Combating the epidemic of "super-bugs"

Lecture by Distinguished Professor Elena Ivanova, RMIT Professorial Academy
Topic:  Combating the emerging worldwide epidemic of “super-bugs”
Presented on 3 July 2019 at RMIT University

Transcript

Speaker 1:       My name is Xinghuo Yu, I'm the Chair of the RMIT Professorial Academy. This occasion is around a very important task of this Academy which is about promoting the excellence on research, education and engagement, and also it is about giving advice and engage within the forum. Today it's my good pleasure to introduce Professor Elena Ivanova. She's going to present the fourth RMIT Distinguished Lecture. Just before I introduce her, I'll just give you a bit of introduction of the RMIT Professorial Academy. This Academy was established by the Vice-Chancellor in 2017. The purpose of it is basically trying to make use of the brands and influence of RMIT Distinguished Professors. At this point in time we have 18. I use them as a think tank to advise the University on future strategic directions, sort of a thought leader, and also as an advocator on behalf of the University. The Lectures are one of the important elements of that mission, but we will do more than just that. In the future we also will hold a public forum on issues, on strategies, on policies, which I think you are more than welcome to participate.

Speaker 1:       Today's talk is presented by Distinguished Professor Elena Ivanova. She joined RMIT in 2018. She's from Swinburne and one of the high flyers. Today's talk is about super-bugs. I think this is one of the highest numbers of participants, just because of interest. But I think it's more because of interest of topic and also, I guess, people want to know you more, about what you're going to do for us.

Speaker 1:       She's going to talk about her research and also her perspective. More than just her own research and perspective, about the future. What do we see for the future. Certainly, we all worry about super-bugs, so you know, that's why we're all very interested in what it is you have to say.

Speaker 1:       Please give a round of applause to welcome Professor Ivanova.

Speaker 2:       Thank you very much for the nice introduction.

Speaker 2:       I'm very honoured to talk to you today about our work and, as you see now, it's one of the major concerns worldwide, the epidemic of super-bugs. And they tell you that we are not losing the battle. There is a light at the end of the tunnel and we will see how we can do it.

Speaker 2:       As an introduction, I would like to talk about some problems imposed by antibiotic resistance, bacterial infections and emerging super-bugs. And then we can see how insect wings, natural bactericidal surfaces could be a potential solution to some of these problems. And I'll show you examples of novel mechanical ways, how we can kill bacteria without any need of antibiotics.

Speaker 2:       The rise of super-bugs. I'm sure you're all well-aware of increasing numbers of reports telling us really dramatic stories. Just a few of them are on the screen. In 2017 the Centers for Disease Control and Prevention reported that a woman in Nevada died from an infection resistant to all available antibiotics in the United States. In the same year, there was another report in China when super-bug Escherichia coli in this case was found resistant to last resort antibiotics carbapenems. That is an antibiotic of broad spectrum of beta-lactam plus. In fact, in Victoria 2016, a 56-year old man from rural Victoria with no history of hospital contact or international travel was reported to have died from Klebsiella pneumoniae and that particular strain was resistant to all antibiotics available in Australia.

Speaker 2:       Just to tell you that the background of this slide, what you see here, is an electron micrograph of Staphylococcus aureus or 'Golden Staph,' which is resistant to Methicillin and you can see how it is colonising, how it can colonise some blood cells. It's growing and colonising the cells.

Speaker 2:       Now, before we go on any further, I thought it will be useful to give you some terminology so that we all know what actually is a super-bug. A definition I found in The Royal Institution of Australia 2017, a 'super-bug' is usually defined as "a microorganism that is resistant to commonly used antibiotics" and of course 'antibiotic resistance', if you look at a major website, you'll find a definition of ‘antibiotic resistance’ which is "an ability of microorganisms and that could be bacteria, fungi or protozoans to grow despite exposure to antimicrobial substances designed to inhibit their growth."

