68. Nobbling the Nonfermentors: Burkholderia cepacia Complex

Did you think the  evidence base for Stenotrophomonas was bad? Well allow  us to introduce you to Burkholderia cepacia complex!

BCC is pretty niche but a key pathogen in people living with cystic fibrosis. We talk through the (very) limited treatment options and evidence base for this difficult bug.

Prep notes for this episode here: https://idiots.notion.site/68-Burkholderia-cepacia-complex-7bba8a7c74df490cb2e91d087a3cdb2a

Transcript

Jame: 0:00

The Idiot's Podcast would like to apologize to Stenotrophomonas Maltophila for implying that it didn't have a lot of good treatment options. Having reviewed Burkhold Area, we now know just how wrong we were.

Callum: 0:12

It's that bad, is it?

Jame: 0:14

Just play the intro callum, how you doing?

Callum: 0:37

I'm good. I really enjoyed summer. And, up here, nae Dors North, in Scotland. All I can say is, brr, cold ur here, eh? Burkholderia? God, that's the best response I've ever gotten, I'm not even sure it's good.

Jame: 0:58

Callum, I just, sometimes you really get me. I don't even know what to say to that. I just, I didn't know what was coming. But yeah. Well done Callum. Because what are we discussing today?

Callum: 1:13

We're the weather no, Burkholderia.

Jame: 1:16

Any Burkholderia in particular Callum?

Callum: 1:20

Now that you ask, I think we're talking about Burkholderia capacia complex.

Jame: 1:26

Indeed we are. Now Callum, before we go on, can I ask you, what do you know about Burkholderia Capatia Complex?

Callum: 1:35

It's not something that I deal with that often, I, so I would say that I know a bit about it. Snippets of information. I think if I was on duty and that result came through, I would be going back to my notes and textbook just to refresh myself because it's not something that's like day to day for me.

Jame: 1:56

To some of the other non fermenters that we've mentioned, they have a preference for colonizing damaged lung, which means that they're important in a particular patient population, which we'll get onto in a sec. But you're right, this is kind of niche and not something that a microbiologist or an infection specialist would necessarily deal with day in, day out, but I think it's worth knowing a little bit about them. I definitely know more having prepped for this episode now than I did before and, particularly about the treatment options that are available for us. So let's dive in, let's do a little quick review of the of the non fermenters. With reference to our oxidase positivity and negativity concerning the Burkholderiae. We've got oxidase positive would be Burkholderia pseudomallei, that's the cause of agent ameliodosis, and then Burkholderia are oxidase negative. And then we've got Burkholderia capacia complex, which isn't one organism, it's 21, 22 and counting separate species. And they are oxidase variable, but actually there are some that are oxidase variable and some that are oxidase negative. And we'll talk about that in a sec. Callum, why don't you take us through the history of the Burkholderia genus?

Callum: 3:23

Burkholderia capatia was discovered by Walter Burkholder as the cause of onion skin rot. Which I think I knew before, because I remember reading that on the

Jame: 3:35

Onion favourite.

Callum: 3:40

Damn, that onion skin rot. I can't remember why that came up. I think it was maybe in a teaching session one time.

Jame: 3:46

Oh yeah,

Callum: 3:47

In 1950, so it was discovered. In this sort of plants area. And 1950s, it was discovered as a human pathogen. And it wasn't until 1977 that it was first isolated in patients with cystic fibrosis. And at this point it was called Pseudomonas cepacia.

Jame: 4:06

In common with almost all the other non fermenters which were turned pseudomonas at one point or another in their lives. Yeah.

Callum: 4:13

And in the 1980s there were reported to be outbreaks of Burkholderia cepacia in cystic fibrosis patients that had around 35% mortality. So really. Significant and there was a specific saying of the complex called Burkholderia Sinocarpatia, and that was associated with outbreaks of Capacia syndrome, as it was termed. So this is something that, was recognized quite early on and has been around and it's always been an issue.

Jame: 4:44

yeah, I think it was in the 80s that when the outbreak started happening that they realized that this was something that they had to deal with and isolate patients from each other to prevent the spread of and all that kind of, all that kind of stuff.

Callum: 4:59

James would you do the taxonomy?

Jame: 5:01

so In the Burkholderia genus, there's about 79 species. They, most of them are plant pathogens, actually, and they're environmental gram negatives. That's their sort of stock and trade. They don't like being in humans. They'll only go into humans when the... The environment has been compromised significantly, but there's this complex of very closely related species of Burkholderia, and these are termed Burkholderia capacia complex or sapacia complex or BCC, and there's, depending on who you ask, there's 21 or 22 species, and they have very Tight homology. So there are 16 SRNA homology is close to 100 percent and RECA. I don't know what that gene is, but that's 94 to 95 percent homologous within the complex. So they used to be identified by they used to get a number that number was their genome of R. And Burkholderia copatia was genome of R1 and then multivorans was genome of R2, senocopatia was genome of R3 and so on. But actually they've now been replaced by actually getting a species name. So we've got them in the prep notes, we've got them in a. A sort of big table. And of course it wouldn't be a nobbling, the non fermentors episode, Callum, without you and me doing an alternate reading of all the species members to send the loyal listeners to sleep. So I'm afraid. Without any further

Callum: 6:36

No,

Jame: 6:37

the first one and then you will take the alternate ones. Cacia,

Callum: 6:41

Multivorans.

