00:00 – Dr. Horsley: One cell here that is almost clean of bacteria. And yet you have the cell directly adjacent to it,
00:06 – which again is covered and encased in a biofilm. Melissa: Hi, I’m Melissa, welcome back to the channel.
00:12 – Today at Live UTI Free we’re talking about bladder biofilms and what they look like under a microscope
00:17 – and with 3D modelling. To share some images and explain what them mean we have another fantastic guest, Dr Harry Horsley,
00:24 – a UK-based researcher who works primarily in the chronic UTI space. If you’re new to our channel, check out the video description
00:30 – as we’ve linked to other resources you might find helpful If you liked this video and want to support us
00:35 – hit subscribe and tick the bell to be notified of future videos. Thanks again for joining us on this journey to bettering women’s health.
00:57 – Melissa: Joining me is Dr Harry Horsley who is a Senior Research Fellow and Principal Investigator for the Bladder Infection and Immunity Group at UCL.
01:04 – Among many other things Dr Horsley founded and runs a state-of-the-art biological imaging suite
01:10 – and that’s why we’re here today. We’re going to cover what biofilms actually look like with real photos and 3D imaging.
01:16 – Thank you for taking the time to share your work with us. Dr Horsley: Thank Melissa, it’s lovely to be here.
01:22 – Melissa: It’s great to have you. Before we get into the imaging side of things, tell us about your background
01:27 – and how you came to be working in this space? Dr Horsley: It’s a bit of a strange… answer to that one.
01:33 – I originally well, I finished my biological degree at King’s College, started as a research associate for a clinical research organisation.
01:43 – which I didn’t enjoy very much at all and wasn’t very good at. and actually was having recurrent UTIs.
01:49 – And this went on for quite a while. I was seeing my GP over and over again.
01:54 – He was saying “oh maybe it’s a psychological issue”, you know. boundless repeats of antibiotic courses
02:00 – that would make it better for a while and got worse. And I ended up being referred to Professor James Malone Lee.
02:08 – I think everyone’s probably already heard of. With that, he wanted me to have a cystoscopy.
02:16 – I managed to get a hold of him on the phone and he said he wanted a biopsy for research.
02:22 – I said I’m happy to give you a biopsy but have you got any jobs? And well…
02:28 – he was a little bit taken a back by this but he said “well, you know, come in”, let’s meet you and…
02:34 – I did. We met, we got on really well and he completely cured my bladder.
02:40 – and then, I started as a research assistant for him then a laboratory manager,
02:46 – then he paid for me to go to the Royal Veterinary College to do my master’s which was with UTIs in cats!
02:53 – Then, I did my PhD at UCL on different delivery methods for drugs in the bladder
02:59 – and pretty much everything chronic UTI and here we are today in my own group. Melissa: We met for the first time at the Urobiome Conference
03:06 – in San Diego recently, which is where I discovered some of your imaging and I thought it might be really interesting to show patients,
03:13 – because we get a lot of questions about what biofilms look like and why a biopsy isn’t enough to find whether there’s a biofilm in the bladder.
03:20 – Your work helps explain those questions and why it’s difficult. It’d be great if you can share some of your images with us
03:29 – and we can talk those through. Dr. Horsley: Yeah, lovely, well I’ll first try to share my screen.
03:35 – So originally, for the first thing to look at is what we see at the clinic.
03:42 – So I’ve been very lucky in my life. well I think so, because,
03:48 – the way we’ve always run things in the way Professor Malone Lee always ran things before he sadly died last year
03:54 – was that scientists… aren’t just in the lab and the clinicians in the clinic.
04:01 – We all mix. So we’re all friends, we all work together, I’m in the clinic every Wednesday, every week,
04:08 – always have been looking at patients’ urine and I find it fascinating.
04:13 – So firstly, on the left here, this is our simple setup, microscope and camera,
04:20 – simple laptop. On the right here is a kind of average picture,
04:25 – or average view of what you would see for a patient’s urine sample, which is this…
04:31 – huge white blood cell population and also these bacterial cells that are shed from the lining of the bladder
04:37 – in response to infection. don’t know if the viewers will be able to see, but there’s also
04:43 – a vast amount of bacteria, filamentous forms and we use…
04:49 – urine microscopy as our kind of point of care test for infection, because as your viewers probably already know,
04:58 – the other tests that are available are simply not up to the job and this way we can track if a patient’s getting better,
05:05 – getting worse, the treatment’s not working and we found it to be accurate up to about 70% of the time
05:13 – which is far better than dipstick. Melissa: Yeah! It definitely is. So this setup on the left, is that what it looks like in the clinic when a patient comes to
05:21 – leave their urine sample? Yeah, it’s very nice room, it’s not just some corner, there’s a lovely room there, a cool room
05:28 – they effectively pass their urine sample and I or another one of the scientists
05:35 – will look at it instantly there’s none of this twenty four forty eight hours wait
05:41 – for it to go to a central laboratory, we look at it there and then within five to ten minutes Melissa: And how do you actually count the cells that you’re seeing?
