Dental caries is one of the most prevalent chronic infectious diseases worldwide, affecting individuals across all age groups. According to the World Health Organization (WHO), dental caries is defined as a disease caused by tooth-adherent bacteria that ferment dietary sugars, leading to acid production and subsequent demineralization of the tooth structure.[1] A recent meta-analysis conducted by the National Health Institute (NIH) in 2021 reported that the estimated prevalence of dental caries in Pakistan is 56.62%, with a 95% confidence interval, underscoring its public health burden.[2] Clinically, GV Black’s classification of caries based on anatomical location continues to guide diagnosis and treatment planning. [3] Dental caries is an infectious disease caused by tooth adherent bacteria, especially Streptococcus mutans.[4] This includes a dynamic interaction between tooth, bacteria, and other factors that starts with the formation of dental plaque; a biofilm, which is the accumulation of debris, bacteria, saliva, and food particles over the tooth's surface.[5] Within hours, the dental plaque matures and becomes colonized by the acidogenic bacteria such as Streptococcus mutans, which in turn start metabolizing carbohydrates on to the tooth surface to produce lactic acid, leading to progressive demineralization of enamel. [6] In 1976 Loesche proposed the “Specific Plaque Hypothesis”, asserting that Streptococcus mutans is the primary etiological agent to dental caries. Nonetheless, this theory did not account for cases where S. mutans was absent despite evident caries [7] Streptococcus mutans, is a gram-positive coccus, alpha-hemolytic facultative anaerobe that thrives in acidic environments, potentiating its cariogenic potential. [8-10] For its identification, S. mutans can be cultured using Mitis Salivarius Agar (MSA) supplemented with bacitracin (2 U/mL), 20% sucrose, and potassium tellurite; a method widely accepted for its specificity. [11] However, a simplified and cost-effective alternative using MSA without additives has also been reported by the Journal of Bacteriology that reliably identifies S. mutans based on the formation of small, pale blue colonies approximately 1 mm in diameter.[12] In this study, we successfully adopted this simplified method, culturing samples under both aerobic and anaerobic conditions. This methodological choice not only reduced the overall cost and complexity but also forms a core component of the study rationale, aligning with our objective to explore efficient and practical identification techniques in resource-constrained settings. Although other bacteria such as Lactobacilli, Streptococcus sobrinus, [8] Actinomyces [13] Bifidobacterium and Veillonella etc [13] have also been implicated in caries development, S. mutans remains central to caries research and prevention strategies due to its high acidogenicity, acid tolerance, and critical role in early lesion formation. In addition to microbial identification, understanding the clinical burden of caries is essential. The DMFT index (Decayed, Missing, and Filled Teeth) remains a globally recognized tool for quantifying dental caries prevalence.[14] Despite its widespread use, limited studies have examined the relationship between S. mutans isolation and DMFT scores in the Pakistani population. Establishing this correlation could provide valuable insights into the microbial etiology of caries and support more targeted prevention strategies. Furthermore, the rising concern of antimicrobial resistance (AMR) among oral bacteria, including S. mutans, presents a significant challenge in dental care. that can potentially compromise therapeutic outcomes, especially in patients having recurrent or complicated infections. With an increased global awareness of antimicrobial resistance (AMR) [15], we need to reassess the effectiveness of commonly used antibiotics in dental practice. To address this, we included three widely used antibiotics in this study: amoxicillin, doxycycline, and clindamycin. These drugs were selected based on their clinical relevance in dental practice and their representation of different antibiotic classes. The concentrations; amoxicillin (10 µg), clindamycin (2 µg), and doxycycline (30 µg), were based on the Clinical and Laboratory Standards Institute (CLSI) guidelines, which recommend standardized disc potencies for antimicrobial susceptibility testing using the disc diffusion method.[16] These concentrations are established through extensive validation to produce zone diameters that correlate with clinical breakpoints, enabling consistent and clinically meaningful interpretation of bacterial resistance patterns.

