[MUSIC] Welcome to this video lecture on beta-lactam resistance in Staphylococcus aureus, with a particular focus on MRSA. My name is Valeria Bortolaia and I have the pleasure to introduce you to this topic on behalf of the EU Reference Laboratory for Antimicrobial Resistance. I would like to start by asking you a question. How many MRSA go undetected in your lab? My aim with this lecture is to bring this number to zero. And for doing so, let's start by defining what is an MRSA. MRSA is methicillin-resistant Staphylococcus aureus. In other words, it is a Staphylococcus aureus that has acquired the ability to to be resistant to methicillin. Methicillin is a drug. It is a beta-lactam antibiotic, and somehow, the name has more historical reasons. Because by definition, an MRSA is resistant to all beta-lactam antibiotics, with the exception of newer cephalosporins with anti-MRSA activity. How does Staphylococcus aureus achieve beta-lactam resistance? If you imagine that this is a schematic representation of a Staphylococcus aureus cell, and the orange circle represents a beta-lactam drug, the drug will enter the cell and it has on the cell wall it's target. But if the Staphylococcus aureus for some reason is able to modify this target, the drug will not be able to interact with it any longer and therefore, the bacterium will be resistant. So this mechanism is defined as alteration of the target site. Another way Staphylococcus aureus can achieve resistance to beta-lactam antibiotics is by inactivating the drug through enzymes. The enzyme interacts with the drug, it transforms into its inactive form, and therefore, the bacterium will be able to grow in the presence of the antibiotic through enzymatic inactivation of the drug. When we talk about MRSA, the methicillin-resistant Staphylococcus aureus, this is the Staphylococcus aureus achieving beta-lactam resistance through alteration of the target site. Let's see how this work in details. This is a very schematic representation of the cell wall of a Staphylococcus aureus. Here we have the plasma membrane, and here we have the peptidoglycan layers. In Staphylococcus aureus, the peptidoglycan layers are cross-linked, so held together by the action of a protein called PBP2, penicillin-binding protein. This protein acts on the peptide chains to hold together the peptidoglycan filaments. If we have a beta-lactam antibiotic, this antibiotic has a very high affinity for the PBP2. Therefore, it will connect to the PBP2 and this protein will not be able to perform the cross-linking of the peptidoglycan layers. And therefore, the cell will not have the correct cell wall and it will eventually die. An MRSA has exactly the same structure, but the difference is that the PBP2a is a new form of the PBP2, it's different. Therefore, if we have a beta-lactam drug, there is no affinity for the drug for the PBP2a. The drug will, therefore, not interact with anything, with any target, and the PBP2a will continue performing the cross-linking of the peptidoglycan layer. Therefore, an MRSA has a different PBP2a and can build its cell wall also in the presence of beta-lactam antibiotics. How can we ensure to detect this in a laboratory? Usually, we perform two kind of test, phenotypic test based on expressed traits, and the genotypic test that are based on DNA. If we want to perform genotypic test, the most classical test to perform when we want to know if bacteria are resistant or not to antibiotics is antimicrobial susceptibility testing. And the beta-lactam drugs that are used for Staphylococcus aureus susceptibility testing are penicillin and either cefoxitin or oxacillin. It should be said that for Staphylococcus aureus, cefoxitin is the drug recommended for testing. And it is used as a surrogate for oxacillin. And oxacillin, especially in the disk test, is not reliable. Another way to phenotypically detect an MRSA is to look for the modified PBP2 proteins, so for the PBP2a. This is done by an agglutination test that is based on the antigen-antibody principle. But you have to be aware, now, that this test will not detect all MRSA, as I will explain in a short while. You should also remember not all penicillin resistant Staphylococcus aureus are MRSA. At the beginning I showed you this slide about mechanism of resistance and I also told you that there are some cases in which an enzyme can inactivate a drug, and therefore the drug will not be active. This enzyme in Staphylococcus aureus is called blaZ. And it confer resistance to penicillin, penicillinase susceptible penicillin. If we want to detect the genotype of an MRSA, we can detect the mecA or the mecC gene, and they can be detected by PCR or by whole genome sequencing. The mecA gene