Tobacco-Induced Oral Dysbiosis and Microbial Shifts: A Narrative Review of Their Role in Systemic Inflammation and Disease

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors congratulations on your manuscript and your contribution to the research. This narrative review focuses on an interesting topic regarding the tobacco-induced oral dysbiosis and systemic inflammation. I recommend major revisions to improve the quality of the manuscript.

Introduction:

This section requires a more detailed description of the oral microbiome, as one of the ecosystems of the human body, whose mechanisms of formation and interaction with other ecosystem are fundamental to understand the correlation between oral and systemic diseases. For example, it would be useful to describe the main biological niches of the oral cavity, the way of adhesins of biofilm to them and the pathological shift in composition. The following article could be useful to elaborate this specific topic: https://doi.org/10.3934/microbiol.2024020

At the end of this section, authors introduced the search strategy: it would be useful to move it in a specific section. In this regard, I suggest adding a “Material and methods” section, including search strategy, inclusion and exclusion criteria, items selection, extracted information organization. This kind of methodology should make the investigation more thorough, although it is a narrative review.

Furthermore, a section should be dedicated to the limits of the study, in order to better outline the future directions.

Conclusion:

This section should be more concise, highlighting the main findings and the future directions.

Author Response

1. Introduction:

This section requires a more detailed description of the oral microbiome, as one of the ecosystems of the human body, whose mechanisms of formation and interaction with other ecosystem are fundamental to understand the correlation between oral and systemic diseases. For example, it would be useful to describe the main biological niches of the oral cavity, the way of adhesins of biofilm to them and the pathological shift in composition. The following article could be useful to elaborate this specific topic: https://doi.org/10.3934/microbiol.2024020

Response:

1. Thank you for this suggestion and reference. We have adjusted the introduction and added the following paragraph to give a more comprehensive picture of the oral biome. (line 39 to 43 and line 46-59)

The human body hosts several distinct microbiomes; the oral microbiome is the third largest after the gut and skin. Like other microbial communities, the oral biota interacts with the host immune system, forming a symbiotic relationship, and plays an essential role in maintaining human physiology and homeostasis [4]

Biofilms form an important part of this ecosystem and are formed when bacteria adhere to oral surfaces. Biofilms are complex, well-organized microbial communities that prevent colonization by pathogenic bacteria. They maintain homeostasis, contribute to immune regulation and assist in metabolic functions. Early colonizers, which are mainly beneficial commensals, initiate biofilm formation and rely on the chemical and physical forces of the oral surface to begin the process. Molecular interactions, such as Van Der Waals forces and Brownian motion, facilitate initial adhesion and an early microbial biofilm is formed. Other bacteria join this initial layer as the biofilm develops, thereby forming a complex and structured community. The microorganisms secrete several molecules, such as polysaccharides, proteins, and nucleic acids, collectively known as extracellular substances. These are important in protecting and maintaining the integrity of the microbial community. Biofilms are an essential part of a healthy oral cavity and necessary to maintain the finely balanced ecosystem; however, pathogenic bacteria can become dominant and cause disease if not appropriately controlled [5].

 

2.At the end of this section, authors introduced the search strategy: it would be useful to move it in a specific section. In this regard, I suggest adding a “Material and methods” section, including search strategy, inclusion and exclusion criteria, items selection, extracted information organization. This kind of methodology should make the investigation more thorough, although it is a narrative review

Response:

We thank the reviewer for this suggestion, and the following section has been incorporated into the manuscript after the introduction. (lines 91-101).

 A literature search up to February 2026 was performed using PubMed, Google Scholar, and Science Direct databases. Original research studies and review articles relevant to the topic were selected and examined to acquire current evidence. Key words used to search the databases included “smoking”, “tobacco”, “cigarettes”, “oral biome”, “dysbiosis”, and “systemic disease”. A Boolean strategy, with words such as “And” and “Or” was employed to retrieve articles specific to the topic. Articles were included if they were peer-reviewed and published in English while non-peer-reviewed manuscripts and pre-prints were excluded. The articles were sorted and separated into those relevant for each of the various subthemes and the information was extracted to formulate a comprehensive review. Original research articles studying the effects of smoking tobacco in various population groups were further separated and analysed to identify similarities and differences.

3. Furthermore, a section should be dedicated to the limits of the study, in order to better outline the future directions

Response:

A section entitled “limitations and future research has been included (lines 450-494

Although there is increasing evidence indicating a risk of systemic diseases linked to smoking related oral dysbiosis, most clinical studies demonstrate an association rather than a causal relationship. Many of the published studies are cross sectional and observational with a lack of longitudinal data, which could track progression of disease over time. To address these challenges multi-omics approaches are required to fully clarify the mechanisms leading to systemic conditions. This may include detecting shifts in microbial communities linked to specific conditions and the identification of possible oral biomarkers, which could predict the risk of disease development. Alternatively, these microbial signatures could be used to formulate targeted therapies to prevent or treat associated conditions [100-102]. As the toxins and chemicals in cigarettes can cause systemic inflammation and disease, further research on the direct effects of these compounds on the immune system and organs is required.  

