A high-throughput cidality screen for Mycobacterium tuberculosis.

Exposure to Mycobacterium tuberculosis (Mtb) aerosols is a major threat to tuberculosis (TB) researchers, even in bio-safety level-3 (BSL-3) facilities. Automation and high-throughput screens (HTS) in BSL3 facilities are essential for minimizing manual aerosol-generating interventions and facilitating TB research. In the present study, we report the development and validation of a high-throughput, 24-well 'spot-assay' for selecting bactericidal compounds against Mtb. The bactericidal screen concept was first validated in the fast-growing surrogate Mycobacterium smegmatis (Msm) and subsequently confirmed in Mtb using the following reference anti-tubercular drugs: rifampicin, isoniazid, ofloxacin and ethambutol (RIOE, acting on different targets). The potential use of the spot-assay to select bactericidal compounds from a large library was confirmed by screening on Mtb, with parallel plating by the conventional gold standard method (correlation, r2 = 0.808). An automated spot-assay further enabled an MBC90 determination on resistant and sensitive Mtb clinical isolates. The implementation of the spot-assay in kinetic screens to enumerate residual Mtb after either genetic silencing (anti-sense RNA, AS-RNA) or chemical inhibition corroborated its ability to detect cidality. This relatively simple, economical and quantitative HTS considerably minimized the bio-hazard risk and enabled the selection of novel vulnerable Mtb targets and mycobactericidal compounds. Thus, spot-assays have great potential to impact the TB drug discovery process

Spot-assay correlation in Msm.

<p>To check suitability of the assay. <b>a</b>. <b>MBC correlation</b> showing inter-assay variability (n = 3) in the cfu-based assay in a 25-μl volume in Msm using RIOE. <b>b</b>. <b>Correlation of spot <i>vs</i>. cfu in Msm</b> for RIOE. MBC values were deduced from this finding (<b>a</b>.) and were compared with the data obtained by the conventional method. The spot-assay MBC of four RIOE reference drugs on Msm was compared with the standard cfu-based plating assay. Cfu-based MBC: Minimum concentration that yielded a ≥2 log₁₀ reduction from the starting cfu. Spot-based MBC: Minimum concentration that resulted in no growth (NG) or countable colonies (upto 30 colonies). The data for all four drugs correlated well for both methods (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117577#pone.0117577.g005" target="_blank">Fig. 5b</a>). <b>c</b>. <b>Correlation of spot <i>vs</i>. cfu of Mtb</b> for RIOE. This method was subsequently replicated in Mtb for all four RIOE reference drugs.</p

Spot-assay correlation in Msm.

<p>To check suitability of the assay. <b>a</b>. <b>MBC correlation</b> showing inter-assay variability (n = 3) in the cfu-based assay in a 25-μl volume in Msm using RIOE. <b>b</b>. <b>Correlation of spot <i>vs</i>. cfu in Msm</b> for RIOE. MBC values were deduced from this finding (<b>a</b>.) and were compared with the data obtained by the conventional method. The spot-assay MBC of four RIOE reference drugs on Msm was compared with the standard cfu-based plating assay. Cfu-based MBC: Minimum concentration that yielded a ≥2 log₁₀ reduction from the starting cfu. Spot-based MBC: Minimum concentration that resulted in no growth (NG) or countable colonies (upto 30 colonies). The data for all four drugs correlated well for both methods (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117577#pone.0117577.g005" target="_blank">Fig. 5b</a>). <b>c</b>. <b>Correlation of spot <i>vs</i>. cfu of Mtb</b> for RIOE. This method was subsequently replicated in Mtb for all four RIOE reference drugs.</p

A plate map for the MIC in a 384-well plate.

<p>A total of 30 test compounds/plate (2 compounds/row) could be tested as a 10-concentration dose response (10c-DR as A2–11 to P2–11 and A14–23 to N14–23). <b>Quality controls (QC)</b>: Columns 1 and 13 = media controls (MC), columns 12 and 24 = culture controls (CC). The 31^st and 32^nd rows = reference drug controls (e.g., isoniazid in rows O&P14 to O&P23) and rows MN14 to MN23 were used for additional reference drug QC, if required. Culture addition was performed with the Multidrop-Combi liquid handling system, and incubation was performed at 37°C for 3–4 days (Msm) and 3–4 weeks (Mtb). Post-incubation, the MIC data were recorded using a spectrophotometer (Spectramax^384Plus) at O.D._600nm.</p

Optimisation of assay conditions.

<p>Initial assay optimization was done in Msm: <b>a. Minimum culturable volume in Msm</b>: Msm culture was dispensed in 1.56-μl aliquots (1.56μlX64) multiple times, up to 50μl twice (50μlX2), on 24-well agar beds and conventional plates. The sum of the cfu obtained for each volume was plotted. The parallel lines of the cfu data plotted against the plating volumes confirmed that there was no variation in the net bacterial counts obtained from plating different volumes. Hence, the 25-μl volume was selected. <b>b. Drug carry-over assay in Msm</b>: Untreated and isoniazid-treated samples yielded a similar number of cfus. Isoniazid exposure yielded similar bacterial counts from both the conventional and 24-well spot-assay over a range of concentrations up to 16 μg/ml.</p

Kinetic screen-spot vs. conventional assay.

<p>The survival kinetics of all three Mtb-AS target genes (<i>ppk</i>, <i>pyrH</i> and <i>coaD</i>) demonstrated quantitative cidality in real time on different days. All findings are reproducible and exhibit comparable overlapping graphs (two-tailed P values <0.001 for all three AS data), with a strong positive correlation (ppk r² = 0.693, <i>pyrH</i> r² = 0.920 and <i>coaD</i> r² = 0.856).</p

The schematics of spotting from an MIC plate to a 24-well plate.

<p><b>a</b>. Culture aspiration from a MIC plate using filter-tips fixed at alternate positions of a 12-channel Pipetman. <b>b</b>. <b>Twenty-four-well plate</b>: Row-wise dispensing into a 24-well plate. Two compounds/row, with a 10c-DR each: e.g., Row A: Compound#1 = 2–11 wells and Compound#2 = 14–23 wells (1 & 13 = media control, 12 & 24 = culture control). One plate of 24 wells = data for 2 compounds (10c-DR) for 1 row of MIC plate: The left half of the 24-well plate contains the 1^st compound (#2 to 11), and the right half contains the 2^nd compound (#14 to 23). <b>c. A picture of a spot-assay in a 24-well plate</b>: colony morphology and countable colonies (e.g., rifampicin on the left half and isoniazid on the right half of the plate).</p

Spot-assay correlation in Mtb and the Manhattan analysis.

<p>The spot-assay was replicated in Mtb for RIOE. <b>a. An MBC correlation between the spot-assay and the conventional method</b>: A strong positive correlation (r² = 0.808). <b>b & c: The Manhattan analysis</b> of isoniazid-treated reference controls exhibited variation within the 2-fold in the spot-assay (<b>b</b>) and conventional MBC methods (<b>c</b>).</p

MBC₉₀ studies: MBC₉₀ testing on <i>M. tuberculosis</i> (sensitive and drug-resistant isolates) is possible.

<p>MBC₉₀ studies: MBC₉₀ testing on <i>M. tuberculosis</i> (sensitive and drug-resistant isolates) is possible.</p