[MUSIC] Hello everyone. My name is Lina Cavaco and this lecture is focused on antimicrobial susceptibility testing with disk diffusion. So if you haven't heard about the lectures before, we have defined the methods, we have introduced them. And let's imagine you have chosen and you are doing disc diffusion, so this lecture is for you when you are decided and you want to pay attention to what you should do and what is actually happening in the test. So in the outline of this lecture, we will introduce to disc diffusion to remember the concepts and the diffusion and the qualitative measurements that you're actually doing. We go through the procedures and how it is going on. And what is happening in the test, and what are the influences you can expect in the results of the factors in the test. So that we also can get a little bit into the reading and interpretation of the results. And if something goes wrong, what can you do and how you can act on it. So just to start with the disk diffusion again, we have explained a little bit before. This method was invented by Kirby and Bauer, and it is basically a very basic method of putting the bacteria in contact with the drug on a plate. And looking at the diffusion property of the drug on the plate, and looking at the reaction of the bacteria to that drug. So it measures the susceptibility based on this diffusion. The result of this is a qualitative result because this diffusion is a little bit variable and is not so easy to standardize, but it should give an idea if the drug will be working on this bacteria or not in [INAUDIBLE]. So in the method itself, you have a lawn of bacteria that you want to put on your plate and you make it in a standardized way. So just to show you how you do this, it is very simple method. You have your agar plate with the Mueller-Hinton agar. Take a swab, a cotton swab and you have your suspension that has been standardized with a 0.5 McFarland density. And you will pass it from one direction, and then you'd rotate the plate a bit and you pass it again, and you rotate again and you make a third time. So that the plate is totally covered with this liquid suspension and very well distributed. Then you put on your discs that you buy from a provider and that are ready to use with a certain concentration of antimicrobials, and you take your plate into the incubator and the next day you can measure the inhibition zone diameters. So that's basis for the method. And as it is a diffusion, it depends on the diffusion of the antimicrobial and the growth of the bacterium. So that's why standardization needs to be followed strictly, because it will affect the diffusion of one and the growth of the other one. So the quality control is essential to use quality control strains to check if these diameters are in order. And we have these strains where we know exactly which diameters are fine and which diameters are not okay. One thing that we should take in consideration, if these fastidious organisms which are basically organisms, bacteria that grow very slow. So these organisms that grow very slow or need a specific media or specific supplements in the media to grow they are not very good for this test. So normally, the standards don't advise to use this for this species, so you don't really have the standards for those species. You should use the standard for the species that you are using. And the results will as I said, be qualitative so that you get the final results in a susceptible or resistant or intermediate. So as I mentioned before it is kind of a race. So you have the growth of the microorganism versus the diffusion of the drug and this is happening overnight. And the only way you can control this is by controlling the media, controlling the disk, controlling the way you put the bacteria on, and which concentration you put them on. So all of this is very important if you want to have this diameter to be right. You want to have the test results that are right and they really depend on all these standardization. If you don't have it very standardize in all the parameters, your results is not okay. If you're just looking at the diameter zones in terms of presence or absence of zone, like if you see the zone or don't see the zone. Well if you don't see the zone and the bacteria are growing up to the disc, it's most likely they are resistant but that's not enough to say, you actually need the measurement. That's what you compare in the tables. And the results are really only valid if you are standardized and if your quality control strains are in the right diameters. Otherwise, your test strains are not valid enough to give the results anywhere. So when we're using this, we need to make all the considerations of which standards do we use and again, we don't have only one standard. We have the American standards, we have the UK standards, and we actually have their own methods. So there's slight differences between what is used in one method and then the other method. And therefore, we need to read them very thoroughly CLSI you have to buy the standards and get them as a booklet or as a file. EUCAST is available online in their website. But you really need to follow the standards very, very precisely because there might be a different media supplement for the species you want. There might be a different concentration, so you need to standardize all of the steps very thoroughly for the standard that you use. Again, as I mentioned in previous lecture, it is a very good idea to have ring tests with other laboratories to compare the results, and check if your results are good in comparison to the expected results. If they are fine in relation to the acceptance criteria of the laboratory that did the ring test, and always use the reference strains for validating the method and checking that the results are in the right range. If something happens and it's outside of the range, you have to document that you did something about it, that you have acted on it and avoid it next time to happen and all of these when we have of course, visitors inspecting what we are doing, we should document everything we are doing. So in relation to the methods in more detail we will be using saline or broth or actually water in some of the standards for suspension of the bacteria. We would start always from a fresh and non-contaminated culture. It's very important to have a pure culture. And from a non-selective media, so that there's no influence of drugs there, so that you don't have any drugs in the media before. And we will standardize with all the standards, the calibration, the inoculum density and this swabbing that I just mentioned before with the three directions. And if we are using spreading, which would be by putting some liquid on and spreading with a spreader kind of tool that should also be in a very standard way. And we would always compare the results we get with the colid control string so that we have the ranges in the right order. So we have the method. Here, just to show you, we have a nephelometer, it's just one model, we can have another nephelometer function. In this case, it's a very simple one where you put in the two in this hole and where you calibrate with the standard that you get, that you buy. And if this indicator is green, the tube is fine. If it's above the green it's because you put in too much bacteria, if it's below if you have too little. So you can adjust it by just getting it in the green range, and then you do the tests from there. So now in this diffusion test, we would also look at the sum of the effects that we can have on how we do things, and