Evolution racing

Why do antibiotics stop working

Konstantin Andreev, "Biomolecule"

Since the discovery of penicillin, mankind has been constantly conducting a kind of "arms race" with the world of bacteria, which have learned to adapt quickly after the advent of new antibiotics. And, alas, today in this race we have begun to gradually lag behind the enemy... In the face of the threat of being left helpless when new, "bulletproof" infections appear, scientists are stocking up on their trump cards up their sleeve. The problem is extremely acute – either to have time to find the optimal replacement for antibiotics in the next decade, or to plunge into the new Middle Ages, risking death from the most insignificant scratch every day.

Enterobacteria (Enterobacteriaceae) treated with carbapenem.
There are antibiotic–resistant colonies in the right Petri dish.
Picture: Centers for Disease Control and Prevention.

Thanks to natural selection, we have developed the ability to resist;
we are not inferior to any bacteria without persistent struggle.
H. G. Wells. The War of the Worlds.Fame and world recognition in science is a capricious and unpredictable thing.

Many have heard the story of Alexander Fleming's discovery of the first of antibiotics, penicillin, in which a seemingly trivial accident played a key role. In 1928, a mold fungus of the genus Penicillium accidentally got into a bacterial culture cup forgotten on the table in his laboratory, and Fleming noticed that microbe colonies were dying (lysing) next to a plaque of mold.

Sometimes you find something you're not looking for at all.
Sir A. FlemmingHowever, not everyone knows that penicillin was discovered several more times before Fleming, though with less success.

60 years before the events described, the English physiologist and part-time medical officer Baronet Sir John Bardon Sanderson, in his report to the Cabinet of Ministers of Great Britain, mentioned a fungal mold that suppressed the spread of "microzymes" (as he called bacteria) in the tissues and fluids of living organisms. But a report is not at all like a publication in a scientific journal. Therefore, the data he received did not become available to the general public.

Photo of a Petri dish from the famous article by Alexander Fleming [2].
The "dead zone" between the plaque of mold fungus and bacterial colonies is clearly visible.

A year later, inspired by the success of his colleague, Joseph Lister – professor of clinical surgery at the University of Edinburgh – thought that Penicillium glaucum (the same blue mold, well known to cheese lovers) could serve him well as an antiseptic during operations. And I even tried it on a patient; then they looked at the ethics of such things a little easier. The patient survived, but for some reason Lister also did not bother to publish the results of his experiment, and the scientific world did not know anything about it.

Since then, penicillium has repeatedly come to the attention of scientists who have systematically observed and described its ability to inhibit the growth of bacteria. Nevertheless, it was Fleming with his perseverance who managed to bring the matter to an end [2].

At the next stage, it was necessary to isolate the active substance in its pure form from the culture of the fungus and learn how to use it correctly. The first attempts to put it into practice resembled a roulette game, since it was unclear who would be the first to kill the drug – the causative agent of the disease or the patient himself. Later, Howard Florey and Ernst Cheyne from Oxford University managed to solve this problem. The determination of the safe dose and the decoding of the chemical structure made it possible to establish mass production of penicillin by the middle of the Second World War, which turned out to be just in time [3].

Starting from the middle of the twentieth century, new antibiotics began to appear one after another, and at first great hopes were pinned on them. However, after some time it became clear that a quick and final victory over infectious diseases with their help, most likely, should not be expected.

What is an "antibiotic"?In fifty cases out of a hundred, even the best of them don't know how to treat you

A. ChristieFirstly, the term "antibiotic" means absolutely not what is often meant by it.

If you literally translate the word, you might think that this is something like DDT – "destroying all living things." In fact, in pharmacology, antibiotics are drugs of natural or semi-synthetic origin that affect bacteria, and only them. Not to mention the fact that in nature antibiotics are most often used not for killing at all. Microorganisms like mold produce them to scare off competitors for an ecological niche, that is, the function of antibiotics is about the same as that of a mosquito repellent. A similar confusion, by the way, turned out with the word "microbe", which is often given a negative connotation, considering it synonymous with "pathogenic bacteria". Although, in general, a microbe is just a microscopic (conditionally: invisible to the naked eye) organism that does not necessarily conflict with a person.