Speaker 2:       You may ask me, how we now come to this point where the antimicrobial resistance is developed. There are several different ways. The major reason is selective pressure through drug use in medicine. Another serious reason is that uncontrollable use of antibiotics in many countries. Australia is not there, not in that list. Also, inappropriate prescriptions. But the major misuse is in agriculture. It's up to 70% of all antibiotics produced worldwide actually used mostly as a prophylactic against disease or as a growth promoter. And, of course, we do not forget about genetic transformation where the genes developed resistance to antibiotics could be easily transferred among different groups of bacteria.

Speaker 2:       Again, you may ask me, is it so difficult to kill bacteria still? It's so small. Well, look at these facts. Temperature: high temperature can destroy proteins, nucleic acids and damage membrane, yet we have bacteria called Pyrolobus fumarii, which can grow at temperatures up to 113 degrees. How about radiation? Can radiation kill bacteria? Yes, it can, but not all of them. There is a bacterium called Deinococcus radiodurans, which can withstand ionizing radiation up to 20 kGy and also UV exposure. What about pressure? There are actually obligatory piezophilic species that can grow at 70 to 80 Mpa. What about reactive oxygen species? In fact, oxidative damage induces production of antioxidants and detoxifying enzymes by bacteria, so they can easily develop resistance to any reactive oxygen species.

Speaker 2:       Now, you can ask me again, why are they so smart? Why are the bugs so smart? And I'm always telling that don't forget that bacteria are the oldest life form on our planet. They have survived an evolution of 3.8 billion years and of course and they've developed versatile metabolic pathways. They can colonise each and every surface. They can survive everywhere, everywhere. Now look at this example. It was estimated that the total number of bacteria in 70 kg (so called ‘Reference Man’), could be 38 trillion. 38 trillion. It could be roughly up to 5 kgs we have in our body. And look at this scanning electron micrograph which I used as the background of this slide. What you see here is a community of bacteria which are found on the surface of our tongue. So, bacteria everywhere and looks like it's not that easy to kill them.

Speaker 2:       Now how do they survive? You may ask me again. A single bacterial cell will not be able to survive because it's very tiny, it's not protected, it’s very difficult for it to survive. What happens, bacteria survives in communities. And these communities are called biofilms. What you see here is just a single bacteria cell, they call it free-floating planktonic cells, settled on the surface. They aggregate, form micro-colonies and excrete extracellular polymeric material. That's how the biofilm is formed. The biofilm growing through that and producing a complex, three-dimensional structure. Something like that, what you see here. And cells they are protected by extracellular polymeric material against host immune cells and antibiotics. When the biofilm reaches a critical mass, the cells could release planktonic cells, could release again and colonise each and every other available surface. That's how the bacteria spread. Once, it forms a biofilm and release from the biofilm.

Speaker 2:       And this is also a reason why they can easily develop resistance to antibiotics because in the biofilm, they are protected from any antibacterial treatments. In fact, and they form infections. Implant-associated infections is number one cause of implant failure in patients. What is also dangerous is once the biofilm reaches the bloodstream, the bacteria can colonise more surfaces in our body. On this little picture, you see size of primary and secondary infections. And in a way that was our own motivation. We wanted to design surfaces which would be free from bacteria. We wanted to fabricate surfaces which could be used for bacterial implants. What you see here is a little movie made in our lab, maybe a good ten years ago now, showing how quickly Golden Staph can colonise the surface of titanium, which is commonly used in implantable materials for the hip replacement you will see here. And here is the real scanning electron micrograph of the biofilm form Staphylococcus pseudointermedius on the orthopaedic bone screw. That's what is happening.

Speaker 2:       Now, as I say, our motivation was to design these surfaces, but how did we rationalise our motivation. We were thinking that, because bacteria survived through colonisation on surfaces, why don't we look at the surface typography and see if we can modify the typography of the surface and stop bacteria from attaching. Once we stop bacteria from attaching on the surface, the biofilm will not be formed and we will be safe. So typography was our focus and the very first experiment we've done, but before we go there, I'll tell you currently what a traditional approach is, where people try to design antibacterial surfaces.

