Skip to Content
MDPIMDPI

Advanced

Applied SciencesApplied Sciences
PDF
  • Article
  • Open Access

17 April 2026

12 Pages

Detection of Cariogenic Bacteria in Pediatric Carious Dentin: Comparative Analysis of Culture and PCR Methods

,
,
,
,
,
,
,
,
…
1
Department of Pediatric Dentistry, Faculty of Dental Medicine, Medical University Sofia, 1431 Sofia, Bulgaria
2
Department of Medical Microbiology, Medical Faculty, Medical University Sofia, 2 “Zdrave” Str., 1431 Sofia, Bulgaria
3
Department of Conservative Dentistry, Faculty of Dental Medicine, Medical University Sofia, 1431 Sofia, Bulgaria
4
Department of Dental, Oral and Maxillofacial Surgery, Faculty of Dental Medicine, Medical University Sofia, 1431 Sofia, Bulgaria
Appl. Sci.2026, 16(8), 3913;https://doi.org/10.3390/app16083913
This article belongs to the Special Issue Recent Advances in Pediatric Orthodontics and Pediatric Dentistry, 2nd Edition
Version Notes
Order Reprints

Abstract

Accurate identification of cariogenic bacteria is crucial for understanding caries development in children. Classical culture methods often underestimate microbial diversity, while polymerase chain reaction (PCR) can detect species that are difficult to cultivate. The aim of this study was to compare culture-based and PCR-based methods for detecting key cariogenic microorganisms in the carious dentin of pediatric patients. Thirty dentin samples were collected from the permanent teeth of children aged 8–14 years. Parallel analyses were performed using standard culture techniques and PCR targeting the gtfB gene of S. mutans and the 16S rRNA gene of Lactobacillus spp. Culture results were quantified as colony-forming units, while PCR results were classified as negative, low-positive, or positive. The results show that culture-based methods identified S. mutans in 16.7% of the samples and Lactobacillus spp. in 3.3%, while PCR identified a signal for S. mutans in 43.3% and Lactobacillus spp. in 100% of the samples. PCR-based methods provide higher sensitivity for detecting key cariogenic bacteria, including S. mutans and Lactobacillus spp. However, PCR detects bacterial DNA and does not indicate bacterial viability or activity. Combining molecular and culture-based approaches allows a more comprehensive assessment of the cariogenic microbiota, supporting accurate microbiological evaluation in pediatric caries research.

1. Introduction

Dental caries develops when biofilms form as a result of complex interactions between the host, diet, and oral bacteria. Recent research shows that dentinal caries progression results from shifts in the balance and composition of the microbial community, not just a single pathogenic species, highlighting the importance of accurate microbiological assessment for understanding pediatric caries [1,2]. Dental caries is seen as an environmental disease, where changes in pH, nutrients, and microbial relationships create conditions that favor the growth of acid-producing and acid-tolerant bacteria. These changes lead to ongoing demineralization and lesion growth, highlighting the important role of the microbial community in dentinal caries development [1,2,3].
Although it is only a small part of the oral microbiota, S. mutans plays a key role in initiating and driving caries due to its unique acidogenic and aciduric properties, allowing it to survive, reproduce, and dominate in low-pH environments. Many people with dental caries lesions do not have high levels of S. mutans, indicating that other cariogenic bacteria should be considered in respect of prevention and treatment [3]. Among these, S. mutans and Lactobacillus spp. are recognized as key contributors to pediatric dental caries due to their metabolic characteristics, making them particularly relevant targets for evaluating microbial detection methods in clinical samples.
To study the diversity of microorganisms in carious dentin, researchers often use classical culture methods for identification. These methods can most often detect viable microorganisms and provide an estimate of their number [4]. Only species that can be grown under laboratory conditions are available for cultivation, while slow-growing or difficult-to-cultivate species that can potentially be associated with caries progression are absent. This greatly limits our ability to further understand the pathogenesis of dental caries and further limits our options for prevention or treatment [5].
Microbial diversity is dynamic and specific across all sites of the same carious lesion. Initial lesions have more facultative Gram-positive bacteria, such as mutans streptococci and lactobacilli, while in deeper lesions more Gram-positive rods and cocci, as well as Gram-negative rods, are identified. This complexity makes comprehensive microbial assessment challenging [6].
However, attempts to isolate anaerobic or difficult-to-cultivate cariogenic bacteria often lead to underestimation and neglect of the total microbial load and microbial diversity.
Molecular diagnostic methods, such as polymerase chain reaction (PCR), can detect bacterial DNA directly from clinical samples, identifying bacteria independent of their ability to grow in culture media [4,7]. PCR is a common molecular tool in microbiology that can identify specific DNA sequences with high accuracy. PCR methods can detect both living and dead bacteria, adding to the information from traditional culture methods, but they cannot confirm if the bacteria are alive. Semi-quantitative PCR results, such as low-positive versus positive, depend on the strength of the DNA signal and should be interpreted with caution when assessing the number of bacteria present [7,8,9,10].
Cariogenic microorganisms such as S. mutans and Lactobacillus species can colonize protected niches in the dentinal tubules. The conditions in these areas, previous treatments, and the way the samples are handled can make it difficult to grow these bacteria in a laboratory. This sometimes leads to negative culture results even when the bacteria are present [7,8]. Although both culture-based and molecular-based methods are widely used, there are few direct comparisons of how effective they are in carious dentin [11]. Understanding the sensitivity, specificity, and limits of each method will help us describe the bacteria that cause caries [12,13].
Recent advances in sequencing-based technologies, including high-throughput 16S rRNA amplicon sequencing and metagenome profiling, have greatly expanded our understanding of the oral microbiome and its role in dental caries. Studies using high-throughput sequencing in pediatric populations have shown shifts in microbial diversity following clinical treatment and have identified taxa beyond classical cariogenic species [14,15], while systematic analyses reveal complex microbiome compositions associated with disease progression [16,17].
The aim of the present study was to compare culture-based and PCR-based methods for the detection of key cariogenic bacteria in pediatric carious dentin, with a focus on S. mutans and Lactobacillus spp., which are recognized as major contributors to caries progression in children. The study further provides a direct parallel analysis of paired clinical samples under standardized conditions and quantifies discrepancies between culture-derived bacterial load (CFU/mL) and PCR detection. These findings provide insights relevant for dental research and clinical diagnostics, emphasizing how integrating molecular and culture methods can improve the evaluation of cariogenic microbiota and highlight the limitations of relying on culture methods alone.