The trick is that the infection can really be caused by evil bacteria, or maybe something else - for example, archaea, fungi or even protists. And against all this splendor, antibiotics are unlikely to be effective. Viruses are even worse. You will not have a single chance to cure any flu or SARS with an antibiotic. Unlike a bacterium – a cellular structure with its own genome, protein–synthesizing apparatus, metabolic enzymes, etc. - a virus from all of the above has only a genome. In fact, it is just a DNA or RNA molecule packed in a protein capsid that replicates inside an infected host cell using its own resource. A kind of saboteur who infiltrated someone else's factory with his drawings and stamped copies of himself on the factory machine. Well, do not break your own equipment, in fact, in order to stop the violator!

According to an article recently published in the Journal of the American Medical Association [4], in the USA, out of ten patients who went to the doctor with complaints of sore throat, six are prescribed antibiotics. Penicillin is still the drug of choice due to its low price and good tolerability. Meanwhile, it is effective only when the infection is caused by group A streptococcus, and this is only one case out of ten [5]. It turns out that the doctor prescribing you an antibiotic for sore throat, most often either is not sure of the diagnosis, or is simply reinsuring himself.

Moreover, even if the cause of the infection is a bacterium, this is not a guarantee that a particular antibiotic will work on it. It all depends on what kind or even strain you have to deal with. On the one hand, this is good, because a healthy body always has its own microflora, which lives with it in a happy symbiosis and which is better not to touch. On the other hand, it is for the same reason that there are no universal drugs that would help equally well from everything at once. In order not to act at random, before prescribing treatment, a culture is usually sown from a sample taken from a patient, and it is alternately checked for resistance to a whole pool of antibiotics, after which the most suitable one is selected. Unfortunately, this procedure requires at least a few days and the presence of a microbiological laboratory at hand, and the infectious process often proceeds much faster. And it may happen that when the results of the tests come... it will be too late to treat.

Of course, researchers are also looking for other ways to diagnose infections at early stages, for example, Alan Jarmusch from Purdue University suggested using mass spectral analysis for these purposes [6]. Great hopes are pinned on DNA diagnostics of pathogens, although the widespread use of these methods in practice is still far away.

How does it all work?The strongest survives

Tsch.
DarwinThe mechanisms of action of modern antibiotics on the target cell can be very different [7].

Representatives of the beta-lactam group (which includes penicillin) inhibit the synthesis of peptidoglycan, which forms the basis of the bacterial cell wall. Without it, osmotic pressure inside the cell destroys the plasma membrane, and the cell bursts like a balloon. Unfortunately, such antibiotics only inhibit the growth of new peptidoglycan chains, but do not destroy the already formed ones. Therefore, they can stop cell division and their active growth, but nothing can be done with bacteria that are at rest, or with the so-called L-forms, which have no cell wall at all, but which have retained the ability to develop. Sulfonamides prefer to hit the intracellular metabolism of the victim, for example, blocking the chemical reactions necessary for the synthesis of folic acid. The bacterium does not know how to absorb vitamins from the outside, so the inability to synthesize them independently is fatal for it. Some antibiotics (aminocoumarins and fluoroquinol compounds) disable bacterial DNA gyrase, an enzyme that unwinds a super-twisted chromosome for its replication. Thus, the cell is deprived of the opportunity to copy its DNA and reproduce. Another way to kill a bacterium is to disrupt the synthesis of its proteins. Tetracycline antibiotics work according to this scheme: they attach to a small subunit of the bacterial ribosome – the organelle responsible for building proteins on the RNA matrix [8].

