The present study has provided two new contributions to the field of human microbial ecology. First, we have performed a comprehensive study of the human distal esophagus microbiome and demonstrated the presence of a complex microbiome about which little prior knowledge was available. The esophageal microbiome is comparable in complexity to those found in the mouth, stomach, colon, vagina, and skin.14–15, 28–32
Collectively, nine phyla were observed, represented by 166 species. The distal esophagus could harbor >200 species, as predicted by the Chao1 richness estimator.20
Second, we have demonstrated by both unsupervised and phenotype-directed analyses that the esophageal microbiome can be classified into two types and that the type II microbiome is the strongest (OR >15) amongst all known environmental factors that are associated with the pathological changes related to GERD (Supplemental Table 5
). Overall, the findings have opened a new approach to understanding the recent surge in the incidence/prevalence of GERD and EA, and suggest the possible role of dysbiosis in their pathogenesis.
The need to compare with risk factors identified by conventional studies promoted us to design a dual assignment scheme for the esophageal samples. This approach enabled testing the independency of two categorical variables, host phenotype and microbiome type. The microbiome types determined from the present study are relevant to pathology in the distal esophagus. There are two possible explanations for the significant association between the type II microbiome and the abnormal histological phenotype. First, the type II microbiome might play a causative role in GERD, which has a complex and not yet completely understood pathophysiology. Abnormal lower esophageal sphincter (LES) pressure and esophageal acidification during transient LES relaxation are believed to be critical, but the etiology of the abnormal LES function is unknown.33
One possibility is that the esophageal microbiome could be intrinsic – each individual might harbor either a stable type I or type II microbiome. Distinct microbiome can be inherited via kinship from mother or caregivers, as suggested in studies of mouse colonic microbiome,34
which can be modified by exposure to antibiotics during or after the postnatal development of microbiome is complete.6
The Gram-negative predominant type II microbiome could serve as a primary or synergistic mechanism in promoting gastric reflux, since lipopolysaccharides (LPS), mainly produced by Gram-negative bacteria induce abnormal relaxation of the lower esophageal sphincter via activation of the inducible nitric oxide synthase (iNOS) pathway.35
Second, the type II microbiome might be secondary to changes caused by gastric reflux. The esophageal microbiome could be transitory: the type I microbiome could represent a direct extension of the normal oral flora via saliva while the type II microbiome could represent regurgitated bacteria in gastric juice. Alternatively, gastroesophageal reflux might modify the esophageal microbiome by selecting against acid-sensitive bacteria in the esophagus. Testing these hypotheses might shed light on the pathogenesis of GERD and lead to new biomarkers for GERD.
Although the present study yielded one of the largest data sets from a single study of microbiome in human diseases,16,29
our understanding of the esophageal microbiome is far from complete. The power of this study might be limited by unrecognized factors unrelated to GERD, but that potentially affect the bacterial microbiome, including diet, medications, and oral and gastric diseases. Similarly, any interpretation of our data in relation to GERD should be cautious, since GERD has a complex definition.36
The GERD phenotype is composed of three heterogenous factors: symptoms, abnormal acid exposure, and mucosal damage.36
While not optimal, this definition is necessary because of the substantial overlap between normal subjects and those with GERD for any of the single factors. Significant acid reflux can occur in 19% of (normal) subjects without reflux-related symptoms.37
Similarly, erosive esophagitis may be found in patients with normal acid exposure.38
Not all patients with abnormal acid exposure have esophagitis, as defined by histology, which can present in subjects without reflux;39
a substantial proportion of patients with BE lack reflux symptoms.40
The significant but nonexclusive association of the type II microbiome with histologically defined changes related to GERD is consistent with the complex GERD phenotype. Notably, our study subjects were generally elderly male veterans. This relatively homogenous cohort helped limit confounding, but also might limit the application of our findings to a more general population. Despite the complex host and environmental factors, the findings in the present analyses make possible design of further studies to directly examine possible causal roles of the type II microbiome in GERD development. If GERD represents a microecological disease, a new type of treatment for reflux might become possible, for example, by converting the type II to type I microbiome through use of antibiotics, probiotics, or prebiotics.