Low-oxygen-recovery assay for high-throughput screening of compounds against nonreplicating Mycobacterium tuberculosis

Abstract

Screening for new antimicrobial agents is routinely conducted only against actively replicating bacteria. However, it is now widely accepted that a physiological state of nonreplicating persistence (NRP) is responsible for antimicrobial tolerance in many bacterial infections. In tuberculosis, the key to shortening the 6-month regimen lies in targeting this NRP subpopulation. Therefore, a high-throughput, luminescence-based low-oxygen-recovery assay (LORA) was developed to screen antimicrobial agents against NRP Mycobacterium tuberculosis. M. tuberculosis H37Rv containing a plasmid with an acetamidase promoter driving a bacterial luciferase gene was adapted to low oxygen conditions by extended culture in a fermentor with a 0.5 headspace ratio. The MICs of 31 established antimicrobial agents were determined in microplate cultures maintained under anaerobic conditions for 10 days and, for comparative purposes, under aerobic conditions for 7 days. Cultures exposed to drugs under anaerobic conditions followed by 28 h of "recovery" under ambient oxygen produced a luminescent signal that was, for most compounds, proportional to the number of CFU determined prior to the recovery phase. No agents targeting the cell wall were active against NRP M. tuberculosis, whereas drugs hitting other cellular targets had a range of activities. The calculated Z' factor was in the range of 0.58 to 0.84, indicating the suitability of the use of LORA for high-throughput assays. This LORA is sufficiently robust for use for primary high-throughput screening of compounds against NRP M. tuberculosis.

FIG. 1.

Drug susceptibility under aerobic (replicating) and anaerobic (nonreplicating) conditions. The vertical lines and shaded arrows are proportional to the incubation times.

FIG. 2.

Growth of an Mycobacterium tuberculosis H₃₇Rv(pFCA-luxAB) inoculum in a 0.5 HSR fermentor culture. The numbers of CFU, RLU, optical density (OD; A₅₇₀), and DOC were monitored during culture in a fermentor. The DOC is expressed as the percent saturation of the medium relative to that of the medium initially equilibrated with air.

FIG. 3.

Proportional distribution of CFU according to the time required to visually detect colonial growth on solid media. The percentage of the CFU value was determined from the proportion of the net number of CFU visible at a given time relative to the total number of CFU after 5 weeks of incubation.

FIG. 4.

(A) Well-to-well variation for positive and negative control well signals from five plates of 31 antimicrobial agents. The calculated Z′ factor is in the range of 0.58 to 0.84. (B) Statistical analysis for reproducibility of duplicates. A significant linear correlation was observed between duplicate sets. Each of the 225 datum points represents the result of one independent experiment.