Hi, everyone. Thank you for joining this session. My name is Krista.

I am an internal medicine specialist, and I'm one of the consultants in the internal medicine and oncology team here at IDEX. So the consulting team here is, is here to help you, get the most out of your lab experience, that can be by helping you, with interpreting the results. You've received.

It can also be with just helping you guide you to what would be the best next step, when you're dealing with a case, the best next sample, how to, get those samples, all those kind of things. As mentioned already, we, we also have an oncology team. So if you have oncology results and you want to discuss those, you want to discuss if you should do immunohistochemistry or those kind of things, you can give us a call.

So, at IDEX we have a, a, a big microbiology laboratory and that processes around 200,000 samples a year, and we actually got a fair amount of calls discussing microbiology results. I think partly this is due because we actually provide a lot of information on our . Microbiology results and, and that can sometimes be a bit confusing.

And we are here to help you with that. But I'm hoping that today I can talk you through some of the things that can help you understand those results better, and can get you more more accurate information from your results. So what I'm hoping to discuss today.

Is, things to consider when you're submitting samples for culture. It's really important to realise that culture does not start in a laboratory, but actually starts at the time of collection. So how you collect it, how you store it, and how you send it to us, will, influence the accuracy of the results.

I will talk through how cultures are actually performed in the lab, so which procedures are, are, are performed and how that ultimately results in an MIC and what an MIC actually is. And then we will talk about how those MIC results can be interpreted and how they can help you in dealing with your patient. So, in this day and age, antimicrobial resistance is one of the biggest threats to global health and food security.

Responsible antimicrobial use, in addition to infection control and hygiene measures, form part of ongoing strategy to minimise the emergence of resistant organisms. Antimicrobial stewardship is important and, and means that veterinarians individually and as a profession as a whole need to take action to preserve the effectiveness and availability of antimicrobial drugs, and we can do that by, making responsible medical decisions. Antimicrobials should be used sensibly, and things like antimicrobial therapy for uncomplicated viral infections or non-septic inflammatory conditions such as pancreatitis.

We really should avoid. SAMSOC, the Small Animal Medical Society here in the UK and the BSAVA have worked together to design the Protect me guidelines. Don't know if any of you've heard of it, but if you haven't, these provide tools for implementing protocols.

Responsible antibiotic use in the practise, and the aim is to protect the antibiotic efficiency, not only for our patients but also for minimising resistance, for pathogens that affect humans as well. The Protect me guidelines, etc. Can be found online and I really encourage you to have a look at it.

Performing cultures is an important tool in the sensible use of antimicrobials. It helps us to establish whether there is an infection present, and if so, it will help us to identify the organism that is causing the infection. Knowing the organism can then aid us in choosing the appropriate antimicrobial, and so we wanna make sure that we choose the right drug for the right bug.

Culture can also be helpful when we're monitoring the effect of treatment. So we can recheck a culture during treatment, or after treatment to make sure that we have, gotten the appropriate effect. Culture is especially important when it has taken some effort to obtain the sample and when there was possible risk to the patient when we took the sample.

So this is a consideration when we're taking CSF for example, or BAL or biopsy and those kind of things. When you're doing a procedure like that, please consider beforehand what you're collecting the samples for, so that, you can, handle the sample appropriately. So, for example, when you're taking a biopsy, consider if it's likely that it would be good to do a fresh tissue culture on that biopsy and if so, make sure that we save that a part of that tissue as fresh tissue before you place it in formalin.

If there are life-threatening infections like pyothorax or peritonitis, it should always be considered to culture. Clearly, we will start empirical treatment as soon as we have taken the sample, because, you know, we need to treat these patients, but The culture can then help to make sure that we are indeed using the right antibiotic or make us change that antibiotic before we get to a stage where we don't have that opportunity anymore because the patient did not do very well. Also, when we have situations where we expect that we have to use antibiotics longer term, so in a pyelonephritis, for example, it is really strongly recommended to do a culture to make sure that we're treating with the right antibiotic.

