On two fronts
How phages and antibiotics reinforce each other
Blog of Rostec, Naked Science
Today, the World Health Organization calls the resistance of infections to antibiotics one of the most serious threats to humanity. In search of alternative strategies for the prevention and control of bacterial infection, doctors are increasingly paying attention to bacteriophage therapy. At the same time, there is an opinion in the medical community that the combined use of antibiotics and phage preparations makes it possible to increase the effectiveness of antibacterial therapy. The potential of a comprehensive campaign in the treatment of bacterial infections with antibiotics and bacteriophages is told by the expert of the holding "Natsibio" of Rostec State Corporation, the head of the project office for the study of bacteriophages Alexander Zharnikov.
The whole history of antibiotics is a lesson in how important it is to trust the scientific approach in medicine, not counting on the "magic medicine". Unfortunately, humanity began to learn this lesson with great delay. Alexander Fleming, the creator of penicillin, also warned that antibacterial drugs should not be used thoughtlessly, otherwise bacteria will quickly become resistant to them. But few people listened to the words of the British microbiologist. Antibiotics began to be used for and without reason in medicine, and even more actively in agriculture.
Penicillin has been widely available since the 1940s, and in the 1950s resistance to it has already become a serious problem. New antibacterial drugs appeared, but a similar fate awaited them. Now the development of new antibiotics has become so complicated and costly that no pharmaceutical manufacturer wants to do it.
In 2014, WHO experts announced that the XXI century will become a post-antibiotic era: sooner or later, antibacterial drugs will lose, for the most part, their effectiveness due to microorganisms with multiple drug resistance. Five years later, in 2019, experts from the American Centers for Disease Control (CDC) said that the post-antibiotic era had already arrived. The creation of new antibiotics is still tight, so scientists are looking for alternatives. And one of them is bacteriophages.
Viruses infecting bacteria were discovered even earlier than penicillin, but lost the competition to it. Now they're back on the radar. In Russia, bacteriophages have been developed and produced on an industrial scale for about 80 years by NPO Microgen (part of the holding company "Natsibio" of Rostec State Corporation). The company's product portfolio includes 14 names of bacteriopreparations from 24 types of bacteria.
Today, phages are spoken of as an alternative to antibiotics. But there is another, no less interesting and effective strategy: to use phages not instead, but together with antibacterial drugs. It turns out that they can reinforce each other, and this has already been proven by many scientific studies.
What will happen if phages are used together with antibiotics?There are three possible outcomes.
In some cases, an additive effect is observed, that is, the addition of the effect of an antibiotic and bacteriophages. In addition to such a "sum", there is often a "multiplication" — synergy. Phages and antibacterial drugs are able to enhance each other, and then the effect of treatment is many times greater than what would be obtained if two drugs were used separately.
Sometimes there is also antagonism — antibiotics and phages interfere with each other. This suggests that their combinations should be used wisely. It is important not only to determine the sensitivity of bacteria to an antibiotic and a phage (there are special tests for this), but also to understand how two specific drugs will interact. How do bacteriophages and antibiotics help each other? Scientists have managed to discover several mechanisms.
Depending on their behavior in the bacterial cell, phages are divided into two large groups: virulent and moderate. Each of the groups has its own mechanisms of interaction with antibiotics.
Virulent phagesBacteriophages of this type act as aggressively as possible.
When a virus injects its DNA into a bacterial cell, it immediately turns into a factory for the production of new viral particles. The host's own vital processes are suppressed, and instead new copies of phage DNA and proteins necessary for the assembly of bacteriophages are synthesized. Eventually, the enzymes destroy the bacterial cell, and new viruses come out in search of the next victims. Such a cycle is called lytic.
