 recording stop? Yeah now it's started, now you can go. Laser pointer so you see that there's a slight evolution also of my title, but I'm glad to be here, although I would have preferred to be in Priests. I already mentioned that we are interested in monitoring the transmission of antibiotic resistant bacteria, their resistant genes and plasmids and we are studying actually the route from organic fertilizer to soil to crops to produce which end up in the human gut microbiome. So as organic fertilizer in this talk I will briefly mention some of the data we have from manure from digest states and from biosolids and here's first my tool set which is pretty different from what is used by most of the colleagues talking here because most of you exclusively focus on isolates and we are trying to use cultivation independent methods and cultivation dependent methods because a majority of bacteria in the environment is not accessible to cultivation so or at least we do not know their conditions they need to form colonies on plates. So what we are doing is that we are extracting DNA directly from our samples and then we can analyze either by PCR, Southern blood hybridization by QPCR or high throughput QPCRs or by analyzing metagenose. In parallel we use a method called exogenous capturing of conjugative or mobilizable elements and this leads really to the plasmids we are all interested in and more recently we are starting also to do plating with or without prior enrichment because we are interested in actually the rare microbiome those plasmids that are not very present in very abundant populations. So to give you a first taste of the kind of analysis we do in the first step this is community DNA extracted from manure from farms from different farms in the northwest of Germany and we detect we target some of the Brotos range plasmids so the W that are a particular interest of Fernando the ING P1 I am very much interested in but also the ING Q plasmids and you can see that in all these farms these plasmids are very abundant so this is not quantitative this is semi-quantitative and you can also see that we have the integrases of class 1 and class 2 integrals highly abundant. The important thing we should always consider is what is the selective pressure we have in our samples and here you can see that for instance in all these farm manuals or in most of them antibiotics in particular the tetra cyclins were detected but also kinolone antibiotics or sulfonamide antibiotics of course the same picture we more or less find when we look at targeted resistant genes and be focused on those that are very frequent on those that confer resistance to antibiotics that are frequently used in animal husbandry so sulfonamide resistance and tetracycline resistance but also the quaternary ammonium compounds when we do a manure metagenome so these are now exactly the same manures analyzed by metagenomic sequencing you see the big advantages here that we can get an idea of the particularly high abundant resistant genes and also of the classes so this is of course great because we get much more detailed insight for instance we never include it on B or so this is something we learned here but when we looked at the metagenome for mobile genetic elements we were pretty frustrated because here you can see how few samples were really where we were detecting an ink P1 or an ink Q plasmid some in w plasmids were detected much more abundant that this was also a surprise were the plasmids from grand positives so if you look at the y-axis you can see this that they were about 10 fold higher in abundance so but of course like this we just know that there are resistant certain resistant genes or mobile genetic elements but when we apply this so-called exogenous capturing so here we have for instance digestate bacteria which reflect more or less also the manure bacteria which will mix this gfp tect E. coli and automate on a filter overnight and then we're plating them and selected for the E. coli plus additional resistances such as tetracycline or sulfa diacine resistant and when you screen them so you right away you get an idea of the type of plasmids and their acquired genes because we know exactly how this E. coli is the backbone or what is the background resistance and a number of resistances were captured together and more or less all of them have these class one integrants and the number of plasmids we have sequenced and you can see here that they differ mainly so this is maybe an interesting case also for the folks that love the sequencing so the backbone of in p1 plasmids is strikingly conserved so they are highly similar and you have acquired genes such just in the hot spots of insertion and here you see that it's mainly the class one integrant jumping into one of these hot spots and some integrants were even empty so they did not have acquired gene resistant genes but others did have so some were pretty complex and this in p1 epsilon group this was only discovered at the end of the 2000 I think it was 2007 the first paper and now we see them everywhere and of course there's not only the animals as a source of resistant genes spread into the environment also the human excretions we have nicely in the biosolids and you just finished a project and published it in