This list is not complete, there are other groups of antibiotics. One way or another, their target is almost always a protein, be it a bacterial enzyme, a metabolite, a cytoskeleton element, a proton pump or something else [9]. Even just to get inside the cell, the antibiotic first needs to pass through its cell wall and membrane through channels that also consist of proteins (porins) [10, 11]. And there are two important points here. Firstly, there are monstrously many proteins, and they are very diverse, hence the variety of antibiotics. Each of them affects its "own" protein – and is harmless to all microorganisms that do not have such a protein. But what is much scarier is that proteins are lighter and faster than other compounds (carbohydrates, phospholipids, etc.), subject to adaptive changes. This is what lies at the heart of a large-scale problem that has not yet manifested itself in full force, but is already looming on the horizon and in the near future threatens to turn into a serious catastrophe. We are talking about the ability of bacteria to develop resistance (resistance) to any antibiotic quickly enough – in a matter of months [12].

Interaction of methicillin-resistant Staphylococcus aureus and human leukocyte.
The MRSA252 strain is one of the most frequent causes of hospital infections in the US and the UK.
Picture: National Institute of Allergy and Infectious Diseases.

Obviously, the simpler the mechanism is, the easier it is to repair it "on the knee" and improve the technical characteristics without spending too much time. Bacteria are very simple devices. When you have only one cell, there is no need to disassemble the nucleus and unpack the chromosomes every now and then, the genome is not overloaded with many regulatory elements, and all genetic material is economically used to maintain only the most necessary vital functions, then it is much easier to adapt it to the changed conditions. The life of the bacterium is short – 20-30 minutes. But if nothing prevents it, it usually ends with mitosis. In other words, every half hour there will already be two cells in place of one, and by the end of the day about seventy generations will have time to change. Therefore, from an evolutionary point of view, time flows faster for bacteria than for us, which means that natural selection also works faster. No matter how powerful an antibiotic is, there is always a chance that among a billion of its victims there will be at least one that will survive thanks to a randomly acquired mutation. And having managed to survive, he will get an exclusive opportunity to reproduce and will transfer this ability to survive to daughter cells, and those, in turn, to their own. The descendants of the surviving bacteria will sooner or later form a population that is completely immune to the old antibiotic – exactly according to Darwin [13].

Higher organisms that are under constant pressure adapt in a similar way. Insects that harm agricultural crops develop resistance to pesticides, except that it takes them much longer. Bacteria, on the other hand, not only multiply quickly, but also know how to "neighborly" exchange genes directly with each other (this is called "horizontal gene transfer"). All this only accelerates their adaptation to new conditions even more.

The world is on the threshold of a post-antibiotic eraMargaret Chan, Head of the World Health Organization
As a result, after the launch of any antibiotic on sale, its effectiveness begins to decrease over time, tending to zero.

Some bacteria today does not take almost anything. In the English–language press, the term "superbugs" was even coined to denote strains resistant to several antibiotics at once. Thus, according to the results of a study published in January 2014 [14], they account for up to half of cases of tuberculosis bacillus infection in the territory of the Russian Federation.

Having once begun the large-scale use of antibiotics in clinical practice, humanity entered into an arms race with the world of prokaryotes, and continues to lead it to this day. Microbiologists, chemists engaged in organic synthesis, and pharmacologists have to constantly look for new molecules with stronger bactericidal properties or change something in the structure of existing ones. Of course, the methods of molecular design [15] allow you to choose a molecule that will be longer or shorter than the original one, change its spatial structure and the number of possible conformations, increase or decrease hydrophobicity and total charge, collapse into a ring or just randomly shuffle structural blocks. With the help of computer modeling (in silico [16]) or artificial model systems, one can even try to calculate its behavior when interacting with the bacterial membrane and predict in which direction to look [17]. However, human imagination is far from limitless, and it is already difficult for the opponent to keep up with updates in time. Further it will only be more difficult, and at some point the development of new antibiotics will simply end with an innovation crisis (some data, however, suggest that it has already come).

Left unarmed, we risk in the future to face a disease for which we will not have an antidote. In terms of the scale of the consequences, such an event is quite comparable to the plague epidemics that raged in medieval Europe. At the same time, it is impossible to predict where the threat will come from. Those species that were completely harmless yesterday can turn into pathogens tomorrow, as soon as they learn to infect a new host. A good example is HIV, which began its existence with the fact that its predecessor, the monkey immunodeficiency virus (VIO), once mutated, jumped the species barrier and settled perfectly on a person [21].

