<i>A NOVEL GENOMIC BLUEPRINT OF THE HEALTHY HUMAN OESOPHAGEAL MICROBIOME</i>

Objectives:
Dysphagia and chest pain are the common symptoms in achalasia. The mechanisms underlying such symptoms are not completely understood. The two symptoms can be triggered by mechanical stimulation via the sensory vagal and spinal afferent nerve fibers. We previously reported that superficial nociceptive mucosal afferent nerves (i.e. calcitonin gene-related peptide-immunoreactive (CGRP-IR) nerves) might contribute to acid hypersensitivity in non-erosive reflux disease. The mucosal nerves can also detect mechanical deformation of the mucosa apart from chemical stimulation. Therefore, the aim of this study was to investigate the association between the severity of esophageal symptoms in achalasia patients and the characteristics of mucosal afferent nerve innervation.

Methods:
This was a muti-center prospective study involving six tertiary centers. Patients with predominant dysphagia and/or chest pain referred to the centers were recruited if they underwent upper endoscopy and high-resolution manometry for the examination of their symptoms. Esophageal motility was diagnosed on the basis of Chicago classification ver 4.0. The severity of dysphagia and chest pain were evaluated using Brief Esophageal Dysphagia Questionnaire (BEDQ) and a self-reported chest pain questionnaire respectively. Biopsies were taken from the proximal and distal esophagus for assessment of CGRP-IR mucosal nerves. The position of such nerves was expressed as cell layers from the esophageal luminal surface. We used a historical cohort of 8 asymptomatic healthy volunteers for comparison with the patients.

Results:
Overall, 61 patients with achalasia (n=57) or EGJ outflow obstruction (n=4) were included (median age, 56 years; 24 female (39%)). All the patients had dysphagia with the median of BEDQ score of 19 (11-26), and 39% of them had concomitant strong chest pain. The mucosal afferent nerves were located significantly closer to the lumen in the patients than in HVs (8 vs 11 cell layers, p=0.046 in the proximal, 9 vs 24 cell layers, p<0.001 in the distal). The position of mucosal afferent nerve negatively correlated to BEDQ score both in the proximal (rho=-0.567, p<0.001) and distal esophagus (rho=-0.317, p=0.021). The patients with strong chest pain showed more superficial mucosal afferent in the proximal esophagus (7 cell layers (4-9)) than those with weak chest pain (9 cell layers (7-14), p=0.022). Multivariate analysis found that the position of the afferent nerves in the proximal esophagus were independently and inversely associated with BEDQ score (-0.013 (-0.020, -0.006), <0.001) and chest pain (-0.054 (-0.101, -0.007), p=0.044). However, there was not such relationship in the distal esophagus.

Conclusions:
The superficial afferent nerves in the proximal, not distal esophagus, were associated with severe dysphagia and chest pain.

Background
The oesophageal microbiome is thought to contribute to the pathogenesis of oesophageal cancer. However, investigations using culture and molecular barcodes have provided only a low-resolution view of this important microbial community. We therefore exploited culturomics and metagenomic binning to generate a catalogue of reference genomes from the human oesophageal microbiome using oesophageal brushes from healthy patients, alongside a comparison set from saliva.

Results
Genomes from cultured isolates revealed twenty-two distinct colonial morphotypes from healthy oesophageal samples. These fell into twelve species clusters, eleven of which represented previously defined species. Two isolates belonged to a novel species, which we have named Rothia gullae.
Metagenome-assembled genomes was performed using metagenomic binning of reads generated from UK samples from this study alongside reads generated from Australian samples in the recent study by Deshpande et al., 2018. Metagenomic binning generated 136 medium or high-quality metagenome-assembled genomes (MAGs). MAGs were assigned to 56 species clusters, eight representing novel Candidatus species, which we have named Ca.Granulicatella gullae, Ca. Streptococcus gullae, Ca. Nanosynbacter quadramensis, Ca. Nanosynbacter gullae, Ca.Nanosynbacter colneyensis, Ca. Nanosynbacter norwichensis, Ca. Nanosynococcus oralis and Ca. Haemophilus gullae. Five of these novel species belong to the recently described phylum Patescibacteria. Although members of the Patescibacteria are known to inhabit the oral cavity, this is the first report of their presence in the oesophagus.
Our non-redundant species catalogue, contained 63 species derived from cultured isolates or MAGs. Mapping revealed that these species account for around half of the sequences in the oesophageal and saliva metagenomes. Although no species was present in all oesophageal samples, 60 species occurred in at least one oesophageal metagenome from either study, with 50 identified in both cohorts.

Conclusions
In recovering over a hundred bacterial genomes through culture and metagenomic analysis, we have obtained the first high-resolution view of microbial diversity within this important environment. Remarkably, we have discovered one new cultured species and eight novel Candidatus species, opening up the way for detailed characterisation of these newfound taxa. We have not only discovered new species within well-characterised genera, such as Strepotococcus and Haemophilus, but also found six species from the enigmatic Patescibacteria, which are thought to live as epibionts in close association with other bacteria in this environment . The genes and genomes that we have released into the public domain will provide a base line for future comparative, mechanistic and intervention studies.