# Antibiotics resistance **Channel:** M A **Source:** https://www.youtube.com/watch?v=xB8cl8Gaw6s **Transcript page:** https://www.withtranscript.ai/video/xB8cl8Gaw6s ## Chapters - 0:00 — Why Antibiotic Resistance Matters - 6:27 — Return to the Post-Antibiotic Era - 10:47 — How Resistance Evolves in Populations - 17:13 — Preventing Resistance Through Proper Use - 25:06 — Medical Practice and Resistance Risks - 34:56 — Hospitals, Sewage, and Global Spread - 41:44 — Plasmids and Gene Transfer - 47:00 — Main Mechanisms of Drug Resistance - 53:24 — Beta-Lactamase Inhibitors Explained - 59:56 — Ribosome Targets and Efflux Pumps - 1:12:43 — NDM-1 and Last-Resort Drugs - 1:19:23 — How to Slow Resistance Development - 1:27:29 — Future Solutions and Phage Therapy - 1:31:20 — Living with Microbial Evolution ## Transcript **[0:00] Speaker A:** In the previous lecture, we concentrated on pathogenic microbes and the way we can stop them by antibiotics. Today, we will see that microbes can fight back against those drugs, a problem that brought us back to the days before antibiotics were available in quite a few cases. **[0:42] Speaker A:** So, in the last lecture, we discussed several antimicrobial drugs. They reduced dramatically the morbidity and the mortality from infectious diseases, especially in the developed world. Let's take tuberculosis as an example. **[1:17] Speaker A:** Before antibiotics, tuberculosis was one of the leading causes of death. People infected each other, but with the introduction of antibiotics, TB, tuberculosis, was practically out of the way in developed countries. By treating the infected people with the appropriate set of antibiotics, we were able to stop the spread of the disease. No effective vaccines is available right now, just antibiotics. **[2:14] Speaker A:** However, today we are back to square one. 40% of Mycobacterium tuberculosis is resistant to some or even all antibiotics that we have against TB. We are back to the time that an effective treatment in those cases is no longer available. Tuberculosis is only one out of many bacteria that become antibiotic-resistant. Just to name a few, Acinetobacter baumannii is a pathogen that came to the United States in 2003 or so with a wounded soldier that got infected with this bacteria in Iraq. **[3:30] Speaker A:** It was found to be a bacteria which is resistant to a lot of different antibiotics, and it was spread in the hospital and later on in other hospitals. **[3:50] Speaker A:** Another example is Pseudomonas aeruginosa, it can pick up DNA from a lot of other organisms and can develop antibiotic resistance. Klebsiella pneumoniae, in 2010, this bacteria was brought from India with drug resistance to make all the beta-lactam antibiotics to be useless. One bacteria made like 70% of the drugs that we are using on a regular basis to be useless. Staphylococcus aureus, we talked about earlier as MRSA, But actually, Staph aureus can be resistant to vancomycin as well. VRSA, so vancomycin-resistant Staph aureus. Since it is a pathogenic organism by itself, having a pathogenic organism that we cannot really treat with antibiotics, making the things to be even more difficult. Now, those are very, very few drug-resistant bacterial cells that have resistance to more than one drug at a time— multidrug-resistant strains— and the list is far longer than what you see here. Every year we have another group of bacterial cells which are resistant to antibiotics. **[5:50] Speaker A:** So even those bacteria that 2 years ago were considered to be okay, we can still treat the patient with antibiotics, well, a year later it is not okay. Dr. Margaret Chan, that was the Director-General of the WHO, said the following: we risk entering into a post-antibiotic era, which means in effect the end to modern medicine as we know it. **[6:27] Speaker A:** Margaret Chan was right And just to show you an example for this, today we need to go back to the old days where the treatment for bacterial infection was surgical operation. Today we have cases that we need to do surgical treatment for infectious diseases like in this one to pick up the infected parts in the body where antibiotics are not working. In the old days, the treatment of tuberculosis in some cases was a surgery to pick up the infected part of the lung. That's of course something that we don't want to do because the other option that we have, antibiotics, it's, it's much more easy to perform and it is more safe. In general, the concept of surgical operation to remove the infected part of the lung in tuberculosis or in this case to take out the Staph aureus infection, that's actually bringing us back to the old days before the time of antibiotics. **[8:05] Speaker B:** Most of the antibiotics that we have at the moment we've been using for almost 50 years, and in 50 years' time microbes have learned how to live with them. There are microbes that are found in hospitals that are causing severe infections in human beings that are resistant to every antibiotic that we know. They cannot be treated. **[8:26] Speaker C:** Tuberculosis, long considered under control in the developed world, is returning. So are many other illnesses we thought antibiotics could cure. **[8:38] Speaker C:** Even though antibiotics work in different ways, microbes are outsmarting them. One by one. **[8:48] Speaker C:** Disease-causing bacteria colonize living tissue. **[8:57] Speaker C:** Penicillin kills the bacteria by weakening their cell walls. When the walls collapse, the bacteria die. **[9:10] Speaker C:** The patient's disease symptoms subside. **[9:22] Speaker C:** Microbes reproduce by dividing in 2, replicating themselves exactly each time. But mutations, the random genetic changes in all living things, are seen more frequently in microbes because they reproduce so rapidly, as often as every 12 minutes. When a mutation enables a microbe to fend off an antibiotic attack, all the better for the new stronger microbe and all the worse for us. As antibiotics kill off the susceptible microbes, the resistant ones can multiply quickly without competition. So the more antibiotics we use, the more the playing field is cleared for antibiotic-resistant bacteria. **[10:12] Speaker B:** People ask for antibiotics when they shouldn't need antibiotics. Doctors prescribe antibiotics when they should not. As much as 30 to 40% of prescriptions written by family physicians are inappropriate. You don't give antibiotics for a