The only thing that is encouraging is that in the event of a global pandemic threatening humanity with extinction, natural selection will begin to work in our favor, according to the principle of the pendulum. Those of the individuals of Homo sapiens who are lucky to stay alive will surely pass on their own immunity to infection to their descendants, and a new round of protracted war will begin. But "winning" it will cost too much.

The latest types of weaponsCan we win this race, in which bacteria have such a clear advantage?

In fact, there are alternatives to antibiotics. Some of these drugs are at the stage of study, others are at the stage of clinical trials. Still others do not require medical intervention at all and are based solely on common sense.

   SOS-repair of bacteriaOf course, the simplest and most logical idea would be, if not to prevent bacteria from evolving, then at least artificially slow down this process [22].

How to implement this in practice was first demonstrated by Floyd Romsberg's group [23]. The task that the researchers set themselves was to learn how to control the bacterial SOS response system, or SOS repair. This phenomenon is well known to microbiologists and was described in the scientific literature thirty years ago [24].

Under normal conditions, genome replication takes place with fairly high accuracy, despite the inevitable errors. Mainly due to the fact that the enzyme that copies DNA (in bacteria it is DNA polymerase III) has, among other things, also (3’, 5’)-exonuclease activity. This means that if a nucleotide that is not complimentary to its partner on the matrix accidentally gets into the growing semantic chain, it will immediately be cut out and replaced with the "correct" one. However, when things start to go from bad to worse for a cell (for example, with a high concentration of an antibiotic in the environment), and too much damage accumulates in its chromosome, it becomes clear that the genome that exists at the moment is not suitable for survival at all. Then, in order to somehow escape, the cell goes all-in and turns on the SOS-repair mechanism. Instead of the "neat" DNA polymerase III, which corrects mistakes, completely different proteins begin to work, which are simply substituted into the damaged areas with whatever came in the calculation of blind luck. The chances of success of such an enterprise are scanty, and most often the cell dies because its vital genes turn out to be hopelessly corrupted. But if it suddenly works, then an ordinary bacterium can turn out to be something completely different from the "ancestors", and possibly extremely tenacious [25].

Two proteins control the SOS system - RecA and LexA. The first plays the role of an activator; the second – a repressor. While everything is calm, LexA constantly sits on the operator area of SOS genes (SOS box), not allowing them to be read and run - until a fragment of single-stranded DNA falls into the possession of his opponent. By contacting it, RecA forms something like a long thread (filament), which causes the repressor to detach from the operator area and triggers SOS repair. As soon as the stocks of single-stranded DNA fragments run out (i.e. when everything is repaired), LexA sits back in its place, and the process stops.

The aim of the work of Romsberg and his colleagues was to search for low-molecular compounds that would not allow the repressor to leave the operator under any conditions, thereby constantly keeping the SOS response genes turned off. They managed to select several such inhibitors capable of working in E. coli cells in the presence of the antibiotics ciprofloxacin and rifampicin, but it never reached clinical trials of the drug.

Horizontal gene transfer in bacteria. Picture: University of North Carolina.

   BacteriophagesAnother approach to solving the problem is to use their natural enemies against infectious agents.

Bacteriophages – bacterial viruses – are themselves able to evolve no worse than their victims and thereby bypass the mechanisms of their resistance [26]. One of the reasons why phage therapy is still poorly represented on the market is not that it is difficult to implement technically, but rather in the confusing patent legislation. There is still no consensus on whether to consider the virus alive. If so, then the right to use it for commercial purposes becomes much more difficult to obtain, so all the efforts of pharmaceutical companies are reduced to legally recognizing the bacteriophage as something like a vaccine or vector for genetic cloning [27]. In any case, working with such a highly specific and changeable medicine and writing instructions for it is not an easy task. There are other controversial points – for example, it is not completely clear how a particular phage will behave when confronted with the human immune system. And in general, there is no guarantee that, having penetrated into the cell of the pathogen, it will immediately destroy it. Instead, many phages prefer to integrate into the bacterial chromosome and remain there indefinitely.