here I'm going to go into what happens in the test. As I said before, it's a race between the diffusion of the drug and the growth of a bacteria. And here we want to look at what if we put too much bacteria or too little bacteria into the test? What happens? Well the normal thing is the upper range where we have this layer of bacteria and this diffusion, and we expect the diameter to be around this. So let's imagine that we have put two little bacteria. So that the drug is diffusing but the concentration of bacteria is too little, and actually the diameter looks much bigger. So this is because we have diluted too much the inoculum. And this ends up increasing the zone so when you're measuring and looking at the you have a result that seems too susceptible. If you do the opposite and when we measure the densities we did something wrong. We put too much bacteria in, just the opposite happens. They grow too much and because they are so dense they get closer to the disc. So what we see is a smaller inhibition zone than what was supposed to, which was the effect of too concentrated which means that it looks more resistant than it should. So all this has a big effect. In the next one, we just have some of the parts that could give an effect on the media disk and that could have an effect on the results. This is something to check in your labs if you're doing this, because it is a good idea to check unless you only check when you already have a problem which is not such a good idea. It's good to prevent the problems. So what can happen is that the agar itself could have an issue. If you buy the agar from a provider that is fine and that checks all it, it should be fine. But there could be problems with the ions in the agar. There could be problems with inhibitors in the agar there could also be problems with you producing the agar when you for example put too much in the plate or when you dry too much the plate or dry them to little and there's a lot of water. So there would be some things that can go wrong here. You can check this with the quality control strains and checking the media, and you can also check the disks if they are fine. So you both check the media and the disks with the quality control strains. Also something could go wrong with the disks themselves, because these are small paper disks. They can get humidity, they can degrade the antimicrobials that are around them. So you want to use the right disks first of all, when you buy them. There's disks in many contents and many amounts. Well they come in a certain micrograms per disk. So if you buy the wrong ones, because you have for example, penicillin, you have several ones, for tetracyclines you have several ones. If you buy the wrong one in relation to your standard than your results are not right ever. So make sure you buy the right disc contents, keep them well. They normally are kept at fridge temperature, and you put them at room temperature just before the use. You need to avoid that they get wet, and then there's something on the plate that you make sure. If you have a big plate, you don't put more than 12 disks on. If you have the normal sized plate that is 90 millimeter, you don't put more than 5, 6 on, 5 is the best even. And also where you put them that they are well distributed so that you can measure it afterwards. Again, similarly to the previous slide where I showed the effect of inoculum, here I would show what happens if you do the agar plate in a wrong way or if you put too much or too little in it. So the normal agar plate should have about 4 millimeters of depth of agar, and that you can measure in your lab if you have 4 millimeters. And you put on your layer of bacteria and you put in your disk, and you expect a certain measurement. This would be the incubated plate afterwards. If you have very thin plates because you want to save in your agar and don't put so much in it, and that happens sometimes when you have little money and want to make many plates. Then you risk to have too big innovation zones because the bacteria will grow. But then the drug is diffusing in such a little amount of agar that it will diffuse more in the broad sense, because it will not diffuse so much in that. So you get a false sense of sensitivity, so the strengths seem more susceptible. If you do the opposite, and you are very generous about the amount of agar you put on then you have a diffusion that goes a lot into depth, but not so much into the broad sense, and you get two little zones. So the strains seem more resistant. So all of this can influence your result besides all these chemical issues and inhibitors and so on. The next step where you are incubating your strains and incubating the test itself you need good incubators. And you don't need just an incubator standing at this temperature. You might need to check this temperature is the correct one, and have an acceptance range for the temperature. And you also want to check the time that you have the plate inside. Another thing is you might want to have a system to know how many plates you can put on top of each other because that influences how much oxygen goes around the plates or not. You also need to check the ventilation systems inside the incubator, if they are running fine or if they are drying too much in one area or the other, and so on. And temperature controls and check the validation of the incubator that's part of your quality assurance system in the laboratory that you need to make sure your equipment is fine. Again, the quality control with the control strains can show you if there is any problem, so you should use them and follow up if there is anything wrong. But let's imagine we have done the tests. We have done the tests, we have results we want to interpret. So we look for the results. Is the zone smaller, the inhibition zone diameter, smaller than the resistance breakpoint we consider it resistant. If it is in between the break points then it should be an intermediate situation. If it's larger than the table says that the susceptible stain should have, then it's the susceptible range. So maybe this one is a susceptible, and then we can classify it. We can also classify it with this if epidemiological cut-off switch is only giving us Wild type versus non-wild type. And I will show you a little bit more how these cut-offs are made, so that we are ahead in the course, we know what we are talking about here. But basically, the wild type are the normal strains susceptible and the non-wild type are the resistant ones. So it's just another classification that is just in two groups. So where the resistance would be the non-wild type and the susceptible the wild type. [SOUND] So, and when we have done this, we report it. We report it to the doctors, to the veterinarians. And we tell it's resistance susceptible or whatever it is. And well, in some exception we might do this regression but we normally don't do this. So we can report the results and they can be used. We normally report right results and that’s what we want to do, but sometimes there are mistakes. They could be mistakes due to other reasons as well not only with the methods could be because we have a mix culture from the start and we didn't notice. And it could be because all of these issues that I told you in the method. So again, and it's not too much to tell it again, it's the standardization that is really necessary. But I'm not going to go in details with that part because I have before. So in regards to this diffusion, I hope you have learned some of the methods, issues and of the particularities and thank you very much. [MUSIC]
