Correlation of Enamel Caries with Occlusal Surface Biofilm: A Quantitative Analysis of Streptococcus mutans, Slackia exigua, and Corynebacterium matruchotii  

Correlation of Enamel Caries with Occlusal Surface Biofilm: A Quantitative Analysis of Streptococcus mutans, Slackia exigua, and Corynebacterium matruchotii | 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 Correlation of Enamel Caries with Occlusal Surface Biofilm: A Quantitative Analysis of Streptococcus mutans, Slackia exigua, and Corynebacterium matruchotii Poonnika Ratanachai, Kemthong Mitrakul, Kadkao Vongsawan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8942470/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Aims To detect S. mutans, S. exigua and C. matruchotii amounts in biofilm from occlusal surfaces of first permanent molars categorized by two clinical diagnostic tools which are the Visible Occlusal Plaque Index (VOPI) and the International Caries Detection and Assessment System (ICDAS) and to assess the correlation between levels of bacterial and enamel caries on occlusal surfaces. Study design Plaque samples were obtained from 160 Thai children (6–8 years-old) and assigned to four groups (n = 40 per group) based on VOPI and ICDAS scores. Methods DNA was extracted and analyzed using real-time PCR quantitatively and specific primers targeting total bacteria (BAC16S), S. mutans, S. exigua , and C. matruchotii. Results Children’s ages were different among the VOPI groups (p<0.001). Quantities of BAC16S, S. mutans and S. exigua, increased significantly with higher VOPI scores (p<0.001). C. matruchotii showed a declining trend when VOPI scores increased. It was detected at 97.5%, 82.5%, 70% and 47.5% in VOPI 0, 1, 2, and 3 groups, respectively. A difference between ICDAS and VOPI scores (p<0.001) was showed. There were positive correlations between VOPI and BAC16S (r =0.504, p<0.001), ICDAS and BAC16S (r = 0.506, p < 0.001), and S . mutans and BAC16S (r = 0.583, p < 0.001). S. mutans /total bacteria and S. exigua /total bacteria ratios exhibited associations (p < 0.001). Using logistic regression, subjects scoring in the VOPI 1, 2, and 3 groups had a 1.97, 3.99, and 6.05 higher chance of having enamel caries compared to those subjects in the VOPI 0 group, respectively. Conclusion Quantities of S. mutans and S. exigua escalated with higher VOPI scores, and associated with greater enamel caries, while C. matruchotii decreased. The inverse relationship between C. matruchotii and VOPI suggests its potential indicator for caries-free or good oral health status. Occlusal biofilms enamel caries lesions S. mutans Slackia exigua Corynebacterium matruchotii Figures Figure 1 Introduction Dental caries continues to be a significant health issue worldwide, ranking among the most prevalent chronic diseases. It affects more than two billion people with permanent dentition and approximately 620 million with primary dentition worldwide (Nyvad et al. 2013; Fakhruddin et al. 2019; WHO 2022). In Thailand, recent national surveys reported caries prevalence rates of 47%, 72%, and 50% amongst children aged 3, 5, and 12 years, respectively (National Oral Health Survey 2023). Caries lesion occurrence is particularly high in first permanent molars in children aged 6–8 years old (Pitts et al. 2017). The occlusal surfaces of these teeth are more vulnerable than smooth surfaces because of their morphological features, including deep pits and fissures, their groove–fossa configuration, prolonged eruption phase, and reduced self-cleansing during mastication, making these areas difficult to clean (Carvalho 2014). These structural features create sheltered niches that facilitate biofilm retention and maturation, which, under altered local environmental conditions, can shift towards cariogenicity. The Visible Occlusal Plaque Index (VOPI) is a straightforward, cost-effective measure for assessing plaque accumulation on occlusal surfaces. It involves visually inspecting and probing pits and grooves, and has been proposed as a valuable indicator for early caries risk assessment (Carvalho et al. 1989; Carvalho et al. 1992). Despite research into the microbiology related to occlusal caries lesions, most studies have focused on advanced dentinal lesions, leaving a gap in understanding of the microbial ecology during the early stages of occlusal caries progression (Mantzourani et al. 2009; Lima et al. 2011). The oralome concept and dysbiosis theory describe the energetic exchanges between oral microbial communities and the host, with interspecies and host–microbe relationships shaping a profile of the microbiome and influencing oral health or disease (Radaic et al. 2021). S. mutans is well established as a key cariogenic organism due to its strong acidogenic and aciduric traits and is consistently associated with severe early childhood caries (S-ECC) (Thitisakyothin et al. 2025; Tantikalchan et al. 2022; Damnoensawat et al. 2024), it has been detected in both active and inactive carious lesions. Other organisms, including Lactobacillus and Bifidobacterium , also show strong associations with S-ECC. In the absence of S. mutans , species such as Bifidobacterium and S. wiggsiae may play critical roles in caries lesion development (Mitrakul et al. 2017; Damnoensawat et al. 2024; Valdez et al. 2016). Slackia exigua , initially identified in 1996 as Eubacterium exiguum before reclassification, is an anaerobic Gram-positive rod that forms part of the oral microbiota. It is mainly linked to periodontal disease and dental caries. Evidence indicates a strong association between S. exigua and S-ECC, secondary caries, and dentinal caries (Tanner et al. 2011; Kalpana et al. 2020). Moreover, recent findings suggest a positive correlation between S. exigua and S. wiggsiae in the advanced stages of carious lesions (Fakhruddin et al. 2019). Corynebacterium matruchotii , also known as Leptothrix buccalis and Bacterionema matruchotii previously, is a Gram-positive aerobic bacterium frequently found in health-associated supragingival plaque (Li et al. 2022). Sequencing studies show that C. matruchotii is more plentiful in individuals without caries, whereas S. mutans predominates in caries-active cases. As S. mutans levels increase, C. matruchotii abundance tends to decline (William et al. 1999; Qudeimat et al. 2021). To assess the occurrence and distribution of occlusal caries lesions VOPI was developed and is used clinically (Carvalho et al. 1989). Longitudinal research shows that erupting molars tend to accumulate thicker plaque because of reduced mechanical cleaning and brushing difficulty, correlating with a higher occurrence of active lesions compared to fully erupted molars, which are more likely to have thin or no visible plaque and inactive lesions (Carvalho et al. 1992; Carvalho et al. 2017). VOPI is recommended as a supplementary diagnostic tool for estimating caries lesion activity, particularly in dentinal lesions in adults and for guiding treatment decisions (Henne et al. 2015; Tanner et al. 2016). To standardise a clinical scoring method for detecting and assessing caries on both coronal and root surfaces, the International Caries Detection and Assessment System (ICDAS) was also used in this study. It is applicable to enamel and dentine lesions, including non-cavitated and cavitated forms, using a 0 to 6 scale, where 0 denotes a sound tooth surface and 6 represents tooth structure destruction where the cavity is both deep and wide and dentine is clearly visible on the walls and at the base. An extensive cavity involves at least half of the tooth surface or possibly reaching the pulp (Ismail et al. 2007). The present study aimed to quantitatively assess S. mutans , S. exigua , and C. matruchotii in occlusal surfaces biofilm from first permanent molars categorised according to VOPI, and to evaluate the associations between bacterial levels and the occurrence of occlusal enamel caries based on ICDAS criteria. We hypothesise that the quantities of S. mutans , S. exigua , and C. matruchotii will differ amongst the VOPI groups. Materials and Methods The Faculty of Dentistry and the Faculty of Pharmacy Human Institutional Review Board, Mahidol University gave it clearance to this cross-sectional study (approval number: COA.No.MU-DT/PY-IRB 2024/042.1807). Based on previous studies, we set the estimated correlation of VOPI and S. mutans at 0.45, anticipating that site-specific sampling would increase the correlation between the bacteria and dental caries (Dikmen 2015). Using a two-sided 95% confidence interval (CI) of 0.45 ± 0.125 (width = 0.25), the calculated sample size amounted to 159 teeth, which was rounded up to 160 teeth (Bonett et al. 2000; Ismail et al. 2009). If a sufficient sample size is not obtained, additional samples are expected to be collected, estimated at 20% of the total sample, which is an additional 32 teeth. Consequently, a total of 192 teeth samples will be collected. We anticipate that each participant will have two qualifying first molars meeting the study criteria, thus estimating that the sample group will consist of 96 primary school students from grades 1 to 3. Using the software named Primer of Biostatistics (McGraw-Hill, NY, USA) performed with α=0.05 and power of 80% obtained at least of 40 children in each group would be enough to reach statistical difference (Bonett et al. 2000). Subject selection: The recruitment was done at Chumchon Prachathipat Wittayakhan School, Pathumthani Province, Thailand. Thai children aged 6 to 8 years old were selected. Consent forms were signed by all parents or legal guardians. Every participant was free to leave the research at any moment . Performed clinical examination full mouth using World Health Organization criteria, the ICDAS and the VOPI. VOPI composed of four scores which are : 0=no plaque was obtained when carefully drawing a dental probe on the groove, 1=slight plaque was detected which is restricted to the groovefossa-system and identified by carefully drawing a dental probe on the groove, 2=abundant plaque: plaque was visually detected on the groove, 3 = dense plaque: occlusal surfaces partially or totally covered with heavy plaque visually. ICDAS used in this study was composed of three scores which are: 0=sound tooth: after 5 seconds of air drying, no caries was detected; 1=early enamel opacity or discoloration (white or brown) is visible at pit or fissure on the occlusal surfaces after prolonged air drying; 2=enamel showed a visible change when wet however, lesion must be visible when dry; 3=enamel breakdown locally (without dentinal involvement) was identified when wet and after prolonged air drying (Ismail et al. 2009; Carvalho et al. 2016). Subjects must have no occlusal dentin caries, sealants or fillings on first permanent molars. Exclusion criteria were children who had received professional fluoride application, has any systemic diseases, taking any kind of antibiotics, had any oral treatment within 2 months before the collection visit or first permanent molars were partially erupted with more than 1/4 of the occlusal surfaces covered with gingival tissue. Clinical examination: Two examiners (PT and PR) who were residents in Pediatric Dentistry at Faculty of Dentistry, Mahidol University were trained and calibrated prior to perform a clinical examination. Weighted Kappa VOPI agreement and ICDAS agreement were 0.91 and 0.83, respectively. Categorized plaque samples into four groups according to ICDAS and VOPI scores. Total of 160 teeth (each group=40). Recorded VOPI and ICDAS scores. Plaque sample collection: Plaque samples were gently collected by drawing a sterile toothpick along the occlusal groove of permanent first molars for collecting plaque samples that were isolated by sterile gauzes to obtain strictly dry areas around the molars in order to prevent the contamination by saliva. Samples were released into 1 ml of Tris-EDTA buffer, immediately stored on ice, transported to the Oral Biology Laboratory, and kept at –20℃ until DNA extraction. DNA extraction: DNA was extracted using a kit (Flavogen, Taiwan) as previously described. 12 Measured DNA concentration and purity at 260 nm/280 nm (Nanodrop 2000C Thermo Scientifc, Delaware, USA). Culture condition and standard strains: S. exigua ATCC 700122 and S. mutans ATCC 25175 were used as a standard . S. exigua was grown in Tryptic Soy Medium with 5% defibrinated Sheep Blood and incubated at 37°C for 48 hr. Grew S. mutans in Brain Heart Infusion broth at 37°C and incubated for 24-48 hr. A Gram stain was tested before the DNA extraction and a ten-fold serial dilution was prepared for a sensitivity test of the employed primers. Quantitative real‑time PCR: All primers used in this study were list in Table 1. Total volume of the reaction mixture was 20 μl as previously described. 