A High-content Screening Assay based on Automated Microscopy for Monitoring Antibiotic Susceptibility of Mycobacterium Tuberculosis Phenotypes

A High-content Screening Assay based on Automated Microscopy for Monitoring Antibiotic Susceptibility of Mycobacterium Tuberculosis Phenotypes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A High-content Screening Assay based on Automated Microscopy for Monitoring Antibiotic Susceptibility of Mycobacterium Tuberculosis Phenotypes Sadaf Kalsum, Blanka Andersson, Jyotirmoy Das, Thomas Schön, Maria Lerm This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-133359/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background Assays enabling efficient high throughput drug screening are necessary for the discovery of new anti-mycobacterial drugs. The purpose of our work was to develop and validate an assay based on live-cell imaging which can monitor growth of two distinct phenotypes of Mycobacterium tuberculosis and to test their susceptibility to commonly used TB drugs. Results Both planktonic and cording phenotypes were successfully monitored as fluorescent objects using the live-cell imaging system Incucyte S3, allowing collection of data describing distinct characteristics of aggregate size and growth. The quantification of changes in total area of aggregates was used to define IC50 and MIC values of selected TB drugs which revealed that the cording phenotype grew more rapidly and displayed a higher susceptibility to rifampicin. A checkerboard approach, testing pair-wise combinations of sub-inhibitory concentrations of drugs, revealed rifampicin, linezolid and pretomanid as superior in inhibiting growth of cording phenotype. Conclusion Our results emphasize the efficiency of using automated live-cell imaging and its potential in high-through put whole-cell screening to evaluate existing and search for novel antimycobacterial drugs. General Microbiology cording planktonic Mycobacterium tuberculosis whole cell screening automated live-cell imaging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Full Text Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the latest manuscript can be downloaded and accessed as a PDF. Supplementary Files Additionalfile1TableS1.xlsx Additionalfile2FigS1.tif Additionalfile3FigS2.tif Additionalfile4FigS3.tif Additionalfile5FigS4.tif Additionalfile6FigS5.tif Additionalfile7TableS2.xlsx Additionalfile8MovieS1.mp4 Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 10 Feb, 2021 Reviews received at journal 27 Jan, 2021 Reviewers agreed at journal 19 Jan, 2021 Reviewers agreed at journal 13 Jan, 2021 Reviewers invited by journal 11 Jan, 2021 Editor assigned by journal 05 Jan, 2021 Editor invited by journal 05 Jan, 2021 Submission checks completed at journal 05 Jan, 2021 First submitted to journal 21 Dec, 2020 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-133359","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":7593738,"identity":"18844ef2-641b-48aa-9b82-c6b308d472ca","order_by":0,"name":"Sadaf Kalsum","email":"","orcid":"","institution":"Linköping University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sadaf","middleName":"","lastName":"Kalsum","suffix":""},{"id":7593739,"identity":"b95b6ad5-9a7e-4751-8214-c548dd2cd3bd","order_by":1,"name":"Blanka Andersson","email":"","orcid":"","institution":"Linköping University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Blanka","middleName":"","lastName":"Andersson","suffix":""},{"id":7593740,"identity":"1e157637-8759-4606-8fe4-2394187f579c","order_by":2,"name":"Jyotirmoy Das","email":"","orcid":"","institution":"Linköping University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jyotirmoy","middleName":"","lastName":"Das","suffix":""},{"id":7593741,"identity":"c1923b1f-a4a3-4d11-b431-1c72bce6a8f9","order_by":3,"name":"Thomas Schön","email":"","orcid":"","institution":"Linköping University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Schön","suffix":""},{"id":7593742,"identity":"f16556c0-04de-4c6b-bfd3-d40d0cea5504","order_by":4,"name":"Maria Lerm","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYJCCAwwMFkCK+eCBBBK0SAAptgTitTBAtPAYHCBKrW772YcHfjBIyJlPO/PhwMM2Bnt+QlrMzqQbHOxhkDCWuZ274UBiG0PizAZCWg6kMRzgYZBInCEN0ZJA0Hlm558xHPzDIFE/QzrnAUiLvT1BLTfSGA4DbUmQkM5hAGlh3EDQLzeeMRyWMZAwnCGdZnAg4RzQhYQdlsb88U2FjbyEdPLDhz/KbOz5GwhZAwYGcJYEUepHwSgYBaNgFBAAADb+P8qAoWPHAAAAAElFTkSuQmCC","orcid":"","institution":"Linköping University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Lerm","suffix":""}],"badges":[],"createdAt":"2020-12-21 15:44:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-133359/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-133359/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":4787294,"identity":"224320c4-e3d6-4174-a883-ae642732bd16","added_by":"auto","created_at":"2021-01-07 19:09:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":268981,"visible":true,"origin":"","legend":"Scheme of experimental layout ","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-133359/v1/5d918052119d452cf2776f16.png"},{"id":4787509,"identity":"46177661-4199-4fbb-8304-79b9c90edfa2","added_by":"auto","created_at":"2021-01-07 