Smoking and Depth-Related Anaerobic Bacteria in Endodontic–Periodontal Lesions: A Pilot Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

PLEASE READ INSTRUCTION TO AUTHORS!!

Title: Title too long but nature of study is unclear. Thus it can be   Smoking and Depth-Related Anaerobic Bacteria in Endodontic-Periodontal Lesions: A Pilot Study

Abstract: I suggest a structured abstract

Introduction: Explicitly state out the research gap and why this study is essential.

Aim: Too long and it looks like a method, be concise,

Method: the small sample size limits the statistical power and may increase the risk of type II error. Hence state that this is  a pilot study.

Smoking has a dose-dependent effect on periodontal tissues and microbial composition. Without quantifying smoking exposure, interpretation of the findings may be limited. Hence, a clear definition of “smokers” and “non-smokers.” Important details include: No of cigarettes smoked per day, Duration of smoking history, Pack-years, Whether former smokers were included or excluded,  Time since smoking cessation, and Whether smoking status was self-reported or biochemically verified

The criteria used to diagnose endodontic-periodontal lesions should be described in detail because different lesion types may have different microbial profiles.

PCR does not provide information about bacterial load or relative abundance. Therefore, not sure about conclusions about “reduced” bacterial presence or co-occurrence.

Authors should justify using five selected anaerobic periodontal bacteria because  endodontic-periodontal lesions are polymicrobial and may include other anaerobes.

Multiple sites may have been sampled from each participant, observations are not fully independent. Statistical methods should account for clustering at the patient level.

Author Response

Comment 1.1 — Title: Title too long but nature of study is unclear. Thus it can be: "Smoking and Depth-Related Anaerobic Bacteria in Endodontic-Periodontal Lesions: A Pilot Study".

Response: We thank the Reviewer for this suggestion. The title has been revised accordingly and now reads: " Smoking and Depth-Related Anaerobic Bacteria in Endodontic-Periodontal Lesions: A Pilot Study", which better captures the exploratory nature of the study. The revision is highlighted in red on the title page of the manuscript.

 

Comment 1.2 — Abstract: I suggest a structured abstract.

Response: Although we also agree that a structured abstract with explicitly named sections—Objectives, Materials and Methods, Results, and Conclusions—would improve clarity, the journal template and recently published articles in the International Journal of Environmental Research and Public Health do not include such explicit section headings. Therefore, the abstract content has been organized according to this structure, but without identifying each section by name.

 

Comment 1.3 — Introduction: Explicitly state out the research gap and why this study is essential.

Response: The Introduction has been revised to explicitly state the research gap. We added the following passage: " […] few studies have specifically investigated how smoking may influence the microbial profile of endodontic-periodontal lesions. In particular, limited attention has been given to the depth-stratified distribution and co-occurrence patterns of key anaerobic periodontal bacteria across periodontal pockets of different depths, healthy gingival sulci, and oral mucosal sites. This gap is clinically relevant, since smoking-related changes in microbial gradients and interspecies associations within these lesions could challenge conventional pocket-depth thresholds used to guide microbiological sampling and treatment decisions in tobacco users."

 

Comment 1.4 — Aim: Too long and it looks like a method; be concise.

Response: The aim statement has been revised to be more concise and clearly distinguishable from the Methods section. The revised aim now reads: "Therefore, this cross-sectional study investigated the depth-related distribution and co-occurrence of selected anaerobic periodontal bacteria in smokers and non-smokers with endodontic-periodontal lesions, across sites of varying pocket depth, healthy gingival sulci, and oral mucosa."

 

Comment 1.5 — Methods: The small sample size limits the statistical power and may increase the risk of type II error. Hence, state that this is a pilot study.

Response: We have included the term “pilot” in the description of the sample at Material and Methods section as follows: "This pilot study was approved by the Institutional Research Ethics Committee (protocol number: CAAE 0104.0.189.000-07). " The word "Pilot Study" has also been incorporated into the title.

