Abstract

Objective Esophageal adenocarcinoma (EAC) and its precursor, Barrett’s esophagus (BE), are associated with a pro-inflammatory, Gram-negative-dominated esophageal microbiome. We investigated whether Lactobacillus casei Shirota (LcS) consumption could shift the microbiome towards a more Gram-positive profile in BE patients.

Design In a single-arm intervention study, 23 BE patients used LcS twice daily for 4 weeks. Endoscopic biopsies from normal squamous epithelium (NSE) and metaplastic columnar epithelium (MCE), both before and after LcS-intervention were analyzed using 16S rRNA gene sequencing for microbiome composition, digital droplet PCR for Gram-positive to negative ratio, and fluorescence in situ hybridization to visualize Eubacteria and Firmicutes.

Results LcS intervention increased Gram-positive Firmicutes in MCE in situ (p<0.01) while the overall abundance of Eubacteria was unaffected. Analysis at DNA level showed an increased Gram-positive to Gram-negative ratio post-LcS. The abundance of Gram-negative Proteobacteria lowered from 74% pre-LcS to 52% post-LcS, while the abundance of Gram-positive Firmicutes, including the genus Lactobacillus, increased from 20% pre-LcS to 31% post-LcS. Overall microbiota diversity significantly increased post-LcS (p=9.9778e-07). LcS intervention also resulted in an increased abundance in the BE associated taxa Prevotella and Haemophilus in both NSE as MCE.

Conclusion LcS consumption significantly altered esophageal microbiome composition in BE patients, increasing Gram-positive taxa and overall diversity. These findings underscore the potential of probiotic-based therapeutic strategies aimed at EAC prevention. The concurrent rise in BE-associated taxa warrants careful consideration and further personalized studies to maximize benefits and minimize unintended consequences for optimal outcomes in BE patients.

What is already known on this subject?

Epidemiological studies point to esophageal microbiota as a risk factor for BE.

Gram-positive bacteria are closely associated with the normal distal esophagus, while Gram-negative bacteria (Veillonella, Prevotella, Haemophilus, Neisseria, and Fusobacterium) are increased in BE and EAC.

LcS has been shown to have potent anti-tumor & anti-metastatic effects on colon and gastric cancer cells and to suppress chemically-induced carcinogenesis in animals.

What are the new findings?

The microbiota of both normal squamous epithelium and post-LcS samples show a higher microbiota diversity, indicating greater richness and evenness in the distal esophagus after LcS intervention.

LcS intervention led, at the phylum level, to a significant increase in the relative abundance of health-associated, Gram-positive Firmicutes, increasing from 22% to 32% (***p ≤ 0.005). Gram-negative Proteobacteria showed a reduction from 72.0% to 54% (***p ≤ 0.005).

Although LcS intervention led to an increase in Gram-positive Firmicutes, including Lactobacillus, it also resulted, in both normal squamous epithelium and metaplastic columnar epithelium, in an increased abundance of bacterial taxa associated with BE and EAC, including the genera Prevotella and Haemophilus.

How might it impact on clinical practice in the foreseeable future?

Our results suggest that the esophageal bacterial composition in BE patients can be modified by consumption of dairy products containing probiotics. Manipulation of microbiota aimed at altering microbiota composition to include higher abundances of health-associated Gram-positive taxa may provide a novel way for disease prevention and therapeutic intervention.

Competing Interest Statement

PDS received unrestricted grants from Pentax, Fuji Film, Norgine, MicroTech, Magentiq Eye, AstraZeneca, Sanofi, and in the advisory board of Sanofi (The Netherlands). The study was partly funded by Yakult Europe B.V. for an amount of 40.000 euro. Yakult Europe B.V. did not have any input in the design, execution or interpretation of the outcome of the study.

Clinical Trial

NL-OMON43164

Funding Statement

The study was partly funded by Yakult Europe B.V. for an amount of 40.000 euro. Yakult Europe B.V. did not have any input in the design, execution or interpretation of the outcome of the study.

Author Declarations

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The study was in accordance with the Declaration of Helsinki, the code of conduct for Health Research, and was approved by ethics committee CMO Arnhem Nijmegen (NL59072.091.16) and registered in the Netherlands Trial Register (NL-OMON43164). All the participants provided informed written consent.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

Data Availability

All data produced in the present study are available upon reasonable request to the authors and microbiome data are available online at ENA under accession number PRJEB89339 and will become available when accepted for publication.

https://www.ebi.ac.uk/ena/browser/view/PRJEB89339

Abbreviations used in this paperLcSLactobacillus case ShirotapreLcSbefore administration of LcSpostLcSafter administration of LcSBEBarrett’s esophagus;EACesophageal adenocarcinomaNSEnormal squamous epitheliumMCEmetaplastic columnar epitheliumRArelative abundanceCIconfidence intervalHGDhigh-grade dysplasiaIQRinterquartile rangeSDstandard deviations