A comparison of methods used to unveil the genetic and metabolic pool in the built environment

Abstract

BackgroundA majority of indoor residential microbes originate from humans, pets, and outdoor air and are not adapted to the built environment (BE). Consequently, a large portion of the microbes identified by DNA-based methods are either dead or metabolically inactive. Although many exceptions have been noted, the ribosomal RNA fraction of the sample is more likely to represent either viable or metabolically active cells. We examined methodological variations in sample processing using a defined, mock BE microbial community to better understand the scope of technique-based vs. biological-based differences in both ribosomal transcript (rRNA) and gene (DNA) sequence community analysis. Based on in vitro tests, a protocol was adopted for the analysis of the genetic and metabolic pool (DNA vs. rRNA) of air and surface microbiomes within a residential setting.ResultsWe observed differences in DNA/RNA co-extraction efficiency for individual microbes, but overall, a greater recovery of rRNA using FastPrep (> 50%). Samples stored with various preservation methods at − 80°C experienced a rapid decline in nucleic acid recovery starting within the first week, although post-extraction rRNA had no significant degradation when treated with RNAStable. We recommend that co-extraction samples be processed as quickly as possible after collection. The in vivo analysis revealed significant differences in the two components (genetic and metabolic pool) in terms of taxonomy, community structure, and microbial association networks. Rare taxa present in the genetic pool showed higher metabolic potential (RNA:DNA ratio), whereas commonly detected taxa of outdoor origins based on DNA sequencing, especially taxa of the Sphingomonadales order, were present in lower relative abundances in the viable community.ConclusionsAlthough methodological variations in sample preparations are high, large differences between the DNA and RNA fractions of the total microbial community demonstrate that direct examination of rRNA isolated from a residential BE microbiome has the potential to identify the more likely viable or active portion of the microbial community. In an environment that has primarily dead and metabolically inactive cells, we suggest that the rRNA fraction of BE samples is capable of providing a more ecologically relevant insight into the factors that drive indoor microbial community dynamics.Electronic supplementary materialThe online version of this article (10.1186/s40168-018-0453-0) contains supplementary material, which is available to authorized users.

Building upon current knowledge and techniques of indoor microbiology to construct the next era of theory into microorganisms, health, and the built environment

“…Microorganisms can be found in every environment, including built environments-our habitat of buildings, roads, vehicles, and other human-associated spaces-and even in ultra-clean rooms and space stations [28]. Collectively, these factors mold the built environment's microbiome [29][30][31]-all the genetic material of the microbial members present, from which can be derived taxonomic identification and metabolic potential, including activities like nutrient utilization and antimicrobial production [30,32]. The indoor microbial community is a blend of microorganisms sourced from various ecosystems which seed each particular built environment.…”

“…An important feature to assess in any microbial community is the determination of which species present are alive and metabolically active [235]; rDNA and rRNA extracted from the same sources resulted in significantly different overall characterization of the microbiota [32,236]. Analyzing both RNA and DNA in combination, even just rRNA and rDNA, can provide further insight than just one method alone, such as the effect of environmental conditions on which microorganisms thrive while others struggle or perish.…”

Rare Taxa Exhibit Disproportionate Cell-Level Metabolic Activity in Enriched Anaerobic Digestion Microbial Communities

“…The ratio between transcribed and genomic 16S rRNA sequences (rRNA/rDNA ratio) is a means of normalizing rRNA transcription against cell count, and it has been used to compare metabolic activity between populations (15, 16). The rRNA/rDNA ratio has been used to study the active microbial communities in aquatic (17–20), ice sheet (21), air (22, 23), soil (24–27), and activated-sludge (28, 29) environments, but it has not yet been widely applied in anaerobic digesters (30). Furthermore, as microbial ribosomal amplification is highly variable across both taxonomy and ecological strategy, the rRNA/rDNA ratio may not always be sufficient to discriminate between microbes in different metabolic states (10), and it may be particularly prone to error in populations with a mixture of amplification levels (31).…”

The environmental risk assessment of cell-processing facilities for cell therapy in a Japanese academic institution

“…Therefore, the need for the identification of microorganisms is described in pharmacopoeias such as Japanese Pharmacopoeia, European Pharmacopoeia, and United States Pharmacopoeia. The microorganisms isolated in this study were reported to live in the GE [ 26 – 28 ]. However, there were multiple candidate species from BLAST, and they had still not been identified as one species.…”