Supragingival dental biofilm microbiomes of tobacco heating system smokers, cigarette smokers and non-smoker

Supragingival dental biofilm microbiomes of tobacco heating system smokers, cigarette smokers and non-smoker | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Supragingival dental biofilm microbiomes of tobacco heating system smokers, cigarette smokers and non-smoker Elvis Božac, Jurica Žučko, Alen Braut, Stjepan Špalj, Romana Peršić Bukmir, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8302044/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Mar, 2026 Read the published version in Clinical Oral Investigations → Version 1 posted 9 You are reading this latest preprint version Abstract Objectives The study compared the bacterial composition of supragingival dental biofilm (SDB) microbiome of THS smokers (THSS), cigarette smokers (CS) and non-smokers (NS). Materials and Methods In the present cross-sectional study a total of 60 subjects were divided in three groups: THS smokers (THSS), cigarette smokers (CS) and non-smokers (NS). SDB samples were collected, DNA was isolated and used for 16s rRNA gene hypervariable region amplicon, next generation sequencing. Determination of SDB bacterial taxa composition was performed through bioinformatic pipelines and analyses. Results Significant differences were found in the bacterial composition of SDB microbiomes between the examined groups on all taxonomic levels. The most significant differences were detected at genus level, specifically among the genera: Actinobaculum (p = 0.004), Avibacterium (p = 0.015), Alysiella (p = 0.016), Propionibacterium (p = 0.025) and Haemophilus (p = 0.030). Significant differences have also been found on the species level, with the most significant regarding Alysiella filiformis (p = 0.006), Avibacterium paragallinarum (p = 0.015), Prevotella multiformis (p = 0.016), Lactobacillus salivarius (p = 0.018), and Actinomyces massiliensis (p = 0.022). Conclusions The compositions of SDB of THSS, CS and NS are different, and due to increased anaerobic bacteria abundance with cariogenic properties, the dysbiosis pattern and pathogenic potential of THSS and CS dental biofilms compared to NS is considerably increased. Clinical Relevance: Due to bacterial composition the SDB of THSS and CS could have a higher pathogenic effect than the SDB of NS dental caries dental plaque microbiota smoking smoking devices Figures Figure 1 Figure 2 Figure 3 1. Introduction The oral cavity is an environment that provides favorable conditions for bacterial growth, as well as hard and soft tissue surfaces suitable for microbial attachment and biofilm formation. Among the oral microbiota (OM) over 700 bacterial species, along with viruses, fungi, archaea and protozoa can be found. An oral sample from a single individual typically contains a fraction of this spectrum, usually between 50 and 200 different bacterial species. OM composition is highly dependent on numerous host factors, such as age, genetics, dietary habits, etc. [ 1 ]. Although we are born with sterile oral cavities, bacteria and other microorganisms begin to establish themselves as commensals during delivery in newborns. Commensal microorganisms in oral cavity serve as a protective role by preventing colonization of pathogenic microorganisms and as such have an important role in maintaining bacterial homeostasis. Furthermore, it is speculated that the constant presence of bacterial biofilm has a role in stimulating the development and proper response of the hosts’ immune system as well as regulating dietary nitrates [ 2 ]. According to the Human Oral Microbiome Database (eHOMD) the prevailing bacterial species presented in oral biofilms belong to the genera Streptococcus, Veillonella, Selenomonas, Gemella, Fusobacterium, Prevotella, Lactobacillus, Neiseria, Dialister, Actinomyces, Capnocytophaga, Granulicatella, Campylobacter, Treponema, Enterococcus, Eubacterium, Atopobium, Bacterioides, Propionibacterium Rothia and Haemophilus [ 1 ]. These bacteria, although normally present as commensals in the oral cavity, are prone to causing microbiome composition shifts – dysbiosis, when exposed to certain favorable conditions. This is most evident in dental biofilms whose prolonged presence on tooth surfaces can cause diseases such as dental caries and/or periodontitis. Even though presence of biofilms is the main prerequisite for dental and periodontal diseases, other environmental factors, such as pH level, availability of nutrients, oxygen concentration etc., also play an important role in disease development [ 1 – 5 ]. Tobacco smoking has been widely researched as a factor influencing OM composition and microbial activity in the oral cavity and has been widely associated with higher rates of caries and periodontitis. Findings of several in vitro studies confirm this. Their results show that components of cigarette smoke, especially nicotine, have adverse effects on the metabolism and proliferation of cariogenic bacteria, depending on their concentration [ 6 – 9 ]. Studies in vivo also confirm these findings since they report an increased incidence of caries lesions in smokers compared to non-smokers [ 10 – 12 ]. Smoking is also considered as a risk factor in the development of periodontal diseases [ 13 ]. Recent 16s rRNA sequencing studies confirm that consumption of tobacco products influences microbial diversity in supragingival and subgingival dental biofilms. Such studies report an increase in bacterial abundance of certain species belonging to the genera Streptococcus , Lactobacillus , and Klebsiella in supragingival dental biofilms (SDB) of smokers. Such studies also report and increased abundance of species belonging to the genera Prevotella, Treponema, Streptococcus, Veillonella , and Tanarella in the composition of subgingival dental biofilms of smokers when compared to non-smokers [ 14 , 15 ]. Tobacco heating systems (THS) are a relatively new product that has been on the market for the last decade. The products are advertised as being less unhealthy than cigarettes since their aerosol, which contains the same components as cigarette smoke, has significantly reduced concentrations. [ 16 , 17 ]. To the authors’ knowledge, their influence on the microbial composition of dental biofilms has not been widely researched. The aim of the present study was to investigate and compare the bacterial diversity and abundance in the SDB of THS smokers (THSS), cigarette smokers (CS) and non-smokers (NS). Since THS aerosol contains the same components as cigarette smoke, a hypothesis was formed which stated that no significant differences in bacterial diversity and abundance would be found among the smoker groups (THSS v. CS) but significant differences would be found between the smokers groups and non-smokers (THSS v. NS, and CS v. NS) 2. Methods 2.1. Subject recruitment In the present cross sectional study, subjects were distributed in 3 different groups: THS smokers (THSS), cigarette smokers (CS) and non-smokers (NS). The recruited subjects were patients referred to the Dental clinic, Clinical Hospital Centre Rijeka, Croatia for consultation and/or treatment from January to December 2023. Subject recruitment was performed in a consecutive method, and since patients that consumed THS were less frequent, they were recruited first and the other groups were matched to them according to age and gender. All participants have read and signed an informed consent form. This research was conducted according to fundamental ethical and bioethical principles which are stated and required by the Nurnberg code and the newest revision of the Declaration of Helsinki. Sample sizes were determined through an online tool: Ristrl, R. Sample Size Calculator Version 1.061. Available online: https://homepage.univie.ac.at/robin.ristl/samplesize.php (accessed on 1 February 2022). The conducted calculations were performed through data obtained from a pilot study regarding clinical parameters (Decayed-Missing-Filled Tooth (DMFT) index, Decayed-Missing-Filled Tooth surface (DMFTs) index, Full Mouth Plaque Score (FMPS) and Full Mouth Bleed Score (FMBS). The pilot study was conducted on 30 subjects (10 THSS, 10 CS and 10 NS; with each group consisting of 9 female (90%) and 1 male (10%) participants) by our study group. The pilot study results were not published. The sample size was determined for one way analysis of variance using mean and standard deviation data of the DMFTs index with a power level of 80% and a significance level of 0.05. The DMFTs index mean values were 17.3 for THSS, 24.3 for CS and 9.8 for NS, while standard deviation values of the DMFTs index were 13.2 in the THSS group, 20.2 in the CS group and 5.5 in the NS group. A mean value of standard deviation values was calculated and used to determine the required sample size, with dropout rates accounted. The calculation determined a minimum of 20 participants per group. 2.2. Inclusion/Exclusion criteria THSS or CS group subjects had to consume either THS or cigarettes exclusively for at least 6 months, and had to have consumed more than 100 cigarettes/tobacco heated sticks [ 18 ]. Since a large variety of THS products and cigarettes are currently present on the market, only subjects that consumed the THS product IQOS (Phillip Morris, Richmond, USA) or Marlboro Gold cigarettes (Marlboro, Richmond, USA) were recruited to participate. Subjects that have declared to never have smoked in their lifetime were considered to be NS. Exclusion criteria were: habits/conditions that have a confirmed effect on microbial proliferation, such as constant or recent use of oral antiseptics, systemic antibiotic treatment which has been taken at least 3 months prior to examination, systemic use of immunosuppressive medications, diabetes mellitus and autoimmune diseases and active orthodontic treatment [ 19 – 21 ]. Furthermore, patients that had fewer than 10 teeth were not eligible for participation [ 15 ]. 2.3. Microbial diversity assessment To determine the microbial diversity and abundance in SDB of the examined groups, hypervariable regions (V2-V9) of bacterial 16s rRNA gene were analysed through Next generation sequencing (NGS) using Ion Torrent technology (Thermo Fisher Scientific, Waltham, MA, USA). 2.4. Collection of supragingival dental biofilm samples SDB sample collection was performed during morning hours (between 8.00 AM and 10.00 AM). To ensure biofilm samples were of similar maturity, subjects were instructed not to brush their teeth in the morning before sample collection and to brush their teeth the evening prior, between 8.00 PM and 10.00 PM. Samples collection was performed using a set of sterile Gracey curettes from all surfaces of maxillary and mandibular first molars on both sides. If subjects were missing any of their first molars, samples were collected from second molars, or alternatively, if those were also missing from second maxillary/mandibular premolars. The samples were stored in a 1,5ml sterile Eppendorf LoBind tubes with storage medium consisting of 750 µL Tris-EDTA buffered solution (pH = 7,4). The collected samples were stored on -20°C prior to DNA isolation. 2.5. DNA isolation DNA was isolated from the collected SDB samples with the Nucleospin Tissue kit (Macherey Nagel, Duren, Germany). A modified protocol for bacterial DNA isolation was used as described in the products instruction manual. The concentration and purity of genomic DNA were measured with a Qubit 4 Flourometer (Thermo Fisher Scientific, Waltham, MA, USA). 