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Iwasaki S, Také A, Uojima H, Horio K, Sakaguchi Y, Gotoh K, Satoh T, Hidaka H, Tanaka Y, Hayashi S, Kusano C. Quantification of Streptococcus salivarius using the digital polymerase chain reaction as a liver fibrosis marker. World J Hepatol 2025; 17:102027. [PMID: 40308822 PMCID: PMC12038421 DOI: 10.4254/wjh.v17.i4.102027] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/06/2024] [Revised: 02/21/2025] [Accepted: 04/09/2025] [Indexed: 04/25/2025] Open
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.
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Affiliation(s)
- Shuichiro Iwasaki
- Department of Gastroenterology, Kitasato University School of Medicine, Sagamihara 252-0375, Kanagawa, Japan
| | - Akira Také
- Department of Microbiology, Kitasato University School of Medicine, Sagamihara 252-0373, Kanagawa, Japan
| | - Haruki Uojima
- Department of Gastroenterology, Kitasato University School of Medicine, Sagamihara 252-0375, Kanagawa, Japan
- Department of Genome Medical Sciences Project, Research Institute, National Center for Global Health and Medicine, Ichikawa 272-8516, Chiba, Japan.
| | - Kazue Horio
- Department of Gastroenterology, Kitasato University School of Medicine, Sagamihara 252-0375, Kanagawa, Japan
| | - Yoshihiko Sakaguchi
- Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8055, Japan
| | - Kazuyoshi Gotoh
- Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan
| | - Takashi Satoh
- Division of Hematology, Kitasato University School of Allied Health Sciences, Sagamihara 252-0373, Kanagawa, Japan
| | - Hisashi Hidaka
- Department of Gastroenterology, Kitasato University School of Medicine, Sagamihara 252-0375, Kanagawa, Japan
| | - Yasuhito Tanaka
- Department of Gastroenterology and Hepatology, Kumamoto University Hospital, Kumamoto 860-8556, Japan
| | - Shunji Hayashi
- Department of Microbiology, Kitasato University School of Medicine, Sagamihara 252-0373, Kanagawa, Japan
| | - Chika Kusano
- Department of Gastroenterology, Kitasato University School of Medicine, Sagamihara 252-0375, Kanagawa, Japan
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Bean EL, McLellan LK, Grossman AD. Activation of the integrative and conjugative element Tn916 causes growth arrest and death of host bacteria. PLoS Genet 2022; 18:e1010467. [PMID: 36279314 PMCID: PMC9632896 DOI: 10.1371/journal.pgen.1010467] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2022] [Revised: 11/03/2022] [Accepted: 10/10/2022] [Indexed: 11/06/2022] Open
Abstract
Integrative and conjugative elements (ICEs) serve as major drivers of bacterial evolution. These elements often confer some benefit to host cells, including antibiotic resistance, metabolic capabilities, or pathogenic determinants. ICEs can also have negative effects on host cells. Here, we investigated the effects of the ICE (conjugative transposon) Tn916 on host cells. Because Tn916 is active in a relatively small subpopulation of host cells, we developed a fluorescent reporter system for monitoring activation of Tn916 in single cells. Using this reporter, we found that cell division was arrested in cells of Bacillus subtilis and Enterococcus faecalis (a natural host for Tn916) that contained an activated (excised) Tn916. Furthermore, most of the cells with the activated Tn916 subsequently died. We also observed these phenotypes on the population level in B. subtilis utilizing a modified version of Tn916 that can be activated in the majority of cells. We identified two genes (orf17 and orf16) in Tn916 that were sufficient to cause growth defects in B. subtilis and identified a single gene, yqaR, that is in a defective phage (skin) in the B. subtilis chromosome that was required for this phenotype. These three genes were only partially responsible for the growth defect caused by Tn916, indicating that Tn916 possesses multiple mechanisms to affect growth and viability of host cells. These results highlight the complex relationships that conjugative elements have with their host cells and the interplay between mobile genetic elements.
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Affiliation(s)
- Emily L. Bean
- Department of Biology Massachusetts, Institute of Technology Cambridge, Massachusetts, United States of America
| | - Lisa K. McLellan
- Department of Biology Massachusetts, Institute of Technology Cambridge, Massachusetts, United States of America
| | - Alan D. Grossman
- Department of Biology Massachusetts, Institute of Technology Cambridge, Massachusetts, United States of America
- * E-mail:
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Lunde TM, Hjerde E, Al-Haroni M. Prevalence, diversity and transferability of the Tn 916-Tn 1545 family ICE in oral streptococci. J Oral Microbiol 2021; 13:1896874. [PMID: 33796228 PMCID: PMC7971310 DOI: 10.1080/20002297.2021.1896874] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
Background: The Tn916-Tn1545 family of Integrative Conjugative Elements (ICE) are mobile genetic elements (MGEs) that play a role in the spread of antibiotic resistance genes. The Tn916 harbors the tetracycline resistance gene tet(M) and it has been reported in various bacterial species. The increase in the levels of tetracycline resistance among oral streptococci is of great concern primarily due to the abundance of these species in the oral cavity and their ability to act as reservoirs for antibiotic resistance genes.Methods: In the current study, we screened 100 Norwegian clinical oral streptococcal isolates for the presence and diversity of the Tn916-Tn1545 family. In addition, we investigated the transferability the elements, and the associated transfer frequencies.Results: We observed that 21 isolates harboured the Tn916-Tn1545 family and that two of these elements were the novel Tn6815 and Tn6816. The most prevalent member of the Tn916 -Tn1545 family observed in the Norwegian clinical oral streptococcal isolates was the wild type Tn916.Conclusion: The detection of other members of this family of ICE and varying transfer frequencies suggests high versatility of the Tn916 element in oral streptococci in Norway.
