hoping yes. As Liz says, it is a very long journey. We’re trying to translate this peptide towards the clinic. We have a long way to go, so the data is very encouraging, but we would be early stage research, and certainly pre-clinical. But I would be hopeful that certainly in the next 5-10 years, we may actually see a worm-derived pill on the shelf in Chemistworks. Everything in science is a team effort – it’s not just me. Over many years, we’ve had lots of people working on this project – people at UTS in the labs here, we have strong collaborators in Queens University in Belfast and also the University of Queensland, as I mentioned, who do the MS [inaudible]. And without the funding that we get from the government and also the charitable organisations, none of this work would be possible, so a big thanks to all the funders. And I would say your donations do make a difference – all of this work was funded by MS Research Australia and the MS Society in the US, which is funded by the public donations, so please do feel that your donations are worthwhile and making a contribution. Thank you very much.

[Applause]

Liz Harry: Wow, what a communicator. Thanks Sheila. I think a worm a day will keep the doctor away, and yes, this is a long, hard road and now is the time where Sheila needs significant investment to get further, and that’s actually a really hard step. But you can see she has the motivation and the patience, and they’re absolutely required. Nural Cokcetin is a postdoctoral fellow at the ithree institute at UTS. She’s a microbiologist, and she has an equal passion for making an impact on the health of society, as well as in communicating science to as broad an audience as possible. In June this year, she was runner up in the world final of the international Fame Lab science communication competition held in the UK. This is a really brilliant achievement.

[Applause]

So she doesn’t get away without giving a talk tonight. Nural completed a PhD at UNSW, University of New South Wales in 2015. Her PhD work was on honey and on testing it as a prebiotic to help with gut health. And this resulted in only 3-4 years of a PhD and the launch of the first prebiotic honey product on the market. She was the first to show that bacteria are not able to become resistant to honey, unlike the situation with antibiotics, where we have a global health crisis due to antibiotic resistance. She’s also coordinating a study to identify active bacterial-killing honey in Australia. Please join me in welcoming Dr Cokcetin to the stage.

[Applause]