2.6. Library preparation, sequencing, bioinformatics and statistical analysis The 16s rRNA gene’s hypervariable regions (V2-V9) were amplified and libraries were created using the Ion 16S Metagenomics Kit (ThermoFisher Scientific, Waltham, MA, USA) according to manufacturers protocol. Quality and quantity of purified PCR products and prepared libraries was controlled on a Agilent 2100 Bioanalyzer instrument with Agilent High Sensitivity DNA kit (Agilent Technologies, Santa Clara, California, USA), according to manufacturers protocol. Purified barcoded library concentrations were diluted to 40 pM and pooled (25 µl of each sample library). Pooled libraries were then loaded into appropriate Ion Chef™ Library Sample Tube on the Ion Torrent Ion Chef (Thermo Fisher Scientific, Waltham, MA, USA) instrument for template preparation and chip loading, with the Ion Torrent Ion 530 Kit –Chef (ThermoFisher Scientific, Waltham, MA, USA). For loading the 400 bp chef protocol was selected in the device settings. Sequencing of the loaded chip was performed on an Ion S5 Ion GeneStudio S5 System (ThermoFisher Scientific, Waltham, MA, USA) instrument. Resulting amplicon sequences were separated into individual variable regions using the MetagenomicsPP plugin in the Torrent Suite software [ 22 ]. Resulting .fastq files corresponding to each variable region were imported into QIIME2 (version 2022.2) [ 23 ] using a manifest file and each data file was quality filtered and dereplicated using QIIME2 DADA2 plugin with pyro method [ 24 ]. Obtained feature tables and representative sequences of each variable region were merged. Taxonomy was assigned to merged representative sequences using the VSEARCH-based consensus taxonomy classifier[ 25 ] implemented in QIIME2. Sequences were searched against SILVA 132 database clustered at 99% sequence similarity. Assigned taxonomies were visualized as bar plots and exported as csv file at species level using the QIIME2 pipeline. Exported .csv file was used for visualization and statistical analysis of microbiome data using the MicrobiomeAnalyst 2.0 platform[ 26 ] and R programming language. Microbiome diversity has been assessed through Alpha diversity metrics (Observed taxa index), Beta diversity metrics (Bray Curtis index along with PERMANOVA for statistical analysis) and Heat tree analysis. Statistical analysis was also done using IBM SPSS Statistics 29 software (IBM, New York, USA) with p < 0.05 set statistical significance level. Conventional descriptive statistical methods have been used for analysis of collected data. Normality of collected data distribution was assessed through the Shapiro-Wilk test. Since data did not have normal distribution, the variables are expressed through median values and interquartile ranges. Kruskall-Wallis test was used for analysis of data on continuous scales. Mann-Whitney test with Bonferroni correction for multiple comparisons was used for post-hoc analysis. 3. Results 3.1. Sample descriptive statistics A total of 60 subject participated in this study. Six were male (10%) and 54 were female (90%). The sample was comprised of predominantly young subjects (median = 29 years). After bioinformatics processing the total number of reads across all samples was 5074107. The average number of reads per sample was 84568. 3.2. Microbial diversity Alpha diversity was tested through the observed taxa index on species level. Mean and standard deviation values of the observed taxa index were: 163.25 ± 31.67 for THSS group, 159.35 ± 24.22 for CS group and 164.2 ± 20.37 for NS group. Statistical analyses were performed through the Mann—Whitney and Kruskal-Wallis test in the MicrobiomeAnalyst 2.0 web tool and revealed no significant significant differences among the THSS, CS and NS groups (p = 0.489). Beta diversity was tested through the Bray-Curtis index on species level and PERMANOVA was used to test statistical significance. No significant differences were found among the examined groups (F-value: 1.0512; R 2 : 0.035571; p = 0.35). 3.3. Taxonomic characterization Through taxonomic identification a total of 8 phyla, 21 classes, 30 orders, 52 families, 88 genera and 232 species were identified across all samples. Among the identified species 205 were identified in the THSS group, 203 in the CS group and 192 in the NS group. The examined groups had a similar composition of bacteria at higher taxonomic levels with the most common being: Firmicutes, Actinobacteria, Proteobacteria, Bacterioidetes , and Fusobacteraia at the Phylum level; Actinobacteria, Bacillli, Bacteroidia, Gammaproteobacteria and Fusobacteria at the Class level; Actinomycetales, Lactobacillales Bacteroidales, Fusobacteriales and Pasteurellales at the Ordo level and Streptococcaceae, Actinomycetaceae, Pasteurellaceae, Prevotellaceae and Corynebacteriaceae at the Family level. Heat tree analyses were performed to assess differences in microbial diversity at higher taxonomic levels (up to Family level) between the examined groups. The results were based on the Wilcoxon rank sum test at the significance threshold of p < 0.05. The results are shown in Figs. 1 – 3 . The nodes represent taxa while their size represents abundance and branches describe taxonomic hierarchy. The colors indicate differences among groups and nodes with written taxa names indicating a statistically significant difference among the compared groups. The THSS group was more abundant in bacteria belonging to the Rhodospirillaceae, Spirochetaceae, Propionibacteriaceae and Ardenscatenaceae families compared to the NS group, although the NS group was more abundant in bacteria belonging to the Campylobacteriaceae family. The CS group was more abundant in bacteria belonging to the Bacillaceae , Thermogemmatisporaceae , Rhodobacteraceae , Rhodospirillaceae , Sphingomonadaceae and Ardenscatenaceae families compared to the NS group. When comparing the two smoker groups the THSS group was more abundant in bacteria from the Rhodobacteriaceae, Carnobacteriaceae and Aerococcaceae family. The CS group was more abundant in bacteria belonging to the Propionibacteriaceae family. Differences in microbial abundance on lower taxonomic levels (genus and species) were tested through the Kruskal-Wallis test. Genera in which significant differences in abundance have been found are expressed in Table 1 , while the results on species level are shown in Table 2 . 1. Differences of bacterial abundance on genus level Table 1 Significant differences in bacterial abundance at genus level Genus Number of samples detected with the genera (at least 1 read) THSS Mean (%) THSS Std. Dev. (%) CS Mean (%) CS Std. Dev. (%) NS Mean (%) NS Std. Dev. (%) Effect size η 2 p Highest abundance in THSS group Propionibacterium 59 1,198 1,413 0,570 0,643 0,439 0,486 0,111 0,025 Alysiella 8 0,016 0,045 0,002 0,006 0,000 0,000 0,073 0,016 Highest abundance in CS group Highest abundance in NS group Haemophilus 60 1,773 3,376 1,641 1,899 2,305 1,640 0,014 0,030 Actinobaculum 53 0,745 0,655 0,248 0,342 1,016 1,539 0,094 0,004 Avibacterium 4 0,000 0,000 0,000 0,000 0,193 0,398 0,135 0,015 * The results are expressed as ratios (% of total reads) On genus level significant differences were found in abundance of genera (Table 1 .): Actinobaculum (p = 0,004), Avibaterium (p = 0,015), Alysiella (p = 0,016), Propionibacterium (p = 0,025) and Haemophilus (p = 0,030). The Actinobaculum genus was predominantin the NS group (1.02% ± 1.54%) and least abundant in the CS group (0.25%±0.34%). Bacteria from the Avibacterium genus were detected only in the NS group (0.19%±0.40%). In the two smoker groups the genus Alysiella was detected with increased prevalence in the THSS group (0.02%±0,05%) compared to CS group (0.002.%±0.006%). The Propionibacterium genus was dominant in the THSS group (1.20% ± 1.41%), and least abundant in the NS group (0.44%±0.47%). Bacteria from the Haemophilus genus were most abundant in the NS group (2.31%±1.64%), and least abundant in the CS group (1.64%±1.90%). 2. Differences of bacterial abundance on species level Table 2 Significant differences in bacterial abundance at species level Species Number of samples detected with the species (at least 1 read) THSS Mean (%) THSS Std. Dev. (%) CS Mean (%) CS Std. Dev. (%) NS Mean (%) NS Std. Dev. (%) Effect size η 2 p Highest abundance in THSS group Alysiella filiformis 7 0,016 0,045 0,000 0,002 0,000 0,000 0,079 0,006 Streptococcus thermophilus 7 0,022 0,089 0,000 0,000 0,009 0,025 0,028 0,025 Propionibacterium propionicum 54 0,649 0,670 0,357 0,383 0,226 0,272 0,123 0,027 Prevotella pleuritidis 25 0,254 0,799 0,105 0,179 0,042 0,121 0,033 0,029 Streptococcus pyogenes 10 0,010 0,032 0,009 0,015 0,000 0,000 0,046 0,035 Kingella oralis 55 0,602 0,573 0,419 0,595 0,573 0,347 0,024 0,038 Highest abundance in CS group Streptococcus lactarius 44 0,030 0,028 0,044 0,028 0,023 0,027 0,088 0,050 Highest abundance in NS group Avibacterium paragallinarum 4 0,000 0,000 0,000 0,000 0,193 0,398 0,135 0,015 Prevotella multiformis 25 0,131 0,409 0,115 0,461 0,134 0,200 0,001 0,016 Lactobacillus salivarius 5 0,000 0,000 0,003 0,013 0,061 0,118 0,146 0,018 Actinomyces massiliensis 59 0,640 0,503 0,643 0,797 1,222 0,845 0,123 0,022 Haemophilus parainfluenzae 53 1,460 3,374 1,165 1,897 1,981 1,612 0,019 0,028 Streptococcus caballi 10 0,005 0,009 0,000 0,000 0,007 0,017 0,064 0,042 * The results are expressed as ratios (% of total reads) Biostatistical analyses discovered significant differences in the abundance of 13 bacterial species among the examined groups (Table 2 .). Alysiella filiformis was detected only in the smoker groups and was found in greated abundance in the THSS group (0.020% ± 0.050%). Avibacterium paragallinarum was detected only in the NS group. Prevotella multiformis was most abundant in the NS group (0.134% ± 0.200%) while being least abundant in the CS group (0.120% ± 0.460%). Lactobacillus salivarius was not detected in the THSS group, and was more abundant in the NS (0.06% ± 0.12%) group than in the CS group (0.003% ± 0.013%). Actinomyces massiliensis was most abundant in the NS group (1.22% ± 0.85%), and least abundant in the THSS group (0.64% ± 0.53%). Streptococcus thermophilus was not detected in the CS group and it had a greater abundance in the THSS group (0.02% ± 0.09%) than in the NS (0.003% ± 0.013%). Propionibacterium propionicum was most abundant in the THSS group (0.65% ± 0.67%), and least abundant in the NS group (0.23% ± 0.27%). Haemophilus parainfluenzae was most abundant in the NS group (1.98% ± 1.61%), and least abundant in the CS group (1.17% ± 1.90%). Prevotella pleuritidis was most abundant in the THSS group (0.25% ± 0.80%), and least abundant in the NS group (0.04% ± 0.12%). Streptococcus pyogenes was not detected in the NS group, and was more abundant in the THSS group (0.010% ± 0.032%) than in the CS group (0.009% ± 0.015%). Kingella oralis was most abundant in the THSS group (0.60% ± 0.57%), and least abundant in the CS group (0.41% ± 0.60%). Streptococcus caballi was not detected in the CS group and was more abundant in the NS group (0.007% ± 0.017%) than in the THSS group (0.005% ± 0.009%). Streptococcus lactarius was most abundant in the CS group (0.044% ± 0.028%), and least abundant in the NS group (0.023% ± 0.027%). 