[MUSIC] Hello everyone, my name is Lina Cavaco and I'm going present you another lecture on this module of antimicrobial susceptibility testing. I have introduced in the previous lecture to the methods in general to what you can do to test the susceptibility of antimicrobials. And this one is again a general presentation where we are going to present the methods in a general group. And it is a presentation intended for the persons that need to choose a method to do some comparisons between them, as we are going then to have more detailed presentations later on with the details inside the procedures for each method. So this one will be to have a general scene on this. So in general for this presentation the outline would be to talk a little bit about the standardization and the methods that are available for testing susceptibility and comparing qualitative methods, and quantitative methods. Again we will brush up again on the definition on the MIC. So that we make sure that we are talking about the right thing and then about the general methods. The disk diffusion, the e-test and the agar dilution. Which will be quizzed on the next presentation. And I'm sorry, also the Broth micro dilution as well, of course as part of the dilution methods. And at last we will make some comparisons between them to see if you have one project or one task. Which methods would you choose? And then you have to take into account several factors. So in general as I told probably a few times before in these lectures. Standardization of methods is quite important in testing for antimicrobial susceptibility. If you want to have results we might have them in quantitative or qualitative terms, but they should be reliable in any case. The qualitative results. That would indicate how it is expected that these drug were work in invivo. So in the patient or in the animal that's going to be treated. We would predict what is expected to happen, and with these qualitative results, which we also can obtain from the quantitative, with the qualitative, we have an idea if it's resistant, if it's intermediate, or susceptible. When we actually have quantitative results, which means that we have an actual concentration an actual MIC. We have something that is really known everywhere in the world. This can be compared. It can be defined. And people around the world know what we are talking about. And again, it is a quantitative. So we have in the scale we know where we are in terms of how resistant. If it's in higher resistance or a lower resistance. But it's not just a classification as the qualitative. Of course, when we have the quantitative results, we can also classify it as qualitative. But then we are reducing our quantitative result to a class. In testing this, the golden number to obtain from this is this concentration, the MIC. And that concentration as I mentioned probably before. Is the lowest antimicrobial concentration that is able to inhibit or kill the microorganism to kill the bacteria. And it will be defined by the lowest one that does that. So this defines the range and the growth rate of the bacteria towards this antimicrobial, showing the effect of this drug. It is definitely the most basic measurement of the activity of an antimicrobial, and many other measurements can be done as well. But it's a measurement both for the pharmaceutical industry, but also for the people testing in the lab for a clinical lab, for example. So well looking at the methods. First I'm starting with the diffusion method, so qualitative method. The agar disk or the tablet diffusion are based on diffusion. They are made on solid agar so on plates. And they are made on a plate that has a lot of bacteria that are spread. On the plate, we are going to describe the procedure ahead. And these antimicrobials, the drug that we are testing is in a certain amount and there are also disk and tablets with different amount. They are in a disk or a tablet. And these disks or tablet can be put on plate and from there it will be diffusing into the agar. The way that we need to prepare for this is quite standard. You need to standardize quite well the medium that you use and the way that you prepare the plates. And the disks or the tablets, you just buy them. It's relatively easy and you need to keep them right, but that's not so difficult. The big advantages for this, it allows you to make many tests because it's quite fast, and it's a very cheap method. These advantages is that you need to standardize quite a lot. But when you see the results and when you read them, you get this inhibition zone diameters that I have shown you before and I will show in the next lectures as well, where you reinterpret the zones and classify the organism as resistant, susceptible or intermediate. So you have a qualitative result. In summary you have your plate with the agar. The agar is quite standardized and need to have the right depth. And you put on your disk that has the antimicrobial drug. When the plate is incubating, this drug will be diffusing, and the bacteria that you put on the plate spread out through out will be growing. So the next day when you incubate this plate, you will see an inhibition zone. Which you can see as a clearing zone around the disk. And that's what you measure. When you measure this you measure the diameter of the zone. And you obtain this number. This millimeter zone. Which can be looked up on table for the results. So when you get these kind of results it should give you a reliable and the producible result if you are doing it very right and standardized and if you're using the standards correctly and it is a qualitative result. If you want to be even more sure, you should have quality assurance systems around working. You should use the quality control strains and even more you can test for your results together with other laboratories doing this kind of ring tests. Where a same bacterial strains are sent around to different labs, and then you compare results to the acceptable results. And again, it is not enough to stretch that you should check with reference strains and quality controls. And if anything happens with quality controls, then you should check that, if anything is wrong and correct it. But in general, it is of course a reliable method, if you are doing it totally correct. But there are several things that are not so easy to standardize. So here again, you obtain these qualitative results. Normally it's this qualitative results that results from the millimeter zone. There are some exceptions to this, and it is if you have a system to do some regression analysis. To correspond this millimeter zone to an MIC. So as I mean quantitative results could be extrapolated if you do a linear regression or use a specific software otherwise it is still a correlated result. Also you need to check what kind of