Characterization of the bacterial microbiome in first-pass meconium using propidium monoazide (PMA) to exclude nonviable bacterial DNA

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Abstract

Numerous studies have reported bacterial DNA in first-pass meconium samples, suggesting that the human gut microbiome is seeded prior to birth. However, these studies have not been able to discriminate between DNA from living bacterial cells, DNA from dead bacterial cells or cell-free DNA. Here we have used propidium monoazide (PMA) together with 16S rRNA gene sequencing to determine whether there are intact bacterial cells in the fetal gut. DNA was extracted from first-pass meconium (n = 5) and subjected to 16S rRNA gene sequencing with/without PMA treatment. All meconium samples, regardless of PMA treatment, contained detectable levels of bacterial DNA; however, treatment with PMA prior to DNA extraction decreased the DNA yield by approximately 20%. PMA-treated meconium samples did not differ significantly from untreated samples in terms of observed number of OTUs (P = 0·945); although they did differ taxonomically, with around one quarter of OTUs identified in untreated samples only, suggesting that they have originated from cell-free/nonviable DNA. The mean Sørensen coefficient for treated vs untreated samples was 0·527. Our findings suggest that the fetal gut is seeded with intact bacterial cells prior to birth. This is an important finding, as exposure to live bacteria during gestation might have a significant impact on the developing fetus. Significance and Impact of the Study: DNA-based microbiome studies performed using 16S rRNA gene sequencing are limited by their inability to discriminate between live bacterial cells, dead bacterial cells and cell-free DNA. Here we use propidium monoazide (PMA) to exclude nonviable bacteria from microbiome analysis of first-pass meconium samples and thereby reveal that the majority of the purported fetal gut microbiome is from intact bacterial cells. This work demonstrates the importance of excluding nonviable bacteria when analysing the microbial community in low-biomass samples such as meconium.

Original languageEnglish
Pages (from-to)378-385
Number of pages8
JournalLetters in Applied Microbiology
Volume68
Issue number5
DOIs
Publication statusPublished - 1 May 2019

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Bacterial DNA
Meconium
Microbiota
DNA
rRNA Genes
Bacteria
propidium monoazide
Parturition
Biomass
Fetus
Pregnancy

Cite this

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title = "Characterization of the bacterial microbiome in first-pass meconium using propidium monoazide (PMA) to exclude nonviable bacterial DNA",
abstract = "Numerous studies have reported bacterial DNA in first-pass meconium samples, suggesting that the human gut microbiome is seeded prior to birth. However, these studies have not been able to discriminate between DNA from living bacterial cells, DNA from dead bacterial cells or cell-free DNA. Here we have used propidium monoazide (PMA) together with 16S rRNA gene sequencing to determine whether there are intact bacterial cells in the fetal gut. DNA was extracted from first-pass meconium (n = 5) and subjected to 16S rRNA gene sequencing with/without PMA treatment. All meconium samples, regardless of PMA treatment, contained detectable levels of bacterial DNA; however, treatment with PMA prior to DNA extraction decreased the DNA yield by approximately 20{\%}. PMA-treated meconium samples did not differ significantly from untreated samples in terms of observed number of OTUs (P = 0·945); although they did differ taxonomically, with around one quarter of OTUs identified in untreated samples only, suggesting that they have originated from cell-free/nonviable DNA. The mean S{\o}rensen coefficient for treated vs untreated samples was 0·527. Our findings suggest that the fetal gut is seeded with intact bacterial cells prior to birth. This is an important finding, as exposure to live bacteria during gestation might have a significant impact on the developing fetus. Significance and Impact of the Study: DNA-based microbiome studies performed using 16S rRNA gene sequencing are limited by their inability to discriminate between live bacterial cells, dead bacterial cells and cell-free DNA. Here we use propidium monoazide (PMA) to exclude nonviable bacteria from microbiome analysis of first-pass meconium samples and thereby reveal that the majority of the purported fetal gut microbiome is from intact bacterial cells. This work demonstrates the importance of excluding nonviable bacteria when analysing the microbial community in low-biomass samples such as meconium.",
keywords = "fetal microbiome, live bacteria, meconium microbiome, NGS, PMA",
author = "Stinson, {L. F.} and Keelan, {J. A.} and Payne, {M. S.}",
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TY - JOUR

T1 - Characterization of the bacterial microbiome in first-pass meconium using propidium monoazide (PMA) to exclude nonviable bacterial DNA

AU - Stinson, L. F.