Jame: 6:43

eno, Cacia.

Callum: 6:44

Stabilis. Delosa.

Jame: 6:49

Amaria

Callum: 6:50

Anthinia.

Jame: 6:52

racia. Arborus, Diffusa, Latens,

Callum: 6:58

Latte. Metallica.

Jame: 7:03

Paludis,

Callum: 7:05

Cytomultivorans.

Jame: 7:07

Seminalis,

Callum: 7:08

Stagnalis.

Jame: 7:09

Territoriae.

Callum: 7:11

Done. Okay, glad

Jame: 7:12

that's all the members of the Capatia complex. And so the loyal listener knows I'm not sure this is worth writing down, but the oxidase variable members of that group are Pyrocinia, Contaminans, and Lata. So yeah. So of all of those, the ones that are most commonly found in CF lung are the ones that are at the top of the genome of our tables. That's capacia, multivulrad, senacapacia, vientnamiensis, and dolosa.

Callum: 7:38

What's the, why, you said the genomic numbers, right? What determines the order? Is it just the order in which they were discovered?

Jame: 7:46

I think it's order of discovery and after they get to 10, they start giving them different genome of our names. Arboris is, it's in brackets after it says BCC3. Now, I'm not really sure about the nomenclature there, but I've left it in because it was in one of the review articles that I,

Callum: 8:04

I don't know. I don't care. It's

Jame: 8:07

That was just what I was about to say. I don't think the loyal listener should care. I think they should just think Burkholder Capatia and Senecapatia and Multivorans and 18 others are in the complex, but those top three are definitely the ones that are causing the most trouble. And really you only need to know about Senecapatia and Multivorans because they are associated with Capatia Syndrome, uh, there. Callum, where are they found? We've hinted this before.

Callum: 8:38

Yeah, so normally they're environmental. So soil, the plant rhizosphere, so that's the bit of the soil near plant roots. Freshwater environments, so river sediment. And marine environments, so marine sponges. So some strains of Burkle Deirugipatia complex can tolerate a high level of salinity.

Jame: 8:59

Yeah. So they can be parasites of marine sponges as well as plants, but that's where they would rather be. Yeah.

Callum: 9:09

But what means that they go from there to human?

Jame: 9:14

The risk factors for human colonization, the number one is cystic fibrosis. So the CF lung is significantly different to, even other bronchiectatic conditions. And, that can get so bad that they can get colonized to the point where person to person spread. Is documented, and that's led to various infection control practices being instituted in CF units to prevent a cross contamination of patients. Prior colonization with Pseudomonas is a particular risk factor. In fact, within the cystic fibrosis population, almost everybody who is infected with BCC has also been colonized with Pseudomonas beforehand. So that may be. An interaction between those two species, or it may just be a confounded by the lung being bad enough that Pseudomonas colonization has occurred beforehand because Pseudomonas is more ubiquitous and easier to acquire than Burkholderia. And then the other interesting risk factors for BCC infection are chronic granulomatous disease. And that's because of a particular pathogenic mechanism that Burkholderia has there, resistant neutrophil killing, in particular, neutrophil mediated non oxidative killing. And they're actually pretty good, they're intracellular and they're good at inducing neutrophil necrosis. This is particularly important with chronic granulomatous disease patients because they're... Whole things that they can't institute the neutrophil oxidative burst, which is the primary mechanism for killing cells that have been fake cytosed in neutrophils. And so they're at risk of this particular pathogen should they get colonized with it. And then the last risk factor is sickle cell disease for the sort of immunological compromise that comes alongside that condition.

Callum: 11:13

So I guess overall, as we're describing there, you need quite a lot of, damage to that sort of mucosal immunity, that innate immunity in your lungs before this becomes a problem. So this is a essentially a low virulence organism. It doesn't have the mechanisms that other sort of more usual human pathogens have to enable them to survive in the human host.

Jame: 11:34

No, it doesn't, but there are some pathogen mechanisms that it does have. So what are they Callum?

Callum: 11:39

So yeah, it produces a couple of chemical enzymes called elastase and gelatinase, and as James just mentioned, it's relatively resistant to neutrophils, so that sort of innate immune system again, and they... Invade and survive inside airway epithelial cells and macrophages. And then I guess alongside that they are, they're good at hearing to plastics like essentially all the non fermenters are and they have a long survival, particularly in a moist environment. On skin, for example, it is reported to survive less than 30 minutes on sputum contaminated surfaces for weeks and still distilled water for many years. It can really stick around in that wet environment.

