Nasal microbiome quantification identifies bacterial features specific to different sampling methods. Nasal microbiome quantification using different sampling methods

The nasal microbiome plays a critical role in health and respiratory diseases. Changes in microbial composition and function have been identified in allergic rhinitis, chronic rhinosinusitis, asthma and chronic obstructive pulmonary disease. However, different methods are used to sample the nasal microbiome, which introduces biases and inconsistencies across studies. Further, current methods for microbiome characterization rely on relative abundances only and do not consider bacterial load, a critical factor for pathogenesis and disease progression. In this study, we investigated the effect of sampling methods and bacterial quantification in the nasal microbiome of 20 healthy subjects. We tested four widely used sampling methods: swabbing or brushing the anterior nares, swabbing the posterior nares, and nasal washings. Using relative abundance estimates, we observed each subject had a characteristic nasal microbiome, but there were no significant differences in bacterial diversity nor composition across sampling methods. However, absolute quantification of the nasal microbiome using flow cytometry revealed a significantly higher bacterial load in posterior swabs compared to other methods, as well as an enrichment in Corynebacteriaceae, Staphylococcaceae and Moraxellaceae families. We found no consistent linear correlation between the relative and the absolute microbiome across the major bacterial taxa and sampling methods, with nasal washings exhibiting the poorest degree of correlation. Our results demonstrate that choice of sampling method and quantification of bacterial load is fundamental to accurately characterize the nasal microbiome. Because correlation between relative and absolute microbiome is poor across all methods, quantification cannot be directly predicted from traditional 16S rRNA gene sequencing methods alone. Swabbing the anterior nares is considered preferable to brushing, as it is less invasive but yields similar microbiome characterization. Posterior nares swabbing, while relevant in diseases such as rhinosinusitis, is highly invasive and results in a distinct absolute microbiome, which has important implications for the design and interpretation of future studies.