10 Annealing temperature at 50°C, 53°C, 55.9°C and 57.4°C for 20 sec for BAC16S, S. mutans , S. exigua and C. matruchotii , respectively. Generated melting curves from 60°C-95°C and read every 0.5°C for 5 sec. Table 1: Primers used in this study Primers Nucleotide sequence (5′ - 3′) Amplicon (Basepair) Ref Universal BAC16S F TGGAGCATGTGGTTTAATTCGA 160 Sinsimer et al , 2005 R TGCGGGACTTAACCCAACA S. mutans F5 AGCCATGCGCAATCAACAGGT T 415 Sato et al , 2003 R4 CGCAACGCGAACATCTTGATCAG Slackia exigua exi557F GCCAAGCGGCCTCGTCGAAG 740 Hashimura et al, 2001 exi1263R GCCGGCTTTAAGGGATTCGCTCG Corynebacterium Matruchotii PP6N5 CGGAATTCATGGAYTAYGGYCARATC 166 van Dijk et al , 1998 P6C5 GCGGA TCCRGTYTCRCCW AGYTCRGA Agarose gel electrophoresis: Checked amplified PCR products using 2% agarose gel (UltraPure Agarose, ThermoFisher Scientifc, USA). Saved gel images with a digital imaging system (Molecular Imager ®Gel docTM Systems, Bio-Rad Laboratories Inc., CA, USA). Statistical analysis Analyzed data using IBM SPSS Statistics Version 28.0 (IBM SPSS Statistics for Windows, Version 28.0. Armonk, NY, USA: IBM Corp.) For table 2, if data was continuous variables, such as the age of children (years), ANOVA was used to compare the average (Mean±SD) between the VOPI group 0-3. If data was categorical variables, such as gender (male/female) and molars positions (top/bottom), the Chi-Square Test was used to compare frequency (%) between the VOPI groups. Testing data normal distribution (the bacteria amount is distributed normally or not) using Kolmogorov-Smirnov and Shapiro-Wilk. Since the data distribution is not normal, Mann -Whitney U Test was used to compare amount of bacteria between two VOPI groups and Kruskal -Wallis H Test was used to compare the median of bacteria between many VOPI groups. For Correlation Analysis, Spearman’s Rho Correlation was used to analyze the relationship between the quantity of bacteria, VOPI scores and ICDAS. Chi-Square test was used to compare the bacteria's detection rate and the distribution of ICDAS between VOPI groups. Logistic Regression was used to analyze the chance of enamel caries at different VOPI group using the VOPI 0 which is a caries-free control group as a reference group. The value of p < 0.05 was accepted as statistically different. Results Among the VOPI groups, ages of the children were significantly different (Table 2). Table 2: Mean age, gender and permanent molar location of participants in four VOPI groups Varibles Group 0 1 2 3 p-value Children's age (years) Mean ± SD Median 6.7± 0.20 6.5 ± 0.24 7.08 ± 0.28 7.08 ± 0.25 <0.001* 6.7 6.8 7 7.1 <0.001* Children's genders (n%) Male Female 0.896 23 (57.5) 20 (50) 20 (50) 20 (50) 17 (42.5) 20 (50) 20 (50) 20 (50) Permanent molar (n%) Upper Lower 0.965 21 (52.5) 19 (47.5) 21 (52.5) 21 (52.5) 19 (47.5) 21 (52.5) 19 (47.5) 19 (47.5) Abbreviations: ANOVA, analysis of variance; SD, standard deviation; VOPI, Visible Occlusal Plaque Index. * p-value < 0.05 were obtained between VOPI groups based on ANOVA or chi-square for continuous and categorical variables, respectively, shown as mean ± SD. The non-parametric Kruskal-Wallis H test was used for median and interquartile range (IQR) which are Median of Children’s age (years) All primers were tested specificity. The standard strain of C . matruchotii was not obtained due to the difficulty of culture in the laboratory. Standard curves were generated for the BAC16S, S . mutans, and S . exigua primers. The detection limit of BAC16S, S. mutans , and S . exigua primers were 10 1 , 10 and 10 3 , respectively. Samples were randomly selected from each VOPI groups to visualized the amplified products from BAC16S (Fig 1A), S. mutans , S. exigua and C. matruchotii. S. mutans was detected in all VOPI group (Fig 1B). S. exigua was presented in the VOPI group 3 while C. matruchotii was not showed in VOPI groups 2 and 3 (Fig 1C and D) Table 3 presents S. mutans , S. exigua , and C. matruchotii prevalences in each of the VOPI scores. S. mutans was detected in nearly all samples, with prevalence ranging from 97.5% in VOPI 0 and VOPI 1 groups to 100% in VOPI 2 and VOPI 3 groups. S. exigua demonstrated an upward trend in detection when VOPI scores increased. It was detected at 65% in the VOPI 0 group, and increased progressively to 82.5% in the VOPI 1 group. It reached 100% in both the VOPI 2 and VOPI 3 groups. Conversely, C. matruchotii showed a declining trend across VOPI levels. It was detected at 97.5% in the VOPI 0 group and decreased to 82.5% in VOPI 1, 70% in VOPI 2, and 47.5% in the VOPI 3 group, respectively. Table 3: Distribution of S. mutans , S. exigua and C. matruchotii prevalences in all VOPI groups (n=40 in each group) Bacterial Group 0 n (%) 1 n (%) 2 n (%) 3 n (%) S. mutans 39 (97.5) 39 (97.5) 40 (100) 40 (100) S. exigua 26 (65) 33 (82.5) 40 (100) 40 (100) C. matruchotii 39 (97.5) 33 (82.5) 28(70) 19 (47.5) Abbreviation: VOPI, Visible Occlusal Plaque Index. Table 4 presents the detection of ICDAS scores in each of the VOPI groups. There was a difference significantly between ICDAS and VOPI scores (p < 0.001), indicating that as the level of visible plaque increases, the severity of enamel caries also rises. Among children with no visible plaque (VOPI 0), 87.5% exhibited sound enamel (ICDAS 0), and only 12.5% showed early enamel changes (ICDAS 1). In contrast, in the highest plaque group (VOPI 3), none had sound enamel, while 15% had ICDAS 1, 47.5% had ICDAS 2 (distinct visual enamel changes), and 37.5% had ICDAS 3 (localized enamel breakdown). Logistic regression showed that subjects in the VOPI 1, 2, and 3 groups had a 1.97, 3.99, and 6.05 higher chance of having caries in the enamel compared to those subjects in VOPI 0, respectively (Table 5). Table 4: ICDAS detection in each VOPI groups (n=40 in each group) ICDAS scores Group 0 n (%) 1 n (%) 2 n (%) 3 n (%) p-value ICDAS 0 35 (87.5) 18(45) 3 (7.5) 0 <0.001* ICDAS 1 5 (12.5) 14 (35) 14 (35) 6 (15) ICDAS 2 0 6 (15) 17 (42.5) 19 (47.5) ICDAS 3 0 2 (5) 6 (15) 15 (37.5) Abbreviations: ICDAS, International Caries Detection and Assessment System; VOPI, Visible Occlusal Plaque Index. *p-value < 0.05, Chi-Square Tests Table 5: Analysis of the Association Between VOPI Groups and Enamel Caries Severity OR 95% C.I. for EXP (B) p-value Intercept (ICDAS 1) 1.97 Intercept (ICDAS 2) 3.99 Intercept (ICDAS 3) 6.05 VOPI 0 Ref. VOPI 1 5.61 (4.30 - 6.91) <0.001* VOPI 2 4.32 (3.09 - 5.54) <0.001* VOPI 3 2.31 (1.19 - 3.42) <0.001* Abbreviations: CI, confidence interval; ICDAS, International Caries Detection and Assessment System; OR, odds ratio; VOPI, Visible Occlusal Plaque Index. *p-value < 0.05, (Logistic regression analysis) The level of BAC16S (p<0.001), S. mutans (p<0.001) and S. exigua (p<0.001) significantly increased when VOPI scores increased. There were significantly different ratios of S. mutans to total bacteria (p<0.001) and S. exigua to total bacteria (p<0.001) amongst the VOPI scores (Table 6). Table 6: Quantities of bacteria and ratios of S. mutans/total bacteria and S. exigua /total Bacteria VOPI 0 Median (Min, Max) VOPI 1 Median (Min, Max) VOPI 2 Median (Min, Max) VOPI 3 Median (Min, Max) p-value Total bacteria 1.63x10 9 (7.85x10 4 ,1.54x10 12 ) 3.75x10 11 (1.50×10², 1.67× 10 14 ) 5.43x10 11 (1.47x10 8 ,7.51x10 14 ) 7.53x10 11 (1.42x10 3 ,3.78x10 12 ) <0.001* S.mutans 3.55x10 4 (0, 1.63x10 6 ) 1.02x10 5 (0, 5.50x10 8 ) 1.05x10 6 (6.27x10 2 ,3.17x10 8 ) 4.53x10 7 (2.14x10 2 ,1.84x10 9 ) <0.001* S.mutans/total bacteria 1.68x10 -5 (0, 1.31x10 0 ) 7.81x10 -7 (0, 3.77x10 6 ) 6.29x10 -7 (2.17x10 -12 ,1.74x10 -1 ) 1.48x10 -4 (1.81x10 -9 , 6.87x10 0 ) <0.001* S. exigua 2.16x10 2 (0, 2.81x10 4 ) 2.30x10 3 (0, 4.5x10 6 ) 1.93x10 5 (1.75x10 2 ,1.93x 10 5 ) 4.98x10 6 (3.19x10 4 , 7.19x10 13 ) <0.001* S. exigua /total bacteria 2.33x10 -8 (0, 3.5 x10 -1 ) 1.08x10 -9 (0, 1.49x10 5 ) 6.29x10 -7 (2.17x10 -12 ,2.58x10 -4 ) 9.06x10 -6 (4.64x10 -8 , 1.34x10 2 ) <0.001* Abbreviation: VOPI, Visible Occlusal Plaque Index. *p-value < 0.05, between VOPI groups based non-parametric Kruskal-Wallis H test Table 7: Comparison of each bacteria quantities and ratios among VOPI Groups Total bacteria S. mutans S. mutans /total bacteria S. exigua S. exigua /total bacteria (I) VOPI (J) VOPI p value (J) VOPI p value (J) VOPI p value (J) VOPI p value (J) VOPI p-value 0 1 < 0.001* 1 0.048* 1 0.01* 1 0.004* 1 0.534 2 < 0.001* 2 < 0.001* 2 < 0.001* 2 0.003* 2 0.802 3 < 0.001* 3 < 0.001* 3 < 0.001* 3 0.222 3 < 0.001* 1 2 0.252 2 0.268 2 0.122 2 0.744 2 0.317 3 0.381 3 < 0.001* 3 < 0.001* 3 < 0.001* 3 < 0.001* 2 3 0.651 3 < 0.001* 3 < 0.001* 3 < 0.001* 3 < 0.001* Abbreviation: VOPI, Visible Occlusal Plaque Index. *p-value < 0.05 (Mann - Whitney test) Table 7 presents significant differences in total bacterial levels between group 0 and the other groups (1, 2, and 3) (p<0.001). For S . mutans , statistically significant differences were observed between VOPI 0 and VOPI 1 (p = 0.048), VOPI 0 and VOPI 2 (p < 0.001), and VOPI 0 and VOPI 3 (p < 0.001). No significant difference was found between VOPI 1 and VOPI 2 (p = 0.268), whereas significant differences were observed between VOPI 1 and VOPI 3 (p < 0.001), and VOPI 2 and VOPI 3 (p < 0.001). These findings showed that S. mutans levels increased significantly with higher plaque accumulation, particularly between lower and higher VOPI scores. For S. mutans /total bacteria ratios, there were significant differences between VOPI 0 and VOPI 1 (p= 0.01), VOPI 0 and VOPI 2 (p< 0.001), VOPI 0 and VOPI 3 (p< 0.001), VOPI 1 and VOPI 3 (p< 0.001), and VOPI 2 and VOPI 3 (p< 0.001). For S. exigua , there were significant differences between VOPI 0 and VOPI 1 (p= 0.004), VOPI 0 and VOPI 2 (p=0.003), VOPI 1 and VOPI 3 (p< 0.001), and VOPI 2 and VOPI 3 (p< 0.001). For S. exigua /total bacteria ratio, there were significant differences between VOPI 0 and VOPI 3 (p<0.001), VOPI 1 and VOPI 3 (p< 0.001), and VOPI 2 and VOPI 3 (p< 0.001). Correlation analysis between levels of bacterial, VOPI, and ICDAS indicated significant positive correlations between VOPI and total bacteria (r = 0.504, p < 0.001), and between ICDAS and total bacteria (r = 0.506, p < 0.001). Additionally, S. mutans showed a positive correlation with total bacteria (r = 0.583, p < 0.001), whereas S. mutans /total bacteria and S. exigua /total bacteria ratios also exhibited significant associations (p < 0.001). These findings indicate strong, statistically significant relationships between bacterial levels, VOPI, and dental caries assessment scores (Table 8). Table 8: Levels of bacteria, VOPI, and ICDAS scores correlation. Total bacteria S.mutans S.mutans /total bacteria S. exigua S.exigua /total bacteria ICDAS ρ P-value ρ P-value ρ P-value ρ P-value ρ P-value ρ P-value VOPI 0.504 <0.001* 0.583 <0.001* 0.734 <0.001* 0.080 0.313 0.423 <0.001* .758 <0.001* ICDAS 0.506 <0.001* 0.425 <0.001* 0.496 <0.001* -0.035 0.663 0.193 0.015* 1 1 Abbreviations: ICDAS, International Caries Detection and Assessment System; VOPI, Visible Occlusal Plaque Index. *p-value<0.05, (Spearman’s Rho) Discussion This study purposely collected plaque samples only from sound and incipient enamel lesions from the occlusal surfaces of first permanent molars in the hope of filling the knowledge gap regarding the microbiology associated with sound and early enamel caries lesions. Most previous studies demonstrated results of the microbiota in occlusal caries focusing on dentinal caries (Lima et al. 2011; Dige et al. 2014; Kressirer et al. 2018). Oral cavity provides optimal conditions for colonization and proliferation of microbiota. It is a unique microenvironment. It is regulated through complex biological signaling systems and interactions with the host. Once the oral microbiota is out of balance or dysbiosis, microorganisms produce virulence factors and metabolites, which will cause dental caries (Rajasekaran et al. 2024; Baker et al. 2024). The highest risk period for caries incidence typically spanned the initial 3 to 4 years following eruption (Zijnge et al. 2010). This susceptibility was attributed mainly to the macromorphology of the occlusal surface, which facilitated the adherence of oral microbiota. This was due to its intricate groove-fossa system, coupled with a relatively extended eruption period during which mechanical oral function was reduce (Zijnge et al. 2010). These protected sites, where mechanical cleansing is hindered, might promote the buildup and maturation of biofilms. These biofilms can develop into cariogenic ones if there is an imbalance in metabolic activities due to changes in local environmental conditions (Zijnge et al. 2010). The bacterial composition of biofilm observed within grooves and at the entry points of fissures was similar to those of supra gingival in vivo biofilm (Zijnge et al. 2010). Bacteria appear on the tooth surface within two hours after cleaning, initiating colonization following the formation of a dental pellicle induced by saliva. Initial colonizers such as Actinomyces , Streptococcus , and Lactobacillus produce acidic byproducts through fermentative metabolism of dietary carbohydrates (Esberg et al. 2020). The oral microbial environment is regularly subjected to external factors both environmental and behavioral factors such as dietary habit and oral hygiene practice might contribute to microbial shifts alongside plaque accumulation. Such conditions are crucial determinants for establishing microbial populations and their ability to endure in this setting, leading to unique interactions between microbes and their hosts that depend on selective pressures. Prolonged acidic exposure suppresses acid-sensitive oral bacteria while promoting acid-tolerant species. Increased acidity in biofilm correlates with enamel surface demineralization and the protective outer layer frail, resulting in roughening surfaces that may clear the way for the colonization of more aggressive microbial pathogens. Secondary colonizers such as S. mutans , Lactobacillus , Bifidobacterium , and Scardovia wiggisiae contribute to climax communities, shaping the environmental conditions of the oral cavity and potentially increasing the pathogenicity of the involved colonizers. S. mutans is considered as a classical cariogenic species . It was present in both inactive and active caries but absent on sound enamel. An abundance of S. mutans was detected in active lesions. Unsurprisingly, this study confirmed the association between S. mutans and initial caries lesions in the occlusal surfaces with a high prevalence. These results are aligned with previous studies (Thitisakyothin et al. 2025; Tantikalchan et al. 2022). Our previous study showed a significant positive correlation between VOPI scores and both ICDAS scores and levels of S. mutans to total bacteria (Thitisakyothin et al. 2025). Similar findings are found in this study, significant quantitative differences in total bacterial load were observed across the VOPI groups, with a consistent upward trend from VOPI 0 to VOPI 3. Median