A major limitation of previous research is the insufficient control of confounding factors, such as lifestyle, dental hygiene, diet, age, and alcohol intake. These all cause dysbiosis and disease, making interpretation and conclusions difficult. Compounding this is the lack of standardization in methodology, study designs, sample type, and sample size [37]. A further inconsistency and barrier to reliable interpretation is the variation in geographical regions and population groups. Ethnicity, socioeconomic factors and access to health care can influence the development of periodontitis and systemic disease [53]. To obtain a more consistent picture of tobacco-induced alterations to the oral biome, large, collaborative, standardized investigations, utilizing the same study design and methodology are recommended. Confounding factors, such as age, sex, type of tobacco, diet, and lifestyle, would need to be recorded and accounted for during statistical analysis to obtain realistic and comparable results.  

One limitation of this narrative review is that it concentrated on the bacterial components of the oral biome. However, there is growing evidence that viruses, parasites, and fungi can also initiate inflammation. Although there is less evidence, in vitro and animal studies have established that smokers are at higher risk of developing fungal infections, such as oral candidiasis, which can lead to periodontitis and systemic inflammation [38,103]. Further research adopting comprehensive approaches including cell culture, microscopy and multi-omics techniques are needed to understand the complex interactions of these microorganisms and how they contribute to the pathogenesis of disease.

As traditional cigarette smoking declines, the popularity of E-cigarettes and vaping has increased, especially among the youth. This review did not address these products; however, there is growing evidence that the aerosols produced from E-cigarettes contain several toxic compounds [68]. These chemicals disrupt the oral microbiome and promote the growth of opportunistic pro-inflammatory organisms. Vaping has been shown to disrupt the epithelial barrier, impair the immune response, and cause oxidative stress. These changes can lead to chronic systemic inflammation [104]. Like the analysis of traditional cigarette smoking, standardized studies are required to account for the variation in products including chemical composition, flavorings, and nicotine concentration.

Finally, there is a lack of longitudinal studies that investigate changes in the oral biota over time, particularly after the cessation of smoking. These studies should include the time to full recovery and the impact of improved oral hygiene, probiotics, and prebiotics on the restoration of healthy micro-organisms [100].

4.Conclusion:

This section should be more concise, highlighting the main findings and the future directions.

Response:

The conclusion of the article has been rewritten to exclude repetition and make it more concise. (lines 496-508).

This narrative review contributes to the existing body of knowledge by providing an up-to-date synthesis of literature examining the effects of cigarette smoking on the oral microbiota, inflammation, and links to systemic disease. The findings emphasize the complexity of characterizing smoking related microbial shifts and highlight the need for large well-designed standardized studies. The inclusion of research focused on Southern African populations emphasizes the importance of expanding investigations across diverse geographical and demographic groups.

Although microbial changes in smokers are well documented, the mechanistic pathways linking these alterations to systemic disease remain poorly understood. Current clinical evidence is limited by small sample sizes and is unable to establish a causal relationship. Addressing this gap will require well-coordinated interdisciplinary research. Advances in this area are essential for the identification of early microbial biomarkers and the development of targeted therapeutic approaches.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript covers an important and timely topic linking tobacco smoking, oral microbiome changes, and systemic disease. It is generally well organized and gives a helpful overview of the literature. However, it would benefit from a more critical and deeper analysis to improve its scientific value.

1. The manuscript summarizes many studies, but the discussion is mostly descriptive. The authors report findings without enough critical evaluation of their quality, limitations, or implications. This is especially visible in sections on microbial diversity, where inconsistent results are presented without clear synthesis. A more critical approach, including discussion of possible sources of these differences (e.g., study design, populations, methods), would strengthen the review.
2. The manuscript acknowledges heterogeneity across studies; however, this issue is not explored in a systematic or analytically meaningful way. A more structured approach - such as grouping studies by sample type, analytical methods, or cohort characteristics - would enhance clarity and allow for more informed interpretation. 
3. The sections on links between oral microbiome changes and systemic diseases are mostly general and based on associations. They would benefit from a clearer discussion of possible biological mechanisms. It would also help to better distinguish between correlation and causation and include mechanistic explanations where possible.
4. The manuscript does not clearly show what is new compared to existing reviews. A review should not only summarize studies but also add value, for example by providing a clearer framework or stronger synthesis of the data. It would help to better explain what makes this review different and why it is needed.

The figure appears overly simplified and does not fully capture the complexity of the discussed relationships. Enhancing its level of detail and ensuring closer alignment with the content of the manuscript would improve its usefulness. The figure legend should also be expanded to provide a clearer and more precise explanation.

Transitions between sections - particularly between microbiome composition and systemic disease - are somewhat abrupt. Adding brief linking statements would improve the overall narrative flow and coherence.

Comments on the Quality of English Language

The manuscript is generally understandable; however, some sentences are overly long and complex, which may hinder readability.

Author Response

1.The manuscript summarizes many studies, but the discussion is mostly descriptive. The authors report findings without enough critical evaluation of their quality, limitations, or implications. This is especially visible in sections on microbial diversity, where inconsistent results are presented without clear synthesis. A more critical approach, including discussion of possible sources of these differences (e.g., study design, populations, methods), would strengthen the review.

Response:

Thank you for this important comment. We have revised sections 4.2, 4.3 and 4.4. and have combined section 4.2 with 4.4 to ensure that the microbial diversity is discussed in the same section. Subsection 4.2.1 goes on to discuss the variation in alpha and beta diversity. (lines 192-231 and 247-292).

