Although the knowledge regarding the human microbiota composition in the gut is vast, the composition of human microbiota in the esophagus is only partially understood. The microbiota in the esophagus under normal circumstances had been considered as a transitional microbiota originating from the oral cavity and oropharynx.1,2 However, as new techniques such as 16S ribosomal RNA (rRNA) gene sequencing have been introduced, it is now known that the esophagus has a remarkably diverse microbiota that depends on several factors.3,4 The genera
Achalasia is a motility disorder that induces aperistalsis and abnormality in lower esophageal sphincter relaxation.10 Therefore, chronic liquid and food stasis causes bacterial fermentation in the esophagus in patients with achalasia.11 Peroral endoscopic myotomy (POEM) is a new option for the treatment of achalasia, and is shown to have excellent efficacy.12 In addition, a previous study reported that POEM may restore the morphologic tortuosity in cases of sigmoid-type achalasia.13 Pajecki et al14 reported that
Twenty-nine patients in total were prospectively enrolled from 4 referral institutions across Korea. Written informed consent was obtained from all patients before their enrollment in the study. The inclusion criteria were as follows: (1) patients who were diagnosed with achalasia and planned to undergo POEM, and (2) patients in the age range of 20-80 years. The exclusion criteria were as follows: (1) patients with a history of previous treatment for achalasia except medication; (2) patients with a history of chest and gastrointestinal surgeries, (3) patients with prior treatment with proton pump inhibitors or antibiotics within 8 weeks, (4) patients with the presence of an active infection in the oral cavity, (5) patients with a history of malignancy, (6) patients with a history of bleeding tendency or intake of antithrombotics, and (7) patients with the presence of a systematic disease who were receiving treatment. The study protocol was approved by the institutional review board of each participating institution (IRB No. 3-2018-0246, KCT0005797, KC18TCDI0507, 2018-07-001, 2019-0109).
General information of patients was obtained in terms of their age, sex, height and weight, body mass index, comorbidities (diabetes, hypertension, pulmonary disease, heart disease, and kidney disease), smoking condition (non-smoker, ex-smoker, and current smoker), and alcohol consumption. The dietary survey was conducted as a face-to-face interview, and the eating habits and food intake questionnaire were designed to be an open-ended survey for reporting various foods using the 3 days 24 hours recall method with various measuring aids.15,16 The consumed food was analyzed using the Computer Aided Nutritional Analysis Program for Professionals 5.0 (CAN-Pro 5.0, The Korean Nutrition Society, Seoul, Korea) based on the intake of individual nutrients. Changes in the nutrient intake pre- and post-POEM were analyzed as the amount of daily energy, energy nutrients (carbohydrate, protein, and fat), fiber, vitamins (vitamin A, vitamin D, vitamin E, vitamin K, vitamin C, vitamin B1, vitamin B2, vitamin B6, vitamin B12, niacin, folic acid, and pantothenic acid), and minerals (calcium, phosphorous, sodium, potassium, magnesium, iron, zinc, and selenium).
We collected 2 samples, esophageal mucosal biopsies and esophageal retention fluid. We developed collecting methods especially for this research project to avoid potential contamination. First of all, we used gloves and followed aseptic procedures with sterile equipment. A standard video-endoscope was passed through the mouth to the esophagus without touching the oropharynx as carefully as possible and we avoided unnecessary suction. The stasis liquid in the esophageal lumen was aspirated via an endoscopic working channel. We connected tubing (disposable specimen trap) which was used in bronchoscopy (HS-SP-50; Hyupsung Medical Co, LTD, Seoul, Korea) for fluid collection at the entrance of the working channel to avoid contamination. After finishing the fluid collection, biopsies were then taken 2 cm above the squamocolumnar junction using sterile disposable biopsy forceps (EndoJaw; Olympus Co, Ltd, Tokyo, Japan). If there was no retention fluid in esophagus after POEM, we aspirated by washing the esophageal lumen with 10 mL of sterile saline solution. We collected samples twice on the day before or on the day of the POEM and 8 weeks after POEM. The biopsy and fluid samples were placed in a test tube (SNP BiomCare; SNP Genetics Inc, Seoul, Korea) and stored at –70°C. We administered broad-spectrum intravenous antibiotics 30 minutes before the start of the POEM. During the POEM procedure, prophylactic antibiotic solution spraying inside the tunnel was performed before closing the mucosal entry. The use of intravenous antibiotics was discontinued 3 days after the POEM and antibiotics were switched to oral intake for another 7 days.
The biopsy and fluid samples (3-10 mL) were centrifuged at 15 000 g for 20 minutes at 4°C to separate the cellular pellet and the cell-free supernatant. DNA was extracted from the pellet using a QIAamp DNA Microbiome Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions.