Speaker 2:       Of course, we are not the only one group trying to design antibacterial surfaces. Currently there are basically two major classes of antibacterial surfaces. Antibiofouling, which will be repelling bacterial cells from the surface, and bactericidal. In this type of surface, the bacteria in contact with the surface will be killed effectively. For that, we use a low molecular weight antibacterial compound including antibiotics, it could be antimicrobial peptides or even silver. Unfortunately, there are some drawbacks associated with these approaches. Toxicity, non-uniformity coatings, decrease in efficacy, environmental health concern and, of course, microbial resistance to antibacterial agents. So back to the start of our work, what was now many years ago, the attachment points or settling points theory was one of the main well accepted norms. What it says is that "the organisms smaller than the scale of the surface micro-texture will attach in larger numbers in micron scale shelters on the textured surface and will have greater adhesion strength because of the multiple attachment points on the surface". So, we said, all right, how about very small surface without many, no attachment point. Would it repel bacterial cells?

Speaker 2:       And the very first, very simple experiment was done by one of our PhD students. Just using the standard microscopic slide, which is actually quite smooth, with the average surface roughness in the range of 2 nanometres, and after the treatment with the buffered solution of hydrofluoric acid, these little peaks are gone and you see this surface even more smooth, with 1.3 nanometre roughness. What you can see here, is just a remarkable response of bacterial cells where you see that the film numbers on the nano-smooth surface, which is really not much change, is even increased. Also changed morphology, morphological transformations and extracellular polymeric substances. So, we re-confirmed this result using different surfaces, including titanium and also using different range of surface roughness, and the results were the same.

Speaker 2:       The main conclusion coming from this work is that the bacteria is far more susceptible to nanometre scale roughness than previously believed and nano-smooth surfaces do not represent a repelling environment for bacterial attachment. So that's why we had to move on and find any other surfaces which may be free from bacteria. And of course, we always look at nature. In nature, what you see in nature, there are lots of examples of bacteria-free surfaces and these are plants or insects, where you can find them free from bacteria. Now, the particular self-cleaning effect associated with these types of surfaces and it's mostly due to the superhydrophobicity of these surfaces, which in turn is a combination of surface chemistry and a particular amount of typography. So, these superhydrophobic surfaces, in a way, are not wet. And the water droplets just bounce on the surfaces and slide, roll off. And on the way of rolling off, it takes all contaminants away from the surface, so it's remaining clean.

Speaker 2:       What we've done next, in collaboration with our colleagues in a Laser Center at Hannover University, we were able to mimic the surface of lotus leaf on titanium and that's how it looks like on your left-hand side. You see an example of what that droplet's behaviour on the glass surface and on the superhydrophobic surface. Obviously, the water contact angle is 153 degrees. Now what happens when we immerse this surface in suspension with bacterial cells? And for that, we used two different types of cells: P. aeruginosa and S. aureus. You see, P. aeruginosa didn't really attach on this surface. However, S. aureus quite successfully colonised this type of the surface. We have to answer the question of why?

Speaker 2:       And it was the brilliant work of our PhD student, Dr Truong, now also a staff member of School of Science. We looked at what happened to surface when they immersed in water. What you see here is a little movie showing you how quickly air replaced by water. So, and that is really followed by the aggregation of S. aureas cells and their colonisation of the surface. Basically, air was one of the most important components in this surface and this surface lost air and it became favourable for attachment or S. aureus cells in particular. So that was quite a disappointing result. So, we looked at other examples of bacteria-free surfaces in nature and there are insects.

Speaker 2:       The next object for our work was actually cicadas. You all know that cicadas are the loudest insect in the world and there are numerous species in the range of 200 species are available in Australia. We used in our work one particular species called Psaltoda claripennis. If you look at the nanoscale, the wing of the cicada will be composed of a tiny, tiny nanopillars of just only 200 nanometres tall and 60 nanometres in diameter. It's a very, very fine pattern which forms in the epi cuticle of insects. What you see here is really the wettability map constructed by another PhD student, showing that the surface maintains high superhydrophobic features with the water conduct angle ranges from 163-173 degrees. What happened with the bacterial cells when we immersed the membrane of the insect wing in the suspension with bacterial cells? And as you can see, we didn't get the results which we expected. We saw that bacterial cells will not be able to attach on this surface. They will be repelled, similar to the water droplet. But what you see here, it was quite the opposite: bacterial cells happily attached on this surface but it looks like they are not so happy. So what you see here is a collage of scanning the electron micrographs, [inaudible 00:20:52] there's also [inaudible 00:20:54] and confocal imaging indicating that the cells are actually dead on the surface.