• Microbial dysbiosis and shifts in diversity caused by Tobacco

The bacterial shifts caused by tobacco smoking across the phylum, genus, and species levels have been well documented [43–52]. These changes affect the well-balanced microbial communities, creating conditions for pathogenic bacteria to become dominant. Studies have been heterogeneous and often conflicting. However, most researchers report an abundance of pathogenic anaerobic bacteria and a reduction in beneficial commensals.

At the phylum level the most common findings in tobacco users were a relative abundance of Firmicutes and Actinobacteria with a reduction in Proteobacteria [43–51]. In contrast, a recent study from South Africa reported a lower abundance of Actinobacteria in smokers [52]. This study was performed on a Southern African mixed ancestry community of low to middle income using subgingival plaque. The ethnicity and sample type differed from other studies and may explain differences in the observed results. Interestingly, the role of genetic diversity has been debated. In another study from Southern Africa the composition of the oral microbiota from several genetically diverse populations was investigated. The authors reported significant variations in the microbial profiles but concluded that this was not related to ethnicity or livelihood but rather socioeconomic factors and poor health conditions [53]. In contrast to this, a study including low-income African Americans and Europeans reported significant differences between the two population groups. However, the effects of smoking were similar [48]. These articles emphasize the influence of the environment, health, and population characteristics on studies of the oral biome and emphasize the need for more standardized and representative research.

Due to differing study designs, methodologies, sample types, and collection sites, the findings reported at the genus and species level are more varied. Although most recent studies use 16S rRNA sequencing for analysis, there are differences in which hypervariable region of the gene is sequenced, the sequencing platform used, and the bioinformatics analysis [37,51]. These differences can all influence the results and final interpretation. In addition to methodology, sample sizes and the type of tobacco also influence the outcome [37]. The articles analyzed in this review describe varying sample sizes, ranging from fifty [46] to over four thousand [33]. In clinical research, small sample sizes can reduce the statistical power of a study, potentially leading to biased or non-representative results.

In this review, despite the varying results, most studies reported differences between smokers and non-smokers. Common to most reports was an abundance of Prevotella, Veillonella, Streptococcus, Actinomyces, and Fusobacterium with a depletion of Neisseria, Haemophilus, and Lautropia [33,45,47,54]. Although inconsistencies were observed at the species level, there is general agreement that smoking results in the growth of anaerobic bacteria, such as P. gingivalis, F. nucleatum, and T. denticola, which are important in biofilm formation [31,52,55]. The differences between “current” and “never” smokers were also investigated. These findings suggest that former smokers and individuals who have never smoked display a similar bacterial profile, indicating that the oral biota have the capacity to recover [48,50,51]. Table 1 summarizes the main studies analyzed in this review.

4.2.1. Alpha and Beta diversity

Alongside the shifts in bacterial communities, it is standard to document microbial diversity. Alpha diversity measures the variation of bacteria in a single sample and indicates the richness and evenness of the microbes present [26].

Studies measuring alpha diversity in tobacco users are often inconsistent and are dependent on several factors. [56]. One explanation is that bacterial diversity and composition vary significantly between oral niches, such as saliva, tongue, subgingival plaque, the palate, and tonsils. In support of this, a recent study compared the bacterial composition in swabs taken from the buccal mucosa, tongue, palate, and the floor of the mouth. 16S r RNA sequencing was used, and the results showed that the samples from the palate and tongue had higher alpha diversity scores compared to the other two sites. The bacterial communities also differed depending on the sample site. For example, buccal mucosa and the floor of the mouth had an abundance of Streptococcus, while the tongue exhibited greater levels of Neisseria and Rothia [57]. One advantage of this investigation was the standardized methodology used for all samples, thus enabling reliable comparisons. Further studies supported this and contrasted samples from the saliva and tongue, and reported differences in both the alpha diversity and microbial profiles between the two sample types [58]. Both examples emphasize the complexity of studying oral microbial shifts and the importance of being aware of the sampling site and type.

In a systematic review of thirty-six articles examining the effect of tobacco on the oral biota, most articles reported an increased alpha diversity in smokers. However, five observed no difference, while four observed a reduction. Despite these differences, all studies demonstrated significant changes to the microbiota in those who smoked [37]. This systematic review highlighted the heterogeneity of study designs, sample types, and methodology as limitations and emphasized the need for more standardized research.

A further explanation for differing measurements is that microbial diversity decreases with advancing age. This was clearly demonstrated in a large study of 4387 adults between 30 and 69 years of age. Both alpha and beta diversity decreased significantly with age, particularly in smokers over 60 years [33].

Unlike alpha diversity, beta diversity determines the differences between groups (E.g. smokers vs non-smokers). Beta-diversity measurements are more consistent than alpha diversity [59].

In this review, we grouped studies according to sample type and examined the variation in both alpha and beta diversity. Although all studies reported a significant difference in beta diversity, the alpha diversity varied significantly and was not dependent on sample type (Table 1). Reasons for the variation in alpha diversity could be explained by other factors, such as sample size and population group, which differed significantly across all studies. This analysis emphasized the importance of reporting both alpha and beta diversity alongside the microbial profile to improve interpretation and arrive at realistic conclusions.

Despite the heterogeneity in published studies, most researchers agree that smoking disrupts the oral microbiota, promotes the abundance of anaerobic pathogenic bacteria, and disturbs the symbiotic relationship between the host immune system and the microbiome.