AU - Keelan, J. A.

AU - Payne, M. S.

PY - 2019/5/1

Y1 - 2019/5/1

N2 - Numerous studies have reported bacterial DNA in first-pass meconium samples, suggesting that the human gut microbiome is seeded prior to birth. However, these studies have not been able to discriminate between DNA from living bacterial cells, DNA from dead bacterial cells or cell-free DNA. Here we have used propidium monoazide (PMA) together with 16S rRNA gene sequencing to determine whether there are intact bacterial cells in the fetal gut. DNA was extracted from first-pass meconium (n = 5) and subjected to 16S rRNA gene sequencing with/without PMA treatment. All meconium samples, regardless of PMA treatment, contained detectable levels of bacterial DNA; however, treatment with PMA prior to DNA extraction decreased the DNA yield by approximately 20%. PMA-treated meconium samples did not differ significantly from untreated samples in terms of observed number of OTUs (P = 0·945); although they did differ taxonomically, with around one quarter of OTUs identified in untreated samples only, suggesting that they have originated from cell-free/nonviable DNA. The mean Sørensen coefficient for treated vs untreated samples was 0·527. Our findings suggest that the fetal gut is seeded with intact bacterial cells prior to birth. This is an important finding, as exposure to live bacteria during gestation might have a significant impact on the developing fetus. Significance and Impact of the Study: DNA-based microbiome studies performed using 16S rRNA gene sequencing are limited by their inability to discriminate between live bacterial cells, dead bacterial cells and cell-free DNA. Here we use propidium monoazide (PMA) to exclude nonviable bacteria from microbiome analysis of first-pass meconium samples and thereby reveal that the majority of the purported fetal gut microbiome is from intact bacterial cells. This work demonstrates the importance of excluding nonviable bacteria when analysing the microbial community in low-biomass samples such as meconium.

AB - Numerous studies have reported bacterial DNA in first-pass meconium samples, suggesting that the human gut microbiome is seeded prior to birth. However, these studies have not been able to discriminate between DNA from living bacterial cells, DNA from dead bacterial cells or cell-free DNA. Here we have used propidium monoazide (PMA) together with 16S rRNA gene sequencing to determine whether there are intact bacterial cells in the fetal gut. DNA was extracted from first-pass meconium (n = 5) and subjected to 16S rRNA gene sequencing with/without PMA treatment. All meconium samples, regardless of PMA treatment, contained detectable levels of bacterial DNA; however, treatment with PMA prior to DNA extraction decreased the DNA yield by approximately 20%. PMA-treated meconium samples did not differ significantly from untreated samples in terms of observed number of OTUs (P = 0·945); although they did differ taxonomically, with around one quarter of OTUs identified in untreated samples only, suggesting that they have originated from cell-free/nonviable DNA. The mean Sørensen coefficient for treated vs untreated samples was 0·527. Our findings suggest that the fetal gut is seeded with intact bacterial cells prior to birth. This is an important finding, as exposure to live bacteria during gestation might have a significant impact on the developing fetus. Significance and Impact of the Study: DNA-based microbiome studies performed using 16S rRNA gene sequencing are limited by their inability to discriminate between live bacterial cells, dead bacterial cells and cell-free DNA. Here we use propidium monoazide (PMA) to exclude nonviable bacteria from microbiome analysis of first-pass meconium samples and thereby reveal that the majority of the purported fetal gut microbiome is from intact bacterial cells. This work demonstrates the importance of excluding nonviable bacteria when analysing the microbial community in low-biomass samples such as meconium.

KW - fetal microbiome

KW - live bacteria

KW - meconium microbiome

KW - NGS

KW - PMA

UR - http://www.scopus.com/inward/record.url?scp=85061613725&partnerID=8YFLogxK

U2 - 10.1111/lam.13119

DO - 10.1111/lam.13119

M3 - Article

VL - 68

SP - 378

EP - 385

JO - Letters in Applied Microbiology

JF - Letters in Applied Microbiology

SN - 0266-8254

IS - 5

ER -