05:51 – Dr. Horsley: So what we do is we have alittle device, a little slide called a haemocytometer,
05:56 – we add urine to the little slide and then we can enumerate exactly
06:03 – how many cells there are of each cell type per microlitre of urine because there’s a little grid for you to count it.
06:11 – And the database is incredible now, we literally have… millions accounts from every patient we’ve ever seen at every single visit.
06:20 – So the database is a treasure trove for UTI research. Melissa: If you’re just taking a microlitre from a urine sample
06:28 – how do you know that it compares to the rest of the sample? Dr. Horsley: Well we have done work where we’ve looked at a microlitre
06:34 – then looked at another microlitre, and another microlitre, and it’s surprisingly good.
06:41 – Actually. Because I think… if you imagine a urine sample and you’ve got a particulate in it
06:48 – which is the cells floating around, they will naturally, once you’ve voided them
06:53 – because you’re effectively mixing them up into the pot, they naturally find an equilibrium. So, yes, there will be differences.
07:01 – But nothing like what one might imagine. Melissa: It’s good to see the actual setup
07:06 – because we receive a lot of questions about how this fresh microscopy works Alright, shall we move on to the next image?
07:13 – Dr. horsley: If we do those counts of those white blood cells which is known as pyorrhoea,
07:19 – so that they’ve been recruited there because there because, your body, your cells have detected an invader.
07:27 – Effectively. So, they’re saying “well, did you come and eat these bacteria or whatever it might be.”
07:33 – And what we did… was we used a relatively simple
07:38 – mathematical model for the linear regression and said well if you look at all the symptoms of all these patients that we’ve looked at…
07:47 – and you look at their pyorrhoea, so the white blood cells what symptom does pyorrhoea, the white blood cell, predict mathematically?
07:57 – And what we found was it was actually voiding symptoms. and any experience of pain,
08:04 – whereas storage symptoms and stress symptoms, so it’s really is what we’re talking about here
08:11 – are likely to be more physical problems. I don’t mean physical as in…
08:19 – in that way, I mean is some sort of damage due to the thumb of whatnot
08:25 – and therefore as quite an interesting thing. So if a person is complaining of voiding symptoms
08:32 – or any pain symptoms whether it be loin pain, iliac fossa pain, pain running down the legs,
08:38 – it’s still very indicative of a loin retracting infection. Melissa: It also lands weight to the fact that
08:43 – these types of symptoms should be considered to potentially be an infection even if standard urine culture is negative.
08:49 – Dr. Horsley: Exactly and this is what drives me around the bend because people think there’s, you know,
08:55 – have you got urgency dysuria, frequency? Melissa: Yeah. Dr. Horsley: That’s not necessarily true.
09:02 – and it’s not- Maths are great because they can only tell the truth.
09:07 – People like to ignore these things but we can’t get around it. Melissa: Patients are pretty adamant.
09:14 – They usually have a UTI when they experience these symptoms so it’s good to see something to support that. Dr. Horsley: And even worse, then you say “you’ve got interstitial cystitis”,
09:23 – Right, which is the next best thing because the test is negative and it’s just it’s an insane scenario.
09:31 – Melissa: Especially given the symptoms overlap between the two. There is basically no difference between the two things.
09:38 – It’s just you’ve used a test that doesn’t work to prove it’s a different disease that doesn’t exist.
09:44 – Yeah it’s a frustrating situation, definitely. Dr. Horsley: So, we’ve talked a bit about the white blood cells
09:52 – but I started off my career and my work being fascinated by these epithelial cells that are…
10:01 – known in both mouse models and in humans to shed in great numbers
10:06 – in response to bacterial infections. So the idea being that you recruit white blood cells
10:12 – but because the bacteria are bound or within the cells that line the bladder and the urinary tract,
10:20 – it makes good evolutionary sense that your bladder and your urinary tract just gets rid of them,
10:26 – just jettisons that layer of cells into the urine because it’s trying to lower the bacterial load.
10:34 – So, obviously, these are wonderful, for me, a wonderful source of research and insight into the disease.
10:42 – Hum, but also if you look at the graph on the left, it can show that there’s a very linear relationship
10:48 – between the number of white blood cells that you have, which is on this x-axis here.
10:53 – and the number of epithelial cells on the y-axis so as the priori goes up so does the number of epithelial cells.
11:01 – Melissa: What are we looking at in the image? This is from human urine? Dr. Horsley: Yeah. Same sort of setup that you saw before.
11:09 – This is just bacteria and a huge number of all of these are epithelial cells.
