Human Practices

iGEM Hamburg: We have all heard about antibiotic resistance. Could you give us a brief overview of the situation: Has it improved, stagnated or even worsened?

Hamprecht: In some respects, it has even improved. For example, the use of antibiotics in veterinary medicine and in animal fattening has decreased due to new regulations. But what worries us most are the increasing resistances in gram-negative bacteria such as Escherichia coli (increase in antibiotic resistances to reserve antibiotics). So, unfortunately, things have worsened overall, despite the improvement in certain areas.

iGEM Hamburg: Where do you see the greatest dangers for humans? Is it really the hospital infections or are there also important other areas?

Hamprecht: Yes, on the one hand it is the hospital infections. In some other countries it is more due to bacteria in food or poor sanitary conditions (e.g. India in lakes and drinking water). In Germany it is associated with the health system. We make it easier for bacteria to develop by using too many antibiotics as therapy. Whether in hospital or outside. This is a point we need to work on.

iGEM Hamburg: On the subject of too much antibiotic therapy: Do you see any way in which this can be improved through better diagnostics?

Hamprecht: Certainly, there is a lot that can be done through diagnostics. But it alone is not enough. There are some studies that have investigated how far better diagnostics can lead to a reduction in antibiotic consumption. But it doesn't have such a big impact. It must always be combined with an antibiotic stewardship approach, so prompt communication and discussion of the results with the treating physicians. This is needed much more, but there are not enough trained doctors for it.

iGEM Hamburg: So would you say that human intervention is what has allowed antibiotic resistance to develop to this point?

Hamprecht: Yes, sure.

iGEM Hamburg: We've been reading up a bit: You've been working on this for a relatively long time, since your habilitation. Maybe briefly about your background: How did you get to the topic, i.e. what interests you most about it? Could you briefly say something about your career?

Hamprecht: Well, I have always found antibiotic resistance very exciting. Antibiotics are simply one of the few methods in medicine where we can treat relatively causally and with few side effects. The fact that they have so few side effects is also the problem that antibiotics are sometimes given very, very lightly. And in this respect I have always been interested, especially in gram-negative bacteria. It's much more complex and scientifically interesting, and that's why I got into it. I've been doing this for a good ten years now, I would say. In this area and with fungi, I've also done a bit of research. I am investigating different aspects of it. First of all, what is the background of these resistances, how can we detect them better? We have also evaluated various tests with manufacturers, in part also together, and also a bit of an understanding of how germs pass on their resistances. Perhaps you have also heard of horizontal gene transfer. Bacteria have the possibility, so to speak, to pass on plasmids, i.e. mobile genetic materials beyond species boundaries and that also contributes a lot to the spread of certain resistances. These are the topics that actually interest me.

iGEM Hamburg: Yes, we read that you mainly deal with the resistance mechanisms, with the tests, also on the resistance mechanisms. We were interested in how you investigate what happens at the molecular level. How do you expose the bacteria to stress? So that they activate the mechanisms?

Hamprecht: In general the mechanisms are always active. So if you have a resistant bacterium, then it has some kind of resistance gene and I can detect that using molecular biological methods, be it through PCR and Sanger sequencing or nowadays also whole genome sequencing, which is actually standard in this area by no. So it is usually not difficult to detect. And then there are some resistances, where we don't know the genes or don't understand the mechanism itself. And then you have to do a bit more functional essays. That means, first you look at whether the bacteria are able to hydrolyse a beta-lactate, to inactivate it, or do they do something else? For example, have they changed their cell wall in such a way that they don't let the antibiotics into the cell at all? Those are the kinds of things we look at. Or, of course, when it comes to the spread of resistance genes, we look at how quickly resistance is transferred when two bacteria are brought together. These are experiments that we do, for example. Then we also look at what effect that has. Sometimes you are “lucky”, and such a resistance gene is naturally also a burden for the bacterium. That means that at the moment when there is no advantage, they might get rid of it. But then there are other resistance genes that are practically no burden for the bacterium and thus naturally remain for many generations. Those are the kinds of things we do.

iGEM Hamburg: Yes, we skimmed some of your publications papers.. You had compared four tests in a paper. Could you perhaps briefly summarize the three colorimetric tests and the modified agar diffusion test (ADT), it's probably about the enzymes, so how do they detect it and convert it colorimetrically? We also have color change as a planned indicator, so we are interested in how they work.

Hamprecht: So with the colourimetric tests, it works via a pH shift. I basically have a pH indicator and incubate the bacterium with the antibiotic plus the indicator. If the bacterium has an enzyme to hydrolyse this antibiotic, which inactivates it, then a proton is produced and I see that in pH value and color change. So that's how the colorimetric tests work. It's relatively simple and works relatively well, I have to say. Not for all resistance mechanisms. There are many enzymes that inactivate antibiotics. And some of them only have a relatively low inactivation and hydrolysis rate. This means that it takes a long time for something to happen, and not all of these tests are suitable for this. The ADT is another, non-colorimetric test. You basically take an antibiotic plate and incubate it with the germ you want to examine. And if the germ now forms an enzyme to inactivate it, I have no antibiotic left in the plates, or only a very small amount. And then I practically put that on an agar plate with a very sensitive strain and incubate that again. And if it's inactivated, I don't see any inhibition zone, and if it's not inactivated, I see a large inhibition zone. And that is usually even more sensitive than these colourimetric tests.

iGEM Hamburg: But then they usually take a little longer.

Hamprecht: That's right, it takes a day, or at least, let's say, eight hours. These colorimetric tests, depending on the case, sometimes take between 30 minutes and two hours.

iGEM Hamburg: Yes, that's quite fast. If I understand correctly, these were tests that already existed before, which you optimized a bit?

Hamprecht: We have optimized various tests, especially for bloodstream infections, as these are very urgent. So if I now have a patient who is in a really poor state and we isolate these bacteria from the blood, we have to be as quick as possible and we have practically developed this further.

iGEM Hamburg: And are you still in the process of developing other tests with other methods? I will call them de Novo tests for the moment.

Hamprecht: Not at the moment. At the moment there are a lot of other projects, but we are not working on any test development.

iGEM Hamburg: I think we can also slowly move on to explaining a little bit what we are doing and maybe talking about it more. We want to detect the RNA of antibiotic resistances. That means that we are now specializing in chloramphenicol resistance in E. coli. Simply so that we can first make a proof of concept. And that's what we use this for. So the main part of our project are the so-called split ribozymes. These are basically ribozymes that can cut themselves out. And the trick is that they were separated at a certain point at the beginning. And then you have these two parts of the ribozyme, each of which has a part of a coding gene, for a transcription factor, for example, and at the bottom a part for a guide RNA that can attach to another RNA. And when there is this antibiotic resistance, both parts accumulate so close to each other that this separate ribozyme becomes functional again, cuts itself out and then a complete gene is created, which can then be read. And in the end, through several steps, we arrive at a situation where, if there is antibiotic resistance, GFP is expressed, so that we get a green solution, and otherwise we get RFP, so that we get a red solution. And the far-fetched vision for the future is that we will develop a test where a sample can be added and you get a red or green solution, like in a Corona test, and that simply works a bit faster and is easier to use. Of course, that's a very broad vision, but that's our big goal. We want to do the whole penetration into the bacteria via phages. And of course we would like to get some feedback, because we haven't been able to talk to experts much yet. You are also the first interview here right now. What do you think of the idea and perhaps we should ask you if it was understandable?

