The end of a beautiful era

Dmitry Gilyarov, N+1 

Last week, a team of Chinese scientists published an article in the Lancet journal in which they summarized the results of many years of observations and reported the discovery of a gene for transmissible resistance to colistin. Thus, the gloomy predictions of many researchers have come true and the world is on the verge of the appearance of bacterial infections, for the treatment of which there is not even formally a single drug. How could such a thing happen, and what consequences does it have for our society? 

Colistin, belonging to the polymyxin group, is a "reserve antibiotic", that is, the last resort used for infections with bacteria that are resistant to all other agents. Like many other antibiotics, colistin was discovered back in the 1950s. But since the 1970s, it has practically not been used in medicine; the reason is simple: this is a very bad antibiotic. In almost half of cases, it causes nephrotoxicity (gives complications to the kidneys), besides, by this time much more effective and convenient carbapenems and fluoroquinolones had already been discovered. Colistin began to be used for the treatment of patients only in the last ten years, when, due to the spread of resistance to carbopenems, doctors had almost no choice left. 

Nevertheless, colistin has never stopped being used in veterinary medicine and until recently was among the top five antibiotics used on farms in Europe and other countries. Scientists have been paying attention to this for a long time and called for a complete ban on the use of an antibiotic critical for the treatment of people in agriculture. Of particular concern was the popularity of colistin in Southeast Asia, where the real scale of turnover could not be tracked, especially since the consumption of antibiotics by farmers is not regulated by law in any way.

How does colistin work? This substance binds to lipids on the surface of bacteria, which leads to the destruction of the membrane and subsequent cell death. Until now, all cases of colistin resistance have been associated with chromosomal mutations, which were usually accompanied by a decrease in the viability of bacteria and, accordingly, could not gain a foothold and spread in the population. 

However, recently, during routine monitoring of drug resistance of bacteria isolated from raw meat samples (the study was conducted in southern China from 2011 to 2014), scientists noticed a suspiciously strong increase in the number of resistant isolates. So, in 2014, up to 21 percent of the studied pork samples contained colistin-resistant bacteria. When biologists began to deal with these strains, it turned out that resistance was determined not by chromosomal mutations at all, but by a previously unknown mcr-1 gene. 

A comparison of the gene sequence with the sequences in the database suggested that it encodes an enzyme that modifies bacterial lipids so that they lose the ability to bind an antibiotic. The gene is located on a plasmid – a separate DNA molecule that can move freely between different strains and even different types of bacteria, giving them additional properties. The presence of plasmid does not affect the well-being of bacteria in any way, and it is stable even in the absence of colistin in the medium. 

The authors' conclusion is disappointing: there is very little time left until the gene spreads around the world and doctors may formally have no options left to treat some infections. In fact, there are almost no options even now: the high toxicity of colistin makes its use in practice difficult, the same applies to other antibiotics of the "last reserve". At the same time, the ability to control bacterial infections with the help of antibiotics is the cornerstone of our medicine: without them, it is impossible to imagine either cancer chemotherapy, organ transplantation, or complex surgical operations – all of them would end in severe complications.

 

The first antibiotic that saved millions of lives during World War II – penicillin – belongs to the beta-lactam group. The success of penicillin was such that it was not only sold without a prescription, but also, for example, added to toothpastes for the prevention of caries. The euphoria disappeared when, in the late 1940s, many clinical isolates of Staphylococcus aureus stopped responding to penicillin, which required the creation of new chemical derivatives of penicillin, such as ampicillin or amoxicillin. 

The main source of resistance was the spread of beta-lactamase genes: an enzyme that cleaves the nucleus of the penicillin molecule. These genes did not reappear, because mold fungi that produce penicillin and bacteria coexisted with each other in nature for millions of years. However, fully synthetic fluoroquinolones, which appeared in clinical practice in the early 1980s, repeated the fate of penicillin ten years later (now the levels of resistance to fluoroquinolones in some groups of clinical isolates reach 100 percent due to the spread of chromosomal mutations and portable resistance factors, such as transporters pumping drug molecules out). 

Over the past 60 years, synthetic chemists and bacteria have been competing: new and new groups of beta-lactam antibiotics (cephalosporins of several generations, monobactams, carbapenems) resistant to cleavage have entered the market, and bacteria have acquired beta-lactamases of a new class with an increasingly wide spectrum of action. In response to the spread of beta-lactamase genes, inhibitors of these enzymes have been developed: beta-lactams, which "get stuck" in the active center of the enzyme, inactivating it. Combinations of beta-lactam antibiotics and beta-lactamase inhibitors, such as amoxiclav (amoxicillin-clavulonate) or piperacillin-tazobactam are now among the main prescribed drugs in clinical practice. Even now, these combinations are often more effective than beta-lactams of the last generation. However, in addition to the evolution of beta-lactamases, which makes them insensitive to a particular inhibitor, bacteria have mastered another trick: the enzyme of cell wall biosynthesis itself, with which beta-lactam binds, may become inaccessible to an antibiotic. It is this form of resistance that is observed in the infamous MRSA (methicillin-resistant Staphylococcus aureus). Such infections are not incurable, but require the use of more toxic and less effective drugs.

