CRI Research focus - Antibiotic resistance

Introduced in the 1940s with the release of penicillin, antibiotics are a class of drugs used to treat bacterial infections. More than one hundred have been developed in the second half of the 20th century and they appeared as a miracle of modern medicine. Today, they are the most common classes of drugs used in medicine. We rely on antibiotics to treat many infections like urinary tract infections, strep throat or some pneumonia. However, even before antibiotics were on the market, scientists observed that antibiotic resistance could happen. In 1945, the year he won a Nobel Prize for his discovery of penicillin, Alexander Fleming warned: “There is danger that the ignorant man may easily underdose himself, and by exposing his microbes to non-lethal quantities of the drug make them resistant.Resistance indeed has emerged rapidly following the use of antibiotics, leading to the emergence of multidrug resistant bacteria, also known as superbugs. Usually, only some bacteria spontaneously mutate their genes to become resistant to drugs, but these germs are in disadvantage in the absence of antibiotics. If people take low doses of antibiotics, non-resistant bacteria are killed while the few mutants that are resistant might survive. They would then multiply and take over.

On 2014, the World Health Organization (WHO) published its first global report on antimicrobial resistance, that revealed “this serious threat is no longer a prediction for the future, it is happening right now in every region of the world and has the potential to affect anyone, of any age, in any country.Experts warned that we are approaching a “post-antibiotic area” in which common infections and minor injuries would once again kill.

If antibiotic resistance occurs naturally, misuses of antibiotics is accelerating the process. Overuse of antibiotics is a typical example. Since antibiotics only treat bacterial infections, taking antibiotic against a viral sore throat will not work and it can create bacteria that are harder to kill. The Center for Disease Control and Prevention (CDC) in the United States estimated that at least one-third of all antibiotic prescriptions are unnecessary. Another phenomenon that encourage antibiotic resistance is underdosage, which occur when treatment is interrupted before the end of the prescription. Moreover, misuses occur also for animals, particularly on industrial farms where breeders give antibiotics to prevent disease and boost animal growth. Resistant bacteria then spread and can be transmitted to humans through the food chain.

In France, a national plan to preserve the efficacy of antibiotics has been developed since 2000. Despite public awareness campaign, levels of antibiotics consumption remains high. According to the WHO, antibiotic resistance is today one of the biggest threats to global health, food security and development. As the antibiotics used to treat become less effective, a growing number of infections, such as pneumonia, gonorrhea, tuberculosis or salmonellosis, are becoming harder to treat.

Tuberculosis, a re-emerging disease

For a long time, tuberculosis (TB) was difficult to cure but antibiotics were a major breakthrough and mortality rates declined in the second half of the 20th century. Many people think that tuberculosis is a disease from the past, but it was never completely eradicated. Even worse, it has re-emerged in drug-resistant forms.

Tuberculosis has been infecting humans for many centuries, it was called phtisie in ancient greece or scofula in middle age. This infectious disease usually affects the lungs but can also affect other parts of the body. Common symptoms of active lung TB are cough with sputum and blood, chest pains, weight loss and fever. The bacteria responsible, discovered by Robert Koch in 1882, is called Mycobacterium tuberculosis. Today, a combination of antibiotics is used to treat TB disease and the full course of drugs requires several months. Causes of drug-resistant TB include incomplete or erratic treatment, wrong treatment or drugs of poor quality. Prevalence of tuberculosis has today increased a such worrying level that the WHO declared it a global health emergency. As the disease is becoming harder to cure, there is an urgent need to improve our understanding of drug-resistant TB, as well as improve TB diagnosis and identify new drugs that are effective against resistant bacteria.

At CRI, this health challenge involves 2 interdisciplinary teams who are at the intersection of teaching and open research. These teams are working on projects to fight antibiotic resistance and drug-resistant TB, as well as improving our knowledge and implicating citizens.

