食道弛緩不能症患者之食道微菌叢特徵及接受治療前後之變化

Characteristics of esophageal microbiome in patients with achalasia and its changes before and after treatment

葉佳衢(Chia-Chu Yeh)

指導教授 ： 曾屏輝 ； 王秀伯

國立臺灣大學/醫學院/臨床醫學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202103011

摘要

研究背景論述：
過去關於食道的微菌叢在各種食道疾病的組成少有研究，而近來有研究使用細胞刷針對食道的微菌叢做採樣分析，研究各種不同食道疾病的微菌叢特徵。食道弛緩不能症是少見的原發性食道蠕動障礙，主要是食道體的蠕動及下食道括約肌的舒張異常，造成患者漸進式的吞嚥困難，體重減輕，病程緩慢進展下可能導致食道極度擴大及扭曲，並可能引發吸入性肺炎或增高未來發生食道癌的機會。食道體的蠕動及下食道括約肌的舒張異常，也會造成食物的滯留、發酵及食道黏膜的慢性發炎，進而影響食道微菌叢的組成，但相關領域仍少有研究。目前針對食道弛緩不能症主流的治療方式是經口內視鏡食道肌肉切開術(POEM)，效果相當好且併發症極低，但長期的效果及安全性仍有待追蹤研究。經口內視鏡肌肉切開術治療前後的食道微菌叢變化目前相關研究甚少，微菌叢的組成改變是否和長期預後有關也待進一步證實。因此，本研究預計研究食道弛緩不能症患者的微菌叢特徵，並分析食道弛緩不能症患者在接受治療前後的食道微菌叢差異，以期做為日後分析疾病特性及提供個人化精準醫療的基礎。
研究方法：
收案族群為經由高解析度食道壓力檢查確診之食道弛緩不能症且預計接受經口內視鏡食道肌肉切開術之患者，在接受治療前和接受治療後3個月安排內視鏡追蹤時，使用細胞刷進行食道微菌叢的採樣及分析，做法為在上食道括約肌下方5公分及胃食道接合部上方3公分分別進行四個象限來回各10次的採樣。另外收案無症狀健康受試者作為對照組，其做法同樣為在上食道括約肌下方5公分及胃食道接合部上方3公分分別進行四個象限來回各10次的微菌叢採樣。進一步使用PCR針對細菌16S rRNA基因定序分析出微菌叢組成，再使用R程式分析比較兩組微菌叢的差異性和治療前後的微菌叢變化。針對食道弛緩不能症患者，進一步分析食道內不同滯留嚴重度、不同程度的食道黏膜變化的患者之微菌叢，並針對不同年齡及食道鋇劑攝影鋇劑滯留高度不同的病人做次族群分析。
結果與討論:
從2019年8月至2021年2月於台灣大學附設醫院共收案46人，其中包含31位食道弛緩不能症患者(男性佔45.2%，平均年齡為53.5±16.2歲)及對照組15位(男性佔46.7%，平均年齡為54.9±14.7歲)。其中20人完成經口內視鏡肌肉切開術術後追蹤，而內視鏡追蹤發現11人(55%)有逆流性食道炎產生。分析食道弛緩不能症患者及對照組的微菌叢組成，發現食道弛緩不能症患者其微菌叢組成在門的級別佔比例最高的依序是Firmicutes (52%)，Bacteroidota (25%)，Proteobacteria (10%)，和Fusobacteroita (5%)，而在屬的級別佔比例最高的依序是Streptococcus (21%)，Prevotella (15%)，Lactobacillus (12%)，Veillonella (10%)，Neisseria (6%)， 和Alloprevotella (5%) (其它佔比均<5%)。而對照組的微菌叢組成，在門的級別佔比例最高的依序是Firmicutes (33%)，Bacteroidota (30%)，Proteobacteria (20%)，和Fusobacteroita (7%)，而在屬的級別佔比例最高的依序是Streptococcus (16%)，Prevotella (15%)，Veillonella (9%)，Neisseria (11%)，Alloprevotella (8%)，和Porphyromonas(6%)(其它佔比均<5%)。進一步比較食道弛緩不能症患者及對照組的微菌叢組成，其alpha多樣性並沒有差異，但beta多樣性卻有顯著差異。使用LEfSe (Linear discriminant analysis Effect Size)進一步分析其菌種組成的差異，發現在門的級別，Firmicutes在兩組間差異最顯著，而在屬的級別，Lactobacillus在兩組間差異最顯著。
