Quantity of the antigens of Streptococcus mutans serotype e and Candida albicans and its correlation with the salivary flow rate in early childhood caries
Endang Winiati Bachtiar1, Vanya A Kusuma1, Ferry P Gultom1, Retno D Soejoedono2
1 Department of Oral Biology, Oral Science Research Center, Faculty of Dentistry, Universitas Indonesia, Jakarta, Indonesia
2 Department of Immunology, Faculty of Veterinary, IPB University, Dramaga Campus, Bogor, Indonesia
|Date of Submission||29-Jun-2020|
|Date of Decision||31-Oct-2021|
|Date of Acceptance||27-Apr-2022|
|Date of Web Publication||30-Jun-2022|
Prof. Endang Winiati Bachtiar
Department of Oral Biology, Oral Science Research Center, Faculty of Dentistry, Universitas Indonesia, Jalan Salemba No. 4, Jakarta 10430
Source of Support: None, Conflict of Interest: None
Background: Streptococcus mutans involved in caries pathogenesis is classified into four serotypes, namely serotypes c, e, f, and k. Candida albicans can be found in the plaque of children with early childhood caries (ECC). Aims: The aim of this study was to analyze the quantity of the antigens of S. mutans serotype e and C. albicans and its correlation with the salivary flow rate in ECC. Materials and Methods: The antigen quantities of caries plaque samples and caries-free were determined using an enzyme-linked immunoassay with 450-nm optical density. Results: There was a significant difference between the quantity of S. mutans serotype e and C. albicans antigens in each salivary flow rate category (P < 0.05). The relationship between the antigen quantity of S. mutans serotype e and C. albicans was r = 0.624 (P > 0.05) for caries plaque samples and r = 0.628 (P > 0.05) for caries-free samples. Conclusion: the antigen quantities of S. mutans serotype e and C. albicans and the salivary flow rate might correlate to the pathogenesis of ECC.
Keywords: Candida albicans, dental plaque, early childhood caries, flow rate, saliva, serotype, Streptococcus mutans
|How to cite this article:|
Bachtiar EW, Kusuma VA, Gultom FP, Soejoedono RD. Quantity of the antigens of Streptococcus mutans serotype e and Candida albicans and its correlation with the salivary flow rate in early childhood caries. J Res Med Sci 2022;27:47
|How to cite this URL:|
Bachtiar EW, Kusuma VA, Gultom FP, Soejoedono RD. Quantity of the antigens of Streptococcus mutans serotype e and Candida albicans and its correlation with the salivary flow rate in early childhood caries. J Res Med Sci [serial online] 2022 [cited 2022 Aug 10];27:47. Available from: https://www.jmsjournal.net/text.asp?2022/27/1/47/349442
| Introduction|| |
According to the American Academy of Pediatric Dentistry, early childhood caries (ECC) is a terminology used when there is a presence of more cavities or missing or filled teeth due to caries in children up to 71 months of age., The etiology of ECC is multifactorial, resulting from interactions between microorganisms, including S. mutans. and sugar on the surface of the teeth. Host factors contributing to caries development are low salivary flow rates, immunological factors, damaged or imperfect enamel, tooth morphology itself, and poor tooth structure. Saliva contains minerals that play an important role in the process of tooth remineralization. Immunoglobulin A contained in saliva and gingival crevicular fluid acts as a immune defense against Streptococcus mutans.
S. mutans is a Gram-positive bacterium and one of seven species of the mutans streptococci. Based on its serotype-specific polysaccharide composition, namely, rhamnose-glucose polymers (RPG), S. mutans is classified into four types: serotypes c, e, f, and k. Polysaccharides in serotypes c, e, and f consist of RPG, whereas, for serotype k, the identified glucose side chains are much reduced with only rhamnose chains present.,
In addition to S. mutans, opportunistic fungal C. albicans also plays an active role in the pathogenesis of caries and is often found in children's oral cavity with ECC., A higher number of C. albicans can be found in the saliva, plaque, and caries tissue of kids with ECC than that in kids without caries.