Speaker 2:       Another experiment is an atomic force experiment. What we showed is that the bactericidal efficiency of the killing process of the cell on the surface of the wing is remarkably fast in the range of 200 seconds, we detect a rupturing point and it was quite a nice match with the height of the nanopillars, which was 200 nanometres. Basically, one single cell could be ruptured in 200 seconds by, I'm talking about Pseudomonas aeruginosa cell, a particular type of the cell. The efficiency, the killing efficiency is also quite good. What you see here is that the number of viable cells remaining in suspension was decreased by 96% per square centimetre over 30 minutes. So that was quite, we were quite happy with the results.

Speaker 2:       Now the question then, again, was how did this happen? Was it a chemistry playing a role in this activity or not? In order to eliminate the fact or understand whether or not chemistry has any effect, we spattered gold on the surface of the wing. And it's a very thin layer of just 10 nanometres gold so that the pattern is not changed so we wanted to maintain the pattern, the same type of the pattern. And what you see here is that in fact, the bactericidal effect remained the same. A conclusion for this work was that the surface chemistry is not really important in the bactericidal effect on the cicada wing, but rather it is typography that is the dominant factor.

Speaker 2:       Another question is to ask, how is it happening? What is the killing process? What is involved in this killing process? The theoretical analysis was done in collaboration with a group of physicists in Spain in University of Rovira i Virgili. That was a PhD student Sergey Pogodin at that time. He modelled the membrane of bacterial cell as elastic layer because the thickness of the membrane in the range of 6, maximum 10 nanometres and the diameter is quite large compared to this one. And what really happens in this particular situation. The bacterial cell is suspended on the array of the nanopillars and the stress which is developed upon this suspension such as high that it breaks, but it breaks, rupturing in between the nanopillars. So in a way these pillars cannot pierce the cell. And this is a very common mistake with some of the people trying to understand what happened on the surface. It's all about dimension. So the large, like this 6 nanometre pillar, it cannot really pierce a really thin layer of the membrane. Rather it stretches and breaks in between the pillars.

Speaker 2:       Unfortunately, this particular type of the nanopattern is only active against Gram-negative bacterial cells. Gram- positive cells, and we tested quite a few of them, Gram-positive bacterial cells remain safe on this type of the nanopattern. The reason for that, we believe that, our hypothesis, we didn't prove it yet, in the different thickness of the Peptidoglycan layer, which adds full Gram-positive bacterial cells additionally GDT for the film, so it's much harder to rupture it. So that's our working hypothesis. Hopefully, we'll try to prove it.

Speaker 2:       Now, the next object in our work was dragonflies. Dragonflies are another numerous group of insects and if you look at the pattern of this insect, it's quite different, that is the pattern of the dragonfly. Maybe it's not that clear here but it's pretty much clear here. It’s very random array of different lengths, quite some different lengths of the nanopillars. Very difficult to describe it. But what is interesting here, this type of the nanopattern is very active. Basically, it ruptured all cell types including B. subtilis spores. This is quite a remarkable observation, though we wanted also to understand, if different types or different species of dragonflies have exactly the same pattern, but looks like they're not, and whether or not they're the same. Well, what is exactly the same, it may be visual similarity. If you look at the surfaces of the dragonfly wings they look quite similar. But yet, they are not identical. On this little movie, you see the behaviour of the water bouncing, taken by a high speed CD camera which takes 28,000 frames per second. So that's what you see here, that's how the water droplets films when it bounces off the surface of the dragonfly wing.

Speaker 2:       On this table, you see the comparison of the nanopillar height and density for this type of species. It's quite similar, quite similar. But when we used three different species of dragonfly and looked at the bactericidal activity, it appears to be different. So that's what you see on this slide that the one Diplacodes bipunctata is the most active one and the two others, which we used, are quite different. AFM topology analysis showed that in fact the curvature, the geometry of the pillars maybe the key when the slight variation in the nanopattern may severely affect the bactericidal performance of the nanopatterns.