antimicrobial you have and which kind of bacteria you have because it could be slightly different if you have bacteria that are growing fast or growing slow. So this needs to be very standardized for making these regressions but it is a possibility. Another method which is also based on diffusion but it's not only a diffusion test is this E-test. And this E-test is different from the tablet or disk test in the way that it's not just a circular disk or tablet. It is a plastic strip that contains a gradient of the antimicrobial. And it contains in a way that the top part contains the highest concentration and the bottom part contains the lowest. Actually some strips are even double. They have two gradients in the same strip. They are divided but the simple test would be like this and when you do the test and have incubated, you get this growth around with an ellipse of clearing around the strip. And this is made in a very smart way that you can actually read the quantity MIC, because you have the gradient and this strip has the concentrations and the place where it intercepts with the ellipse is where you actually can read numerically the MIC. In this case it would be a 12. It has a number of dilutions. Actually it has more dilutions than you would test on a plate because it also has some intermediate dilutions. It is though not a standard method. And it is a quite expensive method, I must say. Another method that we have now in the dilution part, in the dilution methods. Also where you obtain an MIC is this agar dilution. The agar dilution, the principle is that you have two full dilutions of the antimicrobial on the agar, so the solid agar is containing the antimicrobial. You prepare some suspension of your bacteria which is standardized in a certain way with the McFarland standards. You dilute it, and then you spot it in a replicate on these plates, so that the same quantity of bacteria spotted on each plate. And you will be looking for growth or no growth on these agar dilution plates to see which concentration is your MIC in the end. As agar dilution runs, you do it as I said in the solid plates. And it has. This part that is preparing these plates, preparing the solid medium with the antimicrobial. Which I must say, is the hardest part. Because it's quite cumbersome, quite a lot of work. But it is a good standard method. And even though you prepare many plates, you can test a lot of isolates. So it is a good method if you have a project where you quickly have to go and test many isolates in the surgeon range. So it is a good method for that case. The microbroth dilution is not very different from the agar dilution. The main difference is a of course that it's a broth, it is a liquid medium. It can be in this case it's a microtiter plate with small wells, but it could also be in larger tubes. So where it is a microbroth dilution in that case, it also starts from the colonies, the culture. The culture will be standardized making this McFarland suspension and then this is diluted on the medium that is going on the plate. So the liquid medium which is normally a Mueller Hinton broth that is very specifically adjusted for the testing. And then in here you expect to have a certain amount of bacteria that are going to be inoculated on the plate either with the machine or with. And then you have on these plates the antimicrobial normally comes already if you buy these plates commercially. Then you have the antimicrobial already in the bottom of the plate as a solid there. And it will be the when you add the inoculum, so that the concentrations are right when you put in the right volume of inoculum. So from these kind of tests you will always have also to have some controls, to make it purity, to make the incubation as it should. And to cover these plates. But when you get the results you will read the results looking at the growth or no growth. And we have the next presentation with very specifically looking at that. And you get an actual concentration, you get an actual MIC. And these MICs can be used as themselves, as the concentration itself. But it can also be classified qualitatively. Using tables and interpretations. These tables could have the RIS classification. Resistant intermediate susceptible. Or it could be these epidemiological cut-offs which I will explain a bit more later in the lecture about interpretation. But in that case, you will always get some classification out of your MICs afterwards as well. So to finalize in this presentation, we have told about MIC determinations. And here, you can include both agar dilution, and broth dilution. Either micro or macro. And these are very standard methods. They are the golden standard for this kind of testing. The E-test is a little bit outside of the standards, but it also gives you an MIC determination. And you have the advantages that you get through doing an MIC. If you are doing this with commercial plates or with these concentration, these data are quite reproducible. So you get quite reliable data doing an easy test. The separation between resistant and susceptible are quite defined because you have the actual concentration. So it's not just qualitative. And you get much more information as the concentration itself is much more information. Of course the disadvantages of this is that you should beware that sometimes it is a little bit more expensive. Because especially if you have broth cultures, it might be more difficult to detect some contaminations. And actually an advantage to this is some of these methods are very automated. So you can both inoculate and read with machines. But of course you can still do it manually as well. The diffusion techniques, they were the first being invented and the first ones used. They are still used quite a lot around the world. And they are mainly used because they are cheap and they are fast. So they are quite cheap and fast and they work with material that you have in the lab, you don't need to buy much more extra, you need this disks or tablets. You have solid media, so contaminations can be seen and sometimes while some laboratories can do these quick screenings which are not very standard but still it give you qualitative information, it does not give you the concentration as such. And if something is wrong in the standardization, and standardization is a little more difficult in these methods, it's not as reliable, but if you are doing all well then standardization is still a good method. So it depends on the project you have, how many strains, if it's a research or if it is a clinical. This decision has to be taken by who has the right to decide in the laboratory which method to use. And actually many of the labs still use all the three methods, depending on what the intentions are. But thank you very much, and I hope you are liking the course. Bye. [MUSIC]
