 Good evening, wherever you may be. My name is Jeff Lejeune. I'm a food safety officer here at FAO in the Division of Food Systems and Food Safety. And behalf of the FAO AMR Working Group, I'd like to walk you in today's webinar. We have the fortunate opportunity today to hear from Dr. Lawrence Goodrich. We'll introduce in a moment, talking about antifungal resistance. What's all the hype about it? So I have a few introductory comments that I'd just like to go through. And we'll start at all of you. Thank you already for keeping your microphones on mute. So not to disturb the others as we go through. If you could, it would be helpful to rename yourself on Zoom with your organization and your name. So when we refer to you, if you're asking questions, we can get back to you specifically. Caveat here, the views presented today are those of Dr. Lawrence Goodrich and not those necessarily. In terms of the chat, we have the chat there is available to post questions and we'll aim to answer those questions as we go through. Just keep in mind that we'd ask you to refrain from posting any kind of advertisements or commercial services or branding in that chat box there. The last thing I'll mention that this meeting is being recorded, which we will be able to share after the meeting. I would ask you if you do not agree to be recorded, either to not say anything or just leave the meeting and then you will not be recorded. The same goes for the chat discussion. We'll be recorded and shared over your time. So this is for every convention that's at the end. We will, this is a monthly webinar series. We will have another seminar next month on the 8th of September and being enrolled in this, this serve we can inform you of the topic in the speaker. So without any further ado, I would like to introduce the speaker, Dr. Lawrence Goodrich, who is a Leone Family Professor of Food Safety and the Director of the Canadian Research Institute for Food Safety at the University of Guelph, located in Guelph, Ontario, Canada. His primary research is in the development of approaches for risk assessments for food-borne microorganisms, achieving this goal by combining both phenotypic and genotypic analysis and methods to characterize both virulence and antimicrobial resistance in food-borne microorganisms. The specific research studies support, I'm just trying to figure out how to quick share on my screen here. There we go, sorry. This is specific to research areas and identifying mechanisms of resistance in bacteria and in fungi and the role of mobile genetic elements. He also identifies new anti-microbial compounds that may be used to control the growth of pathogenic bacteria. And I know in the last two years, as many people have leveraged this knowledge in terms of addressing the COVID pandemic. With no further ado, I think I'll pass it over to Dr. Goodrich to begin. The format here is about a 30-minute presentation followed by adequate times for questioning and answer. Larry, please, welcome. Great, thank you, Jeff. And I will just share my screen here. Can you see my screen? Looks great, please proceed. Great, thank you. So good afternoon, everyone. Or good morning or good evening, depending on where you're joining us. And I'd like to thank you, Jeff and FAO, for inviting me to share my research today, where we'll be talking about antifungal resistance. So to begin, we know that antimicrobial resistance is an emerging threat to global public health. Currently, there are approximately 700,000 deaths caused by AMR every year. And it's estimated that by 2050, there'll be 10 million deaths caused, which will exceed the deaths caused by various other illnesses or reasons for death, as you can see on the right side of the screen, including cancer. So we really need to begin to study AMR with the goal of trying to understand the mechanisms by which resistance is developed and also with the goal of trying to develop alternative approaches for the treatment of pathogenic microorganisms. So one aspect of AMR is antifungal resistance, which has not received the same attention that antibiotic or I mean antibacterial resistance has received. And so today we will focus on antifungal resistance. And so this is related to the use of fungicides or fungicides that would either kill or inhibit the growth of pathogenic fungi and pathogenic meaning pathogenic to humans. And the issue here is that there's only three types of antifungal drugs. And so because of that, unlike antibiotics that are used to treat bacteria, we really are limited to how we can treat these pathogens. And the concern is that bloodstream infections that can develop in humans, if they are resistant to these, if they should become resistant to these three types of antifungal drugs that can lead to serious health outcomes and even death. And there are several, well, there are actually many different fungi that are resistant to antifungal drugs, but the two main genera of fungi that are of primary concern are Aspergillus and Candida. And within Candida, Candida aureus, which is a new species that is particularly resistant to antifungal drugs and can spread in healthcare settings is of major concern. So when we talk about fungicides or also different types of