bacterial counts increased from 1.63×10⁹ CFU in VOPI 0, to 7.53×10¹¹ CFU in VOPI 3 (p < 0.001), suggesting a clear relationship between plaque visibility and microbial biomass. As expected, S. mutans levels increased significantly with higher plaque accumulation, particularly between lower and higher VOPI scores. However, the proportion of S. mutans relative to total bacterial load increased with increasing VOPI, from 1.68×10⁻⁵ to 1.48×10⁻⁴, indicating that despite the absolute increase in S. mutans , its relative abundance was diluted within the growing polymicrobial community. These findings are consistent with previous studies that have shown S. mutans to be a key contributor in early biofilm formation, with its role diminishing in dominance as the plaque matures and diversifies (Lima et al. 2011; Dige et al. 2014). This phenomenon may be allocated the ecological succession of microbial communities, where later colonizers, particularly anaerobes, become more prevalent as oxygen gradients and nutrient availability change within the thickening biofilm (Marsh et al. 2006). In early biofilm formation, S. mutans plays a dominant role due to its strong adhesion mechanisms, such as the production of extracellular glucans via glucosyltransferase enzymes (GTF), and its ability to thrive in acidic environments through its acidogenic and aciduric properties (Bowen et al. 2011). Its dominance is evident in low-VOPI environments (e.g., VOPI 0), where the microbial community is less complex and less mature. However, as the biofilm matures (VOPI 2–3), environmental conditions such as reduced oxygen, lower pH, and increased substrate availability support the colonization and proliferation of a wider variety of anaerobic and aciduric species. These include S. exigua , Bifidobacterium , and S. wiggsiae , which are more frequently associated with mature carious lesions (Thitisakyothin et al. 2025; Kalpana et al. 2020). S. mutans initiates the caries process but is not the sole pathogen in advanced lesions. Moreover, previous studies indicate that while S. mutans is integral to early colonization and lesion initiation, it eventually shares its niche with other microorganisms as the biofilm thickens and diversifies (Spatafora et al. 2024; Aas et al. 2008). This study initial attempt to quantitatively detect the amount of few novel non-mutans bacteria related to caries in occlusal surface biofilm. The study revealed a significant microbial transition in the VOPI 3 group, with S. exigua exhibiting a considerable rise in both absolute quantity and relative abundance. It is an emerging and less studies species. The observed pattern suggests that S. exigua functions as a late-stage colonizer and may serve as a potential indicator of biofilm maturation and dysbiosis. This interpretation is consistent with prior research that characterizes S. exigua as an obligate anaerobic, Gram-positive, asaccharolytic species, typically absent or found in low abundance in early biofilms, but increasingly identified in mature, low-oxygen, and acidic environments, such as deep dentin caries, secondary caries, and chronic periodontal lesions (Fakhruddin et al. 2019; Tanner et al. 2011; Aas et al. 2008; Sergio et al. 1996; Hashimura et al. 2001). S. exigua , although not typically categorized as acidogenic, is frequently linked to S-ECC and deep dentinal lesions, indicating its role in the advancement of caries rather than their onset (Tanner et al. 2011; Sergio et al. 1996). It is often found alongside S. wiggsiae in advanced lesions, suggesting a possible synergistic effect on biofilm virulence (Fakhruddin et al. 2019). Tanner and colleagues identified S . exigua as a constituent of the ecological consortium associated with active caries, potentially augmenting pathogenicity via microbial interactions and biofilm persistence (Tanner et al. 2011). Its ability to survive acidic, anaerobic environments, like those in necrotic pulp tissue, supports its classification as a secondary colonizer, which contributes to the stability and pathogenic potential of mature biofilms. 38 Previous study found that S. exigu a levels were much higher in children and adult orthodontic patients when compared to non-orthodontic patients. The rates were 71.4% vs. 28.6% in children, and 70.7% vs. 29.3% in adults (Booth et al. 2004). These findings demonstrate that fixed orthodontic appliances can create plaque-retentive, anaerobic environments conducive to the proliferation of S. exigua . Colonization may be facilitated by diminished biofilm clearance and increased microbial retention within these populations. S. exigua has been recognized in systemic infections such as bacteremia, peritonitis, and septic shock, particularly in immunocompromised individuals, underscoring its opportunistic pathogenicity beyond the oral cavity (Shen et al. 2003; Zhou et al. 2025; Rieber et al. 2019). The increasing prevalence of this species in dysbiotic plaque corresponds with the ecological plaque hypothesis, which suggests that environmental alterations, including acidification and reduced oxygen levels, facilitate the emergence of rarer but more virulent species. S. exigua functions as both a microbial biomarker for mature dysbiotic biofilms and a contributor to the advancement of caries. Furthermore, S. exigua has been shown to metabolize amino acids such as arginine and lysine to produce short-chain fatty acids (SCFAs) , including acetate and propionate (Lee et al. 2016). These byproducts may not cause demineralization directly but can contribute to host tissue inflammation and degradation , particularly in endodontic and periodontal contexts. This organism is important in ecological succession and the development of advanced dental caries because it is common in deep carious lesions, it is linked to pathogenic consortia, and it prefers acidic, anaerobic environments. Corynebacterium matruchotii , an aerobic, filamentous bacterium involved in the structural organization of supragingival biofilms, thrives in well-oxygenated, neutral pH conditions characteristic of health-associated plaque (Esberg et al. 2020). Thus, combined with the “pillar” role of C. matruchotii in the structure of dental plaque in healthy people, it could be inferred that C. matruchotii might perform a key part in the formation and stabilization of healthy dental plaque. In contrast, the reduction of C. matruchotii may directly lead to the break of biofilm homostasis. Meanwhile, the decreased abundance of C. matruchotii , based on the close relationship shown in corncob structure of supragingival biofilm, might also be part of the reduction of commensal streptococci , such as Streptococcus gordonii and S. sanguinis , reported before, which showed an antagonistic effect against S. mutans via their capability of producing hydrogen peroxide. The alteration could finally contribute to a replacement of dominant species in the oral ecosystem and induce the formation of diseased-adapted supragingival plaque determined by S. mutans that lead to disorders in acid-base metabolism and eventually cause the demineralization of tooth enamel. In general, C. matruchotii is a potential indicator of a caries-free state, and the reduction of its abundance may indicate the evolution from healthy plaque to caries biofilm (Esberg et al. 2020). The observed decline in C. matruchotii prevalence from 97.5% at VOPI 0 to 47.5% at VOPI 3 suggests a negative association between this species and increasing plaque accumulation. C. matruchotii thrives in well-oxygenated, neutral pH conditions characteristic of health-associated, supragingival plaque (Baker et al. 2024). However, as the biofilm matures and becomes increasingly anaerobic and acidic, conditions become unfavorable for C. matruchotii, while aciduric species like S. mutans and S. exigua are able to proliferate (Lee et al. 2016; Tian et al. 2024). C. matruchotii as potentially associated with health-related biofilms. Supporting this ecological interpretation, previous longitudinal data revealed that C. matruchotii abundance significantly increased at 1 and 3 months after full-mouth caries treatment under general anesthesia in children with S-ECC, compared to pre-treatment and 7 days post-treatment levels (p<0.01) (Esberg et al. 2020). This re-emergence in the post-treatment, caries-free state suggests that C. matruchotii may serve as a biomarker of oral health and microbial homeostasis, reinforcing its association with structurally ordered and disease-resistant biofilms. The study further noted that C. matruchotii was consistently ranked among the top 10 most abundant species in children who had received successful treatment, and its presence coincided with increased alpha diversity and microbial richness in plaque, especially in the later stages of recovery. These findings highlight the potential of C. matruchotii not only as a structural keystone species but also as an ecological indicator of restored microbial balance following caries intervention (Kumaresan et al. 2024; Marsh et al. 2017). These findings suggest that C. matruchotii is highly sensitive to acidic and competitive biofilm conditions, which explains its decreased prevalence in higher VOPI scores. It likely plays a role in maintaining biofilm stability and microbial balance in early-stage plaque (VOPI 0–1) and becomes excluded from advanced dysbiotic communities (VOPI 3). Furthermore, C. matruchotii may facilitate the colonization of non-cariogenic streptococci such as S. gordonii and S. sanguinis , which contribute to oral health by producing hydrogen peroxide that inhibits S. mutans ( Ma et al. 2004). This study has shown a positive correlation between VOPI and ICDAS scores, meaning that as plaque accumulation (higher VOPI score) increases, the severity of enamel caries (higher ICDAS score) also increases. The VOPI is recommended as an additional clinical tool to estimate the activity of caries lesions alongside the clinical characteristics assessed by systems like ICDAS, helping support treatment decisions in daily practice. The constraints of this study include its cross-sectional design, which offers just a momentary view and restricted number of microorganisms tested which does not fully represent a true dynamic progression of the oral biofilm complex community. Additionally, the relatively small sample size may limit the generalizability of the findings to broader populations. Longitudinal research is strongly recommended to understand the dynamic of oral biofilm change over time. In addition, samples have been taken from a single school in Thailand. The ability to generalize findings is maybe restricted at this moment. Further studies by recruiting samples from different areas are highly suggested. Conclusions The findings present that increasing plaque accumulation, as indicated by higher VOPI scores, was significantly associated with greater total bacterial load and higher ICDAS scores, indicating progressive enamel demineralization. Levels of S. mutans and S. exigua increased, whereas C. matruchotii decreased, when VOPI scores increased. S. mutans levels increased significantly with higher plaque accumulation, particularly between lower and higher VOPI scores. Also, significant increases in S. exigua were seen among VOPI scores, but not as noticeably as S. mutans, which showed linear increases when VOPI scores increased. There were significant differences between VOPI and ICDAS scores, indicating that as the level of visible plaque increases, the severity of enamel caries also rises. This can be useful when planning the preventive measures to patients in the future such as using the antimicrobial agents that might specifically target S. mutans and S. exigua. Significant positive correlations are thus observed between VOPI and total bacteria, and between ICDAS and total bacteria. 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PLoS One . 2010;5(2):e9321. doi:10.1371/journal.pone.0009321 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 25 Feb, 2026 Editor assigned by journal 24 Feb, 2026 Submission checks completed at journal 24 Feb, 2026 First submitted to journal 22 Feb, 2026 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8942470","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":596918872,"identity":"b694e46c-a435-40e8-b023-ffa6d502a8ba","order_by":0,"name":"Poonnika Ratanachai","email":"","orcid":"","institution":"Mahidol University","correspondingAuthor":false,"prefix":"","firstName":"Poonnika","middleName":"","lastName":"Ratanachai","suffix":""},{"id":596918873,"identity":"c9685fc1-2141-44df-8d0c-177f07633076","order_by":1,"name":"Kemthong Mitrakul","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYDACHiBmbAAS7A0MzMRrOQjSwnOAZC0SCURq0e05fHTzxx02efKRbww/F1TYMPC3dyfg1WJ2ti3txsEzacWGt3OMpWecSWOQOHN2A34t53nMbhxsO5y4cXaOgTRv22EGA4lcQlr4v0G0zDxj/Js4LWd72MBa5kvwmBFpy5ljZjeA/kncwJNWZs1zJo2HsF/OJD+7Udlmkzi//fDm2zwVNnL87b34tcCBwQEOAxDNQ5xyEJBvYH9AvOpRMApGwSgYUQAAFNxP2Pj/9+wAAAAASUVORK5CYII=","orcid":"","institution":"Mahidol University","correspondingAuthor":true,"prefix":"","firstName":"Kemthong","middleName":"","lastName":"Mitrakul","suffix":""},{"id":596918874,"identity":"a890702c-376f-4af8-8b64-d30176c94147","order_by":2,"name":"Kadkao Vongsawan","email":"","orcid":"","institution":"Siam University","correspondingAuthor":false,"prefix":"","firstName":"Kadkao","middleName":"","lastName":"Vongsawan","suffix":""}],"badges":[],"createdAt":"2026-02-23 03:23:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8942470/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8942470/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104702396,"identity":"1b4da5a1-89f2-4836-85bc-24805934a466","added_by":"auto","created_at":"2026-03-16 08:43:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":317686,"visible":true,"origin":"","legend":"\u003cp\u003eAmplified products from randomly selected plaque samples using (A) BAC16S primers, (B) \u003cem\u003eS. mutans\u003c/em\u003e primers, (C) \u003cem\u003eS. exigua \u003c/em\u003eprimers and (D) \u003cem\u003eC. matruchotii \u003c/em\u003eprimers in four VOPI groups.