Hamprecht: Yes, it is understandable in principle. Now, of course, we have to consider. The big difficulty we actually have is that we have so many different resistance genes. So whenever I use it in everyday clinical practice, I would actually need an extremely large number of different tests in order to find out. Of course, it might be possible to concentrate on a few particularly important ones. Maybe that would be carbapenem resistance or so forth, that would certainly be interesting. Now the question is, there are of course these so-called immunochromatographic tests that can detect resistance genes. It takes about 15 minutes and this is already available, for example, in a whole series of carbapenemases, but also in other enzymes. So the question is, of course, how fast or how cheap is yours in comparison? These immunochromatographic tests are just for orientation. They work within 15 minutes and cost, let's say, ten to 15 euros per test. Of course, the question is whether you can do it with that? I think that would be the first big question. The second question is, of course, about your phages: are they relatively universal phages? I'm going to say that you probably need specialized phages for at least every genus, if not for every species. I don't know how much of a problem that could become. Is this going to be able to infect all, I'm going to say all E. colis or klebsia or whatever? Yes or no? That would certainly be another big question.

iGEM Hamburg: These guide RNAs can be adapted quite quickly. The sequences are very easily exchangeable, which means that if the proof of concept works for us, you can adapt the system relatively well to different sequences and to different things that you might want to detect. For example, one can try to detect the bacteria per se, the resistance RNAs and so on. As long as you have an RNA that you want to detect, you can actually modularise and adapt it relatively quickly. And if you want, you can also check it out. There are indeed phages that can also take a high load in principle and in this respect you can use several ribozymes with different coloured proteins, for example, or enzymes that ultimately produce a coloured solution, so that you can actually test for different resistances with one phage or you have a different coloured output. Of course, that's the proof of concept for now, but that's kind of the idea behind it. The question is, of course, whether our basic idea of detecting RNA is somehow particularly useful in some cases or whether you think that it is smarter to detect the proteins?

Hamprecht: Well, I think it works both ways. Actually, tests that detect proteins are already available via antibodies. But I haven't seen any tests that work according to your procedure yet. Of course, there are a lot of PCR-based tests, but I haven't seen any tests based on this method. So I find it very interesting. I think the difficulty is that you have to try things out. Of course, I am dependent on the bacterium reading the RNA in sufficient quantities. Of course, there could possibly be some differences, depending on how I cultivate the germ. You would have to try it all out. But, in principle, I find it interesting in any case.

iGEM Hamburg: Yes, sensitivity is of course another thing we have to look at. We asked ourselves how much the bacteria express the RNA when there is no need to transcribe it, especially when there are no antibiotics. We also asked ourselves a little bit what ideas you might have about how best to design this so that we somehow get the highest rate of RNA. Do you have any ideas?

Hamprecht: Your test is probably aimed at, let's say, having a cultivated bacterium in the laboratory and wanting to know relatively quickly whether there is resistance or not. Of course, one could also try to add a minimal amount of antibiotics at the same time in order to possibly increase the gene expression. There is relatively little data on this. That might work in some cases, but in some others a little antibiotic probably won't do anything at all. So there is some data that it will not increase the expression. You have to try that out. I think you have to try it out in real life with strains to see whether there is a difference and whether there is a detection problem because perhaps too little RNA is produced.

iGEM Hamburg: This raises the question if the antibiotic is then added and there is a different resistance, that the bacterium is then killed anyway and the resistance can no longer be detected, although there is still a positive test?

Hamprecht: Well, you could try to add antibiotics in sub-inhibitory concentrations, so that it doesn't kill the germ, but stimulates it a bit, or maybe I take an antibiotic that increases the expression without killing it. So a somewhat related antibiotic. You would have to try that.

iGEM Hamburg: Yes, I just remembered I was tasked to ask what are the most human errors in the tests that could be prevented or how they could be prevented? Be it communication design or something similar, because of course we want everything to be applied correctly and we want to be able to do it as safely as possible.

Hamprecht: You mean in the laboratory, what are the mistakes there?

iGEM Hamburg: Yes.

Hamprecht: Well, I would think that it is relatively few or not a big problem. The laboratory is certainly the area in the hospital that has the best quality assurance. You work with EVD tests, you have trained staff. You can control the conditions much better than in other areas, so I wouldn't consider that a big problem. Many tests are highly automated. There are quality controls. So I don't really see a big problem there. Of course, if you were to introduce such a test on a large scale, you would have to automate it as much as possible with quality controls, etc. But that would be something for the later development period.

iGEM Hamburg: Do you have any prior experience? Or have you ever seen how to actually get such a test into practice in general? We have also seen some ideas and papers on how tests could perhaps be made, where researchers have always proposed new ideas, but at least in the discussions I have had with well-known doctors from clinics or others, who have never heard of them and who say that we still somehow have problems in actually getting quick diagnoses. How could we actually bring this closer to practice or where one could perhaps push it more so that a test can actually be implemented and used in practice?

Hamprecht: Yes, well, I think there are of course two things: You need as big an industrial partner as possible, that's just one of those points, that you have as many partners as possible to help you. I have very good contacts in the industry. That would certainly be something, if at some point it goes in that direction, where I could help you. That is one thing. The second point, and this is simply crucial, is more "What does it cost? That is now the problem in hospitals. We have a whole lot of tests that we could actually work with faster than we do at the moment. But that fails because of the costs. That means that if I have a test that costs an additional €100 for a patient and only brings about an improvement in a few percent of them, then that is not so easy to implement in practice. So that's actually the biggest problem, the costs in everyday life, they simply dictate a lot. So you need a test that is as standardized as possible, that goes quickly, that has little hands-on time. That's also a problem, because there are more and more difficulties in getting employees to do these tests. And if it is a very, very labor-intensive test, then it may simply not be used at all for these reasons, because my MTA could have done three other tests in the time available. That is an important point. Standardization, quality control and costs. All in all, these are the points that are important.

iGEM Hamburg: This is a good input for us to deal with this a bit more. Above all, we should start with biotechnology, so we should talk to a few people and talk a bit more about the feasibility. Of course, at the moment we want to look at the biological level first and then go a bit further into implementation. I would say, in any case, thank you very much for the input. There were a few things in there that we hadn't thought of yet and definitely interesting things where we can perhaps still change and work on the final implementation or at least on the outlook that we want to develop at the end of the year. In any case, this has been really great. Thank you very much.

Hamprecht: Sure, you're welcome. I wish you much success. And if I can help in any way, please let me know.

iGEM Hamburg: We will be happy to get in touch with you again.

Hamprecht: Okay. All the best to you then.

iGEM Hamburg: Thank you!

The following questions were answered in advance in writing by Prof. Dr. Heisig.

iGEM Hamburg: How do you assess the current development of antibiotic resistance? Has the problem improved/worsened/stagnated in recent years?