Data Description: This cross-sectional study was conducted at Khyber Medical University (KMU) after receiving approval from the Institutional Ethical Committee, Institute of Pathology and Diagnostic Medicine, KMU under ethical approval number: KMU/IPDM/IEC/2024/21. Samples and data were collected from the Oral and Maxillofacial Surgery Department at Hayatabad Medical Complex, Peshawar, Pakistan. Patients were recruited based predefined inclusion criteria, including adult patients of all genders with grossly carious permanent requiring extraction. A non-probability purposive convenience sampling method was used to enroll 100 patients after taking informed consent from them. The demographic characteristics of the study cohort are given in Table 1. Of the 100 participants, 48 (48%) were female and 52 (52%) were male. Patients were stratified into the following age groups: young adults (18–30 years), middle-aged adults (31–45 years), and older adults (≥46 years). The largest group comprised middle-aged adults (44%), followed by older adults (34%), and young adults (22%) Additional demographic variables such as smoking history, socioeconomic status (SES), and oral hygiene practices were recorded. None of the females reported smoking, while 30 out of 52 male participants (57.7%) had a positive history of smoking. The majority of participants (52%) reported poor oral hygiene, with only 16% maintaining good oral hygiene. Poor hygiene practices were slightly more prevalent among males (53.8%) compared to females (50%) Moreover, socioeconomic status (SES) was assessed using occupation as a proxy indicator, based on Kuppuswamy Scale.[17] Based on the reported employment type, with reference to the locally accepted classification of occupation, participants were categorized into lower or middle SES groups; no participants met the criteria for upper SES. Of all participants, 69% were classified as lower SES, including 35 females (72.9%) and 34 males (65.4%). The remaining 31 participants belonged to the middle SES category (18 males, 13 females). (Figure 1). Methods: The study was conducted in five phases. Clinical examination and DMFT index evaluation Isolation of Streptococcus mutans Identification of Streptococcus mutans Antibiotic Susceptibility testing Statistical analysis Clinical examination and DMFT Index evaluation A detailed history was obtained, including demographics, present illness, past medical and dental history, personal habits, and oral hygiene practices. (Table:1) Each patient’s DMFT score was assessed using a mouth mirror and recorded using the standard formula. (1). DMFT score=〖Teeth〗_Decayed+ 〖Teeth〗_Missing+ 〖Teeth〗_Filled (1) Where, 〖Teeth〗_Decayed, 〖Teeth〗_Missing, 〖Teeth〗_Fillingrepresents the total number of teeth decayed, missing, and filled, respectively. Isolating Streptococcus mutans Following extraction, each grossly carious permanent tooth was put into a labeled, sterile, closed container, containing 20 ml of normal saline and transported to the Microbiology Laboratory at Khyber Medical University. Carious material was aseptically excavated using a sterilized spoon excavator inside a biosafety cabinet, and transferred into labeled Eppendorf tubes containing 700 μL of Brain Heart Infusion (BHI) broth (Oxoid, UK). Samples were incubated at 37°C for 48 hours. Turbidity in the broth indicated bacterial growth Next, bacterial cultures were streaked onto nutrient agar (Alpha biosciences), and incubated both, aerobically and anerobically for 24 hours at 37 °C to confirm facultative anaerobic growth. The colonies were small, usually less than 1mm in diameter and yellowish-white, round colonies, often translucent in appearance. (Figure: 2) Identifying Streptococcus mutans Since nutrient agar is non-selective, Mitis Salivarius Agar (MSA) (TM media) was used as a selective media for confirmation. Several conditions were tested. Plain MSA without any additives MSA supplemented with 10% sucrose, bacitracin 0.2U/ml and potassium tellurite MSA supplemented with 20% sucrose, bacitracin 0.2U/ml and potassium tellurite. All plates were incubated aerobically for 48 hours at 37 °C. Unexpectedly, bacterial growth was observed only on plain MSA, while no growth occurred on supplemented versions. Colonial morphology was compared to a reference S. mutans strain provided by Khyber Medical University, revealing small (less than 1 mm in diameter), multiple raised, blue, opaque colonies with frosted glass appearance. (Figure: 3) Antibiotic