The effects of removing dead bacteria by propidium monoazide on the profile of salivary microbiome

Abstract

Background: Oral microbiome played an important role in maintaining healthy state and might exhibit certain changes under circumstances of diseases. However, current microbiological research using sequencing techniques did not regard dead bacteria as a separate part, causing findings based on subsequent analyses on dynamic equilibrium and functional pathways of microbes somewhat questionable. Since treatment by propidium monoazide (PMA) was able to remove dead bacteria effectively, it would be worth studying how the sequencing results after PMA treatment differed from those focusing on the whole microbiota. Methods: Unstimulated whole saliva samples were obtained from 18 healthy people from 3 age groups (children, adults, and the elderly). After removal of dead bacteria by propidium monoazide (PMA), changes in the profile of salivary microbiome were detected using 16S rRNA sequencing technology, and differences among age groups were compared subsequently. Results: Dead bacteria accounted for nearly a half of the whole bacteria flora in saliva, while freezing had little effect on the proportion of deaths. After treatment with PMA, the numbers of OTUs reduced by 4.4–14.2%, while the Shannon diversity indices decreased significantly (P < 0.01). Only 35.2% of positive and 6.1% of negative correlations were found to be shared by the whole microbiota and that with dead bacteria removed. Differences in significantly changed OTUs and functional pathways among different age groups were also observed between the group of PMA and the control. Conclusions: It was necessary to take the influence of living state of bacteria into account in analytic studies of salivary microbiome.