 

Comment 1.6 — Methods: Smoking has a dose-dependent effect. Without quantifying smoking exposure, interpretation may be limited. A clear definition of "smokers" and "non-smokers" is needed, including: number of cigarettes per day, duration of smoking history, pack-years, whether former smokers were included or excluded, time since cessation, and whether smoking status was self-reported or biochemically verified.

Response: We thank the Reviewer for raising this important issue. The Methods section now includes a clear definition of smoking status: "Participants were classified as smokers or non-smokers based exclusively on self-report. Smokers were defined as individuals who were active smokers at the time of sample collection and reported smoking approximately one pack of cigarettes per day, corresponding to 20 cigarettes, for at least one year. Non-smokers were defined as individuals who reported having never smoked. Former smokers were excluded from the study.”

 

Comment 1.7 — Methods: The criteria used to diagnose endodontic-periodontal lesions should be described in detail because different lesion types may have different microbial profiles.

Response: A detailed description of the diagnostic criteria has been added to the Methods section as follows:

"According to the classification criteria for endodontic-periodontal diseases the lesions included in this study were categorized as primary periodontal lesions with secondary endodontic involvement, meaning that periodontal breakdown represented the pri-mary pathological event, with subsequent retrograde pulpal compromise. This diagnostic characterization should be considered when interpreting the microbial findings and extrapolating results to other endodontic-periodontal subtypes. Eligible teeth showed concomitant evidence of periodontal and endodontic involvement, including localized periodontal pockets deeper than 3 mm, clinical attachment loss at the affect-ed site, signs of pulpal involvement based on pulp vitality testing and endodontic ex-amination, and radiographic evidence compatible with periapical and/or lateral bone involvement. When present, sinus tracts were clinically traced to support the differential diagnosis, and tooth mobility was recorded [12].

Only structurally sound teeth were included, defined as teeth without extensive restorations, previous endodontic treatment, root caries, vertical root fracture, perforations, or other structural alterations that could confound the diagnosis. Root frac-ture and other non-periodontal/non-endodontic causes of attachment loss were excluded through clinical inspection, periodontal probing pattern, radiographic examination, and complementary diagnostic procedures when necessary. Teeth with isolated periodontal lesions, isolated endodontic lesions, or lesions primarily related to fracture, perforation, or iatrogenic damage were excluded.”

 

Comment 1.8 — Methods: PCR does not provide information about bacterial load or relative abundance. Therefore, conclusions about "reduced" bacterial presence or co-occurrence should be reconsidered.

Response: This is an important methodological limitation that we acknowledge. The language in the Results and Discussion has been revised to avoid overinterpretation. We now consistently refer to "bacterial detection" rather than "bacterial presence" or "reduced bacteria." The rationale for using conventional PCR and its limitations are explicitly addressed in the Discussion.

 

Comment 1.9 — Methods: Authors should justify using five selected anaerobic periodontal bacteria because endodontic-periodontal lesions are polymicrobial and may include other anaerobes.

Response: We have added a justification in the Methods section: "The bacterial species selected for analysis — Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Prevotella nigrescens, and Aggregatibacter actinomycetemcomitans — were chosen based on their consistently high prevalence and recognized pathogenic role in endodontic-periodontal lesions, as well as the availability of validated species-specific PCR primers."

 

Comment 1.10 — Statistical methods: Multiple sites may have been sampled from each participant; observations are not fully independent. Statistical methods should account for clustering at the patient level.

Response: We thank the reviewer for raising this important point. We would like to clarify that, although samples were obtained from different anatomical sites in each participant, these sites were not pooled or analyzed together as independent repeated observations in the same statistical comparison. Periodontal pockets, healthy gingival sulci, and oral mucosal samples were analyzed separately, and comparisons between smokers and non-smokers were performed within each anatomical site or pocket-depth category.

