You won't hear too much from me again this afternoon as such. What I'll do this session, I'll hand straight over to Marta, who's part of our. Clinical pathology group in the UK and we'll go straight into the session around microbiology and sample submission, so all yours Marta.

Thank you, everyone. There's a lot to cover. I tend to speak really fast, it's a widely vast subject.

Please feel free to ask any questions in the chat. We'll try to get them at the end and if not, I'll stay behind and answer them in the chat afterwards. So that's great cracking, I'll just share my screen.

OK, so, we're talking about microbiology sample submission, we'll do a little bit more on top of that, but this is going to be the main emphasis. Obviously. I'm a full-time employee of IEX.

It comes with the usual disclaimer. For our learning objectives for this lecture is how to get the most out of microbiology samples, and that really does start with good sample collection. It starts at your end.

And we're gonna do a little bit of more in-depth with urine and ears because it's one of the most common types of samples where we still see some issues with the samples that we receive. And we're gonna address skin, in general, wounds, fluids, biopsies, all sorts of things. And what you collect has an impact on how we process the sample, but also the information that you provide us in terms of what antimicrobial we might have started or, you know, the previous history of this patient, is it entire or nonti, all of that is actually taken into account.

When we're processing the samples, so we really need to make sure that if we're sending things for microbiology, we are including the appropriate collection type collection methods, but also the other clinical findings if you've done any results in-house, if you've done your urinalysis in-house, your in-house cytology, we need to be aware of those results and then. We'll go a little bit at the end if we have time on how we interpret the reports because they are complex, how we deal with polymicrobial or mixed cultures, when, what do we do when we have discordant results, so what we got back is not what we'd expected and why might not be the case. And also we'll talk about the value of a negative report.

So why do we do microbiology testing? Well, we do it mainly because we want to get the patients the right antimicrobial. But also when they need them, there's cost considerations that come into play.

Obviously our patient care is our utmost responsibility, but we also share a very important and common responsibility, so I see myself as part of the lab as sharing that responsibility of being. Antimicrobial guardians, we know that antimicrobial stewardship is vital for human survival, for the global health, and more and more I'm seeing cases where there is nothing that we can offer our patients. So we need to make sure that we're testing.

The right patients that we're giving antimicrobials to the right patients and as soon as possible we're getting the right antimicrobials so we can get a handle on antimicrobial resistance. So when should you submit a sample? Well, ideally is whenever you suspect a bacterial or fungal infection.

And if that needs treatment, then, you know, that's when we should submit it. So it's a bit like voting. We should be doing it early, start early, and we should be doing it as often as we can given that possibility.

But we know that that's not always feasible. It often cost limitations, there is value in empirical medical treatment, and so we can't consider culturing every single case that we provide antimicrobials, even though that's. The most correct thing.

So when should we do it? Well, if we're Doing culture, ideally we need to start it before antimicrobial treatment is instituted. It's been shown on a number of studies that as soon as 12 hours after antimicrobial being started, that you can stop the recovery of organisms from fluids, for example, from BALs and body cavity fluids.

So it's just after your first dose, basically next day, it will have interference on the ability for us to recover. Whenever we're thinking that we might need to do antimicrobials for long periods of time, so whenever the recommended treatment length is more than a week, so if we're talking for some kind of cases of deepyderma, for some cases of pneumonia, for some cases of osteomyelitis, where we know that the recommended length of treatment is longer than a week, then ideally we should be performing antimicrobial susceptibility testing and culture before we start that treatment because it's gonna be much more challenging to do it after we've started. And whenever we're tempted to do antimicrobial cycling.

So it's that patient that we know will come in for recurrent ear infections. Well, let's get ahead of it and start treating. And there are some instances where indeed it is highly recommended that we do it even when it's the first presentation.

And that is if we had any recent previous antibiotic therapy. For a relatively long length of time or relatively recent, and this is because previous antimicrobial therapy will promote antimicrobial resistance, being present from even the commensal flora. So it's more likely that you'll end up with antimicrobial resistant organisms being a part of the problem.

If whenever we have recurrent relapse or reinfections, yes, we're gonna have to address the underlying factor that leads to those relapses or infections, and actually it's really important that we do so because just putting it on antibiotics is not going to sort the issue. We have to address the underlying process. But it's important that in those cases, instead of again cycling or repeating the same antimicrobial and hoping for a good response, that we address it by doing culture, making sure that we're treating the right bug.

If we have immunocompromised patients, we know that they are, will struggle more to be able to fight off infections just on their own and therefore more likely that we might need a longer treatments or addressing that, more directly. If it's life threatening or complicated infections, patients at risk of multi-drug carriage or infections, for example, if a patient has had an issue with MRSA or MRSP in the past. It is very likely that that has become part of their normal flora, so we don't necessarily do treatments to say that we've cleared these patients from these organisms because it's been proven that they can pick it up back again from the environment or from places that we haven't tested.