We see a lot of cases where we have recurring or persistent skin infections, and here we want to do culture especially to look for emerging resistance because we know that MRSP and those kind of things can make treatment very difficult. If we have recurring or persistent urinary tract infections, again, culture can be helpful first of all, diagnostically to establish that there truly is a urinary tract infection and that we're not just dealing with something like feline lower urinary tract disease, and it can help us therapeutically to decide what would be the most appropriate treatment. If we have wounds, draining tracts or subcutaneous lesions that, either, didn't respond or initially responded, but then relapsed again, culture should be considered.

If first line treatment fails for any condition, try to avoid using just another antibiotic to see if that will work, because this can lead to us just using a whole lot of antibiotics without necessarily picking the right antibiotic, and this is a prime reason for bacteria developing multi resistance. And then the other thing to consider is when you have any suspected hospital acquired infections. Those need to be cultured.

That is also important for your sort of hospital environment to know that these are hospital acquired infections and what their resistance pattern is, but lots of these nosocomal bacterial infections are multi resistant and not necessarily easy to treat with empirical antibiotics. The best time for sampling is of course as early in the disease as possible, but that is not always feasible, but ultimately, yes, if we can do this at the start of a disease, we will get the most accurate results. And size matters for your sampling.

So the bigger the sample is that we have often the better results we get. So a biopsy would be better than a superficial swab, fluid is better than just a swab, those kind of things. One of the things to keep in mind is that as disease progresses, necrosis of tissue can occur and microorganisms may actually die in, in that process.

So when we then take samples of the necrosis, we're getting no growth. In addition, it might actually be in an environment where other bacteria like to flourish, so we might actually get a result that shows bacteria that are not the primary cause for the disease to begin with. There's also a substantial risk for false negative cultures if we take the samples while the patient is already on antibiotic treatment.

So ideally we sample prior to treatment, but if the patient is already on treatment, then consider sampling just before the next dose is due to try and have the lowest concentration of antibiotics in the sample. When we have, sam areas where we sample where antibiotics are concentrated, so for example, in the urine, we can get increased concentrations of the antibiotics, then it might actually be best to wait 48 hours after the last dose before collecting the sample to again minimise the effect of that antibiotic on your actual culture result. We also can sample after an antimicrobial course to see if we actually achieved a bacterial cure.

For those, it's generally recommended to wait at least 2 to 3 days after completion of the treatment before you would sample. And again, if you're sampling urine and you've used an antibiotic that concentrates in the urine, I would actually recommend to wait 5 to 7 days before you resample. So we want to avoid contamination with normal flora as much as possible to get again an accurate result.

This is much easier to achieve when we're sampling closed body sites. So for example, think about doing a cystosynthesis for a urine sample. When we do that, we can prepare the skin with antiseptics prior to aspiration, and we can try in that way to avoid any contamination.

Also, if we have things like an abscess or anything else that has a capsule, cystic lesions, those kind of things, we can prepare the surface aseptically. When we have more of a, a, a, a purulent process, it is recommended that you try to take the sample from the capsule or the leading edge of that, process, because again, in the really purulent material, we can have necrosis or a lot of phagocytosis and the bacteria might not be viable, so we won't get a positive culture. When we start looking at communicating body sites, so a little bit like what we're seeing here on the picture where we have fistulous tracts, we want to try and avoid contamination with the bacteria that live here on the surface of the skin.

But still get a representative sample. So we don't want to get too much of the antiseptic into the actual area where we're going to sample. So here we can carefully prepare the surface around the, the wound, avoiding getting any anti within that communicating body site and then we want to sample from the deepest portion of that tract if possible.

Again, trying to avoid the sites here that are on the surface so that again we don't get that normal flora on our swab. If we're dealing with superficial body sites, decontamination should not happen. So don't scrub the skin before you take a skin swab.

