The relationship between the Cbm and Cnm genes in Streptococcus mutans isolated from arches and brackets of patients and antibiotic resistance profiles

Abstract

Purpose: Human dental caries is a troublesome disorder of the teeth, and the bacterium mostly responsible is Streptococcus mutans. The Cbm and Cnm genes, which encode the Cbm and Cnm proteins respectively, are two of the virulence factors associated with Streptococcus mutans strains. Oral appliances such as dental arches and brackets accelerate streptococcal colonization and caries formation. The aim of this study was to determine the gene frequency of Cnm and Cbm in Streptococcus mutans strains isolated from oral arches and brackets in orthodontics patients and their relationship to antibiotic resistance.

Methods: Four hundred seventy-three orthodontics patients were included in this study and 384 Streptococcus mutans strains were isolated from the arches and brackets of these patients. The strains were identified and verified using a Vitek-2 compact system and their antibiotic susceptibility was determined by the disc diffusion method. The presence of the Cbm and Cnm genes was confirmed by the PCR method.

Results: It was found that 2.08% of the strains the Cbm gene and 16.14% the Cnm gene. The rate of co-existence of these two genes was 0.78%.

Conclusion: Strains those harbored the Cbm and Cnm genes are more resistant to antibiotics and more likely to be major risk factors for caries in orthodontic patients.

Introduction

The primary objective of orthodontics is to achieve proper tooth alignment and functional occlusion. Improved facial esthetics and the attainment of an attractive smile are additional benefits of orthodontic treatment. In addition to its mechanical effects, the psychosocial impact of orthodontic treatment plays a significant role in enhancing individual self-confidence in social settings [1]. It is acknowledged that in individuals whose esthetic appearance has improved, their psychological condition is positively affected [2]. On the other hand, due to the prolonged duration of orthodontic treatment, challenges related to maintaining oral hygiene and preventing plaque accumulation often arise. Oral streptococci, classified as viridans group streptococci, account for approximately 20% of the total oral microbiota. This group includes several species, such as Streptococcus mutans (S. mutans), Streptococcus mitis (S. mitis), Streptococcus sanguis (S. sanguis), and Streptococcus intermedius (S. intermedius) [3].

Despite the growing popularity of orthodontic treatment in recent years, the development of white spot lesions following treatment remains a significant concern from an esthetic standpoint [4]. Orthodontic appliances particularly brackets, ligatures, and archwires introduce additional retentive sites that facilitate undesirable plaque accumulation [5]. The accumulation of dental plaque harboring cariogenic bacteria during orthodontic treatment is considered the primary etiological factor in enamel decalcification. This demineralization of dental surfaces can lead to the formation of white spot lesions or dental caries [6,7,8].

The ability of oral streptococci to interact with the host extracellular matrix is considered a key virulence factor facilitating their attachment to and colonization of host tissues, such as heart valves [9]. Streptococcus mutans strains that express collagen-binding proteins (CBPs) on their cell surface demonstrate enhanced pathogenicity [10]. S. mutans is a commensal bacterium of the oral microbiota and plays a key role in the development of dental caries. S. mutans may express collagen-binding proteins (CBPs) on its surface, specifically Cbm and Cnm, which are encoded by the Cbm and Cnm genes, respectively [11,12,13]. These proteins enhance the bacterium’s adhesion to dentin, thereby potentially promoting the development of dental caries [13]. Studies have demonstrated that bonded orthodontic brackets induce specific alterations in the oral environment, including a reduction in pH levels and increased plaque accumulation in the areas surrounding the brackets [14,15]. Another study has reported that individual strains of cariogenic streptococci exhibit distinct adhesion to metal brackets, the degree of adhesion varying according to the bacterial strain and the duration of incubation. These findings collectively suggest that orthodontic brackets represent a potential risk factor for enamel demineralization [16].

The aim of this study was to determine the frequencies of the Cbm and Cnm genes in S. mutans strains isolated from patients undergoing orthodontic treatment, and to investigate the association between these genes and antibiotic resistance. It was hypothesized that the use of fixed orthodontic appliances would increase the predisposition for S. mutans colonization in these patients.