將食道弛緩不能症患者依據食道內滯留程度分為四級，第零級為無滯留，第一級為泡沫滯留，第二級為液體滯留，第三級為食物滯留。分析食道內不同滯留嚴重度的微菌叢組成，其食道菌叢的alpha及beta多樣性並沒有顯著差異。然而使用LEfSe (Linear discriminant analysis Effect Size)進一步分析其菌種組成的差異，當滯留程度較嚴重時，其微菌叢組成和其它級別有特別顯著的差異，在滯留程度為第三級的食道弛緩不能症患者其Lactobacillus的豐富度明顯較其它級別高。食道弛緩不能症患者因長期食物及液體滯留，會產生黏膜增厚且血管相減少等變化，將食道弛緩不能症患者的食道黏膜依據變化程度分為三級，第零級並無黏膜增厚變化，第一級為輕微黏膜增厚變化，第二級則是黏膜完全增厚且血管相減少。進一步分析，黏膜變化越嚴重，其alpha多樣性越低，但仍無統計上顯著差異，而三個分級的菌種組成(beta多樣性)則有顯著差異。進一步分析不同程度的黏膜變化其微菌叢組成，發現當產生黏膜完全增厚時的微菌叢組成和輕微或未增厚時有特別顯著差異。進一步分析，產生第二級黏膜變化(黏膜完全增厚)的食道弛緩不能症患者其Lactobacillus的豐富度明顯較其它級別高。接下來對於食道弛緩不能症患者做次族群分析，針對鋇劑滯留的嚴重度做分析，以食道鋇劑攝影檢查5分鐘10cm的鋇劑高度作為切點分組。比較不同鋇劑滯留嚴重度，其食道菌叢的alpha及beta多樣性並沒有顯著差異。但進一步以LEfSe分析，鋇劑滯留較嚴重的病人，在屬的級別，Bulleidia, Phocaeicola, and Flexilinea比例較高。而將食道弛緩不能症患者以年齡區分，較高齡者(65歲以上)其alpha多樣性較低但無統計上顯著差異，而beta多樣性也無顯著差異。進一步以LEfSe分析，在屬的級別，較高齡者(65歲以上)其Bacteriodes豐富度較高; 而65歲以下的族群其Prevotella 和 Peptococcus豐富度較高。而針對接受經口內視鏡肌肉切開術前後的微菌叢做分析，其食道菌相的alpha及beta多樣性並沒有顯著差異。雖然如此，但用LEfSe進一步分析發現在門的級別，Proteobacteria在兩組間差異最顯著，而在屬的級別，Neisseria在兩組間差異最顯著，另外，Lactobacillus的比例在術後也有明顯下降。
本研究發現，食道弛緩不能症患者之食道微菌叢和對照組相比，Lactobacillus差異最大，可能因為其食道內食物長期滯留發酵，且在酸性環境下更利於此類菌群的生存。而在滯留程度較嚴重和黏膜變化程度較大(完全增厚)的病人，其Lactobacillus的豐富度又顯著地比其它程度較輕微的病人高。這群lactic acid-producing bacteria具有還原nitrate能力，在酸性環境下進一步會產生N-nitroso compounds，進而具有致癌性，長期可能增加食道癌產生的風險，故對於食道弛緩不能症患者，及早發現並治療以改善其瘀滯的食道環境具有重要性。而接受經口內視鏡肌肉切開術後的病人其革蘭氏陰性菌比例上升，尤其是Neisseria增加最顯著，推測是因術後胃酸逆流增加導致。故術後應長期追蹤並適當治療逆流性食道炎，避免長期胃酸逆流造成的食道傷害產生。
結論:
食道弛緩不能症患者因食道體的蠕動及下食道括約肌的舒張異常，造成食物滯留及局部環境改變，因此造就其食道微菌叢特殊的組成特徵，其中以Lactobacillus和對照組的差異最大。此類lactic acid-producing bacteria可能和長期食道癌產生的風險增加相關，但其因果關係仍需進一步驗證。而食道弛緩不能症患者在接受經口內視鏡肌肉切開術後，Lactobacillus比例下降，推論因和食物滯留改善相關。另外，革蘭氏陰性菌(尤其是genus Neisseria)比例增加，可能和術後胃酸逆流增加相關，故應積極治療術後之逆流性食道炎，避免因長期胃酸逆流而造成食道病變，改善食道弛緩不能症患者之長期預後。