Thus, the statistical analyses did not combine observations from periodontal pockets, gingival sulci, and oral mucosa into a single pooled dataset. Instead, each site-specific analysis compared detection frequencies between smoking groups within the same anatomical compartment. Likewise, periodontal pocket samples were stratified according to probing depth, and each depth category was analyzed separately.

To avoid ambiguity, we have revised the Methods section to make clear that the unit of analysis was the site/depth category and that no statistical test treated different anatomical sites from the same participant as independent observations within a pooled model. Therefore, patient-level clustering across different anatomical sites was not applicable to the site-specific comparisons performed in this exploratory study.

Reviewer 2 Report

Comments and Suggestions for Authors

The study aims to categorize key bacterial population in endo perio lesions at different PPD.

The study is interesting and appears sound.

 

Methods

Some issues need to be resolved before publication.

What about other indexes of inflammation, such as BoP. What is the percentage of active bleeding sites on gathered samples?

Another important issue is the oral condition of patients. Was DMFT calculated?

Regarding Endo Perio lesions, all teeth were primary treatments or had a previous failed endodontic treatment? Microbiome may be markedly different in these cases:

The PCR methodology is insufficiently described. The primer table appears to provide only one primer sequence per bacterial species despite referring to primer pairs. Forward and reverse sequences, target genes, primer references, amplicon validation, reference strain identifiers, detection limits, and reproducibility procedures should be reported

The criteria to define smoking are not sufficiently described

 

Results
include demographic information of the population, as well as tooth related information. This could be useful to allow the detection of significant differences between groups. No information is present in result, only samples size definition in methods. This is a matter of concern.

 

The manuscript states that two-tailed Fisher’s exact tests were used; however, some comparisons marked as statistically significant in Table 2 do not appear to be significant when recalculated from the reported counts. For example, P. intermedia and P. nigrescens in 5–6 mm pockets appear to yield two-sided Fisher exact p-values above 0.05. The authors should provide exact p-values for all comparisons and verify the entire statistical analysis.

 

Discussion

Additional comments in the discussion are required concerning recent NGS investigation of periodontal pocket microbiome and oral microbiome. Some studies also analysed the tongue microbiome, revealing a markedly different environment, yet connected to the differences of pocket depth (PPD >4mm). moreover, a number of oral bacteria has clear association with systemic pathologies.   I think it could be a valuable addition to comment the following papers:  doi: 10.3390/antibiotics14080804. doi: 10.1371/journal.pone.0204941

Author Response

Comment 2.1 — Methods: What about other indexes of inflammation, such as BoP? What is the percentage of active bleeding sites on gathered samples?

Response: We thank the reviewer for this relevant comment. Bleeding on probing was considered during clinical site selection, and all periodontal pocket samples were collected from sites that exhibited bleeding on probing, as now clarified in the Methods section. Therefore, among the sampled periodontal pocket sites, the percentage of active bleeding sites was 100% by site-selection criterion. We have revised the manuscript to clarify that periodontal pocket samples were obtained from bleeding sites.

 

Comment 2.2 — Methods: Was DMFT calculated?

Response: We clarify that DMFT was not evaluated in this pilot study. We acknowledge this as a limitation and note that future studies should include a comprehensive oral health assessment to better characterize the study population.

 

Comment 2.3 — Methods: Regarding endo-perio lesions, were all teeth primary treatments or had a previous failed endodontic treatment? Microbiome may be markedly different in these cases.

Response: This is a very relevant point. We agree that teeth with previously failed endodontic treatment may have different microbial profiles. However, the absence of previous endodontic treatment had already been described in the manuscript as one of the inclusion criteria, as stated in the following sentence: “Only structurally sound teeth were included, defined as teeth without extensive restorations, previous endodontic treatment, root caries, vertical root fracture, perforations, or other structural alterations that could confound the diagnosis.”

To emphasize this information, we have additionally added in the Methods section that root fracture and other non-periodontal/non-endodontic causes of attachment loss were excluded through clinical inspection, periodontal probing pattern, radiographic examination, and complementary diagnostic procedures.