fungicides, but really it's the asyl compounds that are of concern here. And they've been used in agricultural practices since the mid 1960s to control the growth of fungi on food crops and to stop spoilage. And you can see on the slide that the crops that asyl compounds have been used on and are currently used. So the situation with the asyl compounds are somewhat analogous to, if we think of bacteria, the use of antibiotics in food production. In that case, the use in food animals. And we've certainly seen the rise of antimicrobial resistance, the rapid rise of antimicrobial resistance and not just antimicrobial resistance, but resistance to multiple drugs in bacteria because of the use in agriculture. And that has led to now global approaches to try to reduce the use of those compounds. And so it's the same with fungi because of the use of fungicides in food production, there is concern that this is driving the emergence and rapid emergence of antifungal resistance. So this slide here just shows the mechanisms by which resistance can develop to asyls in fungi. And so I should also point out that the concern here, again, like the use of antibiotics in bacteria is the fact that once resistance develops to compounds used as fungicides or fungic stats in agriculture, this could also lead to resistance to asyl or fungi. To compounds to antifungals used in human health and even veterinary medicine. So if we look at, if we start here at section A, we see that this is showing the use of fungicides in agriculture. And as I've said, this may lead to development of resistance in fungi, primarily through the development of mutations that allow the fungi to essentially inhibit the compound, the asyl compound. And we'll talk about that in a little bit more. Then if we go down to section B here, we see humans are exposed to the either resistant or sensitive spores of in this case aspergillus fumigatus, which is one of the main species of concern, and they become ill. And then if the patients are then treated in section C with the medical triazole compounds, then this further leads to resistance or selection of resistance strains. Patients can also become infected by inhaling sensitive aspergillus spores. And then if the patients are treated in section C, the aspergillus spores, and then if they were, so in certain people, these spores can cause an opportunistic infection. And so if they receive long-term asyl therapy, then this could drive resistance. And that is section E here. So they've inhaled it, and some of the patients will develop a resistant infection through the long-term exposure of the fungi to the medical triazole compounds. Or in E, the patient could recover. So we're really worried here about section F, and this is the whole basis for this work. So our objective in this project was to determine the prevalence of asyl resistant aspergillus in food crops and their environments, and then also to characterize the resistant islets to identify resistance mechanisms. And really the focus here is on the potential environmental and clinical resistance linkage. So by that, what I mean is it's, you know, the role that agriculture plays in leading to development of fungal resistance to triazole compounds, I would say remains unproven. And by that, what I mean is, while there's been a lot of studies showing a potential linkage, there really hasn't been a definitive concrete proof, at least that the entire scientific community agrees with. And so one way to try to develop that proof is to look for resistance mechanisms that appear to be specific or develop specifically in agricultural islets of aspergillus that are resistant. And then to see if those same resistance mechanisms can then be found in clinical islets of the fungi. And so that was one aspect of our study here. So we looked at several food crops, including tomatoes, wheat and barley, and we sampled them to collect the fungal islets. These foods were produced in North America in various countries in North America. And so we purchased the tomatoes from grocery stores or other retail settings and the barley and wheat came directly from farmers. And we sampled a number of aliquots to isolate fungi. And because there's a diversity of resistance mechanisms and so forth, we collected a large number of islets per plate from per food crop and we did our work that way. So this just shows the methods once we had collected the islets, this just shows two approaches that we used to determine susceptibility to azo compounds. And so we used an azo compound called the phenoconazole which is a broad spectrum azo compound used as a fungicide in agriculture. And so on the panel on the left, we added that to an agar called potato dextrose agar which is typically used to grow fungi. And then we mixed that and put that into a petri plate and once it solidified, then we added, we actually inoculated or we streaked individual fungal islets that we'd isolated from the tomato wheat and barley onto the petri plate. So we used 30 micrograms per mil with the phenoconazole compound on the plate because that's really according to scientific literature what delineates resistance, full resistance from sensitivity. And then in the second approach on the right, we used