Jame: 12:27

Yeah. And in addition to that, there, there are some sort of transmissibility markers, which are explain why some species members are better than others. For example, there's a two things which are found in in particular in Burkholderia senocarpatia, particularly strain ET12, which contains both something called B C E S M and C B L A. So C B L A is a gene encoding a major structural subunit of a cable like and mucin binding pili, so that would help with adherence to to mucosal surfaces. And B C E S M stands for Brocadaria Capacia Epidemic Strain Marker, and it's a transcription regulator that Senecabesha has, as well as CBLA, and that sort of explains why it is associated with these kind of high mortality outbreaks. Possession of one of those will make you more transmissible than others. People have done a lot of. Deep work into this because they're trying to minimize transmission between, CF patients and things like that. LEt's Talk about the lung infections that we get with the Burford Ayer Capacia complex. There's really just there are case reports of it doing other stuff, which I've not included here, like I came across of, a few case reports of it occasionally causing bacteremia and occasionally causing spinal infection, but just one or two case reports and not much else. The main thing that it does is it infects damaged lung.

Callum: 14:03

Cystic fibrosis is a very specific part of medicine, microbiology, and the microbiological sampling is quite different in some ways for their things is a bit of a mismatch between what you grow in culture, what's actually happening in the patient and the resistance and sort of Testing is quite complicated. There's a lot of caveats to this, but essentially, when you go looking for it, about 3 percent of people living with cystic fibrosis will, you'll find it in nirsputum. And 90 percent of the things that are isolated are going to be the burcadouille, sena, capacia are multivorans. And there's one of the main things that we worry about in cystic fibrosis is that when you are colonized of a new organism or, exposed to it is, does it lead to a decline in lung function? So that's a really important kind of outcome marker, which I guess isn't really as relevant in people that aren't living with that disease because. or Maybe it is and we just don't think about it, but in cystic fibrosis, you have, you already got damaged lungs, so any decline in lung function can be really significant and about a third of people that end up with this have a slight decline in lung function. So basically not everybody gets lung function decline, so it's not always pathogenic. And this isn't something that's going to make people septic and sick and bacteremic, but it is going to have an effect on their life. And the ones that are reported to be more harmful are thought to be Burkholderia, seen as sepacia, and dulosa.

Jame: 15:45

So the hominist isolated species are Senecubitia multivorans. But the most harmful ones are Sena Capatia and Dolosa. And in particular the Capatia syndrome is a sort of more aggressive, more fulminant version of this and it's got a few sort of hallmarks, one of which is high fever, high white count, sepsis and severe progressive respiratory failure. And that's mostly associated with Sena ANDS. And doesn't happen the second that you're colonized. The average time in one study from colonization to onset of capacious syndrome was five years. So it's not something that happened right away. And that sort of varies with, what the bug is doing within the, within the lung itself and what stranger you're colonized with because. Just because you were colonized five years ago doesn't mean that you're, that's the only time that you've been colonized with BCC.

Callum: 16:49

cOuld be colonized of one part of the complex and then another

Jame: 16:53

have other members come in? Yeah, that's right. Calum, what effect on lung transplant outcomes does BCC have? Because that's an important thing because, up until recently, our only option for curing CF was lung transplantation.

Callum: 17:07

yeah. So it does have an effect. So if you are in preoperatively you're colonized with BCC, then you're. You've got a higher mortality. The excess mortality be 33% in that group versus the UN colonized would be 12%. And one year post-transplant survival would be 67% with B C, and infected, or 92% of the un unaffected group. So a really pretty massive difference.

Jame: 17:38

Yeah, I agree. When it comes to, declaring somebody an infection free, it is possible to be transiently infected with these. And in fact, a lot of, cases of isolating BCC and sputum, it will be. If the patient doesn't have any symptoms that will be considered colonization until proven otherwise. So if there's no decline in the lung function and no symptoms, then they will, watch it. They may decide to treat it. It depends on which CF center you're in. And of course you're listening to two. Known CF experts here but transient infection is less common with senocarpatia. But it's important to know if somebody is colonized or infected or not, because it determines if you can be cohorted with BCC negative patients on CF wards and CF clinics as well. So the definition is three negative sputum cultures over the course of a one year period. That's the definition that's used in the UK, at least. So if you've not got that, hit that criteria, you have to be treated as if you are BCC positive. What, speaking of transmission risk between CF patients, Callum, what would you consider low risk and what would you consider high risk? This will come as no surprise to anybody who has recently, say, practiced medicine through a pandemic.

Callum: 19:06

Low risk is, briefly encountering someone indoors or outdoors. And it's a sort of transient exposure. The high risk, as James was alluding to we know areas where there's closer contact. So social contacts are like at a pub or a restaurant, somewhere where you're in that same space for a while. Handshaking, interestingly. Contacts involving siblings of cystic fibrosis, which is obviously impossible to avoid. Sharing bedrooms, social kissing. Travelling together in closed conditions like a car or lift, somewhere that's not ventilated. Sports, exercise classes, so you're breathing more heavily. Sharing eating or drinking utensils or intimate contact. And I guess the other thing to think about here is the infection control, One of the risk factors here is going to be contact in a non social situation, like in a hospital. The CF Trust recommends segregating BCC positive patients from... Anybody else, basically, even people with other, with BCC as well, because I guess they might have a different organism they recommend regular microbiological surveillance and centers that are looking after these people and see them in separate days and clinic, see high risk strain patients separately, and avoid. So I guess, historically, there was like, as you would expect, people with CF would We'd congregate and spend time together, support, but we now know that's going to be bad news if you're

Jame: 20:34

Yeah, no longer, although that may change if everybody goes on the novel therapies that we've got.