The V3-V4 region of the 16S rRNA gene was amplified using the 341F (5’ TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG 3’) and 805R (5’ GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C 3’) primers with added Illumina adaptor overhang sequences. The amplicons were purified using a magnetic bead-based clean-up system (Agencourt AMPure XP; Beckman Coulter, Brea, CA, USA). Indexed libraries were prepared by limited-cycle PCR using the Nextera technology, and the samples obtained were further cleaned up and pooled at equimolar concentrations. The final library was denatured with 0.2 N NaOH and diluted to 6 pM with a 20% PhiX control. Sequencing was performed on the Illumina MiSeq platform using a 2 × 300 bp paired-end protocol according to the manufacturer’s instructions.
The primary analysis of the obtained sequences consisted of their demultiplexation with the MiSeqReporter software (Illumina). The paired-end sequences of each sample were then exported from the MiSeq system for analysis in the FASTQ format. Bioinformatic analysis of the sequences was performed using the QIIME2 package.17 For subsequent data analysis, we used a web-based platform called MicrobiomeAnalyst.18 The sequences were then clustered against the 2013 Greengenes (13_5 release) 97% reference dataset of the ribosomal database.19 The UCLUST algorithm was used to cluster sequences that did not match any entries in this reference into de novo operational taxonomic units (OTUs) at 97% similarity. The OTU table was rarefied to a sequencing depth of 20 000 per sample for subsequent analyses.
Taxonomic analyses were performed after collapsing the OTUs at the genus level. The alpha diversity of each sample was assessed using the Shannon index. Beta diversity was determined based on the Bray-Curtis index distance method using permutational MANOVA, and principal coordinate analysis plots were constructed. The statistical significance of the esophageal microbiota structure between different sampling sites and gut regions was assessed by the non-parametric univariate Mann-Whitney/Kruskal-Wallis test. The linear discriminant analysis effect size (LEfSe) was used to identify significantly different abundances in the bacterial taxa. The false discovery rate correction was used for multiple tests. A
Between October 2018 and December 2019, we enrolled 29 patients who underwent POEM for achalasia. The baseline characteristics are shown in Table 1. The subtypes of achalasia were 12 (41.4%), 10 (34.5%), and 4 (13.8%) for type I, II, and III, respectively. The subtype of achalasia in the other 3 patients could not be differentiated. The median pre-POEM Eckardt score was 6.0 (range, 1-11) and the median symptom duration was 24 months (range, 3-300 months).
The results of the change in nutrient intake status are shown in Table 2. The usual energy intake of the patients increased from 1145.9 ± 358.8 kcal to 1645.6 ± 309.6 kcal after POEM. As the overall food intake increased, the intake status of all nutrients significantly increased too, except the intake status of vitamin E.
Among the 29 patients, we collected 105 samples (29 mucosal biopsies and 30 retention fluid samples before POEM and 23 mucosal biopsies and fluid retention samples after POEM). After the quality control, which excluded samples with no amplification or low-quality sequence data, the microbiome of 99 samples were subjected to bioinformatic analysis (25 mucosal biopsies and 30 retention fluid samples before POEM and 22 mucosal biopsies and fluid retention samples after POEM). The observation of the overall microbial composition showed that Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Fusobacteria were the dominant phyla, representing over 95% of the total phyla in all groups (Fig. 1A). Firmicutes was the most abundant bacterial phylum in the pre-and post-POEM fluid samples and the pre- and post-POEM tissues, followed by phyla Bacteroidetes and Proteobacteria (Table 3). At the genus level (Fig. 1B),
We evaluated the alpha diversity using Shannon analysis (Fig. 3A) and the beta diversity using the Bray-Curtis index (Fig. 3B) among the 4 groups. The results indicated that the structure of the esophageal microbiome in the tissue samples was significantly different from that of the fluid samples.