2. Materials and Methods

2.1. Study Design and Sample Collection

The study took place between October 2025 and February 2026 and involved pediatric patients aged 8–14, constituting a pilot cohort intended for preliminary analysis. No formal sample size calculation was performed prior to study initiation due to the exploratory nature of the study. Parents or guardians gave written informed consent, following institutional ethical standards and the Declaration of Helsinki. The Ethics Committee for Scientific Research at the Medical University of Sofia (KENIMUS) approved the study (protocol № 14/28 September 2023).
The object of study was 30 samples of carious dentin from carious lesions (ICDAS 05) of permanent teeth of children between 8 and 14 years old. The inclusion criteria were as follows:
  • Dentin carious lesions with code 05 according to ICDAS II.
  • Visible cavitation of at least 1 mm measured with a periodontal probe;
  • Negative percussion response;
  • Absence of spontaneous pain or radiographic signs of periapical lesions.
From each lesion, dentin material was collected using a sterile excavator. Two parallel samples were collected and immediately placed in transport media and processed within 2 h to maintain bacterial viability. All steps were performed under aseptic conditions to minimize potential contamination.

2.2. Microbiological Cultivation

All specimens were collected with a sterile transport flocked swab (Copan Italia S.p.A., Brescia, Italy) for cultivation and molecular-genetic testing.
One sample from each lesion was processed with standard culture methods. Quantitative cultivation was performed on the following media:
  • Columbia sheep blood agar;
  • Selective Lactobacillus De Man–Rogosa–Sharpe (MRS) agar and broth;
  • Chocolate agar;
  • MacConkey agar;
  • Sabouraud agar;
  • Tryptic soy broth.
All media were supplied by HiMedia Pvt. Limited (Mumbai, India). Columbia blood agar, chocolate agar, and MRS agar were incubated at 36 °C with 5 to 10% CO2 for 24 to 48 h. The other media were incubated aerobically at 36 °C for the same duration. This standard cultivation approach allows comparison with molecular detection but does not fully capture microbial diversity, especially slow-growing or anaerobic species. Anaerobic bacteria and very slow-growing species may not be fully recovered under these conditions, which is a known limitation of culture-based methods.
Bacterial isolates were identified using the RapID CB PLUS System (Thermo Fisher Scientific Inc., Remel Europe Limited, Dartford, UK) and MALDI-TOF MS (Vitek MS, bioMérieux, Marcy-l’Étoile, France). Colony-forming units (CFUs) were counted for each sample to measure bacterial load. Samples were classified as low (<103 CFU/mL), moderate (103–104 CFU/mL), or high (>104 CFU/mL). For each microorganism, the median CFU/mL for positive samples was calculated to show typical abundance.

