Molecular and functional characterization of Streptococcus dentisani 7746: gene expression, biofilm modulation and immunoregulatory effects under cariogenic conditions
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Background: Streptococcus dentisani has emerged as a health-associated oral commensal with potential probiotic relevance due to its inhibitory capacity against cariogenic species and its potential to modulate host immune responses. However, the molecular and functional basis of its ecological role within oral biofilms remains poorly defined. Methods: we performed an exploratory molecular and functional characterization of S. dentisani 7746 by assessing gene expression under varying glucose concentrations and evaluating its impact on mixed-species biofilms and epithelial cell responses. Semi-quantitative RT-PCR and CFU enumeration assays were conducted to quantify gene expression and bacterial abundance, respectively. Results: Adhesion-related genes (fap1, cshA) and immunomodulatory genes (ppiA, atlA) showed stable expression with no consistent glucose-dependent regulation, except for ppiA, which was significantly downregulated at high glucose. In contrast, pox—a gene encoding pyruvate oxidase—was repressed under glucose-rich conditions, suggesting a regulatory link between carbohydrate availability and hydrogen peroxide production. Functionally, S. dentisani reduced S. mutans abundance and promoted Lactobacillus persistence in mixed biofilms. Moreover, pre-exposure to S. dentisani altered pathogen internalization and cytokine responses in HeLa cells, favoring anti-inflammatory signaling. Conclusion: Our findings reveal that S. dentisani maintains a stable molecular profile under cariogenic stress and exerts multifaceted ecological functions—competitive, cooperative, and immunomodulatory—that support further mechanistic evaluation of its ecological and immunomodulatory properties. These findings should be interpreted as exploratory and warrant validation using quantitative and physiologically relevant models.
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Dear Dr. Ortiz Flores, You manuscript has now been assessed by two independent reviewers, bot of which recognised the merit of your work. Yet, some points have been raised regarding the presentation of the results and the underlying data. Most relevantly, (1) representative images of gels used to generate the data presented in the graphs must be presented in every figure as appropriate, and (2) standard deviation of the data must be shown instead of standard error of the mean. Please address the reviewers comments and resubmit your paper for further consideration. Please note that you must provide a point-by-point response to the reviewers comments, as well as a tracked and a clean version of the corrected manuscript.
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Comments to Author
This paper explores various factors that might enable Streptococcus dentisari to protect oral health against the cariogenic bacterium, Streptococcus mutans. The first version of the paper was reviewed critically. Major criticisms were that the data presented were only semi-quantitative and that the presentation of statistical analysis was inconsistent. The authors have revised the paper to answer, t lest in pr, all seven points made by the original reviewer. This new referee therefore focused on checking the changes that have been made. Given the positive response by the authors, the paper should be accepted for publication after only very minor further changes. Figure legends should enable the reader to understand the data presented without reference to the text. Although the authors have mainly …
Comments to Author
This paper explores various factors that might enable Streptococcus dentisari to protect oral health against the cariogenic bacterium, Streptococcus mutans. The first version of the paper was reviewed critically. Major criticisms were that the data presented were only semi-quantitative and that the presentation of statistical analysis was inconsistent. The authors have revised the paper to answer, t lest in pr, all seven points made by the original reviewer. This new referee therefore focused on checking the changes that have been made. Given the positive response by the authors, the paper should be accepted for publication after only very minor further changes. Figure legends should enable the reader to understand the data presented without reference to the text. Although the authors have mainly followed this general guideline, legends to figures 4, 5 and 7 do not include definitions of the blue and grey bars of the histograms. There is also an unexplained insertion, (x1), in the legend to figure 5. Use of punctuation is also erratic. Pairs of commas would be more appropriate that extended hyphens. Examples appear throughout the text, starting with lines 27 and 28. There is also at least one example where a colon is used to introduce a list. However, items in the list should be separated by semi-colons, not by commas. Section 2.3. Formation of biofilms. There is insufficient detail for others to repeat this work. Were disks immersed in flasks, or were flow cells used? Line 159. What is a 1x concentration?