cold. **[10:29] Speaker C:** Antibiotic resistance has reached crisis proportions. **[10:35] Speaker C:** Around the world, thousands of people are dying of infections that existing antibiotics can't touch. **[10:47] Speaker A:** So, how drug-resistant organisms are developed? So, when you are looking on a population of microbes, you are going to see some variability in their ability to become sensitive or resistant to antibiotics. It's not all or none. Are different levels, that microbes are more drug-resistant than others. Some are more sensitive, others are less sensitive. **[11:33] Speaker A:** So, let's say that in this population, the green are drug-sensitive bacterial cells. You can kill them quite easily in— with the short treatment of antibiotics, and the red are more resistant to this drug, and you need a longer period of time in order to kill them as well. The problem is when we are using the drugs not according to the prescription. The prescription is based on the fact that there are drug-resistant mutants in the population, and you need to take the drugs for 7 or maybe 10 days in order to kill them. Now, drug-sensitive bacterial cells are going to be killed much, much, much earlier. **[12:41] Speaker A:** If for some reason you are using low concentration of the drug or using the drug for a shorter duration than prescribed, you are creating a problem, and this problem is not going to be expressed with the majority of the cells. Because the drug-sensitive bacterial cells are going to be killed after 2 days of treatment, even with low concentration of the drug. They are sensitive. The problem here is those bacteria which are more resistant. They have some mutations and they happen to be more resistant than their peers. **[13:34] Speaker A:** And they are going to be left behind. When you stop the treatment early or when you are using low concentration of antibiotics, you're keeping those red drug-resistant mutants in place while all the others are gone. So, low concentration of the drug or using drug for short time is not going to kill the drug-resistant cells, but when the drug is out, they are the only ones that are going to be left behind. Those pathogens that were left behind The drug-resistant mutants continue to grow even better due to the increase in resources that their weaker counterparts, the drug-sensitive bacteria, would have competed for. Now they are all by themselves, all the space, all the nutrients, It's just for them. So, low concentration of antibiotics provides a selective advantage for the resistant bacteria. We are giving them a better environment. We are giving them a better chance to populate our throat. **[15:25] Speaker A:** And next time that you're going to have sore throat, it will be with the red guys, and the treatment that you had here at the first place is not going to be relevant any longer. That's the way that we can develop antibiotic resistance. **[15:50] Speaker A:** And those type of bacteria, if you are infecting someone, they are going to be the majority among the infected agents. So other people will get drug-resistant organism as the infection from you that you were not— took the antibiotics appropriately. **[16:19] Speaker A:** We are talking here about simple evolution. So low concentration of antibiotics induced fast Darwinian evolution towards antibiotic resistance. Overnight. Normally, it should take years for bacterial cells to develop antibiotic resistance from bacteria which is not resistant to the antibiotics. **[16:52] Speaker A:** So, when you are using low concentration of the drug or using the drug for short duration, We are selecting for the drug-resistant strains. **[17:13] Speaker A:** So, how we can slow down the development of drug-resistant organisms in the population? Well, I mean, take the drug according to the prescription. When you are using the drug appropriately, with appropriate concentration, with appropriate duration of the treatment, you're going to kill also the drug-resistant mutants. Again, it's not all or none. If you will put them under a stress of high concentration and long duration, the drug-resistant mutants are going to be killed as well. So, this way you're going to come back to square one when you are going to have the bacterial cells in your throat. Most of them are going to be drug-sensitive bacterial cells, few are going to be drug-resistant mutants that maybe were evolved during the time that those bacterial cell populations is recovered. **[18:33] Speaker A:** So, a high concentration of the drug plus long treatment are likely to kill the drug-resistant pathogens as well. Now, in most of the cases Leave the bacteria alone. Do not use antibiotics unless it is necessary. And if you will go to see the doctor, the doctor will take a sample from the infection, and the doctor will be able also to see whether or not the infection is bacterial infection. Maybe it's a viral infection. **[19:16] Speaker A:** A lot of time they feel more or less the same. So, if this is a viral infection, antibiotics is not relevant, but when it is necessary, treat with high concentration of the drug and long enough duration. In many cases, That should be the decision of the doctor, although many people have leftover antibiotics and, you know, I mean, nobody can stop you to use the drug on your own, and that's probably not the best idea. In this case, you can be involved with development of drug resistance in your own throat. If the treatment is not done appropriately. **[20:15] Speaker A:** So, slow evolution means that it can take years for the antibiotic resistance to be developed, and when a new antibiotic is becoming available for treatment, you can see that after several years, some sort of antibiotic resistance, even if the drug is used appropriately, but it takes time, a lot of time. When you're using the drug inappropriately, the antibiotic-resistant strains are going to take over during one night. 