Introduction

The novel coronavirus (SARS-CoV-2) not only caused respiratory infections but led to gastrointestinal manifestations in a considerable amount of cases, with diarrhea being the most common symptom. Due to the intestinal involvement in the course of the disease, probiotics were suggested as potential adjuvant treatment. Therefore, we conducted a totally remote telemedical study in patients with COVID-19 disease in home quarantine in order to investigate COVID-19-related microbiome changes and test the effect of a multispecies synbiotic on respiratory symptoms, gastrointestinal quality of life and the microbiome composition.

Aims & Methods

Thirty patients were randomized to a 30-day intervention with Ecologic AAD (Winclove, Amsterdam, The Netherlands) or a placebo in a 2:1 ratio. Respiratory and gastrointestinal symptoms were documented in 2-10 day intervals via an online survey tool using the acute respiratory tract infection questionnaire (ARTIQ) and the gastrointestinal quality of life index (GIQLI), respectively. Five stool samples were taken during the study period. Patients were asked to conserve the sample in provided DNA stabilizer tubes and return them via mail at the end of the study for 16S sequencing.

Results

Of 30 recruited patients, 26 finished the study per protocol, 10 of these patients were allocated to the placebo group (4 male/6 female; 34.1±9.6 years), and 16 patients to the synbiotics group (6 male/10 female; 39.4±13.1 years). Patients reported respiratory symptoms and a decreased in their gastrointestinal quality of life which both improved significantly during the study irrespective of the synbiotic intervention. The microbiome composition showed significant alterations in comparison to non-infected controls during the pandemic (p=0.002). These alterations were mainly characterized by an increase in Bacteroidetes and a decrease in Christensenellaceae, Ruminococcaceae and Gammaproteobacteria taxa. Although the synbiotic intervention showed a significant modulation of beta-diversity in treated patients (p=0.001) and a stable introduction of the probiotic strain E. faecium W54, COVID-19-related taxa were not affected.

Conclusion

Patients with mild COVID-19 disease in home quarantine showed respiratory symptoms, reduced gastrointestinal quality of life and changes in the microbiome, but did not benefit clinically from a synbiotic intervention in this phase of the disease.

Disclosure

AH and VS received travel bursaries, speaker honoraria and/or research grants form Winclove

Introduction

The use of proton pump inhibitors (PPI) has been identified as one of the strongest influences on the gut microbiome in the general population. Long term use can lead to an increase of oral bacteria, such as Streptococcus salivarius and Veillonella parvula, in the gut microbiome which has been associated with gut inflammation and worse outcomes in patients with liver cirrhosis. Beside stringent prescription management, a targeted strategy to reduce PPI-induced alterations in the gut microbiome is crucial to reduce associated side-effects but remains an unmet need so far.

Aims & Methods

Here we aimed to design an evidence-based probiotic intervention to reduce PPI-induced dysbiosis of the gut microbiome. We tested 43 probiotic strains on their ability to inhibit the growth of S. salivarius and V. parvula as well as modulate the response of neutrophil granulocytes to the oral bacteria and selected 7 strains for a multispecies probiotic product. The performance of this evidence-based formulation was then tested with and without additional prebiotic in a bioreactor-based in-vitro model of the oralization of the gut microbiome (MyBioScope, CBmed) against two control substances (vehicle and starch) and prebiotics alone for optimization, before it was introduced in a clinical proof-of-concept, single arm study with a total of 61 included and 53 analyzed participants with long-term PPI use as a 3 months intervention.

Results

Based on their inhibitory capacity against S. salivarius, V. parvula and their immune modulatory effect on isolated human neutrophils, the probiotic strains Bifidobacterium animalis W53, Lactobacillus acidophilus W55, Lactobacillus casei W56, Lactobacillus plantarum W1, Lactobacillus plantarum W21, Lactobacillus rhamnosus W71 and Pediococcus acidilactici W143 were selected for their use in the probiotic formulation. When tested in the MyBioScope model for oralization, the probiotic formulation (with and without prebiotic) was able to significantly reduce the abundance of V. parvula and showed a tendency to reduce S. salivarius, which did not reach statistical significance. Prebiotics alone or control (vehicle or starch) did not show an effect on the abundance of the oral bacteria. In the clinical study, probiotic intervention significantly improved the gastrointestinal quality of life index of the participants and reproduced the results of the in-vitro tests by showing a significant reduction of V. parvula and a less effective reduction of S. salivarius in the gut microbiome. Adherence to the study product and the safety profile were excellent.