19:12:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2682438,"visible":true,"origin":"","legend":"Morphological appearance and size of aggregates in planktonic and cording \n \nmodels. H37Rv growing in DMEM as planktonic (A) and cording (B) bacteria are shown at \n \ndifferent time points. H37Rv grown in broth with or without Tween-80 (Tween) as indicated \n \n(C). Frequency plots of the distribution of aggregate sizes at day -2 (D) and day 5 (E). Columns \n \nrepresent size intervals and are logarithmically distributed up to 104 μm2. NP (non-present) \n \nmarks intervals where no objects were identified. Data are presented as mean ±SD (N=3).","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-133359/v1/58f205b27069e72e8c41d435.png"},{"id":4788018,"identity":"9b908c6c-486a-418b-a88a-217ea0d87f0a","added_by":"auto","created_at":"2021-01-07 19:18:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":280515,"visible":true,"origin":"","legend":" Frequency of aggregate sizes during bacterial growth and in response to rifampicin \n \n(RIF). A, C, E) Frequency plots summarizing data from planktonic phenotype and B, D, F) \n \ndata from cording phenotype exposed to different concentrations of rifampicin after 3 and 5 \n \ndays of incubation. G, H) Frequency plots of untreated controls for planktonic and cording \n \nphenotype respectively. Data are presented as mean ±SD (N=3). ","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-133359/v1/ab5fcf656826553427e48db0.png"},{"id":4787298,"identity":"1daed761-4ef5-41fc-80b5-59312a906016","added_by":"auto","created_at":"2021-01-07 19:09:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":273816,"visible":true,"origin":"","legend":"Frequency of aggregate sizes during bacterial growth and in response to isoniazid \n \n(INH). A, C, E) Frequency plots summarizing data from planktonic phenotype and B, D, F) \n \ndata from cording phenotype exposed to different concentrations of isoniazid after 3 and 5 days \n \nof incubation. G, H) Frequency plots of untreated controls for planktonic and cording phenotype \n \nrespectively. Data are presented as mean ±SD (N=3). ","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-133359/v1/fbfb29c1ec9ce8e2cb7b8669.png"},{"id":4787508,"identity":"efb0bd02-64fd-4b32-bc66-0c1510451db2","added_by":"auto","created_at":"2021-01-07 19:12:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":237513,"visible":true,"origin":"","legend":"Dose response to antibiotics. Total area of aggregates in wells treated with antibiotics \n \nnormalized to median of untreated controls (N=33) were used to calculate IC50 values. Black \n \ndotted line represents IC50 value for planktonic and red dotted line IC50 value for cording \n \nphenotype as determined by nonlinear regression (inhibitor vs response) with 4 parameters (A, B) or 3 parameters (C-H). Data are presented as mean of three experiments ±SD. Dotted lines 198\n \ncross x-axes at the point representing IC50 value for planktonic (black line) and cording (red line) models. ","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-133359/v1/965b54b47f6360b453ae2d16.png"},{"id":4787305,"identity":"0e6fac40-87fd-4e7b-8ef0-e4b4dd7fdc95","added_by":"auto","created_at":"2021-01-07 19:09:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":110116,"visible":true,"origin":"","legend":"MIC values for rifampicin (RIF) and isoniazid (INH). Gompertz functions was used \n \nto calculate MIC values at day5 based on total area of aggregates normalized to median of \n \nuntreated controls (N=33) of the A), C) planktonic and B), D) cording model. ","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-133359/v1/788b315b9d6a3c889a3b4d6d.png"},{"id":4787304,"identity":"e5ac71eb-d630-4526-b0e0-546f18cfca82","added_by":"auto","created_at":"2021-01-07 19:09:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":208883,"visible":true,"origin":"","legend":"Inhibition of Rv growth by combination of antibiotics. H37Rv were treated by \n \ncombination of two antibiotics at sub-inhibitory concentrations (details in Table 2). Total area \n \nof aggregates in wells treated with antibiotics normalized to median of untreated controls \n \n(N=33) were used in analysis. Data are presented as mean ±SD (N=3). 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The purpose of our work was to develop and validate an assay based on live-cell imaging which can monitor growth of two distinct phenotypes of Mycobacterium tuberculosis and to test their susceptibility to commonly used TB drugs. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003e\u003c/p\u003e\u003cp\u003eBoth planktonic and cording phenotypes were successfully monitored as fluorescent objects using the live-cell imaging system Incucyte S3, allowing collection of data describing distinct characteristics of aggregate size and growth. The quantification of changes in total area of aggregates was used to define IC50 and MIC values of selected TB drugs which revealed that the cording phenotype grew more rapidly and displayed a higher susceptibility to rifampicin. 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