Don't clean the eye before you take an eye swab, those kind of things. What you can do with the skin is, you can clip the hairs, you can debride the area, so, I mean, if there is a lot of crusts on there, etc. You can make it a little bit easier to get your sample from the area.

Other things to consider with the skin is that pustules are really the preferred lesion for specimen collection. So, try to look for these. So a thorough search for the pustules is recommended.

You can clip the hair to facilitate the examination of the skin. Even using magnification can sometimes help. If you can aspirate the pustule, if it's big enough for an actual fine needle, then that would be great.

Otherwise, we take a swab. If there is no evidence of pustules on the skin, then, your other option is to take the specimens from either beneath crusts, especially if there's a, a little bit of moisture there, that would be a good area. We can also take it from epidermal colorets or from papules that are there as long as these lesions reflect the representative lesions.

If we take swabs of crusts and epidermal choras, there is a higher risk of contamination with the commensal skin surface bacteria. So this needs to be taken into consideration when you get the results. If there is evidence of deep pyoderma, then histology and fresh tissue culture with aerobic and anaerobic culture might be the best option to get an accurate result.

As I already alluded to, specimen management is the one process in clinical microbiology that has the most influence on accurate laboratory results. So specimens that are improperly collected or improperly transported or stored will very likely provide information that is misleading and it could result in inaccurate diagnosis or inappropriate treatment. Accuracy of results is significantly improved if samples are received and processed at the laboratory within 24 hours of collection.

For a culture to show growth, the organism still needs to be viable when it arrives at the laboratory. Tissue that is submitted in formalin or samples submitted on dry sobs are unsuitable for bacterial culture because it will usually lead to the bacteria being dead before they actually arrive. Inappropriate storage, so, keeping them in too hot an environment, .

That is pretty much anything that is outside of a fridge, or too cold. So when you start freezing the sample, this will affect the viability of the bacteria, and prolonged storage will also reduce the viability of bacteria. So try to avoid those things to get accurate results.

We have different things that you can submit your your samples in. So charcoal swabs and E swabs are the things that are most commonly now in practises. Both of these contain transport media for the bacteria and this is to preserve the viability of the organism, while minimising overgrowth by rapid growing bacteria or, or contaminants.

It provides a, a more stable environment for the bacteria that we're interested in. For samples that are collected from normally sterile body sites where we really don't expect a very high yield of bacteria and these are things like CSF, synovial fluid, blood cultures, there it is recommended to actually use an enriched transport medium such as a blood culture bottle. So the main difference there is that these blood culture bottles actually contain nutrients that help to nourish those bacteria and actually can help growth of those bacteria before they reach the lab, to increase the chance that we find them.

Because it provides nutrients, clearly any contaminant will flourish in these things, so it's really important, especially when you're taking those samples that any contamination is avoided. So the charcoal swab as seen here, is also called the M40 swab. It's very good at capturing bacteria in the filaments of of the tip of the swab.

But the disadvantage of that, that is so good at capturing them that it's sometimes more slow at releasing these bacteria into the culture medium and so it reduces sensitivity. On the other hand, if we're looking at the E swab, this is what most human hospitals have switched to now and what we're starting to use more in veterinary medicine. The E swab is also called the FLO swab, and if you've seen these in your practise, the main differences are, first of all, that the, the medium is not dark but it's a clear fluid and that the tip of the swab, is more of a sort of plasticy comb kind of thing.

And it captures bacteria in that area, but then as soon as you place it into this liquid medium, the swab releases those bacteria into that medium, and then we use the liquid to plate it up in the laboratory. Studies have shown that if you look at the bacteria that go into the swab and then are in the medium, if we use the E swab, 92% of those bacteria were released into the medium, whereas with the charcoal swab, it released only 30% of the bacteria and so again, that could reduce the sensitivity of that test. The other advantage that the E swab has is that we can actually use that sample for PCR testing as well.

Whereas if you use a charcoal swab, you also have to send a dry swab in addition to the charcoal swab, if you want to do culture and PCR. So, overall, we recommend using the E swab at the moment if you have it. Another common discussion point is whether boric acid tubes should be used for urine evaluation.

