Discrimination of Bacterial Community Structures among Healthy, Gingivitis, and Periodontitis Statuses through Integrated Metatranscriptomic and Network Analyses

ABSTRACT Periodontal disease is an inflammatory condition caused by polymicrobial infection. The inflammation is initiated at the gingiva (gingivitis) and then extends to the alveolar bone, leading to tooth loss (periodontitis). Previous studies have shown differences in bacterial composition between periodontal healthy and diseased sites. However, bacterial metabolic activities during the health-to-periodontitis microbiome shift are still inadequately understood. This study was performed to investigate the bacterial characteristics of healthy, gingivitis, and periodontitis statuses through metatranscriptomic analysis. Subgingival plaque samples of healthy, gingivitis, and periodontitis sites in the same oral cavity were collected from 21 patients. Bacterial compositions were then determined based on 16S rRNA reads; taxonomic and functional profiles derived from genes based on mRNA reads were estimated. The results showed clear differences in bacterial compositions and functional profiles between healthy and periodontitis sites. Co-occurrence networks were constructed for each group by connecting two bacterial species if their mRNA abundances were positively correlated. The clustering coefficient values were 0.536 for healthy, 0.600 for gingivitis, and 0.371 for periodontitis sites; thus, network complexity increased during gingivitis development, whereas it decreased during progression to periodontitis. Taxa, including Eubacterium nodatum, Eubacterium saphenum, Filifactor alocis, and Fretibacterium fastidiosum, showed greater transcriptional activities than those of red complex bacteria, in conjunction with disease progression. These taxa were associated with periodontal disease progression, and the health-to-periodontitis microbiome shift was accompanied by alterations in bacterial network structure and complexity. IMPORTANCE The characteristics of the periodontal microbiome influence clinical periodontal status. Gingivitis involves reversible gingival inflammation without alveolar bone resorption. In contrast, periodontitis is an irreversible disease characterized by inflammatory destruction in both soft and hard tissues. An imbalance of the microbiome is present in both gingivitis and periodontitis. However, differences in microbiomes and their functional activities in the healthy, gingivitis, and periodontitis statuses are still inadequately understood. Furthermore, some inflamed gingival statuses do not consistently cause attachment loss. In this study, metatranscriptomic analyses were used to investigate the specific bacterial composition and gene expression patterns of the microbiomes of the healthy, gingivitis, and periodontitis statuses. In addition, co-occurrence network analysis revealed that the gingivitis site included features of networks observed in both the healthy and periodontitis sites. These results provide transcriptomic evidence to support gingivitis as an intermediate state between the healthy and periodontitis statuses.

The two reviewers were very enthusiastic about the scope of this manuscript and its findings, however they have a number of comments highlighting areas where this submission could be improved. In addition to addressing each reviewer comment, the authors also need to address the following points: -Ensure that the sequence data is made publicly available. I could not find a record for the accession number DRA011737 on the DDBJ website.
-This manuscript has a large number of supplemental files/figures. Please reduce this to <10 supplemental items as per journal guidelines.
-Figures should include error bars when presenting results averaged from a group. The authors should also more clearly display the number of samples on the figures.
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This is a timely study that aimed to investigate the bacterial characteristics of healthy, gingivitis, and periodontitis statuses through metatranscriptomic analysis. Subgingival plaque samples of healthy, gingivitis, and periodontitis sites in the same oral cavity were collected from 21 patients. 16S rRNA analysis was used to define bacterial composition, whereas the taxonomic and functional profiles of the communities were characterized by mRNA analysis. The results indicate differences in bacterial compositions and functional profiles between healthy and periodontitis sites. Cooccurrence networks were constructed for each group by connecting two bacterial species if their mRNA abundances were positively correlated. The clustering coefficient analysis revealed that the (species-based) co-occurrence network complexity increased during gingivitis development, but it decreased during progression to periodontitis. Notable species displaying greater transcriptional activities associated with disease progression were Eubacterium nodatum, Eubacterium saphenum, Filifactor alocis, and Fretibacterium fastidiosum, in contrast to the 'red complex' bacteria. The study highligts the gingivitis structural and functional microbiome profile as an important transitory stage from health to periodontitis. This is an interesting perspective with a well justified study approach. The comparison of taxonomic profiles of 16S rRNA and taxonomic mRNA (eg. comparison of mRNA/16S rRNA ratios between clinical states) are particularly important, in determining the significantly active taxa. Some points are raised after reviewing if the manuscript. The English language text needs some attention for correction of some grammatical errors. 1. Recent review work on metartranscripomics (PMID: 33226688) and metagenomics of the oral microbiome (PMID: PMID: 33226714) should be acknowledged, and the collective findings should be compared to the present findings. 2. In lines 156-157, the word "expressed" should be introduced "...1,575 bacterial genes were commonly expressed among all three periodontal statuses ..." 3. In lines 183-188 it is reported that five pathways were active only in gingivitis and periodontitis sites, whereas five other pathways were active only in healthy and gingivitis sites. These findings need to be elaborated further in the Discussion section. 4. In lines 264-266 it is stated that "Based on the keystone pathogen theory (24, 25), we presumed that these species 265 might constitute inflammophilic pathobionts and/or keystone species in periodontal diseases". However, the keystone pathogen theory is not commensurate with the inflammophilic pathobiont phenotypic profile of the species, and therefore these terms need to be distinguished from one another. 5. In lines 330-331 it is stated that: "These findings imply an easy transition between healthy and gingivitis statuses". The term "easy" is not well comprehended here, so the authors may choose an alternative one. 6. A limitation of the study as noted in line 331 is its cross-sectional nature, and that future longitudinal investigations of microbiomes and their clinical relationships will elucidate further the bacterial changes during the conversion of health to periodontitis. I would also note that the number of participants is rather limited for this type of study is rather limited and recommend it include this among the limitations.