The prevalence of S. mutans serotype e was found to be 5%. Moreover, still rare study analyzing this serotype e that might has a correlation to ECC. Future research with is required to determine the role of S. mutans serotype e to assess the preventive role against caries development. Analysis of the antigen quantities of S. mutans serotype e and C. albicans in related to the salivary flow rate has not been reported previously.
In this study, we aimed to analyze the quantity of the antigens of S. mutans serotype e and C. albicans and its correlation with the salivary flow rate in ECC.
| Materials and Methods|| |
This study was conducted as an observational study. The samples were obtained through a purposive sampling technique. Sampling was carried out at the TPA Harapan Ibu Ministry of Social Affairs Ministry of Social RI on 50 children: 36 children with ECC and 14 caries-free children. Plaque samples were taken from the first deciduous molars using cotton buds and stored in Eppendorf tubes containing 1 mL of phosphate-buffered saline (PBS).
Salivary flow rate
Salivary flow rate test was performed on each subject. The examination was performed by placing one ply of tissue on the subject with the dried lower lip and observing how much time it took for the tissue to get wet using a stopwatch. The results were categorized into two groups: one with a salivary flow rate <30 s and the other with 30–60 s.
Indirect enzyme-linked immunoassay (ELISA) was carried out in this study., Briefly, the samples that were diluted 1/10 in PBS (Sigma-Aldrich Dorset UK. Cat. P4417) were added to the wells and incubated at 4°C overnight. After incubation and washing, blocking buffer (5% skim milk in PBS-0.1% TweenR20 [Sigma-Aldrich, Cat. P9416]) was added to each well, followed by incubation for 1 h at 37°C. Rabbit sera anti-S. mutans serotype e or anti-C. albicans diluted in PBS at a 1:1,000 ratio were added to each well and bound to the coated antigen of S. mutans and C. albicans. After incubation and washing, tetramethylbenzidine (TMB substrate) (Sigma-Aldrich, Cat. T0440) was added to the wells, followed by the addition of the stop solution. The optical density (450 nm) of the complex antigen antibody was determined using an ELISA reader (AccuReader. M965/M965+ Nangang, Taipei, Taiwan).
The Kolmogorov–Smirnov normality test was used, and the Mann–Whitney U-test comparative test was done to compare the quantity of S. mutans serotype e and C. albicans antigens between the two groups. The correlation of both microorganisms in both ECC and caries-free samples was tested using the Spearman test.
| Results|| |
We have totally 50 subjects included for this study, with 36 caries active and 14 of caries-free aged between 2.4 and 4.5 years. Children with salivary flow rate <30 s in caries active were 20 subjects and 16 children have salivary flow rate of 30–60 s. Whereas 10 children with caries-free have salivary flow rate <30 (P > 0.05).
Quantities of Streptococcus mutans serotype and Candida albicans antigens in children with and without caries
The Kolmogorov–Smirnov test shows that the data were not normally distributed; hence, the Mann–Whitney U comparative test was done. [Figure 1]a shows that the average ± standard deviation of S. mutans serotype e antigens was 2.361 and the average optical density of C. albicans antigens was 1.646 ± 0.23. For caries-free samples and S. mutans, serotype e antigens was 2.028 ± 0.41, and the average of C. albicans antigens was 1.429 ± 0.37 (P < 0.05). In children with active caries, the average of the quantity of S. mutans serotypes e and C. albicans antigens was higher than in the caries-free group.