So first of all, cystocentesis is the recommended collection method for urine culture to avoid contamination, but clearly that is not always feasible. If we don't collect it by cystocentesis, contamination is more likely, and what we have to realise is that urine can be quite a good culture medium or growth medium for some of these bacteria. So if a contaminated urine sample is placed in a plane tube and left outside of the fridge, significant growth of the contaminants can be seen.

Boric acid is a preservative for urine samples. So the brate ion that is in there has bacteriostatic properties. And the addition of boric acid to urine, results again similarly to the swabs in a stable environment for the bacteria that we're interested in, while limiting a contaminant overgrowth of the urine samples.

In veterinary literature, there are only a few studies looking at the effects of boric acid on versus plane samples on culture results. So a study by Dr. Roland and published in the Journal of Small Animal practise showed that submitting urine samples obtained by a cystocentesis in boric acid could result in some false negative culture results, thereby indicating that maybe the bacteriostatic property of a boric acid might reduce the sensitivity.

On the other hand, a study by Dr. Patterson showed that prolonged storage at room temperature of urine in a plane tube and with prolonged storage, we're talking about 5 to 24 hours, so it's not that long. But if we did that, in a plane tube, then that increased the risk for false negative results.

So the bacterial load reduced significantly because those bacteria became non-viable. Whereas if we stored it under the same conditions in boric acid, that was not seen and we still got a positive result on those on those samples. At IDEX, we actually don't receive a lot of cystocentesis samples, so the vast majority of the samples is collected either as a free catch or a catheter sample.

So we performed a study looking at urine samples that were not obtained by a cystocentesis, and for which we received both a plain and a boric acid, sample to compare them and these results were presented at ECVAM and they showed that if we use the plain urine. We had an increased risk of false positive results, so much more growth of contaminants than if we used the boric acid. Also, looking at the samples in the boric acid, we actually still got a good sensitivity of 93%, so only a few false negatives.

We had in the boric acid tubes. So overall, looking at all that information, our current recommendation would be that if you take a cystocentesis sample, you can send it in a plane tube as long as you store it in the fridge after collection and you try to get it to the lab within 24 hours of collection. For any samples that are collected, free catch, any samples where you know that the storage will be a lot longer than 24 hours, so anything that is sampled on the weekend or something like that, and anything where you're, you're sure or where you're not sure that it's gonna be stored appropriately, then the stabilisation of the boric acid is really helpful and we would recommend that you submit that in a boric acid.

And if you want to be on the safe side, you can always submit it in both. So boric acid and a plane tube to cover all the bases. When you're submitting tissue, we want to make sure that those bacteria again, continue to be viable but are not, diluted or anything like that.

So for the bacteria to, stay viable, they need to be in a moist environment but not swimming. So that means that if you have a big enough sample, you can just place it in a sterile. Container and within the sample, the moisture will usually be capped.

But if you have a sample that is very small, you can place that either in a, in a pre-moist gauze, so you can make a a little bit of sterile saline on the gauze or something like that and wrap it in there and then put it in a sterile container or you can add 2 or 3 drops of saline to the sample in the container. Again, try to avoid submitting it in a whole lot of fluid because it just will dilute the sample and it will reduce the sensitivity of the culture. So once the samples arrive at the lab, this is what we do with it.

So first of all, all these samples are plated on a variety of media. This can be done manually as we can see here in the pictures on the left, where we just plate it out. I don't know, he might have done this in vet school, where you have to streak it out over the plate.

But nowadays in our microbiology lab, we have some optimisation that helps. So this is our, machine called the WASP. Pretty much all the machine in microbiology have like a name.

So the WASP is a robotic streaking platform. So basically, it does all the work for you, or for us. Case, and it streaks out, all, all the samples on the right plates.

