[MUSIC] Welcome to this video lecture on resistance to beta-lactam in Escherichia coli, with a particular focus on ESBL, AmpC and carbapenemases. My name is Valeria Bortolaia and I am pleased to introduce this topic to you on behalf of the EU Reference Laboratory on Antimicrobial Resistance. During this presentation, I will explain what are the mechanism of beta-lactam resistance in Escherichia coli, what are ESBL, AmpC and carbopenimases and finally, how do we detect them? Resistance to beta-lactams comes in many forms. If we imagine this as a schematic representation of an E coli cell and we imagine that a purple circle represent a beta-lactam drug, the beta-lactam drug will need to enter into the periplasmic space of the cell and reach its target. A first mechanism of resistance can be given by a decreased cell permeability. In this case, the drug will not be able to enter the periplasmic space and will not be effective. A second mechanism of resistance can be given by a modification of the target site. If this is a normal target site, if the bacteria somehow modified the target site, the drug will not interact with it any longer. A third, very important mechanism of beta-lactam resistance in E coli and other Enterobacteriaceae is enzymatic inactivation of the drug. The cell can produce some specific enzymes that will act on the drug and transform it into an inactive form. And finally, bacteria holds the possibility to export actively drugs outside of the cells. When we talk about ESBL, AmpC and carbapenemases, we're talking about this kind of mechanism. ESBL, AmpC and carbapenemases are all enzymes that are able to inactivate beta-lactam drugs. If we want to simplify and to explain better these terms, we have the ESBL, is an acronym that stands for extended-spectrum beta-lactamase. AmpC indicates a beta-lactamase of the AmpC-type, whereas carbapenemase indicates an enzyme that attacks carbapenems. From biochemistry memories, we should remember that ase is a suffix that form enzymes' names and it is generally added to the end of the substrate name. Therefore, it is almost implicit that an extended-spectrum beta-lactamase is an enzyme that has as a substrate extended-spectrum beta-lactams, and the carbapenemase is an enzyme that has as substrate, carbapenems. The recognition of substrates by the specific enzymes is essential to be able to detect them in the laboratory. In this table, the general information of substrate specificity of the different enzymes is reported. What we can see in this table is different beta-lactam compounds, penicillin early generation cephalosporins, second and third generation cephalosporins, beta-lactam/beta-lactamase inhibitor combinations, and finally, carbapenems. And in here, we observe the different enzymes, ESBLs, carbapenemases and AmpCs, with their relative substrate specificity. This means that an ESBL will recognize as substrate penicillins, early generation cephalosporin, second and third generation cephalosporins. Carbapenems will recognize as substrates all these drugs, since enzymes have a substrate specificity similar to that of ESBLs, but with this important addition of beta-lactam/beta-lactamase inhibitor combination. And finally, there are the oxacillinases, which are in this table only for the sake of completeness. And they represent a large group of enzymes that can behave quite differently. As a general feature, they recognize penicillins, early generation cephalosporin, beta-lactam and beta-lactamase inhibitor combination as substrates, and depending on the enzymes, carbapenemases or second and third generation cephalosporins can also be recognized as substrate. This scheme is extremely important for phenotypic detection of the enzymes. And it is already now time to familiarize with the different compounds that are used in the laboratory. For the third generation cephalosporins, we use cefotaxime ceftazidime. This antibiotic, added with clavulanic acid, are those used for the beta-lactam/beta-lactamase inhibitor combination. And finally, among the carbapenems is meropenem, that is generally used. Why these specific drugs among the many compounds that are present? This is because of a sensitivity and specificity of detection that has been evaluated for many different compounds. Before going deeper into the phenotypic detection of these enzymes, we need to familiarize with some concepts. One is the MIC, minimum inhibitory concentration. Very likely, you already know about this. This is the minimum concentration of an antibiotic able to inhibit growth of bacteria. Here, it is exemplified for drug A, that is present in different wells in concentration from 0.03 milligram per litre, up to 64 milligram per litre. And if the doctor indicates the bacterial growth in the different wells, we can say that the first well in which bacterial growth is inhibited is the well containing 2 milligram per litre of the drug. Which means that the MIC of drug A for this bacterium is two milligram per litre. A second extremely important concept for recognizing ESBL, AmpCs and carbapenemases is the concept of synergy. Synergy can be defined differently if we are performing antimicrobial susceptibility testing by disk diffusion or by broth dilution. When we perform susceptibility testing by disk diffusion, we will use a disk containing cefotaxime, a third generation cephalosporin, and a disk containing the same drug with the beta-lactamase inhibitor in this case. So cefotaxime plus clavulanic acid. Then, we will be interested in the difference between the inhibition zone diameter around the two disks. If the inhibition zone diameter around the disk cefotaxime plus clavulanic acid differs by the inhibitions on diameter around this disk by more than five millimeter, then we talk about synergy. If this difference is less than five millimeter, then we define that there is no synergy. It is very important to remember that this test should be performed not only with cefotaxime and cefotaxime plus clavulanic acid, but also as mentioned before, with another third generation cephalosporin, that is ceftazidime, and with a counterpart with a beta lactamase inhibitor. So ceftazidime plus clavulanic acid. If we are performing broth dilution and we want to define synergy in that way, we here have