Susceptibility test Antimicrobial susceptibility was tested using the Kirby-Bauer disc diffusion method on Mueller-Hinton agar (Oxoid, UK). [18] A lawn of Streptococcus mutans was created and commercially available antibiotic discs containing amoxicillin (10 µg), doxycycline (30 µg), and clindamycin (2 µg) were aseptically placed. The plates were incubated aerobically at 37 °C for 48 hours to allow antibiotic diffusion. (Figure: 6) Zones of inhibition were measured and interpreted according to CLSI (Clinical and Laboratory Standard Institute) guidelines. [19] Statistical Modeling Descriptive and inferential statistical analysis was conducted to assess relationships between variables and validate findings. Descriptive statistics included mean, standard deviation, and distribution plots. Inferential analysis included Levene’s test (for homogeneity of variance), independent t-tests, and regression analysis to evaluate associations between S. mutans presence, DMFT scores, and demographic variables. Data analysis was performed using Python libraries, including SciPy, Seaborn, and Matplotlib.

Table 1. Demographic characteristics of the study cohort. Variable Category Distribution (%) Samples (n) 100 Gender M F 52 (52.0%) 48 (48.0%) Age (group) Young Adult (18-30 years) Middle Aged Adult (31-45 years) Older Adult (≥46 years) 22 (22.0%) 44 (44.0%) 34 (34.0%) Smoking No Yes 70 (70.0%) 30 (30.0%) Socioeconomic Status Low SES Middle SES 69 (69.0%) 31 (31.0%) Oral Hygiene Practices Poor Satisfactory Good 52 (52.0%) 32 (32.0%) 16 (16.0%) Abbreviations: SES: Social Economic Status. M: Male; F: Female. This section briefly discusses the findings of the analysis. We performed statistical analysis to identify Streptococcus mutans from dental caries, and to find its association with DMFT score. Firstly, the descriptive statistics was computed for the DMFT score grouped by the presence or absence of Streptococcus mutans to compare the association between Streptococcus mutans and dental caries. (Table 2). Table 2. Descriptive statistics representing association between DMFT score and Streptococcus mutans Streptococcus mutans (Group) N Mean Median SD SE DMFT Score Not Found 22 5.05 5.00 2.06 0.44 Present 78 7.45 8.00 2.55 0.29 Abbreviations: N: Sample Size; SD: Standard deviation; SE: Standard error. It is shown in Table 2 that the sample with Streptococcus mutans present exhibited a higher mean DMFT score (7.45) compared to the group with Streptococcus mutans not found (5.05). This positive association indicates that the presence of Streptococcus mutans may be associated with a higher level of dental decay (higher DMFT score). Moreover, the estimate of the mean DMFT score is more precise for the Streptococcus mutans present group, indicated by the lower standard error (0.29) as compared to the absence of Streptococcus mutans (0.44). To test the statistical significance of this association, independent t-test was performed. A homogeneity of variances (Levene’s) test, conducted prior to it, yielded significant result (F-statistic = 4.15 and p-value = 0.044), suggesting that the data violates the assumption of equal variance. Consequently, Welch's t-test was employed. Table 3 indicates the results of the independent T-test (both Student’s t-test and Welch’s t-test). Table 3. Independent T-test results for the association between DMFT score and Streptococcus mutans 95% CI Statistic df p Mean difference SE difference Lower Upper DMFT Score Student's t 4.06a 98.0 <.001 2.40 0.591 1.23 3.58 Welch's t 4.58 41.0 <.001 2.40 0.525 1.34 3.46 Note. Hₐ μnot found ≠ μpresent aLevene's test is significant (p < .05), suggesting a violation of the assumption of equal variances Abbreviations: CI: Confidence Interval; df: degree of freedom; SE: Standard error. The results of Welch's t-test (Welch's t-statistic = 4.58, p < 0.001) indicates that the presence of Streptococcus mutans is significantly associated with a higher DMFT score, implying that individuals with this bacterium tend to have more dental issues. Furthermore, despite the violation of equal variances assumption, the results of student’s t-test (Student's t-statistic = 4.06, p < 0.001) indicates a significant difference in DMFT score between two groups of Streptococcus mutans. In summary, the descriptive statistics and independent t-test showed a statistically significant difference in the DMFT scores of Streptococcus mutans (not found) and Streptococcus mutans (present). The 78 bacterial samples