2.The manuscript acknowledges heterogeneity across studies; however, this issue is not explored in a systematic or analytically meaningful way. A more structured approach - such as grouping studies by sample type, analytical methods, or cohort characteristics - would enhance clarity and allow for more informed interpretation

Response:

This is very relevant, and we acknowledge that the discussion of the results is not systematic or easy to read. We have revised the table and discussed the results in a more structured layout. See above comment and re-written section.

3.The sections on links between oral microbiome changes and systemic diseases are mostly general and based on associations. They would benefit from a clearer discussion of possible biological mechanisms. It would also help to better distinguish between correlation and causation and include mechanistic explanations where possible.

Response:

Thank you for this valuable comment. We have expanded in detail on the mechanistic evidence associated with disease and addressed the discussion on observational studies versus causal relationship within the limitation section and section 6.1.

Clinical evidence supporting a causal relationship is inadequate and the mechanisms leading to cardiovascular disease and myocardial infarction require further investigation. Researchers have attempted to study microbial profiles in the peripheral blood of individuals with cardiovascular disease. These have revealed an increase of diverse circulating microorganisms compared to controls [74-76]. However, these studies have been cross sectional with small sample sizes and were unable to establish the source of the organisms. These could have originated from the gut, oral cavity, or skin biome.

Mechanistic studies using well characterized animal models have explored the effects of specific oral bacterial antigens from P. ginivalis and F. nucleatum on endothelial dysfunction and immune regulation. These experiments have shown that bacterial components do cause endothelial dysfunction and systemic inflammation. Experiments have shown that the bacterial components activate pro-inflammatory B2-cells within lymph nodes and can induce a Th1 inflammatory response [77, 78]. This evidence sheds light on the mechanisms leading to inflammation and cardiovascular disease; however, additional comprehensive human studies are required.  

In vitro and animal studies have clarified how F. nucleatum can initiate colorectal cancer.  The adhesion molecules FadA and Fap2 are expressed on the bacterial surface. FadA attaches to the receptor E-cadherin on the epithelial cells of the intestine. This interaction activates b-catenin signaling pathways, which promote the proliferation of tumor cells. Meanwhile, Fap2 inhibits the activation of natural killer cells and CD8+ cytotoxic T cells allowing tumor cells to evade immunity and proliferate [85, 86]

4.The manuscript does not clearly show what is new compared to existing reviews. A review should not only summarize studies but also add value, for example by providing a clearer framework or stronger synthesis of the data. It would help to better explain what makes this review different and why it is needed.

Response:

It is agreed that it is important to state what this review adds to the current literature. This has been included in the conclusion which has been rewritten. (lines 458-470)

This narrative review contributes to the existing body of knowledge by providing an up-to-date synthesis of literature examining the effects of cigarette smoking on the oral microbiota, inflammation, and links to systemic disease. The findings emphasize the complexity of characterizing smoking related microbial shifts and highlight the need for large well-designed standardized studies. The inclusion of research focused on Southern African populations emphasizes the importance of expanding investigations across diverse geographical and demographic groups.

Although microbial changes in smokers are well documented, the mechanistic pathways linking these alterations to systemic disease remain poorly understood. Current clinical evidence is limited by small sample sizes and is unable to establish a causal relationship. Addressing this gap will require well-coordinated interdisciplinary research. Advances in this area are essential for the identification of early microbial biomarkers and the development of targeted therapeutic approaches.

5.The figure appears overly simplified and does not fully capture the complexity of the discussed relationships. Enhancing its level of detail and ensuring closer alignment with the content of the manuscript would improve its usefulness. The figure legend should also be expanded to provide a clearer and more precise explanation

Response:

Thank you for this observation and we agree that the diagram does not do justice to the article. The diagram below has been constructed as a flow diagram depicting six stages. These include from 1. initial cigarette exposure, 2. The change in the oral microenvironment, 3. The altered oral biota favoring pathogenic bacteria, 4. Activation of the immune response and local inflammation. 5. The movement of bacteria into circulation and 6. The development of systemic disease. The figure legend has been expanded to provide a comprehensive explanation. See attached article for new figure.

Figure 1. This flow diagram demonstrates how cigarette smoke alters the oral microenvironment to cause dysbiosis, local immune dysfunction, gum disease, and systemic inflammation. In panel one, the toxic chemicals and gases released from the burning tobacco cause reduced oxygen flow, heat, and oxidative stress. The changes caused to the oral microenvironment by the smoke are shown in panel two. These changes include reduced salivary and blood flow, epithelial damage, and immune suppression, which results in poor healing. In panel three, the effects of these environmental changes result in disruption in the balance of bacteria and other microorganisms, such as fungi and viruses. This dysbiosis leads to an abundance of pathogenic organisms, suppression of beneficial commensals, and virulent biofilm formation. Subsequent inflammation and gum disease are depicted in panel 4. Bacterial toxins activate the innate immune response resulting in the release of proinflammatory molecules, tissue damage, gingivitis, and periodontitis. Panel 5 illustrates how periodontal disease causes epithelial damage, facilitating the movement of bacteria into the circulation causing low grade inflammation and increasing the risk of systemic disease. Panel 6 demonstrates the outcome of this process and how the cycle of chronic inflammation caused by circulating microorganisms can affect distant organs.