an example of a plate in which cefotaxime is present in different concentration. From 0.25 to 32, going up twofold, as it is standard procedure to do. In the following row, we have again cefotaxime plus clavulanic acid, and also in this case, the concentration doubles all the time. If we imagine that we perform the test and what is highlighted in grey here represent bacterial growth, we will define the MIC of cefotaxime at 8 microgram per litre and the MIC of cefotaxime plus clavulanic acid at 1 microgram per litre. And in this case, the synergy is given by the difference between this MIC and this MIC. If this difference is more or equal three twofold concentration decrease, then we will talk about synergy. If there is a less than three twofold concentration decrease, then there is no synergy. As mentioned before for disk diffusion, it is essential to test in parallel also ceftazidime and ceftazidime plus clavulanic acid. If these concepts are clear, and I hope I didn't confuse you too much with all these different drug names, it is actually quite straightforward to apply the criteria set by the European Food Safety Authority to recognize the beta-lactamase phenotypes. The ESBL-phenotype is a phenotype a characterized by four points. The first point is that cefotaxime or ceftazidime MIC should be higher than 1 milligram per litre. The meropenem MIC should be less or equal than 0.12 milligram per litre. The cefoxitin is second generation cephalosporin. The MIC of cefoxitin should be less or equal 8 milligram per litre. And finally, very important, there should be synergy. In this case, we have the so called ESBL-phenotype. The AmpC-phenotype is similar to the ESBL-phenotype for the first two points, but differs in cefoxitin, where we have an MIC higher than 8 milligram per litre, and, very importantly, we do not have synergy. And finally, we can have a carbapenemase-phenotype when we have a meropenem MIC higher than 0.12 milligram per litre. Of course, all this gives an indication. It's based on expressed characteristic of bacteria, and further test to confirm these are usually recommended. These possibilities of MIC and combination of antimicrobials are not exhaustive of all different possibilities. And indeed, we can have ESBL and AmpC phenotypes present together. We can, of course, have a susceptible phenotype, and we can have other phenotypes. Because, as mentioned in the first slide, beta-lactam resistance does not come only as a consequence of beta-lactamases, but several mechanism can play a role. For further information about these other phenotypes or when combination of phenotypes can occur, I would recommend you to follow this presentation, the entire presentation at this website. And if you are noting down the website, please remember about the underscores between ws and april, f11 and efsa, and efsa and criteria. If, for specific reasons, we need to move on to the genotype, we want to detect the genes that are encoding the beta-lactamases. We can do that based on the common ways that are used for detecting genes. That is PCR, that can be complemented by sequencing, microarrays or all genome sequencing. For PCR and microarrays, their limitation is given by the fact that we can target a limited number of genes. And therefore, it is necessary to have some information of the beta-lactamase and that are most common in that area in that period of time. All genome sequencing is definitely the most sensitive method because it virtually allows detection of all beta-lactamases known to date if we are analyzing our sequences against databases are well curated and complete. To give you an example of the complexity of the field, I would like to remember that beta-lactamase comes in thousands. ESBLs, AmpCs and carbapenemases are the three groups we have been mentioning so far. But within each group, there are many enzyme types. Between the ESBLs, we find the CTX-M enzymes, TEM and SHV, just to mention the most frequently encountered ones. But this list can actually be much, much longer. Within the AmpC enzymes, first of all, in the majority of Enterobacteriaceae we found chromosomal AmpC. So, most Enterobacteriaceae have an AmpC enzyme in the chromosome. But this enzyme can be expressed at higher or lower level depending on the bacterial species. Other AmpCs enzymes that are frequently encountered are the CMY, the ACC, DHA and FOX. Again, this list can be much longer with enzymes that have been described more sporadically. And finally, among the carbapenemases, the most frequently encountered enzymes are the IMP, VIM, NDM, and KPC. OXA enzymes have been put in brackets, both among the carbapenemases and among the ESBLs. This is because strictly speaking, these enzymes belong to the oxacillinases, so not to these three groups. But they can have phenotypes comparable with the ESBL-phenotype or comparable with the carbapenemase-phenotype. To further complicate the issue, we should remember that within each type there are many variants. So for example, the CTX-M enzymes have been recognised in over 170 variants, CTX-M-1, 2, etc., and the same apply for each of these enzymes. And if we want to talk about the correct nomenclature, we have a difference between the gene name and the protein name. All those mentioned here are protein names. If we want to indicate the gene, we should indicate is as a bla in italics, beta-lactamase, followed by the protein name as a subscript. To sum up what we have been talking so far, we can say that resistance to beta-lactam antibiotics in Escherichia coli, but also more in general in Enterobacteriaceae, comes in many forms, but it is mainly due to enzymatic inactivation of the drug. ESBLS, AmpCs and carbapenemases are all enzymes that inactivate beta-lactam antibiotics. And they mainly differ for substrate specificity. These enzymes can be detected both by phenotypic and by genotypic methods. And of course, different drugs need to be tested to increase the sensitivity and the specificity of the detection. I would like to thank you very much on behalf of the EU Reference Laboratory on Antimicrobial Resistance, and I invite you to visit the website antimicrobialresistance.dk for further information and further protocols on detection of ESBL, AmpCs and carbapenemases. Thank you. [MUSIC]