2.3. Molecular Analysis (PCR)

The parallel set of samples was subjected to molecular analysis. DNA was extracted using the DNA-Sorb A DNA Extraction Kit (Sacace Biotechnologies Srl, Como, Italy) according to the manufacturer’s instructions, and the DNA concentration and purity were assessed spectrophotometrically prior to PCR. All DNA extracts were stored at −70 °C until PCR analysis. The target genes were the gtfB gene (“F5′-ACTACACTTTCGGGT GGCTTGG-3′” and “R5′-CAGTATAAGCGCCAGTTTCATC-3′”) for S. mutans described by Franco et al. (2007) [14] and the 16S ribosomal ribonucleic acid (rRNA) gene for Lactobacillus spp., using primers “F5′-CATTTGGAAACAGATGCTAATACC-3′” and “R5′-GTCCATTGTGGAAGATTCCC-3′” as previously described by Pahumunto et al. (2019) [18].
DNA amplification was performed using Prime Taq Premix 2× (Genetbio Co. Ltd., Daejeon, Republic of Korea) in a total volume of 15 µL, through conventional PCR. Each reaction included 5 µL premix, 150 ng of extracted DNA, and 10 pmol/µL of each primer. Negative controls (distilled water) and positive controls (purified genomic DNA of S. mutans or Lactobacillus spp.) were included in every reaction. For the gtfB gene, the cycling conditions were as follows: initial denaturation at 95 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, 57 °C for 40 s, 72 °C for 1 min, and a final elongation at 72 °C for 10 min. For the 16S rRNA gene of Lactobacillus spp., the program was the same except that the annealing temperature was 56 °C. Following amplification, the PCR products were separated through 2% agarose gel electrophoresis using GelRed® nucleic acid stain (Biotium, Inc., Fremont, CA, USA) and analyzed semi-quantitatively with the documentation system InGenius 3 (Syngene, Cambridge, UK). Molecular weight markers were used to determine the resulting PCR products: 100–1000 bp DNA Ladder (Meridian Bioscience, Cincinnati, OH, USA). Results were classified as negative, low-positive, or positive. A positive signal was defined by a band at the specified molecular weight, exhibiting a relative optical density that was equal to or exceeded the signal of the positive controls. A low-positive signal was defined as a noticeable band at the expected size, validated by the DNA ladder, showing a relative intensity that was considerably lower than the positive control, yet clearly distinguishable from the background noise. Semi-quantitative PCR results (low-positive vs. positive) reflect relative DNA abundance and should be interpreted in conjunction with CFU/mL counts. Although only S. mutans and Lactobacillus spp. were targeted using PCR, these species are considered primary cariogenic indicators in pediatric caries research, providing a focused comparison with classical culture methods.

2.4. Statistical Analysis

The results were statistically analyzed to summarize microbial loads, detection frequencies, and associations among microorganisms. For each microorganism, the frequencies of cases with low, moderate, and high CFU/mL were determined, and the median CFU/mL per positive sample was calculated to reflect typical microbial abundance. Non-parametric comparisons of CFU/mL counts among species were performed using the Kruskal–Wallis test; however, interspecies comparisons were not conducted for microorganisms with small sample sizes.
PCR results (positive, low-positive, and negative) were summarized. Fisher’s exact test was used to compare detection rates between S. mutans and Lactobacillus spp. Statistical testing for Lactobacillus spp. was not performed because the culture results were nearly constant. Agreement between culture and PCR results for S. mutans was measured using Cohen’s kappa coefficient. McNemar’s test (exact binomial) was used to check whether identification rates differed between culture and PCR results.
Spearman’s rank correlation coefficient (ρ) was used to assess associations among frequently isolated microorganisms. Only pairwise correlations with enough samples were included, and significance was set at p < 0.05.
All analyses were performed considering the semi-quantitative nature of PCR results and the limitations of culture-based counts. Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS), version 19 (IBM, Armonk, NY, USA).

3. Results

3.1. Overview of Analyzed Samples

A total of 30 carious dentin samples were analyzed using both microbiological methods, i.e., culture-based and PCR.

3.2. Microbial Load by Culture

Culture-based microbiological analysis showed varying levels of bacterial growth in the samples. Total CFU/mL counts per sample ranged from 102 to 106 CFU/mL, and counts were grouped as low (<1000 CFU/mL), moderate (1000–10,000 CFU/mL), or high (>10,000 CFU/mL).
Staphylococcus species were the most prevalent, followed by Corynebacterium, Neisseria, and Lactobacillus; Candida. S. mutans was detected in only a few samples, and Lactobacillus spp. was detected in only one. Table 1 summarizes the microbial load, frequency, and median CFU per positive sample.
Table 1. Culture-based detection of microorganisms in carious dentin samples (n = 30).
Percentage frequencies now include 95% confidence intervals (CIs) calculated using the Clopper–Pearson exact method. Non-parametric comparisons using the Kruskal–Wallis test were performed among species with sufficient sample sizes (e.g., H = 5.62, p = 0.060, showing no significant differences in median CFU/mL counts among S. aureus, S. saprophyticus, and S. haemolyticus). Spearman correlation analysis was restricted to species with ≥4 positive samples to ensure reliable pairwise associations. Significant positive correlations were observed between S. aureus and S. saprophyticus (ρ = 0.496, p = 0.005) and S. haemolyticus and S. sanguinis (ρ = 0.501, p = 0.005). Other correlations were not statistically significant (p > 0.05).
Spearman correlation analysis among selected cultivable microorganisms showed significant positive correlations between S. aureus and S. saprophyticus (ρ = 0.496, p = 0.005), and between S. haemolyticus and S. sanguinis (ρ = 0.501, p = 0.005) among less common species. Other pairwise correlations were not statistically significant (p > 0.05).
Overall, culture-based analysis revealed a heterogeneous microbial composition, with variable abundances among positive samples.