11:14 – Melissa: Okay. Dr Horsley: The vast majority of which we have shown will be from the urinary tract
11:20 – Hum, and this… part of the reason I did this is because many hospitals still today
11:26 – if they see epithelial cells down the microscope, they’ll throw the sample away and say it’s contaminated.
11:34 – So it doesn’t- before it’s even got the terrible test, it’s thrown away
11:39 – and we prove that that isn’t the case. So, they may be squamous.
11:45 – which mean by squamous I mean they’re very large and flat,
11:50 – which would be more their characteristics of what you imagine to be skin so vagina, perineum, this sort of thing.
11:59 – But if you do some, oh, I’ll get there in a minute, but if you do some…
12:04 – nice staining… you can prove that they are from the urinary tract.
12:10 – So the red dots here, this is two big cells that are on top of each other
12:18 – the green is DNA, so the large green blobs in the center are the nuclei of the cells
12:25 – so where all of your human DNA is. the tiny dots, which I know, you’re going to get to in a moment
12:32 – are the bacteria, so that’s a biofilm bacteria
12:37 – that’s encased the entire cell. But the red dots are what I’m trying to get out
12:42 – and that’s uroplakin-3. Now uroplakin is a very important protein in the bladder
12:50 – and it’s only expressed in the urinary tract. So if your cell that we look at
12:56 – has uroplakin on it then it’s from the urinary tract it’s not from the perineum skin or the vagina.
13:03 – Melissa: To sum up, we’re looking at two human epithelial cells that are completely covered in bacteria which is a biofilm.
13:12 – Dr.Horsley: Yeah. there’s many definitions for what constitutes a biofilm,
13:17 – But in essence, if there is a sheet of bacteria
13:22 – that you can barely see through because, remember that these bacterial bodies are much larger than what you’re seeing
13:28 – because you’re only seeing the condensed DNA. If you saw this in any other method,
13:34 – like in PNA fish, which I’ll show in a second, you wouldn’t even be able to see the nuclei or anything else
13:40 – because it’s completely encased with bacteria. It’s like a slime that they produce an exopolymeric matrix like a mucus
13:50 – and they all stick together and they protect their bacterial community from antibiotics or any kind of
13:58 – killing from the host immune system. Melissa: It helps to be able to visualize it like this to show the coverage of the biofilm.
14:06 – Dr.Horsley: It’s quite alarming when you first see it and if you know, woo and this picture that I skipped past I just wanted to show was
14:15 – this is actually professor Malone Lee’s urine urine which I’ve still got a picture of which is a control healthy control
14:22 – and as one would expect for a man working in this field
14:27 – you can just see the nuclei of his epithelial cells. Melissa: Right. Dr. Horsley: But nothing else. Clean.
14:33 – But if you looked at a patient sample got the nuclei of their epithelial cells then again you can see these biofilms
14:40 – attached to these cells and sporadic bacteria bound to the other cells as well.
14:46 – And this image again two epithelial cells, actually it could be one because
14:53 – I won’t go into the technicality but it could be an umbrella cell because they have two nuclei, but… let’s not get stuck on semantics.
15:00 – I think this is using a different technique called peptide nucleic acid fluorescent in situ hybridization
15:09 – or PNAFISH. And that’s where we give a designed tiny piece of DNA
15:16 – that’s bound to a little fluorescent probe we then incubate it with the sample
15:22 – and it binds to the bacterial DNA which allows us to give the DNA whatever color we want.
15:29 – So, here these big red rods you can see here are the bacteria that are parasitizing this cell.
15:35 – again the green of the nuclei and the tiny bits of green in the bacteria again, like I said, over here
15:42 – that’s just the nucleolus, so that’s the condensed piece of DNA. whereas when you use PNAFISH
15:48 – you can see the whole bacterial morphology and shape. Melissa: It’s more creepy that way. Dr. Horsley: It is isn’t it and…
15:55 – But this is quite a big thing, really, people have been using PNAFISH for a while
16:00 – but once we start… getting to the nitty-gritty and thinking “well, we think it’s this bacteria
16:08 – along with this other bacteria that are really causing the problem.” Then what we can do is we can design DNA probes
16:14 – to just attach to those, so we can see exactly where they are, and what they’re up to.
16:20 – So I was particularly proud of this slide at Eurobiome which I think Melissa remembers.
16:27 – Melissa: I do! It’s one of my favorites as well. Dr. Horsley: Good, right? And I showed this really because…
16:36 – it was interesting to us as researchers particularly our immunologists that worked with us Dr Catherine Cheng because
16:45 – everyone always thinks “well there’s just one type of epithelial cell or two.” so you’ve got this sort of
16:51 – very spheroid classical eurothelial cell which is what you’d expect to be shed from the bladder
16:57 – and then you’ll see you have this more squamous cell here which again is from the urinary tract
17:03 – but is a completely different shape and size. So I wanted to sort of encompass
17:09 – all of the different types of epithelial cells that we find. And by the way, there’s many more than this.