Heisig This cannot be said in general terms, as it depends in each case on the combination of pathogen and resistance to the antibiotic in question. On the pathogen side, special resistant clones often play a role, which have developed over a certain period of time (accumulation of resistance genes and resistance-associated mutations) and spread under selection pressure.

iGEM Hamburg: What current and future problems do you see coming our way due to resistant germs? What dangers do you see?

Heisig: Problems: Increasing limitation of treatment options especially for immunocompromised and/or elderly patients. Lack of options to switch to other agents if resistance is present.
Hazards: Increasing emergence of multi- (MDR) and even pan-resistant (PDR) pathogens that have a combination of resistance properties on mobile genetic elements and can spread as a clone or transfer the properties as a block to other pathogens.

iGEM Hamburg: Why are these resistant bacteria on the rise?

Heisig: Many factors have an influence: e.g. use without prior testing of pathogen sensitivity (broad-spectrum antibiotics with effect also on stabilizing site microflora, which must be accepted in the case of life-threatening infections, but exerts broad selection pressure. Increase in age-related immune reduction requires more frequent use.
Selection pressure also due to entry of effective antibiotic residues into the environment.

iGEM Hamburg: How and when did resistances start to develop? Has the problem only recently developed in this way or has it always existed?

Heisig: Genes coding for resistance to naturally occurring (="classical") antibiotics are found as prototypes of today's resistance genes to partially synthetically modified antibiotics from very early geological times, i.e. more than 600 million years ago. These genes probably originally evolved in bacterial antibiotic producers to protect them from their own agents. The natural role of antibiotics is as a "weapon" of individual bacteria (e.g. Streptomyces species) against food competitors in a habitat.

iGEM Hamburg: What are you doing in this topic area. What are you doing in your research? Briefly tell us: how did you get into this topic? What interests you most about it?

Heisig: Already during pharmacy studies the realization that with antibiotics usually a cure is achieved, while many other therapies, with the exception of e.g. substitution therapies, mainly only a relief of symptoms is achieved. Furthermore, however, the enormous variety of complex genetic changes to antibiotics.

iGEM Hamburg: To what extent do you deal with antibiotic resistance? What does your research/research group deal with?

Heisig: The focus of our work is on a group of purely synthetically derived antibiotics - the fluoroquinolones - which have not yet undergone millions of years of selection, although highly resistant mutants can also be isolated against them and are also appearing in the clinic. We are investigating which molecular genetic mechanisms contribute to this development and what effects they have.
Of particular interest are those genetic alterations that contribute to the development of resistant mutants into highly effective and pathogenic infectious agents despite impairment of normal cell growth due to the antibiotic resistance mutations.

iGEM Hamburg: What is your overarching goal?

Heisig: The aim is to identify novel targets that are important for maintaining the fitness of resistant mutants and to develop inhibitors against them as novel antibiotics.
Another goal is to use bacteriophages as a "bioweapon" in combination with antibiotics to prevent or at least limit the development of resistance under therapy.advantage of phages is that they can adapt themselves by mutation/selectiuon in the course of combating resistant bacteria.

iGEM Hamburg: What methods do you use to study resistance mechanisms?

Heisig: Main methods are DNA sequence analysis ("genomics") and RNA-based gene expression analysis ("transcriptomics") of laboratory-selected mutants or clinically resistant patient isolates, as well as the development of specific activity/function assays of potential new targets.
The following questions were answered via Zoom Call iGEM Hamburg: Do you think it's a good approach to start with detection so that antibiotic resistance can be stopped?

Heisig: So, I have to say something basic about this first. For this system, you need two prerequisites that are inevitably not always given. The first is that if you want to transfer the system with a phage, then you must ensure that the phage has such a broad spectrum of activity that it can also detect clinically relevant strains. Of course, you don't know in advance whether it's an E. coli, a Pseudomonas, or a Salmonella or Citrobacter or something else. Depending on which phage species you then have, the crucial point is of course the injection of the nucleic acid. The nucleic acid then must be injected into the organism, which works provided that the phage can also bind a specific receptor. This means that it is possible that not all E. coli bacteria, which you want to examine of the different strains of patients, are bound by the phage and that the phage can inject its RNA, in which case you want to inject an RNA, so that it also functions in this way. Phages are also used for therapy, i.e. they can also administer phages directly to humans instead of antibiotics or in combination with them. Here, too, the phage should bind to a corresponding infectious agent in vivo and then destroy it by lytic propagation. But the principle does not always work.

iGEM Hamburg: As we read, you also work with phages as a biological weapon. This means that you first have to detect which bacterium is present and only then you can use phages?

Heisig: That’s right. For this you need to have a very large bank of phages at your disposal, from which you can then check the infectivity and the phage sensitivity via pre-tests.

iGEM Hamburg: Phage sensitivity?

Heisig: Sensitivity in the sense of interaction of the phages with the surface receptor. That’s what I was stalking about earlier. The tail fibers, which you have illustrated in your project diagram, there are also other phages from the structure, often have certain protein components, or LPS components in gram-negative, i. e. hypopolysachharide structures. This is followed by adsorption. Then there is contraction of the tail fiber, and the DNA or RNA is injected from the head part. If these feet or the tail part at the end do not fit exactly, there is no stable connection, and the phage is unable to inject. This is described by the term “host area.”
There are phages with a variable host range. One phage is called bacteriophage Mu, abbreviated for mutator. It can insert itself in different areas of chromosomal DNA, where a corresponding insertion mutation can be induced. This phage has a genetic element, which is invertible. Depending on the orientation of the genetic element – it is part of a reading frame for tail fibers – it can induce one or another type of fibers and infect two different subgroups of bacteria accordingly. But even that will not be enough to dock to many possible infectious agents.
That’s one point. The second point is: You want to use a specific area as a binding site for your split ribozyme and assume that there is a mismatch at one point and then a corresponding attachment is successful. The bacterial RNA should be a ribosomal RNA or a messenger RNA. There is still the question of what you are imagining. There are several different resistance mechanisms for chloramphenicol.

iGEM Hamburg: We have a gene that codes for a protein that causes the lysis of the antibiotic, the chloramphenicol resistance protein. We use its mRNA, no rRNA.

Heisig: In addition, and this is the most common mechanism, there is an acetyltransferase which can modify both free OH groups of chloramphenicol by an acetyl residue. This inactivates the antibiotic. There are then a variety of variants, depending on whether you have Staphylococcus are other organisms. That is, one would first have to identify a conserved structure, abbreviated as CAT enzyme, that is common to all these chloramphenicol acetyltransferases, to capture them all. This is different from using a resistance protein. That’s the one point. The other point is that there are also resistance mechanisms, not only in relation to chloramphenicol but in general, in which not the antibiotic but the actual target structure to which the antibiotic attaches itself can change by mutations. You often have point mutations, but they are not always identical. And there are several places where single or even multiple mutations can occur, but all of them can lead to resistance. With your system, you would probably only discover one. You will then have to inject a mixture of RNA to capture all sorts of RNA then.

iGEM Hamburg: That’s very interesting. That would have been my question as to whether there is a mechanism that extends across all species. That you would find a part everywhere and that you could make it a little easier for yourself, what you bring in everything. It’s going to be a very broad mix.