3.3. PCR-Based Detection of Cariogenic Bacteria

Lactobacillus was detected using PCR in every sample, while S. mutans DNA was detected in 40% of samples, even in some samples where culture methods did not detect it. The results were grouped as negative, low-positive, or positive based on signal strength as presented in Table 2.
Table 2. PCR detection of cariogenic bacteria in carious dentin samples (n = 30).
It should be emphasized that the PCR approach used here is semi-quantitative. Therefore, while it reliably detects the presence of bacterial DNA, it does not directly indicate bacterial viability, activity, or clinical relevance. Interpretation of these PCR results should be made in conjunction with CFU/mL data from culture-based methods.
Percentages for PCR detection are presented with 95% confidence intervals (CIs) calculated using the Clopper–Pearson exact method.

3.4. Direct Comparison of Culture-Based and PCR-Based Methods

When comparing culture-based and PCR-based detection, we found clear differences. Culture-based methods mainly isolated bacteria that grew easily, but PCR consistently found important cariogenic bacteria that culture-based methods often missed. For instance, PCR detected Lactobacillus spp. in every sample, while culture-based methods found them in almost none. PCR also detected S. mutans more often than culture-based methods did.
Table 3 presents a direct comparison of culture-based methods and PCR, highlighting their differences. The results show that culture-based analysis misses many cariogenic bacteria in carious dentin.
Table 3. Direct comparison of culture-based and PCR detection of cariogenic bacteria in carious dentin samples (n = 30).
Statistical analysis was not performed for Lactobacillus spp. due to near-constant culture results. For S. mutans, agreement between methods was assessed using Cohen’s kappa (κ = 0.268, 95% CI: 0.05–0.49), indicating fair agreement. The McNemar test confirmed a statistically significant difference in detection rates between culture-based methods and PCR (p = 0.021, exact binomial test, 95% CI for the difference: 0.10–0.55), highlighting the higher sensitivity of PCR.
Percentages are now reported with a 95% CI, and only microorganisms with ≥4 positive samples were considered for pairwise correlation or direct statistical comparison to ensure the reliability of the analysis.
Overall, PCR detected S. mutans and Lactobacillus spp. more frequently than culture-based methods, highlighting the limitations of culture-based methods in capturing some of the key cariogenic bacteria in pediatric carious dentin.