Conclusion

In conclusion, our screening procedure for an evidence-based probiotic formulation against PPI-induced dysbiosis resulted in a probiotic product containing 7 potent strains and a prebiotic matrix. This formulation was able to reduce the abundance of V. parvula as well as to a lesser degree S. salivarius in a novel bioreactor-based in-vitro model of the gut microbiome. The results of the in-vitro model could be reproduced in a proof-of-concept, single-arm clinical study where the probiotic was able to reduce the abundance of V. parvula in the gut microbiome of long-term PPI-users. This study shows that targeted probiotic interventions can be used to relieve PPI-induced side-effects, including alterations in the microbiome composition.

References

Alexandra Zhernakova et al. ,Population-based metagenomics analysis reveals markers for gut microbiome composition and diversity.Science352,565-569(2016).DOI:10.1126/science.aad3369
Horvath, A., Rainer, F., Bashir, M. et al. Biomarkers for oralization during long-term proton pump inhibitor therapy predict survival in cirrhosis. Sci Rep 9, 12000 (2019). https://doi.org/10.1038/s41598-019-48352-5

Disclosure

This study has been conducted by the Center for Biomarker Research in Medicine (CBmed), a COMET K1 centre, operated as a public-private-partnership funded by the Austrian Research Promotion Agency as well as industry partners including the probiotic companies Institut Allergosan (Graz, Austrtia) and Winclove Probiotics (Amsterdam, The Netherlands).

Introduction

A proportion of patients who contracted a severe COVID-19 infection suffer from post-acute disease.

Aims & Methods

In order to understand the role of the gut-liver axis in this setting, we compared a large panel of patient reported outcomes and biomarkers related to respiratory symptoms, gastrointestinal and health-related quality of life, routine blood work, gut permeability, innate and adapted immune response as well as the intestinal microbiome and metabolome of patients who previously suffered from severe COVID-19 disease (sCOV, n=21) to patients after mild COVID-19 disease (mCOV, n=10) in a crosssectional study.

Results

sCOV patients were studied on average 318 days after the infection, mCOV patients after 221 days (p=0.013) and even after this period of recuperation, sCOV patients reported lower general health (p=0.026), physical functioning (p<0.01) and more lower-airways symptoms (p=0.031) in validated questionnaires compared to mCOV patients. In general, sCOV patients were older (p=0.031), had more comorbidities (p=0.02), higher BMI (p<0.01), and higher rate of arterial hypertension (p=0.023) although being physically more active (p=0.035), had lower serum albumin (p<0.01), total protein (p<0.01), serum calcium (p=0.02), and estimated glomerular filtration rate (p=0.022), but higher fasting glucose levels (p<0.01) compared to mCOV patients. Increased serum diamine oxidase levels (p=0.017) indicated increased intestinal permeability, impaired neutrophil function with impaired directedness of chemotaxis (p=0.035), and lower ROS production in response to E. coli (p<0.01) indicated a dysfunction of the innate immune system. Higher of effector memory cells (p=0.009), terminally differentiated effector memory cells (0.035), CD3+ CD4+ T - cells (p=0.013) and CD45RA negative memory cells (p=0.028) and on the other hand and lower marginal zone B cells (p=0.021), central memory cells (p=0.024), immature T helper cells (p=0.031) and CD161 positive cytotoxic T cells (p=0.04) in sCOV compared to mCOV patients indicated an altered adaptive immune response. Gut microbiome sequencing revealed lower microbial richness (p<0.01) and reorganization of Ruminococcaceae and Lachnospiraceae families, potentially a long-term sequelae of the acute disease. Serum metabolome analysis showed higher glycoprotein to supramolecular phospholipid composite ratio (p<0.05), indicating inflammation, and lower levels of small-medium sized HDL particles (p<0.05) in sCOV patients. Stool metabolome analysis showed lower glycine and lactulose levels (p<0.05), and higher asparagine (p<0.001) and glycocholic acid (p<0.01) levels in sCOV patients. Multi-omics factor analysis showed higher VLDL particle number and predicted higher glucose and mannose metabolism capacity in sCOV patients.