Nural Cokcetin: Thank you Liz, and sorry about those slides – that was me accidentally clicking without realising. Okay. Honey has been used as a medicine throughout all of history by almost every culture that’s had access to it. We’ve seen records of its significance and its use in lots of different rock paintings and carvings and sacred texts all around the world. And an example down here from ancient Egypt is a recipe for a wound salve that heavily features honey, and ancient Egyptians used honey a lot in their medical practices. It’s even used to treat a wide range of ailments, from eye infections to throat infections to gastroenteritis. But it’s been persistently popular as a topical application for wounds. The reason for its continued popularity has no doubt been because it has potent, broad spectrum anti-microbial activity. But with the discovery and then clinical introduction of antibiotics in the 1940s, the medicinal use of honey was largely left in the past. Today, as Liz just explained, we are having a global crisis of antibiotic resistance, which I’ll go into in the next couple of slides, so now we’re looking back to nature and back to honey to help solve some of these issues. Since their discovery, antibiotics have helped us treat lots of diseases that used to be deadly. So they’re really important. And the reason that they’re failing today is because of the huge increase in the number of superbugs. Now, these are bacteria that have learned the ability to fight off the attacks of antibiotics. And in fact, we now have superbugs that are resistant to every single antibiotic that we currently have available, so it is a huge problem all around the world. In fact, by the year 2050, it’s estimated that we’ll be seeing 10 million deaths per year because of antibiotic resistance, so because of these superbug infections, and that’s a number that surpasses the number of deaths that are caused by cancer today. So the problem isn’t that the drugs have simply stopped working, but it’s actually because the bacteria have evolved or changed to fight off the effects of the drugs, just like what’s happening in this graphic here. Now, antibiotic resistance is a natural phenomenon – it does occur naturally. Bacteria, just like every other organism on earth, goes through evolution and natural selection, and in this process, they can randomly mutate or change in accordance to their environmental pressures. So they do this in order to fight off any environmental pressures, and although it is a natural occurrence, this antibiotic resistance, we do do a lot of behaviours that accelerate this process. So some of these behaviours include overusing and misusing antibiotics, like when we treat them to treat viral infections like colds and flus. We also hugely use antibiotics in agriculture, and we use them incorrectly as a growth promoter or to prevent rather than treat diseases. And what that means is that antibiotics are constantly in the environment, so these bacteria can very readily learn how to become resistant to them – how to fight off the effects. And because resistance happens so quickly in the clinic to antibiotics, there’s actually very little interest in investing money to develop new ones, because as soon as we get it out into the clinic, we’ve already seen reports of bacterial resistance. So honey is not going to solve all of the problems of antibiotic resistance, but it can certainly help to alleviate some of the burdens, at least in the wound care sector, so we can use it as a replacement for topical antibiotics to treat things like burns, wounds, skin infections and other skin conditions like eczema as well. In Australia alone, chronic wounds effect 400,000 people, and we’re only going to see this number increase over the coming years, because we’re shifting towards an ageing population, and we’re seeing increased occurrences of obesity, diabetes and other inflammatory conditions as well. So although today I’m focusing mainly on the antibacterial activity of honey, honey also has lots of other wound healing properties that make it a very attractive treatment option – things like creating a moist environment, which is essential for wound healing; it stimulates the immune system, reduces inflammation and reduces wound odour as well. We already have medical honey dressings available for use in the clinic, and over the counter gels that you can purchase from the pharmacy without a prescription and they’re safe to use at home. And these are all approved and regulated by the Therapeutic Goods Administration in Australia and the equivalent regulatory bodies all around the world. So, there are lots of cases where honey has worked and antibiotics haven’t, and just a warning here – I do have a few wound pictures coming up, so if you’re a little bit squeamish you might want to look away when these pop up. Here we’ve got a chronic leg ulcer in a 78-year-old patient, and this was non-healing for eight months, and it was infected for just as long. They tried antibiotics and conventional therapies like compression therapy to help with the wound healing, but these failed for the duration of that eight months. They started honey treatment, and you can see by day 22, the wound had significantly haled, the infection was under control. But this is just one case study of many. Honey works really well in these extreme, chronic wounds that are non-healing, but it works just as effectively in acute wounds, burns and skin conditions like eczema and acne as well. Unfortunately honey is still often looked at as a last resort, and there are a couple of reasons for this. We are underutilising it, and one of the reasons is that there seems to be an attitude towards honey that it’s a complementary or an alternative medicine rather than a real medicine, and another reason is that even though we know it works, we don’t fully understand how it works, so we don’t know its mechanism of action. And that’s what our group focuses on a lot. So an important point to remember is that not all honeys are created equal. In the same way that they look and taste very different, they can also have very different medicinal properties. Now, the antimicrobial activity of honey comes from multiple factors. The first is that it’s high in sugar, and what that means is there’s very little water available for the bacteria to use – and just like us, they need water in order to survive. Another factor is that it’s