obtained were also put through antibiotic susceptibility test to find out the susceptibility and resistance pattern for amoxicillin, clindamycin, and doxycycline. In order to ensure data quality, we excluded samples with inconclusive test results (n=20). This ensures the inclusion of samples (n = 58) with only definitive test results (i.e., resistant, intermediate susceptible, susceptible) for the analysis. Considering the CLSI guidelines, we ran a Chi-square test for Streptococcus mutans against these drugs, respectively (Figure 7). Figure 7. Antibiotic Susceptibility/Resistance Patterns. Chi-square test for susceptibility of Amoxicillin, Doxycycline and Clindamycin against Streptococcus mutans (P < 0.001) Figure 7 shows that 33 (56.9%) of the total isolates of Streptococcus mutans were found susceptible to amoxicillin, whereas 25 (43.1%) of them were resistant. For doxycycline, 24 (41.4%) samples were susceptible, 25 (43.1%) had intermediate susceptibility whereas 9 (15.5%) were resistant. Whereas clindamycin susceptibility test gave us 48 (82.8%) statistically significant resistant cases and 10 (17.2%) intermediate susceptible cases. (P value < 0.001). Moreover, we used multinomial logistic regression (MNLogit) model to find out an association between demographic variables (such as age, gender, oral hygiene practices of the patients, history of smoking, and socioeconomic factors) with the antibiotic resistance pattern of Streptococcus mutans. The fit measures of MNLogit model including deviance, Akaike Information Criterion (AIC), McFadden's R², Chi-Square (χ²), degree of freedom and p-value for each of the antibiotic are presented in Table 4. This MNLogitClindamycin model has lowest deviance (132) which suggests that it fits the data better than the other two models. Likewise, the lowest AIC (168) was achieved by the MNLogitClindamycin model which indicates that it is the best performing model compared to MNLogitAmoxicillin, MNLogitDoxycycline model. In addition, the χ² evaluates the overall significane of the MNLogit models, where the model with p < 0.05 considered as statistically significant. However, the results indicated that none of the models are statistically significant as all the three models showed p-value greater than 0.05. This suggests that the predictors do not significantly improve the model fit compared to a null model (with no predictors). Table 4. Logistic Regression Model (Multinomial) Fit Measures Overall Model Test Model Deviance AIC R²McF χ² df p MNLogitAmoxicillin 67.4 85.4 0.15 11.9 8 0.16 MNLogitDoxycycline 99.9 136 0.15 18.1 16 0.32 MNLogitClindamycin 49.4 67.4 0.074 3.92 8 0.86 Note. Models estimated using sample size of N=58 Furthermore, in order to evaluate the contribution of each predictor (demographic variables) to the model results, we conducted a Likelihood ratio test. Table 5 presents the χ² statistic, degrees of freedom (df), and p-value for each predictor variable. The findings suggest that none of the predictors are statistically significant in any of the models as the p-value for all the predictors in all models are greater than 0.05. In summary, the findings of the analysis indicated that the MNLogit models for antibiotic resistance (Amoxicillin, Doxycycline, and Clindamycin) prediction based on demographic variables (such as Age, Gender, Smoking, SocioEconomic Status, and Oral Hygiene Practices) did not indicate statistically significant results. Likewise, none of the demographic variables were significantly associated with antibiotic resistance. Table 5. Likelihood Ratio Tests for predictors of Amoxicillin, Doxycycline and Clindamycin resistance Amoxicillin Doxycycline Clindamycin Predictor χ² df p χ² df p χ² df p Age 4.99 3 0.17 9.62 6 0.14 2.71 3 0.44 Gender 0.16 1 0.68 0.37 2 0.83 0.55 1 0.46 Smoking 1.23 1 0.26 0.59 2 0.74 1.34 1 0.25 SocioEconomic Status 0.91 1 0.34 4.80 2 0.09 0.42 1 0.52 Oral Hygiene Practices 3.82 2 0.15 6.15 4 0.18 0.45 2 0.79 In addition, we identified the importance of demographic and behavioral variables using random forest algorithm for different antibiotic resistance. Figure 8 illustrates the importance of different features with respected to different antibiotic resistance. It can be seen that poor oral hygiene practices were considered as the most important feature for almost all the three antibiotic resistance followed by the low socioeconomic status. However, good oral hygiene was considered as the least important feature in antibiotic resistance.