Callum: 20:40

And I think there's some stuff around side rooms and these people will be isolated where they're in hospital. And one thing to think about is like the ventilation in your room. We often think about, oh, side room equals safe, but actually what ventilation is going into that room, how many air changes are happening per hour. Because if there's not, if the air isn't changing and someone is in that room and in discharge and someone else goes into that room, there could still be Burkholderia in the air,

Jame: 21:06

So survival in air. So after a BCC positive patient leaves a room, it can survive In air for about 45 minutes. For infection control practices between patients, you need to make sure that there's at least 45 minutes between patients as well. So that the the live bug can die in peace.

Callum: 21:32

Hospitals are very risky places.

Jame: 21:35

So they are micromode engaged

Callum: 21:41

So this is,

Jame: 21:42

out a song for that.

Callum: 21:44

we need to do better. We could do better. Email idiotspodcasting at gmail. com The organisms, the bacteria, it's a gram negative bacilli, which is the whole wheelhouse of where we are in the moment. We've been working our way through all of them. It's Catalase positive. Oxidase, so essentially variable, the SMI says that the oxidase strength varies. So most of them are going to be oxidase positive, the strength of that is variable. They'll be urease positive, but nitrate test may be variable. And how do you grow them? So there's two main agars that are used. One is BCSA, Kel Capacious, selective Agar, and essentially that contains crystal violets. So the same stuff we use in gram stains and bile salts. And the, those inhibit gram-positive co cocke essentially. And then it also contains some antibiotics, ticker soin and poly mix, and B, so they inhibit our gram negatives other than.

Jame: 22:48

yeah,

Callum: 22:48

And the plate contains phenol red pH indicators, so it should turn to pink when Burkholderia produce alkaline by products, so that's, you should get the organism grows on the plate and then you get these sort of pink change. Other things that could grow on that plate would be things like Candida Stenotrophomonas, Ralstonia pachytei, and Pseudomonas aeruginosa. And then there's other, some other Pseudomonas species as well, or other colistin resistant Gram negative bacilli. So that's one way of growing it.

Jame: 23:24

and the alternative agar is O-F-P-B-L, which is oxidation fermentation, poly mixing amtrac and lactose agar. This is less used than BCSA these days. There was a comparison of what grows at best and a subset of BCSA seem to do the best job but the poly mixing is there to kill gram-negative bacilli. The bass trace and kills gram-positive Bai and Sidia, and. A yellow colony is what you'd be looking for there, because that would be a non lactose fermenting organism, and that would be presumed to be Burkholderia, and you would go on to identify it subsequently. And then, if you got another colony that was lactose fermenting, and forgive me, Calum, I don't know what colour that would be, they could be Candida, Stem shofomonas. Or Pseudomonas Florescens, that's the kind of things that can grow on OFPB Alaeba. Once you have plated it out

Callum: 24:20

although one thing to say the growth, so the culture, like when you're doing a plate so most of the time this would be set up at 35 to 37 degrees for 48 hours, which is pretty standard for most plates, to be honest but it's worth noting that some strains will only appear if the colonies are incubated at 30 degrees for up to 5 days. So some of them are lower temperature organisms.

Jame: 24:41

okay.

Callum: 24:42

So there's that like slight note as well. They do prefer 25 to 35 degrees. But they can grow up to 41. And they shouldn't grow at 42 degrees or down to 4 degrees. They're pretty thermal they're pretty good at a range of temperatures.

Jame: 24:57

They're pretty thermotolerant, for something that lives in the ground. Yeah.

Callum: 25:01

So it grows. When it does grow, you'll see it on the plate. 1 2 circular. Medium turns pink, as we mentioned. And we've talked about the tests. The other tests that are used are nitrates. So it should be nitrate negative and ONPG positive. What that stands for I've looked up many times and I always forget. And most of the ID is commercial ID systems. So it's going to be MALDI-TOF. First time is isolated in someone, then it gets sent off to one of the reference labs in the UK, and then they do some further testing that is specific phenotypic tests, and they'll do the wreck a R. E. C. A. Jean mentioned earlier on, and they'll do a P. C. R. And that and essentially what you want to do is look at that from an epidemiology transmission perspective, and they'll also look for So the markers of virulence that were mentioned before, so that was B, C, E, S, M, and C, B, L, A. And basically by doing something called genomic fingerprinting with various methods, they can investigate, those isolates to see, has there been transmission events, essentially.

