A high-throughput cidality screen for Mycobacterium tuberculosis.
Exposure to Mycobacterium tuberculosis (Mtb) aerosols is a major threat to tuberculosis (TB) researchers, even in bio-safety level-3 (BSL-3) facilities. Automation and high-throughput screens (HTS) in BSL3 facilities are essential for minimizing manual aerosol-generating interventions and facilitati...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2015-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4333287?pdf=render |
| _version_ | 1819113378308685824 |
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| author | Parvinder Kaur Anirban Ghosh Ramya Vadageri Krishnamurthy Deepa Gagwani Bhattacharjee Vijayashree Achar Santanu Datta Shridhar Narayanan Anand Anbarasu Sudha Ramaiah |
| author_facet | Parvinder Kaur Anirban Ghosh Ramya Vadageri Krishnamurthy Deepa Gagwani Bhattacharjee Vijayashree Achar Santanu Datta Shridhar Narayanan Anand Anbarasu Sudha Ramaiah |
| author_sort | Parvinder Kaur |
| collection | DOAJ |
| description | Exposure to Mycobacterium tuberculosis (Mtb) aerosols is a major threat to tuberculosis (TB) researchers, even in bio-safety level-3 (BSL-3) facilities. Automation and high-throughput screens (HTS) in BSL3 facilities are essential for minimizing manual aerosol-generating interventions and facilitating TB research. In the present study, we report the development and validation of a high-throughput, 24-well 'spot-assay' for selecting bactericidal compounds against Mtb. The bactericidal screen concept was first validated in the fast-growing surrogate Mycobacterium smegmatis (Msm) and subsequently confirmed in Mtb using the following reference anti-tubercular drugs: rifampicin, isoniazid, ofloxacin and ethambutol (RIOE, acting on different targets). The potential use of the spot-assay to select bactericidal compounds from a large library was confirmed by screening on Mtb, with parallel plating by the conventional gold standard method (correlation, r2 = 0.808). An automated spot-assay further enabled an MBC90 determination on resistant and sensitive Mtb clinical isolates. The implementation of the spot-assay in kinetic screens to enumerate residual Mtb after either genetic silencing (anti-sense RNA, AS-RNA) or chemical inhibition corroborated its ability to detect cidality. This relatively simple, economical and quantitative HTS considerably minimized the bio-hazard risk and enabled the selection of novel vulnerable Mtb targets and mycobactericidal compounds. Thus, spot-assays have great potential to impact the TB drug discovery process. |
| first_indexed | 2024-12-22T04:28:27Z |
| format | Article |
| id | doaj.art-c239f36bee1b49f5b62daa2cac16417d |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-22T04:28:27Z |
| publishDate | 2015-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-c239f36bee1b49f5b62daa2cac16417d2022-12-21T18:39:06ZengPublic Library of Science (PLoS)PLoS ONE1932-62032015-01-01102e011757710.1371/journal.pone.0117577A high-throughput cidality screen for Mycobacterium tuberculosis.Parvinder KaurAnirban GhoshRamya Vadageri KrishnamurthyDeepa Gagwani BhattacharjeeVijayashree AcharSantanu DattaShridhar NarayananAnand AnbarasuSudha RamaiahExposure to Mycobacterium tuberculosis (Mtb) aerosols is a major threat to tuberculosis (TB) researchers, even in bio-safety level-3 (BSL-3) facilities. Automation and high-throughput screens (HTS) in BSL3 facilities are essential for minimizing manual aerosol-generating interventions and facilitating TB research. In the present study, we report the development and validation of a high-throughput, 24-well 'spot-assay' for selecting bactericidal compounds against Mtb. The bactericidal screen concept was first validated in the fast-growing surrogate Mycobacterium smegmatis (Msm) and subsequently confirmed in Mtb using the following reference anti-tubercular drugs: rifampicin, isoniazid, ofloxacin and ethambutol (RIOE, acting on different targets). The potential use of the spot-assay to select bactericidal compounds from a large library was confirmed by screening on Mtb, with parallel plating by the conventional gold standard method (correlation, r2 = 0.808). An automated spot-assay further enabled an MBC90 determination on resistant and sensitive Mtb clinical isolates. The implementation of the spot-assay in kinetic screens to enumerate residual Mtb after either genetic silencing (anti-sense RNA, AS-RNA) or chemical inhibition corroborated its ability to detect cidality. This relatively simple, economical and quantitative HTS considerably minimized the bio-hazard risk and enabled the selection of novel vulnerable Mtb targets and mycobactericidal compounds. Thus, spot-assays have great potential to impact the TB drug discovery process.http://europepmc.org/articles/PMC4333287?pdf=render |
| spellingShingle | Parvinder Kaur Anirban Ghosh Ramya Vadageri Krishnamurthy Deepa Gagwani Bhattacharjee Vijayashree Achar Santanu Datta Shridhar Narayanan Anand Anbarasu Sudha Ramaiah A high-throughput cidality screen for Mycobacterium tuberculosis. PLoS ONE |
| title | A high-throughput cidality screen for Mycobacterium tuberculosis. |
| title_full | A high-throughput cidality screen for Mycobacterium tuberculosis. |
| title_fullStr | A high-throughput cidality screen for Mycobacterium tuberculosis. |
| title_full_unstemmed | A high-throughput cidality screen for Mycobacterium tuberculosis. |
| title_short | A high-throughput cidality screen for Mycobacterium tuberculosis. |
| title_sort | high throughput cidality screen for mycobacterium tuberculosis |
| url | http://europepmc.org/articles/PMC4333287?pdf=render |
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