165- Disease relapse is measured by dividing the numbers of mice
with CFU count positive organs with the total numbers of the
mice. The difference in relapse rates based on CFU count
detection of M. tuberculosis is analyzed using a Fisher’s exact
test between two experimental groups. - Normally for resuscitation of persisters in vitro cultures and in
animal organs, 12 of 10-fold dilutions are needed to ensure the
coverage of growth in culture filtrates. - In the turbid tubes, CFU counts will be performed by plating
a serial of tenfold dilution of the cultures on 7H11 agar plates.
The presence of M. tuberculosis will be confirmed by colony
morphology from single colonies. - Turbid tubes may be due to contamination with other bacteria
or fungi. 50 μL of the cultures from turbid tubes will be placed
on blood agar or Sabouraud dextrose agar plate in triplicate
followed by spreading the cultures on the plates. Blood agar
plates will be incubated at 37 °C for 24 h and Sabouraud dex-
trose agar plates are incubated at 24 °C for 72–96 h.
Acknowledgments
This work was supported by the Innovative Medicines Initiative
Joint Undertaking resources which are composed of financial con-
tributions from the European Union’s Seventh Framework
Programme (FP7/2007-2013) and EFPIA companies’ in-kind
contribution (grant number 115337). This publication reflects
only the authors’ view. The European Commission is not liable for
any use that may be made of the information herein.References
- MRC (1974) Controlled clinical trial of four
short-course (6-month) regimens of chemo-
therapy for treatment of pulmonary tuberculo-
sis. Lancet 2:1100–1106 - RM MC Jr, McDermott W, Tompsett R (1956)
The fate of Mycobacterium tuberculosis in
mouse tissues as determined by the microbial
enumeration technique. II. The conversion of
tuberculous infection to the latent state by the
administration of pyrazinamide and a compan-
ion drug. J Exp Med 104:763–802 - RM MC Jr, Tompsett R (1956) Fate of
Mycobacterium tuberculosis in mouse tissues as
determined by the microbial enumeration
technique. I. The persistence of drug-
susceptible tubercle bacilli in the tissues despite
prolonged antimicrobial therapy. J Exp Med
104:737–762- Lipworth S, Hammond RJ, Baron VO, Hu Y,
Coates A, Gillespie SH (2016) Defining dor-
mancy in mycobacterial disease. Tuberculosis
(Edinb) 99:131–142 - Mukamolova GV, Turapov O, Malkin J,
Woltmann G, Barer MR (2010) Resuscitation-
promoting factors reveal an occult population
of tubercle bacilli in sputum. Am J Respir Crit
Care Med 181:174–180 - Turapov O, O'Connor BD, Sarybaeva AA,
Williams C, Patel H, Kadyrov AS, Sarybaev AS,
Woltmann G, Barer MR, Mukamolova GV
(2016) Phenotypically adapted Mycobacterium
tuberculosis populations from sputum are toler-
A Method to Evaluate Persistent Mycobacterium tuberculosis In Vitro and in the...