The great advantage of this is that you get a real consistency of plates, because we know in the lab that if you work 8 hours, how you streak a plate at the 8th hour versus how you treated at the first hour is gonna be different, whereas the machine does not suffer from this. It also reduces risks of errors, especially in the numbering and, and, locating of the samples, and it frees up our microbiology staff to do the more important interpretive work. So it really helps us to use people more appropriately.

It is important to provide the microbiology lab with some information because based on that information, decisions will be made on what is the most appropriate way to deal with the sample. So yes, please mention the site of collection. Based on that, site of collection, we decide, OK, these are the most likely microorganisms that we're gonna be looking for.

These are the specific organisms that we want to be looking for and we select the plates that are appropriate for this. For example, if you're sending us like a Ear culture, we will always include a plate that is specific for pseudomonas. So that plate will suppress any other organisms and will just support the growth of of pseudomonas because we don't want to miss a pseudomonas infection in the ear.

Whereas for other areas where we're expecting a low amount of growth or not a lot of different bacteria, we actually use media that support growth of all the organisms and don't limit specific growth. So we, for most samples, there will be multiple different plates and we can. Bin the results of all those different plates.

But yes, if you can provide us with the site and, and even provide us with this animal is on antibiotics or we've collected this through cystocentesis, all those things help us in making sure that we do the right culture, the right way of the culture, and that we give you the best interpretation of those results. Once we've plated everything, the plates are incubated at different temperatures and in different environments. So clearly anaerobic environment is different from an aerobic environment.

And again, this is depending on the plate and the specific organism that we're looking for. Generally, the incubation is 24 hours and at 24 hours, all the plates are being looked at and for swabs, we keep them another 24 hours and do the final reading at 48 hours. Following the incubation, we are then reading the plates, so we're, assessing the growth characteristics on the different media that were being used.

If there is a mixed growth or contamination of the plates, then these are reviewed by a microbiologist or a pathologist to determine which organisms are of possible significance. Individual bacterial colonies are then picked for identification. And this is performed by another big and very gla machine.

So this is called the Mittov, this big blue machine that we have here. So I'm gonna say this only once. This Movitov stands for Matrix Assisted laser desorption, ionisation, time of flight.

Ultimately, what it does is it uses ionisation by a laser beam and it ultimately generates a graph which is called the spectrum, which is specific for each bacterium. So the spectrum is what we create here. So it has a very unique, sort of fingerprint for each bacteria.

So this spectrum is then compared to a database, and that leads to the bacterial identification. The introduction of having the Maly has significantly increased the ability to identify the bacteria to species level. This means that we can now identify bacteria that were previously reported as unknown.

So we used to have reports that said gramme negative rod, unable to speciate, but that, is, is a thing of the past. It also means that you might see a report with the name of a bacteria you've never heard of. This can be a new organism or it can be a renamed organism.

Just give us a call when you get a result like that and we can help you figure it out. Most of the time, we have to look it up as well, but at least we have the tools to look up some information about it and help you figure out if this is something that is of clinical significance for your patient. The multi can determine in minutes which bacteria we are dealing with.

So the turnaround time is a lot faster and it's very accurate. So once we have a result, what do we do with this? It's really important to realise that finding bacteria in a sample does not necessarily mean that this bacteria is the cause of disease.

So once you receive the results of a culture, your job really starts because these results should then be interpreted in light of the, the signs of your patient and the lesions. So things to consider are, do the results fit with what you were expecting? Do cytology, histology, and other findings support that there is a bacterial infection?

Could the result reflect the normal flora, which is especially important when we're considering results of skin cultures and other non-sterile areas? Or could this be a contaminant, especially when this was a free catch urine sample? If several different bacteria are isolated, this could indicate contamination or normal flora, whereas if a single organism was isolated, so when we have pure growth, and this was isolated in high numbers, so more than 105 or 100,000, this is more likely to be of significance.

So all those kind of things help us to decide what to do next. Cytology can be really helpful in assessing if an infection is present, and it's an inexpensive thing to do. It could be a first step before considering submitting a sample for culture, especially when you're dealing with ears, for example.