17:16 – Melissa: Okay. Dr. Horsley: Other interesting point, and I think you alluded to it earlier
17:21 – when we spoke a little about biopsies was that if you look at these two cells here,
17:27 – so there’s two cells that are physiologically attached so that means they’ve been shed from the bladder
17:33 – and they were adjacent to each other and yet you have one cell here
17:39 – that is almost clean of bacteria and yet you have the cell directly adjacent to it
17:44 – which again is covered and encased in a biofilm. And this spinning beautiful thing that I produced in the bottom left
17:54 – which is a 3D reconstruction of one of the cells, again, covered in bacteria.
18:00 – Melissa: So that top left one kind of shows why you can’t just take a biopsy to identify if there’s a biofilm present
18:07 – because it may be present just in a very small area and then the rest might be healthy cells.
18:13 – So you could take a biopsy and miss it completely. Dr. Horsley: You could, and this worries me.
18:19 – It’s not to say that I’m not interested in working with biopsies because we are going to have to do it.
18:27 – And we have approval to do so. But you’re right, I mean…
18:34 – unless we can get- it sounds morbid, but unless we can get a cadaver of someone that had a UTI
18:41 – and we can turn the bladder into a pincushion so effectively take a biopsy every couple of millimeters
18:47 – throughout the internal surface area we’re going to really struggle. So, classicaly people will biopsy the trigone area, the bladder neck
18:57 – because that’s, I guess, logically where things will sediment
19:02 – you’d expect at the bottom of the bladder to be where the issue is. But again, you could be half a millimeter less
19:12 – between two biopsies, and completely miss what could effectively be eliciting an immune response.
19:20 – Melissa: Is there a theory about cystitis cystica and that might be actual biofilm formations?
19:26 – Dr. Horsley: Who knows, could be. I’m always… I’ve always, with all of our work, kept a massively open mind
19:35 – because I think it’s really important. I think it’s… particularly important that we don’t make mistakes of the past
19:41 – which is: let’s make a theory, not necessarily find proof for it, but go with it because it makes sense.
19:49 – Then the problem is is the more publicity some of these ideas get, it becomes truth
19:56 – and then the paradigm then someone designs a test based on that. melissa: Yeah. We have enough of those problems already.
20:02 – Dr. Horsley: We do. So… I have which I think you know about
20:08 – and if people showed me evidence or show does evidence to the contrary
20:14 – I’d put my hands up and say “that’s fabulous”, at least we know we’re moving in the right direction
20:20 – but I don’t tend to make any assumptions about anything unless I’ve seen the proof.
20:26 – Melissa: Yeah. That’s a good approach. Dr. Horsley: Served me well up to now. Hahaha!
20:33 – Okay, well, these images are from a early publication
20:39 – which really stemmed from the idea that everything at the time
20:46 – and a lot of it now in fact, is based on the fact that you’ve got
20:52 – E.coli or uro-pathogenic E.coli. This is the cause of all evil.
21:00 – Melissa: Right, haha! Dr. Horsley: Right. There’s likely to be a proportion of people where that’s true.
21:07 – I’m not pretending otherwise. But the other issue being that all of that
21:14 – you know that that idea is based on animal work as well.
21:19 – So, effectively you get E.coli, you put it into a mouse see what happens and then they’ve uncovered this very elegant
21:27 – beautiful life cycle of that bacteria in the bladder which is incredible.
21:33 – However, I’m not sure we’ve ever been able to recapitulate that in people.
21:39 – Melissa: Right. Dr. Horsley: So, we stemmed from that idea because we weren’t finding much E.coli
21:45 – as what we would have expected to because we all thought well this must be true, right?
21:51 – Melissa: Yeah. Dr. Horsley: So we started looking at other supposed uropathogens that are in the bladder
21:59 – one of which is E.coli which is quite a nasty bacteria really but does live happily in your gut and around your body, but…
22:07 – As of all bacteria in right circumstances and the right niche it could opportunistically cause disease.
22:16 – So the idea was here well, not the idea, but we took again the shed cells from our patient, so the shed epithelial cells
22:26 – and we found these amazing colonies of E.colus.
22:31 – And then we use some very nice imaging techniques so these three-dimensional laser scanning confocal microscopy
22:38 – and this bottom left image here is a cross-section of the cell
22:44 – and you can see you’ve got the membrane at the top got the membrane at the bottom and then all this green in the middle
22:50 – and all that green in the middle is the E.faecalis in the cortex of the cell.
22:55 – Then this graph here, you can just see the green sandwich between the two magenta peaks
23:00 – is just a graphical representation of that bacteria gaining access
23:06 – to the cytoplasm in the cell which is quite worrying, you know, because it’s evolved to be able to get inside the cell.