6.Transitions between sections - particularly between microbiome composition and systemic disease - are somewhat abrupt. Adding brief linking statements would improve the overall narrative flow and coherence

Response:

6. Thank you for this observation. We have critically reviewed the entire manuscript to ensure that each section flows into the next. For example, the following statement has been inserted to link the section on microbial shifts and diversity with the section on how tobacco influences the immune system.

Despite the heterogeneity in published studies, most researchers agree that smoking disrupts the oral microbiota, promotes the abundance of anaerobic pathogenic bacteria, and disturbs the symbiotic relationship between the host immune system and the microbiome.

7.The manuscript is generally understandable; however, some sentences are overly long and complex, which may hinder readability

Response:

We have carefully re-read the manuscript ensuring that each sentence and section are readable and flow well. Any grammatical errors have been corrected.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

In your manuscript titled „Tobacco-Induced Oral Dysbiosis and Microbial Shifts: Mechanistic Links to Systemic Inflammation and Disease” you summarizes the current evidence on the impact of tobacco smoking on the oral microbiota (dysbiosis), induced microbial changes, dysfunctional immune response, and mechanistic links to periodontal disease and systemic conditions (cardiovascular disease, diabetes, gastrointestinal disorders, autoimmunity, neurodegenerative diseases, and pregnancy complications). The stated goal of the study is to identify the pathways by which tobacco toxins create a hypoxic, acidic, pro-inflammatory environment, favoring pathogenic anaerobic bacteria, and to propose directions for future research. This is a narrative review, not a systematic one and the assumed objectives are achieved clearly and coherently. The topic is current, original and the multidisciplinary approach is very valuable. The table and figure are well structured and easy to analyze. The references are numerous, from major databases and are well integrated into the manuscript and the analysis.

The study has several issues that need to be answered in order to potentially improve your manuscript:

1. You state that smoking increases the abundance of Firmicutes and Actinobacteria and decreases Proteobacteria, but the South African study by Prince et al. (2024) shows a decrease in Actinobacteria. How do you explain this difference and what role do ethnic/socioeconomic factors vs. sample type (saliva vs. subgingival plaque) play in these differences?
2. What is the specific contribution of bacterial metabolites (e.g. hydrogen sulfide, butyrate, LPS) vs. the direct effect of nicotine/ reactive oxygen species on neutrophil dysfunction in the progression to periodontitis? Are there data from in vitro or animal models that support a direct causality?
3. Why didn't you include data on the viral (bacteriophages) or fungus (Candida) microbiome in tobacco-induced dysbiosis? Don't they play a role in systemic inflammation?
4. Is there evidence that changes in the oral microbiota are fully reversible after smoking cessation or do residual effects persist?
5. Add 1–2 paragraphs on heated tobacco products and e-cigarettes that is an emerging topic in 2024–2026.
6. Add a separate Limitations section before the conclusion.

Author Response

1. You state that smoking increases the abundance of Firmicutes and Actinobacteriaand decreases Proteobacteria, but the South African study by Prince et al. (2024) shows a decrease in Actinobacteria. How do you explain this difference and what role do ethnic/socioeconomic factors vs. sample type (saliva vs. subgingival plaque) play in these differences?

Response:

This is an important observation, and we have added a possible explanation and discussion within section 4.2. lines 198-211.

At the phylum level the most common findings in tobacco users were a relative abundance of Firmicutes and Actinobacteria with a reduction in Proteobacteria [43–51]. In contrast, a recent study from South Africa reported a lower abundance of Actinobacteria in smokers [52]. This study was performed on a Southern African mixed ancestry community of low to middle income using subgingival plaque. The ethnicity and sample type differed from other studies and may explain differences in the observed results. Interestingly, the role of genetic diversity has been debated. In another study from Southern Africa the composition of the oral microbiota from several genetically diverse populations was investigated. The authors reported significant variations in the microbial profiles but concluded that this was not related to ethnicity or livelihood but rather socioeconomic factors and poor health conditions [53]. In contrast to this, a study including low-income African Americans and Europeans reported significant differences between the two population groups. However, the effects of smoking were similar [48]. These articles emphasize the influence of the environment, health, and population characteristics on studies of the oral biome and emphasize the need for more standardized and representative research.

2.What is the specific contribution of bacterial metabolites (e.g. hydrogen sulfide, butyrate, LPS) vs. the direct effect of nicotine/ reactive oxygen species on neutrophil dysfunction in the progression to periodontitis? Are there data from in vitro or animal models that support a direct causality

Response:

2. This is a very relevant and interesting question. We have addressed this in section 5 (lines 314-332)

Several studies have shown that as the oral environment becomes more anaerobic, bacterial components, including lipopolysaccharide, butyrate, leukotoxin, hydrogen sulfide, and gingipains, are released into the gingival tissue and stimulate the innate immune response. Innate mediators, such as complement, neutrophils, macrophages, and natural killer cells, are activated. Bacterial toxins bind to Toll like receptors 2 and 4, expressed on monocytes and granulocytes, leading to the secretion of pro-inflammatory cytokines. These include tumor necrosis factor α (TNF-α), C-reactive protein, interleukins-1, 6, 8, and 17, and nuclear factor kappa B ligand (RANK-L) [64-66]. The persistent release of these molecules results in a vicious cycle of immune activation, which eventually causes immune exhaustion and dysregulation.