It is they who cause such concern among doctors, already claiming tens of thousands of lives every year in the USA and Europe and significantly increasing the cost of treatment. Hospitals, especially intensive care units, are an ideal place for the reproduction and selection of super-resistant bacteria. A person entering the intensive care unit has weakened immunity and requires urgent intervention, so the most powerful drugs of the widest possible spectrum of action are used there. The use of such drugs causes the selection of bacteria resistant to many classes of antibiotics at once.

Microbes have the ability to survive on a variety of surfaces, including bathrobes, tables, gloves. Catheters and ventilators are standard "gates" for hospital pneumonia, blood poisoning, infections of the genitourinary system. Moreover, MRSA is far from the most terrible hospital pathogen: it belongs to the group of gram-positive bacteria, which means it has a thick cell wall, into which molecules of various substances penetrate well. For example, vancomycin. The real horror of doctors is caused by gram-negative Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii: in these bacteria, the cell wall is covered with a lipid membrane into which substances enter through narrow channels. When a bacterium senses the presence of an antibiotic, it reduces the number of such channels, which immediately reduces the effectiveness of treatment; to this we must add plasmid-borne transporters that pump out the drug molecules that miraculously got inside the cell, and beta-lactamase genes (resistance genes are usually transferred by complexes, which further complicates the fight against bacteria). It was to combat such infections that colistin often remained the last means available to doctors.

Nevertheless, as practice shows, the introduction of adequate control procedures inside hospitals (thorough checking of appointments, complex hygiene procedures for all contacts, decontamination of all surfaces, and so on) allows you to limit or even reduce the number of resistant bacteria. This is due to the fact that antibiotic resistance has its own energy price for bacteria. In the absence of selection pressure, resistant microorganisms cannot compete with their faster-growing relatives. Unfortunately, such standards of medicine are only available in some hospitals in developed countries.

The fertile "gold mine" – the study of soil bacteria-streptomycetes, which gave almost all known classes of antibiotics – was almost exhausted: new studies gave only already discovered substances, and laboratories did not have the technologies and resources to conduct large-scale screenings of chemical libraries. But it's not just that. The lack of new antibiotics is a consequence of a real "perfect storm" of coinciding causes, primarily economic. Firstly, new antibiotics, unlike some immunomodulators, are needed by a relatively small number of patients, and these patients live mainly (but not only!) in poor countries. Secondly, the course of antibiotic treatment takes several weeks, not years, as with, say, antihypertensive drugs. Thirdly, resistance can make an expensive drug unprofitable within a few years after the start of use. In general, you can't earn money on them.

Now governments of different countries are trying to find economic incentives to bring large companies back to the antibiotic market: this can be both a reduction in development costs (tax benefits) and an increase in benefits (for example, government procurement obligations). At the same time, more and more scientists are engaged in research on the coexistence of bacteria with each other, antibacterial substances and resistance mechanisms. Unfortunately, the problem of sustainability is a typical problem with delayed consequences: the adequacy or insufficiency of the measures taken becomes apparent only after a long time.

Immediately after the discovery of antibiotics, in the same 1950s, farmers found out that the daily use of sub-therapeutic doses (this means that the dose is slightly lower than that which would be used in case of disease) in animal husbandry allows as much as 20 percent increase in weight gain in terms of the amount of feed consumed. The reasons for this effect are still not clear, but apparently somehow related to the complex community of bacteria in the intestines of the animal and their interaction with the host's immunity. By reducing the number of potentially pathogenic bacteria in the intestine, antibiotics reduce the level of inflammation and activation of the animal's immune system, reducing energy costs. In addition, bacteria directly consume part of the calories coming from food (thereby reducing the amount of calories that the animal itself gets). 

In addition to accelerated weight gain, the intensification of animal husbandry required the inclusion of antibiotics in the diet to prevent all kinds of diseases of livestock and birds. Despite public attention to the problem, the level of antibiotic use in agriculture is increasing every year, and 90 percent of the substance is not used to treat diseases, but as an additive to feed and a growth stimulant. Together with waste products, antibiotics enter the wastewater, causing the selection of resistant pathogens throughout the region.

This may surprise the reader, but even in developed countries (USA, Canada, EU), farmers use for their purposes not penicillin at all, but antibiotics of the latest generations. For example, in the United States, 72 percent of the antibiotics used by farmers are "medically significant", that is, important for the treatment of people. 