Because if you test some of these patients from different areas in the skin, some will be positive and some will be negative for the presence of these multi-drug resistant organisms. So if we know that we have a patient that's had a history of MRSA, I definitely want to test earlier in that patient because I know that there is a risk that that is going to be present there again. And then, as we mentioned, so for long treatments of antimicrobials for when it's a long period of time that's expected like some cases of deep pyoderma.

And I often get asked when should we not submit a sample, it's never, there's not really a contraindication for doing extra microbiology, but there are instances where the results are of limited use, or where it's not that it's the problem with the sample, it's the problem with getting the sample. So it's not so much that we're culturing and it's incorrect to culture. It's because there is a risk of sampling for that patient.

So that sample of costs for getting the sample and the balance of the risk for the patient for getting that sample needs to be considered, and we'll talk a little bit more of that on the urine side of things where we prefer cystocentesis samples, but obviously we cannot get it in every single sample. It's really important that if we're collecting for non-sterile sites, particularly, that we are always evaluating the culture results with other clinical findings, including cytology, if available. And for many of these instances from non-sterile sites, I think cytology should be available at the same time.

Because in those cases, the results may be of, of limited use. It could be that we just have positive growth because of normal commensal flora. That's often the case, for example, for the upper respiratory tract.

I, I get a lot of mouth swabs from ulcers in the mouth and a lot of the time all we're gonna get is commensal mouth flora. It's no non-sterile site. So unless I have the cytology to back up that indeed that is significant, it's gonna be really challenging for us to be able to make that distinction between what's commensal flora and what's a significant opportunistic pathogen.

We can also see as transient colonisation and a classical example for that is when we have asymptomatic bacteria where it's not necessarily an infection is the bacteria is there. Yes, it might be colonising the latter part of the urethra, but it's not necessarily causing an issue. We talked about how invasive procedures, especially if there's risk of contamination, need to be considered against the benefit of sampling.

Sometimes we have fastidious organisms that may not be able to grow easily or even be non-cultural organisms, and so we we're not able to culture these even if we tried. And for those sometimes PCRs might be the best way to go about them and making sure that they're there rather than sending for culture. And then for infections that are treated with topical antimicrobials, identification for the presence of bacteria may be useful, and what type of bacteria may be useful, but we have to take the antimicrobial susceptibility test results with a pinch of salt because they use systemic break points, not, topical breakpoints.

We still don't have those for veterinary medicine presently. And so, even if it is intermediate and we'll see some examples later on, we might not get a we, we might get a clinical response in vivo, even if it came out as intermediate or resistant in vitro, because we're using systemic breakpoints. So, the best microbiology results really start with you.

So let's talk about sampling. Now there's different swabs that you might be having available to you for swabbing, and that could include charcoal swabs, which we recommended for anaerobes previously, AE swabs, which is more of a generic medium. But E swab is really now the preferred sample type for us, IDE.

It allows for several things to be done out of the same swab, so we collect the swab. And the site is swab, the swab is placed in fluid, and then the bacteria is transformed from the swab into the fluid. The virus can be because we can use it for PCR as well.

And so we can use that same sample to culture a lot more different medias and allow for repeat treatment or testing if it's required and also use it for PCR and sometimes even for cytology in the ear cases, but it's not the recommended sample, it's much better to get the glass slides submitted, but we can do it out of, of the swab. It's just not the best. And it's becoming a much more common method and recommended standard method for collecting the swabs using these swabs, not just in veterinary medicine, but also in human microbiology settings.

For fluids and tissues, we mostly use sterile pots. It is important that we do use sterile pots because getting that sweet jar urine sample is really not appropriate for culture. It shouldn't be received for anything, but really not for culture.

My classical example is once I received urine on a wine bottle, and really that's not an appropriate sample and I would say quite dangerous, . If you, it may be for some types of samples, we can use blood culture bottles, obviously for blood cultures, but sometimes it's also useful for places like synovial fluids where we might not be able to get. The, the sample to grow something unless it's put straight away in enrichment.

So using as a type of enrichment fluids such as blood culture bottles, increases the likelihood of getting a positive. Now, when we talk about fluid tissues, and I'll talk about tissue samples, and I'll talk a little bit more about this, when we're saying to put them in sterile pots, please don't just leave them there. They have to be placed in either sterile saline or with a swab that is previously humidified with.

Saline, because, we can't allow for that sample to desiccate. The bacterias need to be reached the lab alive. And then for urine samples we often recommend boric acid, but that is strictly for free catch urine samples, so we shouldn't be using it for cystocentesis samples.

And also really important that if we're using the boric acid that we're filling the tube to the top of the line or in excess, but we're not just putting a couple of drops on a tube that is for a much like higher volume. Now this can be challenging because often our patients express or void their bladder before they reach us in the consult room and therefore we end up with a very small amount of urine to be able to collect, but still in those cases to try and use paediatric tubes which are of lower volume if available. OK.

Oh, just another recommendation is that most of these things, we, we can refrigerate them. The thing is we would recommend if we're specifically looking for aerobic culture that maybe we shouldn't be refrigerated and for specimens that are placed in special transport media again refrigeration might not be necessary. So general recommendations for sampling, we'll talk about collection with a from a sterile site, collections from mucosas or superficial skin or wounds, and then from areas that may be sterile, but there's communication with the outside.