Conclusion

This study highlights the differences in biomarkers of the gut-liver axis in patients who previously had severe COVID-19 disease. The gut-liver axis may be a potential source of biomarkers and a therapeutic target in COVID-19 disease.

Disclosure

AH and VS received speaker honoraria and research grants from Institut Allergosan

Introduction

Poor quality of sleep has detrimental effects on job performance, accident rates and health risks, with a potential link to the gut microbiome composition. First pilot trials have indicated beneficial effects of probiotic modulation of the microbiome on quality of sleep.

Aims & Methods

We conducted a randomized, double-blind, placebo-controlled trial testing the effects of a probiotic intervention on quality of sleep, quality of life and intestinal microbiome.
In total, 130 volunteers with impaired quality of sleep (PSQI>5) were included in the study. They were randomized in a 1:1 ratio to a 28-day intervention with either a multispecies probiotic (Omnibiotic-Stress Repair) or a placebo. Participants completed validated questionnaires to estimate quality of sleep (PSQI), quality of life (SF12, GIQLI) and perceived stress (PSQ20) before, during and after the intervention. Furthermore, stool samples for 16S rRNA gene sequencing were collected before and after the intervention. Ninety-four participants finished the study and could be included in the analysis.

Results

The 50 participants (88.6% female, 41.2±10.6 years old) analysed in the probiotic group and 44 participants (88.0% female, 40.1±10.7 years old) in the placebo group were well comparable at baseline, including the initial PSQI-score (10.1±2.7 vs. 10.5±2.6). After two weeks of intervention, both groups showed a comparable improvement in their PSQI-scores. After four weeks of intervention, the improvement of the PSQI score was significantly more pronounced in the probiotic group compared to the placebo group (6.8±2.9 vs. 7.7±3.1; p=0.036), mainly due to improved sleep efficiency and latency. The intervention did not influence quality of life or perceived stress. Ingested bacteria were partially recovered in the microbiome, causing a slight shift in beta diversity in the probiotic group, while alpha diversity and the overall composition of the microbiome remained unaffected.

Conclusion

In conclusion, the intervention with a multispecies probiotic was able to improve quality of sleep of participants with poor quality of sleep beyond an observable placebo-effect. Participants reported falling asleep more quickly and spending less time awake in bed. This well-powered RCT confirms that the probiotic modulation of the intestinal microbiome can be effective in alleviating sleep disturbances.

Disclosure

VS received research funding and speaker honoraria from Institut Allergosan (Graz, Austria) and Winclove Probiotics (Amsterdam, The Netherlands).

Introduction

Liver cirrhosis is characterized by microbial dysbiosis, intestinal inflammation and a weakened gut barrier and associated with an increased risk of complications. We previously observed a positive influence of L-ornithine-L-aspartate (LOLA) on gut microbiome composition in cirrhosis (1). The current study aimed to investigate the effect of LOLA on microbiome dysbiosis, quality of life, sarcopenia, intestinal permeability, inflammation and metabolite composition.

Aims & Methods

In this monocentric, open-label, phase 4 study, LOLA (18g/day) was administered orally for three months to liver cirrhosis patients with hepatic encephalopathy grades (0-2). We investigated changes in their gut microbiome composition (16s rRNA sequencing, alpha and beta diversity, differential abundance), quality of life (SF36), blood ammonia concentrations, measurements of sarcopenia and frailty (appendicular lean muscle mass, anthropometric measures, liver frailty index, SARC-F), biomarkers of gut permeability, inflammation and sarcopenia as well as metabolic alterations in stool, serum, and urine.