Jame: 26:11

Yeah, they do. They don't do all of this, but they can do it if they need to. Most of the time they'll just do phenotypic tests and maybe the RE APCR and leave it at that. But yeah. Yeah, the Maldi is actually pretty good at identifying the different members of Alder Cacia complex nowadays, so usually that's all that you need at your end too. To meet the to meet the diagnosis let's say.

Callum: 26:37

yeah, I guess before you had MALDI, I presume you'd be doing this on like a API strip, automated biochemical tests.

Jame: 26:44

I think so. I don't know how good the the Vitek 2 was at identifying Burkholderia or if it was any good at all. So don't quote me on that.

Callum: 26:55

So how do you kill them?

Jame: 26:57

How do you treat Burkholderia?

Callum: 26:58

How do you treat this? You alluded to this at the very beginning of the episode where you

Jame: 27:02

Callum we've known about Burkholderia since the 1980s. It's been an important pathogen of a significant patient group. I think you would agree with me that we should probably have some fairly well defined treatments for Burkholderia patient complex by now. Only a a a bunch of silly toll rags would not have. a bunch of well defined treatment options for Burkhardt Area Occupation Complex by now. Isn't that right, Callum? After all, it's 2023.

Callum: 27:32

Yeah, we've, I think we've probably discovered most of science by now, haven't we? There's not

Jame: 27:36

I that's certainly my opinion of it. Callum, would you like to know what you cast have to say on treatment options for for Burkhardt Area

Callum: 27:44

don't want to know. I don't want to know. I've looked at it, so I do know, but

Jame: 27:50

would you like to know if you click on, and I love this document the UCAST clinical breakpoints document, which has the breakpoints for all known bacterial pathogens.

Callum: 28:02

it is a great document. I know where James is going with this, but it is really useful and UCAS do great work. We're not anti UCAS. It's

Jame: 28:09

Oh, I think I'm a little anti

Callum: 28:11

It's

Jame: 28:12

know what it says? And I quote Callum, it says, go read this document on Burkholderia, and so when you do there's a UCAST BCC document from 2013. And it says, and I quote, A recent Cochrane systematic review concluded that there is a lack of trial evidence to guide decision making and no conclusions can be drawn from this review about the optimal antibiotic regimens for CF patients with chronic BCC infections. Clinicians must continue to assess each patient individually, taking into account in vitro antibiotic susceptibility data, previous clinical responses, and their own experience. Unfortunately, evidence to describe a relationship between the in vitro susceptibility to any specific antimicrobial agent and clinical outcome is lacking. This is due to a potential mismatch between in vivo and in vitro expression of resistance, as BCC are known to exist in biofilms in vivo and may also invade and survive inside airway epithelial cells and macrophages. In other words, Callum, you're on your own.

Callum: 29:22

it's not very helpful.

Jame: 29:24

No! It's not very helpful, and it's ten years old, and it's not been updated. But don't worry Callum, Cochrane to the rescue. Okay, so I've got the four most recent Cochrane reviews here, in the chamber ready to pull the trigger, and they're going to have a look at the most recent evidence for Burkholderic Occupation Complex, and they're going to let us know exactly what to do. Maybe I'll start. With the 2019 Eradication Therapy for Burkhardt Area Capacia Complex in People with Cystic Fibrosis review. Would you like to know the results of this

Callum: 30:07

I don't think I'm gonna like it.

Jame: 30:08

No studies were eligible for inclusion in this review. But don't worry Callum, because we've now got the 2020 review, Antibiotic Treatment for Brocaderic Apatia Complex in People with Cystic Fibrosis Experiencing a Pulmonary Exacerbation. So exactly the kind of people that we want to know about. Wouldn't you agree Callum?

Callum: 30:32

Yes.

Jame: 30:34

No relevant trials were identified.

Callum: 30:35

Oh no.

Jame: 30:37

But don't worry Callum, because we have a 2021 review. Antibiotic Therapy for Chronic Infection with Burkholderia Capacia Complex in People with Cystic Fibrosis review. Would you like to know what this... Cochrane Review found.

Callum: 30:52

Go on.

Jame: 30:52

Of 100 participants, lasting 52 weeks, comparing continuous inhaled Aztreonam and placebo in a double blind RCT for 24 weeks, followed by a 24 week open label extension and 4 week follow up period, and that was associated with no improvement. And that, Callum, is the evidence base. dO you see, Callum, why I felt the need to apologise to poor Stenotrophomonas multifilia? Sitting in the

Callum: 31:29

had one antibiotic from UCAS

Jame: 31:31

If you count Kephidergal using the PKPD breakpoint, we had two, and if you count

Callum: 31:35

I was spoiled,

Jame: 31:36

CLSI, we were positively spoiled for choice. But with Burkhardt Incubation Complex, absolutely nothing. It's as if UCAST or any of the breakpoint setting organizations didn't exist. They're basically saying, do in vitro testing, and then guess based on your own experience.

