背景

自酿酒综合症（Auto Brewery Syndrome，ABS），作为一种较为罕见的疾病，其病因学主要为肠道的微生物发酵产生大量内源性乙醇（Endogenous ethanol，EnEth），过量乙醇超出肝脏代谢能力后进入血液导致血液乙醇浓度（Blood Alcohol Concentration，BAC）升高，因此ABS也被称为肠道发酵综合征（Gut fermentation syndrome，GFS）。既往对ABS的病因学研究认为是真菌在肠道的大量增殖导致，因此ABS的治疗方案多为经验性抗真菌治疗。真菌治疗失败的案例提示其他菌群参与ABS发生的可能。课题组前期在一例抗真菌治疗失败的ABS病人中分离到高产乙醇肺炎克雷伯菌（High Alcohol-producing Klebsiella pneumoniae, HiAlc Kpn），后经实验证实其是非酒精性脂肪肝(non-alcoholic fatty liver disease, NAFLD) 的潜在致病因子。HiAlc Kpn的发现证实了细菌也参与了ABS的发生发展，并提出了细菌性ABS的概念。因此我们认为在ABS的发生中，除了真菌，一些可以产生内源性乙醇的细菌，如HiAlc Kpn也参与了ABS的发生和发展，并通过建立人群队列和动物实验进行证实。针对细菌性ABS的治疗，靶向性的抗生素治疗是成功的关键，因此，探索基于整体肠道菌群产乙醇基础上、筛选抗生素靶向性治疗尤为重要。

目的

1 建立细菌性ABS人群队列并通过分析肠道微生物多态性探索病因；

2 通过菌株的分子生物学特征及动物实验阐明克雷伯菌属的作用机制；

3 探索基于整体肠道菌群产乙醇基础上的靶向抗生素治疗方案及治疗效果。

方法

一、自酿酒综合征人群队列建立及病因学研究

设立入组和排除标准，收集病人临床资料信息，建立ABS人群队列。

收集ABS患者及健康对照组的血液样本，采用全外显子测序检测乙醇代谢相关基因ADH（Alcohol dehydrogenase）和ALDH（Aldehyde dehydrogenase）是否存在突变位点。采用试剂盒检测ADH和ALDH酶活力水平，并与健康人群进行比较。

收集ABS患者不同时期的粪便样本，采用宏基因组学方法对其微生物多态性进行分析并与健康人群比较，筛选差异菌属。

收集ABS患者不同时期的粪便样本，采用代谢组学方法对粪便样本中代谢产物进行分析并与健康人群比较，筛选差异代谢产物。

连续性监测病人血液酒精浓度（Blood alcohol concentration，BAC），并与筛选出的差异菌属进行相关性分析，明确起关键作用的菌属。

收集病人不同位置的肠腔灌洗液样本并进行微生物多态性分析及产乙醇能力检测。

采用体外发酵实验观察粪便样本在9中碳源（葡萄糖、果糖、甘露糖、麦芽糖、半乳糖、黏蛋白、糊精、酪蛋白、淀粉）中的产乙醇能力，寻找ABS发作的食物诱因。

    二、克雷伯菌属的分子生物学特征及其在动物模型中的验证

对ABS患者粪便及肠腔灌洗液样本进行差异菌属的分离培养及鉴定，采用气相色谱仪检测不同菌株的产乙醇能力。

采用扫描电镜观察菌株的荚膜、荧光标记染色孵育后采用共聚焦显微镜观察菌株的生物膜形成能力，采用分光光度计检测菌株不同生长时间的OD值并进行生长曲线绘制。

采用体外培养观察三种克雷伯菌属在不同浓度碳水化合物（葡萄糖和果糖）、有氧及厌氧培养条件下产乙醇能力。

提取菌株的基因DNA，分析菌株的基因组学特征，包括基因型，毒力因子以及耐药基因等。

采用兔肠培养模型进行不同克雷伯菌属体内外培养后产生乙醇能力的分析。

采用酒精灌胃组（EtOH阳性对照），YPD灌胃组（Pair阴性对照）、三种克雷伯菌灌胃组、以及三种克雷伯菌属合并果糖饲喂组建立ABS模型，分析不同时间克雷伯菌的丰度变化。

