A crowd of BashTheBug volunteers reproducibly and accurately measure the minimum inhibitory concentrations of 13 antitubercular drugs from photographs of 96-well broth microdilution plates

Abstract

Tuberculosis is a respiratory disease that is treatable with antibiotics. An increasing prevalence of resistance means that to ensure a good treatment outcome it is desirable to test the susceptibility of each infection to different antibiotics. Conventionally this is done by culturing a clinical sample and then exposing aliquots to a panel of antibiotics, Using 96-well broth micro dilution plates with each well containing a lyophilised predetermined amount of an antibiotic is a convenient and cost-effective way to measure the MICs of several drugs at once for a clinical sample. Although accurate, this is still an expensive and slow process that requires highly skilled and experienced laboratory scientists. Here we show that, through the BashTheBug project hosted on the Zooniverse citizen science platform, a crowd of volunteers can reproducibly and accurately determine the MICs for 13 drugs and that simply taking the median or mode of 11-17 independent classifications is sufficient. There is therefore a potential role for crowds to support (but not supplant) the role of experts in antibiotic susceptibility testing.

Data availability

The data tables and a Jupyter notebook that allows the user to recreate the majority of figures and tables in both the manuscript and the supplemental information is freely available here: https://github.com/fowler-lab/bashthebug-consensus-datasetIt is setup so a user can either clone the repository and run the jupyter-notebook on their local computer (the installation process having installed the pre-requisites) or by clicking the "Launch Binder" button in the README, they can access and run the jupyter-notebook via their web browser, thereby avoiding any installation.I've added a short statement to the manuscript -- please advise if you think it needs changing.

The following data sets were generated

Article and author information

Author details

  1. Philip W Fowler

    Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
    For correspondence
    philip.fowler@ndm.ox.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0912-4483
  2. Carla Wright

    Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Helen Spiers

    Electron Microscopy Science Technology Platform, The Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Tingting Zhu

    Institute of Biomedical Engineering, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Elisabeth ML Baeten

    Department of Physics, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  6. Sarah W Hoosdally

    Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  7. Ana L Gibertoni Cruz

    Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9473-2215
  8. Aysha Roohi

    Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  9. Samaneh Kouchaki

    Institute of Biomedical Engineering, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  10. Timothy M Walker

    Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0421-9264
  11. Timothy EA Peto

    Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  12. Grant Miller

    Department of Physics, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  13. Chris Lintott

    Department of Physics, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  14. David Clifton

    Institute of Biomedical Engineering, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  15. Derrick W Crook

    Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0590-2850
  16. A Sarah Walker

    Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0412-8509

Funding

Wellcome Trust (200205/Z/15/Z)

  • Philip W Fowler
  • Carla Wright
  • Sarah W Hoosdally
  • Ana L Gibertoni Cruz
  • Aysha Roohi
  • Samaneh Kouchaki
  • Timothy M Walker
  • Timothy EA Peto
  • David Clifton
  • Derrick W Crook
  • A Sarah Walker

Bill and Melinda Gates Foundation (OPP1133541)

  • Philip W Fowler
  • Carla Wright
  • Sarah W Hoosdally
  • Ana L Gibertoni Cruz
  • Aysha Roohi
  • Samaneh Kouchaki
  • Timothy M Walker
  • Timothy EA Peto
  • David Clifton
  • Derrick W Crook
  • A Sarah Walker

Wellcome Trust (203141/Z/16/Z)

  • Philip W Fowler

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2022, Fowler et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,422
    views
  • 269
    downloads
  • 10
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Philip W Fowler
  2. Carla Wright
  3. Helen Spiers
  4. Tingting Zhu
  5. Elisabeth ML Baeten
  6. Sarah W Hoosdally
  7. Ana L Gibertoni Cruz
  8. Aysha Roohi
  9. Samaneh Kouchaki
  10. Timothy M Walker
  11. Timothy EA Peto
  12. Grant Miller
  13. Chris Lintott
  14. David Clifton
  15. Derrick W Crook
  16. A Sarah Walker
(2022)
A crowd of BashTheBug volunteers reproducibly and accurately measure the minimum inhibitory concentrations of 13 antitubercular drugs from photographs of 96-well broth microdilution plates
eLife 11:e75046.
https://doi.org/10.7554/eLife.75046

Share this article

https://doi.org/10.7554/eLife.75046

Further reading

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Amanda Mixon Blackwell, Yasaman Jami-Alahmadi ... Paul A Sigala
    Research Article

    Malaria parasites have evolved unusual metabolic adaptations that specialize them for growth within heme-rich human erythrocytes. During blood-stage infection, Plasmodium falciparum parasites internalize and digest abundant host hemoglobin within the digestive vacuole. This massive catabolic process generates copious free heme, most of which is biomineralized into inert hemozoin. Parasites also express a divergent heme oxygenase (HO)-like protein (PfHO) that lacks key active-site residues and has lost canonical HO activity. The cellular role of this unusual protein that underpins its retention by parasites has been unknown. To unravel PfHO function, we first determined a 2.8 Å-resolution X-ray structure that revealed a highly α-helical fold indicative of distant HO homology. Localization studies unveiled PfHO targeting to the apicoplast organelle, where it is imported and undergoes N-terminal processing but retains most of the electropositive transit peptide. We observed that conditional knockdown of PfHO was lethal to parasites, which died from defective apicoplast biogenesis and impaired isoprenoid-precursor synthesis. Complementation and molecular-interaction studies revealed an essential role for the electropositive N-terminus of PfHO, which selectively associates with the apicoplast genome and enzymes involved in nucleic acid metabolism and gene expression. PfHO knockdown resulted in a specific deficiency in levels of apicoplast-encoded RNA but not DNA. These studies reveal an essential function for PfHO in apicoplast maintenance and suggest that Plasmodium repurposed the conserved HO scaffold from its canonical heme-degrading function in the ancestral chloroplast to fulfill a critical adaptive role in organelle gene expression.

    1. Microbiology and Infectious Disease
    Francesca Torelli, Diogo M da Fonseca ... Moritz Treeck
    Research Article

    Toxoplasma gondii is an intracellular parasite that subverts host cell functions via secreted virulence factors. Up to 70% of parasite-controlled changes in the host transcriptome rely on the MYR1 protein, which is required for the translocation of secreted proteins into the host cell. Mice infected with MYR1 knock-out (KO) strains survive infection, supporting a paramount function of MYR1-dependent secreted proteins in Toxoplasma virulence and proliferation. However, we have previously shown that MYR1 mutants have no growth defect in pooled in vivo CRISPR-Cas9 screens in mice, suggesting that the presence of parasites that are wild-type at the myr1 locus in pooled screens can rescue the phenotype. Here, we demonstrate that MYR1 is not required for the survival in IFN-γ-activated murine macrophages, and that parasites lacking MYR1 are able to expand during the onset of infection. While ΔMYR1 parasites have restricted growth in single-strain murine infections, we show that the phenotype is rescued by co-infection with wild-type (WT) parasites in vivo, independent of host functional adaptive immunity or key pro-inflammatory cytokines. These data show that the major function of MYR1-dependent secreted proteins is not to protect the parasite from clearance within infected cells. Instead, MYR-dependent proteins generate a permissive niche in a paracrine manner, which rescues ΔMYR1 parasites within a pool of CRISPR mutants in mice. Our results highlight an important limitation of otherwise powerful in vivo CRISPR screens and point towards key functions for MYR1-dependent Toxoplasma-host interactions beyond the infected cell.