 

Comment 2.4 — Methods: The PCR methodology is insufficiently described. The primer table appears to provide only one primer sequence per bacterial species despite referring to primer pairs. Forward and reverse sequences, target genes, primer references, amplicon validation, reference strain identifiers, detection limits, and reproducibility procedures should be reported.

Response: We sincerely apologize for this omission. The PCR methodology section has been substantially revised and expanded. Both forward and reverse primer sequences are now provided for each bacterial species in Table 1. Reference strains, target genes, amplicon sizes, and PCR conditions are now fully described as follows: "PCR-amplification reactions were performed in a reaction mixture containing 1× PCR buffer (10 mM Tris-HCl, pH 9.0, 50 mM KCl, and 15 mM MgCl2), 200 mM of four deoxynucleotide triphosphates, 12.5 pmol of each primer, 2.5 U Taq DNA polymerase, and 5 μL supernatant from the samples submitted for DNA extraction." Standard strains used as controls are also now reported: "Tf ATCC 43037, Aa ATCC 33384, Pg ATCC 33277, Pi ATCC 25611, and Pn ATCC 33563."

 

Comment 2.5 — Methods: The criteria to define smoking are not sufficiently described.

Response: The smoking definition has been expanded in the Methods section, including the number of cigarettes per day, duration, and the self-report basis of classification, as follows:.

"Participants were classified as smokers or non-smokers based exclusively on self-report. Smokers were defined as individuals who were active smokers at the time of sample collection and reported smoking approximately one pack of cigarettes per day, corresponding to 20 cigarettes, for at least one year. Non-smokers were defined as individuals who reported having never smoked. Former smokers were excluded from the study.”

 

 

 

Comment 2.6 — Results: Include demographic information of the population, as well as tooth-related information. No information is present in results, only sample size definition in methods.

Response: We agree this was a significant gap. Demographic data have been added to the Results section, including: "A convenience sample of 26 adult patients of both sexes from 10 (40%) males and 16 (60%) females residents of city of Lavras (MG) and the surrounding region, aged 27 to 54 years and clinically diagnosed with endodontic-periodontal lesions diagnosed through clinical and radio-graphic examinations."

 

Comment 2.7 — Results: Some comparisons marked as statistically significant in Table 2 do not appear to be significant when recalculated from the reported counts (e.g., P. intermedia and P. nigrescens in 5–6 mm pockets). The authors should provide exact p-values for all comparisons and verify the entire statistical analysis.

Response: We thank the reviewer for this careful observation. We have rechecked the entire statistical analysis and agree that some comparisons previously marked as statistically significant in Table 2 were incorrectly labeled. After recalculating the comparisons using two-tailed Fisher’s exact tests based on the reported counts, the differences for Prevotella intermedia in 5–6 mm pockets (3/8 in smokers versus 10/12 in non-smokers; p = 0.062) and Prevotella nigrescens in 5–6 mm pockets (2/8 in smokers versus 9/12 in non-smokers; p = 0.065) did not reach statistical significance. Therefore, the significance markers for these comparisons have been removed from Table 2 and the corresponding statements in the Results and Discussion sections have been revised. The only species-specific comparison that remained statistically significant after verification was Tannerella forsythia in pockets ≥7 mm (1/5 in smokers versus 5/5 in non-smokers; p = 0.048). Exact p-values have now been added to Table 2 for all species-specific comparisons. We also revised the text to avoid overstating non-significant trends and to describe these findings as differences in detection patterns rather than statistically significant differences when p ≥ 0.05.

 

Comment 2.8 — Discussion: Additional comments concerning recent NGS investigation of periodontal pocket microbiome and oral microbiome are required. Some studies also analysed the tongue microbiome. Moreover, oral bacteria have clear associations with systemic pathologies. The following papers should be commented: doi: 10.3390/antibiotics14080804; doi: 10.1371/journal.pone.0204941.