检测不同组别小鼠BAC并进行比较分析。

三、ABS患者抗生素治疗策略及疗效观察

采用体外发酵实验对病人粪便样本进行培养，检测在不同种类、不同浓度抗生素作用下产乙醇能力以及短链脂肪酸生成。

收集患者治疗前后粪便样本，采用宏基因组方法以及数字PCR方法分析粪便样本菌群构成以及靶标菌群的丰度变化。

收集患者治疗前后粪便样本，采用代谢组学分析差异代谢产物。

四、统计学分析

所有数据采用SPSS 21.0软件进行统计学分析，数据结果表示为均值±标准差(SD)。采用Bray-Curtis距离相异矩阵指数检测ABS患者与健康人群间的细菌组成成分差异。采用Kruskal-Wallis分析不同分类级别菌群相对丰度差异性。采用Spearman相关系数分析不同种属的细菌丰度与BAC的相关性，以及BAC的变化并与不同属相关性。采用线性判别分析(Linear discriminant analysis，LDA) 分析ABS组和健康对照组间差异的细菌种类，LDA的临界值设定为2.5。采用T检验或方差分析分析比较组间差异，p值< 0.05被认为有统计学意义。

结果

一、ABS人群队列建立及病因学研究

根据入组标准于排除条件，共有7例ABS患者，最终病例组入组人数5人，根据性别、年龄、BMI匹配5例健康对照。ABS患者组中2例病情较重反复住院，发作时BAC为 40 ~ 90 mmol/L， 3例发作时BAC 为18 ~ 44 mmol/L，可通过严格饮食控制降低。3例患者曾服用1月及以上抗真菌药物治疗无效。

全外显子测序发现，7个ADH基因和4个ALDH基因均未发现与疾病相关的插入、突变和缺失。与健康对照相比，病例组ADH和ALDH酶活力无差异统计学意义。

宏基因组学研究结果显示，ABS患者存在严重的菌群失调，拟杆菌门丰度降低，变形菌门丰度显著升高。在变形菌门中，肠杆菌科菌属丰度最高，组间差异有显著统计学意义。

将差异均属菌属与病人BAC进行相关性分析，埃希氏菌属、沙门氏菌属、志贺氏菌属、肠杆菌属、克雷伯菌属在ABS病人中丰度显著升高且与BAC高度相关，并与BAC变化一致。

不同部位肠腔液样本乙醇浓度检测结果显示，相对于咽部、食管和胃，小肠及结肠的肠腔液产乙醇能力较高，克雷伯菌属丰度较高，肠腔液培养可以分离到克雷伯菌属。

77个代谢产物在ABS组和健康对照组间有显著差异，其中γ-氨基丁酸、多巴、5-羟基多巴胺、鹅脱氧胆酸、酪氨酸、苯丙氨酸、苯甲醇、色氨酸和腐胺在ABS患者种升高，2,3丁二醇在ABS患者中升高。

相对于其他碳水化合物（黏蛋白、糊精、酪蛋白和淀粉），粪便样本在单糖类物质（葡萄糖、果糖、甘露糖、麦芽糖、半乳糖）的作用下产乙醇能力较高，在发作期的产乙醇能力较恢复期及健康对照显著升高。

二、克雷伯菌属的分子生物学特征及其在动物模型中的验证

粪便样本及肠腔液样本中共分离到105株肠杆菌科中的差异菌属，克雷伯菌属（肺炎克雷伯菌、变栖克雷伯菌和类肺炎克雷伯菌）产乙醇能力最高32.31±4.08，其次为埃希氏菌属10.02±1.23、志贺氏菌属9.08±1.13、肠杆菌属7.53±3.17和沙门氏菌属4.73±1.56。

三种克雷伯菌属菌体外有荚膜，与标准株ATCC2146相比，生长速度快，有较强的生物膜形成能力。

三种克雷伯菌属的产乙醇能力随着果糖和葡萄糖浓度升高而增加，6%和8%的糖浓度下产乙醇能力最高，葡萄糖和果糖组间无显著差异，尽管有氧条件下产乙醇浓度高于厌氧条件下，但是两组之间无统计学差异。

基因组分析结果显示，高产乙醇克雷伯菌携带多种耐药基因与毒力基因，其基因组中23种乙醇代谢相关基因与课题组前期分离的HiAlc Kpn（W14）的基因相比同源性较高。

兔肠模型结果表明，三种克雷伯菌属在肠腔内产乙醇能力为~ 30 mmol/L、肠腔粘液中为~ 20 mmol/L，体外培养中为~ 40 mmol/L，与标准菌株ATCC 2146相比，三种克雷伯菌属在体内具有较强的乙醇产生能力。