Speaker 2:       Our next work on trying to understand using simplified forces which are involved in the killing bacterial cells. And this work was about the simplified model of the cell membrane which is really the Giant Unilamellar Vesicles or GUVs, which were constructed from DOPC and we used two other species, other different species of dragonfly.

Speaker 2:       So, on this image you see the Cryo-SEM image of the GUV which was done for the first time, for some reason, despite that this is a very common model of liposome contraction, from variable defined lipids. We couldn't see that before in Cryo-SEM analysis. So, what we'd done at that time was understanding whether or not these GUVs would be ruptured on the surfaces of both species of dragonfly. And, in fact, they both ruptured on this type of the surfaces.

Speaker 2:       What you see here is a confocal scanning of micrographs and cryo-scanning electron micrographs, showing different stages of rupturing, GUVs rupturing on the surfaces of the wing. And that allowed us to estimate that the minimum about of additional tension required to mechanically rupture the GUVs was in the range of 4.3-6.75 mN per metre, but that's all dependent on the relative size of the GUV. That was a pretty good match with the previously recorded data, I think using a micropipette asperation, so we were pretty happy with that, but that's not a direct estimation of the forces. It's still something to study further, so that's a load of work to be done.

Speaker 2:       Now natural bactericidal surfaces are very good and very interesting but the question is, can we fabricate this in synthetic analogue. And the answer is, yes, we can. What you see here is an example of first biomimetic analogue of natural bactericidal surface, or Black Silicon. In fact, it's quite easy, the fabrication of this type of surface is quite easy. It's a matter of optimization of the parameters. So, I'm not going into technical details of the fabrication, just only tell you that its basically plasma assisted reactive ion etching, a quite common technique. Once again, it’s a matter of recipe and optimization of the recipe and you can quite reliably and consistently get the same pattern of your choice.

Speaker 2:       Now on this slide, what you see is a similarity of the pattern which we found on the dragonfly wing and Black Silicon. Once again what you see here, it's quite similar, but it's not an identical pattern. And in a way, it's a good thing. Which means that the pattern which will be bactericidal may not be necessarily identical to the previously found or designed or fabricated. It could be different but of course the variation may be in a very narrow range of the nanofeatures. So that's what you see here, that's a dragonfly and this is a Black Silicon pattern. The table gives you an overview of the bactericidal surfaces which could be of different stability. Black Silicon is not super hydrophobic. The chemical composition could be different, the nanoprotusions or nanofeatures of this surface could be also different, bactericidal effectiveness could be also variable, depending on the pattern. However, what you see here that sometimes synthetic analogue of natural bactericidal surface may achieve a greater performance as a natural template of bactericidal surfaces. So, it’s all about optimization of the surface nanoscale parameters.

Speaker 2:       The mechanism in the context of the dragonfly is not really defined as yet. We believe this is our hypothesis. It's still stretching of the membrane when it's ruptured in between the nanopillars because even the Black Silicon nanopillars appear to be sharper. They're not reaching the dimensions below 10 nanometres. So basically, it's really from a very theoretical point of view, it's very difficult to pierce the cell. Still the membrane will be hard to pierce.

Speaker 2:       What is another remarkable thing about this type of the surfaces, in the way they are self-cleaning? But they are not self-cleaning as it was in the context of the water, when it's bouncing off the surface, but it is self-cleaning when you see that the cell debris actually removing from the surface all the time. The removal of the cell debris from the surface is not as quick as it's rupturing and it's highly dependent on the nanopattern. But in a range, it could be 20-30 minutes, which is required to remove the dead cells from the surface where the debris is probably floating around in the environment.

Speaker 2:       Now you may ask how about eukaryotic cells, what happened in eukaryotic cells? If bacterial cells are effectively ruptured, what about big, large cells? As you can see here, that was our work some time ago where we used fibroblast-like cells, what you see here it's a movie of attachment and settling of COS-7 cell on the Black Silicon surface. And here you see a freeze fracture image where you see the cell and the nanopattern. So, the cell is safe on the nanoscale pattern. Well, if you think from the logical point of view, the nanopillars are too small and I'm always saying that, perhaps it's like a mosquito bite for us, that's what the nanopattern for eukaryotic cell. It’s a matter of different scales of the pattern, which may affect. So for eukaryotic cell bactericidal surfaces are safe. And in a way, it has a very significant, and it’s very important in the context of implantable biomaterials.