got a low pH, which creates an acidic environment that a lot of bacteria can’t tolerate. These two factors are similar in all honeys, but they only make up a very small amount of the total activity we see. In honeys that have significant activity, most of this activity comes from the production of hydrogen peroxide, which is like a weak bleach, and it’s toxic to bacteria. Now, this hydrogen peroxide-based activity is hugely variable from honey type to honey type, and it depends on the floral source. For example, jarrah honey from Western Australia has very high levels of this type of activity. And then we’ve got some very rare unique honeys that have a different type of activity, significant activity, that’s not related to that hydrogen peroxide, and we call this the non-peroxide factor. And it comes from a different floral factor, and I’ll go into this in more detail. The most common, or the most popular example of a honey with this type of activity is manuka from New Zealand, and the Australian equivalents. So we do a lot of work on manuka and the Australia manuka-like honeys here at UTS. And for a long time, we didn’t really understand where this special type of activity was coming from – we just referred to it as a floral factor. But in 2008, two groups of researchers looked at these really active honeys and also found there was a chemical called methylglyoxal, or MGO, that was present in high numbers in these honeys. So they called this the active ingredient, and what they found – and you can see in this graph – is that the more of this active ingredient we have, the higher the activity. So the reason that we are focused on this type of activity is because it’s very potent, it’s very stable and we can sterilise the honey without affecting its activity, and what that means is we can use it in the clinic, because we need to use sterile approaches. So I’ll go through some of the things that we’ve found over the years of research. I started honey research as an honours student 10 years ago now, so this is stuff that we’ve accumulated over 10 years. The first thing is that honey is very good at killing bacteria that cause infections, and these include the superbugs that are resistant to antibiotics – they can be resistant to one antibiotic or multipole; it doesn’t matter. The honey doesn’t discriminate. It also doesn’t matter what the antibiotics are. So some of these super bugs that we’ve shown that honey kills, we’ve got MRSA, which is golden staph; streptococcus, which is the flesh-eating bacteria; you’ve got pseudomonas, which causes nasty infections in burns patients. And we’ve tested lots of different species and strains of these and shown that it works very effectively. Perhaps one of the most exciting things about honey is despite being around for thousands of years, and being used as a medicine for thousands of years, there are no reported cases of bacterial resistance to its killing effects, and we can’t seem to generate resistance in the lab either. So we work a lot on trying to get this bacteria, these superbugs, to become resistant to honey and resistant to antibiotics. And the way that we do that is by training them, so we expose them to non-lethal levels of these things, get them used to living with it in their environment, and then we keep increasing the dose until we get them to become resistant. So here’s an example of some of the results that we get. The red dotted line indicates the resistance threshold, so any time the graph deviates above that line, that means we’ve got resistant bacteria. If we follow the black line, which represents the antibiotic, you can see by day 15 – so just two weeks into the study – we’ve already got bacterial resistance to that antibiotic, and that resistance just keeps going up the longer we continue the experiment. If you follow the blue line, you can see it never reaches that threshold, so we can’t get resistance to honey. So we’ve tried this with a few different antibiotics, a few different types of honey and different types of bacteria, and the results are always consistently like this. So what complicates infections even more is that bacteria actually exist in biofilms. So biofilms are like communities or networks of bacteria – they all come together and they stick to each other and then they usually stick to a surface. And this surface can be living, like a wound bed, or it can be non-living, like a catheter. Now, in this graphic here – I hope you can see that above the sign – you’ve got the free-floating bacteria swimming across the top, and then you’ve got the biofilm, and that’s in a wound bed there. So you’ve got different types of bacteria that have all come together, they’re communicating and they’ve released this slime layer, or this goo, like a protective shield around them. And that protects them from the environment, but it also protects them from our bodies’ natural defence mechanisms, and antibiotics as well. So when you’ve got these biofilm infections, which are 80 per cent of the time, you’ve got to have a treatment option that’s not only killing lots of different types of bacteria, but it needs to be able to penetrate into that biofilm and break it apart, and when that biofilm disperses, that treatment option needs to kill all the bacteria that come out of there as well. And that’s a problem in itself, because bacteria come out of the biofilm a lot more resistant than when they go in. So of course, we were interested in seeing whether honey works on biofilms, and I’ll talk you through these images. So the first thing we found was that not only does honey prevent the formation of biofilms in the first place, but once these biofilms have formed, it’s a very effective treatment option. So in the top panel, you’ve got what a biofilm looks like – these are some microscopy images. That fluorescent green represents live biofilm stains, or cells. And you can see that when there’s no treatment, no honey, they’re growing very [inaudible] of green lawn there. As soon as we add honey into the equation, you’ve got a much lower amount of that fluorescent green, so that means that it’s effectively getting rid of or eradicating that biofilm. And I’d just like to point out here that we’re using 32 per cent manuka honey here and seeing that drastic difference. In the clinic, we’d just be using straight honey, so you’d just get 100 per cent honey, put that on the wound and wrap it up. So even if you had a particularly