New weapons to discover drugs

Despite major advances in the technology of drug research, developing effective antibiotics remains a long, complex and expensive process. New antibiotics have been rare in 30 years. At CRI, the team House of Transgenes works on an open-science technology to simplify the process of drug discovery. “There are many people who have tuberculosis but few of them have money to spend on treatments. And because it is so difficult to develop new drugs, pharmaceuticals companies don’t invest in developing new tuberculosis treatments”, Jake Wintermute explains. This long term fellow, who likes geeking out about transgenes, is excited about a future where biology and bio-technologies play a more direct role in our daily life. He left United States 8 years ago to work at a post-doc with Ariel Lindner, co-founder and research director of the CRI. At that time, CRI didn’t exist in the form that we know it today. It was basically « just a broom closet, at the end of a dirty hallway in Cochin Hospital », as he says. As CRI has grown up over the years to become “an impactful institute”, Jake Wintermute has developed his researches on synthetic biology, working on the biology of aging or metabolism. The idea of using genetic engineering to find new ways of treating tuberculosis came up in 2013, when he mentored a team of students participating in the International Genetically Engineered Machines (iGEM) competition. Besides winning the first prize, the student’s project has evolved into Jake Wintermute’s fellowship proposal. “It was entirely inspired by them. It came just from the natural students passion to help people and to solve problems like tuberculosis drug discovery. And it works. Thanks to them, we are able to show the basic concept of the system”.

The team now works on a safe model organism to make drug discoveries cheaper and faster. Currently, the standard method for discovering a new drug in tuberculosis involves growing the tuberculosis bacteria (Mycobacterium tuberculosis) and testing new drugs directly on them. This method is expensive, first of all because the tuberculosis bacteria is dangerous and requires a lot of expensive equipment to handle safely, and second because the tuberculosis bacteria grows very slowly. It takes about 14 days to do an experiment on the tuberculosis bacteria. So the team House of transgenes has chosen an alternative approach. They move genes from tuberculosis into another bacteria, Escherichia coli (also known as E. coli), in a way that allows them to do drug discoveries directly on E. coli. By this method, the scientists never handle real tuberculosis and never have to be close to it. Even the genes that they used don’t come directly from tuberculosis bacteria. They use only the information from the tuberculosis genome in database and the genes are synthesized chemically, then come to them in a mail. “Instead of testing drugs on a very dangerous and pathogenic bacteria, we can find new drugs using engineered E. coli, which is a harmless lab bacterium that grows faster”, researcher Nadine Bongaerts, who has joined the team for 4 years, explains. This construction that she has developed during her PhD is called TESEC, for “Target-Essential Surrogate E. coli” strains. The vision in term is an easy-to-use toolkit that can carry drug discovery technologies, designed to be replicable and enriched by scientific communities around the world. As Jake Wintermute says, “There are a lot of people who want to participate in the drug discovery process but they don’t have access to the correct facilities. And so, if we can make this technology very cheap and very fast, and completely safe, we can benefit from their enthusiasm and allow them to participate. The image that we have in our head is a medical or a biology student, this may be someone in high school or even in the university, who is just learning the basics of interacting with microbe. Why not design a class for that person where they perform a drug screen against tuberculosis using our toolkit?”

The researcher has also created a free and online course called Synthetic Biology 1. This course is for anyone who is interested in “the art and science of designing DNA sequences for living cells”. If you follow the lessons, you can for example learn how to make yogurt. Along the way, you will learn how to culture, feed and care for bacteria. And “this is something you can do at home, in your kitchen, with no prior training in biology.” This is a part of his vision in making science more accessible, outside of the academic community or the traditional biotechnology industry. He explains that synthetic biology is also the opportunity to have a direct impact on real world problems in the future. “Biological engineering today is in a place similar to where electronic engineering was in the 1960-70’s. At that time, there were very important innovations in computer sciences: cheap electronics that were widely available, open-source software that can be shared. That allows the field of computer science to grow very quickly from something that was expensive and done only by a small number of specialized groups, in something that was cheaper and that was done around the world. So we think that biology is in a similar place today, because biology is becoming much easier to engineer and DNA has become much cheaper to edit or to synthesize. Our understanding of biology is evolving to a place where we can start to make changes in a living system that have predictable results.”

It is indeed thanks to synthetic biology that the team has been able to elaborate their open-science tool. To start, they have focused the project on tuberculosis drug discovery, but the TESEC toolbox could be applied to any pathogen. As explained by Nadine Bongaerts, “people could easily, like lego bricks, take some parts and do other combinations. It is a community toolbox that everyone can share but also build apart”.

Tackling the emergence of antimicrobial resistance

Despite being preventable and treatable, tuberculosis (TB) has become a major threat to global health. “If we look at the incidence of drug resistance TB all across the world, we don’t find a country on the map which doesn’t have a reported case. Which means that it is more or less a global issue”, long-term fellow Anshu Bhardwaj says. She has worked on tuberculosis for the past 10 years, driven by the desire to contribute on public health. One year ago, she associated her experiences in crowdsourcing and genomics with the interdisciplinary of the CRI. The goal of her team AB-Open lab is to address antimicrobial resistance challenge, using genomics and artificial intelligence tools. As defined by the Global Action Plan endorsed by the World Health Assembly, there are 5 objectives to tackle antimicrobial resistance. The two first aim to improve knowledge through surveillance and research, while increasing the awareness of antimicrobial resistance through communication and training.