environmental microbiology where we were looking into 11 different sewage treatment or wastewater treatment plants and you can see here that the size of the wastewater treatment plant was not really important so it was negatively correlated actually to most of the resistant genes or antibiotics detected so where you see red this means positive correlation and this is a nice point I can make here this is correlogram that we have of course in the organic fertilizer we have metal compounds we have antibiotics and we have quarks so this is also what comes out from the hospitals and when you look here we have nice red so correlation of in p1 and for instance the in i1 the in class one integrons and typical components that you can see here and of course you can easily capture from all these biosolids plasmids and the really the waste majority of them belong to the in p1 group and most of them to the one in p1 epsilon group so to sum up this first part of my talk so why are organic fertilizers such as manure digest states of biosolids antibiotics and bacteria carrying plasmid localized multi-drag resistances or introduced into our plant product production systems in the agro ecosystem so another case which I did not show you for which I didn't show your data is irrigation water irrigation water certainly also carries many entero bacteria this conjunctive plasmids and in p1 plasmids in class one integrons they seem to play play a key role in the adaptation of their hosts and this is maybe also important we should always think about the physiology of the host bacteria and when we talk also about how this is affecting transfer frequencies so now I want to move to the plant microbiome and this is where I always argue this could be indeed one of the links that we have between environment and the human gut because we are eating the leaves of lettuce or cilantro for instance without heat processing them so just for those of you who are not so familiar about the plant microbiome it's actually similar to the human microbiome plants have 10 fold more bacterial cells and plant cells and they do play a very important role for the health of the plant so the typical phyla we have there are the proteobacterial firm acute's actinobacterial and actoidatus and the composition depends very much on the plant species the cultivar plant developmental stage and external driver so who is localized or colonizing the leaps they originate from the soil from the seed microbiome but they can also originate from dust and irrigation water now I want to show you a one study which really helps me to make some points about sensitivity of different tools we have so the development of a qpcr system for ink f and ink i1 and ink i2 was actually the basis of this small study because we wanted to apply our systems that were designed in silicone shown with some strains and then we did dna extraction we did exogenous capturing plating a transconjugence or tetracycline resistance and we did plating directly from the leaves from the detached bacteria of the leaves and by enrichment and actually the findings are pretty impressive because we could right away detect our ink f and ink i2 plasmids in isolates this out prior enrichment so these are all coli strains and when we put leaves overnight in a peptone broth as taught them at 37 degree we got more or less like a zoo so it was not a single isolate that came up but when we characterized these e coli's they had very different resistance patterns we detected different genes some of them had for instance a blast CTXM1 and we had loads of plasmids in particular the ink f and plasmids were frequently detected so my colleagues in australia steven georgia which group they sequenced 120 of our e coli's from produce and i just want to show you these class one integral yeah i should say parts of the classical part class one integral so my laser pointer is not really moving as i want so it's a huge diversity and as you can see they are in different sequence types this clearly indicates also horizontal transfer but very much to my disappointment the short read sequences did not allow them to assemble the plasmids that we had detected by pcr but we were successful in capturing plasmids conferring tetracycline resistance so we mixed the bacteria from the leaves together with recipient e coli strains gp tect and then we plated these mixes the matings on to an agar that selected for e coli cd 601 plus tetracycline so and like this we were able to directly capture from the community ink p1 ink f1 plasmids and ink f2 and ink eye plasmids so actually this shows that this is a tool that could be for instance also used in the hospital to analyze plasmids from different yeah micro habitats of human friends so and many of them were conferring multiple resistances now comes my lesson for me very disappointing at the beginning but i realized yes these dna based methods and we talked already about metagenomes they would be not sensitive enough to detect for instance ink f and ink i1 plasmids for instance in salad arugula or cilantro but if we do an enrichment so just without selection putting them into a broth overnight at 37 degree all of a sudden you can detect in all the samples f plasmids you can detect eye plasmids i2 but interestingly the class 1 integrons