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

Abstract

The nasal carriage rate of in human is 25--30% and sporadically causes severe infections. However, mechanisms underlying staphylococcal carriage remain largely unknown. In the present study, we constructed an -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 and were the most predominant viable taxa (average relative abundance: 0.435262 and 0.375195, respectively), and exhibited the highest viability in the nasal microbiota. detection rates from nasal specimens by-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 especially adapted most successfully to human nasal cavity. High detection of DNA by microbiome methods suggests that almost all healthy adults are consistently exposed to in everyday life. Furthermore, the large difference in detection rates between culture and microbiome methods suggests that 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. 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 genus, especialy in human nasal carriage suggests that this taxon has adapted most successfully to human nasal tissue. And also, higher detection of DNA by microbiome methods (84.8%) than that by a culture method (36.9%) suggests that almost all healthy adults are consistently exposed to in the medium- and long-term. Our findings will contribute to a better understanding of the mechanisms underlying carriage of indigenous bacteria.

Faecal freezing preservation period influences colonization ability for faecal microbiota transplantation

“…; Lu et al . ), which has been typically used as a viable cell DNA‐specific PCR technique based on a photoreactive DNA crosslinker that prevents PCR amplification from dead cells (Nocker et al . ).…”

“…Thus, these methods were unsuitable for comparing viable communities in fresh and frozen faeces, we could not perform the bacterial viability analysis unless culture-dependent methods were also used. Recently, Checinska et al, Venkateswaran et al and we conducted a microbiome analysis using samples treated with propidium monoazide (PMA) (Venkateswaran et al 2014;Checinska et al 2015;Lu et al 2018), which has been typically used as a viable cell DNA-specific PCR technique based on a photoreactive DNA crosslinker that prevents PCR amplification from dead cells (Nocker et al 2006). This microbiome method using a PMA dye targeting viable cell DNA (microbiome analysis using PMA (PMA microbiome)) is expected to contribute to a better microbiological interpretation based on viability in future metagenomic studies.…”

Re‐assessing microbiomes in the low‐biomass reproductive niche

“…Microbiome studies using PMA‐treated samples report no change in the overall composition of the bacterial community, but do show the utility of this method reporting increased detection of rare taxa to reflect more accurately the community composition by improving evenness and diversity estimates . When comparing culture‐dependent and cultivation‐independent sample processing, in the presence and absence of PMA treatment, the abundance of even readily cultivable isolates such as staphylococci are overrepresented in molecular analyses, potentially reflecting exposure to a microbe rather than colonisation …”

Comparison of Illumina Versus Nanopore 16S rRNA Gene Sequencing of the Human Nasal Microbiota

“…Unfortunately, traditional culture techniques are unable to detect a wide range of the socalled 'non-culturable' bacteria that DNA sequencing techniques have indicated to be present within the human nasal microbiota [8]. However, to date, accurate species identification using 16S rRNA gene sequencing protocols in combination with the most popular sequencing platform (Illumina sequencing) is currently not universally possible as only short regions of bacterial 16S rRNA genes tend to be sequenced using Illumina technology [9].…”