Response: We are grateful for these valuable suggestions. A new paragraph has been added to the Discussion as follows including the referred references: " The present findings, obtained through targeted PCR-based detection, should be interpreted in light of NGS-based oral microbiome studies showing that periodontal pocket depth is associated with marked shifts in subgingival microbial composition, with deeper sites favoring more anaerobic and dysbiotic communities. These patterns may extend beyond periodontal pockets, as recent evidence indicates that the tongue dorsum, although microbiologically distinct, may also reflect periodontal status and pocket depth, supporting the concept of the oral cavity as an interconnected microbial ecosys-tem [27]. The systemic relevance of periodontal pathogens is also noteworthy. Experi-mental studies have shown that repeated oral exposure to Porphyromonas gingivalis may induce bacterial translocation to the hippocampus and trigger neuroinflammatory and neurodegenerative changes consistent with Alzheimer’s disease-like pathology [28]. In the present study, the consistent detection of P. gingivalis across pocket depths and smoking groups supports its persistent colonization. Therefore, while the targeted PCR approach used here is useful for detecting selected pathogens, future NGS-based studies including other oral reservoir sites may better characterize the microbial ecosystem as-sociated with endodontic-periodontal lesions in smokers and non-smokers.”

Reviewer 3 Report

Comments and Suggestions for Authors

I congratulate the authors on their work.

I have some suggestions for their work:

1) Results

• lines 272-278: "This trend...detection pattern" may be inserted within the discussion section not here

2) Discussion

• Limitations of the study- please insert a bit more clearly and concisely

3) Conclusion

• Please add a more concise and airy variant

4) References

-Please verify and check them to be in accordance with the Journal's recommendations

Author Response

Comment 3.1 — Results (lines 272–278): "This trend...detection pattern" may be inserted within the discussion section, not in the Results.

Response: We agree with this suggestion. The paragraph beginning with "This trend suggests that smoking is associated with reduced co-occurrence of the selected anaerobic periodontal pathogens..." has been moved from the Results to the Discussion section, where it is more appropriately placed as an interpretive statement. The relevant text is highlighted in red in the revised manuscript.

 

Comment 3.2 — Discussion: Please insert the limitations of the study more clearly and concisely.

Response: The limitations of the study have been reorganized and presented more clearly and concisely in the Discussion section, as follows:

“Some methodological considerations should be acknowledged. The relatively small convenience sample reflects the low frequency and diagnostic complexity of endodontic-periodontal lesions in structurally sound teeth, but the strict eligibility criteria con-tributed to a more homogeneous and clinically well-defined sample. Conventional PCR allowed qualitative detection of selected anaerobic periodontal species rather than bacterial quantification or full microbiome characterization; nevertheless, it was suitable for the exploratory assessment of detection patterns and co-occurrence of clinically relevant target pathogens. Although the cross-sectional design limits causal interpretation, standardized sampling across periodontal pocket depths, healthy gingival sulci, and oral mucosa allowed a focused comparison between smokers and non-smokers. Thus, these findings provide clinically relevant data that smoking may influence the depth-related detection and co-occurrence of selected anaerobic bacteria in endodontic-periodontal lesions.”

 

Comment 3.3 — Conclusion: Please add a more concise and airy variant.

Response: The Conclusion has been revised to be more concise: "In this cross-sectional study, smoking was associated with attenuated depth-related bacterial detection and reduced microbial co-occurrence in periodontal pockets of teeth with endodontic-periodontal lesions, a pattern less evident in non-smokers." The revised conclusion clearly summarizes the main finding without restating methods or overextending interpretation.

 

Comment 3.4 — References: Please verify and check them to be in accordance with the Journal's recommendations.

Response: All references have been carefully reviewed and reformatted to comply with the journal's author guidelines.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have revised the paper according to reviewers comments

Reviewer 2 Report

Comments and Suggestions for Authors

paper can be accepted in this form. Revisions are good