Callum: 31:56

At UCAS you have something called PKPD breakpoints. Which are meant to be like, if it's below this then you'll probably get enough drug to that point of action to, to work. But,

Jame: 32:07

but I mean that also, that sort of depends, the issue here with BCC is that it is almost always going to be in a biofilm in the lung. And so that means that it's going to be really difficult for any drug to get there. So if it's coming from the blood, if it's an IV drug or a highly bioavailable oral drug, for example, which is not relevant here, then you must be thinking it's got to get, into the bloodstream. It's going to get to the lung, it's going to get through the basement membrane, it's going to get into the alveolar space, into the alveolar fluid, and then try and penetrate that biofilm. And then you might think maybe I'll inhale it. Maybe I'll get it in the nebulizer, or there's a particular inhaler device for, I think, tobramycin. tHen, it's going to get from the airway spaces. Through the biofilm to where the bugs are actually trying to grow in order to kill them. And that's, if anything, more difficult. So the, the issue with the PKPD breakpoints, and I suppose the reason that in vivo and in vitro are matching up, is that there's so many holes that you can poke. In the PKPD breakpoints. I don't think that you can guarantee that you're going to be getting, CDMI is the PKPD breakpoint two or something like that. I don't think you can guarantee that you can get that with a bunch of these drugs,

Callum: 33:36

Yeah,

Jame: 33:37

but anyway, look,

Callum: 33:37

on each drug by each drug is good, but usually we talk about drug bug combinations for resistance testing. And there's some, I guess you have an idea about whether it's going to work. Yeah. So where do we go from here? Because it doesn't seem great.

Jame: 33:53

well, there are a bunch of drugs, which we do test for kind of a standard and I've included here a. Summary table from the antimicrobe. org website, which is a sort of an online textbook for infectious disease. It's actually a bunch of different textbooks. It's not been updated since, I think, 2017 or so, but I still find it quite useful for referring back to. And the commonly tested groups are beta lactams, and they're I'll come back to them. Beta lactams, quinolones, cotrimoxazole, minocycline, and tegacycline. and chloramphenicol. And of the beta lactams, the Ones that are usually tested for are Pipericillin, Temicillin, which is superior in terms of MIC kill to Meropenem and Keftazidime. Keftazidime, which is superior to Kefapenem and Kefaperazone, two other anti pseudomonal Keflasporins. Carbopenems, and here Meropenem is superior to Doropenem which is superior in turn to Imopenem and Ertopenem. And of all of that I've just mentioned, the ones that appear to be most... active are minocyclin, meropenem, keftazidine, temosilin, and piperacillin, but they're most active. They kill an average of 23 to 38 percent of isolates. So they're not even particularly all that good. It's just that they're better than all the other stuff that I've mentioned. And so it depends on where you are. And usually, thankfully, these patients are being managed in CF centers. And usually the CF centers will have a dedicated, microbiologist. So there's one here in Nados South. There was one up in Nados North as well. And they will have a sort of established anti biogram that they will test for for BCC if it comes up in a, in the sputum culture and then they will get specialized advice, but that's not a lot, is it? Benalatams, quinolones, cotrim, minocycline, ticocycline, and chloramphenicol, and you wouldn't really want to be using chloramphenicol a lot in people, do you know that? It doesn't give you a lot of options.

Callum: 36:16

No.

Jame: 36:18

I have a list here of reasons that, Thanks. The in vivo doesn't reflect the in vitro susceptibility, some of which I mentioned before, so altered pharmacokinetics of the drug in the CF lung. Failure to deliver drug to the bronchiectatic lung. So we've talked about that from the IV and the inhaled space as well. High colony counts, so they, you can get colony counts as high as 10 to the 7 CFUs per mil, biofilm resistance, and certain local factors that affect how antibiotics work, like a low P8, so antibiotics tend not to work in acidic environments, and impaired phagocyte activity. So some antibiotics, like beta latams, can boost phagocytic activity, and that's impaired when. The phagocytes themselves are non functional. when it comes to recommended regimens, you're also tend to be using higher doses and dosing by weight. For example, for Meropenem, you'd be using 40 milligrams per kilogram. 3 times a day. If you think about an, 80 kilogram male, that would be 3. 2 grams, 3 times a day. It's quite a lot. Similarly with tobramycin and amikacin. So for tobramycin, you'd be using 10 milligrams per kilogram per day, as opposed to 5 or 7. So that's again, more than more than normal. There's the dosages of this, That you need to use can be quite high as well, and therefore quite toxic, and you need to use them for quite a long time as well. So the recommended duration is 2 to 3 weeks or until clinical response defined as an improvement in FEV1 or reduced production of sputum. So you're going, the clinical response is quite clinical there, or you just treat for 3 weeks, or 2 weeks as standard. That's, Giving you a lot of time to run into toxicity, particularly if you're using quite high doses of stuff.

Callum: 38:18

yeah, particularly aminoglycosides.

