Dataset Information

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.

SUBMITTER: Ren Q

PROVIDER: S-EPMC8456568 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BackgroundStreptococcus mutans and Streptococcus sobrinus are associated with the development of dental caries in humans. However, previous diagnostic systems are unsuitable for monitoring viable cell numbers in oral specimens. Assessing the relationship between the numbers of viable and dead bacterial cells and oral status is important for understanding oral infectious diseases. Propidium monoazide (PMA) has been reported to penetrate dead cells following membrane damage and to cross-link DNA, thereby inhibiting DNA amplification. In the present study, we established an assay for selective analysis of two viable human cariogenic pathogens, S. mutans and S. sobrinus, using PMA combined with real-time PCR (PMA-qPCR).ResultsWe designed species-specific primer sets for S. mutans and S. sobrinus, generated standard curves for measuring cell numbers, and evaluated the dynamic range of the assay. To determine the effectiveness of the assay, PMA was added to viable and autoclave-killed cell mixtures. PMA treatment effectively prevented DNA amplification from dead cells. No amplification of DNA from dead cells was observed in these organisms. In addition, we applied this assay to analyze viable cell numbers in oral specimens. A significant correlation was found between the number of viable S. mutans cells in saliva and that in plaque among caries-free patients, whereas no correlation was observed between saliva and carious dentin. The total and viable cell numbers in caries-positive saliva were significantly higher than those in caries-free saliva. Finally, we analyzed the usefulness of this assay for in vitro oral biofilm analysis. We applied PMA-qPCR for monitoring viable S. mutans cell numbers in vitro in planktonic cells and oral biofilm treated with hydrogen peroxide (H?O?). In planktonic cells, the number of viable cells decreased significantly with increasing H?O? concentration, whereas only a small decrease was observed in biofilm cell numbers.ConclusionsPMA-qPCR is potentially useful for quantifying viable cariogenic pathogens in oral specimens and is applicable to oral biofilm experiments. This assay will help to elucidate the relationship between the number of viable cells in oral specimens and the oral status.

Project description:BackgroundHigh-throughput sequencing provides a powerful window into the structural and functional profiling of microbial communities, but it is unable to characterize only the viable portion of microbial communities at scale. There is as yet not one best solution to this problem. Previous studies have established viability assessments using propidium monoazide (PMA) treatment coupled with downstream molecular profiling (e.g., qPCR or sequencing). While these studies have met with moderate success, most of them focused on the resulting "viable" communities without systematic evaluations of the technique. Here, we present our work to rigorously benchmark "PMA-seq" (PMA treatment followed by 16S rRNA gene amplicon sequencing) for viability assessment in synthetic and realistic microbial communities.ResultsPMA-seq was able to successfully reconstruct simple synthetic communities comprising viable/heat-killed Escherichia coli and Streptococcus sanguinis. However, in realistically complex communities (computer screens, computer mice, soil, and human saliva) with E. coli spike-in controls, PMA-seq did not accurately quantify viability (even relative to variability in amplicon sequencing), with its performance largely affected by community properties such as initial biomass, sample types, and compositional diversity. We then applied this technique to environmental swabs from the Boston subway system. Several taxa differed significantly after PMA treatment, while not all microorganisms responded consistently. To elucidate the "PMA-responsive" microbes, we compared our results with previous PMA-based studies and found that PMA responsiveness varied widely when microbes were sourced from different ecosystems but were reproducible within similar environments across studies.ConclusionsThis study provides a comprehensive evaluation of PMA-seq exploring its quantitative potential in synthetic and complex microbial communities, where the technique was effective for semi-quantitative purposes in simple synthetic communities but provided only qualitative assessments in realistically complex community samples. Video abstract.