23:13 – If it’s inside a cell, antibiotics are going to be troublesome and also…
23:20 – Also, your immune system may struggle too. So, this is, again, I also want to say that we do not know
23:29 – whether this intracellularity is also just
23:34 – a natural phenomena that’s taking place in everyone day in day out. Melissa: Right.
23:41 – Dr. Horsley: Or whether it’s purely a pathological process. Hum, we don’t know. Wwe just don’t know.
23:49 – Melissa: This is what you’d refer to as an intracellular bacterial community? Dr. Horsley: That’s what they would say with E.coli, yeah.
23:57 – So that’s that phrase IBC, that term,
24:02 – was coined for when you effectively get a colony of bacteria.
24:09 – I mean, technically, it could also be an intracellular biofilm, But the definition of a biofilm doesn’t cover that yet
24:16 – as I know for now but it is. And then if you have single bacteria in lower layers of your tissue
24:26 – so in the basal intermediate levels when you might have one bacteria, again going back to this biopsy issue
24:34 – that’s what would then be referred to as this quiescent intracellular. So this idea of having a single or two bacteria
24:42 – in the middle of nowhere on its own that can reactivate at a later stage and hence causing another cycle of disease.
24:49 – Melissa: That makes sense. Dr. Horsley: Again, this is from an earlier publication but I’m just using this as a segue, really,
24:58 – into some more interesting things about E. faecalis. So, this is a human bladder nematic
25:04 – or human organoid that I designed during my PhD
25:10 – where we took human bladder stem cells, we treated them a particular way
25:15 – and we were able to produce a human bladder-like tissue. So if you look at the bottom here,
25:22 – you have your various steroid cobblestone-like basal cells, you have your intermediate cells, and you have these very big flat
25:30 – thin umbrella cells in the surface. And after we were happy we used lots of key
25:38 – imaging markers to prove that it was expressing the things that it should. We started experimentally infecting it.
25:47 – So, in this next image, you can see that this organoid has been ravaged
25:53 – ever so slightly and effectively its umbrella cell layer and some of its intermediate cell layer
26:00 – has been shed within two hours so that’s again recapitulating what we find in urine of our patients.
26:07 – And these tiny green dots, this is E. faecalis that we’ve infected with.
26:15 – Melissa: I think that’s interesting for our community because a lot of people who have E. faecalis show up on test
26:20 – if they manage to get a positive test. But maybe we can also talk about what shedding looks like to the patient.
26:26 – Can you actually see any of this in your urine? Dr. Horsley: The cells, so if they were to just look at their urine sample
26:33 – So, if you get a lot of shedding, it’s not that easy without a microscope.
26:40 – So it will look cloudy, and it won’t necessarily be odorous.
26:47 – You know this idea that it’s foul smelling if you have an infection isn’t necessarily true.
26:52 – But it will definitely have a particulate in it. So sometimes we see sheets of these cells
26:57 – and you can physically see that, but just be careful and don’t be too alarmed, because even me,
27:07 – there’s a lot of crystalline formation in urine. Particularly amorphous crystals like calcium oxalate
27:14 – blah blah bla. And again, you can produce a sample that looks,
27:19 – you could look at it and be like: “oh my god this is horrendous.” Things are going to get really bad.
27:26 – When in actual fact you look at it down the microscope and it’s just a vast amount of crystal formation
27:31 – which is completely harmless. Melissa: That’s interesting. What about when someone sees the like wispy tissue papery
27:37 – kind of substance in the urine? Dr. Horsley: Yeah, usually, it can be cells,
27:44 – but more likely to be a mucus when you find that. Which again you’ll find down the microscope, all the time.
27:54 – Now… I have my… feelings about what that might be. We are actually looking into it a little bit at the moment.
28:01 – Melissa: I was going to ask. Dr. Horsley: I don’t really want to go down the whole sort of
28:07 – gag layer thing because I’m not a massive believer,
28:14 – well I’m certainly not a massive believer of gag layer replacement. It makes no physiological sense.
28:20 – But obviously your bladder in your urine tract is a mucosal barrier.
28:26 – So, it is producing mucus constantly to protect itself. And whether some people are
28:32 – making a lot more mucus in response to an infection is yet to be seen.
28:38 – But that would probably be my view. It’s a remnant of an inflammatory response.
28:45 – Melissa: Yeah, because people often say that they have that when they have worse symptoms. So it definitely seems related for most people.
28:52 – Dr. Horsley: It does and we see it a lot. So, with our students they’ll look down the sample
28:59 – and they’ll be like Harry, you know Catherine or to the other postdocs or Raj or whatever, like you’ve got to come see this.
29:05 – never seen it before, what is this? And it’s actually just mucus. So, it looks insane down the microscope
29:12 – But it’s very common and you’re right, I don’t personally believe it’s as common
29:19 – in people that don’t have an infection or any symptoms. But we’ll find out this summer.