Reviewer #2 (Comments for the Author):
This is an excellent study utilizing transcriptomics to define bacterial communities in three common stages of periodontal disease. Advantages of the study include sampling within subjects for all three disease states, and generating ratios of gene expression over bacterial counts to create a novel view of bacterial viability/activity and interactions. I have some comments to improve readability for non-dental microbiologists, but otherwise I have no major concerns and find this study to be a valuable contribution to the literature.
Topic: comparing communities of health-gingivitis-perio by gene expression within subjects And network linkage based on gene expression. The authors have previous publications using similar approaches and the writing is generally very clear.
-The authors repeatedly indicate that the composition of the oral microbiome in health, gingivitis, and periodontitis is not well studied, but that is far from the case. The utility of this particular work is the profiling based on levels of gene expression rather than exclusively bacterial numbers. I think the authors need to consider the following list of comments and adjust the emphasis accordingly. The novelty and value in the study is in defining the communities by transcriptomics.
Line 32-35 'However, there remains an inadequate understanding regarding bacteria that are depleted or enriched during the health-to-periodontitis microbiome shift, as well as bacteria associated with gingivitis and/or periodontitis.' This statement is not strictly accurate, as the presence/absence is understood, however the METABOLIC ACTIVITY during these stages is not, so I suggest modifying this line accordingly.

Line 59 regarding the difference in microbiome and it's functional activity in healthy, gingivitis, and periodontitis
Line 65-67 These results provide bacteriological transcriptomic evidence to support gingivitis as an intermediate state between healthy and periodontitis statuses.
-mSystems is not a dental-oriented journal, and the target audience will need careful explanation of dental terms. In that regard, I suggest the following adjustments to the text.
Line 74. Add an additional line defining the periodontium. "The periodontium is comprised of the soft tissue and bone surrounding the tooth, and periodontal diseases are representative oral polymicrobial diseases, which involve a microbiome imbalance known as dysbiosis that triggers periodontal inflammation (1, 2).
Line 79. There is increasing evidence that periodontal disease leads to tooth loss and oral functional decline, as well as the onset or progression of various systemic diseases (5-9). We know that periodontitis leads to tooth loss and oral functional decline, thus I suggest the following adjustment to the wording. Periodontitis leads to tooth loss and oral functional decline, and there is increasing evidence that it is associated with onset or progression of various systemic diseases (5-9).
Other comments: Line 501 : Table 1 legend. Include the information that age and standard deviation are in years.
Line 130-131 Table S1 shows data at the species level, not genus level as described in the text, which is a bit confusing. Please explain or modify.
Lines 123-125 These total numbers of reconstructed OTUs seems low compared to DNAbased methods. Are these values consistent with other transcriptomics studies?  The total number of taxa in H, G, and P sites were 2,058, 2,279, and 2,129, respectively; These seems like an extremely high number, please describe briefly how taxa are defined here (at the genus level, species level, subspecies level, or a mix?) Lines 210 -211. Among active taxa, the activities of eight taxa (F. fastidiosum, Eubacterium nodatum, F. alocis, Actinomyces sp. Prevotella sp., E. saphenum, Porphyromonas endodontalis, and P. nigrescens) were greater than the activities of red complex bacteria in P site. Comparing this text to figure 3AB, only F. fastidiosum, Eubacterium nodatum, F. alocis, and Prevotella sp have a higher mRNA/16srRNA ratio than two of the red species complex. This statement should be modified.
Line 225-227 The significant active taxa (26/36, 45/45, and 20/26 taxa in H, G, and P sites, respectively) were prevalent in all networks ( Figure 4A, Table 2, and 227 Tables S6-S8). I am not sure I understand this statement correctly. The input data for correlation analysis was the ratio data, so wouldn't you expect the highly active taxa to be present in the network? And they were prevalent in their own network, but not ALL networks? For example, there are 45 taxa in the G network, but they are not all present in the H or P networks? This will require additional clarification in the text.
Line 240. Approximately half of adults over 30 years of age exhibit periodontal disease in North America. While this is probably true, the study was done in Japanese adults and in fact periodontal disease is common world-wide. Therefore this is not the best opening statement for the discussion, in my opinion, maybe something about occurrence world-wide is better.
The two reviewers were very enthusiastic about the scope of this manuscript and 3 findings, however they have a number of comments highlighting areas where this 4 submission could be improved. In addition to addressing each reviewer comment, the 5 authors also need to address the following points. Response: We thank the reviewers and editor for their insightful comments, which have 7 helped us significantly improve our manuscript.     Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. For your reference, ASM Journals' address is given below. Before it can be scheduled for publication, your manuscript will be checked by the mSystems senior production editor, Ellie Ghatineh, to make sure that all elements meet the technical requirements for publication. She will contact you if anything needs to be revised before copyediting and production can begin. Otherwise, you will be notified when your proofs are ready to be viewed.
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