|Figure 1: (a) Average of the quantities of Streptococcus mutans serotype e and Candida albicans antigens in the plaque of children with and without caries, (b) quantities of Streptococcus mutans serotype e and Candida albicans antigens in the plaque associated with salivary flow rate|
Click here to view
The average of quantities of Streptococcus mutans serotype e and Candida albicans antigens associated with the salivary flow rate
[Figure 1]b shows the average S. mutans serotype e and C. albicans based on the salivary flow rate in caries and caries-free samples. S. mutans serotype e and C. albicans antigens in caries were higher than those in caries-free in both categories of salivary flow rate (P < 0.05). In caries active and caries-free, the OD 450 of S. mutans in <30 s salivary flow rate 2.331 versus 1.985 and in 30–60 s were 2.463 versus 2.135, whereas C albicans 1.632 versus 1.428 and in 30–60 s were 1.728 versus 1.433, respectively (P > 0.05). Further analysis showed that the average of quantity of S. mutans serotypes e and C. albicans antigens, higher than in the free caries group both in children with a salivary flow rate <30 s and in the group with salivary flow rate of 30–60 s (P > 0.05).
Spearman test yielded a value of r = 0.624, with P < 0.05, indicating that there was a positive correlation between the antigen quantities of S. mutans and C. albicans in the plaque of subjects with ECC [Figure 2]a. A value of r = 0.628, with P > 0.05, was obtained from the Spearman correlation test, suggesting a positive correlation between the antigen quantities of S. mutans serotype e and C. albicans in the plaque of caries-free subjects [Figure 2]b.
|Figure 2: (a) Correlation between antigen quantities of Streptococcus mutans serotype e and Candida albicans in the plaque of subjects with caries, (b) correlation between antigen quantities of Streptococcus mutans and Candida albicans in the plaque of caries-free subjects|
Click here to view
| Discussion|| |
The key finding of our study is the quantity of S. mutans serotype e, and C. albicans antigens on the plaque of children with caries are higher than those caries-free children regardless of the salivary flow rate category.,,,,
Our finding is supported by previous studies that stated that caries was a multifactorial condition where the number of cariogenic microorganisms did not guarantee whether a carious lesion formed. However, caries formation is also determined by a balance of protective and pathologic factors called “caries balance.”
Furthermore, this study showed a strong positive correlation between antigens of S. mutans serotype e and C. albicans. This result is consistent with that of a previous study by Bachtiar and Bachtiar, reporting that there is an association between antigen quantities of both microorganisms. The data on the association between C. albicans and S. mutans serotype e in this study may support the clinical importance of uncovering the pathogenesis of ECC. The results of this study may contribute to find strategies to improve children's oral health. Further in vitro experiment study might need to investigate the molecular interaction between S. mutans and C. albicans under sugar enriched condition.
| Conclusion|| |
The salivary flow involved in S. mutans serotype e and C. albicans existence in ECC patients.
Financial support and sponsorship
This study was supported by HIBAH PUTI from Universitas Indonesia (Project Number: NKB16032020/BA-622/UN2.RST/PPM.00.03.01/2021).
Conflicts of interest
There are no conflicts of interest.
| References|| |
Anil S, Anand PS. Early childhood caries: Prevalence, risk factors, and prevention. Front Pediatr 2017;5:157.
Bezerra DS, Stipp RN, Neves BG, Guedes SF, Nascimento MM, Rodrigues LK. Insights into the virulence traits of Streptococcus mutans
in dentine carious lesions of children with early childhood caries. Caries Res 2016;50:279-87.
Babaeekhou L, Mehrizi AA, Ghane M. Streptococcus mutans
, sugar consumption, and oral hygiene: Which one has more effect on decayed, missing, and filled teeth (DMFT) score in Iranian adults? Dent Res J (Isfahan) 2020;17:134-41.
Borutta A, Wagner M, Kneist S. Early childhood caries: A multi-factorial disease. Oral Health Dent Manage 2010;9:405.
Zafar S, Harnekar SY, Siddiqi A. Early childhood caries: Etiology, clinical considerations, consequences and management. Int Dentistry South Africa 2009;11:24-36.