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8942470/v1/699cf9dd92a31630a9ce2cc0.png"},{"id":104783249,"identity":"15ece886-f54b-49a1-8d15-207bbb9b775a","added_by":"auto","created_at":"2026-03-17 07:58:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1159800,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8942470/v1/56ca7b19-3948-4ce0-aa1c-80d495217ad0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Correlation of Enamel Caries with Occlusal Surface Biofilm: A Quantitative Analysis of Streptococcus mutans, Slackia exigua, and Corynebacterium matruchotii ","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDental caries continues to be a significant health issue worldwide, ranking among the most prevalent chronic diseases. It affects more than two billion people with permanent dentition and approximately 620\u0026nbsp;million with primary dentition worldwide (Nyvad et al. 2013; Fakhruddin et al. 2019; WHO 2022). In Thailand, recent national surveys reported caries prevalence rates of 47%, 72%, and 50% amongst children aged 3, 5, and 12 years, respectively (National Oral Health Survey 2023).\u003c/p\u003e \u003cp\u003eCaries lesion occurrence is particularly high in first permanent molars in children aged 6\u0026ndash;8 years old (Pitts et al. 2017). The occlusal surfaces of these teeth are more vulnerable than smooth surfaces because of their morphological features, including deep pits and fissures, their groove\u0026ndash;fossa configuration, prolonged eruption phase, and reduced self-cleansing during mastication, making these areas difficult to clean (Carvalho 2014). These structural features create sheltered niches that facilitate biofilm retention and maturation, which, under altered local environmental conditions, can shift towards cariogenicity. The Visible Occlusal Plaque Index (VOPI) is a straightforward, cost-effective measure for assessing plaque accumulation on occlusal surfaces. It involves visually inspecting and probing pits and grooves, and has been proposed as a valuable indicator for early caries risk assessment (Carvalho et al. 1989; Carvalho et al. 1992).\u003c/p\u003e \u003cp\u003eDespite research into the microbiology related to occlusal caries lesions, most studies have focused on advanced dentinal lesions, leaving a gap in understanding of the microbial ecology during the early stages of occlusal caries progression (Mantzourani et al. 2009; Lima et al. 2011). The oralome concept and dysbiosis theory describe the energetic exchanges between oral microbial communities and the host, with interspecies and host\u0026ndash;microbe relationships shaping a profile of the microbiome and influencing oral health or disease (Radaic et al. 2021).\u003c/p\u003e \u003cp\u003e \u003cem\u003eS. mutans\u003c/em\u003e is well established as a key cariogenic organism due to its strong acidogenic and aciduric traits and is consistently associated with severe early childhood caries (S-ECC) (Thitisakyothin et al. 2025; Tantikalchan et al. 2022; Damnoensawat et al. 2024), it has been detected in both active and inactive carious lesions. Other organisms, including \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e, also show strong associations with S-ECC. In the absence of \u003cem\u003eS. mutans\u003c/em\u003e, species such as \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eS. wiggsiae\u003c/em\u003e may play critical roles in caries lesion development (Mitrakul et al. 2017; Damnoensawat et al. 2024; Valdez et al. 2016).\u003c/p\u003e \u003cp\u003e \u003cem\u003eSlackia exigua\u003c/em\u003e, initially identified in 1996 as \u003cem\u003eEubacterium exiguum\u003c/em\u003e before reclassification, is an anaerobic Gram-positive rod that forms part of the oral microbiota. It is mainly linked to periodontal disease and dental caries. Evidence indicates a strong association between \u003cem\u003eS. exigua\u003c/em\u003e and S-ECC, secondary caries, and dentinal caries (Tanner et al. 2011; Kalpana et al. 2020). Moreover, recent findings suggest a positive correlation between \u003cem\u003eS. exigua\u003c/em\u003e and \u003cem\u003eS. wiggsiae\u003c/em\u003e in the advanced stages of carious lesions (Fakhruddin et al. 2019).\u003c/p\u003e \u003cp\u003e \u003cem\u003eCorynebacterium matruchotii\u003c/em\u003e, also known as \u003cem\u003eLeptothrix buccalis\u003c/em\u003e and \u003cem\u003eBacterionema matruchotii\u003c/em\u003e previously, is a Gram-positive aerobic bacterium frequently found in health-associated supragingival plaque (Li et al. 2022). Sequencing studies show that \u003cem\u003eC. matruchotii\u003c/em\u003e is more plentiful in individuals without caries, whereas \u003cem\u003eS. mutans\u003c/em\u003e predominates in caries-active cases. As \u003cem\u003eS. mutans\u003c/em\u003e levels increase, \u003cem\u003eC. matruchotii\u003c/em\u003e abundance tends to decline (William et al. 1999; Qudeimat et al. 2021).\u003c/p\u003e \u003cp\u003eTo assess the occurrence and distribution of occlusal caries lesions VOPI was developed and is used clinically (Carvalho et al. 1989). Longitudinal research shows that erupting molars tend to accumulate thicker plaque because of reduced mechanical cleaning and brushing difficulty, correlating with a higher occurrence of active lesions compared to fully erupted molars, which are more likely to have thin or no visible plaque and inactive lesions (Carvalho et al. 1992; Carvalho et al. 2017). VOPI is recommended as a supplementary diagnostic tool for estimating caries lesion activity, particularly in dentinal lesions in adults and for guiding treatment decisions (Henne et al. 2015; Tanner et al. 2016).\u003c/p\u003e \u003cp\u003eTo standardise a clinical scoring method for detecting and assessing caries on both coronal and root surfaces, the International Caries Detection and Assessment System (ICDAS) was also used in this study. It is applicable to enamel and dentine lesions, including non-cavitated and cavitated forms, using a 0 to 6 scale, where 0 denotes a sound tooth surface and 6 represents tooth structure destruction where the cavity is both deep and wide and dentine is clearly visible on the walls and at the base. An extensive cavity involves at least half of the tooth surface or possibly reaching the pulp (Ismail et al. 2007).\u003c/p\u003e \u003cp\u003eThe present study aimed to quantitatively assess \u003cem\u003eS. mutans\u003c/em\u003e, \u003cem\u003eS. exigua\u003c/em\u003e, and \u003cem\u003eC. matruchotii\u003c/em\u003e in occlusal surfaces biofilm from first permanent molars categorised according to VOPI, and to evaluate the associations between bacterial levels and the occurrence of occlusal enamel caries based on ICDAS criteria. We hypothesise that the quantities of \u003cem\u003eS. mutans\u003c/em\u003e, \u003cem\u003eS. exigua\u003c/em\u003e, and \u003cem\u003eC. matruchotii\u003c/em\u003e will differ amongst the VOPI groups.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThe Faculty of Dentistry and the Faculty of Pharmacy Human Institutional Review Board, Mahidol University gave it clearance to this cross-sectional study (approval number: \u0026nbsp; COA.No.MU-DT/PY-IRB 2024/042.1807). Based on previous studies, we set the estimated correlation of VOPI and \u003cem\u003eS. mutans\u003c/em\u003e at 0.45, anticipating that site-specific sampling would increase the correlation between the bacteria and dental caries (Dikmen 2015).\u003csup\u003e\u0026nbsp;\u003c/sup\u003eUsing a two-sided 95% confidence interval (CI) of 0.45 \u0026plusmn; 0.125 (width = 0.25), the calculated sample size amounted to 159 teeth, which was rounded up to 160 teeth (Bonett et al. 2000; Ismail et al. 2009). If a sufficient sample size is not obtained, additional samples are expected to be collected, estimated at 20% of the total sample, which is an additional 32 teeth. Consequently, a total of 192 teeth samples will be collected. We anticipate that each participant will have two qualifying first molars meeting the study criteria, thus estimating that the sample group will consist of 96 primary school students from grades 1 to 3. Using the software named Primer of Biostatistics (McGraw-Hill, NY, USA) performed with \u0026alpha;=0.05 and power of 80% obtained at least of 40 children in each group would be enough to reach statistical difference (Bonett et al. 2000). \u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"525\" height=\"97\" 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\" alt=\"A close-up of a white background Description automatically generated\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSubject selection: \u0026nbsp;\u003c/em\u003eThe recruitment was done at Chumchon Prachathipat Wittayakhan School, Pathumthani Province, Thailand. Thai children aged 6 to 8\u0026nbsp;years old were selected. Consent forms were signed by all parents or legal guardians. Every participant was free to leave the research at any moment\u003cem\u003e.\u003c/em\u003e Performed clinical examination full mouth using World Health Organization criteria, the ICDAS and the VOPI. VOPI composed of four scores which are : 0=no plaque was obtained when carefully drawing a dental probe on the groove, 1=slight plaque was detected which is restricted to the groovefossa-system and identified by carefully drawing a dental probe on the groove, 2=abundant plaque: plaque was visually detected on the groove, 3 = dense plaque: occlusal surfaces partially or totally covered with heavy plaque visually. ICDAS used in this study was composed of three scores which are: 0=sound tooth: after 5 seconds of air drying, no caries was detected; 1=early enamel opacity or discoloration (white or brown) is visible at pit or fissure on the occlusal surfaces after prolonged air drying; 2=enamel showed a visible change when wet however, \u0026nbsp;lesion must be visible when dry; 3=enamel breakdown locally (without dentinal involvement) was identified when wet and after prolonged air drying (Ismail et al. 2009; Carvalho et al. 2016). Subjects must have no occlusal dentin caries, sealants or fillings on first permanent molars. Exclusion criteria were children who had received professional fluoride application, has any systemic diseases, taking any kind of antibiotics, had any oral treatment within 2 months before the collection visit or first permanent molars were partially erupted with more than 1/4 of the occlusal surfaces covered with gingival tissue.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eClinical examination: \u0026nbsp;\u003c/em\u003eTwo examiners (PT and PR) who were residents in Pediatric Dentistry at Faculty of Dentistry, Mahidol University were trained and calibrated prior to perform a clinical examination. Weighted Kappa VOPI agreement and ICDAS agreement were 0.91 and 0.83, respectively. Categorized plaque samples into four groups according to ICDAS and VOPI scores. Total of 160 teeth (each group=40). Recorded VOPI and ICDAS scores.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePlaque sample collection: \u0026nbsp;\u003c/em\u003ePlaque samples were gently collected by drawing a sterile toothpick along the occlusal groove of permanent first molars for collecting plaque samples that were isolated by sterile gauzes to obtain strictly dry areas around the molars in order to prevent the contamination by saliva. Samples were released into 1 ml of Tris-EDTA buffer, immediately stored on ice, transported to the Oral Biology Laboratory, and kept at \u0026ndash;20℃ until DNA extraction.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDNA extraction: \u0026nbsp;\u003c/em\u003eDNA was extracted using a kit (Flavogen, Taiwan) as previously described. \u003csup\u003e12\u003c/sup\u003e Measured DNA concentration and purity at 260 nm/280 nm (Nanodrop 2000C Thermo Scientifc, Delaware, USA).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCulture condition and standard strains: \u0026nbsp;\u003c/em\u003e\u003cem\u003eS. exigua\u0026nbsp;\u003c/em\u003eATCC 700122 and\u003cem\u003e\u0026nbsp;S. mutans\u0026nbsp;\u003c/em\u003eATCC 25175 were used as a standard\u003cem\u003e. S. exigua\u0026nbsp;\u003c/em\u003ewas grown in Tryptic Soy Medium with 5% defibrinated Sheep Blood and incubated at 37\u0026deg;C for 48 hr.\u003cem\u003e\u0026nbsp;\u003c/em\u003eGrew\u003cem\u003e\u0026nbsp;S. mutans\u0026nbsp;\u003c/em\u003ein Brain Heart Infusion broth at 37\u0026deg;C and incubated for 24-48 hr. A Gram stain was tested before the DNA extraction and a ten-fold serial dilution was prepared for a sensitivity test of the employed primers.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eQuantitative real‑time PCR: \u0026nbsp;\u003c/em\u003eAll primers used in this study were list in Table 1. Total volume of the reaction mixture was 20 \u0026mu;l as previously described. \u003csup\u003e10\u0026nbsp;\u003c/sup\u003eAnnealing temperature at 50\u0026deg;C, 53\u0026deg;C, 55.9\u0026deg;C and 57.4\u0026deg;C for 20 sec for BAC16S, \u003cem\u003eS. mutans\u003c/em\u003e, \u003cem\u003eS. exigua\u003c/em\u003e and \u003cem\u003eC. matruchotii\u003c/em\u003e, respectively. Generated melting curves from 60\u0026deg;C-95\u0026deg;C and read every 0.5\u0026deg;C for 5 sec.