This situation is compounded by nicotine, which has been shown to impair neutrophil function, skew monocyte phenotype towards the proinflammatory M1 subtype, and cause bone loss [66-68]. Nicotine increases neutrophil chemotaxis, but impairs phagocytosis and the killing of pathogens. Evidence suggests that nicotine can cause the release of granule content and stimulate neutrophil extracellular traps (NETS). However, this does not improve bacterial killing but rather exacerbates inflammation and tissue damage [69]. The anaerobic environment prevents neutrophils from producing ROS and super oxides which further inhibit the host from clearing infection and increases the risk of periodontitis [63].

3.Why didn't you include data on the viral (bacteriophages) or fungus (Candida) microbiome in tobacco-induced dysbiosis? Don't they play a role in systemic inflammation?

Response:

3.This comment is correct and valid. The other components of the oral biome such as viruses and fungi do play an important role in initiating inflammation. We, however, focused on the bacterial changes as more research has been done in this area. We have, however, included this aspect as a limitation and a focus of future research. We have also added the role of fungi and viruses into figure 1

One limitation of this narrative review is that it concentrated on the bacterial components of the oral biome. However, there is growing evidence that viruses, parasites, and fungi can also initiate inflammation. Although there is less evidence, in vitro and animal studies have established that smokers are at higher risk of developing fungal infections, such as oral candidiasis, which can lead to periodontitis and systemic inflammation [38,103]. Further research adopting comprehensive approaches including cell culture, microscopy and multi-omics techniques are needed to understand the complex interactions of these microorganisms and how they contribute to the pathogenesis of disease

4. Is there evidence that changes in the oral microbiota are fully reversible after smoking cessation or do residual effects persist?

Response

Thank you for this question. The answer is yes; there is evidence that these changes are reversable although they can take many years. Studies comparing former smokers and non-smokers have shown that microbial profiles are similar. This evidence can be seen in references 48,50 and 51. (lines 447-450)

5. Add 1–2 paragraphs on heated tobacco products and e-cigarettes that is an emerging topic in 2024–2026

Response:

It is acknowledged that this review did not address the growing issue of e-cigarettes. This has been addressed in the limitations section where 1-2pararaphs have been inserted.

 As traditional cigarette smoking declines, the popularity of E-cigarettes and vaping has increased, especially among the youth. This review did not address these products; however, there is growing evidence that the aerosols produced from E-cigarettes contain several toxic compounds [68]. These chemicals disrupt the oral microbiome and promote the growth of opportunistic pro-inflammatory organisms. Vaping has been shown to disrupt the epithelial barrier, impair the immune response, and cause oxidative stress. These changes can lead to chronic systemic inflammation [104]. Like the analysis of traditional cigarette smoking, standardized studies are required to account for the variation in products including chemical composition, flavorings, and nicotine concentration

6.Add a separate Limitations section before the conclusion

Response:

Thank you for this very valuable suggestion. A limitations section has been added. See lines 460-504..

Although there is increasing evidence indicating a risk of systemic diseases linked to smoking related oral dysbiosis, most clinical studies demonstrate an association rather than a causal relationship. Many of the published studies are cross sectional and observational with a lack of longitudinal data, which could track progression of disease over time. To address these challenges multi-omics approaches are required to fully clarify the mechanisms leading to systemic conditions. This may include detecting shifts in microbial communities linked to specific conditions and the identification of possible oral biomarkers, which could predict the risk of disease development. Alternatively, these microbial signatures could be used to formulate targeted therapies to prevent or treat associated conditions [100-102]. As the toxins and chemicals in cigarettes can cause systemic inflammation and disease, further research on the direct effects of these compounds on the immune system and organs is required.  

A major limitation of previous research is the insufficient control of confounding factors, such as lifestyle, dental hygiene, diet, age, and alcohol intake. These all cause dysbiosis and disease, making interpretation and conclusions difficult. Compounding this is the lack of standardization in methodology, study designs, sample type, and sample size [37]. A further inconsistency and barrier to reliable interpretation is the variation in geographical regions and population groups. Ethnicity, socioeconomic factors and access to health care can influence the development of periodontitis and systemic disease [53]. To obtain a more consistent picture of tobacco-induced alterations to the oral biome, large, collaborative, standardized investigations, utilizing the same study design and methodology are recommended. Confounding factors, such as age, sex, type of tobacco, diet, and lifestyle, would need to be recorded and accounted for during statistical analysis to obtain realistic and comparable results.  

One limitation of this narrative review is that it concentrated on the bacterial components of the oral biome. However, there is growing evidence that viruses, parasites, and fungi can also initiate inflammation. Although there is less evidence, in vitro and animal studies have established that smokers are at higher risk of developing fungal infections, such as oral candidiasis, which can lead to periodontitis and systemic inflammation [38,103]. Further research adopting comprehensive approaches including cell culture, microscopy and multi-omics techniques are needed to understand the complex interactions of these microorganisms and how they contribute to the pathogenesis of disease.

As traditional cigarette smoking declines, the popularity of E-cigarettes and vaping has increased, especially among the youth. This review did not address these products; however, there is growing evidence that the aerosols produced from E-cigarettes contain several toxic compounds [68]. These chemicals disrupt the oral microbiome and promote the growth of opportunistic pro-inflammatory organisms. Vaping has been shown to disrupt the epithelial barrier, impair the immune response, and cause oxidative stress. These changes can lead to chronic systemic inflammation [104]. Like the analysis of traditional cigarette smoking, standardized studies are required to account for the variation in products including chemical composition, flavorings, and nicotine concentration.