Milgrom P, Riedy CA, Weinstein P, Tanner AC, Manibusan L, Bruss J. Dental caries and its relationship to bacterial infection, hypoplasia, diet, and oral hygiene in 6- to 36-month-old children. Community Dent Oral Epidemiol 2000;28:295-306.
Dawes C, Wong DT. Role of saliva and salivary diagnostics in the advancement of oral health. J Dent Res 2019;98:133-41.
Nakano K, Nemoto H, Nomura R, Homma H, Yoshioka H, Shudo Y, et al.
Serotype distribution of Streptococcus mutans
a pathogen of dental caries in cardiovascular specimens from Japanese patients. J Med Microbiol 2007;56:551-6.
Nakano K, Ooshima T. Serotype classification of Streptococcus mutans
and its detection outside the oral cavity. Future Microbiol 2009;4:891-902.
Widyagarini A, Sutadi H, Budiardjo SB. Serotype c and e Streptococcus mutans
from dental plaque of child-mother pairs with dental caries. J Int Dent Med Res 2016;9:339-44.
de Carvalho FG, Silva DS, Hebling J, Spolidorio LC, Spolidorio DM. Presence of mutans streptococci and Candida
spp. in dental plaque/dentine of carious teeth and early childhood caries. Arch Oral Biol 2006;51:1024-8.
Eidt G, Andrade CG, Negrini TC, Arthur RA. Role of Candida albicans
on enamel demineralization and on acidogenic potential of Streptococcus mutans in vitro
biofilms. J Appl Oral Sci 2019;27:e20180593.
Zhang W, Li Y, Lin J, Abduryim A, Zhao J. Cariogenicity of Candida albicans
of distinct genotypes among 3-5-year-old Uygur children in Kashgar, China – A case-control study. 2018;18:203.
Willems HM, Kos K, Jabra-Rizk MA, Krom BP. Candida albicans
in oral biofilms could prevent caries. Pathog Dis 2016;74:ftw039.
Kavitha M, Prathima GS, Kayalvizhi G, Sanguida A, Ezhumalai G, Ramesh V. Evaluation of Streptococcus mutans
serotypes e, f
, and k
in saliva samples of 6-12-year-old school children before and after a short-term daily intake of the probiotic lozenge. J Indian Soc Pedod Prev Dent 2019;37:67-74.
] [Full text]
Cunha-Cruz J, Scott J, Rothen M, Mancl L, Lawhorn T, Brossel K, et al
. Salivary characteristics and dental caries: evidence from general dental practices. J Am Dent Assoc 2013;144:e31-40.
Mount GJ, Hume WR, Ngo H, Wolff MS. Preservation and Restoration of Tooth Structure, 3rd Edition. 3rd
Editio. West Sussex: John Wiley & Sons Limited; 2016.
Rao AP, Austin RD. Serotype specific polymerase chain reaction identifies a higher prevalence of Streptococcus mutans
serotype k and e in a random group of children with dental caries from the Southern region of India. Contemp Clin Dent 2014;5:296-301.
] [Full text]
Sakamoto S, Putalun W, Vimolmangkang S, Phoolcharoen W. Enzyme – Linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites. J Nat Med 2018;72:32-42.
Gan SD, Patel KR, Elisa S. Enzyme immunoassay and enzyme-linked immunosorbent assay. J Invest Dermatol 2013;133:1-3.
Bachtiar EW, Soejoedono RD, Bachtiar BM, Henrietta A, Farhana NU, Yuniastuti M. Effects of soybean milk, chitosan, and anti- Streptococcus mutans
IgY in malnourished rats' dental biofilm and the IgY persistency in saliva. Interv Med Appl Sci 2015;7:118-23.
Bachtiar EW, Bachtiar BM. Relationship between Candida albicans
and Streptococcus mutans
in early childhood caries, evaluated by quantitative PCR. F1000Res 2018;7:1645.
[Figure 1], [Figure 2]