Speaker 2:       What you see here, it's another experiment. When we try here to infect the surfaces with infectious doses of pathogenic bacteria, and after that seed the surfaces with eukaryotic cells. As you see here within six hours, bacterial cells are effectively ruptured and eukaryotic cells you can see on the next slide are growing very nicely. So what you see here is the COS-7 cells on Black Silicon pre-infected with Pseudomonas aeruginosa and the same green surface pre-infected with S. aureus. And you see on Day 7, eukaryotic cells are growing nicely and reaching confluence stage and on the surfaces, which are flat or the ones which are used for implants, the infected surfaces keep going with the bacterial cells. I mean bacteria pathogen, bacteria happily growing on these surfaces and they, in fact, inhibit and contaminate eukaryotic cells. And eukaryotic cells do not survive on these surfaces.

Speaker 2:       Moving on, the effect of mechano-bactericidal surfaces, as we are trying to now develop this further to show you that it's quite versatile. So there are different ways which we can use or we can use a different type of nanopattern to kill bacterial cell by different way. This is another example when we can cut bacterial cell through using the nanostructured surface. And this surface could be a graphene-like surfaces. What you see here, that you can have a graphene surface with graphene flakes, which is very easy to obtain and we looked at different types of flakes fabricated from graphene and see how bacterial cells will respond on this. On the top you see graphite surfaces. And these two types of graphene surfaces, they are, in fact, highly bactericidal.

Speaker 2:       But until now, the nature of bactericidal activity of graphene flakes is not really resolved. Well there are two hypotheses. One hypothesis says that extraction of the bactericidal effect on graphene flakes. Extraction of the lipids from the membrane, that's what they say what happened when the cell interacts with graphene flakes. What we think it is and that is our work where we are trying to prove it, that actually it is a pore formed due to reorientation of lipid cells to graphene surface while they are interacting with the graphene flakes. What you see here, it's a little bit enlarged images confocal images showing the red cells, which I killed, looking swollen. And that's apparently because the cell, when its cut through, the environment external liquid comes inside and it appeared swollen.

Speaker 2:       Now the theoretical analysis done in collaboration with the same theoretical physicist group in University of Rovira i Virgili, that is Vladimir Baulin you see here, and they apply single chainmail field simulation to show that the pores are actually formed in the interaction. We believe this type of bactericidal activity, the bactericidal efficiency on graphene-like substrata or graphene-like material of this kind depends on the lateral size, shape and interactive angle of exposed sharp edges which are likely to puncture the bacterial cell membrane.

Speaker 2:       Another way we can kill bacterial cells using nanostructure surfaces is this. I'll show you how bacterial cells are torn apart on the nanostructure surfaces. What you see here is exceptionally high aspect ratio up to 3000 of vertically aligned carbon nanotubes. This work was done in collaboration with the group of Michael de Volder in Cambridge University. So what they've done, they grew carbon nanotubes from a catalyst layer and deposited on a silicon wafer using physical vapor deposition. We used two types of the pattern with the one microgram and 30 microgram vertically aligned carbon nanotubes. What you see here is that this type of the surface is also quite bactericidal. The highest bactericidal rates of 99.3% for P. aeruginosa and 84.9% for S. aureus were recorded on one microgram tall vertically aligned carbon nanotubes.

Speaker 2:       Now the question is, again, how this happened? Why? And this is quite interesting. In collaboration with the same group, what we found and they applied so-called engineer's beam theory, or linear theory of elasticity. This same theory was applied for engineering of the Eiffel Tower and this large wheel. It's exactly the same theory used for construction and we applied it to explain the high aspect ratio. So what you see here and what is explained is just stored and released energy. So basically the bending of very tall nanofeatures, when bending they store energy. And when they bend back, they release energy. And that's how it tears the cell apart. And it's quite logical to imagine that, of course, one microgram nanotubes are actually more rigid and store higher energy and that's why they are more effective.