4. Discussion The participants in this study were referred patients for consultation and/or specialist treatment, meaning that due to their dental problems (caries, endodontic pathology and/or periodontitis) their bacterial composition and abundance in OM could be altered from normal [ 1 – 5 ]. Additionally, most of the examined sample were female patients (87%), which is in accordance with a study previously conducted in the same area [ 27 ]. This suggest that the examined sample could not represent the gender ratio of the general population properly, although the gender ratio in the present study confirms the findings of several studies that indicate an increased motivation of female patients towards healthcare compared to males [ 28 , 29 ]. Clinical parameters that could influence or reflect the effect of oral microbiomes on the oral cavity like Decayed-Missing-Filled Tooth index (DMFT), amount of accumulated biofilm and salivary parameters (amount and pH level) were not investigated in this study since our study group already described them and assessed their influence on caries risk. The study model was of the same design as the one reported in this paper [ 30 ]. The bacterial composition of the SDB is influenced by many environmental factors and dysbiotic microbial shifts are contributing to the cause of caries and periodontal disease [ 3 ]. Metrics describing bacterial diversity (Alpha and Beta diversity) of the SDB among the 3 researched groups revealed no significant differences. The underlying reason due to which these metrics have not found significant differences among the examined groups is due to the sample size and data dispersion as it was described in similar metagenomic studies [ 31 ]. Researched groups differed in the number of detected species, with the two smoker groups presented increased bacterial species content of SDB compared to non-smokers. In the present study significant differences in bacterial compositions of SDB microbiomes among the examined groups were found on all taxonomic levels. This can be atributed to rigorous subject selection and a large number of inclusion/exclusion criteria [ 32 ]. Heat tree analyses revealed significant differences in the abundance of several bacterial lineages. Despite this, the consumption of tobacco products (THS or cigarettes) can not be confirmed as the main cause for the detected SDB bacterial composition differences among the examined groups due to study design. The comparison of SDB microbiomes of THSS and NS revealed an increased microbial abundance in THSS. This is predominantly associated to bacteria belonging to the Rhodospirillaceae , Spirochetaceae , Propionibacteriaceae and Ardenscatenaceae families. Bacteria from the Rhodospirillaceae family are facultative anaerobes preferring anaerobic conditions, embracing an adaptable metabolism, which enables them glucose metabolism through multiple metabolic pathways leading to the production of several acids [ 33 ]. Similarly, bacteria from the Spirochetaceae family, which are obligate anaerobes or facultative anaerobes, use saccharose as their fundamental metabolite in order to produce energy [ 34 ]. Microorganisms belonging to the Propionibateriaceae family, also display similar metabolic traits. These anaerobic bacteria produce propionic acid through their metabolism, which causes a pH level drop of biofilms which they inhabit [ 35 ]. An increased presence of the previously mentioned families suggests an increased anaerobic species abundance in THSS compared to NS. This anaerobic microbial shift is apparently, caused due to the early onset of anaerobic and microaerophilic conditions, caused by THS consumption. Such conditions could stimulate early colonization and aggregation of anaerobic bacteria in early stages of biofilm formation. Consequentially, we could consider that biofilms formed in such conditions could have an enhanced pathological – cariogenic potential than biofilms which are formed in non-smoking conditions. Mentioned considerations are further implied by an increased abundance of the Campylobacteriaceae family in NS group compared to THSS. These microaerophilic bacteria are commonly found in the oral cavity when caries activity is absent [ 36 , 37 ]. The THSS group was also more abundant in thermophilic bacteria from the family Ardenscatenaceae . Such finding could imply that THS have a stimulative effect of the growth of certain bacteria due to a temperature increase in the oral cavity while consuming such products [ 38 ]. Similar results were found when comparing the microbiomes of SDB of CS and NS group. In the CS group an increased abundance of bacteria belonging to Rhodobacteriaceae , Rhodospirillaceae and Sphingomonadaceae families was detected. Bacteria belonging to above mentioned families are generally strict or facultative anaerobes, with a distinct capability of producing extracellular polysaccharides which is a key characteristic of cariogenic bacteria [ 33 , 39 , 40 ]. Another finding of this comparison was an increased abundance in thermophilic bacteria from the Ardenscatenaceae and Thermogemmatisporaceae families in the CS group [ 41 ]. Increased abundances of different thermophilic bacteria families in the two smoker groups also indicates that the generated temperature difference, could have adverse effects on the inhabitation of different bacterial species and their microbial shift since tobbacco heating (THS) and tobacco combustion (CS) happens on different temperatures [ 17 ]. Although, when compairing the two smoker groups statistically significant differences regarding abundance of thermophillic bacteria have not been found, mentioned trend is detected Additional comparisons of supragingival microbiomes of the two smoker groups revealed increased abundances of bacterai from the Propionibacteriaceae family in THSS, and increased abundances of Rhodobacteriaceae in CS. Such findings supports the results of previous in vitro studies that suggest an adverse effect of cigarete smoke components on the increased inhabitation and bacterial composition shift of certain bacterial species depending on the concentration of nicotine [ 6 ]. This is further suggested by the increased abundances of bacteria from the Carnobacteriaceae and Aerococcaceae families and the ordo Bacillales ., who produce acids through carbohydrate metabolism which is characteristic for cariogenic bacteria [ 42 , 43 ]. The examined groups revealed significant differences on the genus level. The genera Alysiella and Propionibacterium presented increased abundance in the THSS group, while Avibacterium and Haemophilus were increased in the NS group. Such findings further suggest an adverse effect of different tobacco products consumption on the microbiome of SDB. It is also important to mention that bacteria from the genus Avibacterium was detected in the composition of dental biofilms which to our knowledge was not previously found in published studies. The findings of this study diminish the results of two in vitro studies which suggest that cigarette smoke does not have an effect on the Propionibacterium and Haemophilus genera abundance when analysing SDB bacterial composition [ 44 , 45 ]. A potential cause for this could be the effect of multiple environmental factors affecting the metabolism and activity of these bacteria in vivo conditions. Such results indicate the importance of clinical studies in the research of environmental factors and their effect on the oral microbiome. Comparisons conducted on the species level revealed significant differences in bacterial abundance among the examined groups, although these bacteria make a small fraction of the bacterial composition of the SDB microbiome. Nevertheless, such bacteria have the potential of creating micro niches and present key factors that influence the biofilm's metabolic activity and biodynamics [ 46 ]. Bacterial species which are associated with caries absence, Lactobacillus salivarius, Streptococcus thermophilus, Haemophilus parainfluenze and Kingella oralis have also been detected in the present study. The results indicate a significant difference between researched groups. Since Kingella oralis and Streptococcus thermophilus , were more abundant in the THSS group compared to the NS group we could suspect that consumption of tobacco products does not stimulate the increased inhabitation of exclusively cariogenic bacteria, but stimulates changes in numerous fractions of SDB bacterial microbiome. Furthermore, the study findings confirm previously published results reporting a positive correlation of tobacco smoking with the Prevotella pleuritidis abundance , a bacteria whose presence in oral biofilms could be connected to the early development of rheumatoid arthritis. This suggests that THS consumption could increase the risk for developing the disease since the mentioned bacteria was most abundant in the THSS group [ 47 ]. Furthermore, in this study two bacterial species which were not, to our knowledge, previously described in the composition of SDB microbiome have been identified: Avibacterium paragallinarum and Streptococcus caballi. Such finding of rare bacteria are most probably possible due to methodology of the study, since in the present study 16s rRNA gene sequencing was done on V2-V9 hypervariable regions which is not a general practice in metagenomic studies. Such practice provides an enhanced level of taxa identification leading too deeper understanding of bacterial species composition of SDB. 5. Conclusions To conclude, this study confirms differences in the bacterial composition and abundance of SDB among THSS, CS and NS groups. Differences have been detected on all taxonomic levels. The compositions of SDB of THSS, CS and NS are different, and due to increased anaerobic bacteria abundance with cariogenic properties, the dysbiosis pattern and pathogenic potential of THSS and CS dental biofilms compared to NS is considerably increased. Despite, tobacco products consumption (THS or cigarettes) is confirmed as causative agent for detected SDB bacterial composition shift, specific commercial tobacco product can not be defined due to study design. Nevertheless, tobacco smoking has repeatedly shown negative effects on oral and general health and should be avoided. Declarations Competing interests The authors declare no competing interests. Author Contribution E.B. - data curation, investigation, writing – original draftJ.Ž. - formal analysis, methodology, visualizationA.B. - resources, investigationS.Š. - conceptualization, funding acquisition, project administrationR.P.B. - resources, writing – review and editingN.T. – methodology, investigationM.H. – formal analysisB.M.V. - conceptualization, investigation, methodology, supervision, writing – review and editing Acknowledgments The presented research has been founded by the Croatian Science Foundation through the institutional project „Environmental factors and microbial interactions in the structure of dental biofilm“ (IP-2020-02-4027) https://sites.google.com/view/ip-2020-02/po%C4%8Detna-stranica Data Availability The data presented in this study are available on public repository of Faculty of Dental Medicine, University of Rijeka, Croatia. Špalj S. Okolišni čimbenici i mikrobiološke interakcije u strukturi dentalnog biofilma:istraživački podaci. [Internet]. Fakultet dentalne medicine; 2023.Available from: [**https://urn.nsk.hr/urn:nbn:hr:271:843444**](https:/urn.nsk.hr/urn:nbn:hr:271:843444) Ethics Approval declaration The study design and protocols were reviewed and approved by the Ethical committee of Clinical Hospital Centre Rijeka (Class: 003–05/22 − 1/19; Registry number: 2170-29-02/1-22-2), the Ethical committee of the Faculty of Dental Medicine, University of Rijeka (Class: 035 − 01/22 − 01/150; Registry number: 2170-57-006-01-22-1) and the Ethical committee of the Medical Faculty, University of Rijeka (Class: 007–08/22 − 01/77; Registry number: 2170-24-04-3/1-22-6). Funding Declaration The presented research has been founded by the Croatian Science Foundation (Hrvatska Zaklada za Znanost,) through the institutional project „Environmental factors and microbial interactions in the structure of dental biofilm“ (IP-2020-02-4027) https://sites.google.com/view/ip-2020-02/po%C4%8Detna-stranica References Santacroce L, Passarelli PC, Azzolino D, Bottalico L, Charitos IA, Cazzolla AP et al (2023) Oral microbiota in human health and disease: A perspective. Exp Biol Med (Maywood) 248(15):1288–1301. 