For urine, quiet sediment does not exclude a urinary tract infection and a culture could be considered if there are other indications. But if you have an active sediment, it should prompt you to submit a urine culture. B bacteria can be observed in a smear from a specimen when, when there is a concentration of the bacteria of around 10 to 4th or 105 to the 5th per mil.

So if we're looking at this, slide here, not sure how good everyone can see this, but there are some intracellular bacteria here. So from this cytology, we, we can deduct that there seems to be an infection. However, because these organisms are intracellular, there is the possibility that if you culture this, that the result will be negative because again, these intracellular or phagocytosis bacteria will not be viable and so will not grow on a culture medium.

So if we had not done. We could have missed that infection. And again, this is one of those things that we see actually with ear infections quite regularly when there's very purulent discharge and there's lots of neutrophils, we might see a negative culture just because the neutrophils have done their job and have deactivated or are made bacteria non-viable by the time that it reached the laboratory.

So, overall, I think that cytology is a very useful additional tool, when you're looking for infection and those kind of things. So then once we know what bacteria we're dealing with, we can start looking at the susceptibility and which antimicrobials would be done. So historically, in vitro susceptibility testing was performed by disc diffusion, which is what we're seeing on the left side, the Kirby Bauer plates.

For this, the suspension of bacteria is plated on an ager, and then we add antibiotic impregnated discs. The size of the growth rezone around those discs after incubation determines whether the bacteria is considered to be susceptible, resistant, or intermediate to that antibiotic. Nowadays, IDEX uses mostly automated susceptibility testing with another machine called the Vitek.

So this is based on On the principle of tube dilution, bacteria are inoculated into wells on, on the card that is shown here, so these cards go into the machine. And each well contains a different concentration of the antibiotic that the organism is being tested against. The machine then incubates the sample and measures the turbidity in each chamber.

So if there is no growth, then there will be no turbidity in that, in that chamber. If there is turbidity, then clearly that antibiotic concentration did not stop the growth of the bacteria. The benefits of tube dilution is that it's a quantitative test, so it reports the lowest concentration of an antibiotic in microgram per mL, at which growth is inhibited.

It's an automated test, so it's rapid and accurate, and it's available for many but not all gramme negative and gram-positive organisms. It cannot be used for anaerobic organisms and for some slow growing organisms, it doesn't work either. So for slow growing organisms, we would still use the Kirby Bauer plate and to try and get a sensitivity result.

So antibiotics are really chosen on the, after the identification of the organism. For each individual organism, there are guidelines from the clinical and Laboratory Standards Institute that tell us what these organisms are, Likely to be susceptible to. We know that certain antibiotics do not work for gramme positives, whereas others do not work for gramme negatives and so those are automatically included or excluded depending on the organism.

In addition, once we have identification, certain bacteria are known to have intrinsic resistance to certain antimicrobials and therefore, there's no need to include testing for these. So, for example, enterococcus is intrinsically resistant to cephalosporins and TMPS. So you will never see those on the report for enterococcus because we don't test for it.

So for some bacteria, we have a wider spectrum that we can test for, whereas others are more limited. The most limited spectrum is for pseudomonas. Pseudomonas is resistant to all simple penicillins to 1st and 2nd generation cephalosporins, and to TMPS.

Therefore, the antibiotic choices for pseudomonas are really limited to the ones that we are testing, which is fluoroquinolones, the aminoglycosides, and polymyin B. We get very frequent questions about this, because it's quite frustrating. Amicain, gentamicin clearly are not topical medications.

So for an ear infection, you know, these are not, sorry, Amicain cannot be used. We actually do have a, a topical product for gentamicin. And yes, we have, have limited options, and There are no other options.

This is it. This is actually a good one. We have a fair, fairly, good choice of options here, but Pseudomonas becomes resistant very quickly, so a pseudomonas infection needs to be treated quickly, and, efficiently, and needs to be rechecked because, the failure of treatment could, could be due to developing resistance.