24 hours later, you have drug-resistant strains in your throat. Who should be blamed for antibiotic resistance? **[21:15] Speaker A:** Antibiotics were discovered like 80 years ago, and since that time we abuse them, we misuse them big time. The most important abuse of antibiotics was done by farmers. **[21:46] Speaker A:** 80% of all antibiotics are used on farm animals, but it's not to cure them from infectious diseases. Those antibiotics are used as growth promoters. Here we are taking a very interesting character of antibiotics. If you are using antibiotics at low concentration for long period of time, you are going to gain weight. And we found this first in the farm, in the chicken farms that were treated with antibiotics. **[22:39] Speaker A:** And from here, antibiotics is used pretty much constantly as growth promoters in order to give the farmers some extra financial benefits. Farmers found that if you are giving your farm animals low concentration of antibiotics on a regular basis on their feed, you know, they are going to grow faster, and that's the main reason for the development of antibiotic resistance. We can see a sign that MRSA was developed in a certain farm and distributed from there. If you have development of antibiotic-resistant bacteria in the farm, they are going to move elsewhere. Throughout the water, throughout the manure of the cows or the chickens that is used again in the backyards or in the farms where you're growing vegetables, and there is a chance that you will find those antibiotic resistance elsewhere. In general, The farmers that are using antibiotics, they are doing it for financial reasons, and it's very difficult to stop this process by the lawmakers. **[24:25] Speaker A:** In the United States, it's very, very difficult to do so. In some parts of Europe, we were— we are more successful at this level. But the main problem of antibiotic resistance is about using the antibiotics at low concentration, and when you are talking about 80% of the antibiotic use this way, you can probably get quite a good idea that this is the main place that antibiotic resistance can be developed. **[25:06] Speaker A:** Beside the farming industry, we can also look on some protocols related to the way that we are doing medicine. **[25:20] Speaker A:** I will give you an example: Streptococcus hemolyticus beta, strep A, is a bacterial cell that can infect our throat, can cause a strep throat, and when you are going to see the doctor and the doctor will get the idea that that's the problem, the doctor is going to put you on antibiotics pretty much immediately. You're going to get the first drug in the doctor's office, and then you're going to be sent downstairs to the pharmacy to pick up the prescription and to start the treatment immediately. Why? Why are we so worried about strep throat? The streptococcus have a certain protein on its surface. **[26:28] Speaker A:** It's called the M protein, which happens to have some similarities to proteins that we have on our heart and other places as well. Now, if you're going to be infected with the strep throat for a long period of time, you are going to develop immunity against this pathogen, and the immune system that is fighting the pathogen— after the pathogen is gone, the immune system is going to turn against your heart. This is a disease that's called rheumatic fever. It's an inflammatory disease, autoimmune disease, that may develop after an infection with Group A Streptococcus, such as strep throat or scarlet fever. The disease can affect the heart, joints, skin, and also the brain. **[27:54] Speaker A:** So, the most important way to prevent rheumatic fever is by getting quick treatment for the strep throat and scarlet fever. So, you're going to get the antibiotics to prevent autoimmune disease, and the treatment is going to start pretty much immediately after the doctor gets the idea that you have Streptococcus A, and then the doctor will send the sample to the lab to get some more information For example, to check what is the susceptibility of the bacteria for antibiotics. And there are situations that the antibiotic that you are using for 2 or 3 days, that you get the prescription from the physician, is not the right antibiotic. This bacteria specifically can have some sort of antibiotic resistance against these specific antibiotics, and the doctor is going to ask you to stop to take antibiotic A, the one that he gave you earlier, and to start the treatment with another antibiotic. **[29:20] Speaker A:** Now, all bacterial cells in your body were exposed to antibiotic A, the first antibiotic, And the treatment is very, very short in this case. **[29:35] Speaker A:** Well, antibiotic resistance can be developed in such a situation. Well, that's the protocol, and the protocol is for a reason, but here you are increasing the chance to come with antibiotic resistance. There are situations that the drug is not working for some other reasons. A person that was treated with penicillin for many years, one day is coming with allergy to penicillin. It's when you think that there is an allergic reaction being developed, the treatment must be stopped. **[30:19] Speaker A:** There are examples in tuberculosis that some people are coming to take those treatments when their condition is quite bad, and in some situations the treatment itself, which is done supervised in a medical facility, needs to be stopped in order to let the patients to get better. And that's another situation that you can develop antibiotic resistance against drugs that were used just for short period of time. There is another things that you can find from time to time, and, well, I mean, some doctors are kind of almost forced to give antibiotics by the patient. Let me give you an example. The young mother is coming with her baby to the doctor office. **[31:29] Speaker A:** The doctor office is occupied with a lot of other patients. She needs to wait like 2 hours in the waiting room and the child is driving her crazy. And then she's coming to see the doctor with the child. The doctor gave a quick look to the throat of the child and say, well, I mean, it's a viral infection, take a day or two off, the child is going to be fine really soon on his own. And the mother say, well, I'm not waiting in this waiting room for 2 hours in order to be sent home. I want something, I want antibiotics. The doctor said, well, I mean, antibiotics not going to work in this case. The mother insists, I want antibiotics to treat my child, and if not, I'm going to the nearby walking clinic and I will get it there. And the doctor have some other patients, and there is another thing. **[32:39] Speaker A:** When the doctor can identify viral infection, it's not 100%, it's like, let's say, 95% correct in most cases. There are situations that the bacterial infection is still at the very early stages, so you don't see the pus and other things that normally you can see in cases of bacterial infection. And yes, many doctors