<xhtml:span xmlns:xhtml="http://www.w3.org/1999/xhtml" xml:lang="en">Effect of Sialidase Inhibitors on a Plaque Community Biofilm Model </xhtml:span>
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The oral microbiome is a diverse ecosystem that plays a critical role in health and disease and contains numerous bacterial species capable of metabolizing host-derived glycans, particularly sialic acids. Sialidase enzymes can be produced by both commensal and pathogenic bacteria, influencing biofilm formation and host-interactions. To investigate how sialidase activity might influence the oral microbiome we conducted a series of in vitro polymicrobial biofilm experiments and assessed community composition using 16S rRNA sequencing. As a first step, we tested modified Oxford Nanopore Technology (ONT) primers using an in-house sequencing workflow and compared it to the standard Illumina MiSeq primers. Through in silico and in vitro assessments, we identified primer bias in the standard ONT 16S primers and designed human oral microbiome (HOM) modified primers (HOM_27F-YM/1492R-D) to improve taxonomic resolution, achieving results comparable to the gold-standard Illumina 16S primers, particularly for key oral genera. These HOM-optimized primers had an overall lower error rate (3.4%) and generated community profiles that closely matched to those produced by Illumina. We then used the same ONT workflow and modified 16S primers to evaluate the effect of sialidase inhibitors Oseltamivir and 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA) on Hydroxyapatite (HA)-coated minimum biofilm eradication concentration (MBEC) assay plate derived plaque biofilms from a whole plaque community model. Inhibitor-treated biofilms exhibited differences in relative abundance depending on the inhibitor combination used, with increased abundance of Streptococcus with Oseltamivir alone and Fusobacterium with both inhibitors combined (Kruskal-Wallis cutoff = 0.05, LDA > 2). These findings demonstrate that ONT-based 16S sequencing with HOM-modified primers suggested that sialidase activity can modulate microbial community structure in plaque biofilms.
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The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments.
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Comments to Author
Abstract The absence of empirical data, statistical outputs, and confidence measures in the abstract limit its scientific depth and prevent readers from assessing the robustness and reproducibility of the reported findings, thereby making it appear more narrative than data-driven Introduction The section does not (i) distinguish what is unknown, (ii) Explain existing ONT-based oral microbiome approaches are insufficient and (iii) Explain how the present study advances beyond prior methodological and studies. (iv) Provide a conceptual network focusing on the hypothesis linking primer optimisation, ONT performance, and functional modulation of biofilm communities by sialidase inhibitors. (v) Provide a logical progression toward a single, clearly articulated research gap. (vi) While the authors review …
Comments to Author
Abstract The absence of empirical data, statistical outputs, and confidence measures in the abstract limit its scientific depth and prevent readers from assessing the robustness and reproducibility of the reported findings, thereby making it appear more narrative than data-driven Introduction The section does not (i) distinguish what is unknown, (ii) Explain existing ONT-based oral microbiome approaches are insufficient and (iii) Explain how the present study advances beyond prior methodological and studies. (iv) Provide a conceptual network focusing on the hypothesis linking primer optimisation, ONT performance, and functional modulation of biofilm communities by sialidase inhibitors. (v) Provide a logical progression toward a single, clearly articulated research gap. (vi) While the authors review what is known about sialidase activity and ONT sequencing independently, they fail clearly state what is unknown at the intersection of these areas. (vii) it is not known why existing ONT 16S primers are inadequate for oral microbiome studies. And why this omission reduces the mechanistic coherence of the study and weakens its justification. (viii) The introduction places strong emphasis on taxonomic resolution, yet the biological implications of achieving species-level resolution for understanding sialidase-driven community dynamics are not fully developed. (ix) How does improved primer design directly enable new biological insights beyond methodological optimisation. Methodology The methodological apporach is reproducible. The methodology relies on a restricted number of plaque inocula and pooled technical replicates, which limits the biological representativeness of the findings. Potential amplification and platform-specific biases are not fully controlled or quantified While the authors acknowledge primer bias and sequencing error differences between ONT and Illumina platforms, the methodology does not fully address residual amplification bias, differential error correction, and