Six main mechanisms of synergy with antibacterial drugs have been studied in virulent phages:
1. Antibiotics lead to filamentation. This term refers to the phenomenon when a bacterium repeatedly divides longitudinally, but the daughter cells do not separate from each other, and often no partitions are formed between them. It turns out a long "super cell" with many copies of chromosomes. With the help of filamentation, bacteria try to resist stressful conditions. Resistance to antibiotics themselves increases, and it becomes more difficult for "devouring cells" (phagocytes) in the human body to "swallow" such a large microorganism. But at the same time, the bacterial cell wall becomes more vulnerable to the enzymes of virulent bacteriophages. Due to this, the production of new viruses is significantly accelerated.
2. Antibiotics increase infections caused by phages. To grow bacterial colonies, special flat Petri dishes with a jelly—like nutrient medium - agar are often used. If appropriate virulent phages are applied to such a bacterial lawn, then translucent plaques will appear on it. These are places where microorganisms have been infected and destroyed. In such experiments with Staphylococcus aureus and the phage infecting it, scientists found that some antibiotics contribute to a threefold increase in the diameter of plaques. Other studies have shown that antibacterial drugs significantly increase the number of new phages that come out of cells. Why such effects occur has not yet been fully studied, but it is clearly not only in the aforementioned filamentation.
3. Prevention of resistance to antibiotics and/or phages. Bacteria can become resistant not only to antibacterial drugs, but also to phages. The trick is that phages and antibiotics act differently, and microorganisms must also resist them in different ways. And fighting on two fronts, as you know, is much harder. If there are antibiotic-resistant bacteria among the bacteria that caused the infection, then they will be destroyed by bacteriophages and vice versa. In laboratory experiments, this trick worked even with the most famous "superbug" — methicillin-resistant Staphylococcus aureus (MRSA or MRSA).
4. Phages contribute to the resensitization (restoration of sensitivity) of bacteria to antibiotics. This is also the "effect of a war on two fronts." Bacteria have many defense mechanisms against phage infection, but all this is not free fun. The genome of microorganisms is quite small, and it will not be possible to fit all the useful mutations into it at once. To get resistance to viruses, you need to sacrifice something. Due to this, sensitivity to antibiotics can be restored. And even if this does not happen, the bacterium as a whole may become "weaker", not so aggressive. In the case of an infection, this will also benefit the patient.
5. Bacteriophages reduce the minimum inhibitory concentration (MIC) of antibiotics. MIC is the smallest concentration of antibacterial drugs that can be used to suppress the growth of bacteria. It is determined in laboratories to select doses of drugs for patients. Studies show that some bacteriophages reduce MIC, due to this, a smaller dose of an antibiotic can be used. The mechanisms of such synergy have yet to be studied in more detail.
6. Bacteriophages destroy biofilms. In unfavorable conditions, many organisms produce mucus and the whole colony swim in it, as in jelly. It turns out to be a kind of fortress that provides protection from antibiotics. Phages, unlike antibacterial drugs, are able to evolve, and many of them have learned to "open" biofilms with the help of depolymerase enzymes.
Some of these enzymes are fixed in the envelope of the virus, and some are released freely when the bacterial cell is destroyed. Some of them damage the shells of bacteria, while others dissolve mucus. Both greatly facilitate the work of antibiotics. For example, scientists managed to get good results in experiments with biofilms of Pseudomonas aeruginosa.
Moderate phagesUnlike virulent phages resembling brave paratroopers, moderate ones are more like experienced saboteurs.
When a bacteriophage injects DNA into a bacterial cell, new phage particles are not synthesized immediately. Phage DNA (in this form it is called a profage) is embedded in the bacterial chromosome, and the microorganism can continue to live a normal life. Such coexistence can last for a very long time, but at a certain moment the prophage is activated (this phenomenon is called induction), and the lytic cycle starts. The induction of a profage can occur by itself or under the influence of certain factors, for example, ultraviolet radiation.
Experiments have shown that there may also be synergy between moderate bacteriophages and antibiotics. It is usually provided by two mechanisms:
Moderate phages also increase the sensitivity of bacteria to antibiotics (cause resensitization), but not as virulent. To achieve this effect, we need not ordinary "wild" bacteriophages, but genetically modified in a special way. The most interesting thing is that scientists use their own weapons of bacteria, originally designed to protect themselves from phages.
