[MUSIC] Hello, everyone, I'm Lina Cavaco, and I'm here to present you the lecture on Antimicrobial resistance- introduction to causes and mechanisms. Which is quite related to the previous lecture, if you have seen the previous lecture we went through some mechanisms of resistance that bacteria can use to get rid of antimicrobial drugs and survive them. And here we are going to go into some examples of this. So as an outline we are going into the basis of resistance, how did it happen? And how is it really that is causes resistance? And focusing on point mutations, on the DNA changes, and on resistant genes, and some non-genetic mechanisms with examples to show you how it can work. So as a basic resistance for resistance, there is always some genetic basis. So the strain would have some modification on their genetic environment that causes resistance at some point. It could be on the chromosome. So on their own genome, where this a spontaneous mutation happening, which could be a spontaneous mutation in the target molecule encoding area, or it could be in a drug uptake system. So it could be on a kind of a pump. Or, it could be in the regulatory system so the regulation of the cell and such. These mutations occur spontaneously. Something happened that could be selected by use of drugs but in principle they are occurring in the genomes randomly. And then the will be ending causing some resistance because of these mechanisms. Another thing is when a strain acquires a plasmid, acquires a genetic element that has resistance genes, so something is coming from the outside from another bacteria. And then this bacteria acquires this plasmid which means that they have been transferred, maybe by conjugation or transduction or even a DNA transformation. And then this plasmid has the resistance genes, so it gives the bacteria the possibility to be resistant For this, it means that the plasmid is a genetic element that is mobile. It could also be another genetic element that is mobile, which would be the case of transposons, or integrons, or gene cassettes. Which also are a possibility to transfer resistant genes between different bacteria which could be the same or different species even. But this is not always only genetic, because there are some factors behind this. So the bacteria could also, the infection could also be resistant to the treatment because of some mechanisms behind. That, for example, if an infection is in a place or because it is an abscess and is very surrounded by tissue that doesn't allow the antimicrobial's to get in or it is for example a tuberculosis infection that is a very encapsulated that the drugs don't get into that place. So if the drug doesn't get there, that's resistant by nature, not because it has a specific mechanism. It's just because the drug is not able to get there. It could also be resistant because the strains are in stationary phase. This means the bacteria are not growing and if they are not growing they are not doing this metabolism, this replication that is needed for the drug to act. So then, at this point, they are not susceptible for example, to some of the drugs that inhibit synthesis or replication. It could also be that they just don't have the target I have mentioned it in some of the previous lecture. If these are these protoplasts or spheroplasts which are bacteria that don't have a cell wall. Then of course, if you give a cell wall inhibitor drug, it would not effect them, because they just don't have the target. So these are some basis for reason that are non-genetically. But if we take the genetically ones, the most simple one is, for example, this point mutation. Where you have the chrome of some in the bacteria and there's encoded of course menu of the basal genes for the cell growth and metabolism and one of these house keeping genes that are available in all bacterias these DNA gyrase. And the top summarizes the other top base summarize which are four. And if you have a specific change or some specific changes in this DNA gyrase in some sites, you will have quinolone resistance, because this enzyme is so important in the replication of the DNA that one small change of one nucleotide can make the difference that this quinolone doesn't act anymore. And this happens step wise, so if you have one change in the DNA, one only letter, one only mutation, you have a slightly higher level. But if you have two or three it is increasing in level. So it is step wise increase into resistance. And here as an example we have one strain up here that is susceptible. So quinolone would kill this strain very easily and two strains that are not as susceptible. And the only difference, if you take the genome and separate the genes and separate the parts, that you will look at and just look at, really, the nucleotide level. The only difference between this is there is a cytosine here, and there's an adenine here and a timidine here. The normal one is a cytosine. So these are mutants, these are mutations that occur spontaneously there. Which means that when you find these on this site, you have affected the codon that will be encoding the protein from there. And this will affect the amino acids that are going to be encoded from this protein, which means that in this codon, which is codon 83, and this DNA gyrase, you have this difference in the codons and the normal one is encoding a serine and the other two will be encoding different amino acids. Only this small change, this one amino acid will make the difference and cause the resistance. And if we find this, it's enough for a level of resistance that is shown in even in the tests that we do. So it's a very small