probably will admit to you that in some cases they gave the antibiotics although they were not sure at all that it was necessary. So, the mother got the antibiotics for the child And the child got the first treatment, 1 day, 2 days, and well, I mean, it was a viral infection, and 2 days afterwards the child is playing with friends outside, everything is just fine, as predicted by the physician in the first place, and the mother thinks for herself, maybe Well, I mean, should I give the child the rest of the prescription? **[34:00] Speaker A:** The child is fine. I mean, the child is healthy. **[34:03] Speaker A:** Why should I give the child the rest of the drugs that the child needs to get for 10 days when the child is absolutely healthy? And in some cases, the decision will be to stop the antibiotics. That's shorter duration, which can lead to antibiotic resistance. **[34:27] Speaker A:** So, that's another problem that you can see. Yes, I mean, we are using drugs, oral drugs, under the basic responsibility to follow the rules of getting the drugs, And some people are doing so, many people are not. **[34:56] Speaker A:** And hospitals— yes, in the hospitals we are using a lot of antibiotics, and when you have antibiotic treatments, some of the antibiotics are going to be secreted normally to the sewage of the hospital, and here you have some level of antibiotics that are coming from a lot of other patients as well, and you have a lot of bacterial cells that are part of your gut and everybody else. On top of this, in the hospital you can find sometimes mutagens that we are using as drugs for cancer, for example, and here again they can reach eventually to the sewage. There are some drugs like methotrexate that have a very, very strong color and The patients that are getting this, when they're going to the washroom, you can see the drug in the washroom that is going to the sewage. Now, when you have drugs that are making mutations together with antibiotics, the same environment, you have an excellent place to develop antibiotic resistance. **[36:33] Speaker A:** Bacterial cells are going to be affected by the mutagens by developing mutations. Some of the mutations are going to be involved with antibiotic resistance. You have a lot of bacterial cells that can exchange pieces of genetic material in between themselves. So, we have all the reasons that the bacteria in the sewage of the hospital, and of course in other places, can be the most aggressive bacterial cells in terms of antibiotic resistance. When you are in the hospital, you really don't want to get an infection with hospital bacteria. So that's the reason that today if you are having a surgical operation of some kind, you're going to be kicked out from the hospital very, very quickly to go home where it's more safe. **[37:43] Speaker A:** Who should be blamed for antibiotic resistance? Well, in North America we have quite clear policy about antibiotics. In order to get a prescription drug like antibiotics, you need to go to the physician and it's going to be reported. But in many places around the globe, things are very different. They have no antibiotics policy. **[38:16] Speaker A:** I mean, in some places like India, You can go to the corner store and you can buy any drug that you have in mind. You can get 3 pills instead of the entire treatment. You're going to be treated for 1 day. An excellent way to develop antibiotic resistance. And that's another major issue because when you have antibiotic resistance in one place, it can go quite easily to other parts of the world. **[38:58] Speaker A:** Who else should be blamed for antibiotic resistance? Antibiotics were developed from natural product. Well, I mean, bacterial cells are using antibiotics against each other for billions of years, and they also learned how to fight those antibiotics. So, the antidote for antibiotics is part of the life of bacterial cells. They have the genes to develop antibiotic resistance. **[39:47] Speaker A:** The idea that the antibiotics were developed from a natural product that is part of the evolution of microbes and other organisms made the antibiotic resistance to be quite obvious from day one. Alexander Fleming discovered the first antibiotic resistant against his own penicillin, and he predicted that that's going to be the case for all antibiotics developed. And the reason, well, that's a natural product, and bacterial cells have all the reasons to fight the drug by their antidote. So bacteria are exposed to antibiotics for billions of years. They acquire the ability to be resistant to antibiotics as part of their competition with other microbes. The evolution of microbes is based on their ability to fight antibiotics. It's not our invention. Bacterial cells and other microbes invented antibiotics long, long, long time ago before us. And the selective pressure which makes the microbes that have some better ability to fight antibiotics to make up the majority. **[41:44] Speaker A:** Microbes have their own genetic internet. They invented the share we have in Facebook. Yeah, they know how to share something that they really like— plasmids. Those resistant plasmids, our plasmids, are there to be exchanged very easily. Those plasmids are expressing genes for antibiotic resistance And those type of genes can find their way from non-pathogenic bacteria to pathogenic neighbors. So that's the way that microbes can give each other something that works for them. That's part of the evolution of microbes. **[42:52] Speaker A:** So, yes, we misused, we abused antibiotics quite significantly, and as the result of this, most of the antibiotics were able to stay before antibiotic resistance was discovered only for a short period of time. So, you can see that in some cases the time that the antibiotic was discovered versus the resistance observed can be few years. **[43:36] Speaker A:** So, methicillin, for example, Only one year. So, there are some interesting examples of a drug that was able to stay protected from antibiotic resistance for quite a while, and this is vancomycin. Since the time that vancomycin was discovered, it was used in quite responsible way by using it only when it is necessary and only in the hospitals, and indeed this drug was without any reported resistance for like 30 years or so. And that should be the time that you have antibiotics without antibiotic resistance in order to be able to use antibiotics appropriately. So, as a result of