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. 2017 Jan;2(1):32-42.
doi: 10.1016/S2468-1253(16)30086-3. Epub 2016 Nov 12.

A Non-Endoscopic Device to Sample the Oesophageal Microbiota: A Case-Control Study

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Free PMC article

A Non-Endoscopic Device to Sample the Oesophageal Microbiota: A Case-Control Study

Daffolyn R Fels Elliott et al. Lancet Gastroenterol Hepatol. .
Free PMC article

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Abstract

Background: The strongest risk factor for oesophageal adenocarcinoma is reflux disease, and the rising incidence of this coincides with the eradication of Helicobacter pylori, both of which might alter the oesophageal microbiota. We aimed to profile the microbiota at different stages of Barrett's carcinogenesis and investigate the Cytosponge as a minimally invasive tool for sampling the oesophageal microbiota.

Methods: In this case-control study, 16S rRNA gene amplicon sequencing was done on 210 oesophageal samples from 86 patients representing the Barrett's oesophagus progression sequence (normal squamous controls [n=20], non-dysplastic [n=24] and dysplastic Barrett's oesophagus [n=23], and oesophageal adenocarcinoma [n=19]), relevant negative controls, and replicates on the Illumina MiSeq platform. Samples were taken from patients enrolled in the BEST2 study at five UK hospitals and the OCCAMS study at six UK hospitals. We compared fresh frozen tissue, fresh frozen endoscopic brushings, and the Cytosponge device for microbial DNA yield (qPCR), diversity, and community composition.

Findings: There was decreased microbial diversity in oesophageal adenocarcinoma tissue compared with tissue from healthy control patients as measured by the observed operational taxonomic unit (OTU) richness (p=0·0012), Chao estimated total richness (p=0·0004), and Shannon diversity index (p=0·0075). Lactobacillus fermentum was enriched in oesophageal adenocarcinoma (p=0·028), and lactic acid bacteria dominated the microenvironment in seven (47%) of 15 cases of oesophageal adenocarcinoma. Comparison of oesophageal sampling methods showed that the Cytosponge yielded more than ten-times higher quantities of microbial DNA than did endoscopic brushes or biopsies using quantitative PCR (p<0·0001). The Cytosponge samples contained the majority of taxa detected in biopsy and brush samples, but were enriched for genera from the oral cavity and stomach, including Fusobacterium, Megasphaera, Campylobacter, Capnocytophaga, and Dialister. The Cytosponge detected decreased microbial diversity in patients with high-grade dysplasia in comparison to control patients, as measured by the observed OTU richness (p=0·0147), Chao estimated total richness (p=0·023), and Shannon diversity index (p=0·0085).

Interpretation: Alterations in microbial communities occur in the lower oesophagus in Barrett's carcinogenesis, which can be detected at the pre-invasive stage of high-grade dysplasia with the novel Cytosponge device. Our findings are potentially applicable to early disease detection, and future test development should focus on longitudinal sampling of the microbiota to monitor for changes in microbial diversity in a larger cohort of patients.

Funding: Cancer Research UK, National Institute for Health Research, Medical Research Council, Wellcome Trust, The Scottish Government (RESAS).

Figures

Figure 1
Figure 1
Proportional abundance of microbial taxa (A) Mean proportional abundance of the eight most prevalent phyla and 25 most prevalent families in tissue samples for healthy control patients (n=16), patients with Barrett's oesophagus (n=17), and patients with oesophageal adenocarcinoma (n=15). Significant differences were calculated with linear discriminant analysis effect size (LEfSe), and error bars are standard error of the mean. (B) Mean proportional abundance of representative genera from significantly enriched families identified in (A). Only genera with overall proportional abundances greater than 0·1% are included and error bars are standard error of the mean. *p<0·05. †p<0·01. ‡p<0·001.
Figure 2
Figure 2
Microbial community composition in Barrett's oesophagus The oesophageal adenocarcinoma and control patient groups largely cluster away from each other in this Bray-Curtis cluster dendrogram (p=0·002, parsimony test), but there is no significant difference in clustering for Barrett's oesophagus. Microbial composition is shown at the family level for each tissue sample. Data were sub-sampled at 631 reads per sample.
Figure 3
Figure 3
Microbial alpha diversity in oesophageal adenocarcinoma (A) Observed richness of bacterial operational taxonomic units (OTUs). (B) The Chao estimate of total OTU richness and (C) the Shannon diversity index are shown for tissue samples from healthy control patients (n=16), patients with Barrett's oesophagus (n=17), and patients with oesophageal adenocarcinoma (n=15). Statistical significance was calculated with the Kruskal-Wallis test and Dunns multiple comparisons post test. Data were subsampled at 631 reads per sample. (D) Observed richness of bacterial OTUs for paired normal squamous and tumour tissue samples from 13 patients (26 samples), Wilcoxon signed rank test. Data were subsampled at 656 reads per sample. (E) Overall bacterial abundance using 16S rRNA gene qPCR in matched tumour and normal squamous tissue from 16 patients (32 samples), Wilcoxon signed rank test. Error bars represent standard deviation. *p<0·05. †p<0·01. ‡p<0·001.
Figure 4
Figure 4
Comparison of different methods to sample the oesophageal microbiota (A) Principal coordinate analysis with the Bray-Curtis algorithm for matched endoscopic biopsies, brushes, and Cytosponge samples (31 patients) and 13 throat swabs from a subset of these patients. The first axis (PC1) accounts for 19·6% of the sample variance and the second axis (PC2) accounts for 6·3% of the variance. Data were subsampled at 631 reads per sample. (B) Overall bacterial abundance using 16S rRNA gene-based quantitative PCR in matched endoscopic biopsies, brushes, and Cytosponge samples (20 patients), Friedman test and Dunns multiple comparisons post test. (C) The observed diversity of bacterial operational taxonomic units (OTUs), (D) the Chao estimate of total OTU richness, and (E) the Shannon diversity index for matched endoscopic biopsies, brushes, and Cytosponge samples (31 patients), Friedman test and Dunns multiple comparisons post test. Data were subsampled at 631 reads per sample. *p<0·05. †p<0·01. ‡p<0·001. §p<0·0001.
Figure 5
Figure 5
Microbial alpha diversity in high-grade dysplasia detected with the Cytosponge (A) Observed richness of bacterial operational taxonomic units (OTUs), (B) the Chao estimate of total OTU richness, and (C) the Shannon diversity index for Cytosponge samples taken from normal squamous control patients (n=20), patients with Barrett's oesophagus (n=24), and patients with high-grade dysplasia (n=23). Statistical significance was calculated with the Kruskal-Wallis test and Dunns multiple comparisons post-test. Data were sub-sampled at 19 303 reads per sample. Error bars represent standard deviation. *p<0·05.

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