4. Discussion

In the present study, we found clear differences between culture-based and PCR-based methods for detecting cariogenic bacteria in carious dentin. PCR was more sensitive than traditional culture techniques, detecting S. mutans in 43.3% of samples (13/30) compared to 16.7% using culture techniques (5/30, median 100 × 103 CFU/mL), and Lactobacillus spp. in 100% of samples versus only 3.3% using culture techniques. This quantifies the discrepancy between culture-derived bacterial load (CFU/mL) and PCR detection, highlighting how routine culture-based methods may underestimate key cariogenic bacteria. For instance, 8 samples were negative for S. mutans using culture-based methods but positive using PCR, while 29 samples were detected only using PCR for Lactobacillus spp. These findings provide a more comprehensive view of the microbiota in pediatric carious lesions and are consistent with previous reports demonstrating that molecular methods capture microbial diversity missed by culture-based methods, particularly in pediatric populations [14,15,16,17].
Previous studies, including those by Mohamed Rasheed et al. [15], Su et al. [16], and AlHarbi et al. [14], reported similar discrepancies, confirming that PCR consistently identifies these key species more frequently and that classical culture-based methods underestimate total microbial diversity, particularly for fastidious or anaerobic taxa.
Moreover, molecular methods such as real-time PCR, qRT-PCR, and NGS-based metagenome profiling have repeatedly shown that culture-based techniques underestimate microbial diversity, especially for species that are difficult to cultivate. Recent pediatric sequencing studies further demonstrate shifts in microbial composition beyond classical cariogenic species [14,15,16,17,18,19,20]. These findings support a combined methodological approach to capture both cultivable and non-cultivable cariogenic bacteria [14,15,16,17,21,22,23].
Combining culture-based and molecular methods allows a more comprehensive understanding of microbial interactions within carious lesions and how environmental factors influence bacterial survival and activity. This integrated approach can inform the development of new strategies for preventing and treating dental caries, providing insight into the complex ecosystem of the pediatric oral cavity. Metagenomic studies, including recent pediatric investigations, further support the observation that molecular methods detect bacterial taxa that are often missed or underestimated when using culture-based techniques [14,15,16,17], underscoring the need for complementary methodologies to capture the full microbial profile [7,8,9,10].
Culture-based methods have long been considered the standard in microbiology, but they only measure bacteria that can be grown in laboratory conditions. Many oral bacteria are difficult to cultivate and require special conditions that are not always possible to provide in laboratories. When bacteria face stressors such as oxygen exposure, nutrient limitation, pH changes, or clinical treatments, they can enter a viable but non-culturable (VBNC) state, in which they remain active and pathogenic but do not grow on standard media, leading to false negatives [20,24,25,26]. These limitations demonstrate the need for molecular approaches to better understand the microbial composition of carious lesions.
In the present study, we were unable to recover Lactobacillus spp. effectively, probably due to their fastidious growth requirements and sensitivity to sampling and transport conditions. Lactobacillus spp. were cultured in only 1/30 samples (3.3%), whereas PCR detected them in all 30 samples (100%), highlighting their potential VBNC state. Exposure to oxygen or delays in processing can further reduce viability [27]. Although their DNA remains detectable, these bacteria may not grow under standard laboratory conditions. Their reduced metabolic activity complicates recovery via conventional culture, even though they are known contributors to caries progression [28]. Similar challenges in culturing Lactobacillus spp. have been reported previously, emphasizing the likelihood of underestimation in culture-based studies [18,19].
Non-mutans streptococci and staphylococcal species were more commonly recovered using culture-based methods, likely reflecting their higher tolerance to laboratory conditions. S. aureus was present in 20% of samples, S. sanguinis in 16.7%, and S. salivarius in 23.3%, indicating that these species withstand oxygen and nutrient fluctuations better than more fastidious cariogenic bacteria [29,30]. Consequently, culture results alone may misrepresent the true contributors to caries, reflecting survival in laboratory conditions rather than pathogenic significance. This reinforces the need for multiple methods, including molecular diagnostics, to obtain an accurate representation of microbial communities in carious dentin [2,4,21].
In this study, colony-forming unit (CFU/mL) counts from culture methods provided an estimate of total bacterial burden but did not confirm the activity of specific cariogenic species. While useful for overall bacterial quantification, CFU/mL counts fail to capture full microbial diversity or identify the most clinically relevant species [2,6,21].
In contrast, PCR demonstrated highly sensitive detection of bacterial DNA, including species that are difficult or impossible to cultivate. Our semi-quantitative PCR approach limits direct conclusions regarding bacterial activity or clinical relevance, as PCR cannot differentiate between live cells and DNA from non-viable or lysed bacteria. Consequently, PCR may overestimate biologically active microorganisms [30,31,32]. Nonetheless, PCR enables rapid, specific identification and quantification of target microorganisms, addressing many limitations of conventional microbiology and revealing the true breadth of carious dentin microbiota. These insights can guide future studies investigating microbial viability and functional activity [33,34].
Overall, PCR detected S. mutans 2.6 times more frequently and Lactobacillus spp. 30 times more frequently than culture-based methods, demonstrating its higher sensitivity for key cariogenic bacteria. These findings are consistent with earlier reports [6,7,20,21,22], indicating that molecular methods may detect bacterial DNA not captured by culture-based techniques. The present study should therefore be interpreted primarily as confirmatory evidence rather than demonstrating novel observations, given its pilot design and limited sample size. Culture-based methods can identify viable bacteria capable of growth, whereas PCR reveals the broader presence of bacterial DNA, including clinically relevant but fastidious species [35]. Integrating these approaches allows a more precise representation of the microbial ecosystem in pediatric carious dentin, which may inform future research and methodological approaches [36].
Relying solely on culture-based techniques may result in the underestimation of cariogenic bacterial prevalence and diversity, potentially affecting diagnostic interpretation and experimental conclusions. Using molecular diagnostics alongside classical culture-based methods improves sensitivity and supports evidence-based approaches in pediatric dentistry. Negative culture results should be interpreted with caution, as they do not necessarily exclude the presence of cariogenic bacteria. The integration of molecular diagnostics with classical culture-based methods may improve the overall understanding of microbial detection; however, given the exploratory nature of this pilot study, further validation in larger cohorts is required before drawing conclusions regarding clinical application.
These findings suggest that the combined application of culture- and PCR-based methods enhances the understanding of cariogenic microbiota, bridging gaps between microbial detection and clinical relevance in pediatric caries research. This supports the utility of integrating molecular and culture-based methods for accurate microbial assessment, as consistently highlighted in earlier pediatric studies [14,15,16,17,21,22]. This approach ensures that clinically relevant, fastidious, or VBNC cariogenic bacteria are not overlooked.