Materials and Methods

Clinical samples

Three hundred eighty-four S. mutans strains isolated from a total of 473 patients who received orthodontic treatment for more than 6 months at the Department of Orthodontics, Hatay Mustafa Kemal University Hospital of Dentistry, Hatay, Turkey, were included in this study. Patients were asked to fill in and sign a voluntary consent form so that their samples could be collected. The age range of the patients included in the study was 12 to 27 years, and the gender distribution was 40.63% male and 59.37% female. These data were evaluated as the demographic features of the patients. No limitation of gender or, age was considered as an inclusion criterion except for the existence of oral apparatus in the mouth for less than 6 months. Patients with missing teeth, systemic diseases or various syndromes, and those using aligners, ceramic brackets or, self-ligating braces were excluded. This study was approved as decision number 04 of the Clinical Ethics Committee at its meeting dated 25.02.2021 with the patients’ consent.

The initial incubation medium, Todd-Hewitt Broth (Condalab, Madrid, Spain), was used to enrich the streptococcal colonies at 37°C for 24 h. After the first incubation, Mitis-Salivarius Agar (MSB), which contains 0.2 U/mL bacitracin (Sigma-Aldrich, St. Louis, MO, USA) and 15% sucrose (wt/vol), was used to cultivate the bacteria. The culture conditions were 37°C for 24-48 h. After the process, ‘R’ colonies were selected and verified with a Vitek-2 compact system and a GP identification kit (BioMèrieux, Craponne, France). Streptococcus mutans strains were isolated from 384 of the 473 (81.18%) patients.

Disc diffusion test

Identified S. mutans strains were passaged on Müller Hinton agar (Sigma-Aldrich) and analyzed by the disc diffusion method in terms of antibiotic susceptibility to penicillin (PE), clindamycin (CLIN), erythromycin (ERY), doxycycline (DOX), ceftriaxone (CEFT), cefoxitin (CEF), tetracycline (TE), and vancomycin (VAN). In the bacteriology laboratory, the procedure was carried out by placing a maximum of 6 antibiotic discs on 9-mm Müller Hinton Agar plates inoculated with S. mutans at a McFarland turbidity of 0.5 within 15 min after inoculation. Within 15 min of application of the antimicrobial discs, the plates were inverted and incubated for 16-20 h at 35 ± 1°C in according to the antibiotic susceptibility of the strains on the EUCAST website guide for disc diffusion testing.

Molecular analysis

Genomic DNA extraction

A bacterial genomic DNA extraction kit (Thermo Fisher Scientific, Vilnius, Lithuania) was used for purification of genomic DNA from S. mutans strains in accordance with the manufacturer’s instructions.

PCR amplification and gel electrophoresis

A PCR method was used to determine the Cbm and Cnm genes in S. mutans. The PCR protocol was applied after 4 min of initial denaturation at 95°C, 30 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 2 min, with a final elongation for 7 min at 72°C. To determine the presence of the Cbm gene, GAC AAA CTA ATG AAA TCT AA forward and GCA AAA ACT GTT GTC CCT GC reverse primers were used. To determine the presence of the Cnm gene, GAC AAA GAA ATG AAA GAT GT forward and GCA AAG ACT CTT GTC CCT GC reverse primers were used. The PCR products were electrophoresed in 1.5% or 0.7% agarose gel-Tris-acetate-EDTA buffer solution. For imaging, bands of 1728 bp for the Cnm gene [10] and 1814 bp for the Cbm gene were searched [17]. S. mutans UA159 (serotype c), LM7 (serotype e), OMZ-175 (serotype f), and FT1 (serotype k) were used as positive control strains. All PCR assays were performed in duplicate.

Statistical analysis

G*Power software (Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany) version 3.1 was used for the sample size calculation. This revealed that the minimum number of patients was 429, considering an effect size (w) of 0.15, an alpha value of 0.05, and a power of 0.80. Pearson Chi Squared Test was applied for descriptive statistical analysis (number, percentage) and to compare two rates. Cbm and Cbm/Cnm mutations were not evaluated due to the limited number of strains. For pairwise comparative analysis regarding the presence of mutation, only Cnm mutation was examined.