a micro plate assay. So we took the fungal islets at the top and we inoculated them into broth. And then we had broth with the phenoconazole compound and we added that to the micro tire plate. And then once the fungal islets had independently grown in their individual broth tubes, we then added that to the micro plate as well. Again, the cutoff was 30 micrograms per mil that we use. And so here we incubated the plates for up to 48 hours at 37 degrees Celsius. And we looked for growth, which was determined by optical density. And each micro plate had a control strain or two control strains, one that was sensitive to the compound and one that was resistant. And so resistance was determined by OD as well as comparing to the control. So before we go further, it's in order for you to understand some of the results that I will show today is important to understand the mechanisms of resistance to asal drugs that are currently the current mechanisms that are understood to cause resistance to asal drugs. So there's three major mechanisms in panel A here. And two of them really, I should say, are based around the CYP51A gene. And so in panel A, this is showing the presence of mutations that are responsible for a substitution effect, which is based on causing structural modifications of the CYP51A enzyme. And so what this does, it leads to reduced asal affinity intake. And so there are several amino acid changes that have been reported in the literature. And so these designations here, G54, P216, et cetera, have been identified and associated with that. In panel B, we have overexpression of the CYP51A gene due to various insertions in its promoter region. So here you see the designation TR34, for example, there are other insertions that have been recognized, TR46, TR53, for example. And these insertions in the promoter region are combined with a substitution at codon 98 of the gene, which leads to a substitution of leucine to histidine. So leucine to histidine, hence the designation L98H. So this combination leads to overexpression of the CYP51A gene, which also again leads to reduced or complete elimination of asal uptake. And then finally, panel C, which is a overexpression of efflux transporter. So this is basically a pump that pumps out toxic compounds out of the cell, much like in bacteria, we see efflux transporters playing a major role in antibacterial resistance. And so this basically just pumps out the toxic compound, in this case, the asal compound out of the cell. So A, section A and section B are the main roots of resistance that have received the most attention in the literature. And so we really focused our attention on these two mechanisms of resistance in our work. So this slide shows that those two mechanisms in a bit more detail. So here we see in some more detail, the CYP51A gene and its promoter, showing the locations of the modification. So in panel A, this is a wild type strain, a wild type strain, which is sensitive to asal compounds. And so you can see in the promoter region, we don't have a tandem repeat or inclusion of additional amino acids. And you see the L98 amino acid downstream of the promoter. In panel B, which is a asal resistance strain, we see this TR, the tandem repeat, which could be 34 amino acids long, 46 amino acids long or 53. And we also see this substitution of the leucine to a histidine, and it's both of these compound, both of these changes that lead to the over expression of the CYP51A gene and resistance. So the first thing we did was, what we want to look at these resistance mechanisms and try to understand which, what were the most common ones, so that we could then look for that in our isolates. So we took genome sequences of triase or resistance strains of aspergillus shumigatus from the NCBI database, and we identified the six most common mutations of the CYP51A gene. So we've already talked about the tandem repeats, the L98H mutation, and we looked at two other mutations, M220 mutation and G54. So those latter two would be in panel A of the diagram I showed you, the amino acid substitutions. And then we identified sequences, the sequences as either environmental. So what I mean by that is we identified in the genomes that had these mutations, we identified those genomes that's coming from environmental isolates of aspergillus shumigatus, clinical isolates of aspergillus shumigatus, or paired clinical or environmental isolates. And this identification came from their original publications. So in other words, what were their sources, what were the sources of the isosidium mutations? And again, this was done in an attempt to see whether we could identify any specific mutations that seem to be only associated with environmental isolates and then to check for those in clinical isosidium as a way to try to understand the role that the use of fungicides in agriculture plays in terms of developing resistance in clinical isosidium. So once we had those sequences, then we checked for their presence in our fungal isosidium using polymerase chain reaction. So we designed several primary sets in our lab to amplify a single 744 base pair amplicon that was part of the CYP-51A gene. And the amplicon includes the promoter region and all of the common