29:25 – Melissa: Oh good, we’ll wait to hear about that. Dr. horsley: Hahaha! Again, so these images that are flagged up on the bottom left,
29:35 – again because of the imaging techniques we’re using we’re able to take cross sections through the tissue
29:41 – and you can see where these yellow arrows are that the bacteria’s gained access.
29:47 – inside of the cell. Again we don’t know if this is pathological, but it would certainly not seem like a great thing.
29:55 – Melissa: Haha! It doesn’t seem like it, no. Dr. Horsley: No.
30:02 – And this is again just the same thing, so an organoid that we’ve infected
30:08 – and this is a three-dimensional video of us analysing it so your viewers can get an idea of
30:15 – what biofilms look like and just the process in general.
30:20 – Melissa: Great. Dr Horsley: It’s a bit over the top.
30:30 – So the green here is the bacteria nd the mammalian nuclei. Melissa: Right.
30:35 – Dr. horsley: And the magenta is a filamentous actin, so the skeleton of the cells.
30:44 – Melissa: So there’s quite a bit of bacteria in there, it looks like. Dr. Horsley: A huge amount.
30:50 – We’re all quite shocked to see this. It’s inside the cells, between the cells, on top of the cells.
30:56 – Melissa: Just basically everywhere? Dr. Horsley: Yeah, everywhere that it could even gain any kind of access.
31:05 – Melissa: Kind of gives you an understanding of why it’s so hard to treat chronic UTI if it’s just everywhere like this and it’s so hard for the antibiotics to get to it.
31:13 – Dr. horsley: It does, and I think in essence,
31:18 – the treatment protocol like we use at our clinics, you’re going to find it difficult to penetrate the cells
31:26 – and if the bacteria aren’t actively dividing, they’re just sat there in a biofilm, they’re not dividing,
31:33 – then an antibiotic isn’t going to work great anyway because it requires active division.
31:40 – So in our clinics, the idea being that the tissue is constantly regenerating itself,
31:46 – like your skin does, like everywhere in your body is. And if you have a high enough concentration of antibiotics constantly there
31:54 – even extracellularly, every time the bacteria pop back out again as part of its life cycle,
32:00 – it will have to divide and that’s when you kill it. Those cells will, in the end, will have to have been shed and lost,
32:08 – and then by the end of that, then we should have a healthier, happier person.
32:14 – Melissa: For the people that don’t get better on those longer term protocols, is it because the bacteria are burrowing deeper into the bladder wall
32:22 – or do you think something else is going on? Dr. Horsley: I…
32:27 – again, a bit like what I was saying earlier, we really don’t know. I think it’d be folly to try and hypothesize
32:36 – why that might be. I can say that we’re desperately trying to find out why.
32:41 – I’m putting a huge amount of effort, quite a few of us putting a huge amount of effort into it.
32:49 – So yeah, it could be, I think an important thing I probably should have said a moment ago was,
32:55 – yes, we’ve modelled E. focalis and and yes, people are modelled E. coli and there’s a handful of other opportunistic bacteria
33:02 – that people are looking at. But as Melissa knows, having gone to some of these conferences,
33:11 – it’s been some years now that we… people in this research arena don’t believe it ever to be,
33:19 – it’s not likely to be a single bacteria of people’s entities. It’s likely to be a community of different bacteria
33:25 – and we don’t even know if those bacteria are going to be the same in one person to another.
33:30 – So, if you’re treating everyone in the same way, because it’s the best thing we have
33:36 – and you have one person that’s got a slightly different ecology
33:41 – with a slightly different immune response, then perhaps those two things together make it more difficult to treat.
33:50 – Melissa: And those images you showed earlier with the cells covered in bacteria, were those multiple types of bacteria?
33:57 – Dr. horsley: Yes, that’s the interesting thing as well. Every image that we take, I keep saying we, by the way,
34:04 – but Catherine, our fabulous immunologist, and Peter, our microbiologist, they take these images and
34:14 – we’re finding more and more where we’re looking at three, four, five
34:20 – different species of bacteria all bound to the same cell. So they’re not happily living there in monoculture, it’s a community of bacteria.
34:28 – Melissa: Which also highlights the flaws in standard urine cultures because it rarely comes back with more than one organism
34:34 – and it sounds like it’s just so likely. Dr. Horsley: It’s an insane scenario. I think everyone always goes on
34:42 – gives Cass a hard time, but he didn’t ever design it for this purpose.
34:48 – He designed it for pyelonephritis, which is a kidney infection a very obscure cohort of patients.
34:55 – The problem was, is someone read it and said: “yeah, it looks about right.” And then everyone adopted it.
35:01 – Melissa: Decades later, we’re still stuck with it. dr. horlsey: Yeah. And the problem is, is for people like me,
35:06 – when someone says “what are we going to do about it?”
