Tuberculosis is considered to be the number one public health threat in the world, infecting 9 million people per year and claiming the lives of around 2 million. The World Health Organization (WHO) currently aims to eradicate this deadly disease by 2035, and that means eradicating its causative agent: Mycobacterium tuberculosis, an aerobic bacterium that typically infects the lungs. It’s difficult to do this because tuberculosis is treated with a series of several antibiotics lasting anywhere from 6 to 9 months. There are four first-line antibiotics in use: isoniazid, ethambutol, rifampicin, and pyrazinamide. These drugs are taken in both an intensive treatment phase and a continuation phase, with hope for relief to the patient.
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| A computer-generated artistic rendering of M. tuberculosis cells based on scanning electron microscopy (SEM). Source. |
However, treatment with antibiotics can be hard on patients, especially when they’re taking four at once. Antibiotics can disrupt or upset the microbiota of patients’ guts, often resulting in diarrhea and other unpleasant side effects. Some doctors try to mitigate this by recommending that patients take probiotics or eat yogurt with their antibiotics. Probiotics contain live cultures (often the same found in yogurt) which some consider to have health benefits. There is slim evidence of these benefits, however.
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| An x-ray showing a primary tuberculosis infection in a human lung. Source. |
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| A cartoon depiction of the progression of the disease. Source. |
Treatment is only complicated by the emergence of antibiotic resistance. In cases of antibiotic resistance, bacteria change in response to antibiotics; they are not killed by them. Commonly this occurs when the full course of the drug is not taken, although some bacteria can naturally become resistant to antibiotics. In many cases, this can make diseases harder to treat, increasing the danger to those who may come in contact. This is especially dangerous in situations of antibiotic intolerance or immunodeficiency on the part of the patient. In the case of tuberculosis there are several types of drug resistance.
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| An overhead view of M. tuberculosis colonies grown in a petri dish. Source. |
Mono-resistant tuberculosis occurs when the bacteria is resistant to only one of the first-line drugs; poly-resistance occurs when the bacteria resists more than one first-line drug; multi-resistance occurs with resistance to at least both rifampicin and isoniazid; and extensive resistance occurs with multidrug resistance, including at least one second-line drug. Antibiotic resistance can be dangerous when acquired by many pathogens, but it can be especially so when acquired by a particularly infective and dangerous one. Because of the already-limited options to fully treat tuberculosis, antibiotic resistance presents unique challenges to doctors and their patients. This can lead to much more difficulty in treating tuberculosis, or can even result in resurgence of the illness after the treatment period has ended. Patients with sensitivities or intolerances to certain antibiotics face fewer options.
So, we’ve established that antibiotic resistance in Mycobacterium tuberculosis is bad news. Now what? Well, Walker et al. (2015) have proposed a novel approach to predicting resistance before it is discovered in a clinical setting. Doing so would allow doctors to personalize an antibiotic regimen for each patient diagnosed with tuberculosis, without wasting precious time trying out multiple antibiotics in the hopes of finding one that works. They accomplished this with the help of whole-genome sequencing.
What is whole-genome sequencing? Simply put, whole-genome sequencing means that scientists determine the complete sequence of an organism’s genome. In the case of Mycobacterium tuberculosis, Walker et al. are interested in applying this research technique in the clinical realm. By sequencing the entire genome of an M. tuberculosis strain obtained from a patient’s sample, they suggest, scientists and doctors could pinpoint the genes involved in antibiotic resistance. And if doctors know that a particular strain is resistant to, say, one antibiotic but susceptible to another, they can start their patients on the antibiotic they know will work best.
How did Walker et al. figure this out? All together, they sequenced the genomes of over 2,000 M. tuberculosis isolates. Next, they examined 23 genes that previous research suggested might play important roles in antibiotic resistance. However, they didn’t stop there -- they also looked at mutations that were related in some way to those 23 “candidate” genes. For instance, if a gene was known to be under similar selection pressure as a resistance-determining gene, they examined that gene as well. Their work was complicated slightly by the fact that bacterial “species” are somewhat nebulous and hard to pin down, and even identifying unique strains can be a challenge. Out of all of the isolates, they identified 1,414 different strains.
It turns out that a lot more than just 23 genes are relevant when it comes to antibiotic resistance. Their findings suggest that 120 mutations can code for antibiotic resistance. Meanwhile, 772 mutations are known to be benign. Incredibly, their new knowledge of the M. tuberculosis genome allowed them to predict antibiotic resistance or susceptibility with close to 90% accuracy. Just 12 mutations accounted for 91.7% of rifampicin-resistant isolates, suggesting that although many mutations are associated with antibiotic resistance, a few are especially important. Also intriguing is their discovery that 74% of resistant isolates possessed just one mutation. If a single mutation can code for antibiotic resistance, that means these changes in the M. tuberculosis genome are gain-of-function mutations.
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| This figure displays frequencies of resistant phenotypes associated with mutations to particular genes. The x-axis shows just a fraction of the genes that were examined by the researchers, and the y-axis is a count of the number of phenotypes in the sample containing mutations in those genes. The peaks or “skyscrapers” that we can see in this figure indicate that mutations in a few genes were very common in the sample. Furthermore, blue bars indicate that the associated phenotype has antibiotic resistance. So, when we see a very tall blue bar, we know that resistance-gaining mutations to that gene were common in the sample. |
What does all this mean in the “real world” of public health crises? Currently, the best way of predicting resistance before beginning a clinical antibiotic regimen is to culture M. tuberculosis sampled directly from the patient. Although this is effective, it is time-consuming and labor-intensive. With whole-genome sequencing, researchers might be able to predict with high accuracy which genes that determine resistance to which antibiotics are most prevalent in a region or population. For instance, if scientists identified a gene that encodes rifampicin resistance in a majority of M. tuberculosis samples from Malaysia, that might suggest to doctors that rifampicin is a poor choice for first-line antibiotic treatment regimens in Malaysian settings.
It is critical for doctors to know more about M. tuberculosis, but the value of this research is not limited to the clinical realm. Identifying which genes determine antibiotic resistance or susceptibility is an important step towards stopping the current epidemic in its tracks. If we know that a certain mutation to Gene X results in a resistant phenotype, scientists can also research how that mutation is spread within the population. If Gene X is frequently carried on plasmids, for example, that information would suggest to scientists that more research needs to be done on conjugation in M. tuberculosis.
This post has only just begun to scratch the surface of the fantastic and fascinating research being done on M. tuberculosis by scientists all around the world. As more is learned about M. tuberculosis, doctors are better able to create effective treatment plans and patients have better outcomes. Research on the topic of tuberculosis is critical to lowering instances of the deadly disease, and the whole-genome sequencing done by Walker et al. is just one of many efforts to do so.
Other Sources
WHO-Types of TB drug resistance
CDC-Treatment regimens for TB
WHO-Antibiotic resistance (general)
Harvard-Probiotics
About the Authors:
Sarah Paust '20 is a double major in Anthropology and Biological Sciences. An aspiring medical anthropologist, she is fascinated by plagues, epidemics, and all manner of icky infectious diseases. In her free time, she can be found bird watching, collecting insects and seashells, and reading sci-fi/fantasy novels. Her favorite book is Station Eleven by Emily St. John Mandel, and her favorite place on campus is the Stimson Room.
Michaela Ramsey '20 is a biology major and chemistry minor. She is on the pre-veterinary track and plans to apply to veterinary school in the upcoming application cycle. Michaela hopes to work in large animal medicine and oncology in under-served areas of Maine, where she grew up. She hopes to provide practical and affordable veterinary care to all people. Her favorite place on the Mount Holyoke campus is the student garden.
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