Results

65 patients were enrolled, and 52 patients completed the 3-month study (40.4% female, age 62 (58; 65)). While LOLA reduced the abundance of the genus Romboutsia, it did not significantly change overall microbiome diversity. LOLA led to a significant improvement in the vitality dimension of the SF36 questionnaire (45 (35; 60) to 50 (45; 60), p=0.019) and decreased plasma ammonia levels in patients with elevated baseline ammonia (69.5 (54; 225) to 46.0 (22; 66) µmol/L, p=0.004) Although muscle mass generally declined across the study, this was not observed in patients whose elevated ammonia levels improved. Sarcopenia and frailty scores and the muscle biomarkers myostatin, irisin, fibroblast growth factor 21, insulin-like growth factor 1 were unaffected by LOLA treatment. Diamine oxidase, a marker of gut barrier dysfunction, significantly decreased (from 12.75 U/mL (11.25; 17.45) to 11.15 U/mL (9.9; 15.65), p=0.016), while LPS binding protein, a marker of innate immune response to bacterial translocation, significantly increased (from 17.05 µg/mg (14.7; 19.8) to 17.39 µg/mg (16.2; 21.9), p=0.006). Calprotectin, zonulin and sCD14 did not show significant changes. Metabolomic analysis revealed an increase in serum alanine levels (pFDR=0.014) with additional subtle shifts in lipoprotein and amino acid profiles.

Conclusion

In summary, LOLA was associated with an improvement in vitality, an important patient-reported outcome accompanied by alterations in gut microbial composition and prevented muscle loss in patients with hyperammonaemia. Furthermore, LOLA was associated with improvements in gut permeability and endotoxin handling. The slight increase in alanine observed in the metabolomics analysis may be a consequence of LOLA metabolism. LOLA may be a valuable supportive treatment for enhancing quality of life in cirrhosis by targeting the gut liver axis and may be particularly beneficial in preventing muscle loss in patients with hyperammonaemia.

References

1. Horvath A, Traub J, Aliwa B, Bourgeois B, Madl T, Stadlbauer V. Oral Intake of L-Ornithine-L-Aspartate Is Associated with Distinct Microbiome and Metabolome Changes in Cirrhosis. Nutrients. 2022 Feb 10;14(4):748.

Introduction

Symptomatic uncomplicated diverticular disease (SUDD) is characterized by recurrent gastrointestinal (GI) symptoms in patients with colonic diverticula. Different treatments have been proposed to improve GI symptoms, but with low-level of evidence.

Aims & Methods

We aimed to evaluate the efficacy of a synbiotic formulation combined with a diet rich in fibre (DRF) in improving GI symptoms in SUDD, compared to a DRF and placebo. We conducted a single-centre, no-profit, randomised, double-blind, placebo-controlled trial (Local Ethics Committee n.182 SA_2020). Patients diagnosed with SUDD (colonic diverticula and recurrent abdominal pain with or without altered bowel habits or bloating, in the absence of signs of systemic inflammation) were included. After a 14-day run-in period, all participants completed structured questionnaires [long- and short-lasting abdominal pain, abdominal bloating, Rome IV for IBS, food frequency questionnaire and quality of life (SF-12)], received a dietary counselling, and were randomised to receive either (I) Enterolactis duo (containing the probiotic strain Lacticaseibacillus Paracasei DG and the prebiotic fibre inulin) 1 sachet bid plus advice on DRF (arm A), or (II) Placebo, 1 sachet bid plus advice on DRF (arm B) for 12 weeks. During treatment, patients completed a weekly diary of GI symptoms and bowel habits. At the end of treatment, patients completed all questionnaires again and were followed up after 4 weeks.

Results

After screening 81 patients, 34 (55.9% female; mean age 64±8.6yrs) were included and randomised to arm A (n=17) and arm B (n=17). At baseline, there were no significant differences in terms of clinical characteristics between the two arms. Thirty patients (arm A=14; arm B=16) completed the study. Compliance with the DRF was poor in both arms (0% vs 14.3%). Patients experienced significant improvements in GI symptoms (bloating, short- and long-lasting abdominal pain) and bowel habits compared to baseline in both arms, but no differences were observed between the two arms. Neither arm A nor arm B reported significant changes in the quality of life scores. Mild self-limited adverse events were reported with no differences between arms. Follow-up at 4 weeks showed no clinical changes in either arm, with no differences between arms.

Conclusion

This synbiotic supplementation is as effective as placebo in relieving symptoms in SUDD patients, suggesting the relevant role of the placebo effect also in diverticular disease.