Counting Mycobacteria in Infected Human Cells and Mouse Tissue: A Comparison between qPCR and CFU

Due to the slow growth rate and pathogenicity of mycobacteria, enumeration by traditional reference methods like colony counting is notoriously time-consuming, inconvenient and biohazardous. Thus, novel methods that rapidly and reliably quantify mycobacteria are warranted in experimental models to facilitate basic research, development of vaccines and anti-mycobacterial drugs. In this study we have developed quantitative polymerase chain reaction (qPCR) assays for simultaneous quantification of mycobacterial and host DNA in infected human macrophage cultures and in mouse tissues. The qPCR method cannot discriminate live from dead bacteria and found a 10- to 100-fold excess of mycobacterial genomes, relative to colony formation. However, good linear correlations were observed between viable colony counts and qPCR results from infected macrophage cultures (Pearson correlation coefficient [r] for M. tuberculosis = 0.82; M. a. avium = 0.95; M. a. paratuberculosis = 0.91). Regression models that predict colony counts from qPCR data in infected macrophages were validated empirically and showed a high degree of agreement with observed counts. Similar correlation results were also obtained in liver and spleen homogenates of M. a. avium infected mice, although the correlations were distinct for the early phase (<day 9 post-infection) and later phase (≥day 20 post-infection) liver r = 0.94 and r = 0.91; spleen r = 0.91 and r = 0.87, respectively. Interestingly, in the mouse model the number of live bacteria as determined by colony counts constituted a much higher proportion of the total genomic qPCR count in the early phase (geometric mean ratio of 0.37 and 0.34 in spleen and liver, respectively), as compared to later phase of infection (geometric mean ratio of 0.01 in both spleen and liver). Overall, qPCR methods offer advantages in biosafety, time-saving, assay range and reproducibility compared to colony counting. Additionally, the duplex format allows enumeration of bacteria per host cell, an advantage in experiments where variable cell death can give misleading colony counts

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

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

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