10.1177/15353702231187645 Valm AM (2019) The Structure of Dental Plaque Microbial Communities in the Transition from Health to Dental Caries and Periodontal Disease. J Mol Biol 431(16):2957–2969. 10.1016/j.jmb.2019.05.016 Marsh PD (2010) Microbiology of dental plaque biofilms and their role in oral health and caries. Dent Clin North Am 54(3):441–454. 10.1016/j.cden.2010.03.002 Marsh PD, Zaura E (2017) Dental biofilm: ecological interactions in health and disease. 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Cite Share Download PDF Status: Published Journal Publication published 23 Mar, 2026 Read the published version in Clinical Oral Investigations → Version 1 posted Editorial decision: Revision requested 13 Jan, 2026 Reviews received at journal 13 Jan, 2026 Reviews received at journal 02 Jan, 2026 Reviewers agreed at journal 28 Dec, 2025 Reviewers agreed at journal 19 Dec, 2025 Reviewers invited by journal 17 Dec, 2025 Editor assigned by journal 10 Dec, 2025 Submission checks completed at journal 10 Dec, 2025 First submitted to journal 07 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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1","display":"","copyAsset":false,"role":"figure","size":140476,"visible":true,"origin":"","legend":"\u003cp\u003eDifferences in bacterial abundance between THSS and NS groups – the colour red represents greater abundance in the THS group while the colour blue indicates greater abundance in the NS group. Taxa names indicate statistically significant differences in bacterial abundance among the compared groups.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8302044/v1/ff8a9905cb187e0e7a8f6595.jpeg"},{"id":98602665,"identity":"7be3eaa2-0773-4429-bb55-ed8d6b1440a4","added_by":"auto","created_at":"2025-12-19 12:54:56","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":137112,"visible":true,"origin":"","legend":"\u003cp\u003eDifferences in bacterial abundance between CS and NS groups – the colour red represents greater abundance in the CS group while the colour blue indicates greater abundance in the NS group. Taxa names indicate statistically significant differences in bacterial abundance among the compared groups.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8302044/v1/30a57b3a43706a09433fecfd.jpeg"},{"id":98628945,"identity":"f2c6ce7e-dde8-401b-bf19-d651b752f0db","added_by":"auto","created_at":"2025-12-19 17:12:50","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":133295,"visible":true,"origin":"","legend":"\u003cp\u003eDifferences in bacterial abundance between THSS and CS groups – the colour red represents greater abundance in the THS group while the colour blue indicates greater abundance in the CS group. Taxa names indicate statistically significant differences in bacterial abundance among the compared groups.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8302044/v1/fb8ad9e17e1bd695da3c157c.jpeg"},{"id":105756093,"identity":"a062cc52-49eb-4ef8-b978-5cc74e945df0","added_by":"auto","created_at":"2026-03-30 16:35:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1670995,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8302044/v1/95ce3020-508d-4bfd-9f58-e963740a68cd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Supragingival dental biofilm microbiomes of tobacco heating system smokers, cigarette smokers and non-smoker","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e The oral cavity is an environment that provides favorable conditions for bacterial growth, as well as hard and soft tissue surfaces suitable for microbial attachment and biofilm formation. Among the oral microbiota (OM) over 700 bacterial species, along with viruses, fungi, archaea and protozoa can be found. An oral sample from a single individual typically contains a fraction of this spectrum, usually between 50 and 200 different bacterial species. OM composition is highly dependent on numerous host factors, such as age, genetics, dietary habits, etc. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although we are born with sterile oral cavities, bacteria and other microorganisms begin to establish themselves as commensals during delivery in newborns. Commensal microorganisms in oral cavity serve as a protective role by preventing colonization of pathogenic microorganisms and as such have an important role in maintaining bacterial homeostasis. Furthermore, it is speculated that the constant presence of bacterial biofilm has a role in stimulating the development and proper response of the hosts\u0026rsquo; immune system as well as regulating dietary nitrates [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccording to the Human Oral Microbiome Database (eHOMD) the prevailing bacterial species presented in oral biofilms belong to the genera \u003cem\u003eStreptococcus, Veillonella, Selenomonas, Gemella, Fusobacterium, Prevotella, Lactobacillus, Neiseria, Dialister, Actinomyces, Capnocytophaga, Granulicatella, Campylobacter, Treponema, Enterococcus, Eubacterium, Atopobium, Bacterioides, Propionibacterium Rothia\u003c/em\u003e and \u003cem\u003eHaemophilus\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. These bacteria, although normally present as commensals in the oral cavity, are prone to causing microbiome composition shifts \u0026ndash; dysbiosis, when exposed to certain favorable conditions. This is most evident in dental biofilms whose prolonged presence on tooth surfaces can cause diseases such as dental caries and/or periodontitis. Even though presence of biofilms is the main prerequisite for dental and periodontal diseases, other environmental factors, such as pH level, availability of nutrients, oxygen concentration etc., also play an important role in disease development [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTobacco smoking has been widely researched as a factor influencing OM composition and microbial activity in the oral cavity and has been widely associated with higher rates of caries and periodontitis. Findings of several in vitro studies confirm this. Their results show that components of cigarette smoke, especially nicotine, have adverse effects on the metabolism and proliferation of cariogenic bacteria, depending on their concentration [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Studies in vivo also confirm these findings since they report an increased incidence of caries lesions in smokers compared to non-smokers [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Smoking is also considered as a risk factor in the development of periodontal diseases [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Recent 16s rRNA sequencing studies confirm that consumption of tobacco products influences microbial diversity in supragingival and subgingival dental biofilms. Such studies report an increase in bacterial abundance of certain species belonging to the genera \u003cem\u003eStreptococcus\u003c/em\u003e, \u003cem\u003eLactobacillus\u003c/em\u003e, and \u003cem\u003eKlebsiella\u003c/em\u003e in supragingival dental biofilms (SDB) of smokers. Such studies also report and increased abundance of species belonging to the genera \u003cem\u003ePrevotella, Treponema, Streptococcus, Veillonella\u003c/em\u003e, and \u003cem\u003eTanarella\u003c/em\u003e in the composition of subgingival dental biofilms of smokers when compared to non-smokers [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTobacco heating systems (THS) are a relatively new product that has been on the market for the last decade. The products are advertised as being less unhealthy than cigarettes since their aerosol, which contains the same components as cigarette smoke, has significantly reduced concentrations. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. To the authors\u0026rsquo; knowledge, their influence on the microbial composition of dental biofilms has not been widely researched.\u003c/p\u003e \u003cp\u003eThe aim of the present study was to investigate and compare the bacterial diversity and abundance in the SDB of THS smokers (THSS), cigarette smokers (CS) and non-smokers (NS). Since THS aerosol contains the same components as cigarette smoke, a hypothesis was formed which stated that no significant differences in bacterial diversity and abundance would be found among the smoker groups (THSS v. CS) but significant differences would be found between the smokers groups and non-smokers (THSS v. NS, and CS v. NS)\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Subject recruitment\u003c/h2\u003e \u003cp\u003eIn the present cross sectional study, subjects were distributed in 3 different groups: THS smokers (THSS), cigarette smokers (CS) and non-smokers (NS). The recruited subjects were patients referred to the Dental clinic, Clinical Hospital Centre Rijeka, Croatia for consultation and/or treatment from January to December 2023. Subject recruitment was performed in a consecutive method, and since patients that consumed THS were less frequent, they were recruited first and the other groups were matched to them according to age and gender. All participants have read and signed an informed consent form. This research was conducted according to fundamental ethical and bioethical principles which are stated and required by the Nurnberg code and the newest revision of the Declaration of Helsinki.\u003c/p\u003e \u003cp\u003eSample sizes were determined through an online tool: Ristrl, R. Sample Size Calculator Version 1.061. Available online: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://homepage.univie.ac.at/robin.ristl/samplesize.php\u003c/span\u003e\u003cspan address=\"https://homepage.univie.ac.at/robin.ristl/samplesize.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (accessed on 1 February 2022). The conducted calculations were performed through data obtained from a pilot study regarding clinical parameters (Decayed-Missing-Filled Tooth (DMFT) index, Decayed-Missing-Filled Tooth surface (DMFTs) index, Full Mouth Plaque Score (FMPS) and Full Mouth Bleed Score (FMBS). The pilot study was conducted on 30 subjects (10 THSS, 10 CS and 10 NS; with each group consisting of 9 female (90%) and 1 male (10%) participants) by our study group. The pilot study results were not published. The sample size was determined for one way analysis of variance using mean and standard deviation data of the DMFTs index with a power level of 80% and a significance level of 0.05. The DMFTs index mean values were 17.3 for THSS, 24.3 for CS and 9.8 for NS, while standard deviation values of the DMFTs index were 13.2 in the THSS group, 20.2 in the CS group and 5.5 in the NS group. A mean value of standard deviation values was calculated and used to determine the required sample size, with dropout rates accounted. The calculation determined a minimum of 20 participants per group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Inclusion/Exclusion criteria\u003c/h2\u003e \u003cp\u003eTHSS or CS group subjects had to consume either THS or cigarettes exclusively for at least 6 months, and had to have consumed more than 100 cigarettes/tobacco heated sticks [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Since a large variety of THS products and cigarettes are currently present on the market, only subjects that consumed the THS product IQOS (Phillip Morris, Richmond, USA) or Marlboro Gold cigarettes (Marlboro, Richmond, USA) were recruited to participate. Subjects that have declared to never have smoked in their lifetime were considered to be NS.