A replication clock for Mycobacterium tuberculosis

Abstract

Few tools exist to assess replication of chronic pathogens during infection. This has been a considerable barrier to understanding latent tuberculosis, and efforts to develop new therapies generally assume that the bacteria are very slowly replicating or nonreplicating during latency. To monitor Mycobacterium tuberculosis replication within hosts, we exploit an unstable plasmid that is lost at a steady, quantifiable rate from dividing cells in the absence of antibiotic selection. By applying a mathematical model, we calculate bacterial growth and death rates during infection of mice. We show that during chronic infection, the cumulative bacterial burden-enumerating total live, dead and removed organisms encountered by the mouse lung-is substantially higher than estimates from colony-forming units. Our data show that M. tuberculosis replicates throughout the course of chronic infection of mice and is restrained by the host immune system. This approach may also shed light on the replication dynamics of other chronic pathogens.

Figure 1

In vitro stability of pBP10 in M. smegmatis and Mtb in the absence of antibiotic selection. (a) Total number of M. smegmatis -pBP10, accounting for serial dilutions to show ongoing culture expansion. (b) Frequency of M. smegmatis -pBP10 plasmid-containing bacteria. (c) Frequency of plasmid carriage in M. smegmatis -pBP10 for log-phase cultures versus generations (calculated as (log(OD₆₀₀t / OD₆₀₀(t − 1)) / log(2)). (d) Total number of M. tuberculosis -pBP10. (e) Frequency of M. tuberculosis -pBP10 plasmid-containing bacteria. (f) Frequency of plasmid carriage in M. tuberculosis -pBP10 for log-phase cultures versus generations. Data (means ± s.d.) are shown for log-phase cultures in 7H9, 1:1 7H9 to water, 1:3 7H9 to water, 7H9 without shaking and 7H9 in 2% O₂. Representative data are shown for two to five experiments performed in triplicate, except for the 2% O₂ experiment, which was performed once.

Figure 2

Stability of pBP10 in Mtb in the absence of bacterial replication. (a) Total number of bacteria, as determined by plating for CFUs. (b) Frequency of plasmid-containing bacteria. Data (means ± s.d.) are shown for cultures under starvation, hypoxia or stationary phase. Each independent experiment was performed in triplicate.

Figure 3. Plasmid loss, bacterial replication and cumulative bacterial burden in mouse lungs

(a) Bacterial growth by CFU and the percentage of bacteria carrying plasmid in lungs after low-dose aerosol infection of C57BL/6 mice with H37Rv -pBP10. A subset of mice was treated with dexamethasone (dotted line) starting at week 10 for 8 d; lung CFU and the percentage of bacteria carrying plasmid are shown. Data at each time point are the means ± s.d. of five mice. Representative data from one of three experiments are shown, except for the immunosuppression, which reflects representative data from one of two experiments. (b) Mathematical modeling of cumulative bacterial burden (CBB). Using in vivo CFU, measured by nonselective plating, and the ratio of plasmid-bearing and plasmid-free cells, we calculated the CBB in the mouse lung. 95% confidence intervals (dotted lines) are shown for the bootstrap model of 1,000 synthetic datasets. (c) Depicts the same data as in b, but with a decimal y axis to highlight the cumulative bacterial killing during the first 3 weeks of infection. CFUs are the means ± s.d. of five mice. CBB is shown with the 95% confidence interval (gray).