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Table 3 Distinct types of “microbiome”

From: Toward a microbial Neolithic revolution in buildings

Microbiome type

Characteristics

(a) Microbial ecosystem

Active metabolism with or without growth

(b) Seed microbiome

Metabolically inert but can “wake up”

(c) Dead microbiome

Epitopes and chemicals, irreversibly inert

(d) Extracellular intact DNA

Intact sequence from the once-alive

(e) Never-living recovered sequence

Pre-mutagenically lesioned DNA

  1. The word “microbiome” currently conflates several categories that can be considered as distinct but related: (a) A “microbial ecosystem” is an actively metabolizing and growing microbial community, the intestinal microbiome being the exemplar. (b) A “seed” microbiome. Consider the relationship of the seed rack at a garden store to the fields in which plants are growing. Many microbes (inclusive of viruses, bacteria, and micro-eukaryotes such as fungi) in the dry state remain viable and able to grow when conditions allow. (c) The “dead sequence” microbiome from irreversibly non-viable cells, spores, eukaryotes, and viruses. In one study, the ratio of total to colony-forming fungal spores was 100:1 in indoor samples [83]. (d) Extracellular DNA. Approximately half of the microbial DNA in soil is extracellular [84, 85]. (e) Extracellular DNA is chemically stable but not informationally unchangeable. Without the enzymatic repair processes present in living cells, premutagenic lesions accumulate due to physical-chemical processes such as heat, light, ionizing radiation, and oxidation [86]. When lesioned DNA is a template for polymerase, novel sequences are generated by a variety of mechanisms. 8-Oxoguanine, the most common product of reactive oxygen damage, leads predominantly to G > T transversions [87]. Deamination of cytosine leads after a couple of rounds of replication to C > T mutations [88]. Abasic sites and chain breaks can lead to bridging PCR which creates new sequences as copy-choice assemblages of templates in the original sample [89–91]. By the hypothesis proposed here, some of the sequences seen only once in NGS studies [92, 93] were never present in even a single living cell and in fact were never present as an intact sequence but are consequent to reading premutagenic lesions on damaged and largely extracellular templates. This possibility could be tested by exhaustive DNase treatment followed by heat inactivation of the DNase prior to normal extraction protocols. More than 90 % of the microbial DNA sequences reported from nature have never been cultivated, [94] and a large fraction of sequences seen only once have not been proven to have ever existed inside a living microbe. Some of this unique sequence may be an artifact consequent to recovering damaged DNA. The prediction is that a fraction of the “seen only once” sequence will disappear with prior DNase treatment. Treatment with DNase plus proteinase K is reported as able to distinguish between DNA that is present in live cells from cells that are dead or extracellular DNA [95]. Extracellular DNA is vulnerable to DNase treatment alone [96]. RNA analysis also has potential to differentially note growing cells in “omics” style sampling but with caveats. In Escherichia coli, the proportion of rRNA to rRNA-encoding genes increases as a linear function of growth rate [97]. This intriguing property whose functional basis remains uncertain [98] is not universal and it is unsure how general the effect is. Vibrio has a much smaller change in rRNA as a function of growth rate [99]. As speculation, more ribosomes in stationary phase may allow cells to enter rapid growth with less lag phase. ATP analysis is used as a marker of bacterial viability and activity [100], but again, there are caveats because ATP is sometimes quite stable [101]. One only has to recall PCR reaction conditions to note the stability of triphosphate nucleotides through multiple cycles of heating and cooling
  2. Intermediate cases: Some microbes are able to undergo repeated cycles of wetting and drying without specialized forms such as spores. Desiccation tolerance can be a property either of individual cells [102] or of microbial communities [103]. Viable but not culturable (VBNC) microbes are a class based on physiological state first characterized and named by Rita Colwell in the context of aquatic Vibrio cholera [104]. VNBC cells are not spores but require specific conditions to revive. Once revived growth and metabolism are normal, natural transformation can resurrect “dead” DNA and is an important mechanism of horizontal gene transfer [105, 106]. An indeterminate fraction of the microbiome sequence in, e.g., household dust is either “seed,” “dead,” extracellular DNA, or generated sequence
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Microbiome

ISSN: 2049-2618

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