To identify the specific bacterial taxa associated with the tissue and the fluid samples, we compared their esophageal microbiota using LEfSe. Figure 4A shows that 15 genera or species distinguished the esophageal microbial communities between the 2 groups pre-POEM according to the criteria of linear discriminant analysis ≥ 2.5 at
Achalasia causes esophageal stasis and delayed esophageal clearance. The stagnated residues in the esophagus create a propitious environment for the growth of microbiota. In particular, a previous study reported that
The study of esophageal microbiota has advanced from a culture to a culture-independent method. In addition, the methods used to collect esophageal microbiota samples are varied – from biopsies, aspirations, brushes, and esophageal string tests.20 In this study, we collected esophageal samples from patients with achalasia in 2 ways: biopsy and aspiration. Most studies have evaluated the esophageal microbiome using tissue biopsies, which would be the most suitable method, considering the presence of an adherent microbiome at mucosa of esophagus. However, sampling by brushes or aspirates was adopted and this was a less invasive method and used as an alternative to esophageal biopsies. However, esophageal biopsies are always necessary for a direct histological assessment of the diseased tissues in order to avoid misclassification and decrease the risk of contamination by oropharyngeal or gastric secretions. In this study, the bacterial communities in the esophageal tissue and fluid groups pre- and post-POEM were structurally different, with the alpha diversity (OTU number) decreasing from the fluid to tissue samples. Previous studies have reported that reduced microbial diversity, in itself, is a disease status, such as inflammation or cancer.21,22 Therefore, the microbiota of the esophageal tissues may play a pivotal role in patients with achalasia, as the microbiota of the esophageal fluid group is translocated from the oropharynx. Using LEfSe, we identified specific bacterial taxa associated with the esophageal tissue in patients with achalasia, which were the genera
This is the first study to show the esophageal microbiota in patients with achalasia using 16S rRNA sequencing to the best of our knowledge. The most common bacterial phyla in normal esophagus are Firmicutes (70-87%), Bacteroidetes (5-20%),
Many factors affect the bacterial composition in the esophagus such as aging, dietary fiber intake, proton pump inhibitors, and diseases that cause esophageal dysbiosis.26,27 We conducted a dietary survey of patients with achalasia in this study. It is reported that many patients with achalasia complain of discomfort such as dysphagia, vomiting, decreased appetite, and chest pain, which can lead to weight loss and nutritional deficiencies.28,29 As a result of the survey analysis, nutrient intake pre-POEM was insufficient, and the nutrient intake was significantly improved after POEM in most patients. However, a long-term lack of energy and protein intake can have a direct effect on weight and muscle loss.30 In addition, even after POEM, there were still many nutrients (energy, vitamin A, vitamin C, vitamin B2, niacin, calcium, and magnesium) whose levels in patients did not meet nutritional recommendations.31 Although this study did not analyze the relationship between food intake and the microbiota, the nutrient intake status results show the need for early nutritional intervention and management in patients with achalasia. In addition, decreased acid reflux by PPI administration could affect the esophageal microbiomes. Amir et al32 reported that esophageal microbiome in patients with Barrett’s esophagitis, or a normal distal esophagus, was assessed from distal esophageal biopsies, comparing results before versus after PPIs. After PPIs were administered, Lachnospiraceae, Comamonadaceae, and unclassified Clostridial families significantly increased in distal esophagus.
In this study, the median Eckardt score decreased from 6.0 to 0.0, and all patients achieved clinical success (Eckardt score ≤ 3). However, there were no significant alterations of the esophageal microbiota in patients with achalasia pre- and post-POEM. We hypothesize that the small sample size and short follow-up period (8 weeks) in our study may have contributed to the results observed concerning the esophageal microbiota in patients with achalasia. Improved esophageal clearance and acid reflux has been observed in patients after POEM. According to the survey conducted, we know that there is an alteration of food intake in patients post POEM. Therefore, we think that there is a possibility of alterations in the esophageal microbiota in the long term. However, the follow-up period of 8 weeks may not be sufficient to induce esophageal microbiota alteration. In addition, although POEM treatment improves esophageal clearance, the unique esophageal microbiota composition in patients with achalasia may not change because food stasis may still persist to a small extent even after POEM. Therefore, further research is needed to demonstrate whether the esophageal microbiota in patients with achalasia changes in the long term after POEM.
This study has some limitations. First, we did not investigate the microbiome in the oral cavity of the patients and healthy controls. Therefore, we did not determine the origin and role of the oesophageal microbiota in patients with achalasia. Second, the sample size was not sufficient for subgroup analysis according to the type, symptom duration, and severity of disease. Finally, we performed the rarefraction. Rarefying has been criticized as a normalization technique because data can be omitted through the exclusion of either excess sequences or entire samples, depending on the rarefied library size selected.33 However, from another perspective, rarefying enables (1) characterization of the variation introduced to diversity analyses by this random subsampling, and (2) selection of smaller library sizes where necessary to incorporate all samples in the analysis.34 In this study, subsampling via rarefying was also a necessary process to include all possible samples in the analysis.
In conclusion, this study determined the unique oesophageal microbial composition of patients with achalasia by 16S rRNA gene sequencing, and found that this unique esophageal microbial composition did not significantly change in the short-term after POEM despite a significant improvement in the nutritional intake. These esophageal microbial data by 16S rRNA gene sequencing in patients with achalasia will provide novel insights for further research on achalasia.
The sequencing output was uploaded to NCBI SRA archive (Accession No. PRJNA756810).
This study was financially supported by a faculty research grant from Yonsei University College of Medicine (6-2017-0154), and by a grant from the Korean Society of Neurogastroenterology and Motility for 2019.
In planning and/or conducting the study, and collecting and/or interpreting data: Da Hyun Jung, Young Hoon Youn, Do Hoon Kim, Chul-Hyun Lim, Hee-Sook Lim, Hee Seok Moon, Ju Yup Lee, Hyojin Park, and Su Jin Hong; and drafting the manuscript: Da Hyun Jung, Young Hoon Youn, and Su Jin Hong.