 correct yes it is elita i see apologies i have a bit of a cold so my voice will be a bit cracking but hopefully um being in the uk have you tested sorry being in the uk have you tested yes latter flow negative but um yeah can i just confirm that you can see my slides yes we can brilliant so just before i start i just want to really thank all the um organizers for this workshop as i think it's been brilliant brilliant week and we all have learned lots of lots of things and i'm really really pleased to be able to share today with all of you some really beautiful multidisciplinary work that we have done with collaborators here at imperial college as well as uk health security agency work university and a couple of other places and today my talk will be about looking into genomics of plasmids and how analyzing these plasmids that we actually identified that the outbreak that we were investigating was spread across multiple species and all of them had the same plasmid carrying these imp and mcr9 genes um so i'm elita onikaitem advanced research fellow in bacterial genomics and epidemiology at imperial college as well i am research lead for priority pathogens theme as part of our health protection research unit um in health care associated infections and antimicrobial resistance so just a little bit of a background but i'm gonna keep it short as we already had a beautiful presentation as well from alvaro um so carbapenemies producing into bacterialis are able to break down most common antibiotics used in hospitals to treat patients and these are penicillin cephalosporins as well as carbapenems most common carbapenemies gene families that are included in current diagnostic routine clinical tests to detect kpc and m oxa 48 uh vim and imps and most common species carrying these carbapenemies resistant genes um are ecolycloxial anemone as well as entrobacter cloakiae in recent years as well resistant to colostin which is the last line um therapeutics um choices especially for treating ccp infections is rising resistance to colostin is rising globally and this is partly due to this um fairly new mobilized colostin resistant gene um called mcr and there's already 10 alleles uh numbered 1 to 10 that have been reported for for this gene and here on on the right just to indicate that um cp is really an endemic currently and here from european center for a disease um for disease center report um they have actually looked at european countries and try to highlight using these different colors how how well these cps different species of cps are spread within the countries and you can see like majority of them are actually looking at interregional spreads as well in some places there's already endemic situation and only very few countries that are in green were on just sporadic report sporadic cases are reported in this so what we had in in our hospital um nhs trust which comprises of five hospital sites um between the 2016 and um 2019 um as part of um introduction of enhanced cp screening program which started in 2015 um a cp carrying in gene was first observed in the hospital in june 2016 and that was done from a routine rectal screening sample and from november 2016 increasing numbers of cp isolates carrying in gene were identified across a diverse intravacterial species in at the three of the acute hospital sites that were being tested so here in in this figure it's just to summarize that really the first case that started um the longitudinal investigation of this the this bacterial group um within our hospital and it's just to show that there was like a diversity of species involved as well a different number of um days that actually patients were carrying was they were still testing positive um for cp bacteria and just to indicate how long they actually spent um within the hospital um so we also looked at the data on patients who did test positive for cp to try to identify which of the months of particular year we actually had the majority of the cases and we can see that in january and april 2019 we really can see very clear peaks of cp carrying in gene um and it also shows that actually a continuous burden of patients colonized with such cp isolates um were identified throughout the hospital is kind of like you see like nearly consistent fairly high number um constantly identified and particularly those patients were identified um were colonized with intravacterial species which is indicated with red color and in total um 424 inpatient bed days per month were at the peak of of the of the cases of cp cases that we that we had so just to really like highlight in how epidemiological epidemiological investigation have identified there was a problem with cps and carrying in particularly within our hospital so what we did next with our brilliant phd students doing mathematical modeling is very interested in patient networks we used electronic healthcare records for 116 patients that were colonized with this cp positive isolates and he has drawn pathways for all of these patients across the hospital network and the patient contacts clearly split into 12 separate clusters so each of the clusters indicated with this gray shading apart from one of this the biggest cluster which involved 45 patients and we were able to further partition this cluster into seven sub clusters and and here each of the nodes is colored in specific cpe species identified and any ward contact between the patients just indicated with the um black edge and such analysis of cpe blind positive contacts at regional individual hospital as well as ward levels suggested that different species of our cpe positive in genes were involved in particular transmission events and potentially there could have been multiple transmission events present within our hospital um network so what we did to try to investigate this in more details we collected the all available cp isolates from those patients so we were able to collect and hold genome sequence 85 cp isolates from 82 out of 116 patients and most patients carried only one species but there were four patients who were colonized with two different species and we sequenced both of them hence the number is not exactly the number of the patient and as expected all cpe um blind isolates carried multiple um betalactam resistant genes in addition to antimicrobial resistant genes but here mainly we concentrated to illustrate the imp gene present which is indicated in the middle of the circle here in pink and the lighter pink presents imp allelic the imp one allelic version and the darker pink indicates imp allelic version four the blue outline here across the phylogeny tree indicates presence of mcr9 genes we were surprised to see that majority of our isolates were also positive for mcr9 and that would be a approximately 81 percent so 69 out of 85 isolates also carried mcr9 gene um as i mentioned this is phylogeny tree for all of our cpe isolates so as expected um species specific species were clustering together but of course this is not the uh the tree that we were we're looking into particular individual uh associations between um the individual samples as we really were looking interested to see that those isolates that carried imp as well as mcr9 gene actually carried the same had the same plasmid present which is indicated by this most inner circle in in green and blues and this plasmid um carried by majority of the isolates was the ink hi2 plasmid so what we did further we looked at the phylogenetic analysis of this ink hi2 plasmid we had one of our isolates previously sequenced with long weeds using monions and we were really lucky to have like a full reference sequence of our own um hi2 plasmid as well carrying um um gene and as you can see here