5. Limitations

This study represents a pilot investigation with a small sample size, which limits the generalizability and statistical power of the findings. The analysis targeted only Streptococcus mutans and Lactobacillus spp., two well-established cariogenic bacteria, while dental caries is a complex, polymicrobial disease. This limited microbiological scope may underestimate the diversity and interactions of other cariogenic species, and results should be interpreted within this context. Additionally, the choice of target species and semi-quantitative PCR limits the assessment of broader microbial diversity. Secondly, species-specific PCR cannot distinguish live from dead bacteria, and we did not directly assess bacterial viability in this study. CFU/mL counts provide a rough estimate of bacterial load but do not capture the functional activity or pathogenic potential of the detected microorganisms. Thirdly, culture-based methods might miss some microbial diversity because certain species have strict growth needs or are sensitive to their environment. Our methodology did not include strict anaerobic cultivation, focusing instead on facultative anaerobes, which play a crucial role in the formation of dental caries. The absence of strict anaerobic culture conditions reduced the recovery of obligate anaerobes, which often dominate in deep carious dentin and severe lesions. Consequently, our study may not completely reflect the total diversity of the anaerobic species involved in the progression of caries. Future research utilizing specific anaerobic conditions is crucial for thoroughly elucidating the role of these oxygen-sensitive organisms alongside facultative anaerobes. Taken together, these factors underline that our findings should be interpreted cautiously. Future studies using broader metagenomic or metatranscriptomic methods could offer deeper insight into microbial diversity, function, and activity.
Even with these limitations, our results emphasize that integrating culture-based and molecular methods provides a more comprehensive assessment of cariogenic bacteria, supporting microbiological evaluation in pediatric caries research.

6. Conclusions

In this study, PCR consistently detected Streptococcus mutans and Lactobacillus spp. in pediatric carious dentin more frequently than culture-based methods. Combining culture-based methods and PCR revealed discrepancies in microbial detection, showing that relying solely on cultivation can underestimate the presence of these cariogenic bacteria. PCR detects DNA regardless of bacterial viability, so while it is more sensitive, it does not directly indicate active infection. These findings demonstrate that integrating molecular and culture-based approaches provides a more comprehensive representation of microbial presence in carious lesions.

Author Contributions

Conceptualization, N.M. and M.G.; methodology, N.M., R.G., V.B. and A.A.; validation, R.G., V.B. and A.A.; formal analysis, N.M. and R.G.; investigation, N.M., R.G., V.B., A.A., E.K., N.G., V.D., Z.M., M.M.-T., K.H., D.K., I.T.-I. and M.G.; resources, E.K., N.G., V.D., Z.M., M.M.-T., K.H., D.K. and I.T.-I.; data curation, N.M., R.G. and V.B.; writing—original draft preparation, N.M.; writing—review and editing, M.G., N.M., R.G., V.B., A.A., E.K., N.G., V.D., Z.M., M.M.-T., K.H., D.K. and I.T.-I.; visualization, N.M.; supervision, M.G.; project administration, N.G., E.K. and Z.M.; funding acquisition, N.G., E.K. and Z.M. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financed by the European Union—NextGenerationEU, through the National Recovery and Resilience Plan of the Republic of Bulgaria, project BG-RRP-2.004-0004-C01 “Strategic research and innovation program for development of the Medical University—Sofia, Bulgaria”.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Ethics Committee of the Medical University of Sofia (protocol code № 14/28 September 2023).

Data Availability Statement

The data are not publicly available due to privacy and ethical restrictions.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
PCRPolymerase chain reaction
CFU/mLColony-forming units per mL
ICDASInternational Caries Detection and Assessment System
MRSde Man, Rogosa and Sharpe (medium for Lactobacillus)
DNADeoxyribonucleic acid
rRNARibosomal ribonucleic acid
SPSSStatistical Package for the Social Sciences
VBNCViable but non-culturable