Finally, there is a lack of longitudinal studies that investigate changes in the oral biota over time, particularly after the cessation of smoking. These studies should include the time to full recovery and the impact of improved oral hygiene, probiotics, and prebiotics on the restoration of healthy micro-organisms [100

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

manuscript can be now accepted 

Author Response

1. manuscript can be now accepted

Response: Thank for this and all the valuable comments which led to an improvement in this article.

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you to the Authors for revising the manuscript and addressing the previous comments. The paper has clearly improved.

The sections on mechanisms linking the oral microbiome with systemic diseases are now more detailed and helpful. It is also good that the Authors have pointed out the difference between association and causation. The revised figure and its description are clearer and better reflect the content of the manuscript.

The reorganization of the sections on microbial diversity has also improved the readability. The Authors now mention several factors that may explain differences between studies, such as methodology, sample type, and population, which is important.

However, the manuscript is still partly descriptive. Although the sources of heterogeneity are mentioned, the discussion could be more critical. It would be helpful if the Authors more clearly indicated which results are more consistent across studies and which should be interpreted with caution.

In addition, the explanation of what is new in this review is still quite general. Please more clearly state what this paper adds compared to existing reviews.

I also suggest indicating in the title and keywords that this is a narrative review, for better clarity.

Importantly, the methodological section still lacks key information. It is not clear how many studies were included, what time period was covered (it is stated that studies up to February 2026 were included, but the starting point is not specified), and how the literature search was conducted in detail. Information such as the number of records identified in each database, duplicates, and the final number of included studies is missing. Even for a narrative review, providing at least basic transparency in the search strategy would strengthen the manuscript.

Overall, the manuscript is improved and close to being acceptable, but some further strengthening of the discussion and clarification of the methodology would be beneficial.

Author Response

1.However, the manuscript is still partly descriptive. Although the sources of heterogeneity are mentioned, the discussion could be more critical. It would be helpful if the Authors more clearly indicated which results are more consistent across studies and which should be interpreted with caution.

Response:

We thank the reviewer for this valuable comment and writing this review has been a steep learning curve. We have strengthened critical appraisal throughout the manuscript and included a table indicating which results are consistent and those which are not and should be interpreted with caution. We have also added a dedicated discussion paragraph highlighting the robust findings compared to the more inconsistent findings.

Although the evidence for tobacco associated oral biota changes is moderately strong at the phylum and genus level, species level shifts and diversity measurements are less consistent across studies (Table 2). After section 4.2, lines 239-241

In summary, while most studies reported microbial alterations towards anaerobic pathogenic bacteria in smokers, alpha diversity measurements are inconsistent and should be interpreted with caution as they are influenced by several factors including sampling sites, population diversity and methodology. (Table 2) After section 4.2.1 lines 294-297

Table 2: Summary of Consistency and Strength of Evidence in Studies of Tobacco-Associated Oral Dysbiosis

Finding	Consistence	Interpretation & Influencing factors
Abundance of anaerobic bacteria	High	Robust finding
Phylum changes	Moderate	Population differences
Genera level shifts	Moderate	Sequencing method, sample site, study design.
Species level shifts	Low	Methodology, sample site, sample type, study design
Beta diversity differences	High	Reliable findings
Alpha diversity measurements	Low	Sample site, method, population diversity, age

Together these studies support the role of pathogenic bacteria moving from the oral cavity to the peripheral blood and causing inflammation, endothelial dysfunction and atherosclerosis. However, the specific role of tobacco associated dysbiosis remains less defined as the current evidence is mostly associative and does not demonstrate a causal relationship. Lines. Section 6.1, Lines 388-392.

9. Discussion

 Despite the limitations, several findings in smokers compared to non-smokers are consistent and robust. These include the shift to gram-negative anaerobic bacteria and the abundance of genera including Prevotella, Veillonella and Fusobacterium. The significant differences in beta diversity are similar across all studies however alpha diversity measurements and changes at the species level are diverse and inconsistent, making interpretation difficult. These results are most likely influenced by study designs, sampling sites, population differences and methodology approaches highlighting the need for large standardized collaborative studies. Lines 529-537.  

2.Thank you for this suggestion and yes, we agree that the title should be changed to provide more clarity.

The new title is:

Tobacco-Induced Oral Dysbiosis and Microbial Shifts: A Narrative Review of Their Role in Systemic Inflammation and Disease.

3.We agree that what is new and different in this review is not strongly stated. A new conclusion section has been written. See lines 539-551

This review provides a mechanistic integrated synthesis of literature linking tobacco-related changes to the oral microenvironment and dysbiosis, immune dysregulation and systemic disease. Unlike previous reviews which focus largely on microbial shifts and periodontal inflammation, this review critically examines how the environment, the host and microorganisms converge to initiate systemic disease. It further advances the topic by providing data from diverse, underrepresented Southern African populations demonstrating the importance of socioeconomic and environmental factors in the variability of oral microbial profiles.  

Microbial changes in smokers are well documented but the mechanistic pathways linking these alterations to systemic disease remain poorly understood. Addressing this gap will require well-coordinated interdisciplinary research. Advances in this area are essential for the identification of early microbial biomarkers and the development of targeted therapeutic approaches.

