Quantification of Streptococcus salivarius using the digital polymerase chain reaction as a liver fibrosis marker

Shuichiro Iwasaki; Akira Také; Haruki Uojima; Kazue Horio; Yoshihiko Sakaguchi; Kazuyoshi Gotoh; Takashi Satoh; Hisashi Hidaka; Yasuhito Tanaka; Shunji Hayashi; Chika Kusano

doi:10.4254/wjh.v17.i4.102027

Quantification of Streptococcus salivarius using the digital polymerase chain reaction as a liver fibrosis marker

World J Hepatol. 2025 Apr 27;17(4):102027. doi: 10.4254/wjh.v17.i4.102027.

Authors

Affiliations

¹ Department of Gastroenterology, Kitasato University School of Medicine, Sagamihara 252-0375, Kanagawa, Japan.
² Department of Microbiology, Kitasato University School of Medicine, Sagamihara 252-0373, Kanagawa, Japan.
³ Department of Genome Medical Sciences Project, Research Institute, National Center for Global Health and Medicine, Ichikawa 272-8516, Chiba, Japan. kiruha@kitasato-u.ac.jp.
⁴ Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8055, Japan.
⁵ Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan.
⁶ Division of Hematology, Kitasato University School of Allied Health Sciences, Sagamihara 252-0373, Kanagawa, Japan.
⁷ Department of Gastroenterology and Hepatology, Kumamoto University Hospital, Kumamoto 860-8556, Japan.

Abstract

Background: The Streptococcus salivarius (S. salivarius) group, which produces the enzyme urease has been identified as a potential contributor to ammonia production in the gut. Researchers have reported that patients with minimal HE had an increased abundance of the S. salivarius group, which is a specific change in the gut microbiota that distinguishes them from healthy individuals. The correlation between the aggregation of specific bacterial species and fibrosis progression in chronic liver disease (CLD) is yet to be fully elucidated.

Aim: To quantify S. salivarius using digital PCR (dPCR) as a liver fibrosis marker of CLD.

Methods: This study retrospectively analysed 52 patients with CLD. To quantify S. salivarius in patients with CLD using dPCR, we evaluated the specificity and sensitivity of S. salivarius bacterial load using dPCR for a type strain. Next, we evaluated the clinical usefulness of dPCR for S. salivarius load quantification for detecting liver fibrosis in patients with CLD. The liver fibrosis stage was categorized into mild and advanced fibrosis based on pathological findings.

Results: The dPCR assay revealed that S. salivarius was highly positive for the tnpA gene. The lower limit of quantification for dPCR using the tnpA gene with a 1 μL template comprising 1.28 × 10² CFU/mL was 4.3 copies. After considering the detection range in dPCR, we adjusted the extracted DNA concentration to 5.0 × 10^-4 ng/μL from 200 mg stool samples. The median bacterial loads of S. salivarius in stool sample from patients with mild and advanced fibrosis were 1.9 and 7.4 copies/μL, respectively. The quantification of S. salivarius load was observed more frequently in patients with advanced fibrosis than in those with mild fibrosis (P = 0.032).

Conclusion: Quantifying of S. salivarius load using digital PCR is a useful biomarker for liver fibrosis in patients with CLD.

Keywords: Chronic liver disease; Digital PCR; Liver cirrhosis; Liver fibrosis; Quantification; Streptococcus salivarius.