Evaluation of Bacteria and Fungi DNA Abundance in Human Tissues

Abstract

Whereas targeted and shotgun sequencing approaches are both powerful in allowing the study of tissue-associated microbiota, the human: microorganism abundance ratios in tissues of interest will ultimately determine the most suitable sequencing approach. In addition, it is possible that the knowledge of the relative abundance of bacteria and fungi during a treatment course or in pathological conditions can be relevant in many medical conditions. Here, we present a qPCR-targeted approach to determine the absolute and relative amounts of bacteria and fungi and demonstrate their relative DNA abundance in nine different human tissue types for a total of 87 samples. In these tissues, fungi genomes are more abundant in stool and skin samples but have much lower levels in other tissues. Bacteria genomes prevail in stool, skin, oral swabs, saliva, and gastric fluids. These findings were confirmed by shotgun sequencing for stool and gastric fluids. This approach may contribute to a more comprehensive view of the human microbiota in targeted studies for assessing the abundance levels of microorganisms during disease treatment/progression and to indicate the most informative methods for studying microbial composition (shotgun versus targeted sequencing) for various samples types.

Keywords: 16S; metagenomics; microbiome; mycobiome; qPCR; shotgun.

Figure 1

Standard DNA dilution curves for human, bacteria (V1-16S) and fungi (5.8S) qPCR, showing expected (red) and observed (blue) Ct-values considering the genome size and target copy number of the bacteria and fungi present in the mock community. Calculations do not consider the variable ploidy observed in many fungi species. Values used for plotting are shown in Table 1. In the equation, y denotes the genome copy number while x denotes the mean Ct value.

Figure 2

Effects of varying DNA mass over amplification capabilities of bacteria (A) and fungi (B), as compared to human DNA amounts. Horizontal axes indicate the Ct value for human DNA and vertical axes indicate Cts for bacteria (16S) or fungi (5.8S). Each dot represents the mean of a triplicate experiment.

Figure 3

Genome-ratios for bacteria:human (A) and fungi:human (B) and total genome copies for bacteria (red), fungi (green) and human (blue) (C) in different human sample types. Mann–Whitney test * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 significant.

Figure 4

Correlation between the ratios of bacteria:human reads observed by shotgun sequencing data and the relative bacteria:human genome copies calculated by qPCR (A). The same is shown for fungi (B). Each circle represents a distinct sample. For statistical analysis, Spearman’s correlation was used, and the closer it is to 1, the greater the correlation between the variables. The shaded area represents the confidence interval. Correlations were considered as statistically significant when p < 0.05.