Heisig: So, you have basically three basic mechanisms. One is the enzymatic modification of the antibiotic – the classic example is the β-lactamase that cleaves the β-lactam ring, of which there are hundreds, thousands, that also play a clinical role. The second point is what we were talking about, namely a modification of the target structure itself by mutation. There are different places, after all, the binding site is formed by a part of different protein chains, and then the antibiotic docks with different ways of interacting with each other. Depending on where there is a disturbance, there is a reduced binding and thus resistance. This can happen in two ways: the first is to prevent the antibiotic from getting into the cell. This happens in the case of gram-negative via so-called purine, pore-forming proteins, which may be reduced in length or may be altered by a mutation in such a way that they can no longer be. And on the other hand, it is possible to transport the antibiotics out of the cell again via so-called efflux pumps. Here, too, there are a whole range of different types. In both cases, the amount and not the type of pump or purine plays an important role. Often, a regulator, a repressor, for example, is used for an efflux pump. Every mutation that leads to a stop codon, every insertion, every deletion destroys the repressor, and the pumps are upregulated.
In other words, the mechanism you’re investigating relates to very specific constellations. A suitable example would be the change leading to resistance to streptomycin. Streptomycin resistance is at least mainly due to a change in one of the small ribosomal proteins, the S10 protein. It’s a point mutation. If it is present, the binding site is blocked, and the structure of the small ribosomal subunit slightly changes. There are not so many different variants, because the partly double-stranded RNA structure of the ribosomal RNA stabilizes the ribosomal RNA. This means that if there is an additional point mutation in the double-strand region, it is destabilized, and the ribosome usually stops working because it doesn’t fold properly. Therefore, these double mutations fallout from the outset. You can only detect those who have this specific mutation and, of course, that’s not 100% the case, the ribosomal RNAs are but not in all areas either. Especially in the double-stranded areas you would have a good opportunity to bind your split ribozyme parts to conserved parts. But that would have to be tried.

iGEM Hamburg: This is very interesting. We’ll bring that in again. What I also found very interesting in your interview sheet are pan-resistant germs. Are they resistant to everything or what exactly distinguishes them?

Heisig: The terms come from the treatment of tuberculosis; there you have mycobacteria. Tuberculosis is usually treated with a combination of three to four antibiotics. This is because the tuberculosis pathogens grow very slowly and antibiotics that interfere with the metabolism do not work immediately, so the bacteria have a certain time to continue to multiply slowly. In vivo there is a doubling every 12-24 hours. Thus, during the time when the antibiotic is taken, absorbed from the stomach, transferred to the bloodstream to the site of infection and then flushed out again, excreted, and metabolized by the liver, some bacteria may not be fully affected, and the effect may not last long enough in the dividing phase. That’s why you give such a combination. And what has been observed is that because of the long-term therapy, usually over months, the chance of developing resistance is also greater. Even if you give several – two or three – antibiotics, a corresponding resistance may develop during the time of treatment, which then affects several antibiotics. This means that during therapy, if you would notice this, you would also change and give other, so-called reserve antibiotics that are not given in advance. This can continue though. Pan-resistance in this context would mean that the clinically usable antibiotics no longer work. But these are still different classes and correspondingly different mutations. As a rule, you have the MDR phenotype, the multidrug resistance phenotype, especially in gram-negative patients, when they upregulate the pumps because they are nonspecific. These efflux pumps can pump out different molecules up to a certain size regardless of structure – to a certain extent, some a little better and some a little worse. Then with a mutation, you suddenly have resistance to four, five different antibiotics. If you call it pan-resistance, it’s usually a combination of different mechanisms, but they exclude the important clinically usable antibiotics. It’s not necessarily all there is.

iGEM Hamburg: And then you wrote something about pathogen sensitivity. How is that being tested now? Is the Hemmhof method structured in such a way that you can see how strong the bacteria are resistant by looking at the plate?

Heisig: This is a method called the agar diffusion method, which you see in Hemmhof. The second method, which is more commonly used, is the MIC (minimum inhibitory concentration). You do not specify a concentration gradient, as you do with the Hemmhof, but you create a series of dilutions from the beginning; always diluted 1:2 from a relatively high concentration. You must define a range for each antibiotic beforehand, dilute this further down, add the so-called inoculum to each dilution of the antibiotic, let it incubate overnight and then see the next day: In the low-concentration dilutions I find growth up to a concentration of 4 μg/mL. And if I look further back, I see that the less diluted one, which is then 8 μg/mL, is already inhibiting. Then 8 μg/mL is the lowest concentration that gives you an inhibition, because if you go further up – 16, 32 and so on – you still have the inhibition. That’s the procedure. Basically, both last overnight between 18-20 hours. In the meantime, however, there are optical systems that can detect the growth behavior of bacteria after just a few hours, e.g., by turbidity measurement. So, you can already see after six hours: Under the conditions I enter, i.e. certain antibiotic concentrations, are there any cells still growing/dividing to be observed? If that is the case, resistance is still to be expected. If that is no longer the case, then I have a finiteness, then there is my inhibiting concentration.

iGEM Hamburg: And are there also connections with the resistance mechanisms, in our case proteins? Is there a correlation there too? It is of course interesting for us that we can also include a quantification of the resistance in our test at the end.

Heisig: If you mean by correlation, how the type of inhibition, the extent of the inhibition and the concentration of the antibiotic are related – this is not actually tested by the test approach. You always add the same amount of bacterium, the so-called inoculum, in an amount so that you don't see any turbidity at all and the cells have a lot of time to grow or divide until you have visible growth. This is the prerequisite for you to be able to conclude that there is an inhibition at all. First, this is independent of the mechanism. The type of mechanism is not important, you always mix the same number of bacteria with a certain predetermined amount of antibiotic. Inhibition means no more growth, no turbidity. No inhibition means further growth, turbidity. There is no more difference. It's just that over a period you can see if the number of cells increases enough to tell you the antibiotic isn't working yet.

iGEM Hamburg: Can you think of anything that we could use to quantify that? We don't really know if the level of resistance is related to the amount of mRNA or how we can include that into the test. In the end we have the color signal and of course we can see how quickly the color signal develops. But that also depends on many other factors, such as the transcription factor that we express first.

