title: Serial image analysis of Mycobacterium tuberculosis colony growth reveals a persistent subpopulation in sputum during treatment of pulmonary TB.
abstract: Faster elimination of drug tolerant 'persister' bacteria may shorten treatment of tuberculosis (TB) but no method exists to quantify persisters in clinical samples. We used automated image analysis to assess whether studying growth characteristics of individual Mycobacterium tuberculosis colonies from sputum on solid media during early TB treatment facilitates 'persister' phenotyping. As Time to Detection (TTD) in liquid culture inversely correlates with total bacterial load we also evaluated the relationship between individual colony growth parameters and TTD. Sputum from TB patients in Malawi was prepared for solid and liquid culture after 0, 2 and 4 weeks of treatment. Serial photography of agar plates was used to measure time to appearance (lag time) and radial growth rate for each colony. Mixed-effects modelling was used to analyse changing growth characteristics from serial samples. 20 patients had colony measurements recorded at ≥1 time-point. Overall lag time increased by 6.5 days between baseline and two weeks (p = 0.0001). Total colony count/ml showed typical biphasic elimination, but long lag time colonies (>20days) had slower, monophasic decline. TTD was associated with minimum lag time (time to appearance of first colony1). Slower elimination of long lag time colonies suggests that these may represent a persister subpopulation of bacilli.
authors.affiliation: Wellcome Trust Liverpool Glasgow Centre for Global Health Research, University of Liverpool, 70 Pembroke Place, Liverpool, L69 3GF, UK; Brownlee Centre for Infectious Disease, Gartnavel General Hospital, 1053 Great Western Road, Glasgow, G12 0YN, UK. Electronic address: David.Barr@liverpool.ac.uk.
authors.forename: David A
authors.initials: DA
authors.lastname: Barr
authors.affiliation: Department of Microbiology, College of Medicine, University of Malawi, Mahatma Gandhi Road, Blantyre 3, Malawi. Electronic address: mkamdolozi@medcol.mw.
authors.forename: Mercy
authors.initials: M
authors.lastname: Kamdolozi
authors.affiliation: Queen Elizabeth Central Hospital, P.O. Box 95, Blantyre 3, Malawi; Malawi Liverpool Wellcome Trust Clinical Research Programme, College of Medicine, University of Malawi, Mahatma Gandhi Road, Blantyre 3, Malawi. Electronic address: dr.yo@me.com.
authors.forename: Yo
authors.initials: Y
authors.lastname: Nishihara
authors.affiliation: Department of Microbiology, College of Medicine, University of Malawi, Mahatma Gandhi Road, Blantyre 3, Malawi. Electronic address: vndhlovu@medcol.mw.
authors.forename: Victor
authors.initials: V
authors.lastname: Ndhlovu
authors.affiliation: Department of Microbiology, College of Medicine, University of Malawi, Mahatma Gandhi Road, Blantyre 3, Malawi. Electronic address: mkhonga@medcol.mw.
authors.forename: Margaret
authors.initials: M
authors.lastname: Khonga
authors.affiliation: Department of Microbiology, College of Medicine, University of Malawi, Mahatma Gandhi Road, Blantyre 3, Malawi; Institute of Infection and Global Health, The Ronald Ross Building, University of Liverpool, 8 West Derby Street, Liverpool, L69 7BE, UK; Malawi Liverpool Wellcome Trust Clinical Research Programme, College of Medicine, University of Malawi, Mahatma Gandhi Road, Blantyre 3, Malawi. Electronic address: gerrydavies@doctors.org.uk.
authors.forename: Geraint R
authors.initials: GR
authors.lastname: Davies
authors.affiliation: Liverpool Heart and Chest Hospital, Thomas Drive, Liverpool, L14 3PE, UK; Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK. Electronic address: Derek.Sloan@lstmed.ac.uk.
authors.forename: Derek J
authors.initials: DJ
authors.lastname: Sloan
mesh_terms: D000995:Antitubercular Agents / Q000627:therapeutic use*; D058491:Bacterial Load; D024881:Drug Resistance, Bacterial; D006801:Humans; D007090:Image Interpretation, Computer-Assisted; D008295:Malawi; D008853:Microscopy; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000254:growth & development; D010641:Phenotype; D011237:Predictive Value of Tests; D013183:Sputum / Q000382:microbiology*; D013997:Time Factors; D016896:Treatment Outcome; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis / Q000188:drug therapy* / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents
keywords: Biomarkers; Drug tolerance; Persisters; Pharmacodynamics