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1:\u003c/strong\u003e\u0026nbsp; Primers used in this study\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"708\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 207px;\"\u003e\n \u003cp\u003ePrimers\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 312px;\"\u003e\n \u003cp\u003eNucleotide sequence (5\u0026prime; - 3\u0026prime;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;Amplicon\u003c/p\u003e\n \u003cp\u003e(Basepair)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eRef\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 136px;\"\u003e\n \u003cp\u003eUniversal BAC16S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 312px;\"\u003e\n \u003cp\u003eTGGAGCATGTGGTTTAATTCGA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e160\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 113px;\"\u003e\n \u003cp\u003eSinsimer \u003cem\u003eet al\u003c/em\u003e, 2005\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 312px;\"\u003e\n \u003cp\u003eTGCGGGACTTAACCCAACA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 136px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. mutans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003eF5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 312px;\"\u003e\n \u003cp\u003eAGCCATGCGCAATCAACAGGT T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e415\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 113px;\"\u003e\n \u003cp\u003eSato \u003cem\u003eet al\u003c/em\u003e, 2003 \u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003eR4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 312px;\"\u003e\n \u003cp\u003eCGCAACGCGAACATCTTGATCAG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 136px;\"\u003e\n \u003cp\u003e\u003cem\u003eSlackia exigua\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003eexi557F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 312px;\"\u003e\n \u003cp\u003eGCCAAGCGGCCTCGTCGAAG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e740\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 113px;\"\u003e\n \u003cp\u003eHashimura \u003cem\u003eet al,\u0026nbsp;\u003c/em\u003e2001\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003eexi1263R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 312px;\"\u003e\n \u003cp\u003eGCCGGCTTTAAGGGATTCGCTCG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 136px;\"\u003e\n \u003cp\u003e\u003cem\u003eCorynebacterium Matruchotii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003ePP6N5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 312px;\"\u003e\n \u003cp\u003eCGGAATTCATGGAYTAYGGYCARATC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e166\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 113px;\"\u003e\n \u003cp\u003evan Dijk\u003cem\u003e\u0026nbsp;et al\u003c/em\u003e, 1998\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003eP6C5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 312px;\"\u003e\n \u003cp\u003eGCGGA TCCRGTYTCRCCW AGYTCRGA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAgarose gel electrophoresis: \u0026nbsp;\u003c/em\u003eChecked amplified PCR products using 2% agarose gel (UltraPure Agarose, ThermoFisher Scientifc, USA). Saved gel images with a digital imaging system (Molecular Imager \u0026reg;Gel docTM Systems, Bio-Rad Laboratories Inc., CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnalyzed data using IBM SPSS Statistics Version 28.0 (IBM SPSS Statistics for Windows, Version 28.0. Armonk, NY, USA: IBM Corp.) For table 2, if data was continuous variables, such as the age of children (years), ANOVA was used to compare the average (Mean\u0026plusmn;SD) between the VOPI group 0-3. If data was categorical variables, such as gender (male/female) and molars positions (top/bottom), the Chi-Square Test was used to compare frequency (%) between the VOPI groups. Testing data normal distribution (the bacteria amount is distributed normally or not) using Kolmogorov-Smirnov and Shapiro-Wilk. Since the data distribution is not normal, Mann -Whitney U Test was used to compare amount of bacteria between two VOPI groups and Kruskal -Wallis H Test was used to compare the median of bacteria between many VOPI groups. For Correlation Analysis, Spearman\u0026rsquo;s Rho Correlation was used to analyze the relationship between the quantity of bacteria, VOPI scores and ICDAS. Chi-Square test was used to compare the bacteria\u0026apos;s detection rate and the distribution of ICDAS between VOPI groups. Logistic Regression was used to analyze the chance of enamel caries at different VOPI group using the VOPI 0 which is a caries-free control group as a reference group. The value of p \u0026lt; 0.05 was accepted as statistically different.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eAmong the VOPI groups, ages of the children were significantly different (Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2:\u003c/strong\u003e\u0026nbsp; Mean age, gender and permanent molar location of participants in four VOPI groups\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"604\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 217px;\"\u003e\n \u003cp\u003eVaribles\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eGroup 0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp; 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u0026nbsp;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 122px;\"\u003e\n \u003cp\u003eChildren\u0026apos;s age (years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 95px;\"\u003e\n \u003cp\u003eMean \u0026plusmn; SD\u003c/p\u003e\n \u003cp\u003eMedian\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e6.7\u0026plusmn; 0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e6.5 \u0026plusmn; 0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e7.08 \u0026plusmn; 0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e7.08 \u0026plusmn; 0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e6.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e6.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e7.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" rowspan=\"3\" style=\"width: 217px;\"\u003e\n \u003cp\u003eChildren\u0026apos;s genders (n%)\u003c/p\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 321px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.896\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e23 (57.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e20 (50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e20 (50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e20 (50)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e17 (42.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e20 (50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e20 (50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e20 (50)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" rowspan=\"3\" style=\"width: 217px;\"\u003e\n \u003cp\u003ePermanent molar (n%)\u003c/p\u003e\n \u003cp\u003eUpper\u003c/p\u003e\n \u003cp\u003eLower\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 321px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.965\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e21 (52.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e19 (47.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e21 (52.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e21 (52.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e19 (47.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e21 (52.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e19 (47.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e19 (47.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviations: ANOVA, analysis of variance; SD, standard deviation; VOPI, Visible Occlusal Plaque Index.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e* p-value \u0026lt; 0.05 were obtained between VOPI groups based on ANOVA or chi-square for continuous and categorical variables, respectively, shown as mean \u0026plusmn; SD. The non-parametric Kruskal-Wallis H test was used for median and interquartile range (IQR) which are Median of Children\u0026rsquo;s age (years)\u003c/p\u003e\n\u003cp\u003eAll primers were tested specificity. \u003cem\u003e\u0026nbsp;\u003c/em\u003eThe standard strain of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ematruchotii\u003c/em\u003e was not obtained due to the difficulty of culture in the laboratory. Standard curves were generated for the BAC16S, \u003cem\u003eS\u003c/em\u003e. \u003cem\u003emutans,\u003c/em\u003e and \u003cem\u003eS\u003c/em\u003e. \u003cem\u003eexigua\u0026nbsp;\u003c/em\u003eprimers. The detection limit of BAC16S, \u003cem\u003eS. mutans\u003c/em\u003e, and \u003cem\u003eS\u003c/em\u003e. \u003cem\u003eexigua\u003c/em\u003e primers were 10\u003csup\u003e1\u003c/sup\u003e, 10 and 10\u003csup\u003e3\u003c/sup\u003e, respectively.\u003c/p\u003e\n\u003cp\u003eSamples were randomly selected from each VOPI groups to visualized the amplified products from BAC16S (Fig 1A), \u003cem\u003eS. mutans\u003c/em\u003e, \u003cem\u003eS. exigua\u0026nbsp;\u003c/em\u003eand \u003cem\u003eC. matruchotii. S. mutans\u0026nbsp;\u003c/em\u003ewas detected in all VOPI group (Fig 1B). \u003cem\u003eS. exigua\u003c/em\u003e was presented in the VOPI group 3 while \u003cem\u003eC. matruchotii\u003c/em\u003e was not showed in VOPI groups 2 and 3 (Fig 1C and D)\u003c/p\u003e\n\u003cp\u003eTable 3 presents \u003cem\u003eS. mutans\u003c/em\u003e, \u003cem\u003eS. exigua\u003c/em\u003e, and \u003cem\u003eC. matruchotii\u003c/em\u003e prevalences in each of the VOPI scores. \u003cem\u003eS. mutans\u003c/em\u003e was detected in nearly all samples, with prevalence ranging from 97.5% in VOPI 0 and VOPI 1 groups to 100% in VOPI 2 and VOPI 3 groups. \u003cem\u003eS. exigua\u003c/em\u003e demonstrated an upward trend in detection when VOPI scores increased. It was detected at 65% in the VOPI 0 group, and increased progressively to 82.5% in the VOPI 1 group. It reached 100% in both the VOPI 2 and VOPI 3 groups. Conversely, \u003cem\u003eC. matruchotii\u003c/em\u003e showed a declining trend across VOPI levels. It was detected at 97.5% in the VOPI 0 group and decreased to 82.5% in VOPI 1, 70% in VOPI 2, and 47.5% in the VOPI 3 group, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3:\u003c/strong\u003e\u0026nbsp; Distribution of \u003cem\u003eS. mutans\u003c/em\u003e, \u003cem\u003eS. exigua\u003c/em\u003e and \u003cem\u003eC. matruchotii\u0026nbsp;\u003c/em\u003eprevalences in all VOPI groups (n=40 in each group)\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"577\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 137px;\"\u003e\n \u003cp\u003eBacterial\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eGroup 0\u003c/p\u003e\n \u003cp\u003en (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003cp\u003en (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003cp\u003en (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003cp\u003en (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 137px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. mutans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e39 (97.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e39 (97.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e40 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e40 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 137px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. exigua\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e26 (65)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e33 (82.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e40 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e40 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 137px;\"\u003e\n \u003cp\u003e\u003cem\u003eC. matruchotii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e39 (97.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e33 (82.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e28(70)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e19 (47.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviation: VOPI, Visible Occlusal Plaque Index.\u003c/p\u003e\n\u003cp\u003eTable 4 presents the detection of ICDAS scores in each of the VOPI groups. There was a difference significantly between ICDAS and VOPI scores (p \u0026lt; 0.001), indicating that as the level of visible plaque increases, the severity of enamel caries also rises. Among children with no visible plaque (VOPI 0), 87.5% exhibited sound enamel (ICDAS 0), and only 12.5% showed early enamel changes (ICDAS 1). In contrast, in the highest plaque group (VOPI 3), none had sound enamel, while 15% had ICDAS 1, 47.5% had ICDAS 2 (distinct visual enamel changes), and 37.5% had ICDAS 3 (localized enamel breakdown). Logistic regression showed that subjects in the VOPI 1, 2, and 3 groups had a 1.97, 3.99, and 6.05 \u0026nbsp; higher chance of having caries in the enamel compared to those subjects in VOPI 0, respectively (Table 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4:\u003c/strong\u003e\u0026nbsp; ICDAS detection in each VOPI groups (n=40 in each group)\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"604\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003eICDAS scores\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eGroup 0\u003c/p\u003e\n \u003cp\u003en (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003cp\u003en (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;2\u003c/p\u003e\n \u003cp\u003en (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;3\u003c/p\u003e\n \u003cp\u003en (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003eICDAS 0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e35 (87.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e18(45)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e3 (7.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003eICDAS 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e5 (12.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e14 (35)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e14 (35)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e6 (15)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003eICDAS 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e6 (15)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e17 (42.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e19 (47.