areas for the mutations and tandem repeats of the six most common mutations that we had studied. And we just use a standard protocol with a 40 cycle amplification of the DNA and then a kneeling temperature of 56 degrees Celsius. So let's get to some results. So we identified 85 isolates or we chose 85 isolates from three different countries in North America from which we obtained the food crops and isolated the fungi. So you can see in this chart that the male is barley and wheat and the number of isolates coming from each country. So of the 85 isolates approximately 31% were observed to be resistant to the 30 micrograms per male of the phenoconazole and we identified resistant isolates regardless of whether we streaked them out on the plate containing the phenoconazole or whether we use the microplate assay. And so in the pictures on the right, you're seeing the growing fungi. So in picture A1 here and D1, but these are two different isolates grown on the pateodectros agar without the phenoconazole and plates A2 and B2 are the same isolates grown on the pateodectros agar with the 30 micrograms of the phenoconazole. So you can see A1 and A2 are the two isolates B1 and B2 are the same isolates. So you can see A1 in particular had no problem growing in the presence of the azole compound whereas B1, there was a few resistant colonies but nothing as prominent as A2. So this shows our bioinformatic analysis which is assessment of the gene mutations in the NCBI database. So you can see in the row labeled mutation or sorry, in the column labeled mutation, you can see the six most common mutations that we focused on the tandem repeats either 34, 46 or 53 amino acids long and then the amino acid substitutions including the L98H which is a very common one. And so in the clinical assets, we saw the majority of, and I should say that the tandem repeats and this L98H mutation are linked if you can remember that diagram that showed a few slides ago. So we can see that the majority of the clinical mutations tend to have this TR34 and L98H. If we look at environmental isolates, we see that there are a number of them that have the TR34 mutation and the L98H mutation but we see for this TR46, this is 46 amino acid substitution, we do see a much higher number in environmental isolates than in clinical isolates. And then this represents papers that looked at both environmental and clinical isolates that were resistant. So it appears that this TR46 could be used as a marker for environmental resistance as compared to clinical resistance. So what this slide shows here is the PCR analysis or PCR analysis of the CYP51A mutations. So again, we amplified the primers that we designed were initially designed to amplify a 744 base pair region which includes the promoter region and all the region of the CYP51A mutation that could contain the mutations and tandem repeats. So the way this works is that, so here if we look on the left side of the screen, this is a, and I should add that we were looking for either the absence of a band or a band that had increased size above the 744 predicted size. So 744 somewhere around here. And what I wanna point out is that, and these are different isolates. The ones in the red box are sensitive isolates that we included. And so if we saw, for example, an increase in the molecular weight above 744 base pairs, that could mean that that was due to the presence of tandem repeats in the promoter region which would lead to an increased molecular weight. If we saw the absence of a band, there are faint bands here, for example, which could mean that the amplicon did not amplify, this might be a nonspecific band because of the presence of mutations. These amino acid substitution or substitutions in the codon leading to differential amino acids, which would cause the PCR reaction to fail because the primers would not be recognized by the DNA strands. So those were the two mechins we were looking for here. On the right side, this just shows why we use capillary electrophysicists. It's a great approach to see small changes in the molecular weight of an amplicon that one could not see with conventional gel electrophysicists, for example. So this is showing isolate A12, M28, so which looks to be, here you see the size of the molecular weight. So that looked to be around 744, but actually when we look at it, it's 787, as you can see by the peak over here. So that would seem to indicate that there's a tandem repeat there. So the original PCR that amplifies the 744 base spray region worked well, but we want to really try to drill down and see if we could really tease out the different resistance mechanisms. So again, remember that the main one seems to be a tandem repeat in the promoter region, which could either be 34, 46, or 53 amino acids in size, coupled with a mutation downstream leading to a transfer of leucine to histidine. And so we designed, well, we obtained primers, which had been previously designed in the literature, in the scientific literature, and we use those to tease out these differences. So this is what you're seeing here. So at the top is the L98H PCR assay, where we're expecting a 143 base per band. So this is what you're seeing here at this size. And if there was a