So if you get a result like this, where we just have the antibiotic NAID sensitive or resistant, it indicates that we have used the curvy bower plate instead of the Vitek. And again, this is sometimes done because the organism is slow growing or for some other reason doesn't want to grow in the Vitek machine. For most Oh yeah.

So, for for these results, if we're looking at what susceptible actually means, generally speaking, susceptible infections respond to, appropriate therapy approximately 90% of the time. Whereas if we're looking at resistant infections, they respond to, to appropriate antibiotics in less than 60% of the time. Of course, patient-specific situations and pharmacokinetic factors will play a role in that as well.

But if we have an organism that is susceptible, there is a high likelihood of therapeutic success if we're using a standard dosing of that antibiotic. When we have an organism that is intermediate, the microorganism could be susceptible to that antibiotic if we lead to increased exposure. We can do increased exposure, for example, by increasing the dose or by increasing the dosing interval.

So instead of giving it once a day, we could give the medication twice a day. We could give it as an intravenous infusion instead of giving it. All those kind of things could play a role, or we could look at what the excretion pattern is, make sure that, you know, we want an excretion pattern that leads to a high concentration in that area.

So for example, when we're talking about urine tract urinary tract infections, we can, we can use things that would show up in the urine more than elsewhere. Also, when we're using topical medication, we know that we will far exceed what we would commonly achieve based on the serum levels and so again, topical medication will lead to increased exposure and might still be very effective for intermediate organisms. When an organism is resistant, there is a high likelihood of therapeutic failure, even when there is an increased exposure.

So generally, we avoid using those resistant patterns. So if we're getting a result like this, where we not only provide you with the actual interpretation of the results, so whether this is intermediate resistant or sensitive, but also provide you with a numerical result, so, true MIC then this will be a result that has been created by the Vitech. So, again, we, we show here which antibiotic we've tested.

We show here what the interpretation of the result is. We show you what the actual MIC result was and then this last column, which is probably the column that leads to most confusion, supposed to help you by showing you what range we have tested against. So for each antibiotic and each organism, there will be a different sense.

Range. And so, for example, for ampicillin, we are testing a concentration of antibiotics from 2 to 32, whereas for something like doxycycline, we test, a range from 0.5 to 16 in concentration of the antibiotic.

So there is a difference there. And then the middle column is actually a visualisation of what we're doing. It's it's corresponding to the wells that we use here on the Vitech plate and it shows you that if there was no growth in the first well, then this would have been a sensitive result, but the growth stopped in the 4th well, it's an intermediate result.

We'll look at this in, in different ways just to to see if we can get that concept a little bit clearer. But it, I know that it leads to a lot of confusion. So the minimum inhibitory concentration reflects the lowest concentration of an antibiotic that inhibits the growth of the bacteria.

Once we know what this lowest concentration is, we then interpret that in light of the internationally agreed breakpoints for susceptibility and resistance. These break points are established by the CLCLSI, which is a global institute that studies the break points for the bacteria for the different antibiotics that are available, and Ucast is a European. That does the same.

So these, provide all this information. All the different labs use the same information, well, either the information from the COSI or Ucast to determine if a bacteria is sensitive or resistant. The goal of in vitro antimicrobial susceptibility testing is to accurately predict in vivo therapeutic efficacy, but of course, it doesn't always completely correspond.

We have a limit of being able to perform this only on aerobic bacteria, so we can't do this for, for anaerobic bacteria. And we also have to realise that an MIC is based on the serum concentrations that can be achieved, and therefore is less useful when we're starting to, consider if topical treatment would be useful. So this is a visualisation of the Vitek plate and it shows you that on the Vitec plates, we use these different concentrations of the antibiotic in twofold .

Dilution. So we start with a concentration of 2 that goes to 48, 16, and 32. For each antibiotic, these concentrations are slightly different.

Again, the Vitech will then look at whether there is growth or no growth in each of these concentrations. And the break points that have been provided by CLSI, then give us an interpretation of these results. So if the growth stopped in the first plate, in the first well, we would call it sensitive.