与酒精灌胃组以及单纯菌株灌胃组相比，三种高产乙醇克雷伯菌属合并高浓度果糖灌胃可以在动物体内产生大量的内源性乙醇，诱导小鼠ABS发生。

三、ABS病人抗生素及噬菌体筛选、治疗策略及疗效观察

ABS-2患者粪便样本在体外培养条件下，亚胺培南和左氧氟沙星可以有效的抑制粪便样本的产乙醇能力以及短链脂肪酸（乙酸，丙酸，丁酸和戊酸）生成。

ABS-2患者口服左氧氟沙星治疗后，血液样本BAC含量显著降低，粪便样本中克雷伯菌属的拷贝数显著降低，抗生素合并益生菌联合使用可有效缓解症状。

治疗后肠道菌群中，拟杆菌门中的细菌丰度升高，变型菌门、肠杆菌科的丰度降低；代谢组学分析显示，乙醇代谢相关通路以及多种合成神经递质类氨基酸的水平降低。

结论

ABS病人存在菌群失调，变形菌门、肠杆菌科丰度显著升高。肠杆菌科中克雷伯菌属可产生大量的内源性乙醇，是ABS发作的重要病因。

ABS病人肠道代谢产物中，克雷伯菌属代谢产物2，3丁二醇和神经递质类物质显著升高。

单糖类碳水化合物的大量摄入是ABS发作的主要诱因。

克雷伯菌属具有较厚的荚膜、较快的生长速度，较强的生物膜形成能力，在有氧及厌氧条件下均可发酵糖类产生乙醇。

动物实验证实，高糖饮食下，克雷伯菌属在肠道过度繁殖产生大量内源性乙醇，是ABS发作的重要病因。

基于整体肠道菌群产乙醇基础上的靶向抗生素治疗可以有效缓解ABS的临床症状甚至治愈。

 

Background

Auto brewery Syndrome (ABS), also known as Gut fermentation syndrome (GFS), is a rare, underdiagnosed, and understudied condition. The etiology of this syndrome was excess endogenous ethanol (EnEth) produced by intestinal microbial fermentation. Fungi proliferation in intestine was regarded as the main cause of ABS in previous studies, so the treatment of ABS is mostly based on empirical antifungal therapy. The failure of anti-fungal treatment suggests that other bacteria may be involved in ABS. In our previous study, high alcohol-producing Klebsiella pneumoniae (HiAlc Kpn) was isolated from a severe ABS patient who had failed in antifungal therapy, this HiAlc Kpn was proved to be a potential pathogenic factor of non-alcoholic fatty liver disease (NAFLD). This confirmed that bacteria were also involved in the pathogenesis of ABS, and we proposed the concept of bacterial ABS. Based on the previous studies, we speculate that in addition to fungi, some bacteria, such as Klebsiella, play an important role in the pathogenesis of ABS. These overgrown bacteria in the gut of ABS patients can produce large amounts of endogenous ethanol, leading to the patient's state of intoxication. In order to verify this hypothesis, we established a clinical cohort and their gut microbiota were analyzed to seek the potential pathobiont, which was finally verified with animal experiments. For the treatment of bacterial ABS, targeted antibiotics use is the key point of successful treatment. Therefore, it is important to explore a microbiome-guided selection of antibiotics treatment strategy.

Aim

1. To explore the potential “pathobiont” of bacterial ABS in a clinical cohort by Metagenomics and Metabolomics analysis.

2. To analyze the molecular characteristics of genus Klebsiella and verify the mechanisms of this bacterial in ABS through animal experiments.

3. To explore the microbiome-guided selection of antibiotics treatment strategy and efficacy in ABS patients.

Methods

I. Clinical cohort and the “pathobiont” of ABS investigation

1. ABS cohort was enrolled according to inclusion and exclusion criteria. Clinical information was also collected.

2. Blood samples of ABS patients were collected and whole exon sequencing was used to detect the mutation of adh and aldh genes related to ethanol metabolism. The enzyme activity of ADH and ALDH were detected and compared with healthy control.

3. Fecal samples were collected from ABS patients at different stage. Metagenomic analysis was performed to analyze their gut microbiota and compared with healthy control.

4. The blood alcohol concentration (BAC) of patients were continuously monitored, and analyzed the correlation with the differential bacteria genera.

