Three Klebsiella species as potential pathobionts generating endogenous ethanol in a clinical cohort of patients with auto-brewery syndrome: a case control study

Abstract

Background: Patients with auto-brewery syndrome (ABS) become inebriated after the ingestion of an alcohol-free, high-carbohydrate diet. Our previous work has shown that high-alcohol-producing (HiAlc) Klebsiella pneumoniae can generate excessive endogenous ethanol and cause non-alcoholic fatty liver disease (NAFLD). Therefore, it is reasonable to speculate that such bacteria might play an important role in the pathogenesis of ABS.

Methods: The characteristics and metabolites of the intestinal flora from a clinical cohort of patients with ABS were analysed during different stages of disease and compared to a group of healthy controls. An in vitro culture system of relevant samples was used for screening drug sensitivity and ABS-inducing factors. Rabbit intestinal and murine models were established to verify if the isolated strains could induce ABS in vivo.

Findings: We observed intestinal dysbiosis with decreased abundance of Firmicutes and increased of Proteobacteria in patients with ABS compared with healthy controls. The abundance of the genus Klebsiella in Enterobacteriaceae was strongly associated with fluctuations of patient's blood alcohol concentration. We isolated three species of HiAlc Klebsiella from ABS patients, which were able to induce ABS in mice. Monosaccharide content was identified as a potential food-related inducing factor for alcohol production. Treatments with antibiotics, a complex probiotic preparation and a low-carbohydrate diet not only alleviated ABS, but also erased ABS relapse during the follow-up observation of one of the patients.

Interpretation: Excessive endogenous alcohol produced by HiAlc Klebsiella species was an underlying cause of bacterial ABS. Combined prescription of appropriate antibiotics, complex probiotic preparation and a controlled diet could be sufficient for treatment of bacteria-caused ABS.

Funding: The funders are listed in the acknowledgement.

Keywords: Auto-brewery syndrome; Diagnosis; Gut microbiota; Klebsiella; Treatment.

Fig. 1

Gut microbiome characterization and bacterial diversity of ABS patients. (a) PCoA based on the Bray–Curtis dissimilarity index shows the between-subjects (β) diversity across groups, in which the blue and red dot represent HC and ABS subjects, respectively. (ANOSIM, p = 0.007) (b) The top fifteen abundant families of intestinal microbiota community between HC and ABS subjects shows the intestinal dysbiosis based on Kruskal–Wallis test. (c) The Spearman's correlations analysis showing that correlation of average BAC level of each patient and abundance of Enterobacteriaceae. (d) Heatmap shows the abundance of 30 difference taxa in genus enriched in HC and ABS group (Kruskal–Wallis test). (e) A cladogram to identify the specific bacteria associated with ABS patients (Linear discriminant analysis with cutoff value 2.5). Red nodes indicate microbial taxa that play an important role in ABS group, green nodes indicate microbial taxa that play an important role in HC group, and yellow nodes indicate no difference taxa.

Fig. 2

Correlations of intestinal microbiota and blood alcohol concentration of patient. (a) Timeline of observation and treatment for the patient ABS 2. The stool and blood samples of patient ABS-2 were collected during different stages and marked as from S1 to S26 (1 mmol/L = 4.6 mg/dL). (b) The correlation between different genus abundances and blood alcohol concentration (BAC) was measured by Spearman's rank correlation coefficient. (c) The variation abundance of different genus and BAC fluctuation during the entire monitoring period from November 16, 2020 to December 19, 2020. (d) Microbial network showed strong correlation among different genus in Proteobacteria. (e) The alcohol-producing ability of the isolated bacterial in different genus of Enterobacteriaceae, including Klebsiella (n = 35), Escherichia (n = 39), Shigella (n = 10), Salmonella (n = 3) and Enterobacter (n = 15). Each genus is presented with means ± standard deviations (left) and scatter plot (right). Blue dot for Escherichia, pink triangle for Klebsiella, yellow dot for Shigella, red triangle for Salmonella, and green square for Enterobacter.

Fig. 3

Alcohol producing capacity of gastrointestinal lavage fluid from different parts of ABS patients under esophagogastroduodenoscopy (EGD) and colonoscopy. And characteristics of HiAlc Klebsiella isolated from ABS patients. (a) Gastrointestinal lavage fluid from different parts of ABS-2 were collected under EGD and colonoscopy. Concentration of alcohol (AC) in these parts of patient ABS-2 were tested by headspace gas chromatography method. Bacterial in these parts were also isolated and the alcohol producing capacity were tested. (b) Concentration of alcohol from different parts of patient ABS-2. (c) Intestinal microbiota community from different parts of patient ABS-2. (d) Images of typical colonies and capsules (marked with arrows), growth curves, images of biofilm formation of the three HiAlc Klebsiella isolates (K. pneumoniae, K. quasi pneumoniae, K. variicola named as K-1, K-2, and K-3). (e) Ethanol producing capacity (alcohol concentration Y-axis) in different concentrations in glucose and fructose under aerobic (different shades of red) and anaerobic (different shades of blue) conditions.

Fig. 4

Effect of different antibiotics on intestinal microbiota. (a) Capabilities of alcohol-producing (Panel 1) as well as short chain fatty acid-producing intestinal microbiota under various antibiotic stresses in an in vitro microbiota fermentation system. Acetate (Panel 2), propanoic (Panel 3), butanoic (Panel 4), and pentanoic acid (Panel 5) are shown. Two concentrations (low and high) of different antibiotics were used which were cefixime with 1.0 mg/L and 4.0 mg/L (line 3), imipenem with 4.0 mg/L and 16.0 mg/L (lines 4), metronidazole with 1.0 mg/L and 4.0 mg/L (line 5), levofloxacin with 1.25 mg/L and 2.5 mg/L (lines 6), and vancomycin with 4.0 mg/L and 32.0 mg/L (line 7). Fermentation of faecal samples from this patient and a normal individual in YPD medium without antibiotic were used as the controls (Lines 1 and 2). Samples were collected at 8, 16, 24 and 48 h, respectively. Data are expressed as the mean ± SD of three biology replicates. (b) The detail treatment strategy of ABS patient C. Amino acid and vitamin C injection were first used for 3 days, followed by oral antibiotic 7 days and complex probiotic preparation for 15 days. (c) The copy numbers of Klebsiella in faecal samples were tested by digital PCR. The copy numbers of Klebsiella in faecal samples were significantly decreased accompanying the decreasing of blood alcohol concentration after the antibiotic use. (d) Dynamic changes of intestinal microbiota community before and after treatment. The abundance of genus of Klebsiella matched well with the treatment and symptoms.

Fig. 5

The hypothesized mechanism of bacterial ABS. The intestinal dysbacteriosis resulted proliferation of HiAlc Klebsiella after eating alcohol-free high-carbohydrate food and produced a large number of endogenous ethanol, which increases the permeability of intestinal mucosa. Then the metabolites of HiAlc Klebsiella and other microbiotas, especially ethanol, enter intestinal blood through lamina propria. The endogenous ethanol diffuses rapidly and uniformly throughout the body water. Some of them were excretion in expires air and urine. The majority of them entered in liver through portal vein and metabolized by Adh and Aldh. Once the amount of endogenous ethanol exceeds the metabolic capacity of the liver, excess ethanol accumulates in the body, resulting in elevated BAC and auto-brewery syndrome. Some metabolites, such as neurotransmitters, could also act on the brain via the gut–brain axis, causing neurological symptoms.