Performance characteristics of qPCR assays targeting human- and ruminant-associated bacteroidetes for microbial source tracking across sixteen countries on six continents

Abstract

Numerous quantitative PCR assays for microbial fecal source tracking (MST) have been developed and evaluated in recent years. Widespread application has been hindered by a lack of knowledge regarding the geographical stability and hence applicability of such methods beyond the regional level. This study assessed the performance of five previously reported quantitative PCR assays targeting human-, cattle-, or ruminant-associated Bacteroidetes populations on 280 human and animal fecal samples from 16 countries across six continents. The tested cattle-associated markers were shown to be ruminant-associated. The quantitative distributions of marker concentrations in target and nontarget samples proved to be essential for the assessment of assay performance and were used to establish a new metric for quantitative source-specificity. In general, this study demonstrates that stable target populations required for marker-based MST occur around the globe. Ruminant-associated marker concentrations were strongly correlated with total intestinal Bacteroidetes populations and with each other, indicating that the detected ruminant-associated populations seem to be part of the intestinal core microbiome of ruminants worldwide. Consequently tested ruminant-targeted assays appear to be suitable quantitative MST tools beyond the regional level while the targeted human-associated populations seem to be less prevalent and stable, suggesting potential for improvements in human-targeted methods.

Figure 1

Concentrations of markers measured in target and nontarget fecal DNA extracts for each assay. Results were measured in the 1:4 dilution of the DNA samples and transformed into logarithmic format after addition of 1 to each value. Boxes, 25th and 75th percentile; lines within the boxes, median; whiskers, 10th and 90th percentile, respectively; n, number of samples in each category.

Figure 2

Quantitative relationship of AllBac concentration vs BacH (a), BacHum (b), BacCow (c), BacR (d), and BoBac (e) in target samples. The figure only includes samples yielding positive results with both assays compared in a plot; dashed lines denote the first median while solid lines are linear regression curves. Equations and coefficients of determination for regressions are given in the figure.

Figure 3

Percentages of the host-associated marker concentrations in relation to AllBac concentrations in the respective target samples. The y-axis is scaled logarithmically.