we could detect this out prior enrichment so they must be abundant much more abundant in populations that occur more frequently and maybe also as we know they occur on different plasmids so the conclusions of my talk and maybe some implications for food safety so wire organic fertilizer and irrigation water bacteria carrying plasmid localized antibiotic resistant genes are introduced and this is the important thing together this pollutants so this antibiotics this metal compounds this disinfectants or pesticides into the agro ecosystem and they are nutrients because this is also a point in the environment we do not have growth like you observe typically in your tubes this lb so they are growing very differently in the environment and still you can detect transfer events in soils where you introduce for instance a labeled ink p1 plasmid so mobile genetic elements such as plasmids do play a very important role for this rapid adaptation of bacterial host to diverse micro pollutants and the rare microbiome of plants that carries transferable transferable resistant genes this might proliferate under selective conditions and transfer resistances required for instance by the human gut microbiota the direct DNA based methods they are often not sensitive enough to detect ARGs and MGEs that occur not in the dominant populations but in the rare microbiome but that's this we really have to keep in mind in addition they are also not suitable to show or to study resistance gene transferability or the genetic context so we need to use a polyphasic approach using the different tool sets and we propose that the transferable resistance of produce might be a major link between the environment and humans and as that I thank you for your attention and the gentleman on the left hand side is Julius Kuhn because this is he gave his name to the institute I'm working in so and here are a few collaborators on the left corner up there you see even top also we have many collaboration over the year or Heike Schmidt from the Netherlands and the rest these are my former and present co-worker thank you very much thank you very much Connie do we have any questions for Connie speak up because I don't see who wants to speak from here just ask your question there's one thing in the chat and I know I'm just trying to read it so thank you for this interest this may be a very specific question but I was struck by your statement that the size of the wastewater treatment plant was negatively correlated with the presence of with the presence of AMR genes do you have an hypothesis for what is causing this is this related to the retention time in the wastewater treatment plant so actually the point is that we had this project financed by the German environmental protection agency and they wanted to find out whether the small treatment plants are less polluted and because there's a new law that from a certain size the biosolids need to be incinerated or and the phosphorus needs to be recovered but not from the smaller ones and actually the answer is pretty clear that the bigger sewage treatment plants they use all anaerobic sludge stabilization techniques while for the small ones there's only aerobic treatments and what we did not envisage when we did our experimental design obviously also whether in the catchment of the sewage treatment plant or hospitals this is influencing very likely the type of pollutancy you have and also the abundance of of so-called risk or high risk bacteria based on 16S we have to be very careful using this term but still I found this very interesting it was from Jana Hussmann the question and there was a question of course it was a request to share the slides I said the the the talk is recorded so yeah okay so we can go ahead thanks now and let me check so is the shiru here apparently not no so we so I'm starting to think that I probably missed an email now I will check okay so so we go to the next speaker who is FIFA me and she will talk about I guess an antibacterial activity of terminalia superva mark extracts against multi-drug resistance extended spectrum vital actamases producing antibacteria say strains you have 15 minutes are you there FIFA me are you there oh my god she was here yesterday yeah yeah I saw I think I saw her name okay next one is yeah let's let's go ahead and then if they come back they should say something you know so the the next one is a bright I get it is bright there to talk about implications of plasmids and exogenous or cell-free DNA distribution on microbial resistance is bright I get it there oh my god Alice what did they do no no I don't know because the the emails were sent for from the secretaries but we've already had contributed talk and they contributed the talk and Rohan meta told me he couldn't and I moved him although he's not in there I need to move him but okay but I don't know what happened really so we had we had three contributed talks and nobody's here so so the only thing we can do now is to allow Connie to speak for an hour more how about that Connie we can have a longer discussion because there are now two additional questions yeah so there was another there was another there are now two additional ones yeah and before I have to write that's a good idea yeah so so which one do you want to ask is