— Igor, what made you study tuberculosis for such a long time?
— I'm not a doctor, and I see TB not as much of a disease, but as a biological species, Mycobacterium tuberculosis, which has coexisted with us for tens of thousands of years. The microbial nature of the disease was not clarified until the end of the 19th century when Robert Koch discovered the tubercule bacillus itself.
"Tuberculosis is just another infection, it's boring", a colleague epidemiologist once said to me. For her, it was just another infection on the long list, a row in a table: diphtheria, tetanus, tuberculosis... But the history, the origin, and evolution of the microbe are what interest me the most. That's a lot more fascinating.

— What is the most intriguing thing that has happened in the research of this biological species recently? Any milestones?
— Well, the first tubercule bacillus genome was sequenced in 1997. In the past 10 years, new, high-throughput technologies have arrived, enabling us to sequence hundreds of bacterial strains in a matter of hours. And, finally, the bioinformatics hardware has become more affordable, user-friendly (at least, for tuberculosis research) and powerful.

— Which research questions are the most important for you?
— First of all, I’d like to see if all of our assumptions and speculations about the changes in the genome of Mycobacterium tuberculosis are valid: what those changes are exactly, when and how they occurred and why they became preserved. For instance, there's the Ural genotype, which was initially assumed to have originated in the Urals. Years later, it was established by analyzing the available data that this genotype is more prevalent in the North Black Sea region. There's another related family in the South Black Sea region, TUR (named after Turkey). Apparently, their common ancestor originated somewhere in the Black Sea region.
The Latin American-Mediterranean family (LAM) of M. tuberculosis is interesting in its own way. It has this branch, LAM-RUS, unique to Russia. It covers a vast territory — in Russia and beyond, across Northern Eurasia, including places like Mongolia. All LAM-RUS strains are very close to each other. It's unclear when this branch emerged, but our guess is, it goes way back. Its long-term existence is hinted at by the fact that the Mongolian strains, for instance, do not demonstrate drug resistance. This suggests that these LAM-RUS strains had originated and started to spread before antibiotics came on the scene. We do not know how this genotype spread as well. We assume it had spread through human migration, very slowly and gradually, possibly over the course of decades, even centuries. To answer this question, we would have to study ancient bones (which would be ideal but is very difficult for many reasons) or collect a larger and more geographically representative sample, and use more sophisticated methods of bioinformatics analysis.
Working on our recently concluded RSF project, we discovered another, highly unusual genetic variant of M. tuberculosis. Its genetic profile shows six distinct mutations in drug resistance genes, making it resistant to four antibiotics. This variant, although found in small percentage rates, is present in different parts of Russia (Northwest Russia, Siberia), in Greece (brought by migrants from the former USSR), and rare cases in Albania, Serbia, and recently, also Poland. Its resistance profile was formed pretty long ago – our guess is, some time in the 1970s – but we have no idea where it happened. My personal hypothesis is that this could have happened in the Soviet penitentiary system, where the unhealthy and overcrowding conditions would favor spread of the more contagious strains. Another famous Russian epidemic strain, Beijing B0/W148, was first identified in the late 1990s in the prisons of four different Siberian cities. As part of the same RSF project, we have described for the first time another peculiar strain, which I've called "Buryat genotype". This strain is characterized by high mortality rate in mice. The intriguing thing about this strain is, it is found pretty much exclusively in Buryatia, although nothing would stop it from spreading outside it via the migration processes in the past 30 years for instance.
In 2012, we made an attempt to study ancient DNA. My colleague and friend from Irkutsk, Oleg Ogarkov, gathered a box full of bones with potential traces of bone tuberculosis during an archaeological expedition at a 19th-century church graveyard. PCR tests showed the presence of TB pathogenic DNA, or so we thought. However, it is critically important in this kind of analysis to take into account the risk of contamination, that is, pollution of ancient DNA with modern DNA fragments of the same TB strain. When I replicated the experiment in St. Petersburg, using highly sensitive real time PCR, the findings were less than ideal (negative control, a sample with no biological material of interest, also showed a weak signal). Even though I was confident that some of these bones did contain M. tuberculosis DNA (specifically the ancient strain), the findings, to my regret, weren't conclusive enough to be fit for publication, at that time. It would be far more illuminating to identify the exact gene variant of those Siberian strains, and see whether it was, or wasn't related to the modern strains. We have recently revisited those findings. At some point in our conversations, Oleg suggested we do a specific analysis of the fluorescence signal accumulation curves for the real time PCR. We went ahead and did it, and our initial findings were fully validated! There was no trace of contamination. The bones collected from the late 18th — early 19th century historical church graveyards in Irkutsk did reveal evidence of lesions caused by TB mycobacteria. Interestingly, these were not the strains that emerged in Russia in the 20th century and are currently circulating (the Russian epidemic and endemic variants of the Asian genotype Beijing). They were some other strains brought by settlers from the European part of Russia in the 18th century.

— Does your lab work with live or inactivated TB bacilli? Where do you get them?
— The Institute doesn't have the facilities to work with live tuberculosis pathogens. This would require a higher level of biosafety. We receive the heat-killed strains from our collaborators, mainly from the St. Petersburg Research Institute of Phthisiopulmonology. They have a reference laboratory overseeing 11 regions in Northwest Russia. They put the bacteria in a test tube, inactivate them thermally, and then hand them over to us. The material we get is not infectious. We then extract the DNA, and we can do whatever we want with this DNA. In our new project (with RSF support) we'll be studying RNA and proteins as well as DNA. No single part of this material is infectious.

