Identifying resistance genes in tuberculosis

Newly published in PLOS Biology is our work identifying genes that confer resistance to common and last-resort antibiotics in bacteria that cause tuberculosis. Resistance to these drugs contributes to mortality and sickness on a pandemic scale every year, and disproportionately affects the poorest people in the world.

This new article is one of a series presenting results generated by more than 100 scientists across 23 countries across 5+ years as part of a collaboration called CRyPTIC.

Our role in CRyPTIC was the discovery of genes and mutations likely to cause drug resistance by applying a tool known as a genome-wide association study (GWAS), an approach we helped adapt to bacteria.

Using GWAS, we identified previously uncatalogued genes and mutations underlying resistance to every one of the 13 drugs we investigated. These include new and repurposed drugs, as well as the first- and second-line drugs more often used to treat tuberculosis.

Thanks to its generous funders, CRyPTIC dedicated scale (10,000+ genomes) and technical innovation (new high-throughput MIC assays) to help decode the DNA blueprint of antibiotic resistance. Pushing these boundaries has yielded a steep increase of up to 36% in the variation in resistance attributable to the genome for the important and previously understudied new and repurposed drugs.

Science at this scale can produce a seemingly overwhelming wealth of new information. We avoided the temptation to over-emphasize any individual result for the sake of simple narrative. Instead, we highlighted discoveries of uncatalogued genes or genetic variants that we found for every drug investigated:

The amidase AmiA2 and GTPase Era for bedaquiline.

The cytochrome P450 enzyme Cyp142 for clofazimine.

The serine/threonine protein kinase PknH for delaminid.

The antitoxin VapB20 for linezolid.

The PPE-motif family outer membrane protein PPE42 for amikacin and kanamycin.

The antibiotic-induced transcriptional regulator WhiB7 for ethionamide.

The rRNA methylase TlyA for levofloxacin.

The DNA gyrase subunit B GyrB for moxifloxacin.

The putative rhodaneses CysA2 and CysA3 for rifabutin.

The tRNA/rRNA methylase SpoU for ethambutol and rifampicin.

The multidrug efflux transport system repressor Rv1219 for isoniazid.

All these hits passed stringent evidence thresholds that take into account the large amount of data crunched. For each hit, we identified possible relationships between gene functions, such as they are known, and the mechanism of action of the antibiotics.

Beyond the biological discoveries of primary interest, this new paper unveils methodological advances in bacterial GWAS. We introduced a systematic, whole-genome approach to analysing not just short DNA sequences (so called oligonucleotide or “kmer”-based approaches), but also short sequences of the proteins that the DNA codes for (an oligopeptide-based approach). We have released our software on an open-source GitHub repository.

We also discovered a relationship that may help disentangle a technical issue in bacterial GWAS where the co-occurence of traits can trick us into thinking that a gene influences one trait when it influences another instead. For antimicrobial resistance, this issue is known as artefactual cross resistance. We observed that true associations tended to produce larger associations (as measured by the 'coefficient', rather than the p-value), providing a possible way to prioritize signals in the future.

This paper was published alongside the CRyPTIC Data Compendium in PLOS Biology, in which we released our data open source to the community, with resources provided by the European Bioinformatics Institute.

Some of the results of CRyPTIC have already been rushed into service by the World Health Organization on the grounds of exceptional importance based on a candidate gene approach; this includes the DNA gyrase subunit B – moxifloxacin association spotlighted above (Walker et al 2022). However, the new results go beyond a candidate gene approach, detecting a range of previously uncatalogued genes via its agnostic, whole-genome strategy.

Unpicking the genetics of antimicrobial resistance is a priority for improving rapid susceptibility tests for individual patients, selecting drug regimens that inhibit the evolution of multidrug resistance, and developing improved treatment options. The need is particularly great in M. tuberculosis, which killed 1.4 million people in 2019, owing to the slow (6-12 week) turnaround of traditional susceptibility testing, and the alarming threat of multidrug resistant tuberculosis. The discovery of many new candidate resistance variants therefore represents an advance that we hope will contribute to progress in reducing the burden of disease.

CRyPTIC: rapid diagnosis of drug resistance in TB

The Modernising Medical Microbiology consortium has announced a new worldwide collaboration called CRyPTIC to speed up diagnosis of antibiotic resistant tuberculosis (TB).