\u003c/p\u003e \u003cp\u003eExclusion criteria were: habits/conditions that have a confirmed effect on microbial proliferation, such as constant or recent use of oral antiseptics, systemic antibiotic treatment which has been taken at least 3 months prior to examination, systemic use of immunosuppressive medications, diabetes mellitus and autoimmune diseases and active orthodontic treatment [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Furthermore, patients that had fewer than 10 teeth were not eligible for participation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Microbial diversity assessment\u003c/h2\u003e \u003cp\u003eTo determine the microbial diversity and abundance in SDB of the examined groups, hypervariable regions (V2-V9) of bacterial 16s rRNA gene were analysed through Next generation sequencing (NGS) using Ion Torrent technology (Thermo Fisher Scientific, Waltham, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Collection of supragingival dental biofilm samples\u003c/h2\u003e \u003cp\u003eSDB sample collection was performed during morning hours (between 8.00 AM and 10.00 AM). To ensure biofilm samples were of similar maturity, subjects were instructed not to brush their teeth in the morning before sample collection and to brush their teeth the evening prior, between 8.00 PM and 10.00 PM.\u003c/p\u003e \u003cp\u003eSamples collection was performed using a set of sterile Gracey curettes from all surfaces of maxillary and mandibular first molars on both sides. If subjects were missing any of their first molars, samples were collected from second molars, or alternatively, if those were also missing from second maxillary/mandibular premolars. The samples were stored in a 1,5ml sterile Eppendorf LoBind tubes with storage medium consisting of 750 \u0026micro;L Tris-EDTA buffered solution (pH\u0026thinsp;=\u0026thinsp;7,4). The collected samples were stored on -20\u0026deg;C prior to DNA isolation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. DNA isolation\u003c/h2\u003e \u003cp\u003eDNA was isolated from the collected SDB samples with the Nucleospin Tissue kit (Macherey Nagel, Duren, Germany). A modified protocol for bacterial DNA isolation was used as described in the products instruction manual. The concentration and purity of genomic DNA were measured with a Qubit 4 Flourometer (Thermo Fisher Scientific, Waltham, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Library preparation, sequencing, bioinformatics and statistical analysis\u003c/h2\u003e \u003cp\u003eThe 16s rRNA gene\u0026rsquo;s hypervariable regions (V2-V9) were amplified and libraries were created using the Ion 16S Metagenomics Kit (ThermoFisher Scientific, Waltham, MA, USA) according to manufacturers protocol. Quality and quantity of purified PCR products and prepared libraries was controlled on a Agilent 2100 Bioanalyzer instrument with Agilent High Sensitivity DNA kit (Agilent Technologies, Santa Clara, California, USA), according to manufacturers protocol. Purified barcoded library concentrations were diluted to 40 pM and pooled (25 \u0026micro;l of each sample library). Pooled libraries were then loaded into appropriate Ion Chef\u0026trade; Library Sample Tube on the Ion Torrent Ion Chef (Thermo Fisher Scientific, Waltham, MA, USA) instrument for template preparation and chip loading, with the Ion Torrent Ion 530 Kit \u0026ndash;Chef (ThermoFisher Scientific, Waltham, MA, USA). For loading the 400 bp chef protocol was selected in the device settings. Sequencing of the loaded chip was performed on an Ion S5 Ion GeneStudio S5 System (ThermoFisher Scientific, Waltham, MA, USA) instrument. Resulting amplicon sequences were separated into individual variable regions using the MetagenomicsPP plugin in the Torrent Suite software [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Resulting .fastq files corresponding to each variable region were imported into QIIME2 (version 2022.2) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] using a manifest file and each data file was quality filtered and dereplicated using QIIME2 DADA2 plugin with \u003cem\u003epyro\u003c/em\u003e method [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Obtained feature tables and representative sequences of each variable region were merged. Taxonomy was assigned to merged representative sequences using the VSEARCH-based consensus taxonomy classifier[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] implemented in QIIME2. Sequences were searched against SILVA 132 database clustered at 99% sequence similarity. Assigned taxonomies were visualized as bar plots and exported as csv file at species level using the QIIME2 pipeline. Exported .csv file was used for visualization and statistical analysis of microbiome data using the MicrobiomeAnalyst 2.0 platform[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and R programming language. Microbiome diversity has been assessed through Alpha diversity metrics (Observed taxa index), Beta diversity metrics (Bray Curtis index along with PERMANOVA for statistical analysis) and Heat tree analysis. Statistical analysis was also done using IBM SPSS Statistics 29 software (IBM, New York, USA) with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 set statistical significance level. Conventional descriptive statistical methods have been used for analysis of collected data. Normality of collected data distribution was assessed through the Shapiro-Wilk test. Since data did not have normal distribution, the variables are expressed through median values and interquartile ranges. Kruskall-Wallis test was used for analysis of data on continuous scales. Mann-Whitney test with Bonferroni correction for multiple comparisons was used for post-hoc analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Sample descriptive statistics\u003c/h2\u003e \u003cp\u003eA total of 60 subject participated in this study. Six were male (10%) and 54 were female (90%). The sample was comprised of predominantly young subjects (median\u0026thinsp;=\u0026thinsp;29 years). After bioinformatics processing the total number of reads across all samples was 5074107. The average number of reads per sample was 84568.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Microbial diversity\u003c/h2\u003e \u003cp\u003eAlpha diversity was tested through the observed taxa index on species level. Mean and standard deviation values of the observed taxa index were: 163.25\u0026thinsp;\u0026plusmn;\u0026thinsp;31.67 for THSS group, 159.35\u0026thinsp;\u0026plusmn;\u0026thinsp;24.22 for CS group and 164.2\u0026thinsp;\u0026plusmn;\u0026thinsp;20.37 for NS group. Statistical analyses were performed through the Mann\u0026mdash;Whitney and Kruskal-Wallis test in the MicrobiomeAnalyst 2.0 web tool and revealed no significant significant differences among the THSS, CS and NS groups (p\u0026thinsp;=\u0026thinsp;0.489).\u003c/p\u003e \u003cp\u003eBeta diversity was tested through the Bray-Curtis index on species level and PERMANOVA was used to test statistical significance. No significant differences were found among the examined groups (F-value: 1.0512; R\u003csup\u003e2\u003c/sup\u003e : 0.035571; p\u0026thinsp;=\u0026thinsp;0.35).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Taxonomic characterization\u003c/h2\u003e \u003cp\u003eThrough taxonomic identification a total of 8 phyla, 21 classes, 30 orders, 52 families, 88 genera and 232 species were identified across all samples. Among the identified species 205 were identified in the THSS group, 203 in the CS group and 192 in the NS group.\u003c/p\u003e \u003cp\u003eThe examined groups had a similar composition of bacteria at higher taxonomic levels with the most common being: \u003cem\u003eFirmicutes, Actinobacteria, Proteobacteria, Bacterioidetes\u003c/em\u003e, and \u003cem\u003eFusobacteraia\u003c/em\u003e at the Phylum level; \u003cem\u003eActinobacteria, Bacillli, Bacteroidia, Gammaproteobacteria\u003c/em\u003e and \u003cem\u003eFusobacteria\u003c/em\u003e at the Class level; \u003cem\u003eActinomycetales, Lactobacillales Bacteroidales, Fusobacteriales\u003c/em\u003e and \u003cem\u003ePasteurellales\u003c/em\u003e at the Ordo level and \u003cem\u003eStreptococcaceae, Actinomycetaceae, Pasteurellaceae, Prevotellaceae\u003c/em\u003e and \u003cem\u003eCorynebacteriaceae\u003c/em\u003e at the Family level.\u003c/p\u003e \u003cp\u003eHeat tree analyses were performed to assess differences in microbial diversity at higher taxonomic levels (up to Family level) between the examined groups. The results were based on the Wilcoxon rank sum test at the significance threshold of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The results are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The nodes represent taxa while their size represents abundance and branches describe taxonomic hierarchy. The colors indicate differences among groups and nodes with written taxa names indicating a statistically significant difference among the compared groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe THSS group was more abundant in bacteria belonging to the \u003cem\u003eRhodospirillaceae, Spirochetaceae, Propionibacteriaceae\u003c/em\u003e and \u003cem\u003eArdenscatenaceae\u003c/em\u003e families compared to the NS group, although the NS group was more abundant in bacteria belonging to the \u003cem\u003eCampylobacteriaceae\u003c/em\u003e family.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe CS group was more abundant in bacteria belonging to the \u003cem\u003eBacillaceae\u003c/em\u003e, \u003cem\u003eThermogemmatisporaceae\u003c/em\u003e, \u003cem\u003eRhodobacteraceae\u003c/em\u003e, \u003cem\u003eRhodospirillaceae\u003c/em\u003e, \u003cem\u003eSphingomonadaceae and Ardenscatenaceae\u003c/em\u003e families compared to the NS group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen comparing the two smoker groups the THSS group was more abundant in bacteria from the \u003cem\u003eRhodobacteriaceae, Carnobacteriaceae and Aerococcaceae\u003c/em\u003e family. The CS group was more abundant in bacteria belonging to the \u003cem\u003ePropionibacteriaceae\u003c/em\u003e family.