Results

The age range of the patient cohort was 12-27. One hundred seventy-two patients (44.8%) were male and 212 (55.2%) were female. Clinical examination for regulatory of toothbrushing and oral hygiene revealed that 81.25% (312/384) of the patients were regular and the rest (72/384; 18.75%) non-regular. A sugar-rich diet was found in 44.8% of the patients. One hundred forty-two S. mutans strains (44.8%) isolated from these patients lacked the Cbm and/or Cnm genes. Two Cbm-positive (Cbm+) (1.16%), 27 Cnm-positive (Cnm+) (15.69%) and one Cbm- and Cnm-positive (Cbm/Cnm+) S. mutans strains (0.58%) were isolated from these patients. The prevalence of a hereditary caries predisposition among the patients was 13.2% (51/384). When the patients were asked about their caries history in the previous year, 6.77% (26/384) of them stated that they had developed caries during the orthodontic treatment. Ten isolates obtained from these 26 cases were analyzed, and 3 were Cbm+, 5 were Cnm+, and 2 were Cbm/Cnm+ Three hundred eleven of the 384 isolates (80.9%) were negative for Cbm and/or Cnm. The prevalence of Cbm + isolates was 2.08% (8/384) and that of Cnm + isolates was 16.14% (62/384). Only 3 isolates (3/384; 0.78%) harboring both Cbm and Cnm were found (Fig. 1). The demographic features of the patients are shown in Table 1.

A total of 384 S. mutans isolates were analyzed and verified both bacteriologically and molecularly in terms of strain identification and Cbm/Cnm positivity. All the strains were subjected to the disc diffusion antibiogram test and their antibiotic susceptibility was determined on the basis of Clinical and Laboratory Standards Institute (CLSI) methodology. Among a total of 311 Cbm- and/or Cnm-negative isolates, the antibiotics to which they were most susceptible were CEF (89.1%), ERY (81.3%), and CLIN (79.1%). The rate of resistance to TE was 60.7% (189/311). The number of Cbm + S. mutans strains was 8 (2.08%). Cbm + strains had a high rate of susceptibility to CEF 75% and 62.5% were resistant to TE. Sixtytwo isolates were Cnm + and most of them were resistant to TE (77.4%). CEF was the most effective antibiotic agent against Cnm + S. mutans strains at 53.2% (33/62). Three of these isolates (3/384; 0.78%) were found to harbor both Cbm and Cnm. All of these three isolates were mostly susceptible to VAN and completely resistant to PE and TE. Overall, the antibiotic resistance rates of Cbm +, Cnm + and Cbm/Cnm + isolates were higher than other isolates and the most effective antibiotic agents against S. mutans strains were CEF, ERY, and CLIN (Table 2).

PCR analysis showed that the prevalence of Cbm + strains was 2.08% (8/384) and that of Cnm + strains was 16.14% (62/384). Only 0.78% (3/384) of these strains had co-existing Cbm/Cnm genes (Fig. 1).

Gel electrophoresis images showing the Cbm and Cnm genes detected by conventional PCR are presented in Fig. 2. Product sizes of 1814 bp and 1728 bp for the Cbm and Cnm genes, respectively, were investigated.

The PE and CLIN resistance rates of Cbm + strains were 37.5%. The same strains showed resistance rates of 50% for ERY, 25% for DOX, 12.5% for CEFT, and 62.5% for TE. CEF and VAN resistance was not detected in Cbm + S. mutans strains. However, these results were not statistically significant due to the limited number of isolates. The PE resistance rate of Cbm/Cnm-negative strains was 9.1%, being significantly higher in Cnm + strains with a rate of 33.8% (P < 0.001). In Cbm/Cnm-negative strains, CLIN resistance was 6.3%. On the other hand, the CLIN resistance rate was 23.1% in Cnm + strains (P < 0.001). While the ERY resistance rate was 12.9% in CBP genenegative strains, it was 24.6% in Cnm + strains (13.8%) was nearly seven-fold higher than in CBP-negative strains (2.2%) (P < 0.001). The TE resistance rates of CBP-negative strains and Cnm + strains were 60.8% and 78.5%, respectively (P = 0.004). Resistance to VAN was evident in 3.4% of CBP-negative strains and 23.1% of Cnm + strains (P = 0.001) (Table 3).