is definitely by far the most common background of resistance for MRSA. But also the mecC gene is increasingly recognized. Both genes code for modified PDP2 protein. But the proteins encoded by these two genes are less than 70% similar. Therefore, this is the reason for which in the agglutination test that I mentioned before, it is possible to detect the product of mecA, but not the product of mecC. And therefore, an MRSA whose resistance is mediated by mecC will be undetected by the PBP2 test. Now, let's imagine that you have confirmed that you have an MRSA in your la and you want to perform some further characterization. I will just give you some brief explanation of the methods that you can use for characterizing an MRSA, and then further details will be referenced in the slides. You can start by typing the SCCmec element, the Staphylococcal Cassette Chromosome mec. And this element is the genetic fragment that contains the mec gene. In a very schematic representation of the SCCmec element, we can recognise three joining regions, 3, 2 and 1. The ccr gene complex, that is the cassette chromosome recombinase complex, that is responsible for the movement of the entire genetic region into or from chromosome, and then there is the mec gene chromosome. There are different PCR reactions targeting this part to give you a ccr type and targeting this part to give you a mec class. And combining the results of the two experiments, you will define an SCCmec type, and for more information about this is in the website sccmec.org. When you talk about MRSA, you might have heard different terminology, both in scientific literature, but also in the news. I should emphasize already from now that often we hear about this terminology in connection with the MRSA, but this would be actually valid for all and not only MRSA. If we hear a say t011, t899 or t followed by other numbers, what does that mean? This refer to the spa type. The spa type can be defined by PCR and sequencing of one gene, the staphylococcal protein A gene, to recognize the type and the order of repeats. And for protocols and information about this, you can visit the webpage spaserver.ridom.de. When we read about MRSA, we can also hear something about MRSA ST398/CC398 or ST9/CC15, or other number combinations. What does this mean? Here we are talking about sequence type and clonal complex. This is a characterization that is based on a method called multilocus sequence typing that is based on PCR and sequencing of seven genes. Again, for full schemes and protocol, you can visit saureus.mlst.net. Why do we perform these characterizations defining STs, defining spa types? This is because there are many groups of MRSA that can be differentiated also epidemiologically. Historically, the first MRSA were recognised in the hospitals, in hospitalized patients, therefore we have the hospital-associated MRSA. But after some time, it was possible to recognize MRSA also in the people with no contact to health care settings. And we talk, therefore, about community-associated MRSA. And in other areas, in livestock production, there are also MRSA associated for example with pigs, with chickens or with other animal species, and we therefore call them livestock-associated MRSA. Usually, these three categories are different lineages based on sequence types and the spa types, but transfer of strains between these settings is increasingly recognized. Therefore, the differentiation becomes more and more blurry. To sum up and to conclude what a MRSA hunter should always remember. Testing penicillin and either cefoxitin or oxacillin allow deduction of susceptibility or resistance to a wide range of beta-lactam antibiotics. An MRSA is resistant to penicillin and cefoxitin, and also oxacillin, but oxacillin disk testing is not always reliable. Staphylococcus aureus, it is resistant to penicillin only, is not an MRSA. The PBP2a agglutination test is a very useful phenotypic test, but detects only mecA MRSA. And finally, we should keep into consideration that although right now very rarely, it is possible to find MRSA that are negative for mecA and mecC, and this implies that phenotypic test is to be considered the gold standard for detection of MRSA. I would like to emphasize that everything I've said in this lecture refers specifically to Staphylococcus aureus. It is possible to recognize methicillin resistance in other Staphylococcus species, but that was not the topic of this lecture. I would like now to thank you for your attention on behalf of the team of the EU Reference Laboratory for Antimicrobial Resistance. And I invite you to visit our website antimicrobialresistance.dk for protocols and for the specifications. Thank you. [MUSIC]