In the case of tuberculosis, one of the challenge is the need to identify infections from nontuberculous mycobacteria (NTM). NTM is a class of 180 different species present naturally in the environment. They live in water or soil and can infect humans or animals. Clinically, they are very similar to TB. Symptoms include cough, fever, shortness of breath, weight loss or lack of appetite, feeling tired... As the disease presentation is similar to tuberculosis, it is crucial to know NTM species and their drug resistant profiles for prescribing an appropriate treatment.

Currently, tuberculosis diagnosis was improved because clinicians use a precise method to identify Mycobacterium tuberculosis, based on unique fragments of the bacterial genome. The same markers are not known, neither established for NTMs yet. “When you speak to clinicians on what they really do for diagnosis, you realize that only a few labs right now are trying to characterize the NTMs.” Anshu Bhardwaj explains. She adds that therapy for NTM infections is still empiral. “The drugs that we give to a TB patient are the same set of drugs that we give to a NTM patient. But in the case of TB, we have a fixed regimen, so you know which drug and what combination for how long. There is no global standard for NTM infections that everybody in the world follow, so clinicians try and mix and match. You see how the patient responds and you keep changing treatment. The problem is : if you don’t match the right antibiotic to the right pathogen, instead of curing the patient, you actually contribute to increase the resistance.”

Therefore, the aim of her project at CRI is to “identify markers we can use to first identify if it is TB or NTM. Secondly, if it is NTM, which one, and once you know which one, can you say if this specie or this particular strain will or not respond to X Y Z antibiotic but may respond to A B and C?”

Rania Assab, postdoctoral researcher in AB-openlab, works on a computational software to better delineate NTMs species : “We compare whole genome sequences of NTMs and work on identifying commons or unique fragments. Beyond these genes, which one are signatures of resistance to a particular antibiotic?” Among NTM species, they work in particular on Mycobacterium abscessus. It is is the most pathogenic and drug-resistant germ, that is why scientists have named it “incurable nightmare”. This mycobacteria has intrinsic and acquired resistance to commonly used antibiotics, which limits the therapeutic options for infections.

The project is crowd-sourced, as researchers work closely with partners from all across the world. They want to get feedback from the community who is at the interface of seeing patients and developing protocols. “Drug discoveries are made usually in a very confidential environment. So when things fail, you never know why they fail. Even if it’s a success, only the outcomes are shared and not the process. There are a lot of learning gaps, so we thought : why not make the process open for everyone to participate? Anshu Bhardwaj explains, “we are setting up with reference laboratories the algorithms we are developing. They can test it in clinic, and if it is works, they can use it. And if it doesn’t, they can give a feed back to us on where to improve. We are not doing this work in isolation.”

Another part of the work is focused on education. Because antimicrobial resistance is a complex concept to communicate, the idea was to start by educating kids and teenagers. And the most exciting thing for them is to play video games, so the post-doctoral researcher in AB-openlab Raphael Goujet is developing a gaming application to understand the concept and challenges of antimicrobial resistance : “The aim is not only to learn something. It will not be an educational game where you have to memorize things, this game is designed to encourage people to use antibiotics wisely”. The first prototype is already available, with 9 game levels. “We are introducing concepts which are not very easy to communicate”, Anshu Bhardwaj says. “For example, if we take a look at behaviors, when a doctor prescribes an antibiotic regimen for 5 days, many people don’t finish that regimen because they feel good after 3 days. Another example, in my home, somebody else is sick and the doctor gave an antibiotic prescription. A week after I got sick and maybe it is the same infection. So I self prescribe antibiotics to myself. All of these things are bad practices and lead to misuses of antibiotics.” The game is strategical : if you don’t kill the right pathogen with the right antibiotic, you will see more resistant pathogens and you are going to lose the game. If you don’t give the right amount of doses of antibiotics, the pathogen will come back. To win, the player needs to have a correct understanding of how antibiotics work. A chatbot is developed in parallel with the power of artificial intelligence and will be integrated into the game. It will advise the player and tell him specific details about different pathogens, their life cycles, symptoms and treatments… To Anshu Bhardwaj, this project was made possible by the unique environment of the CRI, which promotes interdisciplinarity with many collaborations :“It allows me to integrate various skills in an easy fashion. That makes it feasible for somebody like me who is a biologist, to basically work with somebody who did his PhD in game design”.