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003eICDAS 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e2 (5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e6 (15)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e15 (37.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviations: ICDAS, International Caries Detection and Assessment System; VOPI, Visible Occlusal Plaque Index.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e*p-value \u0026lt; 0.05, Chi-Square Tests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5:\u003c/strong\u003e\u0026nbsp; Analysis of the Association Between VOPI Groups and Enamel Caries Severity\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"425\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 146px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 61px;\"\u003e\n \u003cp\u003eOR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003e95% C.I. for EXP (B)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 146px;\"\u003e\n \u003cp\u003eIntercept (ICDAS 1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 61px;\"\u003e\n \u003cp\u003e1.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 146px;\"\u003e\n \u003cp\u003eIntercept (ICDAS 2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 61px;\"\u003e\n \u003cp\u003e3.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 146px;\"\u003e\n \u003cp\u003eIntercept (ICDAS 3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 61px;\"\u003e\n \u003cp\u003e6.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 146px;\"\u003e\n \u003cp\u003eVOPI 0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 61px;\"\u003e\n \u003cp\u003eRef.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 146px;\"\u003e\n \u003cp\u003eVOPI 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 61px;\"\u003e\n \u003cp\u003e5.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e(4.30 - 6.91)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 146px;\"\u003e\n \u003cp\u003eVOPI 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 61px;\"\u003e\n \u003cp\u003e4.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e(3.09 - 5.54)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 146px;\"\u003e\n \u003cp\u003eVOPI 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 61px;\"\u003e\n \u003cp\u003e2.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e(1.19 - 3.42)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviations: CI, confidence interval; ICDAS, International Caries Detection and Assessment System;\u003c/p\u003e\n\u003cp\u003eOR, odds ratio; VOPI, Visible Occlusal Plaque Index.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e*p-value \u0026lt; 0.05, (Logistic regression analysis)\u003c/p\u003e\n\u003cp\u003eThe level of BAC16S (p\u0026lt;0.001), \u003cem\u003eS. mutans\u003c/em\u003e (p\u0026lt;0.001) and \u003cem\u003eS. exigua\u003c/em\u003e (p\u0026lt;0.001) significantly increased when VOPI scores increased. \u0026nbsp;There were significantly different ratios of \u003cem\u003eS. mutans\u003c/em\u003e to total bacteria (p\u0026lt;0.001) and \u003cem\u003eS. exigua\u003c/em\u003e to total bacteria (p\u0026lt;0.001) amongst the VOPI scores (Table 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6:\u003c/strong\u003e\u0026nbsp; Quantities of bacteria and ratios of \u003cem\u003eS. mutans/total bacteria\u003c/em\u003e and \u003cem\u003eS. exigua /total\u0026nbsp;\u003c/em\u003e\u003cem\u003eBacteria\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"642\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 122px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eVOPI 0\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eMedian\u003c/p\u003e\n \u003cp\u003e(Min, Max)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003eVOPI 1\u003c/p\u003e\n \u003cp\u003eMedian\u003c/p\u003e\n \u003cp\u003e(Min, Max)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eVOPI 2\u003c/p\u003e\n \u003cp\u003eMedian\u003c/p\u003e\n \u003cp\u003e(Min, Max)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eVOPI 3\u003c/p\u003e\n \u003cp\u003eMedian\u003c/p\u003e\n \u003cp\u003e(Min, Max)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003eTotal bacteria\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e1.63x10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(7.85x10\u003csup\u003e4\u003c/sup\u003e,1.54x10\u003csup\u003e12\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003e3.75x10\u003csup\u003e11\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(1.50\u0026times;10\u0026sup2;, 1.67\u0026times; 10\u003csup\u003e14\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e5.43x10\u003csup\u003e11\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(1.47x10\u003csup\u003e8\u003c/sup\u003e,7.51x10\u003csup\u003e14\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e7.53x10\u003csup\u003e11\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(1.42x10\u003csup\u003e3\u003c/sup\u003e,3.78x10\u003csup\u003e12\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e\u003cem\u003eS.mutans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e3.55x10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(0, 1.63x10\u003csup\u003e6\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003e1.02x10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(0, 5.50x10\u003csup\u003e8\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e1.05x10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(6.27x10\u003csup\u003e2\u003c/sup\u003e,3.17x10\u003csup\u003e8\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e4.53x10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(2.14x10\u003csup\u003e2\u003c/sup\u003e,1.84x10\u003csup\u003e9\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e\u003cem\u003eS.mutans/total bacteria\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e1.68x10\u003csup\u003e-5\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(0, 1.31x10\u003csup\u003e0\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003e7.81x10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(0, 3.77x10\u003csup\u003e6\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e6.29x10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(2.17x10\u003csup\u003e-12\u003c/sup\u003e,1.74x10\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e1.48x10\u003csup\u003e-4\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(1.81x10\u003csup\u003e-9\u003c/sup\u003e, 6.87x10\u003csup\u003e0\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. exigua\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e2.16x10\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(0, 2.81x10\u003csup\u003e4\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003e2.30x10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(0, 4.5x10\u003csup\u003e6\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e1.93x10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(1.75x10\u003csup\u003e2\u003c/sup\u003e,1.93x 10\u003csup\u003e5\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e4.98x10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(3.19x10\u003csup\u003e4\u003c/sup\u003e, 7.19x10\u003csup\u003e13\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;S. exigua /total bacteria\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e2.33x10\u003csup\u003e-8\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(0, 3.5 x10\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003e1.08x10\u003csup\u003e-9\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(0, 1.49x10\u003csup\u003e5\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e6.29x10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(2.17x10\u003csup\u003e-12\u003c/sup\u003e,2.58x10\u003csup\u003e-4\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e9.06x10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(4.64x10\u003csup\u003e-8\u003c/sup\u003e, 1.34x10\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviation: VOPI, Visible Occlusal Plaque Index.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e*p-value \u0026lt; 0.05, between VOPI groups based non-parametric Kruskal-Wallis H test \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 7:\u003c/strong\u003e\u0026nbsp; Comparison of each bacteria quantities and ratios among VOPI Groups\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"642\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 42px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003eTotal bacteria\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. mutans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. mutans\u003c/em\u003e/total bacteria\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. exigua\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. exigua\u003c/em\u003e /total bacteria\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e(I) VOPI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 42px;\"\u003e\n \u003cp\u003e(J) VOPI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003ep value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e(J) VOPI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003ep value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e(J) VOPI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003ep value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e(J) VOPI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003ep value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e(J) VOPI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 42px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u0026lt; 0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e0.048*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.01*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.004*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.534\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 42px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u0026lt; 0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026lt; 0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026lt; 0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.003*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.802\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 42px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u0026lt; 0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026lt; 0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026lt; 0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.222\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026lt; 0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 42px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e0.252\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e0.268\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.122\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.744\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.317\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 42px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e0.381\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026lt; 0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026lt; 0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026lt; 0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026lt; 0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 42px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e0.651\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026lt; 0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026lt; 0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026lt; 0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026lt; 0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eAbbreviation: VOPI, Visible Occlusal Plaque Index.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e*p-value \u0026lt; 0.05 (Mann - Whitney test)\u003c/p\u003e\n\u003cp\u003eTable 7 presents significant differences in total bacterial levels between group 0 and the other groups (1, 2, and 3) (p\u0026lt;0.001).\u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003eS\u003c/em\u003e. \u003cem\u003emutans\u003c/em\u003e, statistically significant differences were observed between VOPI 0 and VOPI 1 (p = 0.048), VOPI 0 and VOPI 2 (p \u0026lt; 0.001), and VOPI 0 and VOPI 3 (p \u0026lt; 0.001). No significant difference was found between VOPI 1 and VOPI 2 (p = 0.268), whereas significant differences were observed between VOPI 1 and VOPI 3 (p \u0026lt; 0.001), and VOPI 2 and VOPI 3 (p \u0026lt; 0.001). These findings showed that \u003cem\u003eS. mutans\u003c/em\u003e levels increased significantly with higher plaque accumulation, particularly between lower and higher VOPI scores.\u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003eS. mutans\u003c/em\u003e/total bacteria ratios, there were significant differences between VOPI 0 and VOPI 1 (p= 0.01), VOPI 0 and VOPI 2 (p\u0026lt; 0.001), VOPI 0 and VOPI 3 (p\u0026lt; 0.001), VOPI 1 and VOPI 3 (p\u0026lt; 0.001), and VOPI 2 and VOPI 3 (p\u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003eS. exigua\u003c/em\u003e, there were significant differences between VOPI 0 and VOPI 1 (p= 0.004), VOPI 0 and VOPI 2 (p=0.003), VOPI 1 and VOPI 3 (p\u0026lt; 0.001), and VOPI 2 and VOPI 3 (p\u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003eS. exigua\u003c/em\u003e/total bacteria ratio, there were significant differences between VOPI 0 and VOPI 3 (p\u0026lt;0.001), VOPI 1 and VOPI 3 (p\u0026lt; 0.001), and VOPI 2 and VOPI 3 (p\u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003eCorrelation analysis between levels of bacterial, VOPI, and ICDAS indicated significant positive correlations between VOPI and total bacteria (r = 0.504, p \u0026lt; 0.001), and between ICDAS and total bacteria (r = 0.506, p \u0026lt; 0.001). Additionally, \u003cem\u003eS. mutans\u003c/em\u003e showed a positive correlation with total bacteria (r = 0.583, p \u0026lt; 0.001), whereas \u003cem\u003eS. mutans\u003c/em\u003e/total bacteria and \u003cem\u003eS. exigua\u003c/em\u003e/total bacteria ratios also exhibited significant associations (p \u0026lt; 0.001). These findings indicate strong, statistically significant relationships between bacterial levels, VOPI, and dental caries assessment scores (Table 8).