misusing antibiotics, they usually have quite a short life before resistance kicks in. **[44:59] Speaker A:** And vancomycin, on the other hand, was treated with more respect. This drug was used only when it was necessary. As a result of this, the drug was available without any reported resistance for over 30 years. The development of resistant organisms in the population. Resistance by bacteria acquired in 2 ways: new mutations of chromosomal genes. This process, as we talked earlier, is quite slow. You need to accumulate several mutations in a certain gene before you will have an antibiotic-resistant organism. So it's multiple steps of mutations. And the gene transfer, where the acquisition of the resistant plasmid, our plasmid, via transformation, transduction, and conjugation. And this process is fast. **[46:29] Speaker A:** You're getting the entire package at once. It is single step. All you need is that the bacterial cell will be able to transmit the DNA to another bacteria. There are several ways that antibiotic resistance can be acquired. **[47:00] Speaker A:** So, just to mention some of the strategies that microbes use to develop resistance to antimicrobial drugs: Let's start with the inactivation of the drug. **[47:19] Speaker A:** Beta-lactam antibiotics can destroy the beta-lactam ring. Aminoglycosides can be inactivated. **[47:36] Speaker A:** Other examples is the efflux pump. Bacterial cells, especially gram-negative, have the ability to pick up the antibiotics that just enter the cell and to throw it out by using a certain pump. So bacterial cells like gram-negative are going to pick up the antibiotics in the periplasmic space, and over there, there are a lot of different pumps that can put this drug back outside of the cell. We have an efflux pump to tetracycline, that was discovered first, but then we realized that we have efflux pumps to pretty much every other antibiotic. **[48:40] Speaker A:** Bacterial cells can sense what is going to kill them and they're going to resist. **[48:49] Speaker A:** Bacterial cells can block the penetration of some drugs And bacterial cells can modify the target of the drug. Every drug needs to have a certain target. **[49:14] Speaker A:** Drugs that are going to inhibit a protein need to bind to this protein in some way. So, their target protein can be modified so the drug will not be able to bind to the target protein. And that's another thing that bacterial cells know how to do it, by mutations in most of the cases. They can change the recognition site of the drug in such a way that the drug will not be able to be recognized, and then it's going to be not effective against this microbe. **[50:03] Speaker A:** So, let's go to some examples. Inactivation of microbes— bacterial cells can come with enzymes that alter the antibiotic to inactivate its function. For example, they can simply modify the drug by adding a certain chemical group to the drug. Chloramphenicol acetyltransferase, the name telling us that We have an enzyme that knows how to add an acetyl group to chloramphenicol, stopping the chloramphenicol to be active on ribosomes. Or degradation— beta-lactamases or penicillases. Well, in each case, You're talking about enzymes that know how to destroy the beta-lactam ring. And the idea is working as follows. Earlier we discussed the importance of the beta-lactam ring in all the beta-lactam antibiotics. Penicillin, cephalosporins, monobactam, carbapenems— all of them have beta-lactam ring, which is the active portion of the drug. The way that beta-lactamases are working, they are cutting the bond over here. **[52:01] Speaker A:** In the beta-lactam ring. In other words, turning it to be not a closed ring. So this is the side of the penicillases' action. They are simply breaking the beta-lactam ring, making these antibiotics to be not effective. In general, this is penicillin, an active penicillin. **[52:34] Speaker A:** This is inactive penicillin when the beta-lactam ring was broken. **[52:46] Speaker D:** Gram-positive bacteria release the beta-lactamase enzyme from the cell into the extracellular environment where it inactivates the drug before it enters the bacterial cell. In contrast, gram-negative bacteria retain the beta-lactamase enzyme within the periplasmic space, resulting in a more efficient mechanism than gram-positive bacteria. Ultimately, the destruction of the beta-lactam ring of the antibiotic renders it incapable of binding to the penicillin-binding protein, and thus the bacteria become resistant to that drug or class of drugs. **[53:24] Speaker A:** We have a very interesting trick how to deal with beta-lactamases. **[53:33] Speaker A:** Carbonic acid is actually penicillin that was not working so well as penicillin, but it was found that it can bind to beta-lactamases, to the enzymes that can destroy penicillin at a very high affinity. **[54:00] Speaker A:** So, how to reduce the effect of beta-lactamases? By using carbonic acid. **[54:11] Speaker A:** So, There are 2 enzymes that can bind to the antibiotics. One is the penicillin-binding protein, or the transpeptidase, and the other one is the beta-lactamases. Both of them can bind to antibiotics, right? So calvonilic acid, which is a penicillin, is going to bind strongly to beta-lactamases. It's not working well on the transpeptidase, but it can block the beta-lactamases, stopping them from destroying the antibiotics, the penicillin. So, uh, Today we can use the carvonylic acid in combination with penicillin, and one drug is going to do the real job inhibiting the transpeptidase, and the other one is going to bind to the beta-lactamases stopping their activity. Buying more time for the real penicillin to work against the transpeptidase. Here you can see an example for such a combination. There is a synergism between carbonic acid with penicillin and with cephalosporins as well. Here it is amoxicillin, which is supplemented by carbonic acid. **[56:10] Speaker A:** What we have here, it's almost a checkmate where we have an enzyme that makes the bond between D-alanine and L-lysine. Penicillin can block this enzyme. However, beta-lactamases can destroy penicillin and the enzyme can work again. But carbonic acid can inhibit the beta-lactamases stopping the enzyme after all from working. So when carbonylic acid is working appropriately, the transpeptidase is not going to work, is not going to make enough bonds between the alanine and the lysine, the aminopimelic