So for the first sight, what we should be doing is that surgical prep of the skin surface that is gonna be put where we're gonna put the needle through. And that will go for anything that's listed here. So the skin blisters or pustules, they can be quite small, but actually it's a great way to get a very good sample.

If we do that skin prep, allow it to dry and then just rupture the tip of that pustuol with the sterile needle, and then get the sample and get the swab and get that little tiny bit of material onto the swab and put it in the E swab fluid. Because that means that we will not be contaminated with that superficial skin flora. For things like the mucosis, conjunctiva, ear, and general open wounds, there will be contamination of that normal for us.

So it's really important that we get the superficial material cleared out before we actually get the swab to collect the bacteria that are most closely related with that mucosa or with that open wound. And this is because there's more likely that we're going to find the significant organisms than if we get all of the gunk where the other organisms might be present. Now when I'm saying this, I want to make it very clear that for the years we're not saying that you're going to use the topical cleaners because a lot of those have antiseptics and so even if they're not anti.

Biotics, they can have an antimicrobial effect, so may have an impact on our ability to recover organisms, especially if they're present in relatively low number. And therefore what we're saying is that we're taking a sterile swab, and we're getting rid of the majority of that superficial material, not necessarily that we're doing a skin prep in the same way as this. And then for anything where we can have material to be expressed to the surface, ideally we reject the first, bit of that material and we recover the material from the culture that is not that first expressed material because that is more likely to have that superficial flora.

For example, for voided urines, the recommendation. Is that we reject the 1st 5 mLs. Again, very aware that that's often not possible because we don't have 5 mils to reject.

But just to give you that idea that ideally we want to try and get rid of that superficial flora that will be there and that might impact on our ability to recover the right culprit for that infection. So we've talked about the skin, the skin and doing the swabs from either epidermal colorettes or ruptured pustules, but there's also the possibility of doing skin biopsy. And if that is the case, then we do prep it like we would for any type of biopsy, so sterile prep, so that we again avoiding that contamination from the normal flora, because what we want for those cases that we're addressing with biopsy is for the deep pioderma, not for that superficial flora.

From the conjunctiva, ideally we want to swab material from the cul de sac or the medial cantus, and not necessarily from all all around, because that's we're more likely it's going to accumulate that material to the ears, it's really important that we address mostly the the horizontal canal, so deep in that ear canal, not the vertical ear canal. Again, that's where, more likely to have secondary growth rather than the organisms of interest. And like I mentioned before, we want to make sure that we're taking away that debris out of the ear canal before we get that sample, but without using the air cleaners.

If we're doing middle ears, it's important that we sample it separately from the external ear canal. So we're looking at the material that is sampled with a needle through the tympanic membrane, and that is submitted separately from the external ear because they can and often do have different organisms involved. The same with if we have both ears affected, we need to sample separately the left and the right ear because they often do have different organisms, taking in.

Place of tis is mostly not a primary bacterial infection. It's secondary to something else, so different organisms might be appearing in different ears, not necessarily the same. So it's another type of sample that we would say don't pull it together because we really want to look at it separately.

Because it might be the case that you end up putting one type of eardrops in one ear canal and another type of eardrops in another one, which might be tricky but might be necessary in some cases. And then for the wounds and fistulas again, like I've mentioned, just swab after removing the debris, we want to make sure that we're collecting the sample that is close to the open mucosa or the open wound skin. Please always use sterile containers we've mentioned and do not submit syringes with needles.

It's, it's a big health hazard. We once had this submission. Which had to be rejected unfortunately, because there's nothing that I was able to do with that.

Not just would this be a health hazard in terms of processing the sample, but this material here is going to be desiccated and therefore I won't be able to recover anything from it that is of use. In cases like this, what I would recommend you just got a little bit with that syringe chip is just squirt it into that swab, put it in the e swab method and then. Describe to us what you've done.

There was just a small amount of material that we could get, and, and this is what we've done so that we can then in the lab, react appropriately and select if we put it through enrichment or if we do another type of selective culturing. For the tissues of biopsies, we've talked about how we need to minimise dehydration and use that plain sterile container with a small amount of sterile saline or the involve it's, if it's very small and it's gonna potentially break, involving it in a small amount of sterile saline in a swab might be the best way to go about it so it doesn't get lost or dissolved into that sterile saline. Remember to always try an aseptic, prepare the collection site to avoid contamination with the flora.

And again for some fluids, they can be submitted in plain sterile pots. Ideally, we would like a minimum of 1 mL of fluid. The best are 5 mL of fluid.

If we have a smaller amount of fluid, it's going to reduce the sensitivity ability to perform more tests. And also it's for some organisms where they're more fastidious or if there's anaerobes, it's more likely that we're going to receive a false negative because there is some exposure to air, to oxygen within that gradient microanaerlos. So if we're considering anaerobes, we're gonna try and minimise the exposure to air, fill the tube as much as we can, or use appropriate transport medium or enough volume of sample that that's not gonna be a problem.