35:12 – That’s not great, but it’s all right saying it’s not great. So what’s next?
35:18 – So we are trying to look at different methods and different ways of doing this, but until we can get down
35:23 – to the nitty gritty of what might be causing disease, and whether it’s a mixture of bacterial ecology
35:31 – and the host immune system or just bacterial ecology until we know that, then constantly designing new tests
35:39 – to pick up lots of different types of bacteria isn’t going to help anyone. Melissa: Talking about which organisms are found,
35:44 – how do we even know which one is causing your symptoms and which ones might just be part of your normal flora?
35:51 – Dr. horsley: That’s a massive problem at the moment. And a thing of great interest to our group.
35:58 – So this idea of causation, which is, you know, what bacterial, what groups of bacterial potentially causing disease in that particular person.
36:08 – So what we’re trying to wrap up at the moment, there’s a great big study,
36:13 – just so much work you wouldn’t believe, but we’re hoping to publish this in the next few months or so.
36:21 – And we’re just waiting for the sequencing results. The idea being that if white blood cells, which you can see here,
36:27 – each one of these is a white blood cell that we found in the urine of our patients,
36:33 – are being recruited to a site of infection. Then they are attacking the bacteria that’s causing disease.
36:41 – and the inflammation, which is these white cells being recruited there,
36:46 – again, perpetuating the symptoms. So if you look at these white blood cells,
36:53 – so this is like a neutrophil or granular site. Firstly, I want to mention, by the way,
36:58 – classically, everyone’s always thought that you only have an innate immune response that is
37:05 – neutrophils, granular sites, they go to the site of infection, they gobble up the bacteria,
37:11 – you get better. And then there’s no memory of that ever happening again. Unlike the adaptive immune response, the reason you get vaccinated for things.
37:21 – But if we look at our patient samples, we do actually find lymphocytes, so T and B cells, which are part of your adaptive
37:29 – immune system. We also have macrophages, neutrophils, granular sites, which are part of your
37:35 – innate immune system. But if you look at these little green dots,
37:40 – and these rods here, that’s attached to this neutrophil, these are bacteria that these white blood cells are actively trying to kill.
37:50 – Which got us to thinking, well, if we can separate these white blood cells, from the epithelial cells,
37:57 – sorry, this is just a lovely picture of a white blood cell eating some PNA fish probes. Melissa: I can see, yeah.
38:05 – Dr. horsley: but if we take agar plates, so this is very basic, we get our urine, and we split it.
38:12 – So we’ve been using what’s called magnetic cell sorting. So, you use an incredibly powerful magnet with magnetic beads with little antibodies.
38:22 – And it splits your urine into your epithelial cells that I’ve shown you earlier, and your white blood cells, which I’ve shown you earlier as well.
38:31 – And then you stick the epithelial cells on one agar plate, and you stick the white blood cells on another agar plate.
38:37 – And then you see what bacteria grow. And what we found quite clearly is the things that are
38:44 – the bacteria that are associated with your white blood cells, so hence, potentially causing disease,
38:50 – that’s why they’re there. And the things that are found on your epithelial cells might not necessarily be the same.
38:58 – Now, this brings lots of other questions. But the idea being that once we’ve finished this work,
39:05 – we should get an idea of is it the same bacteria causing immune response
39:11 – in all these people with a chronic or recurrent UTI? Or people that don’t have any symptoms,
39:17 – because you’ll still have some white blood cells in your urine? What are they trying to kill?
39:23 – People that have come on, and they’re on antibiotics, are the things that were there when they weren’t on antibiotics no longer there?
39:31 – And are they feeling better? And are the bacteria that are there, are they the same
39:37 – between each patient that has the same symptoms? So this is where we’re hoping, although on the grand scheme
39:43 – of it, it’s a small study, but a huge amount of work that we are going to begin drilling down into this idea of causation.
39:52 – Melissa: Such an important part of the puzzle for patients, especially the ones that are on antibiotics and are not getting better.
39:58 – Dr. Horsley: Yes, yeah. And it’s… in its essence, it’s quite a simple idea.
40:05 – But I think in science, the simple ideas can often yield the most beautiful things.
40:11 – Because if we keep it simple, and we just see if there’s a difference, if there’s a difference, we know that there’s something important happening.
40:18 – And yeah, we desperately hope that we’ll get something out of this that’s going to be very helpful to you.
40:25 – Melissa: That would be amazing. And this work is going to be published this year sometime? Dr. horsley: Yeah, well, everyone keeps promising me that we’re hoping for the end of August.
40:36 – But so effectively, the work is done, everyone’s been recruited, we have all the samples.