Heisig: Yes, at the moment I also see the difficulty in the fact that the phage never infects efficiently, depending on the amount of template, i.e. bacterial RNA, that is already available from the outset. You also need to check the Multiplicity of Infection. It is possible that one phage per cell infects, but it can also be the case, if you use too many phages from the beginning, that the phage does not infect with one but with a large number, let’s say 10 phages per cell. Then you have significantly more split ribozyme RNA from the outset and, accordingly, a faster reaction. The correlation you mention should, of course, as in the other methods, provide some concentration that you can use to estimate: What is an effective concentration that I can use or not? And since you can create an exact correlation via a logarithmic scale via a good correlation between the inhibition zone diameter in milliliters and the MIC determined in parallel, you can exchange the two methods for one another. Of course, the MIC only provides you with one range due to the gradual grading of the concentration. You cannot say exactly: This is now 24.5. It's either 16 or 32. That's where the range lies. To do this, however, you would have to use the same strains, the same antibiotic and the same medium conditions to determine this inhibition zone diameter for the various insensitive or sensitive strains and do this with the same collective with the MIC determination. Then you get a correlation line. And then, which is technically easier, you can do this agar diffusion test where you inoculate the plate with the cells once and then put 6 or 8 discs on top. Then the next day you have results for 8 different antibiotics. That's tricky here because of course you have a variable in there. First, how much of the ribozyme comes in initially, depending on the multiplicity of infection. Second, the other question is the signal intensity: what is your zero value, what is your 100% value and how much does that count?

iGEM Hamburg: Yes, that's another big question. It's still a question of how exactly we want to measure this, because we can't always rely on the naked eye to estimate how much signal comes out in the end. These are all just a few more hardware things that we still must discuss.
Now on a molecular-genetic level everything is done from my side. I have one more question about the test at the end. You said you work with patient isolates. Are these blood tests or how does it work?

Heisig: It depends on what type of infection you have. If you have a urinary tract infection, then you are isolated from the urine, i.e. you take a urine sample - we don't do that directly ourselves, but of course that happens in the clinic, because the results should of course be available there immediately. Then you can centrifuge part of the urine – it's not about the number of cells, it's about isolating some in the first place. You can then buffer neutralize, you can also filter if necessary. You then isolate the cells and first place them on a culture medium so that they can grow, and you can separate them. Normally one assumes that one has a monospecific infection. This means you don't have 25 different germs that then all do something, but you usually have one germ that causes the symptoms. However, and this has to do with the way in which the urine sample is taken, there is always the chance that normal germs, e.g. from the skin flora, will also be in your sample. And if you now want to differentiate further, you can also use certain selective media, or universal media on which all can grow first, except for the anaerobes, and then select according to appropriate criteria. Then you only have the pure isolate. If you have a blood isolate, of course there is, like with sepsis, i.e. blood poisoning. You have the germs in your blood, you must take a blood sample first, then you must ensure that inhibitory substances – you also have the immune system which is active – are removed, you can also filter them and then the germs are isolated put on plates again. You must assume a pure culture. You must isolate beforehand; you cannot take the mixture.

iGEM Hamburg: I don't think we can do that anymore, but of course it would be very nice to test the system on real infections at some point.

Heisig: Well, then you need to talk to someone from the clinic, i.e. medical microbiology. For example, address Mr. Rode, he does this every day. Patient isolates, urine samples, skin swabs, everything from surgical areas, wound swabs, etc. or also from the ear, nose and throat area, samples are taken and pre-processed accordingly so that you can see the colonies without being disturbed by endogenous substances. And from these you would then create a culture, a pure culture. And that would then be your system, which you then must examine. And then the first thing to check is: is the phage you want to use or the phage mixture capable of infecting such germs? That would be the requirement. If that doesn't work, then of course you can't test anything. This is the first point. And the second would be: Do you have a target sequence in there that fits? That's the other story then.

iGEM Hamburg: Alright, thanks. Then the last point: Are you also involved in the process of approving antibiotics, we would of course be interested in approving detection tests, or do you know how that works?

Heisig: No, we don't do that ourselves. Basically, when it comes to antibiotics themselves, there are of course authorities. You can't do that in this frame. To do this, you would need a chemist who would then check these tests on the patient afterwards. This is less of a problem for the tests, because you are not treating the patient directly in any way or taking samples from them. You only must provide the technical specifications. That means that another authority would probably do the same. There is an institute for medical products where the testing would probably be evaluated at least and then test results would be available, which would be carried out under certain comprehensible and plausible conditions. And then that will be assessed if you intend to put that on the market. The other way for antibiotics is a hugely complex process through the clinical trials because they must go through several phases. You can count on an average of 8 to 10 years. When it comes to substances. When it comes to such tests, things can go relatively quickly. But you must apply for a clinical application. I think it also costs a little to prepare all these registration documents and to classify reports, etc.

iGEM Hamburg: Well, that's still a long way off for us anyway.

Heisig: I was just about to say that this cannot be done so quickly.

iGEM Hamburg: Yes, I've checked off all my questions. Do you have any comments that you could think of as feedback?

Heisig: So, if I see this correctly, your detection would be a fluorometric determination of a gene product that you release when the split ribozyme is activated.

iGEM Hamburg: Exactly, we might want to move away from the fluorescence signal again, so of course you need a device to be able to detect it, and we are still considering working with chromoproteins so that you can see the signal with the naked eye. But we want to test the system first. If everything works like this with GFP, then we want to move on to other proteins.

Heisig: And the expression then requires at least corresponding ribosomal binding sites, so basically you have already finished the whole thing at the mRNA level. You no longer need an additional promoter.

iGEM Hamburg: Yes. I don't even know if we're working with DNA or RNA. In any case, we have prepared a large construct. We do this with a Golden Gate assembly, so we can always switch promoters, ribosomal binding sites and everything and see when we have best rates. In any case, we have a complete construct so that the cell only has to read it.

Heisig: Good. This is yet another variable to consider, as classical E. coli promoters do not necessarily work in all endobacteria. You also must take that into account. Ultimately, the T7 system is structured in such a way that you have a specific interaction with the T7 promoter for the T7 RNA polymerase, so that you can then also achieve specific gene expression. Pairing that is another thing. However, the extent of expression would not only depend on how much of the corresponding transcription factors or transcript is released, but also on how far subsequent reactions are then expressed in the respective species via the regulatory sequences provided. So, it's not necessarily the case that if you were to process or test E. coli and another gram-negative organism in parallel, then using the same system, even if the same amount of RNA was present in both cells, you would see the same signals. It depends on how strong the expression of your test gene is and how well it works in the respective species via transcription, if you work with DNA, and then translation. Shine Dalgarno sequences are also suitable for the ribosomal conversion, which is not identical for every species.

iGEM Hamburg: Good objection, we have to talk about that again. You may notice that we are still very much in the planning phase and still have a lot to think about. Heisig: It's right that you look through it again beforehand and then examine it from different angles, because that's also a bit time-consuming. The question that I would otherwise ask myself from practice: What time factor do you have in mind? Because it is a great advantage if it can be done quickly.

iGEM Hamburg: Exactly, that's our goal, that we get a quicker test than with Hemmhof because you always have to wait for the cells to grow. We want to try to get that right without waiting too long. Our goal is a few hours at most. In the end, of course, it depends on how quickly the signal can be seen so that you can use it. We have also heard from another side, from another interview, that it is important that not so much work has to be done and that it runs relatively smoothly, because otherwise it is not efficient. These are the two factors that we want to combine. Our goal is that you schedule the test, then go away for a short time and get your result after 1-2 hours. That is our goal; we mainly want to improve the time factor.

Heisig: This is certainly an important point. The second is sensitivity. What is your minimum inoculum that you need to put in before you can even see any signal and distinguish it from background signal? That would certainly also be something that you would have to work out beforehand through series of experiments if you really wanted to allow something like this.

iGEM Hamburg: Yes. We are also in the process of considering how to amplify the signal so that it can be seen more quickly. But of course, that goes hand in hand with quantification. You must be able to relate it every time so that it can be used again and again. We are still facing a big question mark as to how we should do it.