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 8:\u003c/strong\u003e\u0026nbsp; Levels of bacteria, VOPI, and ICDAS scores correlation.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"661\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eTotal bacteria\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u003cem\u003eS.mutans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u003cem\u003eS.mutans\u003c/em\u003e/total bacteria\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. exigua\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u003cem\u003eS.exigua\u0026nbsp;\u003c/em\u003e/total bacteria\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eICDAS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026rho;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 49px;\"\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026rho;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 49px;\"\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026rho;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 49px;\"\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026rho;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 49px;\"\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026rho;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 49px;\"\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026rho;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 56px;\"\u003e\n \u003cp\u003eP-value \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003eVOPI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026nbsp;0.504\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026nbsp;0.583\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026nbsp;0.734\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e\u0026nbsp;0.080\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026nbsp;0.313\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026nbsp;0.423\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e\u0026nbsp;.758\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003eICDAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026nbsp;0.506\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026nbsp;0.425\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026nbsp;0.496\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e-0.035\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026nbsp;0.663\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026nbsp;0.193\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e\u0026nbsp;0.015*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviations: ICDAS, International Caries Detection and Assessment System; VOPI, Visible Occlusal Plaque Index.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e*p-value\u0026lt;0.05, (Spearman\u0026rsquo;s Rho)\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study purposely collected plaque samples only from sound and incipient enamel lesions from the occlusal surfaces of first permanent molars in the hope of filling the knowledge gap regarding the microbiology associated with sound and early enamel caries lesions. Most previous studies demonstrated results of the microbiota in occlusal caries focusing on dentinal caries (Lima et al. 2011; Dige et al. 2014; Kressirer et al. 2018).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOral cavity provides optimal conditions for colonization and proliferation of microbiota. It is a unique microenvironment. It is regulated through complex biological signaling systems and interactions with the host. Once the oral microbiota is out of balance or dysbiosis, microorganisms produce virulence factors and metabolites, which will cause dental caries (Rajasekaran et al. 2024; Baker et al. 2024).\u003c/p\u003e\n\u003cp\u003eThe highest risk period for caries incidence typically spanned the initial 3 to 4 years following eruption (Zijnge et al. 2010). This susceptibility was attributed mainly to the macromorphology of the occlusal surface, which facilitated the adherence of oral microbiota. This was due to its intricate groove-fossa system, coupled with a relatively extended eruption period during which mechanical oral function was reduce (Zijnge et al. 2010). These protected sites, where mechanical cleansing is hindered, might promote the buildup and maturation of biofilms. These biofilms can develop into cariogenic ones if there is an imbalance in metabolic activities due to changes in local environmental conditions (Zijnge et al. 2010). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe bacterial composition of biofilm observed within grooves and at the entry points of fissures was similar to those of supra gingival \u003cem\u003ein vivo\u003c/em\u003e biofilm (Zijnge et al. 2010). Bacteria appear on the tooth surface within two hours after cleaning, initiating colonization following the formation of a dental pellicle induced by saliva. Initial colonizers such as \u003cem\u003eActinomyces\u003c/em\u003e, \u003cem\u003eStreptococcus\u003c/em\u003e, and \u003cem\u003eLactobacillus\u003c/em\u003e produce acidic byproducts through fermentative metabolism of dietary carbohydrates (Esberg et al. 2020). The oral microbial environment is regularly subjected to external factors both environmental and behavioral factors such as dietary habit and oral hygiene practice might contribute to microbial shifts alongside plaque accumulation. Such conditions are crucial determinants for establishing microbial populations and their ability to endure in this setting, leading to unique interactions between microbes and their hosts that depend on selective pressures. Prolonged acidic exposure suppresses acid-sensitive oral bacteria while promoting acid-tolerant species. Increased acidity in biofilm correlates with enamel surface demineralization and the protective outer layer frail, resulting in roughening surfaces that may clear the way for the colonization of more aggressive microbial pathogens. Secondary colonizers such as \u003cem\u003eS. mutans\u003c/em\u003e, \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eBifidobacterium\u003c/em\u003e, and \u003cem\u003eScardovia\u003c/em\u003e \u003cem\u003ewiggisiae\u003c/em\u003e contribute to climax communities, shaping the environmental conditions of the oral cavity and potentially increasing the pathogenicity of the involved colonizers. \u003cem\u003eS. mutans\u003c/em\u003e is considered as a classical cariogenic species\u003cem\u003e.\u0026nbsp;\u003c/em\u003eIt was present in both inactive and active caries but absent on sound enamel. An abundance of \u003cem\u003eS. mutans\u003c/em\u003e was detected in active lesions. Unsurprisingly, this study confirmed the association between \u003cem\u003eS. mutans\u003c/em\u003e and initial caries lesions in the occlusal surfaces with a high prevalence. These results are aligned with previous studies (Thitisakyothin et al. 2025; Tantikalchan et al. 2022). Our previous study showed a significant positive correlation between VOPI scores and both ICDAS scores and levels of \u003cem\u003eS. mutans\u003c/em\u003e to total bacteria (Thitisakyothin et al. 2025). \u0026nbsp;Similar findings are found in this study, significant quantitative differences in total bacterial load were observed across the VOPI groups, with a consistent upward trend from VOPI 0 to VOPI 3. Median bacterial counts increased from 1.63\u0026times;10⁹ CFU in VOPI 0, to 7.53\u0026times;10\u0026sup1;\u0026sup1; CFU in VOPI 3 (p \u0026lt; 0.001), suggesting a clear relationship between plaque visibility and microbial biomass. As expected, \u003cem\u003eS. mutans\u003c/em\u003e levels increased significantly with higher plaque accumulation, particularly between lower and higher VOPI scores. However, the proportion of \u003cem\u003eS. mutans\u003c/em\u003e relative to total bacterial load increased with increasing VOPI, from 1.68\u0026times;10⁻⁵ to 1.48\u0026times;10⁻⁴, indicating that despite the absolute increase in \u003cem\u003eS. mutans\u003c/em\u003e, its relative abundance was diluted within the growing polymicrobial community. These findings are consistent with previous studies that have shown \u003cem\u003eS. mutans\u003c/em\u003e to be a key contributor in early biofilm formation, with its role diminishing in dominance as the plaque matures and diversifies (Lima et al. 2011; Dige et al. 2014). This phenomenon may be allocated the ecological succession of microbial communities, where later colonizers, particularly anaerobes, become more prevalent as oxygen gradients and nutrient availability change within the thickening biofilm (Marsh et al. 2006). \u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eIn early biofilm formation, \u003cem\u003eS. mutans\u003c/em\u003e plays a dominant role due to its strong adhesion mechanisms, such as the production of extracellular glucans via glucosyltransferase enzymes (GTF), and its ability to thrive in acidic environments through its acidogenic and aciduric properties (Bowen et al. 2011). Its dominance is evident in low-VOPI environments (e.g., VOPI 0), where the microbial community is less complex and less mature. However, as the biofilm matures (VOPI 2\u0026ndash;3), environmental conditions such as reduced oxygen, lower pH, and increased substrate availability support the colonization and proliferation of a wider variety of anaerobic and aciduric species. These include \u003cem\u003eS. exigua\u003c/em\u003e, \u003cem\u003eBifidobacterium\u003c/em\u003e, and \u003cem\u003eS. wiggsiae\u003c/em\u003e, which are more frequently associated with mature carious lesions (Thitisakyothin et al. 2025; \u0026nbsp;Kalpana et al. 2020). \u003cem\u003eS. mutans\u003c/em\u003e initiates the caries process but is not the sole pathogen in advanced lesions. Moreover, previous studies indicate that while \u003cem\u003eS. mutans\u003c/em\u003e is integral to early colonization and lesion initiation, it eventually shares its niche with other microorganisms as the biofilm thickens and diversifies (Spatafora et al. 2024; Aas et al. 2008).\u003c/p\u003e\n\u003cp\u003eThis study initial attempt to quantitatively detect the amount of few novel non-mutans bacteria related to caries in occlusal surface biofilm. The study revealed a significant microbial transition in the VOPI 3 group, with \u003cem\u003eS. exigua\u003c/em\u003e exhibiting a considerable rise in both absolute quantity and relative abundance. It is an emerging and less studies species. The observed pattern suggests that \u003cem\u003eS. exigua\u003c/em\u003e functions as a late-stage colonizer and may serve as a potential indicator of biofilm maturation and dysbiosis. This interpretation is consistent with prior research that characterizes \u003cem\u003eS. exigua\u003c/em\u003e as an obligate anaerobic, Gram-positive, asaccharolytic species, typically absent or found in low abundance in early biofilms, but increasingly identified in mature, low-oxygen, and acidic environments, such as deep dentin caries, secondary caries, and chronic periodontal lesions (Fakhruddin et al. 2019; Tanner et al. 2011; Aas et al. 2008; Sergio et al. 1996; Hashimura et al. 2001). \u003cem\u003eS. exigua\u003c/em\u003e, although not typically categorized as acidogenic, is frequently linked to S-ECC and deep dentinal lesions, indicating its role in the advancement of caries rather than their onset (Tanner et al. 2011; Sergio et al. 1996). It is often found alongside \u003cem\u003eS. wiggsiae\u003c/em\u003e in advanced lesions, suggesting a possible synergistic effect on biofilm virulence (Fakhruddin et al. 2019). Tanner and colleagues identified \u003cem\u003eS\u003c/em\u003e. \u003cem\u003eexigua\u003c/em\u003e as a constituent of the ecological consortium associated with active caries, potentially augmenting pathogenicity via microbial interactions and biofilm persistence (Tanner et al. 2011). Its ability to survive acidic, anaerobic environments, like those in necrotic pulp tissue, supports its classification as a secondary colonizer, which contributes to the stability and pathogenic potential of mature biofilms. \u003csup\u003e38\u0026nbsp;\u003c/sup\u003ePrevious study found that \u003cem\u003eS. exigu\u003c/em\u003ea levels were much higher in children and adult orthodontic patients when compared to non-orthodontic patients. The rates were 71.4% vs. 28.6% in children, and 70.7% vs. 29.3% in adults (Booth et al. 2004). These findings demonstrate that fixed orthodontic appliances can create plaque-retentive, anaerobic environments conducive to the proliferation of \u003cem\u003eS. exigua\u003c/em\u003e. Colonization may be facilitated by diminished biofilm clearance and increased microbial retention within these populations. \u003cem\u003eS. exigua\u003c/em\u003e has been recognized in systemic infections such as bacteremia, peritonitis, and septic shock, particularly in immunocompromised individuals, underscoring its opportunistic pathogenicity beyond the oral cavity (Shen et al. 2003; Zhou et al. 2025; Rieber et al. 2019). The increasing prevalence of this species in dysbiotic plaque corresponds with the ecological plaque hypothesis, which suggests that environmental alterations, including acidification and reduced oxygen levels, facilitate the emergence of rarer but more virulent species. \u003cem\u003eS. exigua\u003c/em\u003e functions as both a microbial biomarker for mature dysbiotic biofilms and a contributor to the advancement of caries. Furthermore, \u003cem\u003eS. exigua\u003c/em\u003e has been shown to metabolize amino acids such as arginine and lysine to produce \u003cstrong\u003eshort-chain fatty acids (SCFAs)\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e including acetate and propionate (Lee et al. 2016). These byproducts may not cause demineralization directly but can contribute to \u003cstrong\u003ehost tissue inflammation and degradation\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e particularly in endodontic and periodontal contexts. This organism is important in ecological succession and the development of advanced dental caries because it is common in deep carious lesions, it is linked to pathogenic consortia, and it prefers acidic, anaerobic environments.