References

  1. Ribeiro, A.A.; Paster, B.J. Dental caries and their microbiomes in children: What do we do now? J. Oral Microbiol. 2023, 15, 2198433. [Google Scholar] [CrossRef] [Scilit]
  2. Spatafora, G.; Li, Y.; He, X.; Cowan, A.; Tanner, A.C.R. The Evolving Microbiome of Dental Caries. Microorganisms 2024, 12, 121. [Google Scholar] [CrossRef] [Scilit]
  3. Aas, J.A.; Griffen, A.L.; Dardis, S.R.; Lee, A.M.; Olsen, I.; Dewhirst, F.E.; Leys, E.J.; Paster, B.J. Bacteria of dental caries in primary and permanent teeth in children and young adults. J. Clin. Microbiol. 2008, 46, 1407–1417. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  4. Zhang, J.S.; Chu, C.-H.; Yu, O.Y. Oral Microbiome and Dental Caries Development. Dent. J. 2022, 10, 184. [Google Scholar] [CrossRef] [Scilit]
  5. Cugini, C.; Ramasubbu, N.; Tsiagbe, V.K.; Fine, D.H. Dysbiosis From a Microbial and Host Perspective Relative to Oral Health and Disease. Front. Microbiol. 2021, 12, 617485. [Google Scholar] [CrossRef] [Scilit]
  6. Martin, F.E.; Nadkarni, M.A.; Jacques, N.A.; Hunter, N. Quantitative microbiological study of human carious dentine by culture and real-time PCR: Association of anaerobes with histopathological changes in chronic pulpitis. J. Clin. Microbiol. 2002, 40, 1698–1704. [Google Scholar] [CrossRef] [Scilit]
  7. Wen, Z.T.; Huang, X.; Ellepola, K.; Liao, S.; Li, Y. Lactobacilli and human dental caries: More than mechanical retention. Microbiology 2022, 168, 001196. [Google Scholar] [CrossRef] [Scilit]
  8. Zhang, S.; Li, X.; Wu, J.; Coin, L.; O’Brien, J.; Hai, F.; Jiang, G. Molecular Methods for Pathogenic Bacteria Detection and Recent Advances in Wastewater Analysis. Water 2021, 13, 3551. [Google Scholar] [CrossRef] [Scilit]
  9. Long, K.; Cai, L.; He, L. DNA Sequencing Data Analysis. Methods Mol. Biol. 2018, 1754, 1–13. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  10. Paruch, L. Molecular Diagnostic Tools Applied for Assessing Microbial Water Quality. Int. J. Environ. Res. Public Health 2022, 19, 5128. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  11. Gheorghiu, I.M.; Ciobanu, S.; Roman, I.; Păunică, S.; Dumitriu, A.S.; Iliescu, A.A. Deep Caries Lesions Revisited: A Narrative Review. J. Mind Med. Sci. 2025, 12, 37. [Google Scholar] [CrossRef] [Scilit]
  12. Cherkaoui, A.; Schrenzel, J. Total Laboratory Automation for Rapid Detection and Identification of Microorganisms and Their Antimicrobial Resistance Profiles. Front. Cell. Infect. Microbiol. 2022, 12, 807668. [Google Scholar] [CrossRef] [Scilit]
  13. Hassan, A.F.; Yadav, G.; Tripathi, A.M.; Mehrotra, M.; Saha, S.; Garg, N. A Comparative Evaluation of the Efficacy of Different Caries Excavation Techniques in reducing the Cariogenic Flora: An in vivo Study. Int. J. Clin. Pediatr. Dent. 2016, 9, 214–217. [Google Scholar] [CrossRef] [Scilit]
  14. AlHarbi, S.G.; Almushayt, A.S.; Bamashmous, S.; Abujamel, T.S.; Bamashmous, N.O. The oral microbiome of children in health and disease-a literature review. Front. Oral Health 2024, 5, 1477004. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  15. Mohamed Rasheed, Z.B.; Sheng, C.W.; Norfitriah, E.; Nasruddin, N.S.; Yazid, F. Oral Microbiome Dynamics in Treated Childhood Caries: A Comparative Study. Life 2024, 14, 1576. [Google Scholar] [CrossRef] [Scilit]
  16. Su, S.; Qi, T.; Wang, W.; Salama, E.S.; Li, Y. Investigation of the oral microbiome of children associated with dental caries: A systematic study. Arch. Oral Biol. 2023, 154, 105776. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  17. Gong, S.G.; Gong, S.G.; Switzer, J.; Nainar, S.M.H.; Lévesque, C.M. Microbiome in Early Childhood Caries: Caries Severity-Dependent Insights. Caries Res. 2025, 59, 394–405. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  18. Pahumunto, N.; Sophatha, B.; Piwat, S.; Teanpaisan, R. Increasing Salivary IgA and Reducing Streptococcus mutans by Probiotic Lactobacillus paracasei SD1: A Double-Blind, Randomized, Controlled Study. J. Dent. Sci. 2019, 14, 178–184. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  19. Franco e Franco, T.C.C.; Amoroso, P.; Marin, J.M.; Ávila, F.A. Detection of Streptococcus mutans and Streptococcus sobrinus in Dental Plaque Samples From Brazilian Preschool Children by Polymerase Chain Reaction. Braz. Dent. J. 2007, 18, 329–333. [Google Scholar] [CrossRef] [Scilit]
  20. Jeong, J.; Mun, S.; Oh, Y.; Cho, C.-S.; Yun, K.; Ahn, Y.; Chung, W.-H.; Lim, M.Y.; Lee, K.E.; Hwang, T.S.; et al. A qRT-PCR Method Capable of Quantifying Specific Microorganisms Compared to NGS-Based Metagenome Profiling Data. Microorganisms 2022, 10, 324. [Google Scholar] [CrossRef] [Scilit]