Heisig: So, what you need is a standardized initial bacterial count. This is also crucial for the other tests. You must prepare a so-called inoculum from the outset, because it has been found with the methods of agar diffusion or serial dilution tests that you can get different results by increasing or decreasing the number of germs. That's logical too. If you use very little, even a small amount of antibiotic is enough to show a greater inhibitory effect. And conversely, if you set the inoculum too high, you won't be able to kill all the bacteria with the standard amount of antibiotic, you'll have to use more. Accordingly, the inhibitory concentrations that you determine go up or down. It would be similar here. That means you must have a threshold signal somewhere where you say I must reach at least that. But I must relate it to a certain starting cell number, which I must have at least to get this signal.

iGEM Hamburg: Thank you. Well, I think that would be it from my side, we already have a lot of objections for our next meetings. That was very interesting.

Heisig: With pleasure. It's just always good to check and think about it, because this is a real clinical problem to know in good time: When can I start therapy? In that case, as I said: compare commercial systems that can ultimately photometrically show growth inhibition within a few hours with the classic methods. We also have such a device that only tests cell growth, and you can use microtiter plates to follow in real time how the cells are multiplying and how fast it is. And then, of course, with this simpler approach, just throwing in antibiotics, you have the option of testing many different types of antibiotics in parallel. A high-resolution camera then runs over it and shows you a picture every 15 seconds. That can be absorbed, and you get a growth curve afterwards. You can then compare that to their standard, an antibiotic-free approach. And if there's a significant reduction in growth, you already have an indication that it seems to be working. You could build that in somehow, depending on how many phages you're using relative to the cells. And you would need a corresponding control.

iGEM Hamburg: Yes, exactly. We are currently assembling the system and we want to start to carry out the tests soon and then compare them with conventional ones. We haven't done that yet because we haven't gotten that far yet. But that's something we must do.

Heisig: So, you must establish it as such first. What you could do, at least from the clinically relevant resistance mechanisms, is to select individual genes, from the so-called CAT genes, a few representatives and then introduce genes into the cell first. And of course, that still depends on what kind of plasmid you have. Multicopy, single-copy, large, small, etc. You will then have more or fewer templates for the ribozymes accordingly.

iGEM Hamburg: Thank you. I'll bring that up. Otherwise, the fundamental question: Can we come back to you for further questions?

Heisig: You are welcome to do so, preferably via e-mail and, if necessary, you can also do something over the phone. Otherwise, you are welcome to use Zoom again.

iGEM Hamburg: That was a lot of helpful input. Many Thanks.

Heisig:Gladly, then I'll keep my fingers crossed that you could establish the proof of principle first. I'm curious to see what comes out of it if it works. If you still want to test clinical strains, we have some. Although chloramphenicol is not the best example, that is hardly ever used. If so, then in eye drops. Because it does have several side effects, it is now not the prime example of applicable antibiotics whose effectiveness would be tested. But it's a way to make it work.
I'm curious!

iGEM Hamburg: How do you evaluate the current development of antibiotic resistances and did the problem have improved so far or did it get worse since you started working as a doctor?

Montero: Since I was working as a doctor, I have been educated in this topic by other doctors. The base is that it’s going further. Nowadays we are trying to introduce new antibiotics because there are no more. The resistances are so strong that there are almost no new ones. But the bacteria are going further but we don’t. So we need to find out what to do to make a mix of many antibiotics. We are very late in that way.

iGEM Hamburg: Do you think that too many antibiotics are being prescribed to the patients despite there might be other ways to cure a sickness.

Montero: The problem with that is how some doctors are used to using antibiotics to center the patients' empiric treatments. One example for the problem with empiric treatments is that Cephalexin used to work well against bacteria. But nowadays almost everyone is resistant to this antibiotic because the doctors prescribed it too much.

iGEM Hamburg: Which future problems might develop due to antibiotic resistances?

Montero: I think the fear of doctors and teams in hospitals is to run out of options to help the patients. That we can not just solve it with antibiotics. So we need to invent new combinations of antibiotic treatments that may or may not work. But it should be something directly that you are sure of that it will work. Sometimes the doctors have no other solutions and have to find out what works.

iGEM Hamburg: Do you have any statistics that you can share about E. coli or antibiotic resistances in general?

Montero: For example there are so many resistances of the bacteria Streptococcus pneumoniae, Streptococcus pyogenes or Staphylococcus aureus. They and many other bacteria are very famous because they get resistant very fast even after surgeries and tracheostomies. Resistances occur to around 70%. Resistances can occur so fast and that’s why you always need to be careful with these patients. You need to be prepared and almost suspect what is coming. There are many mechanisms that do not work anymore.

iGEM Hamburg: Do you think that a new diagnostic method/tool can actually solve this problem?

Montero: Yes I do think that. It’s not enough to just hand out an antibiotic. The main part is to make a really good exploration. To get as further as you can to find out. We as doctors learn that 80 – 90% of the whole diagnosis should be clinical and the rest should be laboratory or different kinds of things. Trying to find it out with the clinic, that’s how you give this treatment. Sometimes the treatment is not good enough. Mostly we just do what we know and do an empiric treatment. It may work for a couple of weeks but then comes again and you treat again with another antibiotic but then this one also doesn’t work. That would be solved with a good exploration. Also looking at what is exactly there. There is also an antibiogram. It's this blood test that may also be of assistance of this bacteria resistance with antibiotics how it works all together. So of course it's like a whole paper with all the antibiotics you can use which are resistant and which are not and try to start from the base and go higher and higher. Not just like the most powerful because I know that will work. Because if this person creates resistance to this powerful antibiotic then what do you do? So it’s a good thing to make a good exploration and start from the base and never with the highest.

iGEM Hamburg: In which department are you currently working or did you work so far?

Montero: I was working in the emergency room. I was looking at general things in the patients. For example, infections. Every day I see patients infected by bacteria. Of course you don’t have to make a big treatment but you also need to use antibiotics.

iGEM Hamburg: Since when did you work as a doctor?

Montero: Since November last year (2021).

iGEM Hamburg: How do you diagnose a bacterial infection in your patients? How does that procedure work and how long does it take to diagnose?

Montero: It depends on many different things. Firstly it depends on which infection but the main part are symptoms that the patients come with. That could be fever for example which is pain and these kinds of things. Then you make this expiration if the patient has pain or not. If it’s positive, we always try to send blood tests. If we send the blood test, with special marks. We call them ‘bands’. It has marks onto which you can look and see if the patient has an infection or not. These bands give you a factor. This kind of factor says you can find out how the person has the infection. It depends on how high it is to send an antibiotic. Make an acute expiration and good diagnosis. And to send an antibiotic. When it is so high, try to send an empiric antibiotic. A very low one. So I will send an antibiotic that I know that works for this ‘flora’. It means how the bacteria grows from where it comes from. For example in the throat you think it would be staphylococcus aureus. In the stomach: E. coli. You know that there will be these types of bacteria. You do the test as best as you can and then send what’s right.

iGEM Hamburg: How long does the diagnosis take?