Speaker 2:       And I think, I just would like to finish up with some aspects of the nanofabrication which is currently in this field and give you just one example of our recent work on the hydrothermal treatment of titanium. So basically there are different types of nanofabrication techniques which could be applied to produce nanopatterns. And the nanopatterns could be quite different. It still will be active but the range of the activity could be quite different, so that is a change. These are techniques you can use: laser irradiation, lithography, plasma etching, electrospinning, and even simple data of nanoimplant lithography template methods. It's all about the resolution, but with advances in nanofabrication where we can reach nanoscale resolutions, then the results will always be promising.

Speaker 2:       The hydrothermal treatment is one of the examples of nanofabrication using a very simple technique, but again, it's a matter of optimization. Optimization of parameters and time of the treatment where you can fabricate the surface of different patterns. So what you see here is the formation of asymmetrical nanosheets with sharp nanoedges and in a way, I would say the mechanism of bactericidal efficiency here could be similar to the sharp edge of graphene nanoflakes. It's in the same type of bactericidal effect as we believe it is. If you look at the bactericidal activity you see the P. aeruginosa could be up 100% cells killed and a quite large range of the treatment conditions, however S. aureus is much more difficult to kill, as all this. But it could be a high rate of killing could be achieved after six hours treatment of these surfaces.

Speaker 2:       And I would like to finish up again with this particular antibiotic resistance problem and show you if nanostructured surfaces, and we call them mechano-bactericidal surfaces, can kill antibiotic resistant strains. What you see here, I just wanted to emphasize that we used and there is a report from the Centers for Disease Control in the United States, 2013. And this year, in autumn they will break news in new edition of the same report. They identify the top 18 antibiotic resistance threats in the United States and Methicillin-resistant S. aureus is one of those threats regarded as a "Serious Threat". What I also found quite upsetting data that in the way there was epidemics that in our community, it's up to 5% of healthcare workers in the United States are carrying this Methicillin-resistant Staph aureus. And up to 2% of the population are actually carrying the same thing.

Speaker 2:       Now I probably wouldn't stop here except just to tell you that the antibiotic resistance and mechanisms are quite different, for different types of antibiotics in the case of Methicillin-resistant bacteria. What happens with this strain. First of all, the drug cuts the bonding, the close-linking that is again Peptidoglycan. So once the drug cuts this close link between the Peptidoglycan layer, the inner work is damaged and the cell is dead. What is happening in the Methicillin-resistant strain? It is actually building the bonding again and that's how it survives. That's how the resistance is developed. And now what we did in this work, we used Methicillin-resistant strain and Methicillin-susceptible strain (our regional standard strain) and we put this strain on the hydrotreated titanium surfaces and what you see here, they are dead. They are dead. It doesn't matter in the presence of antibiotics, I should say, the build up of the reconstruction of the bonding of the peptidoglycan layer triggers by the presence of antibiotics. That's why we run experiments in standard physiological conditions and in the presence of antibiotics. And you can see both types of the strains are successfully killed by the surface.

Speaker 2:       So, yes the conclusion we arrived to that highly bactericidal nanotopologies of insect wings represent the first reported example of purely physical antibacterial activity and opened a new era of biomedical antimicrobial nanotechnology. These mechano-bactericidal surfaces will be potentially useful when applied to implants reducing the risks for post-operative bacterial infection, and minimizing the demand for antibiotics, thereby helping to combat antibiotic resistance. Mechano-bactericidal can, of course, be exploited in various industrial and bionic applications.

Speaker 2:       Future directions. The target now is to fabricate the surface with dual-functionality so there will be repelled bacterial cells and the ones that will not be able to repel and settle on the surface will be killed by this surface. This is our next goal and hopefully we can achieve it with the help of a group of students.

Speaker 2:       And I think that was all from me. I would like to acknowledge many, many people without the excellent contribution of these people, this work wouldn't be finished, confirmed, performed. And of course, Professor Russell Crawford, Khanh, lots of people from Swinburne University of Technology and our colleagues all over the world, and this last slide with the students. Oh! with the students, and I was hoping that the movie would run, showing the work of the students on the left, but it looks like it's not. But thank you very much for your kind attention.