And again, if those cases, if you're using the media or have enough volume of sample, we might not need to refrigerate it, otherwise we would refrigerate. And here we got an example of the blood culture bottle, that again, really routinely we tend to use for blood cultures or for some synovial fluid, . Collections.

So this is just, in general, we're gonna start with the urines and going a little bit into more detail. I hope you're in with me because we really need to improve when we collect urine samples and how we look at these urine samples for culture. So 3 big large collection methods that are described as being available for urine cultures, which are free catch, catheter and cystosynthesis, and I would have to say that free catch as it was designed, is very different than from collecting from the literature.

A free catch. I like the, the name says, free catch. It's catched with a little kidney dish under this, this flow of urine so that we're catching it while it's on air before it touches other surfaces and might be contaminated.

And I know that there's lots of urine signs out there for urine collection, and they're very useful for us to collect samples for other types of urinalysis, but not necessarily for culture. Because the truth is that those will become more likely to be contaminated. Again, for the free catch sample, the idea is that we reject at at least the first few mLs of urine, so that that bit of the commensal flora from the skin and from the lower or the latter part of that urinary tract is expelled, and then we get to the bit that is indeed in the urine.

Obviously, ideally we always prefer cystosynthesis for culture. And unless there is a specific contraindication, this is what we would recommend. So things if you have an animal which has a coagulopathy, or if you have an animal that has potentially a bladder mass, which you're suspicious for TCC, then cystocentesis might not be the way to go forward because you do have that problem.

In those cases, you might choose to do a catheter collection. Actually, traumatic catheter collection might also provide you more. Cells for the cytology bit for looking at that suspected mass.

So you can do that type of collection, but sometimes that is a clearly challenging to do in some of our patients to get a catheter through and in those situations, then yes, we can use free catch. I'm gonna show you a little bit later on how the type of urine collection method impacts on how we evaluate the samples in the labs. So it's really important that you tell us what is the method that you used for the collection of those samples.

Because it impacts on how we address and how we interpret those samples. So one of the things that we often say as well for urine is that we receive a lot of urines that are coming in for culture where there when you've put the clinical signs or the clinical picture, there are no clinical signs compatible with UTI. It's really important that we are culturing urine mainly when we have a suspicion of UTI because we shouldn't be culturing subclinical bacteria urine.

There isn't a single test in the lab that can differentiate between. A UTI and a subclinical bacteria. The organisms are the same and the counts or the amount of colony forming units per millilitre also do not allow to make that differentiation between subclinical and clinical bacteria.

So it's really important that you take that into account when you're interpreting the sample. And so it's important that ideally we get that information as well, because once you get then a positive, it's, it's really challenging to say, oh, I got a positive culture from the lab with the antimicrobial susceptibility testing, but I'm choosing not to treat. It's like, why did you culture it to begin with?

Always try cystocentesis unless contraindicated, and sometimes we even when we're reporting out the results, recommend, oh, I'm not sure about the significance of this organism. You might want to try again with the cystocentesis sample, which obviously leads to potentially delays in treatment. Again, if we're doing free catch samples, we, recommend boric acid.

We do not recommend that for cystocentesis samples. The reason being that boric acid is a preservative. And it holds the population stable of those bacteria for about 48 hours, and so.

For longer than that, they don't no longer remain stable, so that's an important information because it means that it's gonna impact when we have our clients bring us the samples, because if we receive our sample on a day after it was collected and then it reaches the lab a day after it was collected, we're reaching that limit of the 48 hours. So ideally we want to get those samples into the lab as quickly as possible. Because it is a preservative, it's been shown that in cystocentesis samples where the colony forming unit counts might be lower, it can actually kill off the few significant bacteria that might be there if they're present in low numbers, so it decreases the sensitivity.

So really it's recommended for free catch samples and as I've said before, we really need to fill up the tube up to the right level. A sterile container for the cystocentesis and ideally refrigerate and process within 24 hours. So again, ideally this would be a sample that would be sent to the lab same day as it's collected so that the urine culture is still appropriately validated.

I've included here the definition of subclinical bacteria, just that reinforcing that information that we really can't differentiate. This from a bacterial cystitis based on the quantification or the type of organisms and also that the presence or absence of pyuria or leukocytes in the urine does not define subclinical versus clinical bacteria because you can have and often do have presence of leukocytes, for example, in cats with cystitis without necessarily having a bacterial culture behind, or a bacterial infection behind that presence of the leukocytes. So when do we consider culturing if there's clinical signs compatible with UTI?

And then treatment follow-up is really only recommended in cases of recurrent or complicated UTIs and that's mostly if we're doing longer courses of antimicrobials. One of the recommendations is you're doing during that treatment and then after treatment, particularly in cases that we know that. Are recurrent, so that we're having trouble getting on top of, or there may be underlying predisposing factors that predispose to the retention of those, example would be a presence of uroys that we're waiting to be dissolved by diet because we don't want to intervene, surgically.