5. Gastrointestinal lavage fluid of patient ABS-2 was collected via upper and lower endoscopy during hospitalization and their alcohol producing ability were tested. Gut microbiota was analyzed with metagenomic sequencing.

6. Fecal samples from the onset and recovery stage of the five ABS patients were cultured in media contained carbohydrates or proteins (glucose, fructose, galactose, mannose, maltose, dextrin, casein, starch, or mucin) at various concentrations, while corresponding samples from the five healthy individuals were used as controls.

II The molecular characteristics of genus Klebsiella and its mechanisms in ABS in animal models

1. The differential genus bacterial from fecal samples and gastrointestinal lavage fluid samples were isolated and their alcohol producing ability were tested.

2. The scanning electron microscopy was used to observe the capsule of the strains, and confocal microscopy was used to observe the biofilm of the strains. The growth curves of the strains were evaluated with OD values detected by spectrophotometer at different culture period.

2. The alcohol producing ability of three Klebsiella species under different concentrations of carbohydrates (glucose and fructose) and different culture conditions was tested with an in vitro culture system in Yeast Extract Peptone Dextrose (YPD) medium.

3. The genomic DNA of the strain were extracted and the characteristics of the strain, including genotype, virulence and drug resistance genes were analyzed.

4. An in vivo assay of the isolated alcohol-producing strains were evaluated in a rabbit intestinal culture model. The alcohol concentrations were measured in vivo, in vitro, and in colonic mucus.

5. ABS mouse model was established with gavage with three bacterial with or without fructose in SPF male C57BL/6J mice.

6. The BAC of ABS mouse model were detected by GC-MS.

III. Evaluation of microbiome-guided selection of antibiotics treatment strategy

1. The effects of various antibiotics in inhibition of alcohol producing abilities in ABS patient, including cefixime (1.0 mg/L and 4.0 mg/L), imipenem (4.0 mg/L and 16.0 mg/L), metronidazole (1.0 mg/L and 4.0 mg/L), levofloxacin (1.25 mg/L and 2.5 mg/L), and vancomycin (4 mg/L and 32 mg/L) and short chain fatty acids were tested using an in vitro culture system.

2. Fecal samples were collected before, during and after treatment. The digital PCR method was used to analyze the copies of Klebsiella spp. The BAC of the patient were monitored throughout the treatment period.

3. The gut microbiota and metabolite were analyzed by metagenome sequencing and metabonomic.

III. Statistical analysis                                            

Data are expressed as means ± standard deviations (SD), and performed statistical analysis by using SPSS 21.0 software. Bray-Curtis dissimilarity index was performed to detect the significant difference in bacterial composition between ABS samples and healthy controls. The Kruskal-Wallis test was employed to analyses the statistical significance of the different taxonomic (phylum, family, genus) levels. The relative abundance of these features was subjected to statistical analyses. Spearman’s rank coefficients were used to test for correlation of bacterial abundance at family or genus level with BAC and correlation among different genera. Linear discriminant analysis (LDA) and effect size (LEfSe) analysis were used to determine the organisms most likely to explain differences between the ABS and healthy control group. Different features with an LDA score cut-off of 2.5 were identified. Student’s t-test or one-way analysis of ANOVA was used for group comparisons. P-values < 0.05 were considered statistically significant.

Results

I. Clinical cohort and the “pathobiont” of ABS investigation

1. According to the inclusion and exclusion criteria, a total of 7 suspected ABS patients were enrolled, finally 5 cases were included in this study. Five healthy controls were matched according to gender, age and BMI. Two patients with a severe onset of inebriation (BAC 40-90 mmol/L) experienced frequent hospitalizations. The BAC of the other three patients (BAC 18-44 mmol/L) could be reduced through strict diet control. Three patients had been used for one month or more anti-fungal treatment prior to sampling and failed in therapy.

2. Whole exon sequencing revealed that no disease-related insertions, mutations or deletions were found in 7 adh genes and 4 aldh genes. When compared with healthy controls, there was also no significant difference in enzyme activity.

3. Metagenomics indicate microbiota dysbiosis in the ABS cohort, which was characterized by increased abundance of phylum Proteobacteria and decreased Bacteroidetes, when compared with HCs. Among Proteobacteria, Enterobacteriaceae had the highest abundance and showed a significant difference between the ABS and HCs.

