 Thank you very much for the, thank you very much for the introduction, I'm just gonna time myself to make sure that I don't run like over time. So, as Ali said, I'm a research fellow working with examine, but DBI, but also at the Sanger Institute. So, let's just get started with this. As many of you know, so the monoceridinosa is one of the escape pathogens, and this is largely due to the very broad arsenal of antibiotic resistant factors encoded in each genome. But unlike many other pathogens, the contributions of plasmids to the so the monoceridinosa system has been historically underestimated to say the least. Plasmids are mostly associated into the monoceridinosa with extended spectrum beta lactamases and mostly associated with a few number of small plasmids. But so the monoceridinosa along with many other pathogens are responsible for the global crisis of antimicrobial resistance, but something that is quite important to remember is that this problem is actually uneven because there are some countries that are more affected than others. And one of these countries is Thailand, where back in 2012, around 32, sorry, 38,000 deaths caused by antimicrobial resistant infections were already recorded per year. And it's in this context where we started our study tracking the origin of resistant infections caused by certain monoceridinosa in a clinical environment. This environment is the rheumatic body hospital part of the middle university located in Bangkok, and it's one of the largest in the region. Well, back in 2013, there were several reports of a variety of so the monoceridinosa resistant infection circulating in the hospital. So we decided to use genomics to investigate, to investigate the drivers of such resistance. So we sampled the hospital, and we gather a set of clinical isolate displaying different levels of antibiotic resistance. We sequence them all using Illumina, but something really different is that those isolates displaying the highest level of resistance to multiple antibiotics we also sequence them using pack value. So in finding long and short read sequencing data, we were able to complete the genome of the multidrug resistant strains. And in doing so, we discovered these very large plasmids, about 400 kb, that we refer to as mega plasmids, that are quite remarkable not only because of their size, but because they carry those sense of antibiotic resistant genes, located in very complex featuring large duplications in rearrangements. In one of the plasmids, we actually found a complete efflux pump. And it's important to mention, couple of places these mega plasmids are within the PTU PSP13. And so because these plasmids and mega plasmids were associated with the most resistant isolates in the hospital, we wondered whether we could find them somewhere else. And the answer was yes, we found other 13 cases in databases, mostly associated with seromonas aeruginosa, but also in three other seromonas species, which are very important to our study. I'm going to mention a bit of this afterwards. Well, when we compare the genomes of these mega plasmids, we define core components on their genomes, in which functions modally related to the biology of the plasmid could be detected, and I mean that was expected. But we also identify a very open pan genome and reach in many different and adaptive traits. Here on the slide on the right side I'm listing some resistant to antibiotics, metals and but also some disinfectant compounds, and not only resistant but also integration transposition and even metabolism or plant derived compounds. They are very diverse, their pan genome is quite open and still with 15 members. But due to the focus of our study we were quite interested on the diversity of antibiotic resistant genes we could find. And when we look in detail into the diversity, we found that only 15 mega plasmids carry 56 AMR genes that are resistant against antibiotics of at least nine different classes. This is quite a scary picture if you're thinking of this mega plasmid circulating in a clinical environment. And when we look at the distribution so the pattern of the distributions of the different genes amongst all the plasmids we found very interesting patterns, for instance, here on the left side, the, the profile puts together all of these mega plasmid that come geographically from the same place, and they were reported by the same group, and as we can see their profile of resistant genes is quite similar. And the same story goes for the two mega plasmid that we identified in the hospital in Thailand. So this implies that the precision of the resistant factors was local. And something equally relevant is that the only two mega plasmids that we got, or we identified in natural environments carry no antibiotic resistant genes. Instead, the regions where the AMR genes are so are usually encoded are replaced for other metabolic pathways that are relevant to that particular niche where these plasmid were isolated from. Previously we use now the complete sequences as a reference, trying to identify more plasmids in Illumina data that we got from the hospital. And because that is totally was successful then we decided to apply it to allow their broader scale, and pretty much go fishing for these mega plasmids in the databases. So back then we use all this of the monas genomes that were available, not only aeruginosa because we have found some mega plasmids in different non aeruginosa species, and that number was around 5000 genomes. And when we analyze them and we use this reference we actually identified other 71 mega plasmid that have been there for decades overlooked. Quite interestingly, some of these were already reported so they were part of publications where the authors mentioned they didn't find a signature of any plasmid content in serdomonas which is widely accepted pretty much is thought that serdomonas aeruginosa really don't like, doesn't like plasmids. But analyzing these new overlooked mega plasmids we identified these very broad distribution not only geographically, but also temporal because one of the oldest isolates are from 1970. And then, obviously we identified the mega plant missing multiple species, and in broader solution sources so we have pretty much the column of clinical environments, any kind of infections, but also natural environments it goes everywhere from sewage soil, and even by your reactor these mega plasmids are really very widely distributed in environment, and they seem to hint a link between an environmental reservoir and how those can invade a clinical environment and start spreading quite successfully. Because these mega plasmids are large one may assume and we would be correct to do so that they represent a board them to their host. And that's why we investigated the fitness cost of carrying one of these big plasmids and how frequently they could transfer into their plasmid so we did this and all credits to James Hall, Jamie did his experiments with us. So, the fitness we measure via competition assays, first we transfer the mega plasmid from one of the environmental isolates, the serdomonas corensis and one of the clinical isolates are the ones aeruginosa so one mega plasmid carry no a margins and the other one does, and we transfer those to serdomonas fluorescence and it wasn't this model that they know very well as you heard yesterday, it wasn't this model when we were with the competition assays. So, pretty much the summary of their results is that we really didn't