Quantifying Live Microbial Load in Human Saliva Samples over Time Reveals Stable Composition and Dynamic Load

Evaluating microbial community composition through next-generation sequencing has become increasingly accessible. However, metagenomic sequencing data sets provide researchers with only a snapshot of a dynamic ecosystem and do not provide information about the total microbial number, or load, of a sample. Additionally, DNA can be detected long after a microorganism is dead, making it unsafe to assume that all microbial sequences detected in a community came from living organisms. By combining relic DNA removal by propidium monoazide (PMA) with microbial quantification with flow cytometry, we present a novel workflow to quantify live microbial load in parallel with metagenomic sequencing. We applied this method to unstimulated saliva samples, which can easily be collected longitudinally and standardized by passive collection time. We found that the number of live microorganisms detected in saliva was inversely correlated with salivary flow rate and fluctuated by an order of magnitude throughout the day in healthy individuals. In an acute perturbation experiment, alcohol-free mouthwash resulted in a massive decrease in live bacteria, which would have been missed if we did not consider dead cell signal. While removing relic DNA from saliva samples did not greatly impact the microbial composition, it did increase our resolution among samples collected over time. These results provide novel insight into the dynamic nature of host-associated microbiomes and underline the importance of applying scale-invariant tools in the analysis of next-generation sequencing data sets. IMPORTANCE Human microbiomes are dynamic ecosystems often composed of hundreds of unique microbial taxa. To detect fluctuations over time in the human oral microbiome, we developed a novel workflow to quantify live microbial cells with flow cytometry in parallel with next-generation sequencing, and applied this method to over 150 unstimulated, timed saliva samples. Microbial load was inversely correlated with salivary flow rate and fluctuated by an order of magnitude within a single participant throughout the day. Removing relic DNA improved our ability to distinguish samples over time and revealed that the percentage of sequenced bacteria in a given saliva sample that are alive can range from nearly 0% up to 100% throughout a typical day. These findings highlight the dynamic ecosystem of the human oral microbiome and the benefit of removing relic DNA signals in longitudinal microbiome study designs.