Whole lung tissue is the preferred sampling method for amplicon-based characterization of murine lung microbiota.

Background: Low-biomass microbiome studies (such as those of the lungs, placenta, and skin) are vulnerable to contamination and sequencing stochasticity, which obscure legitimate microbial signal. While human lung microbiome studies have rigorously identified sampling strategies that reliably capture microbial signal from these low-biomass microbial communities, the optimal sampling strategy for characterizing murine lung microbiota has not been empirically determined. Performing accurate, reliable characterization of murine lung microbiota and distinguishing true microbial signal from noise in these samples will be critical for further mechanistic microbiome studies in mice.

Results: Using an analytic approach grounded in microbial ecology, we compared bacterial DNA from the lungs of healthy adult mice collected via two common sampling approaches: homogenized whole lung tissue and bronchoalveolar lavage (BAL) fluid. We quantified bacterial DNA using droplet digital PCR, characterized bacterial communities using 16S rRNA gene sequencing, and systematically assessed the quantity and identity of bacterial DNA in both specimen types. We compared bacteria detected in lung specimens to each other and to potential source communities: negative (background) control specimens and paired oral samples. By all measures, whole lung tissue in mice contained greater bacterial signal and less evidence of contamination than did BAL fluid. Relative to BAL fluid, whole lung tissue exhibited a greater quantity of bacterial DNA, distinct community composition, decreased sample-to-sample variation, and greater biological plausibility when compared to potential source communities. In contrast, bacteria detected in BAL fluid were minimally different from those of procedural, reagent, and sequencing controls.

Conclusions: An ecology-based analytical approach discriminates signal from noise in this low-biomass microbiome study and identifies whole lung tissue as the preferred specimen type for murine lung microbiome studies. Sequencing, analysis, and reporting of potential source communities, including negative control specimens and contiguous biological sites, are crucial for biological interpretation of low-biomass microbiome studies, independent of specimen type. Video abstract.