difference. The good thing about these chromosomal mutations is that they are in their own chromosomes so they are not transferred to another bacterial cell. Of course the cell when it divides and gets daughter cells it still takes along this mutation and the daughter cells will have the same, but it doesn't transfer to the neighbors. So, there would be the difference between the resistant and the susceptible. And when you do this, well, you have this system where you have the target and you have the drug fitting in. And it would be acting on the drug. Of course, if you have modified the target, even so with such a small difference, you will not have an effect and the effect is stopped. Sometimes these mutations, it's not the case of the one I just showed, cause some up-regulation in pumps as well. It could be that these mutations occur in a regulation system and then the drug is pumped out more heavily than it should be normally and that's happened in some cases with mutations as well. Another thing is when a bacteria acquires genes, and the genes are not spontaneously created in a bacteria. They have to come from somewhere. So these are genes that are acquired. And they will lead to a production of a certain enzyme or a certain product the will effect the resolution. And one of the things in this system that can happen is that the target is replaced or protected so that the drug is not able to act and that's the replacement or protection of the target. There is a possibility of this gene encoding an enzyme for example that breaks down the drug or that modifies the drug so that it doesn't fit with the target. Or that it actually encodes a whole pump that is able to lower the concentration of the drug. So the gene itself encodes the pump as well. It could also be the mechanism. So to give you some examples and now we've went through the basic mechanisms. We take just the tetracycline family. It's a very bit family of antimicrobials, it's very used. It's used in animals, it's used in humans. And if we think that the tetracycline is a good example, we also can think that it is a broad spectrum antimicrobial that affect's many species. And the mode it act's, is act's on the proteins in to this, so it would be affected both by the entry of the drug in cell because it has to act inside the cell in the protein synthesis, and it will be effective, anything happens here in the process. And if we look at the modes that the bacteria find to resist to tetracycline, we actually find many ways. So if you take this tet A, B, C, D, actually as researchers in this area we call it the tet alphabet because there are so many genes in the tetracycline resistance world that we can name them with numbers and with letters and we have many of them. And they actually cover several mechanisms of the ones that haven't been explaining. So only for tetracyclines and similar drugs they are not so different. We have drugs, we have mechanisms of resistance acting on the active efflux so these pumps that pump the drug out really many of them. So they act in different ways, but they act more or less the same. We have ribosomal protection, so the drug would act on the ribosomal production of protein. If you protect the ribosomes, it wouldn't work so well, so we have several genes as well in this family, on this area, acting for resistance. We also have some that produce enzymes that modify the drugs, that break down the drugs, so that they are not active. And we even have some mechanisms where the mechanism of action of resistance, we know they cause resistance, but we know very little about them. So, only for the one family of drugs which is very similar we have many mechanisms and that happens quite frequently with other families of drugs. That we have 100s sometimes of genes, that cause resistance to just one. So, it is quite a big world, so many times we have actually one drug. Various mechanisms which could be 100s actually, and we actually have many species as well, because these drugs would target many species so the mechanisms could vary between the different species. And when we look at the effect, the phenotype, we call that is the what we see of the effect of the drug. It could be very variable. It could be very different. And also when we look at what happens in the patient, what happens in the person that is infected that could also be very different. So for researchers, for doctors, it is quite puzzling and it is quite difficult to figure out in all of this because there is many mechanisms around. And that's what it makes quite interesting. Just taking an example of beta-lactam resistance. We do have for the same group of drugs also different mechanisms. It could depend on which species you are treating or which groups of bacteria, and also it could be interfering on how the bacteria gets these mechanisms into them. If it is reduced permeability, it actually maybe, they just didn't acquire anything. They're just like that. If it is some enzymes and enzymatic modification genes, it is probably something they got by conjugation or transduction. If it is a target replacement, that is a very specific situation where there is a big gene cassette. Which is not so easily transferred by conjugation. So the mechanism is quite unknown how they get it. But there's also different ways of acquiring resistant genes or acquiring resistant encoding. Genetic elements that makes the strain become resistant. And it's just one group. So if we take one group we still have many things to look at. So thank you very much I hope you, you like this area of antimicrobial resistance because we are going to talk very much more about it. [MUSIC]