Jame: 38:21

Yeah, and I don't know, for aminoglycides the, I've included the, that summary table on, on what the MIC90 is for aminoglycides and the MIC90 for topramicin is 256. Now, you can nebulize it and give it IV at the same time, but I doubt you're going to be getting 256 a plasma level of 256 milligrams per mil, at the best of times, I think that's fairly optimistic. So yeah, I don't know what part aminoglycides have to play really with this

Callum: 39:03

you just have to hope you have one of the organisms that's got a low MIC, because MIC is 90, that's how much you need to get 90 percent of them dead at that level. And so there's gonna be, it sounds like there's a big range. It's one of those things that's like a bit odd because then you're saying that there's no break points to test against, but actually the in vitro stuff might be quite useful. I know it doesn't correlate well with in vivo, but I would always feel like more data is useful, more useful than none, but

Jame: 39:31

I guess the, that brings me on to the next thing that people sometimes do, and I'm not sure how commonly this is done these days, but the, people are obviously at their wits end trying to determine what to use. And of course, if you test something and the MIC is sky high, obviously you can't use it. But if you test something and it's within a susceptible range or a range that you might you think you might be able to achieve in the patient's lung, temptation would be to use it. But because we've got so few options the tendency is to use two agents. And so for that reason, people have been checking combination therapy by something called checkerboard MIC testing. So what this is, Calum, is you may have, you might do this in, a 96 well plate. You might not use all 96 wells, but what you'll do is say you'll have antibiotic A on the columns. And so for the columns, you'll put in a set concentration. So one, one of the column will be like one mic or 25, then 5, then 1, then 2, then 4, then 8, then 16. And on the rows, you'll have another antibiotic, and then you'll add that as a concentration of, 1, then 2, then

Callum: 40:50

a really, it's quite a complicated thing to set up and it's pretty laborious as well.

Jame: 40:56

Yeah, and it's old school. It's really old school. And the reason that people... Did this historically was they were looking for synergy or synergistic kill, and they were looking to avoid antagonism. So antagonism is when one antibiotic interferes with the function of another. So say tetracyclines, for example, this has been documented, tHat inhibits growth. It's static because it interferes with the way that ribosomal RNA works in the bacterial cell. Then if the bug's not growing, it's not trying to increase the size of its cell wall. And that means then that beta lactams are less effective. There's a old historical trial that sort of showed that, and it's a well recognized in vitro phenomenon. And the checkerboard MIC testing is trying to look for synergy and also look for antagonism so that they can avoid it. And typically, when you do this, between 1 and 15 percent of isolates are have some sort of. Synergistic combination of antibiotics that you can use, but 9 percent of tested combinations will be antagonistic as well. sO there's

Callum: 42:17

Doesn't seem like a slam dunk

Jame: 42:19

not really

Callum: 42:19

the answer. I think it's a marker of the desperation that, that you're in, because you're trying to find something, anything to help, and,

Jame: 42:29

luckily, this bug is not all that virulent, so usually you've got a bit of time but if you don't, or, even if you do, but you've got a range of options, people tend towards using a double treatment composing of meropenem plus one of anamoglycide, minocycline, or equinolone, And then for more serious infection, people tend we tend to use cotrimoxazole plus either a benalatam or a quinolone. And this is, again, this is all taken from, my, my references, one of which is antimicrobe. org. And then there was another review article. But, uh, I'm fully aware that I could be out of date with this. And, again, none of us are experts. And then there is the option for triple therapy, and that would usually be meropenem. Tobramycin and a third agent, and that third agent can be Piptaz, Keftazidime, Cotramoxazole, or Amikacin. iNterestingly, the third agent can be a Betelatam, despite the fact that Meropenem is a Betelatam, or Amikacin, despite the fact that Tobramycin is an Amiklaxide as well. And they that triple combination will be sidal against 85 percent of in vitro isolates. Thanks for watching! So that's good, you can just give them three highly toxic agents and we've solved the problem, haven't we Callum?

Callum: 44:01

Yeah, and it's difficult to know, and I guess you're just in a situation where you're just trying things and you get to a point where you grow the organism and then we know those antibiotics that may work and you're not really relying on antimicrobial susceptibility results, you're just giving it and seeing what happens and seeing if they feel better. And that's all you can do really, which I guess goes against a lot of what we do in clinical microbiology. So it's feels a bit weird.

Jame: 44:32

And I don't know if you remember this, but I remember we had a, almost a spate, Callum, of BCC... Cultures coming through that were resistant to all tested antimicrobials, and we were having to add on, tests for other things that we were, discussing with with local CF microbiologist, I, my, my memory of this is that this is a really tricky bug to

Callum: 45:02

Yeah, it's not good. So why is it so tricky? What are the resistance mechanisms that we normally see? Because we know that people with CF are more likely to have resistance strains than people who don't have CF, potentially because of they're getting more antibiotics or they're more likely to have biofilm going on. Okay. So yeah, it's a big

Jame: 45:23

and resistant rates are high as well so about, in one study, half of all isolates tested were resistant to 10 commonly tested agents. I don't know what those 10 were and in CF patients, rates of resistance to carbapenems are about 74 percent and cotrimoxazole about 95%. And if you think I was just talking about the double therapy sort of standard of care, they were based around a backbone of either meropenem or cotrimoxazole. So very quickly you can write, you can run into problems

Callum: 45:56

One of the caveats there is that we're saying they're resistant, but we don't have validated breakpoints. So it is we test it in the lab, but we don't really rely on that. So we truly say

Jame: 46:05

No, I disagree. Callum, I think if you've got, a resistance that's, unmeasurable or, 256 or above, that's not level that you're going to be able to get in plasma. And so if it's not doing anything to inhibit growth in vitro, I don't think I'll do anything in vivo either.