Caries is one of most highly prevalent oral pathologies. According to the World Health Organization, oral diseases are one major concern for public health, and the reason for chronic oral pathologies such as dental caries depends upon the risk factors such as diet and lifestyle factors.[20] The results obtained about the age distribution of carious patients show us that equal number of adult patients were suffering from dental caries, irrespective of their gender. However, patient’s compliance in practicing good oral hygiene was found more in females as compared to males. This is supported by a study conducted by Australian Research Centre for Population Oral Health, according to which 98% of women and 94% of men brushed their teeth daily. [21] However, majority of the population (52 patients) in our study showed to practice poor oral hygiene and only 16 of them had good oral hygiene practices. This could be possible because of the low socioeconomic status of people, as also outlined by Folayan in a survey, where he stated that children coming from low socioeconomic background had 44.44% incidence of early childhood caries. [22] Since, majority of the patients recruited in our study belonged to lower SES (69 participants), hence, it can be deduced that the socioeconomic status of an individual does have an impact on oral hygiene practices and ultimately on developing carious lesions in that individual. Thus, there is a need for awareness regarding the importance of preventive oral care in lower SES communities so that progression of oral diseases such as dental caries could be prevented. Also, Streptococcus mutans are notorious for being the primary cause of dental caries. [23] However, we were only able to isolate it from 78 samples out of the total 100 samples. This implies that there may be other causative organisms of dental caries, rather than Streptococcus mutans. The same concept was indicated in 1976 by Loesche when he proposed the “Specific Plaque Hypothesis” which, however, lacked the potential to explain that Streptococcus mutans were, in fact, absent from certain carious sites. [24] According to the T independent test conducted to find an association between presence of Streptococcus mutans and DMFT scores (n=100), a significant association was seen. This could be attributed to the fact that Streptococcus mutans is the principal causative agent of dental caries, thus highlighting the critical role of microbial factors in dental health, particularly in populations suffering from high levels of dental caries. Liu R in 2025 said that Streptococcus mutans is the key pathogen in causing dental caries and that a dysbiotic microbial community results in the initiation of dental caries. [25] Correspondingly, in 2024, Narjess Bostanghadiri also claimed that oral microbiota is very diverse and a number of factors influence disease progression in oral cavity. [26] Similarly, in 2023, Omotuyole AS, states about preschool children that the level of Streptococcus mutans was significantly higher in caries active individuals.[27] This is plausible as Streptococcus mutans are agents that initiate dental caries. Concurrently, according to an American study published in 2024, it was found that the group of people who had a high DMFT index also had more caries associated bacteria mainly Streptococcus mutans and Lactobacilli.[28] To sum it up, this study highlights the need for public health interventions focused on controlling Streptococcus mutans in populations at high risk for dental caries, that includes oral health education programs and early detection of Streptococcus mutans. Antibacterial mouthwashes or fluoride treatments could also be used in high risk population. For Antibiotic Susceptibility and Resistance Pattern, statistically significant results were obtained and it was found that the antibiotics under our investigation were not effective for all patients. These findings also coincides with a Mexican study about children that gave us 43% amoxicillin resistant strains of Streptococcus mutans.[29] On the contrary, in 2018, Loyola-Rodriguez JP also found out that in adults, amoxicillin showed susceptibility that was more than 99.1%. Nonetheless, he did report that Streptococcus mutans, out of all the other oral microflora, had an increased frequency of antimicrobial resistance. [30] The same studies also reported highest resistance observed for Clindamycin with values of 85.9% and 59.4% in children and adults, respectively.[29, 30] Another research by Al-Ahmad A, 2014, states that a number of Streptococcus mutans isolates were high biofilm producers, which infers that there was high resistance observed for those cases. [31] All these researches are aligned with our outcome of an enhanced resistance pattern observed by Streptococcus mutans against amoxicillin, clindamycin and doxycycline, as these drugs were not found to be effective for all cases. Hence, there is a need for further study to explore alternative treatment options for dental patients.

