Development of new application for comprehensive viability analysis based on microbiome analysis by next-generation sequencing: insights into staphylococcal carriage in human nasal cavities.

The nasal carriage rate of Staphylococcus aureus in human is 25--30% and S. aureus sporadically causes severe infections. However, mechanisms underlying staphylococcal carriage remain largely unknown. In the present study, we constructed an rpoB -based microbiome method for staphylococcal species discrimination. Based on a microbiome scheme targeting viable cell DNA using PMA dye (PMA microbiome), we also developed a new method to allow the comprehensive viability analysis of any bacterial taxon. To clarify the ecological distribution of staphylococci in the nasal microbiota, we applied these methods in 46 nasal specimens from healthy adults. PMA microbiome results showed that Staphylococcaceae and Corynebacteriaceae were the most predominant viable taxa (average relative abundance: 0.435262 and 0.375195, respectively), and S. epidermidis exhibited the highest viability in the nasal microbiota. S. aureus detection rates from nasal specimens by rpoB -based conventional and PMA microbiome were 84.8% (39 of 46) and 69.5% (32 of 46), respectively, which substantially exceeded the values by a culture method using identical specimens (36.9%). Our results suggests that Staphylococcaceae especially S. epidermidis adapted most successfully to human nasal cavity. High detection of S. aureus DNA by microbiome methods suggests that almost all healthy adults are consistently exposed to S. aureus in everyday life. Furthermore, the large difference in S. aureus detection rates between culture and microbiome methods suggests that S. aureus cells frequently exist under the viable but non-culturable state in nasal cavities. Our method and findings will contribute to a better understanding of the mechanisms underlying carriage of indigenous bacteria. Importance Metagenomic analyses, such as 16S rRNA microbiome, have provided new insight in various research fields. However, conventional 16S rRNA microbiome dose not permit taxonomic analysis of only viable bacteria, and has poor resolving power below the genus level. Our new schemes allowed for viabile cell specific analysis and species discrimination, nasal microbiome data using these methods provided some interesting findings regarding staphylococcal nasal carriage. According to our comprehensive viability analysis, high viability of Staphylococcus genus, especialy S. epidermidis in human nasal carriage suggests that this taxon has adapted most successfully to human nasal tissue. And also, higher detection of S. aureus DNA by microbiome methods (84.8%) than that by a culture method (36.9%) suggests that almost all healthy adults are consistently exposed to S. aureus in the medium- and long-term. Our findings will contribute to a better understanding of the mechanisms underlying carriage of indigenous bacteria.