\u003c/p\u003e \u003cp\u003eDifferences in microbial abundance on lower taxonomic levels (genus and species) were tested through the Kruskal-Wallis test. Genera in which significant differences in abundance have been found are expressed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, while the results on species level are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e1. Differences of bacterial abundance on genus level\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSignificant differences in bacterial abundance at genus level\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eGenus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNumber of samples detected with the genera (at least 1 read)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTHSS Mean\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTHSS Std. Dev. (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCS Mean (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCS Std. Dev. (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNS Mean (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNS Std. Dev. (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eEffect size\u003c/p\u003e \u003cp\u003eη\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"10\" nameend=\"c11\" namest=\"c2\"\u003e \u003cp\u003eHighest abundance in THSS group\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePropionibacterium\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1,198\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1,413\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,570\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,643\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,439\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0,486\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,111\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0,025\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAlysiella\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,045\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,073\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0,016\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"10\" nameend=\"c11\" namest=\"c2\"\u003e \u003cp\u003e\u003cb\u003eHighest abundance in CS group\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"10\" nameend=\"c11\" namest=\"c2\"\u003e \u003cp\u003e\u003cb\u003eHighest abundance in NS group\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHaemophilus\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1,773\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3,376\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1,641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1,899\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2,305\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1,640\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0,030\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eActinobaculum\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,745\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,655\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,248\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,342\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1,016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1,539\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,094\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0,004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAvibacterium\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,193\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0,398\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,135\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0,015\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"11\"\u003e* The results are expressed as ratios (% of total reads)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eOn genus level significant differences were found in abundance of genera (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.): \u003cem\u003eActinobaculum\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0,004), \u003cem\u003eAvibaterium\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0,015), \u003cem\u003eAlysiella\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0,016), \u003cem\u003ePropionibacterium\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0,025) and \u003cem\u003eHaemophilus\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0,030). The \u003cem\u003eActinobaculum\u003c/em\u003e genus was predominantin the NS group (1.02% \u0026plusmn; 1.54%) and least abundant in the CS group (0.25%\u0026plusmn;0.34%). Bacteria from the \u003cem\u003eAvibacterium\u003c/em\u003e genus were detected only in the NS group (0.19%\u0026plusmn;0.40%). In the two smoker groups the genus \u003cem\u003eAlysiella\u003c/em\u003e was detected with increased prevalence in the THSS group (0.02%\u0026plusmn;0,05%) compared to CS group (0.002.%\u0026plusmn;0.006%). The \u003cem\u003ePropionibacterium\u003c/em\u003e genus was dominant in the THSS group (1.20% \u0026plusmn; 1.41%), and least abundant in the NS group (0.44%\u0026plusmn;0.47%). Bacteria from the \u003cem\u003eHaemophilus\u003c/em\u003e genus were most abundant in the NS group (2.31%\u0026plusmn;1.64%), and least abundant in the CS group (1.64%\u0026plusmn;1.90%).\u003c/p\u003e\n\u003ch3\u003e2. Differences of bacterial abundance on species level\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSignificant differences in bacterial abundance at species level\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNumber of samples detected with the species (at least 1 read)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTHSS Mean\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTHSS Std. Dev. (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCS Mean (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCS Std. Dev. (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNS Mean (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNS Std. Dev. (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eEffect size\u003c/p\u003e \u003cp\u003eη\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"10\" nameend=\"c11\" namest=\"c2\"\u003e \u003cp\u003eHighest abundance in THSS group\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAlysiella filiformis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,045\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,079\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0,006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStreptococcus thermophilus\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,089\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0,025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,028\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0,025\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePropionibacterium propionicum\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,649\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,670\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,357\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,383\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,226\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0,272\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,123\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0,027\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePrevotella pleuritidis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,254\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,799\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,179\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,042\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0,121\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,033\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0,029\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStreptococcus pyogenes\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,032\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,046\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0,035\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eKingella oralis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,602\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,573\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,419\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,595\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,573\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0,347\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0,038\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"10\" nameend=\"c11\" namest=\"c2\"\u003e \u003cp\u003e\u003cb\u003eHighest abundance in CS group\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStreptococcus lactarius\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,028\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,044\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,028\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0,027\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,088\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0,050\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"10\" nameend=\"c11\" namest=\"c2\"\u003e \u003cp\u003e\u003cb\u003eHighest abundance in NS group\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAvibacterium paragallinarum\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,193\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0,398\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,135\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0,015\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePrevotella multiformis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,409\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,461\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0,200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0,016\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLactobacillus salivarius\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,061\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0,118\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,146\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0,018\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eActinomyces massiliensis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,640\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,503\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,643\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,797\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1,222\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0,845\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,123\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0,022\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHaemophilus parainfluenzae\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1,460\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3,374\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1,165\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1,897\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1,981\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1,612\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0,028\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStreptococcus caballi\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0,017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,064\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0,042\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"11\"\u003e* The results are expressed as ratios (% of total reads)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eBiostatistical analyses discovered significant differences in the abundance of 13 bacterial species among the examined groups (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.). \u003cem\u003eAlysiella filiformis\u003c/em\u003e was detected only in the smoker groups and was found in greated abundance in the THSS group (0.020% \u0026plusmn; 0.050%). \u003cem\u003eAvibacterium paragallinarum\u003c/em\u003e was detected only in the NS group. \u003cem\u003ePrevotella multiformis\u003c/em\u003e was most abundant in the NS group (0.134% \u0026plusmn; 0.200%) while being least abundant in the CS group (0.120% \u0026plusmn; 0.460%). \u003cem\u003eLactobacillus salivarius\u003c/em\u003e was not detected in the THSS group, and was more abundant in the NS (0.06% \u0026plusmn; 0.12%) group than in the CS group (0.003% \u0026plusmn; 0.013%). \u003cem\u003eActinomyces massiliensis\u003c/em\u003e was most abundant in the NS group (1.22% \u0026plusmn; 0.85%), and least abundant in the THSS group (0.64% \u0026plusmn; 0.53%). \u003cem\u003eStreptococcus thermophilus\u003c/em\u003e was not detected in the CS group and it had a greater abundance in the THSS group (0.02% \u0026plusmn; 0.09%) than in the NS (0.003% \u0026plusmn; 0.013%). \u003cem\u003ePropionibacterium propionicum\u003c/em\u003e was most abundant in the THSS group (0.65% \u0026plusmn; 0.67%), and least abundant in the NS group (0.23% \u0026plusmn; 0.27%). \u003cem\u003eHaemophilus parainfluenzae\u003c/em\u003e was most abundant in the NS group (1.98% \u0026plusmn; 1.61%), and least abundant in the CS group (1.17% \u0026plusmn; 1.90%). \u003cem\u003ePrevotella pleuritidis\u003c/em\u003e was most abundant in the THSS group (0.25% \u0026plusmn; 0.80%), and least abundant in the NS group (0.04% \u0026plusmn; 0.12%). \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e was not detected in the NS group, and was more abundant in the THSS group (0.010% \u0026plusmn; 0.032%) than in the CS group (0.009% \u0026plusmn; 0.015%). \u003cem\u003eKingella oralis\u003c/em\u003e was most abundant in the THSS group (0.60% \u0026plusmn; 0.57%), and least abundant in the CS group (0.41% \u0026plusmn; 0.60%). \u003cem\u003eStreptococcus caballi\u003c/em\u003e was not detected in the CS group and was more abundant in the NS group (0.007% \u0026plusmn; 0.017%) than in the THSS group (0.005% \u0026plusmn; 0.009%). \u003cem\u003eStreptococcus lactarius\u003c/em\u003e was most abundant in the CS group (0.044% \u0026plusmn; 0.028%), and least abundant in the NS group (0.023% \u0026plusmn; 0.027%).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe participants in this study were referred patients for consultation and/or specialist treatment, meaning that due to their dental problems (caries, endodontic pathology and/or periodontitis) their bacterial composition and abundance in OM could be altered from normal [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Additionally, most of the examined sample were female patients (87%), which is in accordance with a study previously conducted in the same area [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This suggest that the examined sample could not represent the gender ratio of the general population properly, although the gender ratio in the present study confirms the findings of several studies that indicate an increased motivation of female patients towards healthcare compared to males [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e Clinical parameters that could influence or reflect the effect of oral microbiomes on the oral cavity like Decayed-Missing-Filled Tooth index (DMFT), amount of accumulated biofilm and salivary parameters (amount and pH level) were not investigated in this study since our study group already described them and assessed their influence on caries risk. The study model was of the same design as the one reported in this paper [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe bacterial composition of the SDB is influenced by many environmental factors and dysbiotic microbial shifts are contributing to the cause of caries and periodontal disease [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Metrics describing bacterial diversity (Alpha and Beta diversity) of the SDB among the 3 researched groups revealed no significant differences. The underlying reason due to which these metrics have not found significant differences among the examined groups is due to the sample size and data dispersion as it was described in similar metagenomic studies [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Researched groups differed in the number of detected species, with the two smoker groups presented increased bacterial species content of SDB compared to non-smokers. In the present study significant differences in bacterial compositions of SDB microbiomes among the examined groups were found on all taxonomic levels. This can be atributed to rigorous subject selection and a large number of inclusion/exclusion criteria [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHeat tree analyses revealed significant differences in the abundance of several bacterial lineages. Despite this, the consumption of tobacco products (THS or cigarettes) can not be confirmed as the main cause for the detected SDB bacterial composition differences among the examined groups due to study design.\u003c/p\u003e \u003cp\u003eThe comparison of SDB microbiomes of THSS and NS revealed an increased microbial abundance in THSS. This is predominantly associated to bacteria belonging to the \u003cem\u003eRhodospirillaceae\u003c/em\u003e, \u003cem\u003eSpirochetaceae\u003c/em\u003e, \u003cem\u003ePropionibacteriaceae\u003c/em\u003e and \u003cem\u003eArdenscatenaceae\u003c/em\u003e families. Bacteria from the \u003cem\u003eRhodospirillaceae\u003c/em\u003e family are facultative anaerobes preferring anaerobic conditions, embracing an adaptable metabolism, which enables them glucose metabolism through multiple metabolic pathways leading to the production of several acids [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Similarly, bacteria from the \u003cem\u003eSpirochetaceae\u003c/em\u003e family, which are obligate anaerobes or facultative anaerobes, use saccharose as their fundamental metabolite in order to produce energy [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Microorganisms belonging to the \u003cem\u003ePropionibateriaceae\u003c/em\u003e family, also display similar metabolic traits. These anaerobic bacteria produce propionic acid through their metabolism, which causes a pH level drop of biofilms which they inhabit [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. An increased presence of the previously mentioned families suggests an increased anaerobic species abundance in THSS compared to NS. This anaerobic microbial shift is apparently, caused due to the early onset of anaerobic and microaerophilic conditions, caused by THS consumption. Such conditions could stimulate early colonization and aggregation of anaerobic bacteria in early stages of biofilm formation. Consequentially, we could consider that biofilms formed in such conditions could have an enhanced pathological \u0026ndash; cariogenic potential than biofilms which are formed in non-smoking conditions. Mentioned considerations are further implied by an increased abundance of the Campylobacteriaceae family in NS group compared to THSS. These microaerophilic bacteria are commonly found in the oral cavity when caries activity is absent [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The THSS group was also more abundant in thermophilic bacteria from the family \u003cem\u003eArdenscatenaceae\u003c/em\u003e. Such finding could imply that THS have a stimulative effect of the growth of certain bacteria due to a temperature increase in the oral cavity while consuming such products [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSimilar results were found when comparing the microbiomes of SDB of CS and NS group. In the CS group an increased abundance of bacteria belonging to \u003cem\u003eRhodobacteriaceae\u003c/em\u003e, \u003cem\u003eRhodospirillaceae\u003c/em\u003e and \u003cem\u003eSphingomonadaceae\u003c/em\u003e families was detected. Bacteria belonging to above mentioned families are generally strict or facultative anaerobes, with a distinct capability of producing extracellular polysaccharides which is a key characteristic of cariogenic bacteria [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Another finding of this comparison was an increased abundance in thermophilic bacteria from the \u003cem\u003eArdenscatenaceae\u003c/em\u003e and \u003cem\u003eThermogemmatisporaceae\u003c/em\u003e families in the CS group [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIncreased abundances of different thermophilic bacteria families in the two smoker groups also indicates that the generated temperature difference, could have adverse effects on the inhabitation of different bacterial species and their microbial shift since tobbacco heating (THS) and tobacco combustion (CS) happens on different temperatures [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Although, when compairing the two smoker groups statistically significant differences regarding abundance of thermophillic bacteria have not been found, mentioned trend is detected Additional comparisons of supragingival microbiomes of the two smoker groups revealed increased abundances of bacterai from the \u003cem\u003ePropionibacteriaceae\u003c/em\u003e family in THSS, and increased abundances of \u003cem\u003eRhodobacteriaceae\u003c/em\u003e in CS. Such findings supports the results of previous in vitro studies that suggest an adverse effect of cigarete smoke components on the increased inhabitation and bacterial composition shift of certain bacterial species depending on the concentration of nicotine [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This is further suggested by the increased abundances of bacteria from the \u003cem\u003eCarnobacteriaceae\u003c/em\u003e and \u003cem\u003eAerococcaceae\u003c/em\u003e families and the ordo \u003cem\u003eBacillales\u003c/em\u003e., who produce acids through carbohydrate metabolism which is characteristic for cariogenic bacteria [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe examined groups revealed significant differences on the genus level. The genera \u003cem\u003eAlysiella\u003c/em\u003e and \u003cem\u003ePropionibacterium\u003c/em\u003e presented increased abundance in the THSS group, while \u003cem\u003eAvibacterium\u003c/em\u003e and \u003cem\u003eHaemophilus\u003c/em\u003e were increased in the NS group. Such findings further suggest an adverse effect of different tobacco products consumption on the microbiome of SDB. It is also important to mention that bacteria from the genus \u003cem\u003eAvibacterium\u003c/em\u003e was detected in the composition of dental biofilms which to our knowledge was not previously found in published studies.\u003c/p\u003e \u003cp\u003eThe findings of this study diminish the results of two \u003cem\u003ein vitro\u003c/em\u003e studies which suggest that cigarette smoke does not have an effect on the \u003cem\u003ePropionibacterium\u003c/em\u003e and \u003cem\u003eHaemophilus\u003c/em\u003e genera abundance when analysing SDB bacterial composition [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. A potential cause for this could be the effect of multiple environmental factors affecting the metabolism and activity of these bacteria \u003cem\u003ein vivo\u003c/em\u003e conditions. Such results indicate the importance of clinical studies in the research of environmental factors and their effect on the oral microbiome.