Montero: The test only takes minutes to one hour because sometimes it’s a mess in the emergency room labs. So with the laboratory you get the first results within one hour for the normal blood test to see if the person has an infection or not. If the person has this infection, you take another test that you already think about. When you realize that there is something strange, I would take this cool tip plus the normal general test. The general test detects an infection. Then the cool tip takes days. 1 to 2 days depending on what grows in this test. Sometimes it takes even longer. When there is really a resistance, this patient comes again one week after the treatment with the same problem, despite me sending this antibiotic. But then you have the test results of the cool tip. With this antibiogram-test you for example have 0.25 on resistance but it has already a bit. So you use it. Maybe create with complete resistance to this antibiotic. Then you have the results on what you can use for that plus what it growed before.

iGEM Hamburg: Which illnesses are caused by resistant bacteria that you might know about?

Montero: Mostly respiratory sickness. For example people with asthma or pocks or problems with the stomach intestines. The most illnesses appear in the respiratory system; Streptococcus in one year, staphylococcus aureus. These are famous for creating resistance very fast. There is even a whole one treatment of the last ones for this kind of this stem because this bacteria gets so far that we need to have one exact weapon that we could have to use for this which is called separolin. And of course you only use it when you can use nothing else.

iGEM Hamburg: Have there ever been bacteria resistant against this this antibiotic?

Montero: Yes

iGEM Hamburg: What do you do then?

Montero: You play with another kind of antibiotics. There are many formulas for antibiotics. There are penicillins for example; there are many. The problem is you need to play because the bacteria are divided into gram positive and gram negative. Some antibiotics only work only for gram positive and other antibiotics only work for gram negative. So you make a mix between for example gram positive – I need to use this separoline. It has a resistance but a bit. So you use it and combine it with another one. But of course there is always the fear that they might get resistant. But sometimes there is not more to do. That’s always the fear of these procedures or these moments.

iGEM Hamburg: Are the pathogens in the patients automatically or usually tested for antibiotic resistance?

Montero: Yes

iGEM Hamburg: Do you have any idea how often patients do get the wrong antibiotic prescribed? With wrong I mean that there was a resistance in the bacteria.

Montero: It happens every day. Sometimes it’s hard because there is not enough time to treat the patients. Sometimes we doctors need to work fast because the system obligates us to have some quantity of patients per hour. So we need to find it out fast which should not be. We should have enough time to help the patients the best we can. Around 20 – 30 % of the patients with a bad treatment will create a resistance.

iGEM Hamburg: Had any treatment with the wrong antibiotic had consequences when given the wrong antibiotic?

Montero: Usually there is no problem. However there might be secondary effects. For example allergies or low tolerance that affects another part. Antibiotics do not work against resistances and have an effect like water in this case. You lose your time there.

iGEM Hamburg: Which alternatives to antibiotics do you know about? Do you use any alternatives for your patients?

Montero: We use a special combination of antibiotics. For example we use “martyred” antibiotics. One will sacrifice itself and the other one works. We try to play with that. When we observe a resistance, we use these kinds of combinations. One antibiotic immediately stops or fights the bacteria and there is a special window that the other antibiotics go. That’s how we somehow try to manage to help a patient with that.

iGEM Hamburg: You might hear in the news that harmless infections can be harmful due to resistant bacteria. Do you have any experiences or examples of your workplace?

Montero: For example some people that come to the hospitals have infections in their skin or on the foot. It happens with Streptococcus or Pseudomonas which also develop so much resistance that sometimes it’s hard to control. The problem is that when the patient comes and has a bad treatment or patients with wounds can spread the infection. We try to control it and to know when this happens and try to prepare the place. Sometimes mistakes happen because of poor management. Sometimes the patient has to be isolated in a special room.

iGEM Hamburg: Where do you see the most significant malfunction or failure of antibiotics?

Montero: The biggest problem is after surgeries. There is always the fear of complications during surgeries. They could get infected with bacteria. The problem with tracheotomy and bandages and you will not change it every day because it’s painful. You always clean it but sometimes even that is not enough. A high percentage could catch something. That’s why you always give the patient a treatment with antibiotics, which is a factor in the development of antibiotic resistances. The internist and the surgeons have different opinions. The surgeons send an antibiotic thinking “this will work” but the doctor of the internist always asks “why?”. There is never a good connection because one doctor creates resistance and the other one must solve it. Of course you need to send an antibiotic because there is the risk of infection but sometimes this will create a resistance.

iGEM Hamburg: Do you have any idea on which actions might be necessary to reduce the increase of antibiotic resistances?

Montero: I would say from the primary attention. From the base when the patient comes to the consultation with an infection. That could be solved with something other than an antibiotic. Of course you give a treatment and help the patient with their pain but some colleges are used to just instantly sending an antibiotic because that might solve it. And yes it will solve it but a resistance might follow. Sometimes it’s not necessary. The whole body works alone to create antibodies to work against the bacteria. First it’s good to use something else and give the body some days to solve it. Also natural things are good, they won't affect the body. I think it’s good to try to use something else first and if the infections stays and then slowly begin to give antibiotics to the patient.

iGEM Hamburg: Do you see any way of fighting the problem of antibiotic resistances in a general way?

Montero: Some patients save antibiotics at home. It happens around the world that patients stop taking antibiotics because they feel better. This means that the patient didn’t finish the antibiotic. When they feel sick again, they will use it again. This is also a way to create resistance. The bacteria “learn” how the antibiotic works and it doesn’t work anymore.

iGEM Hamburg: What do you think are the biggest challenges for the future when it comes to diagnostic of antibiotic resistances?

Montero: The biggest challenge is to find out new antibiotics because the bacteria develop faster than we do. Also detection of antibiotic resistances should be faster because it can take up to one or even two weeks.

iGEM Hamburg: We want to detect antibiotic resistances by RNA detection by using a split ribozyme, which will cut itself out when it detects the mRNA of the antibiotic resistance. This should lead to an easy and fast test which can be implemented in hospitals. What do you think of the idea of research towards the detection of bacteria and known antibiotic resistances?

Montero: I think it’s good trying to find new ways to detect it and not just the ones we have because maybe it works. Even if that means that it will have higher costs because we are dealing with the lives of patients and that has no cost. It’s good to have alternatives and new ways to solve this problem.

iGEM Hamburg: Do you see any advantages of the very early detection of antibiotic resistances?

Montero: Yes that will replace the empiric treatment which has a generalized problem of only detecting gram positive bacteria in a general and not specific ones. This way I could send a specific antibiotic that will work in this bacteria.

iGEM Hamburg: Do you see any problems in our project or any failure that could occur?

Montero: There are always risks that it could not work but that doesn’t mean it’s not smart to try it. It's not a way that would have a big impact. Maybe it won’t work at the start or it will take longer than expected or even spend money for your project. When it does not work, there is no difference for the patients, but if it does, then that’s perfect.

iGEM Hamburg: Do you have any tips for us or do you know any other research groups that might work on something similar?