TB infects nearly 10 million people each year and kills 1.5 million, making it one of the leading causes of death worldwide. Almost half a million people each year develop multidrug-resistant (MDR) TB, which defies common TB treatments. Time consuming tests must be run to identify MDR-TB and which drugs will work or fail. This delays diagnosis and creates uncertainty about the best drugs to prescribe to individual patients.

CRyPTIC aims to hasten the identification of MDR-TB using whole genome sequencing to identify genetic variants that give resistance to particular drugs. The project is funded by a $2.2m grant from the Bill & Melinda Gates Foundation and a £4m grant from the Wellcome Trust and MRC Newton Fund.

CRyPTIC aims to collect and analyse 100,000 TB cases from across the world, providing a database of MDR-TB that will underpin diagnosis using WGS. Samples from across Africa, Asia, Europe and the Americas will be collected by teams at more than a dozen centres They will conduct drug resistance testing and much of the genome sequencing. Read more information here.

Science for the People: Understanding Neuroscience

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Blue Pyramidal Neuron - original watercolor painting on clayboard by Michele Banks (All Rights Reserved - Used with Permission)

Blue Pyramidal Neuron – original watercolor painting on clayboard by Michele Banks (All Rights Reserved – Used with Permission)

This week, Science for the People is we’re looking at the ways we try to understand the inner workings of the brain. They talk to University College London researcher Cliodhna O’Connor about patterns in the way the public interprets neuroscience news. And they’ll ask Duncan Astle, Program Leader at the MRC Cognition and Brain Sciences Unit, about “neuromyths,” popular misconceptions about the way the brain functions.

*Josh provides research help to Science for the People and is, therefore, completely biased.


Filed under: Follies of the Human Condition, This Mortal Coil Tagged: Cliodhna O'Connor, Duncan Astle, Medical Research Council, Michele Banks, MRC, Neuroscience, Podcast, sciart, science for the people, University College London

Neighbors Helping Neighbors

Busy Gym Testing for TB

When the Fairfax County Health Department (FCHD) in Virginia put out a call for volunteers to help conduct a tuberculosis (TB) contact investigation, Rosalia Parada, a long time Medical Reserve Corps (MRC) volunteer, jumped at the chance to serve her community.  The investigation was sparked when news of three students from Robert E. Lee High School acquired TB around the same time.

As a nurse and a resident of the Lee High School community, Parada saw firsthand the need for care and support throughout her neighborhood.  With a variety of ethnic groups and languages, the community is very diverse, lending itself to some challenges during a public health response of this scope.  For example, many families living in the community were from Spanish-speaking backgrounds.  In the same situation herself, , Parada felt that volunteering was an opportunity to provide interpretation, as well as nursing services for the contact investigation – when contacts like teachers, other students, and people who interacted with the students would be tested for TB.  “Many people don’t have access to health care,” Parada expressed, adding to the need for proper communication about the situation through interpretation. 

In the second phase of the contact investigation, screening and testing was provided at many sites around the community, one of which was located at Lee High School.  During the second phase, Parada volunteered as a screener.  She gathered information, distributed learning materials about what to do if someone has TB, and provided encouragement to those with concerns.  Her skills as a nurse permitted Parada to assist with TB testing, such as performing tuberculin skin testing (TST)

Although Parada is a practicing nurse, this was the first time she was involved in a response of this size.  During screening and testing, Parada saw the importance of providing comfort and reassurance for families, making them aware and helping build their knowledge about TB and the broad size and scope of signing upthe investigation.  Parada also stressed community awareness about TB vaccinations as a preventive measure for acquiring TB – and making the community aware of the consequences of not being tested, especially for those who were in close contact with the students who had acquired the active TB disease.

Among the many projects that Parada worked on as an MRC volunteer and as a practicing certified nurse, she says the Lee High School TB contact investigation is the largest and most unique. Parada explained that the gymnasium was very well organized for the screening and testing process, as volunteers kept the flow of students steady.   As a mother of three boys who graduated from Lee High School, she understood the worry, questions, and concerns from parents whose children may have been exposed to TB; but as a volunteer nurse, she knew the importance of making parents aware by gathering and providing accurate, helpful information while still being able to ease their worry.

Parada was extremely encouraged by the involvement of the community during the TB response.  She said the  investigation was a great learning experience for her community.  It combined the responsibility of the health department, along with the school and community, to help make people aware of good public health practice.