And then in some cases, even if there's no clinical signs of UTIs, the patients may have specific risk factors that means that we might want to culture the urine. And if there's suspected pyelonephritis, you won't have the traditional clinical signs of UTI. You'll have the clinical signs that are maybe pyrexia and renal failure.

Even referred pain, but not necessarily your polyuria type of thing. If we are suspected of an animal as being sepistemic with bladder or prostate as a source of the septicemia, or again, like I said, with the dogs with rightists, there's also a mentioning of paraplegic dogs that might be predisposed with lack of bladder control that might be predisposed to UTIs. So when we look at this quantitative bacterial culture, we take account the numbers of bacteria present, and there's different cutoffs that are considered significant depending on the collection type, which is why it's really important that you provide us with that information with the collection type.

Now, I have to say the majority of our urine samples that we receive are still free catch, and we definitely want to move those that come for culture. Into the cystocentesis, not the others, not the other type of urinalysis, but the ones that come for culture, ideally move more into the cystocentesis group. And the other thing is that if we don't have the information, Of what type of collection method was used, we assume it's free catch because it's the most frequent one that we have.

In this case, that means that we might be incorrectly interpreting these cutoffs because you haven't told us that it's a cystocentesis. So please, if you're sending us urine for culture, always tell us what's the collection type. Now, at ADE we also provide a test for antibacterial activity, and this is substances within the urine that are not necessarily antibiotics, but they they will inhibit in vitro growth of bacteria.

And we use those to explain maybe some of the false negatives that we might be expecting, but also to make sure that we're not taking these cutoffs too strictly on animals that might be under treatment, for example. If you are testing under treatment or after treatment from a recurrent infection, also always give us that information because again, we will be less strict with this type of cutoffs and we'll take that information on board when we're assessing the results. So take a whole message for the urines, make sure that you're selecting the cases that you cultured to the ones that have clinical signs or to the exceptions that we've mentioned.

Always give it the relevant clinical infant and the collection method, and we do prefer cystocentesis unless it's contraindicated. Remember that white blood cells indicate inflammation, not necessarily infection, so we can't use that to differentiate. And it's also important to remember that not all UTIs will have increased white blood cell counts in the urine, in the sediment, so that also does not differentiate between the two.

And because asymptomatic bacteria can have the same type of bacteria and the same number of bacteria growing as bacterial cystitis, we can't use just the fact that we've grown something as the, it needs to be treated with antibiotics. Right. Next topic, I said that there was a lot of information is ears.

With ears, it's really important that again we're assessing the culture alongside your clinical exam, but also the cytology. It's really important because the ears are non-sterile. That means that if I have a urine, an ear, sorry, with no growth, immediately I go, what's going on here?

Because the ear is meant to grow something, we're supposed to have bacteria and flora and fungals as part of the normal flora. So the ear exam and the smear exam for the cytology are essential. And again, we're happy that you're doing that in-house, but please provide us that information so that we can use it when assessing the the results that we get.

So ear cytology provides information that can help treatment immediately while the patient is still within the practise. So it's a good thing to do in-house and again, we know that that's sometimes challenging to do. So IDEX is looking into alternatives to be able to support that good piece of diagnostics to be done in-house, but if you're not doing it in-house and you're submitting the culture, please do submit smears for cytology as well.

And also make sure that particularly in cats where it tends to be more frequent, we're looking at things like parasitic causes, which are primary causes, and they will literally walk off the slides that you sent us, so it's best that it's done in-house. Most cytologist will have variable amounts of keratinaceous material, yeasts or and bacterial organisms and variable information. So again, collecting from the ear canal can be really, really challenging when I told you to collect from the horizontal ear canal because pain is a big factor in some of these patients.

And so we might need to address that pain and inflammation first. I heard the great tip from our dermatologist Arianne that she says sometimes what she will do is she'll get behind the dog with fingered hands, so with gloved hands, and we'll start massaging the canal and then the ear canal and then the dogs will, you know, because they're itchy, they kind of find that that's kind of not as displeasant and then she will put her ear down. And her finger down the ear canal and get the sample out of that.

So it doesn't have to be a swab that might be more aggressive in a way or interpreted as such. It can be part of your thing and then you use that material, you put it in the swab. Obviously, again, we're trying to use the best material that we can.

So ideally we kind of get the most material out, as I've mentioned before. For doing ear cytology, it's really important that we take the swab or that material and that we're rolling it on the slides rather than spreading it like this because it will lie cells and so we'll miss some of the inflammation there. And again, we do recommend that the ear swab, but ideally, we want the smear to be submitted separately from the ear swab, E swab.

We've talked about how the ear is not sterile, and the majority of the organisms that we do grow are just this biosis, so they're overgrowth of the normal flora. We see that all the time with the staphs and with the malaces. They're part of the normal flora, but they can be overgrown.

And they can cause significant issues. When we're assessing this, we assess, do we have just one type or multiple types, and if I have lots of different types of bacteria growing on a plate and they are all from all different sorts of organisms, then we say, well, mixed commensal organisms, you know, bacteria is not your main issue here. We're going to need to find other underlying causes.