  21. Baker, J.L.; Morton, J.T.; Dinis, M.; Alvarez, R.; Tran, N.C.; Knight, R.; Edlund, A. Deep metagenomics examines the oral microbiome during dental caries, revealing novel taxa and co-occurrences with host molecules. Genome Res. 2021, 31, 64–74. [Google Scholar] [CrossRef] [Scilit]
  22. Obata, J.; Takeshita, T.; Shibata, Y.; Yamanaka, W.; Unemori, M.; Akamine, A.; Yamashita, Y. Identification of the microbiota in carious dentin lesions using 16S rRNA gene sequencing. PLoS ONE 2014, 9, e103712. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  23. Choudhary, K.; Gouraha, A.; Sharma, M.; Sharma, P.; Tiwari, M.; Chouksey, A. Clinical and Microbiological Evaluation of the Chemomechanical Caries Removal Agents in Primary Molars. Cureus 2022, 12, e31422. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  24. Liu, Y.; Wang, J.; Dong, B.; Zhai, Y.; Zhou, L.; Sun, S.; Li, X.; Wu, L. Prediction and validation of microbial community function from normal pulp to pulpitis caused by deep dentinal caries. Int. Endod. J. 2023, 56, 608–621. [Google Scholar] [CrossRef] [Scilit]
  25. Rajasekaran, J.J.; Krishnamurthy, H.K.; Bosco, J.; Jayaraman, V.; Krishna, K.; Wang, T.; Bei, K. Oral Microbiome: A Review of Its Impact on Oral and Systemic Health. Microorganisms 2024, 12, 1797. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  26. Wasfi, R.; Abd El-Rahman, O.A.; Zafer, M.M.; Ashour, H.M. Probiotic Lactobacillus sp. inhibit growth, biofilm formation and gene expression of caries-inducing Streptococcus mutans. J. Cell. Mol. Med. 2018, 22, 1972–1983. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  27. Ari, M.M.; Scholz, K.J.; Cieplik, F.; Al-Ahmad, A. Viable but non-cultivable state in oral microbiota: A critical review of an underexplored microbial survival strategy. Front. Cell. Infect. Microbiol. 2025, 15, 1533768. [Google Scholar] [CrossRef] [Scilit]
  28. Radaic, A.; Kapila, Y.L. The oralome and its dysbiosis: New insights into oral microbiome-host interactions. Comput. Struct. Biotechnol. J. 2021, 19, 1335–1360. [Google Scholar] [CrossRef] [Scilit]
  29. Perera, D.; McLean, A.; Morillo-López, V.; Cloutier-Leblanc, K.; Almeida, E.; Cabana, K.; Welch, J.M.; Ramsey, M. Mechanisms underlying interactions between two abundant oral commensal bacteria. ISME J. 2022, 16, 948–957. [Google Scholar] [CrossRef] [Scilit]
  30. Dewhirst, F.E.; Chen, T.; Izard, J.; Paster, B.J.; Tanner, A.C.R.; Yu, W.; Lakshmanan, A.; Wade, W.G. The Human Oral Microbiome. J. Bacteriol. 2010, 192, 5002–5017. [Google Scholar] [CrossRef] [Scilit]
  31. Khalaf, M.E.; Karched, M.; Shawaf, N.A.; Qudeimat, M.A. In vitro investigation of the impact of contemporary restorative materials on cariogenic bacteria counts and gene expression. J. Dent. 2023, 133, 104486. [Google Scholar] [CrossRef] [Scilit]
  32. Sereti, M.; Zekeridou, A.; Cancela, J.; Mombelli, A.; Giannopoulou, C. Microbiological testing of clinical samples before and after periodontal treatment. A comparative methodological study between real-time PCR and real-time-PCR associated to propidium monoazide. Clin. Exp. Dent. Res. 2021, 7, 1069–1079. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  33. Schwendener, S.; Flury, M.; Jenzer, J.; Thurnheer, T.; Karygianni, L. PMA-qPCR to quantify viable cells in multispecies oral biofilm after disinfectant treatments. Biofilm 2025, 9, 100281. [Google Scholar] [CrossRef] [Scilit]
  34. Naumann, A.A.; Elmorr, E.M.; Lamont, E.I.; Hendrickson, E.L.; Holmes, T.; Nelson, T.M.; McLean, J.S.; Kerns, K.A. DNA and RNA-based amplicon sequencing of paired supragingival and dentin lesion plaque in children with severe early childhood caries. Int. J. Oral Sci. 2026, 18, 19. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  35. Zhang, R.R.; Zhang, J.S.; Huang, S.; Lam, W.Y.-H.; Chu, C.-H.; Yu, O.Y. The oral microbiome of root caries: A scoping review. J. Dent. 2025, 160, 105899. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  36. Rudkjøbing, V.B.; Thomsen, T.R.; Xu, Y.; Melton-Kreft, R.; Ahmed, A.; Eickhardt, S.; Bjarnsholt, T.; Poulsen, S.S.; Nielsen, P.H.; Earl, J.P.; et al. Comparing culture and molecular methods for the identification of microorganisms involved in necrotizing soft tissue infections. BMC Infect. Dis. 2016, 16, 652. [Google Scholar] [CrossRef] [Scilit] [PubMed]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Article Metrics

Citations

Article Access Statistics

Journal Statistics
Multiple requests from the same IP address are counted as one view.
Influence of Follicular vs. Luteal Phases on Sweat Rate and Estimated Sodium Loss in University Female Football Players: A Field-Based Within-Subject Study
Previous
Hydrolytic Stability and Optical Properties of 3D-Printed, Milled, and Conventional Interim Resins After Thermal Aging
Next