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. 2025 Apr 30:16:1551824.
doi: 10.3389/fmicb.2025.1551824. eCollection 2025.

Transcriptome analysis of calcium hydroxide tolerance in Enterococcus faecalis

Zhibo Xu  1   2 , Haonan Ma  3 , Xinmiao Jiang  1 , Quzhen Baima  1 , Yuqi Zhen  1 , Shipeng Yang  1 , Xiuping Meng  1
Affiliations

Transcriptome analysis of calcium hydroxide tolerance in Enterococcus faecalis

Zhibo Xu et al. Front Microbiol. .

Abstract

Calcium hydroxide (Ca(OH)2) is commonly used as a root canal disinfectant, but its effectiveness against Enterococcus faecalis is limited, likely due to the bacterium's ability to tolerate it. This study aimed to investigate the underlying mechanism of E. faecalis tolerance to repeated exposure to Ca(OH)2. Initially, E. faecalis was exposed to Ca(OH)2 for 10 successive passages. The survival rate of the bacteria increased progressively, suggesting the development of tolerance to Ca(OH)2. Crystal violet staining revealed that calcium hydroxide-tolerant strains formed more robust biofilms compared to standard strains. To delve into the mechanisms of Ca(OH)2 tolerance in E. faecalis, RNA sequencing was employed for a preliminary investigation. Transcriptome sequencing identified 683 differentially expressed genes (DEGs) in the calcium hydroxide-tolerant strain, with 368 genes upregulated and 315 downregulated compared to the standard strain. Bioinformatics analysis revealed that the upregulated genes were associated with carbohydrate transport and metabolism, starch and sucrose metabolism, quorum sensing, aminoacyl-tRNA biosynthesis, and two-component systems signaling pathways. In contrast, the downregulated genes were primarily linked to lipoic acid metabolism, degradation of valine, leucine, and isoleucine, and the citric acid cycle (tricarboxylic acid cycle) signaling pathways. The findings suggest that prolonged exposure to Ca(OH)2 induces tolerance in E. faecalis. RNA sequencing further revealed that this tolerance involves multiple interconnected signaling pathways, providing a critical foundation for future research into therapeutic strategies for E. faecalis infections.

Keywords: Enterococcus faecalis; calcium hydroxide; persistent apical periodontitis; tolerance; transcriptomics.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
The process of tolerance development in E. faecalis exposed to Ca(OH)2.
Figure 2
Figure 2
Study on E. faecalis tolerance to Ca(OH)2: (A) E. faecalis ATCC 29212 was exposed to Ca(OH)2 and regular BHI medium over 10 repeated passages, with survival rates recorded for each passage after 24 h. The red line represents survival in Ca(OH)2 BHI medium, and the blue line shows survival in regular BHI medium. (B) The experiment also measured the pH of the bacterial supernatant after each passage in both media. Data are presented as mean ± SD.
Figure 3
Figure 3
The crystal violet staining method was used to compare biofilm formation between E. faecalis and EF-CHs. A microplate reader measured the optical density of crystal violet dissolved in 95% ethanol at 550 nm. Higher values indicate greater biofilm formation. Results represent the average of three replicates and are shown as mean ± SD. *p < 0.05.
Figure 4
Figure 4
DEGs between the EF-CHs and E. faecalis groups were analyzed. (A) A volcano plot shows the distribution of DEGs, with red indicating up-regulated and green indicating down-regulated genes. (B) A histogram displays the number of up- and down-regulated genes. (C) A heat map illustrates DEG clustering, where each row is a gene and each column is a sample. Colors indicate gene expression levels, with red showing higher expression and green showing lower. Darker shades represent higher expression levels.
Figure 5
Figure 5
Gene Ontology (GO) functional categories of DEGs. The y-axis lists the functional categories, while the x-axis indicates the number of genes from the differential gene set in each category.
Figure 6
Figure 6
COG functional enrichment heatmap of DEGs. (A) Enrichment for up-regulated genes. (B) Enrichment for down-regulated genes. The y-axis shows functional annotations, and the x-axis shows the ratio of differentially expressed genes in each functional category to the total annotated genes in that category. The size of each point indicates the number of differentially expressed genes in a functional entry, while color intensity reflects the significance of enrichment.
Figure 7
Figure 7
KEGG pathway enrichment map of DEGs. (A) Enrichment for up-regulated genes. (B) Enrichment for down-regulated genes. The y-axis displays functional annotations, while the x-axis indicates the ratio of DEGs in each pathway entry to the total annotated genes in that entry. Dot size reflects the number of DEGs in each pathway, and color intensity indicates the significance of the enrichment.
Figure 8
Figure 8
Molecular mechanisms of E. faecalis tolerance to Ca(OH)2.
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References

    1. Abe Y., Honda M. (2023). A novel control method of Enterococcus faecalis by co-treatment with protamine and calcium hydroxide. Pharmaceutics 15:1629. doi: 10.3390/pharmaceutics15061629, PMID: - DOI - PMC - PubMed
    1. Anija R., Kalita C., Bhuyan A. C., Hussain M. D. I., Saikia A., Das L. (2021). Comparative evaluation of the concentration-dependent effect of proton-pump inhibitor in association with calcium hydroxide and chlorhexidine on Enterococcus faecalis: an in vitro study. J. Oral Maxillofac. Pathol. 25:198. doi: 10.4103/jomfp.JOMFP_303_20, PMID: - DOI - PMC - PubMed
    1. Bassler B. L., Losick R. (2006). Bacterially speaking. Cell 125, 237–246. doi: 10.1016/j.cell.2006.04.001, PMID: - DOI - PubMed
    1. Benjamini Y., Hochberg Y. (1995). Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. B 57, 289–300. doi: 10.1111/j.2517-6161.1995.tb02031.x - DOI
    1. Carbonetto P., Luo K., Sarkar A., Hung A., Tayeb K., Pott S., et al. . (2023). GoM DE: interpreting structure in sequence count data with differential expression analysis allowing for grades of membership. Genome Biol. 24:236. doi: 10.1186/s13059-023-03067-9, PMID: - DOI - PMC - PubMed

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