acid. **[57:21] Speaker A:** Altered target genes. Bacterial cells can alter or eliminate the target site for antibiotics. **[57:37] Speaker A:** The penicillin-binding protein, the transpeptidase, can make mutations which are not going to affect the transpeptidase activity, but they are going to affect the ability of this protein to bind penicillin. **[58:00] Speaker A:** Bacterial cells can separate between the susceptibility of this protein to inhibitors and the ability of this enzyme to do its job. In order for an antibiotic to effectively inhibit a cellular mechanism, it must be recognized at a target site on the bacterial cell. If an organism do not have the target site for the antibiotics, this organism is going to escape the effect of the antibiotics. It is resistant to the effect of the antibiotics, and that's exactly what bacterial cells are trying to do. They are trying to escape the activity of antibiotics by making mutations that stop the penicillin-binding protein from binding penicillin, and the transpeptidase is still working despite this mutation. This is a mutation which is not affecting the transpeptidase activity. **[59:24] Speaker D:** If this remodeled DNA segment codes for cross-linking enzymes, i.e., penicillin-binding proteins, The result is the production of altered penicillin-binding proteins. These altered penicillin-binding proteins can still cross-link the peptidoglycan layers of the cell wall, but have a reduced affinity for beta-lactam antibiotics, thus rendering the bacterium resistant to the effects of penicillin and other beta-lactam agents. **[59:56] Speaker A:** Another example described by another group of antibiotics that are inhibiting the protein synthesis machinery. This is a group called macrolides, like erythromycin, for example, and the bacteria can be resistant to macrolides when they produce a certain enzyme called methylase that causes the methylation of ribosomal RNA, make the 50S ribosome subunit resistant to the binding of the macrolide antibiotics. The drugs are no longer able to interfere with ribosomal RNA process, but this ribosome is still going to make proteins. If an organism gains resistance to an antibiotic that acts on the ribosome, the target site of binding for the drug is changed and the drug no longer binds. Another mechanism that I mentioned earlier is antibiotic efflux pump. **[1:01:15] Speaker A:** Here we are talking about a certain pump which is able to move antibiotics outside of the cell. So bacterial cells can kick tetracycline out and pretty much every other antibiotic, each of them by different type of efflux pump. So, following the entry into the cell, tetracycline-resistant bacteria can quickly expel the drug, preventing it from doing any harm. We can divide the antibiotic resistance into 2 different types. Multi-drug resistance: the pathogen can acquire resistance to more than one drug at a time, and our plasmids, the resistant plasmids, express several genes for resistance for drugs with different chemistry that work against different targets. Multi or extensive drug resistance can be found, for example, in tuberculosis, Tuberculosis is a disease which is treated by 5 antibiotics. First, we have a combination of 3 antibiotics, and then the next step, combination of other 2 antibiotics. The treatment can last for over a year, and can lead to the recovery, to eradication of the pathogen from the body of the infected person. **[1:03:33] Speaker A:** But you need the 5 drugs for this, and we started to see some antibiotic resistance to one drug, but still we have the other 4 Then we found organisms that had an antibiotic resistance to more than one drug, and this is the multidrug resistance. Multidrug resistance in tuberculosis is determined when the 2 important drugs are not working with the same pathogen. **[1:04:16] Speaker A:** Isoniazid, which is important to stop the cell wall formation of the tuberculosis, and rifampin, which is an inhibitor of the RNA polymerase. Those are the 2 frontline treatments for tuberculosis, and when both of them are not working the tuberculosis is considered multidrug-resistant. You still have the other 3 drugs that can do something in order to control the disease, but recently we realized that this MDR, multidrug-resistant tuberculosis, can come with a resistance against another antibiotics, and that's getting to be much more significant problem. It's called extreme drug resistance, where we have the effect of the MDR and the other one or two drugs against tuberculosis are not working as well because of antibiotic resistance. Now, those drugs are very, very different from one another. **[1:05:45] Speaker A:** Isoniazid, very, very different in the chemistry, in the target, as compared to rifampin. We have a situation here that the bacteria learn how to deal with with drugs that have different chemistry. Cross-resistance is when the pathogen can acquire resistance to several antibiotics, but here those antibiotics have a similar chemical structure, and I'm going to aim towards beta-lactam antibiotics. And to tell you about an enzyme, NDM-1 is an enzyme that makes bacteria resistant to a broad range of beta-lactam antibiotics. These include the antibiotics of the carbapenem family, which is a new beta-lactam antibiotics. **[1:07:00] Speaker A:** We are talking here about an enzyme that was found initially in New Delhi, so the name of this enzyme is New Delhi metallo-beta-lactamase 1. That was the report from 2010. Now, that was the first time that we realized that we have such a problem of one enzyme that can make all the beta-lactam antibiotics to be useless. A person from England came after cosmetic surgery in India She picked up the NDM-1 resistant bacteria over there, and now you found NDM-1 in England. This is 2010, and we were able to find cases of the NDM-1 in India, Pakistan, and the United Kingdom. **[1:08:21] Speaker A:** But before we were able even to report this further, we realized that we have the superbug here in the GTA area code 905, here around us. And the date, it's 2010. Bacterial resistance is running very, very quickly. **[1:08:50] Speaker A:** Around. Today you are going to find it in a lot of different places, and as the result of this, all the beta-lactam antibiotics are going to be subjected to a destruction by the NDM-1, including Carbapenems that happen to be really, really new drugs that became available by making different type of modifications around the