there a way to ascribe the antibiotic resistant profiles obtained uh from wastewater treatment plant to the human population alone so I'm actually not sure whether I completely understand whether he talks about antibiotic profiles these are the antibiotics we detected or resistance profiles maybe you can specify this yeah I think she means if you can ascribe these antibiotics to the to be produced by humans yeah resistant profile yeah okay so what is quite interesting is that for some of the plasmids and that's why I thought also the previous talks were very interesting for me you see that you have a very stable backbone depending of course on the plasmid types but in these hot spots for integration of acquired resistances for instance of acquired DNA you see of course integrons having more or less exactly the same resistance genes as for clinical ones but class one integrons are nice case to show that you need sometimes very detailed analysis so my gillings showed that the promoter region of this class one integron from the hospital or 751 is different just in a very few mutations from those from the environment and so there is an evolution under the hospital conditions but very likely the reservoirs are in the environment so then there is a question from Olivia asking what techniques do you use to link plasmids to the host in the microbiome data so and this is indeed a big challenge I can say and of course the best is you do plating but because then you have the isolates and you know the host we do sometimes capture the plasmids but then we use the E. coli carrying the plasmid as donor introduce it into soil or in the rhizosphere and look for potential recipients and then identify them by plating all the other techniques such as those used by Uli Klumper or also by Massa Shintani a lot using flow cytometry and fish they give an idea of the transfer range as already stated yesterday by Fernando the transfer range is one thing the replication range is something else and so like this for instance in P1 plasmids were even detected in grand positive bacteria but we so I'm actually not aware of isolates carrying in P1 plasmids that are grand positive so I wouldn't say that this is too for for because I don't know I once saw a paper from Brazil about an autobacter carrying an in P1 but I was at that time not very convinced okay so it's it is a challenge and this linking methods that are used for instance also an ever top slip I think they are not easy let's say it like this thank you so the top organizer Alice Leda what do you think we should do now final call to the speakers today I think we close this I don't know we can discuss something or we can go have a proper dinner right I get it are you there if I'm a goon you mom are you there I mean the shiru are you there it really feels we are doing one of these you know the the beginning of ghost when they call for yeah yeah yeah yeah yeah spiritism yeah but they vanished in thin air so I think we scared the people with the thing of presenting themselves because yeah yeah yeah they yesterday there were 70 people today that are here here thank you oh well uh here is uh Esperance hello sorry I can do screen sharing I have a problem that I do not control but maybe she can send her talk and then somebody of you can share it yeah she cannot do anything oh she can send the talk to somebody to Alice you can send your talk to Alice I put my email but I don't know if can you speak because if she cannot speak can you speak your does your microphone work we don't hear anything so probably the problem is she has not good contact here right so even if she sends the presentation there's nothing we can do because yeah yeah okay well at least you know we know you're there okay thank you very much for appearing on this call so I can invite the name of the contributed and emails and abstracts that are also the abstracts but I think she was even in the session of when we prepared before the meeting today because I remember that I saw her name and she shared so we were able to share but maybe the do you want to go out and re-log in maybe it works or is it a more serious problem Alice is talking to Esperance yeah I was Esperance yeah I was looking at her name in the yeah yeah yeah yeah I don't know anyway we scared a lot of people well I think yesterday we had peaks of 90 participants 90 connected people which was out of the total is 126 hey it was a lot and today we yeah now it's 48 was about yeah so I think Alice we should finish this yes and start tomorrow fresh I think because so we reconvened tomorrow at 3pm central European time that's 4pm UK time right I don't know you don't know okay I don't live in the UK never right okay 3pm european time it should be earlier in UK earlier 2pm yeah that's why I sent all the emails with Rome time because I didn't want to mess up anyone so it's Rome time and then you change to your time and whatever uh yeah we cannot do anything else okay if you wonder you can move the and show you the sunset you would be seeing now you took a photo during my talk I thought okay so I'm leaving okay bye bye thanks to all the speakers and thanks to all the oh yes very beautiful yeah thanks to all the participants and we reconvene here tomorrow okay bye bye have a nice time sorry so bye