Montero: I don’t know other groups working on this approach. Regarding tips, you should find out how all the bacteria and antibiotics work, try to make the statistics of the factors that are creating more resistance nowadays. I think after surgeries the first reason that gets resistance is because of the treatment afterwards. It’s a good start because many people are trying to find out how to solve this problem. I think there are some fears of what could happen. Your approach will be very useful when it works. It won’t have disadvantages for the patients because it’s from the same sample that we take from the patients already. In the end your approach can be compared to the one we are using in hospitals and that’s how you find out new different ways to help the patients. But overall, I think it’s very good.

iGEM Hamburg: How do you assess the current development? Has the problem improved/worsened/stagnated in recent years?

Tomczyk: Antibiotic resistance is one of the greatest current challenges. The implementation of the German antibiotic resistance strategy by the German Ministry of Health or the international antibiotic stewardship programs take this into account. If you look more closely at the data, the situation with Klebsiella seems to be getting worse, while resistance from pseudomonas or MRSA is declining ¹. The topic of antibiotic resistance will certainly remain highly topical in the years to come.

iGEM Hamburg: Do you feel that too many antibiotics are being prescribed even though there are other ways?

Tomczyk: We often experience in the inpatient sector that infections of the upper respiratory tract, i.e. colds, which usually take a mild and self-limiting course and are also >90% of viral origin, are treated with antibiotics on an outpatient basis. However, due to the viral genesis of the infections, these cannot develop any effect and contribute to a deterioration in the resistance situation.
In the hospital setting, complete diagnostics are very important for unclear infections. Depending on the focus of infection, narrow-spectrum antibiotics such as penicillin W/V are sufficient for erysipelas. In the case of a pure increase in the laboratory-chemical inflammation parameters, in particular an increase in CRP, it is important to differentiate this from viral infections, mycoses, diseases of the rheumatic type or tumors. Unnecessary antibiotics can be reduced in this way.

iGEM Hamburg: What current/future problems do you see for us due to resistant germs?

Tomczyk: If antibiotics are not used in a targeted and rational manner, the resistance situation threatens to worsen. The result would be that common antibiotics would not work in critically ill patients, e.g. those with Klebsiella pneumonia. If the bacterium develops resistance to, for example, the frequently administered aminopenicillins, escalation to reserve antibiotics such as carbapenems is necessary. However, this costs crucial time in fighting an infection. The situation is even more serious if the microbiological findings also show resistance to reserve antibiotics. Early effective antibiotic therapy can make a significant contribution to a patient's survival in critical patients.

iGEM Hamburg: Do you know some statistics on E.coli and resistance in Germany?

Tomczyk: The resistance situation in E.coli currently seems to be stabilizing with a slightly declining trend in the resistance rate. ² A high proportion of E. coli pathogens is resistant to aminopenicillins, almost 50%, with cephalosporins the rate is around 10-15%, fortunately there is still resistance to carbapenems today Rarity.

iGEM Hamburg: When did resistance start to develop? Did the problem develop this way recently or has it always been there?

Tomczyk: A distinction must be made between natural and acquired resistance. Natural resistance existed before the introduction of antibiotics³.
With the introduction of antibiotics, however, the bacteria developed increasing resistance. Already at the beginning of the antibiotic era it was seen that bacteria are capable of developing resistance within 5 years³.
Today, overuse of antibiotics in animal husbandry as well as in veterinary/veterinary medicine increases the resistance problem.

iGEM Hamburg: What alternatives to antibiotics do you see?

Tomczyk: On the one hand, it is possible to reduce the risk of infection with suitable behavioral measures. To prevent enteral infections, this includes washing food, regular hand disinfection and stool hygiene, urinary tract infections, intimate and sexual hygiene, and in the case of severe respiratory infections, respiratory masks reduce the risk of infection, and not just since Covid-19. On the other hand, infections such as asymptomatic bacteriuria do not require any pharmacological therapy if risk factors are not present. Vaccinations play an essential role in prophylaxis against infections, from the bacterial spectrum the pneumococcal vaccination should be mentioned here.

iGEM Hamburg: In which department do you work?

Tomczyk: I work as an internist in a maximum care hospital, currently in the central emergency room, previously two years as a ward doctor in various specialized internal medicine clinics.

iGEM Hamburg: How long have you been working as a doctor?

Tomczyk: 2 ½ years. Before that, I worked on my doctorate for a long time.

iGEM Hamburg: How do you diagnose bacterial infection in your patients? What is the procedure for this?

Tomczyk: For standard diagnostics in suspected infection, with a comprehensive anamnesis and a precise physical examination. Serological examinations (inflammation parameters, liver and pancreas enzymes or kidney retention values), taking blood cultures, urine cultures, determining the urine status, and often a chest X-ray after a previous indication check, usually include an abdominal sonography as well Procedure. Depending on the clinical symptoms, further diagnostic steps such as a sputum examination or an echocardiography are required, especially if the findings are unclear. If the focus of infection is clinically clear, the number of diagnostic procedures can be reduced. The microbiological examination of the blood cultures is of essential importance. iGEM Hamburg: How long does the diagnosis take?

Tomczyk: It often takes 5-7 days to receive final blood culture results. If the result is positive, we usually receive preliminary results about the detected bacterium earlier, but we wait days for an antibiogram to be available, since the incubation of the bacteria in the laboratory incubator takes this time.

iGEM Hamburg: What diseases are caused by resistant germs?

Tomczyk: In principle, resistant bacteria can occur at any infection focus and create difficult therapeutic conditions there. Common infections include urinary tract infections, pyelonephritis, cholangitis, colitis, pneumonia, deep and upper respiratory tract infections, sinusitis, pelvic inflammatory disease, appendicitis, spondylodiscitis, or abscesses. Particularly severe infections can take a septic course regardless of the site of infection.

iGEM Hamburg: Are the pathogens automatically tested for antibiotic resistance?

Tomczyk: The pathogens tested in the blood and urine cultures are automatically tested for antibiotic resistance, as is the case with skin swabs.

iGEM Hamburg: How often is it that patients are prescribed an antibiotic to which they are resistant?

Tomczyk: I don't have exact data on the resistance rate. I would estimate that we have to switch about 15-20% of the antibiotics after we have the results of a culture breeding because of resistance in the antibiogram.

iGEM Hamburg: What can happen if the patient receives an antibiotic to which the bacterium shows resistance before the antibiogram is known?

Tomczyk: If the patient has an infection, e.g. of the bile ducts, pneumonia or a urinary tract infection and only receives sensitive antibiotics late due to resistance, life-threatening sepsis can develop with changes in vigilance, dyspnea or circulatory depression. Such patients must then be transferred to an intensive care unit, receive intubation with mechanical ventilation or catecholamine perfusor therapy as required. These patients fight for their survival, even with maximum therapy, the prognosis is often critical. Unfortunately, not all patients make it, the lethality of sepsis is high.

iGEM Hamburg: It is often said that harmless infectious diseases can cause problems due to resistant bacteria. Can you give an example?

Tomczyk: A large part of the population shows non-pathological colonization of the skin, e.g. in the nose and throat area, by S. aureus. Especially in the inpatient sector with increased use of antibiotics, resistance develops, the so-called MRSA. If an endogenous infection occurs, for example triggered by immunosuppression, or an exogenous infection through transmission, the diverse resistances can pose a real therapeutic problem.

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