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCorynebacterium matruchotii\u003c/em\u003e, an aerobic, filamentous bacterium involved in the structural organization of supragingival biofilms, thrives in well-oxygenated, neutral pH conditions characteristic of health-associated plaque (Esberg et al. 2020).\u003csup\u003e\u0026nbsp;\u003c/sup\u003e Thus, combined with the \u0026ldquo;pillar\u0026rdquo; role of \u003cem\u003eC. matruchotii\u003c/em\u003e in the structure of dental plaque in healthy people, it could be inferred that \u003cem\u003eC. matruchotii\u003c/em\u003e might perform a key part in the formation and stabilization of healthy dental plaque. In contrast, the reduction of \u003cem\u003eC. matruchotii\u003c/em\u003e may directly lead to the break of biofilm homostasis. Meanwhile, the decreased abundance of \u003cem\u003eC. matruchotii\u003c/em\u003e, based on the close relationship shown in corncob structure of supragingival biofilm, might also be part of the reduction of commensal \u003cem\u003estreptococci\u003c/em\u003e, such as \u003cem\u003eStreptococcus gordonii\u003c/em\u003e and \u003cem\u003eS. sanguinis\u003c/em\u003e, reported before, which showed an antagonistic effect against \u003cem\u003eS. mutans via\u003c/em\u003e their capability of producing hydrogen peroxide. The alteration could finally contribute to a replacement of dominant species in the oral ecosystem and induce the formation of diseased-adapted supragingival plaque determined by \u003cem\u003eS. mutans\u003c/em\u003e that lead to disorders in acid-base metabolism and eventually cause the demineralization of tooth enamel. In general, \u003cem\u003eC. matruchotii\u003c/em\u003e is a potential indicator of a caries-free state, and the reduction of its abundance may indicate the evolution from healthy plaque to caries biofilm (Esberg et al. 2020).\u003c/p\u003e\n\u003cp\u003eThe observed decline in \u003cem\u003eC. matruchotii\u003c/em\u003e prevalence from 97.5% at VOPI 0 to 47.5% at VOPI 3 suggests a negative association between this species and increasing plaque accumulation. \u003cem\u003eC. matruchotii\u003c/em\u003e thrives in well-oxygenated, neutral pH conditions characteristic of health-associated, supragingival plaque (Baker et al. 2024). However, as the biofilm matures and becomes increasingly anaerobic and acidic, conditions become unfavorable for \u003cem\u003eC. matruchotii,\u003c/em\u003e while aciduric species like \u003cem\u003eS. mutans\u003c/em\u003e and \u003cem\u003eS. exigua\u003c/em\u003e are able to proliferate (Lee et al. 2016; Tian et al. 2024). \u0026nbsp;\u003cem\u003eC. matruchotii\u003c/em\u003e as potentially associated with health-related biofilms. Supporting this ecological interpretation, previous longitudinal data revealed that \u003cem\u003eC. matruchotii\u003c/em\u003e abundance significantly increased at 1 and 3 months after full-mouth caries treatment under general anesthesia in children with S-ECC, compared to pre-treatment and 7 days post-treatment levels (p\u0026lt;0.01) (Esberg et al. 2020). This re-emergence in the post-treatment, caries-free state suggests that\u003cem\u003e\u0026nbsp;C. matruchotii\u0026nbsp;\u003c/em\u003emay serve as a biomarker of oral health and microbial homeostasis, reinforcing its association with structurally ordered and disease-resistant biofilms. The study further noted that \u003cem\u003eC. matruchotii\u003c/em\u003e was consistently ranked among the top 10 most abundant species in children who had received successful treatment, and its presence coincided with increased alpha diversity and microbial richness in plaque, especially in the later stages of recovery. These findings highlight the potential of \u003cem\u003eC. matruchotii\u003c/em\u003e not only as a structural keystone species but also as an ecological indicator of restored microbial balance following caries intervention (Kumaresan et al. 2024; Marsh et al. 2017). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese findings suggest that \u003cem\u003eC. matruchotii\u003c/em\u003e is highly sensitive to acidic and competitive biofilm conditions, which explains its decreased prevalence in higher VOPI scores. It likely plays a role in maintaining biofilm stability and microbial balance in early-stage plaque (VOPI 0\u0026ndash;1) and becomes excluded from advanced dysbiotic communities (VOPI 3). Furthermore, \u003cem\u003eC. matruchotii\u003c/em\u003e may facilitate the colonization of non-cariogenic streptococci such as \u003cem\u003eS. gordonii\u003c/em\u003e and \u003cem\u003eS. sanguinis\u003c/em\u003e, which contribute to oral health by producing hydrogen peroxide that inhibits \u003cem\u003eS. mutans (\u003c/em\u003eMa\u003cem\u003e\u0026nbsp;et al. 2004).\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study has shown a positive correlation between VOPI and ICDAS scores, meaning that as plaque accumulation (higher VOPI score) increases, the severity of enamel caries (higher ICDAS score) also increases. The VOPI is recommended as an additional clinical tool to estimate the activity of caries lesions alongside the clinical characteristics assessed by systems like ICDAS, helping support treatment decisions in daily practice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe constraints of this study include its cross-sectional design, which offers just a momentary view and restricted number of microorganisms tested which does not fully represent a true dynamic progression of the oral biofilm complex community. Additionally, the relatively small sample size may limit the generalizability of the findings to broader populations. Longitudinal research is strongly recommended to understand the dynamic of oral biofilm change over time. In addition, samples have been taken from a single school in Thailand. The ability to generalize findings is maybe restricted at this moment. Further studies by recruiting samples from different areas are highly suggested.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe findings present that increasing plaque accumulation, as indicated by higher VOPI scores, was significantly associated with greater total bacterial load and higher ICDAS scores, indicating progressive enamel demineralization.\u003c/p\u003e\n\u003cp\u003eLevels of \u003cem\u003eS. mutans\u003c/em\u003e and \u003cem\u003eS. exigua\u003c/em\u003e increased, whereas \u003cem\u003eC. matruchotii\u003c/em\u003e decreased, when VOPI scores increased. \u003cem\u003eS. mutans\u003c/em\u003e levels increased significantly with higher plaque accumulation, particularly between lower and higher VOPI scores. Also, significant increases in \u003cem\u003eS. exigua\u003c/em\u003e were seen among VOPI scores, but not as noticeably as \u003cem\u003eS. mutans,\u003c/em\u003e which showed linear increases when VOPI scores increased.\u003c/p\u003e\n\u003cp\u003eThere were significant differences between VOPI and ICDAS scores, indicating that as the level of visible plaque increases, the severity of enamel caries also rises. This can be useful when planning the preventive measures to patients in the future such as using the antimicrobial agents that might specifically target \u003cem\u003eS. mutans\u003c/em\u003e and \u003cem\u003eS. exigua.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSignificant positive correlations are thus observed between VOPI and total bacteria, and between ICDAS and total bacteria. \u003cem\u003eS. mutans\u003c/em\u003e shows a significant positive correlation with total bacteria, whereas \u003cem\u003eS. mutans\u003c/em\u003e/total bacteria and \u003cem\u003eS. exigua\u003c/em\u003e/total bacteria also exhibit significant associations. Our study indicated strong, statistically significant relationships between these bacterial levels, VOPI, and dental caries assessment scores\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eP.R.: Methodology, investigation, formal analysis, and writing-original draft preparation. K.M.: Methodology, writing-review and editing, and supervision. K.V.: Conceptualization, methodology, and supervision.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003e\u003cspan data-teams=\"true\"\u003e\u003c/span\u003e\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eAas JA, Griffen AL, Dardis SR, Lee AM, Olsen I, Dewhirst FE, et al. 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Oral biofilm architecture on natural teeth. \u003cem\u003ePLoS One\u003c/em\u003e. 2010;5(2):e9321. doi:10.1371/journal.pone.0009321\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-archives-of-paediatric-dentistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"EAPD","sideBox":"Learn more about [European Archives of Paediatric Dentistry](https://link.springer.com/journal/40368)","snPcode":"40368","submissionUrl":"https://submission.springernature.com/new-submission/40368/3","title":"European Archives of Paediatric Dentistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Occlusal biofilms, enamel caries lesions, S. mutans, Slackia exigua, Corynebacterium matruchotii","lastPublishedDoi":"10.21203/rs.3.rs-8942470/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8942470/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eAims \u003c/strong\u003eTo detect \u003cem\u003eS. mutans, S. exigua \u003c/em\u003eand \u003cem\u003eC. matruchotii \u003c/em\u003eamounts in biofilm from occlusal surfaces of first permanent molars categorized by two clinical diagnostic tools which are the Visible Occlusal Plaque Index (VOPI) and the International Caries Detection and Assessment System (ICDAS) and to assess the correlation between levels of bacterial and enamel caries on occlusal surfaces.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy design \u003c/strong\u003ePlaque samples were obtained from 160 Thai children (6–8 years-old) and assigned to four groups (n = 40 per group) based on VOPI and ICDAS scores.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e DNA was extracted and analyzed using real-time PCR quantitatively and specific primers targeting total bacteria (BAC16S), \u003cem\u003eS. mutans, S. exigua\u003c/em\u003e, and \u003cem\u003eC. matruchotii.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eChildren’s ages were different among the VOPI groups (p\u0026lt;0.001). Quantities of BAC16S, \u003cem\u003eS. mutans\u003c/em\u003e and \u003cem\u003eS. exigua,\u003c/em\u003e increased significantly with higher VOPI scores (p\u0026lt;0.001). \u003cem\u003eC. matruchotii\u003c/em\u003eshowed a declining trend when VOPI scores increased. It was detected at 97.5%, 82.5%, 70% and 47.5% in VOPI 0, 1, 2, and 3 groups, respectively. A difference between ICDAS and VOPI scores (p\u0026lt;0.001) was showed. \u003cem\u003e\u0026nbsp;\u003c/em\u003eThere\u003cem\u003e \u003c/em\u003ewere\u003cem\u003e \u003c/em\u003epositive correlations between VOPI and BAC16S (r =0.504, p\u0026lt;0.001), ICDAS and BAC16S (r = 0.506, p \u0026lt; 0.001),\u003cem\u003e \u003c/em\u003eand \u003cem\u003eS\u003c/em\u003e. \u003cem\u003emutans\u003c/em\u003eand BAC16S (r = 0.583, p \u0026lt; 0.001). \u003cem\u003eS. mutans\u003c/em\u003e/total bacteria and \u003cem\u003eS. exigua\u003c/em\u003e/total bacteria ratios exhibited associations (p \u0026lt; 0.001). Using logistic regression, subjects scoring in the VOPI 1, 2, and 3 groups had a 1.97, 3.99, and 6.05 higher chance of having enamel caries compared to those subjects in the VOPI 0 group, respectively.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion \u003c/strong\u003eQuantities of \u003cem\u003eS. mutans\u003c/em\u003e and \u003cem\u003eS. exigua\u003c/em\u003e escalated with higher VOPI scores, and associated with greater enamel caries, while \u003cem\u003eC. matruchotii\u003c/em\u003edecreased. The inverse relationship between \u003cem\u003eC. matruchotii\u003c/em\u003eand VOPI suggests its potential indicator for caries-free or good oral health status.\u003c/p\u003e","manuscriptTitle":"Correlation of Enamel Caries with Occlusal Surface Biofilm: A Quantitative Analysis of Streptococcus mutans, Slackia exigua, and Corynebacterium matruchotii ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-16 08:40:31","doi":"10.21203/rs.3.rs-8942470/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-25T12:16:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-24T07:39:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-24T07:39:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Archives of Paediatric Dentistry","date":"2026-02-23T03:18:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-archives-of-paediatric-dentistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"EAPD","sideBox":"Learn more about [European Archives of Paediatric Dentistry](https://link.springer.com/journal/40368)","snPcode":"40368","submissionUrl":"https://submission.springernature.com/new-submission/40368/3","title":"European Archives of Paediatric Dentistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"59db6918-e9ea-47ea-8905-439a6e1463a5","owner":[],"postedDate":"March 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T08:40:37+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-16 08:40:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8942470","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8942470","identity":"rs-8942470","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}