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Comparative Study
. 2009 Aug;137(2):588-97.
doi: 10.1053/j.gastro.2009.04.046. Epub 2009 Apr 23.

Inflammation and Intestinal Metaplasia of the Distal Esophagus Are Associated With Alterations in the Microbiome

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Free PMC article
Comparative Study

Inflammation and Intestinal Metaplasia of the Distal Esophagus Are Associated With Alterations in the Microbiome

Liying Yang et al. Gastroenterology. .
Free PMC article

Abstract

Background & aims: Gastroesophageal reflux causes inflammation and intestinal metaplasia and its downstream sequelum adenocarcinoma in the distal esophagus. The incidence of esophageal adenocarcinoma has increased approximately 6-fold in the United States since the 1970s, accompanied with a significant increase in the prevalence of gastroesophageal reflux disease (GERD). Despite extensive epidemiologic study, the cause for GERD and the unexpected increases remain unexplainable. Microbes are among the environmental factors that may contribute to the etiology of GERD, but very little research has been done on the esophageal microbiome, particularly in its relation to GERD. This is the first comprehensive reported correlation between a change in the esophageal microbiome and esophageal diseases.

Methods: Biopsy samples of the distal esophagus were collected from 34 patients. Host phenotypes were histologically defined as normal, esophagitis, or Barrett's esophagus (intestinal metaplasia). Microbiomes from the biopsy samples were analyzed by bacterial 16S ribosomal RNA gene survey and classified into types using unsupervised cluster analysis and phenotype-guided analyses. Independence between host phenotypes and microbiome types were analyzed by Fisher exact test.

Results: Esophageal microbiomes can be classified into 2 types. The type I microbiome was dominated by the genus Streptococcus and concentrated in the phenotypically normal esophagus. Conversely, the type II microbiome contained a greater proportion of gram-negative anaerobes/microaerophiles and primarily correlated with esophagitis (odds ratio, 15.4) and Barrett's esophagus (odds ratio, 16.5).

Conclusions: In the human distal esophagus, inflammation and intestinal metaplasia are associated with global alteration of the microbiome. These findings raise the issue of a possible role for dysbiosis in the pathogenesis of reflux-related disorders.

Figures

Figure 1
Figure 1
Typing of esophageal microbiome. (A) Detection of natural microbiome groups by unsupervised cluster analysis. The dendrogram was constructed using the average linkage algorithm and cosine measure of the genetic distance calculated from samples of the microbiome. Samples are represented by colored rectangles (green for normal, red for esophagitis, and black for Barrett’s esophagus). (B) Phenotype-directed classification of the microbiome by genetic distance-based normal reference range. First, the mean genetic distance between each normal sample and other 11 normal samples were calculated. A single outlier was identified and the mean distance for each remaining 11 normal samples was recalculated after excluding the outlier. The 95% NRR was defined as mean distance ± 1.96 S.D. based on the 11 normal samples. The mean distance for each sample in the esophagitis and BE groups is the mean distance between the sample and the 11 normal samples. The dotted line (0.1693) is the upper limit of the 95% NRR, which separates the 34 samples into the normal (inside the NRR) and abnormal microbiome (outside the NRR). (C) Double principal coordinate analysis (DPCoA) of the microbiome. Samples are represented by circles. Microbiome types are indicated by fill colors (blue for type I and brown for type II). Host phenotypes are indicated by edge colors (green for normal, red for esophagitis, and black for Barrett’s esophagus). Within-sample diversity is proportional to circle size, determined by Rao’s analysis. The location of a sample in the plot was determined by the first two orthogonal principal axes. The percentages shown for each axis represents the percent of total dissimilarity captured by the axis. The samples from the two types of microbiome are separable along the first principal coordinate, as indicated by the dividing line at x ≈ 0.015.
Figure 2
Figure 2
Bacterial phylogenesis in the distal esophagus. (A) DNA sequences were pooled according to host phenotypes: normal, esophagitis, and Barrett’s esophagus. The number of taxa at a specific identity level (ID), every 1% between ID46 and ID80 and every 0.1% between ID80 and ID100, were calculated using DOTUR, showing changing in the number of taxa with increasing mutations over the entire hierarchy of domain Bacteria. The rates of the changes and their turning points were analyzed by linear regressions. Values from the domain level to the species level are represented by filled circles while those at the species level and below are indicated by open circles. (B) DNA sequences were pooled according to microbiome types: type I and type II using the same methods as Figure 2A.
Figure 3
Figure 3
Differential representation of genera between the two types of microbiome. Pooled 16S rRNA gene sequences from type I samples were compared at the genus level (or the lowest classifiable rank above genus) with those from type II samples using LIBRARY COMPARE in RDP II. Relative abundances of a genus in the two types are shown in the table and by the horizontal bars, with genera that are significantly different between the two types of microbiome highlighted in red. Unclassified taxa are marked with a ¶. Distribution of the genera in the taxonomic hierarchy of domain bacteria is shown in the phylogenetic tree, with alternating black and green brackets to contrast neighboring phyla. Bootstrap values were based on 500 replicates.
Figure 4
Figure 4
Taxonomic definition of microbiome types. (A) Microbiome-abundance correlation (MAC) analysis. The first principal coordinates (PC1) in DPCoA were correlated with the relative abundance of Firmicutes, Streptococcus, or S. mitis, for every sample, by linear regressions. (B) Classification of microbiome by the relative abundance of Streptococcus. An outlier (solid circle) was excluded using a box plot in which the upper whisker length is 1.5*IQR. The 95% normal reference range (NRR) (mean ± 1.96 S.D.) was calculated by the relative abundance of Streptococcus after excluding the outlier. The dotted line (50.3%) is the upper limit of the 95% NRR, which separates the 34 samples into normal (inside the NRR) and abnormal taxonomic types (outside the NRR).
Figure 5
Figure 5
Taxonomic characterization of microbiome by population of main bacterial groups. (A) Microbiome-abundance correlation analyses between the first principal coordinates (PC1) in DPCoA and the relative abundance of anaerobic/microaerophilic bacteria (AN/M). (B) Correlation between the first principal coordinates (PC1) in DPCoA and the relative abundance of Gram-negative (G-) bacteria. (C) Correlations between the relative abundance of Streptococcus and that of anaerobic/microaerophilic bacteria. (D) Correlations between the relative abundance of Streptococcus and that of Gram-negative bacteria. (F) Comparisons of microbiome types according to culture conditions. (E) Comparisons of microbiome types according and staining properties.
Figure 6
Figure 6
Difference between the two types of microbiome in biological diversity. (A) Shannon-Wiener diversity index. (B) Shannon-Wiener evenness index. (C) Richness by observed and estimated SLOTUs. Mean ± 1.96 S.D. is indicated by horizontal lines.

Comment in

  • Microbiome analysis in the esophagus.
    Suerbaum S. Suerbaum S. Gastroenterology. 2009 Aug;137(2):419-21. doi: 10.1053/j.gastro.2009.06.017. Epub 2009 Jun 27. Gastroenterology. 2009. PMID: 19563840 No abstract available.

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