substitution in the given fungal respective isolate, then we would expect to see a failure here. Because the primers wouldn't find. So in lane seven, that's what you're seeing here. We do see increased banding here, increased molecular weight banding. So that was as unclear why we're seeing that. And we'll get to that in more detail later. And then here is a TR that stands for tandem repeat. So in this case, the band is supposed to be 100 base pairs in length or in size, the amplicon. And so we're seeing that here. So if there's a tandem repeat, we'd expect to see an increase in the molecular weight. So we're seeing that in lane five and also in lane seven. So again, these could indicate the presence of tandem repeats in the promoter region. So, we thought that we had evidence of mutations in these isolates. So we wanted to take a closer look at that. So what we did was we sequenced three isolates. We actually sequenced four isolates, but I'm presenting the results of three because one of them was actually, once we analyzed the sequences, we found that it was contaminated. It was a mixture of two fungal species. So we sequenced the genomes of three isolates. So to do that, we isolated DNA using the kaigen DNA power soil pro kit. And we subjected the genomes to long read DNA sequencing using the Oxford nanopore grid ion platform. And you can see the flow cells that we used. And so this generated anywhere from 150 to 300,000 reads, sequencing reads with a median length of 20 kb. So following sequencing, we assembled the sequence reads using a pipeline called fly version 2.9.1. And long read sequencing can be prone to error correction. So we used a program called Medaka to correct for those errors. And then we wanted to then extract two targets we focused on in the genomes, the CYP 51A gene that we've talked about, extensively here, and another gene ERG 11, which I'll talk about later. Excuse me. And then once we'd extracted those, we aligned the different sequences with a program called muscle. So here we see the results of our sequencing. So we picked a nice set from tomato, one from wheat and one from barley. Now the first thing I wanna point out is if we go to the far right column, you'll see that the goal here was to work with aspergillus fumigatus. However, when we sequenced these fungal acids, they did not turn out to be aspergillus, but rather penicillium and to be more precise, penicillium lumens. So penicillium and aspergillus are actually very closely related. And so, and as we'll see, the fact that these acids are penicillium that did not really subtract from our analysis. What we're also seeing is some statistics here that really show the quality or can be used to determine the quality of the sequencing. So the overall size of the genomes range from 28.6 to 28.7 gigabases, which is good because they're all identified using a program called Kraken II as the same, not only the same species, but really the same islet. The number of contigs range from seven for the tomato islet to 10 for barley, which again shows that the sequencing was high quality. And then we see other statistics that can be used like the end count and gaps, which are really indications of missing data and completeness. So there's no missing data here. So again, it really shows that the sequencing was very high quality and the end 50, this column here really denotes the median content length, which is an indicator of the completeness and overall quality of the assembly, which is about 4.5 gigabase for all of them. So again, overall just a very good sequencing approach and good sequencing data, which gives us confidence that when we extracted the various gene homologs, we could be confident in what we're seeing. So I just put this slide up here again to highlight the fact that we had aimed to isolate Aspergill's fumigas, but we got Penicillium Rubens. This is a recent paper from 2021 that came out in which the authors report two new ASL resistance mechanisms in the CYP51A gene in Penicillium and they show that one of the mutations could be transferred to ASL sensitive Aspergill's fumigatus isolates, which subsequently became ASL resistance. So we see that these again, these two species are closely related and resistance mechanisms in the Penicillium can cause resistance in the Aspergillus. So even though we identified Penicillium and not Aspergillus, interrogating their genomes for their resistance mechanisms can certainly be applied to Aspergillus as well. So what this shows here, this is a genomic tree and of Aspergillus as well as 16 other different fungal species. So as we've talked about point mutations in the CYP51A gene, it is really a predominant mechanism of resistance to ASL drugs in Aspergillus. So the CYP51 proteins belong to what is called the CYP super family and are conserved across different fungal genera. So this is what we've done here is assess the diversity of the CYP51A gene by constructing a phylogenetic tree of all protein sequences from fungi and the NCBI database. And what we did was we can destructed the tree from multiple sequence alignments of amino acid sequences of approximately 1,100 gene sequences from the fungi. And so in the tree sequences can originate from the same genus have