At the moment, only in the European Union the use of antibiotics to accelerate the weight gain of animals is completely prohibited (since 2006), which, of course, required the introduction of protectionist measures in agriculture. However, antibiotics are still widely used for preventive purposes. In the USA, the use of cephalosporins in agriculture has been restricted only since 2012. But, unfortunately, the ban on the use of antibiotics in animal husbandry in one country does not prevent the penetration of resistance genes from other countries where such prohibitions do not apply.

Generally speaking, intensive animal husbandry without the use of antibiotics is possible, but requires a high level of control and organization of production, which makes it even more expensive. As alternatives to antibiotics, it is proposed to use probiotics – cultures of "beneficial" bacteria, and substances that stimulate their growth to normalize the intestinal microflora, vaccination or even the use of bacteriophages.

 

The military can be understood: it is difficult to organize adequate procedures in the field, and wounded soldiers returning from Iraq or Afghanistan often brought difficult-to-treat infections with them. More recently, DARPA supported the project "stimulating host defense mechanisms" – it is assumed that if you understand the mechanisms of natural immunity (why some people get infected and others don't), you can protect anyone from infection (even unknown). Such studies, of course, are not without meaning: according to immunologists, it is the degree of reaction of the immune system to a pathogen (virus or bacterium) that determines the outcome of the course of the disease. Too strong a response ("cytokine storm") destroys healthy tissues, and too weak is insufficient to destroy the pathogen.

Unfortunately, we still do not understand well enough how the immune system works and it is unlikely that we can expect rapid success in this area. On the other hand, the classic vaccines developed against a specific bacterium have proven their effectiveness, allowing to eradicate many terrible diseases during the XX century. And vaccination of livestock against common diseases would reduce the use of antibiotics in agriculture.

Bacteriophages (from the Greek "devouring bacteria"), or bacterial viruses, were discovered almost 100 years ago by a French doctor of Canadian origin, d'erel. He was the first to use bacteriophages in the treatment of infections. Despite the huge (at first) public interest associated with large losses from infection of wounds and typhus in the First World War, d'erel failed to achieve significant success: the procedures for isolating viruses active against a specific culture of bacteria, their storage and transportation, as well as the results of the treatment itself could not be controlled, systematized and not really reproduced. 

Nevertheless, the Institute of Bacteriophages, founded by d'erel in Tbilisi in 1933-35, exists to this day, and is one of the few places in the world where it is possible to receive treatment with therapeutic phages. The growth of antibiotic resistance has naturally revived interest in phages: having a narrow specialization, they can "devour" infectious agents without affecting normal intestinal inhabitants, as well as destroy biofilms inaccessible to drugs. At the same time, from the point of view of selection, the use of phages is no different from the use of tablets: a single mutation in the receptor protein on the surface of the bacterium is enough for the phage to stop sitting on it. And the problems that existed back in d'erel's time have not gone away: the procedure for selecting the right phages (or rather, their mixtures) takes at least a few days, only the surfaces of the body or intestines accessible from the outside can be treated, besides, as it turned out, phages multiply effectively only with a sufficiently large concentration of bacteria, mass lysis of which causes toxic shock in the patient. 

All this leaves no place for phage therapy as a standard ubiquitous method of treatment. However, phages can be useful in narrow niches, and enthusiasts of the use of bacteriophages do not give up trying to come up with effective ways to use them. For example, targeted destruction of resistant bacteria using a CRISPR system targeting specific resistance genes.

The use of antibacterial peptides also faces similar problems: animals, plants and even humans are in service (our skin is covered with antibacterial peptides), they show high efficiency in laboratory conditions, but are unstable in the blood or toxic to human cells. Most of the agents developed in the last decade have not yet passed clinical trials.

In any case, the use of any complex "personalized" medicines will require ultra–fast diagnostics - after all, with many bacterial infections, it is vital to start treatment within the first day or even the first 12 hours of the disease. This year, the European international program Horizon 2020 has awarded a prize of 1 million euros for the creation of a "diagnostic tool for bacterial infection within 1-2 hours". The British charity Nesta went even further, establishing in 2014 the Longitude Prize of 10 million pounds for solving the problem of rapid diagnosis of infections and determining the spectrum of antibiotic resistance.

As we can see, despite all the apparent diversity of approaches, there is no worthy alternative to "low molecular weight inhibitors" (this is what traditional antibiotics are called in scientific circles), and it is not expected in the near future. This means that we will continue to live with sustainability. And it should be taken very seriously. The good news is that it seems that "superbugs" can be brought under control, but it requires the efforts of the whole society. 

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25.11.2015