Development of a 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 ofStaphylococcus aureus in human is 25 to 30%, andS. aureus sporadically causes severe infections. However, the mechanisms underlying staphylococcal carriage remain largely unknown. In the present study, we constructed anrpoB -based microbiome method for staphylococcal species discrimination. Based on a microbiome scheme targeting viable cell DNA using propidium monoazide (PMA) dye (PMA microbiome method), 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 thatStaphylococcaceae andCorynebacteriaceae were the most predominant viable taxa (average relative abundance: 0.435262 and 0.375195, respectively), andStaphylococcus epidermidis exhibited the highest viability in the nasal microbiota.Staphylococcus aureus detection rates from nasal specimens byrpoB -based conventional and PMA microbiome methods were 84.8% (39 of 46) and 69.5% (32 of 46), respectively, which substantially exceeded the values obtained by a culture method using identical specimens (36.9%). Our results suggest thatStaphylococcaceae species, especiallyS. epidermidis , adapted most successfully to human nasal cavity. High detection ofS. aureus DNA by microbiome methods suggests that almost all healthy adults are consistently exposed toS. aureus in everyday life. Furthermore, the large difference inS. aureus detection rates between culture and microbiome methods suggests thatS. aureus cells frequently exist in a viable but nonculturable 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 methods, have provided new insights in various research fields. However, conventional 16S rRNA microbiome methods do not permit taxonomic analysis of only the viable bacteria in a sample and have poor resolving power below the genus level. Our new schemes allowed for viable cell-specific analysis and species discrimination, and nasal microbiome data using these methods provided some interesting findings regarding staphylococcal nasal carriage. According to our comprehensive viability analysis, the high viability ofStaphylococcus species, especiallyStaphylococcus epidermidis , in human nasal carriage suggests that this taxon has adapted most successfully to human nasal tissue. Also, a higher detection rate ofS. aureus DNA by microbiome methods (84.8%) than by a culture method (36.9%) suggests that almost all healthy adults are consistently exposed toStaphylococcus aureus in the medium and long term. Our findings will contribute to a better understanding of the mechanisms underlying the carriage of indigenous bacteria.