clustering thresholds that could influence taxonomic assignments. Amplification and bias, although not completely controllable, are not completely accounted for though the presence of primer bias, as well as the difference in sequencing error rates between ONT and Illumina platforms. The application of separate denoising and clustering strategies for Nanopore and MiSeq datasets, for example, differing base difference threshold values, raises a potential confounding factor in the comparative evaluation of the platforms. . Results This section is ell structured and represents key major findings. To what extent might the higher base-calling error rates of Nanopore sequencing, particularly in homopolymeric regions, together with the use of different filtering criteria for ONT and MiSeq data, confound cross-platform comparisons of abundance and richness, such that the reported improvements in species-level resolution reflect increased analytical sensitivity rather than genuine gains in accuracy? 2.In vitro biofilm model simplicities undermine the ecological validity and generalisability of functional effects. How can this be avoided? The discusiion: a good but mostly repeatition of results. Discuss results ith current literature.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly 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
In its current form, the manuscript is not suitable for direct publication. While the study concept is promising, the manuscript suffers from major issues Several claims are not adequately supported by data, and key experimental and analytical details required for reproducibility are missing or incomplete. The manuscript claims to present an "optimized" ONT protocol for oral microbiome profiling, yet no systematic comparison is performed against: Existing ONT oral microbiome protocols Standard 16S rRNA short-read sequencing Shotgun Illumina metagenomics Without head-to-head benchmarking, it is unclear: What exactly is optimized? Whether improvements are statistically or biologically meaningful If gains are due to protocol changes or sequencing depth alone. Critical metadata are missing or …
Comments to Author
In its current form, the manuscript is not suitable for direct publication. While the study concept is promising, the manuscript suffers from major issues Several claims are not adequately supported by data, and key experimental and analytical details required for reproducibility are missing or incomplete. The manuscript claims to present an "optimized" ONT protocol for oral microbiome profiling, yet no systematic comparison is performed against: Existing ONT oral microbiome protocols Standard 16S rRNA short-read sequencing Shotgun Illumina metagenomics Without head-to-head benchmarking, it is unclear: What exactly is optimized? Whether improvements are statistically or biologically meaningful If gains are due to protocol changes or sequencing depth alone. Critical metadata are missing or insufficiently described: Number of participants, Inclusion/exclusion criteria, Oral sampling site (saliva, plaque, tongue dorsum, gingival crevice), Health status (caries, periodontitis, systemic disease), Antibiotic usage history Oral microbiome composition varies dramatically by site and host condition; without this information, results are not interpretable or generalizable. Host (human) DNA contamination is a major issue in oral metagenomics. The manuscript does not convincingly demonstrate: Host DNA depletion efficiency Differential lysis efficiency for Gram-positive bacteria ,Preservation of microbial diversity. Claims of protocol "optimization" are undermined without quantitative evaluation of these biases. The manuscript lacks: Technical replicates Biological replicates justification Sequencing depth normalization Observed differences may simply reflect stochastic variation rather than protocol superiority. The manuscript suggests improved species- or strain-level resolution, but:No validation against mock communities is provided,No error-rate analysis of ONT reads is shown,No confirmation using reference genomes or orthogonal methods. Functional potential is discussed, yet:No robust functional annotation pipeline is described, ONT error profiles can inflate false positives in gene prediction. The discussion section is overly optimistic and does not adequately address: ONT error rates Sample-to-sample variability Cost, scalability, and clinical applicability constraints. Following questions are not addressed or satisfied while reading manuscript. 1. How does your protocol perform on mock oral microbial communities? 2. What is the host DNA proportion across samples? 3. Can your method reliably distinguish Streptococcus species with high genomic similarity? 4. How reproducible is the protocol across different operators and runs? 5. Why was Oxford Nanopore chosen over hybrid long-short read approaches? Minor issues. grammatical correction needed . No use of full form at first use of abbreviations.
Please rate the manuscript for methodological rigour
Satisfactory
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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