find a significant difference between the fitness of the control strain with no plasmid and that one carrying the mega plasmid from natural environment, quite interestingly, the one that carries the AMR mega plasmid seems to have a slightly better fitness, and this is compared to something very well known, the other two, a very big plasmid that Mike and Jamie talked about yesterday which we know represent a fitness cost. And then when we look at the frequency of a conjugation between these mega plasmids and their new recipient, the frequency for both mega plasmids is actually quite high. And we take as reference again the other PQR sorry PQBR plasmids that Jamie and Mike have been studying for for many years now. So, summary, these mega plasmids that we call PBT-24-Li are involved in the dissemination of AMR genes against multiple antibiotics in classes in clinical environments. They are flexible and dynamic because the pan genome as I mentioned is open and highly flexible so they feature that position of a complex, sorry, it features a complex gene acquisition patterns and interactions with other mobile elements. It has been neglected for decades, despite having a global distribution and being associated with very diverse isolation sources. But they seem to be very efficient because at least in our experimental conditions, these seems to be stable in non-selective conditions, and they transfer really to other sedomonas. They seem to pose no fitness cost to their hosts. And something that I was really intrigued by back then for two many different reasons is how can we broaden our view of the mega plasmids evolution. And this is in part because I got some emails from other researchers saying, you know, I found a plasmid that is quite large that is in sedomonas. I read your paper but we haven't seen, we haven't found a match with the one that you are reporting. So I knew there were other mega plasmids that were pretty much being discovered and they weren't similar to ours. So I asked myself that the same question, how can we broaden our perspective of the evolution and I believe it's all about surveillance. But I would argue is about plasmid-oriented surveillance rather than only looking for whatever is resistant because resistance is quite dynamic, it can be transitory. Right, so not long ago we published this paper with Jamie again in other quarters on what makes a mega plasmid. And so if you're interested on how we think you can define what a mega plasmid is, we invite you to read this paper. But what I want to point out here is that we present this plot that shows the percentage of publications on plasmids that contain the term mega plasmid. So as you can see, it's becoming popular in the last few years and that usually comes along with more sequences and that is good for us because we can analyze the sequence space in a deeper way. And in Zedemona-Cyruginosa in particular, this plot represents the plasmid distribution that is different depending on the family and taxonomically. And in Zedemona-Cyruginosa, this component that is the large plasmid is starting to increase in the last few years. And so what I did now is kind of update on analysis. And it was quite shocking for me that not even two years after our publication in the last time that we check in our analysis, we found now many more of these mega plasmid that are complete. And something really different this time is that we were more relaxed regarding the threshold that we will use to identify similarity between these plasmids. Now we were looking for anything that was similar to at least 10% coverage, so this is very, very low. What we found now is that the number of complete mega plasmids has more than quadrupled in less than two years. And this network of similarity represents how they are related to each other. So this is still diversity. Here I mark the names of the kind of reference mega plasmid that we've known for some time. So you can see there are different clusters in this similarity network. But something that is quite shocking is these plasmids are colored here by the number of AM margins they contain. And all together, these mega plasmids in the network and got a 571 AM margins and 56 was the number of our previous record. So at least eight of the species are present in this network. So the monas a full band introducing seeing they are newly reported as mega plasmid carries so we didn't see this before. And in terms of where they are coming from blood urine and feces of omega 30 bear also featured among the isolates that we haven't seen before sorry the isolation sources we haven't seen before. The replicant times are very complicated because they feature multiple of them, but at least five different ones. Nearly 70% of these plasmids and I think that's very important are predicted by different programs to be non movable despite we know they can move and they can conjugate because of our experiments. So it's kind of a cautionary tale on how to read the different predictions in the end they are predictions and they remain to be tested. One remaining question here is because we said our threshold of similarity very low, we might just be catching things randomly, and they are actually not related to each other. So we wonder whether the only thing in common in the plasmid that are present in this network is that they are actually big, because don't let this small guy fool you these are the smallest is 168 KB and now the largest is 636 KB. So, we knew from this network that many of these are not going to be similar the nuclear level to other plasmids, because we don't see those connections in the network. So what we did now was going one step beyond and start comparing at the level of protein. So what you can see here is just a comparison of kind of representative of different clusters in the network, and how similar they are to each other. But this is adamino acid level so you can see there are some regions, mostly related with a transference conjugation. And another variety of functions that actually are conserved back to the level of protein, so you can see those similarities to the level of nucleotide but there's something in common beyond being large. So you want to finish this talk by saying, this is the picture when you take only one mega plasmid as reference as you can see here, their sizes are very large, but what if we start looking at the pendulum of a mega plasmid super family. So those plasmid that are very large, that can also be connected by a moloji, but the spider virgins, and then you put them all together and then you make a pendulum of those. Well, the result is they also share very complex patterns of sequence similarity. And now the pendulum of these mega plasmid goes above. So it's quite similar to the size of a small group of a small bacteria chromosome. So with that I just want to finish and thank everyone involved in my current supervisors for letting me personally in this, and also for your time and the organizers for having. Thank you very much. Happy to have any questions. Wow, interesting talk. And to ask is there more to them, are they just classes but you ask yourself the question and you answer so I let's check if somebody has hands on no. No, I think everyone is ready for a break. I think everybody. I just have an announcement on the one met mystery. He was late because he had something at no so you couldn't make it, and he will be speaking at six 30. Rome time after our last speaker. Hopefully, let's take. That's all.