So is culture beneficial in these cases, like we mentioned, it may be of limited benefit intitis external. That's why a lot of the times you will get these commensals recovered, non-significant growth type of things. If we only have that mixed flora, go and find what's the underlying problems.

In most cases, you will see when you have two bacterial infections, usually secondary, it's part of that dysbiosis, and it's malaci Staphylococcus or pseudomonas. That cytology will be very helpful in guiding you because if you have cocca, you might go for something like with fluorinnacle. But that is not gonna be effective if it's a pseudomonas, or if it's bacilli, you might go for something with polymyin or with quinolones type of thing.

So just having that information, is it mostly yeast, is it mostly cocka? Is it mostly bacilli can be helpful. Now, the majority of your cocci will fall within the staph strep groups.

The the bacilli, you can have growth of many different things. The pseudomonas is the one that is mostly causing us problems and it may be associated with biofilm, but we also have coinobacteria growing, which can be challenging to address as well, and sometimes lots of enterobacteria that found their way into the ear of the dog and then found a good environment and start growing there. So we always do cytology when doing culture, so we can make sense of what was grown.

Like we've talked about, the ASDs are the antimicrobial susceptibilities testing is based on our systemic breakpoints, so it may not be so useful for topicals. And so even if it's resistant, it may still respond in vitro, but it does tell me one thing. If it's susceptible on the systemic break points and it's not working on your clinical case, it's not because of antimicrobial resistance.

It's because we need to deal with either compliance or with the other local factors that are impacting on our, on our clinical response. It's not because of antimicrobial resistance. So I think that they still provide some useful information and definitely more useful when we have the eyes on cytology.

So when is culture beneficial, when it's chronic otitiss, definitely it's more beneficial when we have rods on cytology. If we have any organisms of unusual morphology, because sometimes we get weird and wonderful things growing out, and here I sent the result out yesterday of Aspergillu growing on a deer stalk. It was secondary to a foreign body, found the right environment in the ear, and started growing there.

It's a secondary opportunistic organism that is still causing significance. Whenever I have otitis media, because then systemic therapy may be indicated in those cases. If I have those histories of previous infections, with multidrug resistant bacteria, your MRSPs, your MRSAs, your pseudomonas, if I know that I've been giving long term antimicrobial therapy to that patient because I know it's predisposed to having that flora with antimicrobial resistance, and if there's bacteria persisting on cytology despite us putting on appropriate therapy.

So, always look for underlying causes, always do your cytology, make sure that you're definitely culturing any recurrent cases, and remember that because just because we've grown it doesn't mean that it's the primary problem there, it often isn't. So what happens after that, you've collected the sample, you've given us your patient identification, you've given us our site, you've given us the collection methods. Remember all of that emphasis on the urines.

Please also give us any results of other diagnostic findings like cytology, if you've done it in-house, or imaging, for example, if you're sending a BAL. Please also mention any ongoing or prior antimicrobial therapy so that we can make sure that we're testing for what you've started giving it if we can. Sometimes we can't test the antimicrobial organism combination that that we have because we have no break points for it.

But if we have no information on what the animal has been started with, then we can't make sure that we're addressing it. And then if it's a repeat sample, please always mention the previous results and give us the previous information so we can go and look it up to make sure that we're not missing, that this had a previously multi-resistant E. Coli and there's still a few E.

Coli that we would otherwise dismiss from a non-sterile site. So how do we set up the plates? Well, we look at your clinical history and we look at the site of collection to see what we're going to set up for.

So there's different growth media and different growth conditions that are applied to different types of samples. So if you just say swab, for us it's like, I don't know what you want me to culture, but if you say, oh, it's a, an ear swab, then I know which organisms your target. I'm Include a a pseudomonas Agar, so I can make sure that I'm going to find those pseudomonas, or if you tell me that it's a a a sample from a flush, if you tell me that it's a prosthetic wash versus a BAL, I'm gonna be looking for different target organisms, so I'm gonna set up different media and different growth conditions.

Wherever it is required, like for the urines, we do provide quantitative results, but most of the times you will have a semi grading of, scant, moderate, profuse growth in peer culture or in mixed culture. So, when do we assess these organisms? Well, you can expect results from from 24 hours from urines to 48 hours from most samples if they're negative.

Some are incubated for longer, so you might get results later than that, saying, you know, extended culture was positive now, but you only get that if it's positive. At 48 hours if we see something, it might take up to 48, sometimes 96 hours after that for us to be able to recover the full result of that one specific organism that then has the AST because they have to be in pure culture, so sometimes they need subcultures. If we're looking for a specific type of organisms like dermatophytes or other fungal, we might have extended cultures.

And in those cases, it's mostly because they're slow growing organisms. What we need to do verify unusual results might extend our usual turnaround time. So how do we interpret the results, we look at what has grown and consider the following.

Is this an organism that is likely to be a pathogen? Is it part of the normal flora for the species and site, or is it an environmental or contaminant? Then we look at how the sample was collected.