It is concluded that Streptococcus mutans is the principal cause of dental caries and it is significantly associated with DMFT score. This highlights the importance of managing microbial count in the prevention of dental caries through better education, improved access to care, and effective treatments targeting Streptococcus mutans. Future scientific advancements may lead to certain vaccines, probiotics, or antibacterial therapies that would be specifically designed for the prevention of dental caries by Streptococcus mutans in high-risk populations. The findings could also inspire the development of more precise diagnostic tools to detect high levels of Streptococcus mutans, that would allow more tailored and early interventions before caries have progressed. Moreover, the emergence of antibiotic resistance in treatment of dental infections has been a matter of concern. This was particularly evident with significant results obtained for Amoxicillin, Doxycycline, and Clindamycin when tested against Streptococcus mutans. This leads to a greater emphasis on the preventive oral measures. Strict guidelines should be made and followed by dental professionals for the responsible prescribing of antibiotics, which should include avoiding unnecessary prescriptions and using the right antibiotic for the right condition. Alternate treatment options should also be explored to improve oral health. Thus, we can achieve better oral health outcomes, reduce health disparities, and ultimately lower the public health burden associated with dental diseases. 2. Ethical approval This study was approved by the Institutional Ethical Committee, Institute of Pathology and Diagnostic Medicine, Khyber Medical University. Ethical approval number: KMU/IPDM/IEC/2024/21, dated 12th May 2024. All study procedures were conducted in accordance with relevant guidelines and regulations. All participants provided written informed consent to publish identifying information/images and clinical records. 3. Informed Consent Statement Written informed consent was obtained from all participants before inclusion. The study adhered to ethical guidelines, ensuring confidentiality and voluntary participation 4. Acknowledgment We would like to express our sincere gratitude to the staff of the Outpatient Department at Hayatabad Medical Complex, Peshawar, for their invaluable support and assistance in data collection for this study. 5. Author’s contribution Conceptualization, and study design: AZ, BA, S Ahmad, MK, MAA, MM, S Ali, SI, HRA Project Supervision: BA Methodology: BA, S Ahmad, SI, MAA, HRA Acquisition of data: BA, AZ, MK Analysis and interpretation of data: S Ali, BA, HRA Writing the manuscript, drafting the article: AZ, BA, S Ahmad, MK, MAA, MM, S Ali, SI, HRA Revising content: AZ, BA, S Ahmad, MK, MAA, MM, S Ali, SI, HRA Final approval of the version: AZ, BA, S Ahmad, MK, MAA, MM, S Ali, SI, HRA All authors reviewed and contributed to the manuscript, verified the manuscript and gave consent for its publication. 6. Conflict of interest The authors declare no conflict of interest. 7. Funding Statement This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. 8. Data Availability Statement The data supporting the findings of this study are available upon reasonable request from the corresponding author.

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