4.Importantly, the methodological section still lacks key information. It is not clear how many studies were included, what time period was covered (it is stated that studies up to February 2026 were included, but the starting point is not specified), and how the literature search was conducted in detail. Information such as the number of records identified in each database, duplicates, and the final number of included studies is missing. Even for a narrative review, providing at least basic transparency in the search strategy would strengthen the manuscript.

Response:

Thank you for this comment and we have extended the methodology section to explain how the articles were sourced and selected. Lines 90-108

A literature search from 2010 up to February 2026 was performed using PubMed, Google Scholar, and Science Direct databases. Original research studies and review articles relevant to the topic were selected and examined to acquire current evidence. Key words used to search the databases included “smoking”, “tobacco”, “cigarettes”, “oral biome”, “dysbiosis”, and “systemic disease”. A Boolean strategy, with words such as “And” and “Or” was employed to retrieve articles specific to the topic.

For some sections of the article such as those examining the effects of tobacco toxins and nicotine, the search had to go back to 1995 to obtain original articles.  Population statistics on the global prevalence of smoking and in South Africa were obtained from World Health Organization (WHO) statements and fact sheets.

In the first search there were several duplicates and pre-prints across the three databases. These were excluded before proceeding. Publications were further refined and included if they were peer-reviewed and published in English while non-peer-reviewed manuscripts were excluded. The articles were then sorted into those relevant for each of the various subthemes and the information was examined to formulate a comprehensive review.

Original research articles addressing the effects of traditional cigarette smoking on the oral biota were obtained from an additional search using PubMed spanning 10 years (2016 to 2026). Articles focused on other forms of tobacco including E-cigarettes were excluded. Twelve manuscripts which were representative of studies from diverse regions of the world were further analyzed for similarities and differences.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have adequately addressed all my comments and revised it accordingly.

The manuscript is significantly improved upon the readability and clarity of the manuscript. It is well-structured, designed, and referenced. Therefore, I have no further comments.

Author Response

The authors have adequately addressed all my comments and revised it accordingly.

The manuscript is significantly improved upon the readability and clarity of the manuscript. It is well-structured, designed, and referenced. Therefore, I have no further comments.

Response:

Thank you very much for all the very helpful and valuable comments that have led to the improvement of this article.

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for your revisions and for addressing many of the previous comments. The manuscript has improved; however, a few issues still require clarification.

First, the methodology section would benefit from greater transparency. While you have expanded the description of the search strategy, it is still unclear how many records were identified, screened, and ultimately included in the review. Please further clarify the study selection process by providing these numbers, even in a simplified form.

Second, I recommend reconsidering the structure of Section 9, currently labeled as “Discussion.” In its present form, this section serves more as a summary of key findings than as a full discussion. It would be more appropriate to incorporate this paragraph into the preceding section as a concluding summary, rather than presenting it as a standalone Discussion.

Author Response

1.First, the methodology section would benefit from greater transparency. While you have expanded the description of the search strategy, it is still unclear how many records were identified, screened, and ultimately included in the review. Please further clarify the study selection process by providing these numbers, even in a simplified form.

Response:

Thank you once again for highlighting the importance of a comprehensive methodology section in a review article of any type. We have rewritten this section to be as transparent as we can. We used three databases but unfortunately did not keep a record of how many articles were obtained from each one. This is a lesson learned and in future will record those numbers. However, we have gone back and recorded the total articles identified and the ones that were excluded. This has now been written into a new methodology section which describes how we arrived at the final 104 articles. Please see below and lines 89-111.

A comprehensive literature search was performed for publications from 2005 to February 2026 using PubMed, Google Scholar and ScienceDirect databases. The initial search identified 258 articles which included original research studies and review articles relevant to the topic. Key words used to search the databases included “smoking”, “tobacco”, “cigarettes”, “oral biome”, “dysbiosis”, and “systemic disease”. A Boolean strategy, with words such as “And” and “Or” was employed to refine and retrieve articles specific to the review.

Additional targeted searches were performed to identify original studies investigating tobacco-toxins and their effects on the oral environment published between 1995 and 2026 as well as recent population statistics (2020-2026) on the global and South African prevalence of smoking. These searches identified a further nine original research articles and five statements and fact sheets published by the World Health Organization and the South African Department of Health resulting in a total of 272 manuscripts.

Non-peer-reviewed articles, duplicate manuscripts, preprints and articles focused on alternative forms of tobacco or E-cigarettes were excluded. The documents were further refined by excluding review articles containing substantial overlapping information. Following this process, 106 manuscripts remained including 40 review articles, 62 original research articles, three fact sheets and 1 consensus report. The selected review articles comprised of narrative reviews, systematic reviews and meta-analyses. The articles were subsequently grouped according to thematic subtopics, and information was extracted to formulate a comprehensive review of the literature. After extensive revision of the manuscript a total of 104 manuscripts were included in the final review.

2.Second, I recommend reconsidering the structure of Section 9, currently labeled as “Discussion.” In its present form, this section serves more as a summary of key findings than as a full discussion. It would be more appropriate to incorporate this paragraph into the preceding section as a concluding summary, rather than presenting it as a standalone Discussion.

Response:

Thank you for this comment and we created this section as a suggestion from another reviewer. We have now included this in the previous section and agree that it flows much better. See lines 528-535.