title: A Flow Cytometry Method for Rapidly Assessing Mycobacterium tuberculosis Responses to Antibiotics with Different Modes of Action.
abstract: Current methods for assessing the drug susceptibility of Mycobacterium tuberculosis are lengthy and do not capture information about viable organisms that are not immediately culturable under standard laboratory conditions as a result of antibiotic exposure. We have developed a rapid dual-fluorescence flow cytometry method using markers for cell viability and death. We show that the fluorescent marker calcein violet with an acetoxy-methyl ester group (CV-AM) can differentiate between populations of M. tuberculosis growing at different rates, while Sytox green (SG) can differentiate between live and dead mycobacteria. M. tuberculosis was exposed to isoniazid or rifampin at different concentrations over time and either dual stained with CV-AM and SG and analyzed by flow cytometry or plated to determine the viability of the cells. Although similar trends in the loss of viability were observed when the results of flow cytometry and the plate counting methods were compared, there was a lack of correlation between these two approaches, as the flow cytometry analysis potentially captured information about cell populations that were unable to grow under standard conditions. The flow cytometry approach had an additional advantage in that it could provide insights into the mode of action of the drug: antibiotics targeting the cell wall gave a flow cytometry profile distinct from those inhibiting intracellular processes. This rapid drug susceptibility testing method could identify more effective antimycobacterials, provide information about their potential mode of action, and accelerate their progress to the clinic.
authors.affiliation: Public Health England, National Infection Service, Porton Down, Salisbury, Wiltshire, United Kingdom.
authors.forename: Charlotte Louise
authors.initials: CL
authors.lastname: Hendon-Dunn
authors.affiliation: National Institute for Biological Standards and Control, South Mimms, Potters Bar, Hertfordshire, United Kingdom.
authors.forename: Kathryn Sarah
authors.initials: KS
authors.lastname: Doris
authors.affiliation: Public Health England, National Infection Service, Porton Down, Salisbury, Wiltshire, United Kingdom.
authors.forename: Stephen Richard
authors.initials: SR
authors.lastname: Thomas
authors.affiliation: Public Health England, National Infection Service, Porton Down, Salisbury, Wiltshire, United Kingdom.
authors.forename: Jonathan Charles
authors.initials: JC
authors.lastname: Allnutt
authors.affiliation: Public Health England, National Infection Service, Porton Down, Salisbury, Wiltshire, United Kingdom.
authors.forename: Alice Ann Neville
authors.initials: AA
authors.lastname: Marriott
authors.affiliation: Public Health England, National Infection Service, Porton Down, Salisbury, Wiltshire, United Kingdom.
authors.forename: Kim Alexandra
authors.initials: KA
authors.lastname: Hatch
authors.affiliation: Public Health England, National Infection Service, Porton Down, Salisbury, Wiltshire, United Kingdom.
authors.forename: Robert James
authors.initials: RJ
authors.lastname: Watson
authors.affiliation: InCytometry, Todenham, Gloucestershire, United Kingdom.
authors.forename: Graham
authors.initials: G
authors.lastname: Bottley
authors.affiliation: Public Health England, National Infection Service, Porton Down, Salisbury, Wiltshire, United Kingdom.
authors.forename: Philip David
authors.initials: PD
authors.lastname: Marsh
authors.affiliation: Public Health England, National Infection Service, Porton Down, Salisbury, Wiltshire, United Kingdom.
authors.forename: Stephen Charles
authors.initials: SC
authors.lastname: Taylor
authors.affiliation: Public Health England, National Infection Service, Porton Down, Salisbury, Wiltshire, United Kingdom joanna.bacon@phe.gov.uk.
authors.forename: Joanna
authors.initials: J
authors.lastname: Bacon
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D002939:Ciprofloxacin / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D007538:Isoniazid / Q000494:pharmacology; D007612:Kanamycin / Q000494:pharmacology; D008826:Microbial Sensitivity Tests / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000187:drug effects*; D012293:Rifampin / Q000494:pharmacology
chemical_list: D000995:Antitubercular Agents; D007612:Kanamycin; D002939:Ciprofloxacin; D007538:Isoniazid; D012293:Rifampin