Regression models for predicting log(CFU) from log(qPCR) in macrophage cell cultures.

<p>Regression models for predicting log(CFU) from log(qPCR) for <i>M. tuberculosis</i>, <i>M. a. avium</i> and <i>M. a. paratuberculosis</i> in <i>in vitro</i> infected macrophage cell cultures derived from the data in respective training subset for each mycobacteria. Regression line in the middle with 95% prediction limits for an <u>individual log(CFU)</u> on each side. As it is customary to do multiple replicate CFU measurements from the same biological sample, note that the 95% prediction limits for an individual log(CFU) are wider apart than the corresponding 95% prediction limits for the predicted mean log(CFU) (not shown) for multiple measurements. Hence, the regressed point estimate for the predicted log(CFU) will be the same, but using the models as presented will tend to give wider and in fact more conservative estimates of the confidence intervals if used to predict the mean log(CFU) of multiple measurements as compared to the individual log(CFU).</p

Standard Curves for the Mycobacterial, Human and Mouse targets used.

<p>Typical standard curves for the qPCR assays generated from serial dilutions of genomic <i>M. tuberculosis H37Rv</i> with slope −3,255 (A.), human genomic DNA with slope −3,141(B.) and mouse genomic DNA with slope −3,225(C.). The PCR reactions display similar efficiency (E) of near 100% as given by the equation E = 10^(−1/slope)−1.</p

Experimentally determined regression equations for prediction of log(CFU) from log(qPCR) in infected macrophage cultures.

<p>Experimentally determined regression equations for prediction of log(CFU) from log(qPCR) in infected macrophage cultures.</p

Quantification of <i>M.a.avium</i> in infected mouse tissue as measured by colony counting and qPCR.

<p>Experimentally determined quantification curves of <i>M. a. avium</i> in infected mouse spleen (A) and liver (B) as measured by viable CFU counts (solid line) and total qPCR counts (dashed line). Values are means of log(CFU) (○) or log(qPCR) (□) from 3 to 4 mice at each time point. Analysis of the mean CFU to qPCR ratio (▴) in spleen (C) and liver (D) shows that the geometric mean ratio decreases significantly between early and later time points.* p-value for difference in mean log(CFU/qPCR) for Early vs. Later time points in spleen p<0.0001 and liver p<0.0001. All error bars are ± 2×SEM.</p

Correlation between colony counts and qPCR in <i>M.a.avium</i> infected mouse tissue.

<p>The linear relationships are similar in spleen (A) and liver (B), but distinct for the early (○, solid line) and later phase of infection (□, dashed line).</p

Mycobacterial growth in infected Macrophages as measured by colony counting and qPCR.

<p>Growth of <i>M. tuberculosis</i> (A), <i>M. a. avium</i> (B), and <i>M. a. paratuberculosis</i> (C) in <i>in vitro</i> infected human macrophages as monitored over time by colony counting (solid line) and qPCR (dashed line). Error bars represent 95% confidence intervals.</p

Key Characteristics of the Mycobacterial <i>GroEL2 gene</i> qPCR Assay.

<p>Key Characteristics of the Mycobacterial <i>GroEL2 gene</i> qPCR Assay.</p

Comparison of Duplex qPCR with corresponding Singleplex qPCR in three simulated situations.

<p>C_T = Treshold cycle.</p

Benzoic Acid-Inducible Gene Expression in Mycobacteria

<div><p>Conditional expression is a powerful tool to investigate the role of bacterial genes. Here, we adapt the <i>Pseudomonas putida</i>-derived positively regulated XylS/<i>Pm</i> expression system to control inducible gene expression in <i>Mycobacterium smegmatis</i> and <i>Mycobacterium tuberculosis</i>, the causative agent of human tuberculosis. By making simple changes to a Gram-negative broad-host-range XylS/<i>Pm-</i>regulated gene expression vector, we prove that it is possible to adapt this well-studied expression system to non-Gram-negative species. With the benzoic acid-derived inducer <i>m</i>-toluate, we achieve a robust, time- and dose-dependent reversible induction of <i>Pm</i>-mediated expression in mycobacteria, with low background expression levels. XylS/<i>Pm</i> is thus an important addition to existing mycobacterial expression tools, especially when low basal expression is of particular importance.</p></div

<i>Pm</i>–mediated basal expression is low and compares favorably to <i>Ptet</i>-mediated basal expression in <i>Msmeg</i>.

<p>(A-B) <i>Msmeg</i> transformed with pMDX-zeo or pTET-zeo was diluted to OD₆₀₀ 0.005 in the presence or absence of m-toluate (1.5 mM) or atc (200 ng/ml), respectively, and increasing amounts of zeocin (0, 0.5, 2.5, 5, 7.5, 10, 15, 25, 50, 100, 150, 200, 250 or 500 μg/ml). Samples were grown in triplicates and monitored for 120 hours by a Bioscreen, registering OD₆₀₀ every other hour. Basal expression from <i>Pm</i> and <i>Ptet</i> is presented by growth of <i>Msmeg</i> pMDX-zeo and <i>Msmeg</i> pTET-zeo in increasing concentrations of zeocin, when the respective sample grown in the <i>absence</i> of zeocin reached mid log phase (A) or stationary phase (B). (C) Induced and uninduced samples of pMDX-zeo strain in late log phase. (D) Induced and uninduced samples of pTET-zeo strain in mid log phase. The results represent two independent experiments.</p