However, if it stopped in the 4th well, it would be intermediate and otherwise on 32, it would be resistant. So again, this is how the wells are being read. So the machine sees that there's turbidity in all these wells, even at the highest concentration, and so the machine will say, OK, the result is either equal to or greater than this highest concentration which is 32, and that will then be interpreted as a resistant, result.

Whereas here for gentamicin, where we have measured from a concentration of 1 to a concentration of 16, and there is already no growth in the first well, so the result will be less than or equal to 1, because at the concentration of 1 growth is already inhibited. And so that will be the MIC result. In our sensitivity range, we show you that we have measured from a concentration of 1 to a concentration of 16, and knowing that each one of these wells is twofold, we know that the first well would have been concentration of 1.

The second is a concentration of 2, then 4, then 8, then 16. The capital letter indicates where there was no growth anymore. So the capital letter here indicates that this was no growth in the first well and therefore it's a sensitive result.

When we start looking at the results of the MICs as already mentioned, we can't compare the numbers of the MICs of different antibiotics to each other, but what we can compare is how far the actual MIC is from the breakpoint. So an antibiotic breakpoint again is the dilution where bacteria begin to show resistance. And if we're looking at antibiotic A, The MIC for this bacterium is 4 dilutions away from the breakpoint or the R, whereas for antibiotic B, the MIC is only 2 dilutions away.

And therefore, if we're just looking at the MICs, this bacterium is more susceptible to antibiotic A. But how do we select which antibiotic to use? This is not only something that we base on the MICs because again, it's just an a way to predict in vivo therapeutic effects, but we need to take other things in, in consideration.

We need to take the site of infection into consideration and the pharmacodynamics, pharmacokinetics. So how is the medication absorbed and distributed? Is it actually distributed to the site of infection?

How is it excreted? Medications might not be able to get to the site of infection. So, we, we need to consider that and concentrations can be either higher in urine, so it can be quite beneficial, to, use even an intermediate antibiotic, for urine infections, whereas CSF, might be difficult to reach for some antibiotics.

We also have differences in whether an antibiotic is time dependent or, or concentration dependent and of course, we need to take into consideration age because certain antibiotics should not be given in a young age. If the immune function is reduced, then we might need longer courses of antibiotics, and then again, biofilm could significantly reduce the effectiveness of antibiotics and so we would need additional medications to treat the biofilm situation. So the protect guidelines help us in using the right antibiotics.

If antimicrobial treatment is indicated, we should really use first line antimicrobials first, such as amoxicillin, amoxicla, cephalexin, clindamycin, etc. Fluoroquinolones and 3rd and 4th generation cephalosporins should only be used if first-line antimicrobials are inappropriate or ineffective. And then there are a whole list of antibiotics that are restricted in human medicine and we in veterinary medicines should really try to not use these.

We generally do not show these on our sensitivity profiles, so, they shouldn't really appear in our results, but Try to avoid those as as best you can. And this is just a poster that you can get from the BSAVA to help you provide some protocols for your practise. So in summary, antimicrobial resistance is one of the greatest challenges of our generation, and performing cultures is an important tool to use antibiotics appropriately.

How you obtain and handle the sample for culture can have an impact on the results, so keep this in mind. But the culture can help to determine if bacterial infection is present, and if so, what organisms are involved and what antimicrobials are appropriate. It also can help you to reassess if your treatment has actually worked.

If your treatment is not working clinically, then go back through the drawing board sooner rather than later. Try to avoid just dispensing more and more antibiotics in the hope that over time it will work. But assess if maybe we're using the wrong antibiotic, maybe an antibiotic is not needed, and those kind of things.

Again, if you want more information about the Protect me guidelines, you can go to the BSAVA website. I hope that you found some or all of the information in this lecture. Helpful.

If there are any questions, again, feel free to call the internal medicine service, and I hope you have a good rest of the day. Thank you very much. Take care.

Bye-bye.