40:42 – We have the results from ADAR. But as you know, like with the MSU, on ADAR, you’re not going to grow
40:51 – a large proportion of the bacteria that are actually there. so, Peter, our molecular microbiologist is now
40:58 – in the midst of prepping the samples, and they’ll be off sequencing. And then so once we get the genetic fingerprint of the bacteria that are there,
41:06 – we’re going to have a vast amount of data to start unpicking. Melissa: Yeah, amazing.
41:11 – Dr: And yeah, you’ll be the first to know. Melissa: Yeah, do share it with us. And we can add it to the video. And we can also share it with our community.
41:18 – Dr: Will do. Melissa: So we’re talking about causation and the… like testing just doesn’t work at the moment.
41:24 – what do you think the future of UTI diagnosis might look like given the advances in technology that we have now with like machine learning, even AI,
41:32 – everyone’s into chatGPT? You know, what’s the future of diagnosis? Dr: Well, obviously, you know, this work that we’ve just spoken about,
41:41 – if we find something, hum… This is the wonderful thing about science, right? You have a hypothesis, but…
41:48 – we could all be wrong. So once we get the data for this and say, oh, it is these particular
41:54 – bacteria when they are together in this particular scenario, then great, we can design something to look for them.
42:01 – I think it’s unlikely, but it could happen.
42:06 – But that’s this in itself is very good for research, but as it would stand would not be a practical way to diagnose something.
42:14 – Melissa: Yeah. Dr: I’m glad you mentioned artificial intelligence, Melissa, because this is something that’s very, very dear to my heart.
42:23 – And myself, and this incredible man here, Dr. Arto Dr. Arthur Yakimovich, who’s a machine learning engineer,
42:31 – data scientist, and principal investigator at CASUS, in Germany.
42:36 – We’ve been friends for many years years. And we had this idea, genuinely, I think it was four years ago now, three, four years ago,
42:45 – and to start seeing if we could use image-based deep learning to diagnose urinary tract infection.
42:56 – We knew it was going to be tough. And the first thing that Arto said was that we’re going to need an unbelievable amount of data to even,
43:04 – you know… look at the tip of the iceberg for this.
43:09 – the first thing for us to do, I’d also like to do a little shout out to Dr. Natasha Liu,
43:14 – who’s our PhD student, who’s been primarily working on this, and done some absolutely
43:20 – fabulous work. So I hope when your viewers see all these things, that they don’t think this is me.
43:27 – Melissa: Haha, they know now! Dr: So I think it’s very important that these people get a shout out because they’re doing
43:33 – some incredible things. So if you remember when we were taking pictures of the urine samples
43:41 – from the clinic, we use it for diagnostic purposes by looking at the white blood cells, bacteria, the epithelial cells, and many
43:48 – many other wonderful things that we can find there. What we started doing, or Natasha mainly, was
43:56 – taking many, many images. So we now have a data set of over 40,000 images from going on 4,000 samples,
44:06 – and now I believe just over 2,000 patients. So what we’re trying to do is get a…
44:14 – very, very wide net of information from many, many different people,
44:20 – with many, many different types of symptoms. And the idea being that once this is all built,
44:26 – we can start looking at the symptoms and what we find in the urine, and seeing what kind of
44:32 – connections can be made. But already this is showing huge promise. And again, I know I keep
44:38 – saying August, but this paper is about to be submitted. So yeah, watch this space.
44:49 – Yeah, we’ll keep an eye out for it. We’ll talk about this in more depth another time.
44:55 – Melissa: Yeah, sounds good. Dr: Yeah. But effectively, we’ve got… a computer to be able to recognize all these different cell types,
45:03 – along with bacteria, yeasts, whatever. And it it would be able to do what I would do. First of all,
45:10 – it would do it far, far, far better than I could ever do it. And it would do it in about 0.2 of a second rather than five minutes. And it would be
45:18 – incredibly cheap, which I think is very important. Melissa: Very important. It sounds very promising for patients.
45:25 – Dr.: I hope so. Melissa: That was also fascinating. And I look forward to seeing more of your work, especially if you publish more later this year.
45:31 – And for everyone who’s interested in following along with that, we’ll put your Twitter handle in the video description so they can find you. And we’ll also add the links when those are available.
45:39 – Again, thank you so much for your time. and for sharing your work with our community. Dr.: Thank you so much, Melissa. And thanks ever so much
45:47 – for everyone who listened. So I’ll see you soon. Thanks so much for watching. I hope you found this discussion as interesting as I did,
45:53 – especially where we talked about what biofilms look like within a bladder epithelial cell. If you’d like to learn more about this topic, be
46:00 – sure to check out our other videos or head over to liveutifree.com to have a look at our articles. In particular, we have one on chronic UTI and how
46:07 – infection may become embedded in the bladder wall. We’ll add a link to this in the video description. If you like what we’re doing on
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46:19 – Thanks so much for watching. And until next time, keep asking questions and pushing for better solutions.