weepy wound, where it diluted the honey from 100 per cent, all the way down to 32 per cent, you’re still having a significant clearing effect. So I’ve got another wound picture coming up. Yeah, this one’s a bit horrible. So this is an 80-year-old lady who had these really chronic infected wounds for 20 years, or longer than 20 years. She had recurrent infections, including the biofilm infections. She couldn’t walk, she was in extreme pain, and the doctors had decided that amputation was the best way forward. One of the nurses suggested that they try honey as a last resort, and you can see here, at five weeks, the infection’s under control and the wound’s mostly healed. By 10 weeks, the infection was cleared and the wound healing had really begun. She’d begun to walk again, and her quality of life, as you can imagine, was hugely improved. So in this woman’s case, the honey literally saved her limbs. So some of the other work we look at is whether we can use honey as a combination treatment with antibiotics. And one of the reasons that we’d like to do that is because combined, they might be able to have a better effect, and one example of a better effect is by reducing the dose of antibiotics that we use, because that helps slow down antibiotic resistance. So here we’ve got an agar plate, and that cloudy stuff is bacteria growing there. We’ve got staph. In the middle, you’ve got a disc and that’s soaked with the antibiotic. And around the disc you can see there’s a zone of clearing – I hope you can see that. And what that means is that the antibiotic’s working – the bacteria can’t grow in that zone. But we do have a few single colonies – they’re the dots in that zone, and that means the bacteria are resisting the killing effect of the antibiotic. In the second panel, we’ve got the same set up but this time we’ve added honey into that agar plate. And you can immediately see that that the zone of clearing is much bigger, so the antibiotic and honey together are working really well and clearing more bacteria. So that’s great, but this was in the free-floating bacteria, which we know is not necessarily representative of the clinical situation, so we also looked at this in biofilms, and this is the same as the image types before, where you’ve got the biofilm growing on its own in that first panel. Just next to that, you’ve got the biofilm treated with honey; that’s at a level we would expect not to have any effect, so it’s just a very low dose. Then you’ve got two low doses of antibiotic in the next screen, and when we combine those low doses that had no effect on their own, you can see a huge clearing of the biofilm. So it’s very exciting – it means we can use it in combination therapies as well. So our PhD student Daniel, just in that corner, and another honours student, Simon, have ben looking at understanding how honey works to kill bacteria. And they’re looking at this, at genetic changes in the bacteria. And what we’ve already identified is because honey has lots of different modes of action – so lots of different antibacterial factors – it’s targeting lots of different things in the bacteria itself that are really essential, like making DNA, making proteins and forming these biofilms. So when we figure all this out, it means that we can use the information to develop new drugs that work in the same way as honey, but it also means that we’ll just be able to promote the use of honey in the clinic. If we can understand how it works, we explain that to clinicians and they’re more likely to use it. But currently New Zealand is the main producer of these medical grade honeys, and already they’re struggling to keep up with the demand, and that’s where our Oz Honey Project fits in. So we’re surveying the Australian equivalents of these manuka honeys, of which we have over 80 types growing all around the country, as you can see in that map there. And we’re looking at the chemistry of these honeys and the biological activity, so we’re looking at how it affects not just bacteria but fungi as well. So in a previous study, we had access to 80 of these Australian manuka-like honeys, or the leptospermum honeys that we went back to test. These were originally collected in 2007 when we didn’t know about that active ingredient. So the first thing we did was go back and test whether we had that active ingredient. And yes, we did. And you can see here that the relationship between that active ingredient and the activity of these special honeys is the same as the manuka, and what that means is we can supplement our variety of the honey in that medical honey field. We were also in this unique position to compare how the activity had changed over that seven year period, and we were actually really surprised to see that it hadn’t changed at all, so it’s very stable under these storage conditions. And that has huge implications for extending the shelf life of medical honey products, which is especially useful in remote areas. The new collection is still ongoing; we’ve received 1000 samples so far from all around the country, as you can see by the dots on the map there. And we’ve already identified a number of species that have very high levels of activity that we can use in the clinic. So, just to sum everything up: honey is an attractive treatment option for topical infections, and we know that it works against superbugs resistant to multiple different antibiotics, and that it’s effective against biofilms as well. It can be used on its own in the clinic, and also in combination therapies too. The best bit is that there’s no resistance to honey, so we’re helping the antibiotic resistance crisis in that way as well. These manuka-like honeys are still the primary choice for medical-grade honeys around the world, and we’re not going to run out anytime soon, because Australia has this untapped resource of these as well. But this is just the tip of the iceberg, so we’ve focused a lot of one certain type of honey ,but we also know that there are lots of different Australian honeys and honeys around the world that have just as important antimicrobial activity. I’d just like to finish by thanking everyone that was involved in the project, especially the Oz Honey team, and of course the hundreds and hundreds of bee keepers who have donated samples – we would not have been able to do any of this research without your support. Thank you.

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