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Affiliation(s)
- Tracy Munthali Lunde
- Department of Clinical Dentistry, Faculty of Health Sciences, UiT the Arctic University of Norway, Tromsø
| | - Erik Hjerde
- bCenter for Bioinformatics, Faculty of Science and Technology, UiT the Arctic University of Norway, Tromsø, Norway
| | - Mohammed Al-Haroni
- Department of Clinical Dentistry, Faculty of Health Sciences, UiT the Arctic University of Norway, Tromsø
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Detection of a Novel, and Likely Ancestral, Tn 916-Like Element from a Human Saliva Metagenomic Library. Genes (Basel) 2020; 11:genes11050548. [PMID: 32422869 PMCID: PMC7288454 DOI: 10.3390/genes11050548] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2020] [Revised: 05/01/2020] [Accepted: 05/12/2020] [Indexed: 01/22/2023] Open
Abstract
Tn916 is a conjugative transposon (CTn) and the first reported and most well characterised of the Tn916/Tn1545 family of CTns. Tn916-like elements have a characteristic modular structure and different members of this family have been identified based on similarities and variations in these modules. In addition to carrying genes encoding proteins required for their conjugation, Tn916-like elements also carry accessory, antimicrobial resistance genes; most commonly the tetracycline resistance gene, tet(M). Our study aimed to identify and characterise tetracycline resistance genes from the human saliva metagenome using a functional metagenomic approach. We identified a tetracycline-resistant clone, TT31, the sequencing of which revealed it to encode both tet(M) and tet(L). Comparison of the TT31 sequence with the accessory, regulation, and recombination modules of other Tn916-like elements indicated that a partial Tn916-like element encoding a truncated orf9 was cloned in TT31. Analysis indicated that a previous insertion within the truncated orf9 created the full length orf9 found in most Tn916-like transposons; demonstrating that orf9 is, in fact, the result of a gene fusion event. Thus, we hypothesise that the Tn916-like element cloned in TT31 likely represents an ancestral Tn916.
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Wei T, Yao H, Sun P, Cai W, Li X, Fan L, Wei Q, Lai C, Guo J. Mitigation of antibiotic resistance in a pilot-scale system treating wastewater from high-speed railway trains. CHEMOSPHERE 2020; 245:125484. [PMID: 31864053 DOI: 10.1016/j.chemosphere.2019.125484] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/27/2019] [Revised: 11/15/2019] [Accepted: 11/25/2019] [Indexed: 06/10/2023]
Abstract
Wastewater from high-speed railway trains represents a mobile reservoir of microorganisms with antibiotic resistance. It harbors abundant and diverse antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). This study investigated the removal of ARB and ARGs in a pilot-scale reactor, which consisted of an anaerobic/anoxic/oxic process, anaerobic/anoxic/aerobic process, and ozone-based disinfection to treat 1 m3/day wastewater from an electric multiple unit high-speed train. Further, the high prevalence of two mobile genetic elements (intI1 and Tn916/615) and five ARGs (tetA, tetG, qnrA, qnrS, blaNDM-1, and ermF) was investigated using quantitative PCR. Significant positive correlations between ARGs (tetA, blaNDM-1, and qnrA) and intI1 were identified (R2 of 0.94, 0.85, and 0.70, respectively, P < 0.01). Biological treatment could significantly reduce Tn916/1545 (2.57 logs reduction) and Enterococci (2.56 logs reduction of colony forming unit (CFU)/mL), but the qnrS abundance increased (1.19 logs increase). Ozonation disinfection could further significantly decrease ARGs and Enterococci in wastewater, with a reduction of 1.67-2.49 logs and 3.16 logs CFU/mL, respectively. Moreover, food-related bacteria families which may contain opportunistic or parasitic pathogens (e.g., Moraxellaceae, Carnobacteriaceae, and Ruminococcaceae) were detected frequently. Enterococci filtered in this study shows multi-antibiotic resistance. Our study highlights the significance to mitigate antibiotic resistance from wastewater generated from high-speed railway trains, as a mobile source.
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Affiliation(s)
- Ting Wei
- Department of Municipal and Environmental Engineering, Beijing Jiaotong University, Beijing, 100044, China
| | - Hong Yao
- Department of Municipal and Environmental Engineering, Beijing Jiaotong University, Beijing, 100044, China.
| | - Peizhe Sun
- Department of Municipal and Environmental Engineering, Beijing Jiaotong University, Beijing, 100044, China; School of Environmental Science and Engineering, Tianjin University, Tianjin, 300072, China
| | - Weiwei Cai
- Department of Municipal and Environmental Engineering, Beijing Jiaotong University, Beijing, 100044, China
| | - Xinyang Li
- Department of Municipal and Environmental Engineering, Beijing Jiaotong University, Beijing, 100044, China
| | - Liru Fan
- Department of Municipal and Environmental Engineering, Beijing Jiaotong University, Beijing, 100044, China
| | - Qingchao Wei
- Department of Municipal and Environmental Engineering, Beijing Jiaotong University, Beijing, 100044, China
| | - Cai Lai
- Department of Municipal and Environmental Engineering, Beijing Jiaotong University, Beijing, 100044, China
| | - Jianhua Guo
- Advanced Water Management Centre (AWMC), University of Queensland, St Lucia, Brisbane, QLD, 4072, Australia
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