\u003c/p\u003e \u003cp\u003eComparisons conducted on the species level revealed significant differences in bacterial abundance among the examined groups, although these bacteria make a small fraction of the bacterial composition of the SDB microbiome. Nevertheless, such bacteria have the potential of creating micro niches and present key factors that influence the biofilm's metabolic activity and biodynamics [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Bacterial species which are associated with caries absence, \u003cem\u003eLactobacillus salivarius, Streptococcus thermophilus, Haemophilus parainfluenze\u003c/em\u003e and \u003cem\u003eKingella oralis\u003c/em\u003e have also been detected in the present study. The results indicate a significant difference between researched groups. Since \u003cem\u003eKingella oralis\u003c/em\u003e and \u003cem\u003eStreptococcus thermophilus\u003c/em\u003e, were more abundant in the THSS group \u003cem\u003ecompared to\u003c/em\u003e the NS group we could suspect that consumption of tobacco products does not stimulate the increased inhabitation of exclusively cariogenic bacteria, but stimulates changes in numerous fractions of SDB bacterial microbiome.\u003c/p\u003e \u003cp\u003eFurthermore, the study findings confirm previously published results reporting a positive correlation of tobacco smoking with the \u003cem\u003ePrevotella pleuritidis abundance\u003c/em\u003e, a bacteria whose presence in oral biofilms could be connected to the early development of rheumatoid arthritis. This suggests that THS consumption could increase the risk for developing the disease since the mentioned bacteria was most abundant in the THSS group [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, in this study two bacterial species which were not, to our knowledge, previously described in the composition of SDB microbiome have been identified: \u003cem\u003eAvibacterium paragallinarum\u003c/em\u003e and \u003cem\u003eStreptococcus caballi.\u003c/em\u003e Such finding of rare bacteria are most probably possible due to methodology of the study, since in the present study 16s rRNA gene sequencing was done on V2-V9 hypervariable regions which is not a general practice in metagenomic studies. Such practice provides an enhanced level of taxa identification leading too deeper understanding of bacterial species composition of SDB.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eTo conclude, this study confirms differences in the bacterial composition and abundance of SDB among THSS, CS and NS groups. Differences have been detected on all taxonomic levels. The compositions of SDB of THSS, CS and NS are different, and due to increased anaerobic bacteria abundance with cariogenic properties, the dysbiosis pattern and pathogenic potential of THSS and CS dental biofilms compared to NS is considerably increased. Despite, tobacco products consumption (THS or cigarettes) is confirmed as causative agent for detected SDB bacterial composition shift, specific commercial tobacco product can not be defined due to study design. Nevertheless, tobacco smoking has repeatedly shown negative effects on oral and general health and should be avoided.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eE.B. - data curation, investigation, writing \u0026ndash; original draftJ.Ž. - formal analysis, methodology, visualizationA.B. - resources, investigationS.Š. - conceptualization, funding acquisition, project administrationR.P.B. - resources, writing \u0026ndash; review and editingN.T. \u0026ndash; methodology, investigationM.H. \u0026ndash; formal analysisB.M.V. - conceptualization, investigation, methodology, supervision, writing \u0026ndash; review and editing\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThe presented research has been founded by the Croatian Science Foundation through the institutional project \u0026bdquo;Environmental factors and microbial interactions in the structure of dental biofilm\u0026ldquo; (IP-2020-02-4027) \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://sites.google.com/view/ip-2020-02/po%C4%8Detna-stranica\u003c/span\u003e\u003cspan address=\"https://sites.google.com/view/ip-2020-02/po%C4%8Detna-stranica\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data presented in this study are available on public repository of Faculty of Dental Medicine, University of Rijeka, Croatia. Špalj S. Okolišni čimbenici i mikrobiološke interakcije u strukturi dentalnog biofilma:istraživački podaci. [Internet]. Fakultet dentalne medicine; 2023.Available from: [**https://urn.nsk.hr/urn:nbn:hr:271:843444**](https:/urn.nsk.hr/urn:nbn:hr:271:843444)\u003c/p\u003e\u003cp\u003e \u003cstrong\u003eEthics Approval declaration\u003c/strong\u003e \u003cp\u003e The study design and protocols were reviewed and approved by the Ethical committee of Clinical Hospital Centre Rijeka (Class: 003\u0026ndash;05/22\u0026thinsp;\u0026minus;\u0026thinsp;1/19; Registry number: 2170-29-02/1-22-2), the Ethical committee of the Faculty of Dental Medicine, University of Rijeka (Class: 035\u0026thinsp;\u0026minus;\u0026thinsp;01/22\u0026thinsp;\u0026minus;\u0026thinsp;01/150; Registry number: 2170-57-006-01-22-1) and the Ethical committee of the Medical Faculty, University of Rijeka (Class: 007\u0026ndash;08/22\u0026thinsp;\u0026minus;\u0026thinsp;01/77; Registry number: 2170-24-04-3/1-22-6).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFunding Declaration\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe presented research has been founded by the Croatian Science Foundation (Hrvatska Zaklada za Znanost,) through the institutional project \u0026bdquo;Environmental factors and microbial interactions in the structure of dental biofilm\u0026ldquo; (IP-2020-02-4027) \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://sites.google.com/view/ip-2020-02/po%C4%8Detna-stranica\u003c/span\u003e\u003cspan address=\"https://sites.google.com/view/ip-2020-02/po%C4%8Detna-stranica\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSantacroce L, Passarelli PC, Azzolino D, Bottalico L, Charitos IA, Cazzolla AP et al (2023) Oral microbiota in human health and disease: A perspective. 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Microorganisms 9(8):1657. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/microorganisms9081657\u003c/span\u003e\u003cspan address=\"10.3390/microorganisms9081657\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"clinical-oral-investigations","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cloi","sideBox":"Learn more about [Clinical Oral Investigations](http://link.springer.com/journal/784)","snPcode":"784","submissionUrl":"https://submission.nature.com/new-submission/784/3","title":"Clinical Oral Investigations","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"dental caries, dental plaque, microbiota, smoking, smoking devices","lastPublishedDoi":"10.21203/rs.3.rs-8302044/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8302044/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eThe study compared the bacterial composition of supragingival dental biofilm (SDB) microbiome of THS smokers (THSS), cigarette smokers (CS) and non-smokers (NS).\u003c/p\u003e\u003ch2\u003eMaterials and Methods\u003c/h2\u003e \u003cp\u003eIn the present cross-sectional study a total of 60 subjects were divided in three groups: THS smokers (THSS), cigarette smokers (CS) and non-smokers (NS). SDB samples were collected, DNA was isolated and used for 16s rRNA gene hypervariable region amplicon, next generation sequencing. Determination of SDB bacterial taxa composition was performed through bioinformatic pipelines and analyses.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSignificant differences were found in the bacterial composition of SDB microbiomes between the examined groups on all taxonomic levels. The most significant differences were detected at genus level, specifically among the genera: \u003cem\u003eActinobaculum\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.004), \u003cem\u003eAvibacterium\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.015), \u003cem\u003eAlysiella\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.016), \u003cem\u003ePropionibacterium\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.025) and \u003cem\u003eHaemophilus\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.030). Significant differences have also been found on the species level, with the most significant regarding Alysiella filiformis (p\u0026thinsp;=\u0026thinsp;0.006), \u003cem\u003eAvibacterium paragallinarum\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.015), \u003cem\u003ePrevotella multiformis\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.016), \u003cem\u003eLactobacillus salivarius\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.018), and \u003cem\u003eActinomyces massiliensis\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.022).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe compositions of SDB of THSS, CS and NS are different, and due to increased anaerobic bacteria abundance with cariogenic properties, the dysbiosis pattern and pathogenic potential of THSS and CS dental biofilms compared to NS is considerably increased.\u003c/p\u003e\u003ch2\u003eClinical Relevance:\u003c/h2\u003e \u003cp\u003eDue to bacterial composition the SDB of THSS and CS could have a higher pathogenic effect than the SDB of NS\u003c/p\u003e","manuscriptTitle":"Supragingival dental biofilm microbiomes of tobacco heating system smokers, cigarette smokers and non-smoker","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-19 12:54:13","doi":"10.21203/rs.3.rs-8302044/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-14T03:30:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-13T08:16:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-02T13:33:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"164724762326647388036815390142135789426","date":"2025-12-28T08:41:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"332958410066661840418933898851969063513","date":"2025-12-19T07:06:56+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-17T06:42:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-10T07:59:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-10T07:57:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Clinical Oral Investigations","date":"2025-12-07T21:37:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"clinical-oral-investigations","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cloi","sideBox":"Learn more about [Clinical Oral Investigations](http://link.springer.com/journal/784)","snPcode":"784","submissionUrl":"https://submission.nature.com/new-submission/784/3","title":"Clinical Oral Investigations","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"856d30f3-eebb-43ff-b170-a3b9f0cdfa5d","owner":[],"postedDate":"December 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T16:34:15+00:00","versionOfRecord":{"articleIdentity":"rs-8302044","link":"https://doi.org/10.1007/s00784-026-06844-5","journal":{"identity":"clinical-oral-investigations","isVorOnly":false,"title":"Clinical Oral Investigations"},"publishedOn":"2026-03-23 16:09:13","publishedOnDateReadable":"March 23rd, 2026"},"versionCreatedAt":"2025-12-19 12:54:13","video":"","vorDoi":"10.1007/s00784-026-06844-5","vorDoiUrl":"https://doi.org/10.1007/s00784-026-06844-5","workflowStages":[]},"version":"v1","identity":"rs-8302044","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8302044","identity":"rs-8302044","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}