the same color. And we can see the Aspergillus in the gray here formed most of the sequences in the NCBI database which isn't really surprising because this is the genus that has been the most studied for ASL resistance. Although, and then we see that in different genomes though the CYP51A gene is conserved across different fungal genera. So we see the different ones here, the different colors and you can see we've labeled them here but you can also see what they are here. And even though they come from different genera they cluster together in the tree. And so since this tree was constructed based on point mutations this could suggest a similar mutation or similar mutation patterns in the gene sequences from different fungal genera. So again, in terms of our work with penicillium this shows that our analysis would be is still useful in terms of trying to delineate these mutations that are related to fungal resistance. Larry, I can jump in here if we can try to wrap things up quickly but we're getting a lot of questions and we'd like to make sure we have time for to answer those questions. Yeah, okay, I only have, I think it's two or three more slides. So this is a phylogenetic tree that was constructed from the multiple sequence alignments of amino acid sequences of six CYP51A sequences in the NCBI database. This is aspergillus. What you're seeing in the pink with the star are three islets that we sequenced the penicillium rubens and their CYP51A genes. And we can see that they cluster nicely with the aspergillus although they do form a separate clade within the tree. So what this is showing is the alignment of our three islets with another islet. And this is where we did this alignment to try to identify known CYP51A mutations that have been associated with azoresistence. So what you're seeing are the alignments at the top. This is a sequence from NCBI that came from our resistant islet of aspergillus from McGillus and then here the remaining lines are or in the rows are islets. And so this is a partial sequence of CYP51A genes. So what I want to highlight is the mutations in red. We found a number of mutations in red particularly in this islet, BM32, which comes from Barley. And you can see amino acid substitutions. These have been previously associated or reported, I should say in azoresistence fungal islets. So we are seeing some mutations in these islets, these resistant islets that have been previously reported. So the last thing I want to talk about is a gene called ERG11, which is also mutations of which lead to azoresistence in Candida albicans. So you might remember at the start of the talk, I said that aspergillus and Candida are two main genera of concern with respect to azoresistence and development of human infection. So we want to see, there are homologs of ERG11 in our penicillin mysid. So we want to see if we could identify any resistance mutations in that gene. And so we did the alignments again. Here you see Candida albicans or I mean Candida auris islet from NCBI and our three islets that we aligned and we looked for mutations associated with this gene in the penicillin, but we did not see any there. So to summarize, azoresistence continues to emerge, which we know as a threat to human health. And as I said at the start of the talk, the link between environmental fungicide use and clinical azoresistence while likely remains unproven definitively. We did identify azoresistence and penicillin rubens based on known CYP51 mutations. And in future work, since we have the entire genome sequences will be aimed at identifying novel azoresistence mechanisms in food and environmental fungal islets that have not previously been reported. And this will include sequencing additional islets from our collection. So with that, I'd like to acknowledge the funding for this project from FAO, as well as the two people who did primary, the majority of the work and the primary work on this, Dr. Opiemi Lawal, who's a postdoctoral fellow in my lab, as well as Jesse Huffman, who's a PhD candidate. And with that, I thank you for your time and we'll take some questions. All right, thanks, sir. If you could stop sharing your screen, that'd be helpful. We don't need to look at it, a question mark. And I'll share mine. A number of questions in the chat, and maybe I'll start with this first one, kind of moderators privilege a little bit too. You talked about the CYP51 gene. Do you know what that does in a normal, what is it's role in a normal fungi? And the question is, mutations in that gene, does that add to any fitness cost to the fungi and maybe even specifically from the chat, is there relations between mitochondrial dysfunction? And obviously someone knows a little bit more about this than I do and the mutations in that CYP51. Okay, so CYP51A is a gene coding for cytochrome P450, 14 alpha sterile demethylase. So it's a complex gene. So it's really there to demethylate compounds. And so this is how it breaks down. Well, so it will do that at a low level in azole compounds. However, if you have these mutations and what that leads to is, as I said, overexpression of the gene, which therefore increases its demethylase activity and therefore it leads to breakdown of the azole compound. And