oops sorry uh from the phylogenetic analysis we identified the two major sublineages that clearly split the plasmid so one was small as lineage A which further could be subdivided into sublineages 1A and 8.1 and 8.2 well for these we had only three isolates in total and then lineage B which was the major lineage um in our in our study and it could further clearly be subdivided into lineages B.1 and B.2 and each of the lineages as well were associated with particulate microbial genes as well identified in the associated associated species where the plasmid has been found and especially um I we saw the co occurrence with B.1 lineage that um also carried the isolates having plasmid of this lineage also carried resistance genes such as tet D, qnrb2, oxa1 and mph we did also the um backdating analysis to try to date the potential um expansion of this plasmid and we got the results that it was around um 1949 this plasmid could have emerged in total and the most recent common ancestor for the lineages A and B dated back in 1965 and 1967 and this estimate suggests that not only our plasmid engage high two have been circulating potentially been circulating among interbacterialias for many decades um but they also have undergone an extensive evolution in in the antibiotic era um so after we have done this analysis and really break down and identify these particular lineages of of the plasmid we overlaid again that uh the data with a patient um patient network and interactions um map that we had and here in this figure you can see that in this case the contact between the two patients is indicated by the specific color um and we have mainly four of our hospital sites and we have um h our plasmid ink hi two lineages indicated by the coloring in of the um of the edges of the circles and lineage one point A is is red and you can see that we did have only two isolates of this um only two isolates that had carried this plasmid sub lineage and it was confined to cluster five where only two patients were positive for this isolate and then any sub lineage of B point one was in dark blue and B point two which was our largest um number of isolates that belonged to this sub lineage are indicated with a light blue and you can see that again they were quite confined to our biggest cluster one and even the sub clusters within within this patient network cluster were having a lot of interactions because the the more interactions potentially the the patients had the thicker the thicker was the line between them and there was a couple of smaller clusters confined to the clusters carrying the sub lineage B point two isolates with sub lineage B point two um plasmid um and we know that there were two cases within the cluster one point two that were identified as real in real time as related cases and um that also allowed us that these two cases were linked to multiple other cases across across the the other clusters just by by looking how how they were interacting and it potentially could have helped this sub sub lineage B point two to spread and um we know that predominant plasmid plate B or lineage B um was represented by 69 out of 72 of plasmids that we have sequenced engage it to plasmids that we have sequenced and it was identified in approximately 60 percent of all of the cases identified in the study and it was distributed widely across across the hospital net networks even though it was dominating within the cluster one and um I hope this as well shows like we found it extremely interesting that it was also like captures your interest that these integrated genomics spatial temporal analysis actually can show the spread of plasmids containing not only in genes um as we showed here but also they can um potentially show some different interactions some different potential clusters of any other plasmids carrying AMR and to do that we just you know had to follow the patient movements for a specialty care around around the our hospital network and and try to investigate as well the possible even multiple introductions of these species carrying the in K2 plasmid with positive or imp and mcr9 so just to overlook again in slightly different way of the patient network looking at the spatial analysis and the slightly different grouping so you can see here we looked at really like the potential transfers between the hospital based on a particular sub lineage of the plasmids and you can see again that for a hospital hospital five there was only hospital level data available that the four the four only intra-hospital movements are shown and actually very little movement um we were able to show here just due to lack of data but um it does seem that there's some repeated transfers of patients between the wards within each of of the other clusters um are aggregated um and they are indicated in in in this um in this figure with proportional greater edge width as well at any transfers in between um between the hospitals the more patients were transferred between the hospital sites the thicker the line is and for patients with multiple cpi's list transfers that duplicated as well just to match each eyes isolated uniquely in the visualization which kind of like allows you as well to really see there's not only patients within one hospital site that are connected and have interactions but actually that a lot of interactions are happening as well in between hospitals that are looked after by the same um NHS trust and just to summarize um I hope again like as has been discussed before but just strengthens to to see that whole genome sequencing is a really powerful tool to detect not only novel antibiotic resistance mechanisms but as well to to track the potential evolution of this and spread in between different bacterial species and also it it is able to develop novel rapid detection um assays in case a new um antimicrobial resistance mechanism is reported but there's no currently a routine diagnostics test for it and um use the whole genome sequencing to characterize in detail the strains and plasmids as as shown in in the study I have presented today benefits the control and management of the outbreak and in-depth genomic analysis of plasmids carrying um airmar genes that might be not as commonly suspected to be present as commonly um as initially was thought really helps to reveal the wide distribution of the plasmids as well as genes within the species in general and multi-layered method methodology as we have used them in this study incorporating plasmids phylogeny with contact network analysis provides an invaluable tool for outbreak investigations and the mentioned study as well highlights the importance of vigilance for unusual carbapenemies activity and further investigations how um how these things work and as I mentioned there's like a huge huge multidisciplinary team involved in the study and um across a lot of different different sites and we do have our as well paper on medarchi as well as under revision currently with Lancet microbe as well um so hopefully we'll we'll be able to share it soon as as peer review publication as well and thank you very much for for the attention and giving me time to share this lovely story I'm very happy to to take any any questions. Thanks Lita for being on time and for presenting this nice story that I didn't know joking. Yeah. Uh are there meanwhile while you were talking there has been a discussion going on in the chat are there any questions for Lita any hand sorry now I know that I have to check for raised hands so maybe we take the last talk because we are already late we take the last talk because Clement is very far away and this is the middle of the night for him and then we can have a discussion later if there are questions or