Bacterial cells have a special "antiviral immunity" – the CRISPR-Cas system. In the chromosome of microorganisms there are special areas in which fragments of phage DNA are encoded. With the help of them, the bacterium is able to recognize the DNA of the virus and destroy it with enzymes-endonucleases. When scientists understood the principle of the CRISPR-Cas system, they began to edit the genes of plants and animals with it. After all, the system can be designed so that it can precisely "cut" any given gene in the right place.
A CRISPR-Cas system can be inserted into a moderate bacteriophage, which will target the bacterial genes associated with antibiotic resistance. At the same time, it increases resistance to lytic phages and, thus, bacteria sensitive to antibiotics gain an evolutionary advantage, displacing resistant ones.
With the help of moderate phages, genes can also be introduced into the bacterium, which themselves will make it sensitive to an antibiotic. For example, streptomycin resistance often occurs due to a mutation in the rpsL gene. If you introduce another gene into a bacterium without mutations, it will start working as a dominant one, and antibiotics will work again.
Antibiotics are able to activate a moderate phage. For example, some of them act through an SOS response. This is the name of a special system in bacterial cells that is activated when DNA is damaged and starts its "repair". But at the same time, the phage DNA "wakes up" and the lytic cycle starts.
Synergy in action: Patient stories
No matter how encouraging the experiments "in vitro" are, the human body is more complicated, and everything can go completely wrong in it during infection. Fortunately, the synergy between bacteria and phages is also confirmed by the examples of real patients. Two vivid stories have been published on this topic.
One story tells about a 76-year-old American who underwent surgery for an aortic aneurysm in 2012 and had an implant installed. As a result, a complication developed — an infection caused by Pseudomonas aeruginosa. This microbe circulates in many clinics, causes nosocomial infections and is resistant to many antibiotics.
For several years, surgeons regularly "cleaned" the man's chest, pumped out pus, conducted courses of antibiotic therapy, but stubborn bacteria did not give up. Then scientists from Yale University proposed an experimental method of treatment: bacteriophage in combination with the antibiotic ceftazidime. OMKO1 phage was chosen as an assistant to antibacterial therapy.
It effectively dissolves biofilms of Pseudomonas aeruginosa. In addition, bacteria that have developed resistance to phage simultaneously become sensitive to antibiotics.
The patient was injected with a bacteriophage into the chest cavity, and after a short course of treatment with ceftazidime was performed. The infection went away and did not recur over the next 18 months, as the scientists reported in their article.
The second story can be found not only in the scientific literature, a whole popular science detective story "The Perfect Predator" has been written on it. In her book, epidemiologist Stephanie Strathdy tells how she saved her husband from necrotizing pancreatitis, a severe inflammation of the pancreas caused by the antibiotic—resistant bacterium Acinetobacter baumannii. 68-year-old Tom Patterson picked up an intestinal infection in 2015 while in Egypt with his wife.
When he started vomiting, Stephanie gave him an antibiotic and was sure that Tom would be on his feet the next morning. But there was no improvement. The man's stomach swelled, he began to rave, and then fell into a coma. Egyptian doctors just shrugged. When it became clear that everything was very serious, the patient was taken to one of the leading clinics in San Diego. There a terrible microbe was revealed. The luminaries of American medicine also practically could not help: not a single antibiotic acted on the malicious bacterium.
Stephanie began to search for information herself and found out that bacterial infections can be fought with the help of special viruses. She used her connections in the scientific world to get a cocktail of the necessary bacteriophages. When they began to be used in parallel with antibiotics, the man went on the mend. Now Stephanie and Tom are actively promoting phage therapy.
To date, phage therapy scientists have yet to answer many questions: which antibiotics and bacteriophages should be used together, in which cases, in what dosages and what regimens. But one thing is already clear unambiguously — a "union" between two fighters against bacteria is not just possible, but exists in reality, and this "friendship" can potentially save many lives: phages do not displace antibacterial drugs — they complement them, and sometimes give them a second life.
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