— Did you ever get anxious about getting infected?
— No, never. Never even thought about it. We've been dealing with TB since 1996. Perhaps earlier, maybe in the 1970s, when safety standards weren't so strictly enforced, such a fear might have been justified. My older colleagues told me some 10 or 15 years ago that many staff members of TB research institutes and bacteriological labs had had TB in one form or another and recovered. On the other hand, there was no such problem as drug resistance back in those days.
Nowadays, a person can contract a strain that is already resistant to, let's say, 10 antibiotics, and then we have to rack our brain devising an extra ingenious treatment program that may take as much as 2 years to complete. The patient needs to take pills every day. At some point, the patient figures they've had enough and they are tired. They give up treatment. Now the environment is perfect for the bacilli. It's classic Darwinian evolution and natural selection in action. They liven up and jump into action. A small number of resistant mutants gain an advantage, evading the effects of the antibiotic. This resistant population begins to dominate in the patient who now is unable to recover and may give TB to their healthy companion.

— How quickly can bacteria develop antibiotic resistance?
— It can happen rapidly, literally in a few weeks. One of the major improvements in our field is that we no longer study individual markers or resistance genes, but everything all at once, thanks to whole-genome sequencing. The genome of a tuberculosis bacillus consists of 4.5 million nucleotides, and roughly 4,000 genes. To do PCR and then sequence every gene we want to parse would be way too much work. With whole-genome sequencing, we can instantly see how a resistance mutation begins to appear with some bacteria in a specific genome position.
For example, let's take a look at how the second-line antibiotics — Fluoroquinolones - and the very new drug Bedaquiline impact the mycobacterial genome. We know now that bacteria are quick to develop resistance to these drugs. Whole-genome sequencing reveals more than just the evidence of resistance mutations in the strain's genome; it shows how the mutations have become numerous in the strain isolated three months after treatment versus one month after. From this, we can infer that there's hardly any point in treating the patient with these antibiotics.
That being said, it's important to understand that antibiotics do not act on DNA directly. They are not mutagens, otherwise they would affect human DNA as well, which would be unacceptable. As mentioned earlier, antibiotics contribute toward an environment where bacterial cells with certain random yet beneficial mutations gain an advantage and survive.
There's another method of fighting bacteria, when the patient receives a massive dose of antibiotic in an attempt to overwhelm the resistance. It is very interesting to study these overdoses and their short-term action. A drop of blood is taken and immunochemistry is applied to see how much of the antibiotic has entered the bloodstream. It is possible that the drug gets promptly expelled from the body, the patient takes the medication, but it never gets into the bloodstream.

— Do we know how the different TB strains spread?
— People get around and transmit TB bacteria to one another. The Industrial Revolution gave a powerful boost to the incidence of TB and mortality as well. There were no drugs or vaccines. The mortality rate in Russia was as high as 400 per 100,000 people at the beginning of the 20th century. That's incredibly high. Europe had experienced the same phenomenon earlier, in the 18th century. Before, the disease had likely spread inside households, between family members. I call it vertical transmission, vertical in a broad biological sense. People simply lived together, slowly infecting each other. Some died, and some had the covert, asymptomatic variant of TB. It passed from generation to generation, and that's how TB spread around across neighborhoods. When people relocated to another area, they brought their strains with them, and the strains took root in the new area. In regions with high population density, horizontal spread of TB increased dramatically, sometimes reaching the scale of an epidemic. The genetic susceptibility of some ethnic communities to TB certainly played a part, but this is its own fascinating subject. Let me just say that this is not about a single gene or mutation, but about numerous weakly interacting human genes.
In studying tuberculosis, its origins and the spread of its strains, it's always useful and exciting to track the concomitant historic events. My colleagues from South Africa once put forward a hypothesis that strains of the Beijing genotype (originated in China) had been introduced into their country by the Dutch East India Company, which had imported Malaysian and Indonesian slaves for its plantations and mines since the 17th century. Later on, when we examined the genetic diversity of Russian strains in the context of a global sample, we found that the genotype identified in Shanghai indeed is quite common in Cape Town and in the south of South Africa.

— It seems that this work could benefit from highly unusual interdisciplinary collaborations — perhaps STEM and humanities?
— History is incredibly fascinating. Overall, TB geography largely mirrors the history and geography of human populations, though there are some weird exceptions. Common sense is the true north in everything we do. There exist some highly sophisticated bioinformatics strategies today. They make it possible to pinpoint the dates when TB strains branched out. Let’s say it was 100 years ago. But in statistics, there are are confidence intervals. The computer-generated estimate may differ from reality. Basically, when it says "100 years ago  plus-minus 300 years", such an estimation is just useless, in my opinion. History is filled with events. It is often possible to link the findings of bioinformatics research with historical evidence, and find a suitable event that could confirm your hypothesis. On the other hand, conclusions of this nature should be taken with  a caution, a healthy dose of self-criticism and self-irony.

— How do you envision the further progress of the research field you work in?
— Thus far, I can say that research will become ever more productive in terms of systems biology, where all knowledge is consolidated and applied, for instance, toward analyzing the interaction networks of diverse genes or interaction between the human organism and a germ, both on individual and population level, over short and long periods of time. In collaboration with our Russian and international colleagues, we plan to investigate how different genes manifest in a living germ (transcriptomics, proteomics), with a special focus on the process of interaction between the micro and macro organism (experimental tuberculosis on a model of infected lab animals).
In addition, we have high hopes for the arrival of increasingly powerful computers and neural networks. This will further advance our research. It would be desirable for all of this to happen through interaction between many stakeholders — medical professionals, microbiologists, immunologists, biochemists. This is the only way to achieve a full and accurate interpretation of the findings (although with this approach, however, some particularly wild ideas will likely be nipped in the bud). To me, science is first and foremost about debate, explanation, and interpretation of knowledge rather than mere acquisition and accumulation thereof.