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
This is a revised version of a manuscript which has already gone through one round of reviews. The authors have attempted to characterise the effect of Streptococcus dentisani on the interaction of both pathogens and commensals found in the oral tract with epithelial cells and modulation of mixed species biofilm formation. They also look at the effect of glucose concentration on gene expression by S. dentisani and its effect on cytokine gene expression by epithelial cells. While the aims of the study are excellent the design of the experiments and the presentation of the results could be improved. Response to reviewers comments Point 4 Supplemental Figure 1 is supplied as a representative image for rtPCR results. However, the image supplied is of positive control products not of products amplified …
Comments to Author
This is a revised version of a manuscript which has already gone through one round of reviews. The authors have attempted to characterise the effect of Streptococcus dentisani on the interaction of both pathogens and commensals found in the oral tract with epithelial cells and modulation of mixed species biofilm formation. They also look at the effect of glucose concentration on gene expression by S. dentisani and its effect on cytokine gene expression by epithelial cells. While the aims of the study are excellent the design of the experiments and the presentation of the results could be improved. Response to reviewers comments Point 4 Supplemental Figure 1 is supplied as a representative image for rtPCR results. However, the image supplied is of positive control products not of products amplified under different experimental conditions which were used to generate data supplied in Figures throughout the manuscript. A representative image that was used to generate data supplied in figures should be shown. Point 7 The authors state that error bars have now been added to all figures. However, the error bars added are standard errors of the mean (SE) not standard deviations (SD) as stated in point 7. The small and similar error bars in the figure suggest that it is in fact SEs that have been used and not SDs. SDs should be supplied for all figures. Abstract Line 28 and 29 "These results encourage further in vivo investigations to validate its clinical applications" The results presented are very preliminary and certainly do not suggest that in vivo investigations are the next step. Further, more detailed molecular and biological functional investigations are required before in vivo work is done. I would modify this sentence. Introduction Lines 58 and 59 "To address these gaps, we performed a comprehensive molecular and functional analysis" The investigation presented in this paper is very preliminary and I would suggest that this sentence be modified to reflect this. Materials and methods More detail could be supplied in the Methods section to enable the reader to better understand how the experiments were conducted. Section 2.1 Streptococcus dentisani were grown in BHI broth or agar under aerobic conditions for 48 h. When grown in broth were the bacteria grown until the OD reached 1.5 or for 48 h or after 48 h was the OD always 1.5? Please give growth conditions for all other bacterial strains used. Please describe how the media was modified to increase the glucose concentrations. BHI contains 2g/l of glucose, it is considered a high glucose concentration media. A salivary glucose concentration above 6.64 mg/dL has been identified in some studies as correlating with moderate to high severity of dental caries and increased cariogenic activity. How did the authors decide what concentrations of glucose use in experiments? 5-15% seems extremely high. Section 2.2 Trizol was used to extract bacterial RNA. How was RNA extracted from epithelial cells? Results Section 3.3 Lines 165-167 "This downregulation was statistically significant at 5% and 15% glucose when compared to the baseline condition. A significant downregulation of pox expression was observed at 5% and 15% glucose (p
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Dear Dr. Ortiz-Flores, I performed an initial assessment of your manuscript "olecular and functional characterization of Streptococcus dentisani 7746: gene expression, biofilm modulation and immunoregulatory effects under cariogenic conditions", and although the subject seems to be of interest, there a few amendments that need to be done before it can be sent out for review. One major concern is that a great part of the work is based on semi-quantitative RT-PCR analysis obtained using 40-cycle PCR reactions. This number os cycles is normally saturating. Please explain how you standardised your reaction conditions so that the PCR remains at the exponential phase (prior to plateau) after 40 cycles. You can also include the primer sequences used for the amplification of 16S rRNA gene in Table 1. The inexistence of any quantitative data …
Dear Dr. Ortiz-Flores, I performed an initial assessment of your manuscript "olecular and functional characterization of Streptococcus dentisani 7746: gene expression, biofilm modulation and immunoregulatory effects under cariogenic conditions", and although the subject seems to be of interest, there a few amendments that need to be done before it can be sent out for review. One major concern is that a great part of the work is based on semi-quantitative RT-PCR analysis obtained using 40-cycle PCR reactions. This number os cycles is normally saturating. Please explain how you standardised your reaction conditions so that the PCR remains at the exponential phase (prior to plateau) after 40 cycles. You can also include the primer sequences used for the amplification of 16S rRNA gene in Table 1. The inexistence of any quantitative data (RT-qPCR) for any of the genes under study, including for those where a difference in expression was observed by semi-quantitative PCR was observed is a down side; it would be of interest to have some RT-qPCR data included as this would strengthen the conclusions. Likewise, quantification of cytokines by ELISA would be of interest. Finally, if only semi-quantitve data is used, you should show at least one representative gel used in the analysis. Moreover, I have to request that you check the statistical analysis presented in the figures: - In Figure 1, all bars display error bars of the same size, is this correct? - In Figures 2 and 3, the group used for normalisation is marked as "ns". Why? - In all other figures, there are no error bars. Please check. Please address the comments above and amend the text as necessary. After you resubmit your paper, it be reassessed prior t o be sent out for review. Best regards, Gustavo
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