then sorry, you asked the second part. Well, is there a fitness cost or an advantage to survival and the environment or potentially survival and clinical hosts? Well, if you remember the slide that I showed of the plate with the isolates where we showed, we streaked them out in the presence and absence of the azole compound and you could see one of them grew just as well. So it depends on the isolate, it depends on the type of mutation like the tandem repeats in the primordial region depending on how overexpressed that gene is. So I would say depending on the isolate, you can see that there could be a fitness cost or there may not be. And given the fact that these are the most common mutations that have been shown and there are a number of clinical isolates that have them, I would say that there really isn't a fitness cost in the most successful isolates. And of course, I think the advantage, the obvious advantage of this is that it can survive in the environment with these compounds. One thing I didn't say about azole compounds is because they've been widely used in agriculture, the compounds are really everywhere in that environment. So not just the soil, but even the air. And so these fungi really have the ability to interact with the compounds and then become resistant. So I think these mutations are a direct result of that and it does improve their ability to survive in that given environment. Great, thank you. Another question is, and unlike bacteria, well, as you said, like bacteria, these mutations that you're talking about are not located on mobile genetic elements. And so the question is, how important are they in terms of, is it total expansion due to selective pressure that's the main issue as compared to transmission? Do you anticipate a continued expansion or if we were to reduce the use of azoles in agriculture, do you think the prevalence of these organisms would decrease in the environment over time? Yeah, so that's a great question. So the first part was, you know, unlike bacteria, these mutations, at least the ones I presented are not on mobile genetic elements, which is true. I would, however, remind the audience that in bacteria, there are mutations, particularly in efflux pumps that are not located on mobile genetic elements. And I did show briefly that that is one mechanism, albeit not the main one that is present also in these fungi. So with respect to what we're seeing here, yeah, I think what we're seeing are clones from resistant islets that developed, and then they've just been disseminated. You know, one thing that would be interesting to see, which we haven't looked at and we may try, it depends on what information we can glean from the scientific literature, but it'd be very interesting to see, and I see there's a question here about information regarding farmers, and I was just about to discuss this. So it'd be very interesting to see if farmers tend to have a higher, if the number of, if the resistant fungal infections tend to be higher in farmers than in the general population, because they're there and potentially breathing in the spores, the fungal spores and so forth, which we've just said, tend to become resistant in that environment because of their interaction with the compounds. But yes, I think what we're seeing are clones of these. So if we remove the azo compounds, I think that we may not, I don't think we'd see necessarily a decrease. Again, it looks like these isos once they become resistant, there's not really a fitness trade-off. So it'd be interesting to see, but I think the goal of removing the compounds is to stop the emergence of new clones and new mechanisms. In the scientific literature, there have been some groups that are now starting to look at the entire genomes like we plan to. And some groups have found, in preliminary evidence, some new mechanisms for resistance. So I think that's what we would try to do. All right, so you have several different things talking about the problem for human health. One is the transmission problem from the potential, from the environment and humans, and what is the attributable fraction that causes the resistant infections. And then the other one, and what is the impact of using azols to the prevalence of these and magnitude in the environment. We'd like to stay on time here. We're one minute over, I need to close up. I think we appreciate your presentation, Larry. And when we attack antimicrobial resistance, we need to go from farm to table. We need to go from the molecular to the global scale. And I think your presentation here really go into some of the molecular sequence-based data and how that can inform the questions for developing more practical on farm or agriculture or in clinic solutions that address this problem. And so I'd like to thank you for your time. I'd like to remind the audience that there will be another webinar related to antimicrobial resistance on the same time. There will be a 12.30 from our headquarters time in Rome on September 8th. And we look forward to having you join us again. The recordings should be available of this presentation future on our YouTube channel. Thank you very much for everyone, for your time and your participation. Have a good day. Goodbye.