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Absolute quantitation of microbiota abundance in environmental samples.

ABSTRACT: BACKGROUND:Microbial communities (microbiota) influence human and animal disease and immunity, geochemical nutrient cycling and plant productivity. Specific groups, including bacteria, archaea, eukaryotes or fungi, are amplified by PCR to assess the relative abundance of sub-groups (e.g. genera). However, neither the absolute abundance of sub-groups is revealed, nor can different amplicon families (i.e. OTUs derived from a specific pair of PCR primers such as bacterial 16S, eukaryotic 18S or fungi ITS) be compared. This prevents determination of the absolute abundance of a particular group and domain-level shifts in microbiota abundance can remain undetected. RESULTS:We have developed absolute quantitation of amplicon families using synthetic chimeric DNA spikes. Synthetic spikes were added directly to environmental samples, co-isolated and PCR-amplified, allowing calculation of the absolute abundance of amplicon families (e.g. prokaryotic 16S, eukaryotic 18S and fungal ITS per unit mass of sample). CONCLUSIONS:Spikes can be adapted to any amplicon-specific group including rhizobia from soils, Firmicutes and Bifidobacteria from human gut or Enterobacteriaceae from food samples. Crucially, using highly complex soil samples, we show that the absolute abundance of specific groups can remain steady or increase, even when their relative abundance decreases. Thus, without absolute quantitation, the underlying pathology, physiology and ecology of microbial groups may be masked by their relative abundance.

SUBMITTER: Tkacz A

PROVIDER: S-EPMC6009823 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Absolute quantitation of microbiota abundance in environmental samples.

Tkacz Andrzej A Hortala Marion M Poole Philip S PS

Microbiome 20180619 1

<h4>Background</h4>Microbial communities (microbiota) influence human and animal disease and immunity, geochemical nutrient cycling and plant productivity. Specific groups, including bacteria, archaea, eukaryotes or fungi, are amplified by PCR to assess the relative abundance of sub-groups (e.g. genera). However, neither the absolute abundance of sub-groups is revealed, nor can different amplicon families (i.e. OTUs derived from a specific pair of PCR primers such as bacterial 16S, eukaryotic 18 ...[more]

PMID: 29921326

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Project description:The quantitation of human milk oligosaccharides (HMOs) is challenging because of the structural complexity and lack of standards.The objective of our study was to rapidly measure the absolute concentrations of HMOs in milk using LC-mass spectrometry (MS) and to determine the phenotypic secretor status of the mothers.This quantitative method for measuring HMO concentration was developed by using ultraperformance LC multiple reaction monitoring MS. It was validated and applied to milk samples from Malawi (88 individuals; 88 samples from postnatal month 6) and the United States (Davis, California; 45 individuals, mean age: 32 y; 103 samples collected on postnatal days 10, 26, 71, or 120, repeated measures included). The concentrations of ?(1,2)-fucosylated HMOs were used to determine the mothers' phenotypic secretor status with high sensitivity and specificity. We used Friedman's test and Wilcoxon's signed rank test to evaluate the change in HMO concentration during the course of lactation, and Student's t test was used to compare secretors and nonsecretors.A decrease (P < 0.05) in HMO concentration was observed during the course of lactation for the US mothers, corresponding to 19.3 ± 2.9 g/L for milk collected on postnatal day 10, decreasing to 8.53 ± 1.18 g/L on day 120 (repeated measures; n = 14). On postnatal day 180, the total concentration of HMOs in Malawi milk samples from secretors (6.46 ± 1.74 mg/mL) was higher (P < 0.05) than that in samples from nonsecretors (5.25 ± 2.55 mg/mL ). The same trend was observed for fucosylated species; the concentration was higher in Malawi milk samples from secretors (4.91 ± 1.22 mg/mL) than from nonsecretors (3.42 ± 2.27 mg/mL) (P < 0.05).HMOs significantly decrease during the course of lactation. Secretor milk contains higher concentrations of total and fucosylated HMOs than does nonsecretor milk. These HMO concentrations can be correlated to the health of breastfed infants in order to investigate the protective effects of milk components. The trials were registered at clinicaltrials.gov as NCT01817127 and NCT00524446.

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Absolute quantitation of microbiota abundance in environmental samples.

Publications

Absolute quantitation of microbiota abundance in environmental samples.

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