   Antimicrobial peptidesAnother effective, but still little–known weapon is antimicrobial peptides [28, 29].

Since the entire defense of bacteria is based on changes in their protein component, then why would potential antibiotics not choose something else as their target? Among non-protein compounds, this "something else" is likely to be phospholipids – universal building blocks for any cell membrane. They have changed little since the origin of life on Earth to the present day, since their task is extremely simple: to create a film that is hydrophilic on the outside and hydrophobic on the inside, and with the help of this film to separate the inner world of the cell from the entropy surrounding it.

(A much more "intelligent" target for antibiotics in bacterial membranes are lipid-II molecules, which are unique for microorganisms, a substance that is an important intermediate in the synthesis of the cell wall. It is affected by bactericidal substances called lantibiotics, and this target is considered extremely promising for the development of a new class of antibacterial substances that are not subject to the burden of resistance development: "Computer modeling of membranes, lipid-II and Penelope blanket" [17]. – Ed.)

It is clear that the bacterium will die if the integrity of its membrane is violated. This is exactly what most antimicrobial peptides do (although not all). A molecule capable of making a hole in the membrane must, firstly, be small and compact in order to slip through the outer peptidoglycan shell without hindrance, and secondly, have a total positive charge sufficient for electrostatic attraction to the membrane surface. (Phosphoric acid residues in phospholipids are negatively charged, which gives a negative charge to the bacterial membrane from the outside. But the outer side of our cells, compared with a bacterium, practically does not carry a negative charge.) And most importantly, one half of this molecule should be hydrophobic, and the other hydrophilic.

Antimicrobial peptides are involved in the innate immunity system of many organisms, from humans to bacteria fighting other bacteria. They are short oligopeptides consisting of an average of 20-40 amino acids and are called antimicrobial for their ability to fight almost any microorganisms and even viruses if they are covered with phospholipid supercapsid. A wide spectrum of action is one of the main trump cards of antimicrobial peptides, but the downside of a wide spectrum is low selectivity: such a weapon hits everyone indiscriminately. Because of this, as well as because of the rapid cleavage of proteases in the bloodstream, it makes no sense to inject antimicrobial peptides. It remains only to apply them externally in case of infection of the mucous membranes or in the form of tablets for stomach infections [30]. Therefore, despite the fact that a lot of research has been conducted over the past decade, only two antimicrobial peptides are now commercially available – ramoplanin and enfuvirtide (T20). Ramoplanin is produced by actinomycete fungi; this, to be precise, is a glycopeptide - an amino acid chain closed in a ring and with a "suspension" of two mannose residues. It prevents bacteria from building a cell wall and is effective against respiratory tract infections, primarily staphylococci, as well as against gastrointestinal infections. Enfuvirtide is also used as an antiretroviral drug that prevents the fusion of the HIV virion with human cells.

Phages attack the Escherichia coli cell. Picture: Environmental Health Perspectives.

It is necessary to convey to the public that not every disease needs a pillPeter Swinyard, Chairman of the Association of Family Physicians of Great Britain
Finally, the cheapest and most widely available, but therefore no less important measure: common sense and the implementation of simple rules.

It just so happened that there is no single agreement between doctors in the world that would limit the use of antibiotics and leave some of them in reserve. In those countries where antibiotics can be purchased without a prescription, they are often kept in a home medicine cabinet "just in case" and begin to be taken at the first signs of malaise. Self-medication together with ignorance of the correct dosages create excellent conditions for bacteria to develop resistance. It is better to save antibiotics as a last resort, giving the immune system the opportunity to do its job. But if you really had to take them, follow the doctor's instructions – drink the course to the end, and do not quit at the first signs of improvement.

Surprisingly, the epic battle of bacteria and antibiotics with an unclear ending was predicted by Fleming himself, speaking with a Nobel speech in 1945. He is also credited with a rather philosophical phrase that "penicillin, of course, helps, but wine makes you happier." And I must say, in some ways he is subtly right here.

The original version of the article was published in Chemistry and Life [31].

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