Was this from an aseptic collection, from a sterile site, or was it from a non-sterile site? Is this pure or mixed growth? If I have pure and abundant growth, then I'm more likely to find this as significant than if I have mixed growth, particularly if that mixed growth comes from non-sterile sites.

So how much has it grown? Has it grown only from enrichment, and how do these results correlate with the other information that we have? So we took it does, it's a very involved and hopefully you'll be able to understand why we do need that information in the submission form.

So let's quickly look at an anatomy of a result before we finish. This is from a urine example, and so you will have urinalysis, but on a urine, you will get the organism that was recovered and you will get the UFCs or colony forming units per millilitre. And I've shown you before the cutoffs that we use for for significance for these are different depending on collection site.

You'll also get that urine antibacterial activity test which is shown just below the . Culture result. And then underneath it, it will come the list of antimicrobials, their qualitative result, which is resistant, intermediate or sensitive.

You will get the MIC, so the minimum inhibitory concentration for that specific organism, drug combination, and then the sensitivity range that has that was tested and where within that sensitive range. Is the MIC of that break. So ampicillin here, we have an MIC of 32, 32 is the top that we're testing, so it comes out as resistant.

Here, for example, we have a sensitive of 8, that's 48, 1632, 64, the 64, it would be where it became resistant, so that S capitalised indicates where your MIC for this. Antimicrobial organism combination lies within that sensitivity range. So susceptible means MIC or below a zone diameter or a concentration where the usual drug regiment.

So a concentration of antimicrobial that is given and timings for that antimicrobial will be effective at the site of the infection, and we might have different breakpoints for urine versus systemic breakpoints for antimicrobials that are concentrated in the urine. Intermediate means that I might need higher dosages or more regular administration. And in those circumstances, it might be effective.

And this is where, for example, if I'm looking at my topicals and I have intermediates, I think, well, because we're using much higher concentrations than the one to determine systemic breakpoints, this is going to be effective. And then resistance, which is even with those increased concentrations, it's unlikely to be effective at that site. So the MIC is the lowest number of an antibiotic, dilution that inhibits the growth of a given bacteria.

It's really important that we don't look at the number as face value because we can have the same number being as resistant or susceptible depending on the breakpoints that are, so we only use it like this, and for example, that we're not using at 2 compared to less than 0.12. For example, here we have the same number, breakpoint, but one is very susceptible and the other one is resistant.

One of the most frequent questions is can we test for this other antimicrobial or for the other combination is sometimes why don't we have an MIC and you're still giving us the result? It's because we can't test for all antimicrobials that are present in the market, so we often use surrogate or class reference antimicrobials, where we're using one antimicrobial to predict how it will be susceptible or resistant for all of the other antimicrobials. We've talked about MICs and how they can be used if they're all sensitive, you choose the narrow spectrum and avoid the high priority, critical important antimicrobials, and we now provide some guidance for where those lie, the different types of classes in our reports.

If we have restricted choices, consider using those intermediate or susceptible dose dependent, something that we're not currently still reporting, but we're looking into to start incorporating in the reports. But also remember that if it's topical, some of these intermediates might actually be effective. Sometimes we can't perform MICs because the organism doesn't allow us to do so by their growth characteristics or because we don't have break points for that organisms.

If there's no criteria available, we might provide to you that . What is known from the literature as a form of a comment. OK, so look for those as well, because sometimes they're kind of in the bottom bits of the comments.

If you don't have an ASD, it might be that it's covered as a comment in terms of what is in the literature that may be used for that anti antibiotic for that organism. Treatment. We talked about how we use more than the MICs that you have here for references.

Just very quickly before I finish, discordant results, sometimes we have cultural negatives and we see bacteria or infection in cytology or the opposite, and there's a number of instances where this can happen. Obviously, we can have also incorrect interpret. Or errors, but if we have all of the clinical information, we can deal with that.

And we've listed the causes for it's more common, and one of the most common one is that the bacteria are non-viable. So we're seeing them on the cytology. They're there, but we're unable to grow them, and that happens usually because of prior antimicrobial use.

So sometimes if we think it's not being effective clinically. And it might not be effective clinically, but the antimicrobial that is there may be enough to inhibit the growth of the bacteria in vitro in the plate where it's not as nice environment as it is in the body of the patient. If the sample has been submitted to extreme conditions like temperature or pH, it might kill the bacteria.

We have also white blood cell inhibition. Which is when we collect the fluids onto the sterile pods, the cells are still alive. If we have a lot of white blood cells and not that many bacteria, they'll still be doing their job on the way to the lab.

So we might have the bacteria there, but they're non-viable because the white blood cells did their job and. You know, made them, killed them ineffectively. We have a lot of pseudobacteria, lots of growing things in those sediments that look like bacteria and they're not bacteria.

Sometimes we have, contaminated reagents, so our diffquake will have bacteria growing in it and it's not, so it appears on the cytology, but it's not in the sample. But often as well what we have in those is no insignificant growth and people get really frustrated with this, but it's just if we can get a better sample initially, we'll see less and less of those coming through. And then sometimes we have cultures that are positive and no bacteria on the cytology or no inflammation.