4. Some genera in Enterobacteriaceae were enriched in the ABS group, including Escherichia, Klebsiella, Shigella, Salmonella, and Enterobacter. These genera were highly correlated with BAC value and consistent with BAC changes.

5. High concentrations of alcohol (>10 mmol/L) were detected in intestinal lavage fluid, along with the high abundance of Klebsiella spp.

6. The abundances of 77 metabolites differed between ABS samples and HC samples. Multiple neurotransmitters and specific metabolites (gamma-aminobutyric acid, dopa, 5-hydroxydopamine, 2,3-butanediol, chenodeoxycholic acid, tyrosine, phenylalanine, benzyl alcohol, tryptophan, and putrescine) significantly increased during the onset of an ABS episode.

7. Compared with other carbohydrates (mucin, dextrin, casein and starch), fecal samples with monosaccharides (glucose, fructose, mannose, maltose, galactose) had higher alcohol producing ability, and the concentrations of alcohol during the seizure period was significantly higher than that during the recovery period and healthy controls.

II The molecular characteristics of genus Klebsiella and its mechanisms in ABS in animal models

1. A total of 105 strains of differential Enterobacteriaceae bacteria were isolated from fecal samples and intestinal fluid samples, and Klebsiella had the highest ethanol production capacity, followed by Escherella, Shigella, Enterobacteriaceae, and Salmonella.

2. These HiAlc Klebsiella strains constituted typical mucoid lactose fermenters, and had clear capsules and biofilms, along with higher growth speed compared with the standard strain ATCC2146.

3. The alcohol producing ability of the three Klebsiella species increased with the increasing of fructose and glucose concentrations. The concentration of alcohol was highest at 6% and 8% sugar concentrations. There was no significant difference between glucose and fructose, aerobic than anaerobic, although the alcohol concentration was higher under aerobic conditions than anaerobic conditions.

4. Genomic analysis showed that Klebsiella species harbored a variety of drug resistance and virulence genes. The 23 ethanol metabolism-related genes in these three species were highly homologous with the genes of HiAlc Kpn (W14) isolated in our previous study.

5. The rabbit intestinal culture model showed that alcohol concentrations were comparable among in vivo (~30 mmol/L), colonic mucus (~20 mmol/L), and in vitro (~40 mmol/L) samples. All three HiAlc Klebsiella strains had a strong alcohol-producing ability in vivo, when compared with standard strain ATCC 2146.

6. Compared with the pair-fed group, the BAC in HiAlc Klebsiella plus fructose-fed mice increased, which could induce the ABS onset.

III. Evaluation of microbiome-guided selection of antibiotics treatment strategy

1. The usage of levofloxacin and imipenem significantly reduced the alcohol-producing abilities of intestinal microbiota. Additionally, high-performance liquid chromatography revealed that short chain fatty acids (e.g., acetate acid, propanoic acid, butanoic acid, and pentatonic acid), which are the main byproducts of fermentation, were also significantly reduced in the presence of levofloxacin and imipenem.

2. After oral levofloxacin treatment in ABS-2 patients, both BAC and Klebsiella copies were significantly reduced, and antibiotics combined with probiotics could effectively relieve symptoms.

3. After treatment, the abundance of Proteobacteria and Enterobacteriaceae decreased in ABS-2 patients while bacterial in Bacteroidetes increased. Metabolomic analysis revealed that the pathways associated with ethanol metabolism and more amino acids related with neurotransmitter decreased.

Conclusion

1. There was significant intestinal dysbiosis in ABS patients, which was characterized by increased relative abundance of Proteobacteria and Enterobacteriaceae. Klebsiella species in Enterobacteriaceae can produce a large amount of endogenous ethanol, which is the major important pathobiont of ABS.

2. Multiple neurotransmitters and specific metabolite-2,3 butanediol were significantly increased, which indicate the potential of neurotransmitter-mediated changes in the patient's mood via the gut-brain axis, similar to the neural signaling that occurs during alcohol intoxication.

3. Monosaccharide was identified as a potential food-related inducing factor for alcohol production in ABS patients.

4. Three HiAlc Klebsiella species showed a fast growth rate, strong biofilm formation ability, and could ferment sugars to produce ethanol at aerobic and anaerobic conditions.

5. Animal experiments confirmed that the excess EnEth produced by the over proliferation of Klebsiella high-carbohydrate diet in intestine is the important cause of ABS.

6. The microbiome-guided selection of antibiotics treatment strategy could effectively relieve the clinical symptoms of ABS.