A non-endoscopic device to sample the oesophageal microbiota: a case-control study

Abstract

SummaryBackgroundThe 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.MethodsIn 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.FindingsThere 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).InterpretationAlterations 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.FundingCancer Research UK, National Institute for Health Research, Medical Research Council, Wellcome Trust, The Scottish Government (RESAS).

Risk factors for esophageal cancer: emphasis on infectious agents

“…in 2009, identified two distinct patterns of EM: type I, associated with normal esophagus and characterized by a predominance of Gram‐positive taxa, with a high abundance of the genus Streptococcus (79%); and type II, associated with erosive esophagitis and BE, and characterized by a predominance of Gram‐negative taxa (mostly from Bacteroidetes, Fusobacteria, and Proteobacteria phyla), with a marked reduction in the Streptococcus abundance (30%). Similar findings were reported in a subsequent study by Liu et al ., whereas recognition of the type I and II patterns was not confirmed by most recent studies employing NGS methodologies, possibly due to differences in patient characteristics (i.e., symptomatic controls, treatment with proton pump inhibitors).…”

“…Information about healthy EM remains limited, with very few studies enrolling healthy asymptomatic subjects and using culture‐independent techniques. Previous findings were confirmed by recent studies based on next‐generation sequencing (NGS), which demonstrated that the healthy esophagus is dominated by the genus Streptococcus (phylum Firmicutes), in association with Prevotella (phylum Bacteroidetes) and Veillonella (phylum Firmicutes), although at less abundance. Other frequently identified genera included Fusobacterium (phylum Fusobacteria), Haemophilus , Neisseria (phylum Proteobacteria), and Porphyromonas (phylum Bacteroidetes) (Table ), reinforcing the notion that the esophageal microenvironment is phylogenetically closely related to that found in the oropharynx …”

“…analyzed the composition of the EM in a cohort of patients with BE and identified positive and negative correlations for the Streptococcus–Prevotella ratio with waist‐to‐hip ratios and hiatal hernia length, respectively, two known risk factors for BE and EAC . The incidence of EAC has been rapidly increasing over the past two decades in Western countries, but only a few culture‐independent studies have investigated the potential role of EM in the progression to esophageal malignancies . Blackett et al .…”

“…The authors hypothesized that this condition could eventually induce metaplastic changes in a dysbiotic and previously injured esophageal mucosa (i.e., columnar metaplasia characteristic of BE), ultimately favoring malignant transformation . The most recent study on this topic did not confirm an enrichment of any Campylobacter species in EAC patients, but instead showed decreased bacterial richness and diversity compared to controls and BE patients, along with a significant increase of Lactobacillus fermentum and lactic acid bacteria in almost half (47%) of EAC patients. Moreover, the authors did not identify any differences between the microbiota of BE and EAC patients and suggested that bacterial taxa with very low relative abundances, being more difficult to detect, may potentially contribute to alteration of the EM in these patients …”

“…Little information is available about the stability of EM over time and about any homogeneity of the EM throughout the esophagus. Rare studies have investigated the latter and evaluated upper and lower esophageal biopsy samples in healthy subjects, normal squamous and metaplastic mucosa in BE patients, or normal squamous and malignant mucosa in EAC patients . In all reported cases, the authors did not identify significant differences between various esophageal segments or histotypes.…”