Abstract

Hendon-Dunn CL, Thomas SR, Taylor SC, Bacon J.
Public Health England, National Infection Service, Porton Down, Salisbury, SP4 0JG

Tuberculosis (TB) is one of the most serious bacterial infections, infecting 10 million people world-wide and causing 1.4 million deaths a year. There is an urgent need for more effective interventions for the disease, including improved antibiotics. Standard antibiotic therapy for tuberculosis is lengthy and includes 3 or more drugs. Lengthy treatment is required because of the development of MDR-Mycobacterium tuberculosis strains and the continued persistence of drug tolerant bacteria that survive initial treatment. Current methods for assessing the drug susceptibility of M. 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. Fluorescent marker calcein violet with an acetoxy-methyl ester group can differentiate between populations of M. tuberculosis growing at different rates, while sytox green can differentiate between live and dead mycobacteria. Similar trends in the loss of viability are observed when comparing flow cytometry and colony forming units. However, the flow cytometry analysis captures information about cell populations that were unable to grow under standard conditions. The flow cytometry approach provides insights into the mode of action of antibiotics and we are now adapting the method as a screen for novel antibiotics against MDR M. tuberculosis.
Hendon-Dunn CL, Doris KS, Thomas SR, Allnutt JC, Marriott AA, Hatch KA, Watson RJ, Bottley G, Marsh PD, Taylor SC, Bacon J. A Flow Cytometry Method for Rapidly Assessing Mycobacterium tuberculosis Responses to Antibiotics with Different Modes of Action Antimicrobial Agents & Chemotherapy. 2016 Jun 20;60(7):3869-83.