關鍵字

食道弛緩不能症 ； 致癌性 ； 經口內視鏡食道肌肉切開術 ； 微菌叢

並列摘要

Background:
Achalasia is a rare primary esophageal motility disorder involving smooth muscle of esophageal body and lower esophageal sphincter (LES). Patients often suffer from dysphagia, chest pain, regurgitation, and weight loss. Moreover, chronic achalasia has been associated with increased risk of developing esophageal cancer. We hypothesized that chronic food stasis and fermentation in achalasia may lead to alterations of the esophageal microbiome and associated inflammation and/or dysplastic changes of the esophageal mucosa. Therefore, this study aimed to evaluate the characteristic of esophageal microbiome in achalasia and its changes before and after POEM.
Method:
We conducted a prospective study from Aug 2019 and enrolled achalasia patients and asymptomatic subjects as the control group in a tertiary medical center. Esophagogastroduodenoscopy (EGD) was performed in all subjects and additional follow-up endoscopy was performed 3-months after POEM in achalasia patients. We collected esophageal microbiome samples by endoscopic brushing over 5 cm below the upper esophageal sphincter and 3 cm proximal to the squamo-columnar junction in each subject. The composition of esophageal microbiome was determined and compared between 1) achalasia vs. controls; 2) achalasia patients with different grades of esophageal rentention; 3) achalasia patients with various severities of esophageal mucosal change; 4) achalasia patients with different barium column height on timed barium esophagogram; 5) achalasia patients with different age; 6) achalasia patients before and after POEM.
Results:
Thirty-one achalasia patients (mean age 53.5 ± 16.2 years; male 45.2%) and 15 asymptomatic subjects (mean age 54.9±14.7 years; male 46.7%) were analyzed. We observed a distinct esophageal microbial community structure in achalasia patients, with an increased abundance of phylum Firmicutes and decreased abundance of phylum Proteobacteria compared with control group. The discriminating enriched genera in achalasia patients was Lactobacillus, which belongs to phylum Firmicutes. Lactic acid generated by lactic acid bacteria may contribute to microbiota related carcinogenesis. In achalasia patients, we further investigated the influence of different grades of esophageal rentention on microbiota and found that genus Lactobacillus significantly enriched in those with food retention compared to those with minor retention. Regarding the influence of various severity extents of esophageal mucosal change on microbiota, alpha diversity decreased in those with complete mucosal thickening although no statistically different. Lactobacillus was the relatively enriched esophageal microbial genera in achalasia patients with complete mucosal thickening compared to those with partial or no thickening. For subgroup analysis of achalasia patients, there was no difference in the alpha diversity and beta diversity between achalasia patients with different severity extent of barium stasis on timed barium esophagogram. However, discriminant analysis using LEfSe revealed the significantly enriched esophageal microbial genera in achalasia patients with the height of barium stasis less than 10cm at 5th minutes were Alloprevotella, Stomatobaculum, Solobacterium, Atopobium, and Mogiobacterium. The significantly enriched esophageal microbial genera in achalasia patients with the height of barium stasis more than 10cm at 5th minutes were Bulleidia, Phocaeicola, and Flexilinea. Comparing achalasia patients above and below 65 years of age, the most significantly enriched genera in achalasia patients above 65 years of age was genus Bacteriodes and the most significantly enriched microbiota in achalasia patients below 65 years of age was Prevotella loescheii and genus Peptococcus. Further analysis of achalasia patients before and after POEM revealed that the relative abundance of phylum Firmicutes decreased and phylum Proteobacteria increased after POEM. Relative abundance of genus Neisseria increased after POEM. 55% of patients suffered from erosive esophagitis by endoscopic examination after POEM. The increased of relative abundance of Neisseria might be related to post-POEM reflux esophagitis caused by pathological acid exposure.
Conclusion:
Altered local environment of esophagus in achalasia patients lead to distinct esophageal microbial composition in this special population. Higher abundance of lactic acid-producing bacteria in achalasia patients may be related to further carcinogenesis in achalasia. Howevere, the causal relationship needs further longitudinal follow-up and investigation. Increased abundance of Gram-negative bacteria is probably caused by high prevalence of pathological acid exposure in post-POEM achalasia patients. Therefore, adequate acid control is crucial to improve long-term outcome.

並列關鍵字

Achalasia ； Carcinogenesis ； Peroral endoscopic myotomy ； Microbiota

參考文獻

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