Speaker 1:       Thank you very much for a very interesting and entertaining talk. Any questions? We can have a few minutes for a few questions. Yes.

Speaker 2:       Did I go too long?

Speaker 1:       It's all right.

Questions and Answers

Speaker 3:       So beautiful and eye-opening. So one potential problem, one potential opportunity in terms of [inaudible 00:49:21] or targeting all the type of lipid-enveloped organisms. So Pox viruses, the largest viruses, around 500 nanometers and they're lipid-enveloped. So I was just wondering as you were talking if there might be some utility also in that area? And the problem, the eukaryotic cells at least you've shown you've tested in this presentation, they have very nice mechanisms for recycling their membrane, for keeping membrane stability. But red cells, we have a red cell expert sitting there at the front, do not have those same capacities. So how would you see first in the opportunity in the Pox viruses and perhaps the in vivo utility in terms of perhaps negatively affecting red cells?

Speaker 2:       That's a very good question. I'm impressed you know this, the possible effect on the red blood cells. You are absolutely right: red blood cells are very specific and they will not be safe on the pattern and we actually did this work and we showed that they are not safe. But again, what I want to emphasize all over again, it's a matter of the pattern. So, you can optimize the structure, the nanofeatures of the pattern and use the pattern which will be bactericidal to bacterial cells, say even the super high aspect ratio features which kills a bacterial cell by a different way. But by this way the eukaryotic cells will remain safe again, including red blood cells. We simply didn't test it. But that will be beautiful work, actually to compare different types of bactericidal patterns and see how they affect red blood cells. But I think that not all patterns will be similarly affecting red blood cells.

Speaker 2:       As for the viruses, it’s a matter of, again, the nanopattern and the fabrication. I remember when we just started working on this, the resolution of the nanopattern seven years ago, was so poor that we couldn't even dream about what we can do now. So, for the viruses you need a pattern with more fine features, nanofeatures to see. But I can't guarantee. So there needs to be some work done.

Speaker 1:       Thank you very much.

Speaker 4:       It's been such a fantastic presentation and beautiful work. I'm also wanting to follow on from Magdalena's question about how you think this technology might work inside the gastrointestinal tract to, you know, engineer the microbial population. So really targeting and dealing with that mucus and biofilm, and that sort of environment.

Speaker 2:       I don't have any answers on this question because no work was done. I don't know. There's no perfect solution, because that's the nature of science. As long as we go on our way, we find something else. So, so far, I don't know the answer. But I'm sure that we can handle in the future and find a solution.

Speaker 4:       And we can discuss together.

Speaker 1:       [inaudible 00:53:00]

Speaker 5:       Can you put the surfaces on the cactus?

Speaker 2:       Yes. It could be... like I said, maybe five, seven years ago, it was basically impossible to put this particular nanoscale resolution in the range of 10-100 nanometres on the plastic. Now it is possible. I can tell you that there is a company in Japan, they are very close to fabricating that type of a nanopattern in a large commercially up-scaling area. So yes, it is coming.

Speaker 1:       Any more questions? It's okay, we have another second, it's fine.

Speaker 6:       I went to a beautiful talk recently [inaudible 00:54:01] where she was describing on the smaller scale how polymers by their nature and flexibility could avoid the binding of proteins and how their movement and their typography was very, very important. As you were talking I was wondering, because you were saying there was the issue of attachment and then there is the issue of the clearance. And I was wondering when you were presenting your talk, all the surfaces you were showing us were rigid. And whether there were any plans of also exploring motility.

Speaker 2:       Yes. And it is work also about to be completed. Like I said before, it was very difficult to run a systematic study with changing only one parameter on the surface like the length of the nanofeatures. It was only possible to achieve using nanotypography which is very expensive and very, kind of, difficult to achieve. We still managed to have this particular type of the samples and about to finalise this work where indeed the bending could be a part of the effect. Yeah.

Speaker 1:       All right. I think we might just stop here. We’ve got refreshments next door. So you can continue to talk to Elena and mingle with each other. So thank you very much for coming and hopefully we'll see you next time in September for another one. So thank you very, very much.

Speaker 2:       Thank you. Thank you.