Callum: 46:24

that's true. Yeah, I'd say 95 percent is above the threshold where UCAST would just say it's intrinsically resistant.

Jame: 46:32

Don't bother. Oh I suppose they would if they ever gave any recommendations for BCC, which, as we've covered above, they don't. But what are the mechanisms? So for polymyxins and amyglyxides, your nephrotoxic agents, which you would prefer not to use if you didn't have to. It's interesting that they're... Both those molecules are quite polar, and there's a lack of cationic binding sites on the lipopolysaccharide of BCC. And so that means that they're not able to really attach onto the cell to then be transported in. The outer membrane isn't particularly permeable anyway, and if you get through all that, an efflux pump might throw you out into the plasma anyway. ANd then for beta lattans, again, there's they're not particularly permeable, but they do have... Beta lactamases too, inducible chromosomal ones, plasmid mediated cephalosporinases, mostly TEM, and metallobeta lactamases as well. And particularly they're active against imipenem. And then lastly, if you don't have any of that, there's an efflux pump that can throw you out into the plasma. For chloramphenicol, ciprofloxacin, tetracyclines, and ticacycline, there's an efflux pump. And for cotrimoxazole they can hyperproduce dihydrofolate reductase, so to, to overcome the mechanism of of cotrimoxazole. And there's an efflux pump that works for them as well. lOts of reasons for it to be resistant, not many treatment options, even if it is sensitive. And no breakpoints to help you. Good luck.

Callum: 48:12

We need to do some, we need to go and do a bug episode on something that's really easy to treat and really straightforward. I don't know about a run of, I think maybe we were slightly dreading these episodes because they are organisms which are not straightforward, complicated, not easy treatment options.

Jame: 48:33

Callum, I didn't know how bad it was going to be. Seriously, I had no idea how in the dark you were about particularly this organism. This is just ridiculous. But hopefully, next week it will be a little bit better with, or the next episode it will be a little bit better with

Callum: 48:52

Will there be a next episode? Because as I was saying to you privately before, I've now sat my part two exam and I was saying to Jayme that really this whole podcast thing was just me essentially manipulating Jayme into providing me two years of free coaching. towards this exam. So now I've sat it, if I've passed then, I'm done.

Jame: 49:16

All right, so the secret's out, is it? 100,

Callum: 49:18

Yeah, that's all it was. I just wanted to learn stuff.

Jame: 49:22

000 downloads, all for naught. Seeing as you brought it up, Camel, and if you want to mention it on the podcast, how'd it go?

Callum: 49:31

The exam was very difficult, but I think it was a fair exam that was, actually quite clinically relevant.

Jame: 49:38

It's good to hear you say that, because the part two has got a bit of a reputation for not being particularly clinically relevant, but that's something that I think they've

Callum: 49:46

Yeah, I think, yes, I've heard that perception as well but I, and I think the exam has changed a lot. So I would say I was pleasantly surprised it wasn't easy, but particularly the infection control scenarios were like. Okay, yeah, this makes sense. And recently I've done, had been involved in some things at work where I've been like, oh, actually the revision for the exam was really useful and the exam questions were relevant, but there's one of the papers is complex scenarios and oh my God, it was one of those things where the stem kept expanding and there was more and more information. And you're like, what? What is happening? This is awful. This is terrible. It was just, an absolute nightmare situation.

Jame: 50:24

Yeah what did you, you gave me an example what was it again?

Callum: 50:28

I can't tell you.

Jame: 50:29

chickenpox exposure, it was a pregnant, Baby no suppressed, Jehovah's Witness,

Callum: 50:34

was just yeah, exactly.

Jame: 50:35

one leg.

Callum: 50:36

It just got worse and worse. And you're like, what's happening? I think I'll just be crying at this point of work or phoning everybody. And so yeah,

Jame: 50:45

But I suppose that's the point if it was actually happening at work, you'd be like, I need some help. Like you, you'd be getting an MDT together. You'd

Callum: 50:52

and the role they're doing is they're testing your knowledge of the most complex, the flowchart and like the bottom bit of it not just the basic stuff, but the, the real complex stuff. Yes thankfully, Bercondelier Copacia complex did not come up. I think I'm allowed to say that. Because that would have been really annoying for something to come up in the exam that we hadn't covered yet.

Jame: 51:12

Yeah oh, so you specifically just answer questions

Callum: 51:15

No,

Jame: 51:16

covered in the first...

Callum: 51:19

You're not getting me. I'm not telling what was in the sound. But yes. Good luck. Didn't believe this, is it?

Jame: 51:25

Aye, yeah, true. Yeah, cool. Lovely.