beta-lactam ring. But this enzyme cares only for beta-lactam ring, doesn't care so much about the modifications around. So carbapenems happen to be irrelevant in those cases as well when we have antibiotic resistance from NDM-1. Again, the idea is that the enzyme which can recognize the beta-lactam and really don't care so much about the other parts of this molecule is going to cut the beta-lactam ring. **[1:10:11] Speaker A:** When we have beta-lactam antibiotics, a lot of time we are going to say that we don't have a lot of things to offer to the patient with this type of an infection. Again, penicillins, beta-lactams are the most important antibiotics that we have around. And as the result of this, scientists have to look for drugs that normally they are not going to use. In some cases, they can see some activity where other drugs are not working on certain pathogens with NDM-1. And one of those drugs is Colistin. **[1:11:05] Speaker A:** Colistin is a polymyxin antibiotic. It's polymyxin E. Now, this is an old drug, like decades, and we were not using it because of its toxicity. It's a drug that fell out of favor due to its high toxicity, but with NDM-1 multidrug-resistant bacteria, it became one of the last resort antibiotics for resistant bacteria. So today we are using drugs that are more toxic in cases that the regular drugs are not working. So, a toxic drug, colistin, is now back. And don't think for a second that bacterial cells are not going to have some resistance to colistin as well. Bacterial cells are fighting anything. That you are trying to use as antibiotics. So, even the plasmid-mediated colistin resistance is also found in some of those bacterial cells. **[1:12:43] Speaker A:** What else? Another bad news is that this NDM-1 is not the only protein which belongs to this family of proteins that know how to destroy all beta-lactam antibiotics. Soon afterwards, we had NDM-2, NDM-3, NDM-4, NDM-5, and NDM-6. And here we are just in 2012. The bacteria that was the carrier of the NDM-1 was Klebsiella pneumoniae. Klebsiella pneumoniae is not a major pathogen. **[1:13:29] Speaker A:** It is a bacterium that can live harmlessly in the large intestine. Most of the time, they are not causing any major health issues. However, in some patients, the known superbug version of Klebsiella pneumoniae can cause urinary tract infection, pneumonia— and that's the name it's coming from— and even sepsis, which is a life-threatening infection of the blood. The treatment of this bacteria is with a class of phenyl antibiotics called the carbapenems. And unfortunately, the new version of the Klebsiella is the superbug that is resistant to carbapenems, and it's not going to stay just with the Klebsiella pneumoniae. **[1:14:25] Speaker A:** The plasmid is going elsewhere as well. And in general, you can say quite a lot of pathogens that are developing antibiotic resistance. Again, this is not even 20% of the list, but we have a lot of organisms that are simply not there. Sexually transmitted diseases today— syphilis, gonorrhea, in many cases is by antibiotic-resistant bacterial cells, and today we can see a huge number of cases, much more than we had in the last 20 years, of sexually transmitted infections by those bacterial cells that is antibiotic-resistant. You don't have the same type of treatment that was done in the past. **[1:15:32] Speaker A:** Which can be one injection and you're going to be cured. And there is another problem: making antibiotics not seems to be like an excellent business plan for most of the drug companies. The recent ill approval of new antimicrobial drugs by the FDA Federal Drug Administration have steadily fallen, went down. In the 5-year period of 1983 to 1987, 60 new antimicrobial drugs were approved compared to just 2 from 2008 to 2012, and the reason is that there is a conflict between the need of the drug companies to satisfy their shareholders, to make profit, and our need for drugs that are not likely to give very high revenues. I mean, this is a drug that you are going to take for a week or so and that's it. **[1:17:08] Speaker A:** There are other options for anti-cholesterol drugs that you're going to take every day, mood stabilizer drugs you will take it also every day, antibiotics that are going to be used in very few occasions, and when they are working well, we will try to hide them and to use them like vancomycin just when it is really necessary. That's not a great business plan for most of the companies. So, what they are doing? They are making the decision not to make drugs. And that's the problem that we have. We have more cases of antibiotic resistance, as we discussed earlier, and we have less drugs that are available for us. Even the drugs that were developed during the '70s, the '80s, most of them were derivatives of existing drugs. All the invention that we had for antibiotics was done during the '40s with beta-lactam drugs, sulfa drugs in 1936, and then other drugs, one phenicol, aminoglycoside, etc., were developed more or less towards 6 days or so. **[1:18:43] Speaker A:** And from this point, we started to make derivatives to existing drugs. What I mean by derivatives? **[1:18:54] Speaker A:** Well, you can add some side chain to the beta-lactam antibiotics, you can make changes over here, And a lot of time it's going to work, it's going to give you a better drug for many reasons. **[1:19:11] Speaker A:** And well, I mean, we have a lot of better lactam antibiotics, but all you need is one NDM to destroy all of them. **[1:19:23] Speaker A:** So let's go back to the words of Dr. Margaret Chan, and she also recognized this problem And she called for intervention. We need to do something in order to deal with such a problem. **[1:19:45] Speaker A:** So what should we do in order to reduce resistance, to limit the use of antimicrobial to necessary cases? So, although we have antibiotic resistance, we are still in the position to use those drugs in many cases. Antibiotic resistance can be found, but a lot of time the drugs are working. We need to make sure that we are using those drugs only to the time that it's really necessary, and when it is necessary to use the drugs appropriately, high concentration of drug, and long treatment. High concentration of the drug maintained in patients for long time to kill all sensitive and drug-resistant cells. **[1:20:46] Speaker A:** Use antimicrobial agents in combination. We realize that combination therapy works. It's