Culture is more sensitive than cytology to determine the presence of bacteria. It could be the bacteria is obscured by the breeze, so it doesn't seem like it's there, but it is there. But it also could be that there is growth of contaminants or mental flows, so we really need to look at what has grown in there.

It's really important that we value a negative result for bacteriology because it might tell us that we might not need antimicrobials and help us, in terms of our diagnostic tree to get closer to what's the underlying process. So it might mean that we need to go and look for allergic skin disease. A lot of the times I have this with my anal gland cultures where we get, obviously it's not sterile, so we do get growth, but we get nothing predominating, and I always think this animal clearly has anal gland problems.

So what's the underlying problem? Does it have a mass? Does it have allergic disease or atropy or problems with the the faecal consistency that predisposes to issues with those animal glands because it's not gonna be the bacteria there.

So look for underlying causes if you have a no growth or non-significant growth for a commensal environmental organisms only. So absence of growth does not indicate absence of infection. Growth does not necessarily indicate infection.

And then there's this 9060 rule that transpires from the human medicine that says that 90% of your susceptible cases will respond to the antimicrobial. There's 10% that don't because it's all of the other factors that we have to take into account. It's not just the antimicrobials, it's the patient.

And then the 60 comes from some of the resistance, up to 60% of the resistance, actually clinically they're responding because the antimicrobial is just a helper. It's your patient that is responding to the infection. That means that always treat the patient first, not the numbers that you get on your report, not the results necessarily.

So if you have an otitis, it comes back as, as resistant to the antimicrobial that you put it in to begin with, but the animal is responding, you don't necessarily have to swap it. OK, treat the animal, not the numbers, not the results. And by all means, consider us a part of our team and always reach out for any questions that you might have when any thing does not fit your clinical picture.

Please always come to us and ask for any question you may have. Thank you, I hope we still have time for questions, David. Yeah, that's great.

If there's a few questions coming in here, so I'll just work through some of those from the top. So the first one that's come in is in terms of, you mentioned obviously about making sure that we consider our previous results when we're submitting samples. Is noting down just the IDEX reference number adequate for that, or do you need more information?

Yes, that's fine, noting that there's a previous submission and saying there is enough, but if you've had, for example, previous pseudomonas, if you add that immediately it will trigger everyone that's looking at that submission form that that's what you're looking for. But yes, if you have the previous submission number, we can look up the previous results. Right, and the next one is, this will test me.

Er which test is best to request for dermatophytes? But we haven't, oh, well, how long is a piece of string? So we have the basic culture, which is fine for 99% of the cases.

Well, obviously, a culture for dermatophytes takes a long time, so it can take up to 4 weeks to turn positive. And so it might be that you need the results quicker than that. It also might mean that you're screening an animal for a carrier status.

And in those cases, or that you have presence on a biopsy sample, for example, from a carrion, from an abscess caused by, by fungal infection by dermatophytes, and in those cases, in those three cases, the PCR might be a better a better option for you and we also offer that, so we offer dermatophyte PCR. So culture and PCR are the two, the, obviously there's other methodologies like doing the wood lamp or looking at for the dermatophytes in the fur, but they have lower sensitivity than the aforementioned tests and therefore that would not be my recommended test. Fantastic.

There's a couple more come through here. We'll take this one and we'll see how we're doing for time. For UTI cases, would you do another urine sample post treatment if the bacteria is resistant to a lot of antibiotics for both, if the animal is and isn't showing signs of a UTA after the treatment.

Oh, so if it's the first time that the animal has a problem. And you've treated with the right antimicrobial and the animal doesn't have clinical sign, then I wouldn't retest it. That's it.

If it's a recurrent case, or we've had previous issues, and that's defined as repeat infections in the previous. 3 months or more than 3 episodes within 1 year, then definitely we need to retest it after treatment to make sure and look for underlying predisposing factors. Don't just test to see if it's still there.

Look for why is it still there. Great, OK, right, we'll take this one more and then, if you're happy to stay on and just answer the last couple on the, on the chat, that would be great. So this one from Lydia, when using urine cytology for an animal showing UTI signs, would seeing a particular bacteria, so rods versus cockey, for example, make you more likely to recommend culture for clients with cost concerns?

Not necessarily, and I'm going to tell you the big difference with that is the presence that we often recover enterococci from urine and enterococci do have a lot of intrinsic resistances, even for antimicrobials that we might reach for first with the neurons. So I would not, I would culture both of them. Yeah, that's my recommendation.

I wouldn't necessarily choose the antimicrobial different from years based on is it cocci versus bacilli. Fantastic, thank you. Right, well, I won't attempt this next one because I don't know whether we'll have time to answer it cos I'm clearly not the expert in knowing what the answer's gonna be.

So there's 3, there's 3 questions we've not managed to answer, Marta, on the, on the chat there if you're happy to have a look at those. Thank you so much. Thanks, Marta.