working against a lot of different things, viral diseases for example, and the folic acid seems to be a great target for antibiotics. But you cannot rely only on sulfadragone and trimethoprim that are now used in combination. At certain stage, you will see some antibiotic resistance there as well, to the level that this combination is not going to work so well. **[1:21:33] Speaker A:** As a student, I learned about combination therapy. The example that I've heard in this time is the sulfa drugs, sulfonamides, trimethoprim combination. This is a combination which is around for really, really long time. It's working in most of the cases, but if this is working, maybe we can use the principles to expand this further because there are other type of drugs that can inhibit other enzymes and we can increase the ability to block this pathway, whether we are using more than 2 drugs at the time, maybe 3 drugs together, which make this inhibition to work even better, or to find other type of combinations which are going to work well. So right now We have a combination therapy against this pathway, but we need to extend the inhibition of this pathway and to come with combination therapies to other pathways as well. **[1:23:01] Speaker A:** What else we can do in order to reduce the antibiotic resistance? **[1:23:10] Speaker A:** Developing of new variations of existing drugs, and there are quite a few cases that changing the side chain and adding some other chemical groups to antibiotics is making the antibiotics to work much better. Tetracycline is not considered a great antibiotic, and it's not being used a lot. But tigecycline, which is tetracycline plus some modification, is an excellent drug. It is active against tetracycline-resistant bacteria. It's active against MRSA and most gram-negative as well. **[1:23:59] Speaker A:** So, we Here you see that by modification of existing drugs, which is around for quite a while and it was abused dramatically by the pharma industry, we came with a new drug which is working much, much, much better. To develop drugs for new targets, since the '60s we learned a lot about bacterial cells? Well, I mean, we sequence all bacterial cells that we probably know about them, and we learn about them a lot of things that we didn't know during the '60s. So, today you can use new targets. And another thing which is quite important is to use better diagnostic tools. **[1:25:00] Speaker A:** If you're going today to the family doctor with something that looks like tuberculosis, the test is going to last for quite a long time. There are DNA-based tests for tuberculosis and other types of pathogens, but it's still not part of the regular way that we are making this type of diagnosis. Diagnosis in a lot of cases is the old type of diagnosis. Doctors are treating infections empirically, Initially, at least, they do not know what is the pathogen. They prescribe initially a drug that is working in other patients with the same symptoms, and better diagnostic tools like PCR-based or sequencing can be very helpful in reducing antibiotic resistance because you are going to get the answer quickly enough. **[1:26:24] Speaker A:** The article about the next-generation sequencing that was used in order to do this type of diagnosis on a bacteria which is quite similar to the tuberculosis. Again, I mean, It's not a standard tool, it's a unique tool which is just at the very, very beginning. And that's an example that the next-generation sequencing is working in those cases. You can get the answer very quickly. I mean, next-generation sequencing can give you the identity of a pathogen really, really, really quickly. I mean, in hours. You don't need to wait for the regular tests to be done that are taking long time and they're not as accurate as sequencing. **[1:27:29] Speaker A:** And what else? We need to think out of the box. Maybe we need to make some decisions about reducing the conflict between the need of drug companies to satisfy the shareholders and our need for a drug that is not likely to give them very high revenues. Is the idea that drugs is made only by drug companies that can make their choice what they want to do and what not is the best idea? Should we use more government-related systems in order to make certain drugs directly? **[1:28:23] Speaker A:** And, well, another thing is maybe we want to go back to the future. Before antibiotics was discovered, scientists suggested that bacteriophage can be used for therapy. The idea was that if you have the appropriate bacteriophage and this bacteriophage can destroy the pathogen, You're going to do better. We are exposed to bacteriophages all the time. When you're swimming in the lake, for every bacteria that you are swallowing to your gut, you have probably 100 bacteriophages over there as well. **[1:29:18] Speaker A:** So, they are simply ignoring us, we're ignoring them, everything is fine. But if you are using them as a therapy when you know that there is a certain pathogen and you can fit the appropriate bacterial phage to this pathogen, maybe you can get a better response here. And there is another thing. When drugs are constant in their structure. Bacteriophage have their own evolution. **[1:30:00] Speaker A:** So, drugs do not have evolution, it's constant. The bacterial cells can have their evolution and they can come with mutations against the drug that can do nothing because it cannot be changed. But bacteriophages need the bacterial cell in order to survive, so bacteriophages are going to do their best to feed their evolution to the bacteria. So we have 2 forces over here that both have an evolution capacity of their own, and each of them want to live. So, bacteriophage can be much better than regular drug. Bacterial cells will have very, very difficult time to become resistant to the bacteriophages after all. The same evolutionary power that bacterial cells have, the bacteriophages have them as well. **[1:31:20] Speaker C:** We are destined to live in a contaminated world. We are not going to live in a sterile environment. **[1:31:28] Speaker C:** The evolution of our interaction between microbes and humans is in a constant state of flux. **[1:31:38] Speaker E:** And so I think that we will, as we have in the past, constantly be surprised by new kinds of interactions that we will view as disease and the microbes will view as survival. **[1:31:59] Speaker A:** That's what I had to tell you about bacteriology. Thank you.