Fig. 1 Cbm, Cnm, and Cbm/Cnm gene frequency in the isolated S. mutans strains

Table 1 Demographic and numerical data for the patient cohort

Pattern	S. mutans strains without the Cbm and/or Cnm genes	Cbm + S. mutans strains	Cnm + S. mutans strains	Cbm/Cnm + S. mutans strains
Number of isolates	311	8	62	3
Age (12-27)	12-26	23-27	15-24	17-26
Gender male n = 172 female n = 212	male = 136 female = 164	male = 3 female = 5	male = 28 female = 34	male = 3 female = 0
Toothbrushing regular n = 312 non-regular n = 72	regular = 267 non-regular = 44	regular = 6 non-regular = 2	regular = 53 non-regular = 9	regular = 2 non-regular = 1
Sugar-rich diet and oral sugar intake n = 172	142	2	27	1
Hereditary predisposition to caries n = 51	43	1	7	0
History of tooth decay within the last year n = 35	16	3	5	2

Table 2 Antibiotic susceptibility of the isolates

Strains	Susceptibility	Antibiotics
		PE	CLIN	ERY	DOX	CEFT	CEF	TE	VAN
S. mutans strains lacking Cbm and/or Cnm genes n = 311	S	202 (64.9%)	246 (79.1%)	253 (81.3%)	181 (58.2%)	239 (76.8%)	277 (89.1%)	85 (27.3%)	171 (54.9%)
	I	83 (26.7%)	48 (15.4%)	21 (6.75%)	113 (36.3%)	49 (15.7%)	29 (9.3%)	37 (11.9%)	129 (41.4%)
	R	26 (8.3%)	17 (5.4%)	37 (11.9%)	17 (5.4%)	23 (7.4%)	5 (1.6%)	189 (60.7%)	11 (3.5%)
Cbm + S. mutans isolates n = 8	S	2 (25%)	4 (50%)	2 (25%)	2 (25%)	4 (50%)	6 (75%)	1 (12.5%)	6 (75%)
	I	3 (37.5%)	5 (62.5%)	2 (25%)	4 (50%)	3 (37.5%)	2 (25%)	2 (25%)	2 (25%)
	R	3 (37.5%)	3 (37.5%)	4 (50%)	2 (25%)	1 (12.5%)	0	5 (62.5%)	0
Cnm + S. mutans isolates n = 62	S	23 (37.1%)	26 (41.9%)	29 (46.8%)	25 (40.3%)	28 (45.1%)	33 (53.2%)	7 (11.3%)	28 (45.1%)
	I	20 (32.2%)	22 (35.4%)	18 (29%)	19 (30.6%)	23 (37.1%)	20 (32.2%)	7 (11.3%)	19 (30.6%)
	R	19 (30.6%)	14 (22.5%)	15 (24.2%)	18 (29.1%)	11 (17.7%)	9 (14.5%)	48 (77.4%)	15 (24.2%)
Cbm/Cnm + S. mutans isolates n = 3	S	0	2 (66.6%)	2 (66.6%)	0	2 (66.6%)	2 (66.6%)	0	3 (100%)
	I	0	0	1 (33.3%)	1 (33.3%)	0	1 (33.3%)	0	0
	R	3 (100%)	1 (33.3%)	0	2 (66.6%)	1 (33.3%)	0	3 (100%)	0

Fig. 2 Collagen binding protein-encoding genes

a. Cbm gene positivity electrophoresis image; b. Cnm gene positivity electrophoresis image. Line 1. DNA ladder 1k bp; Line 2. Gene positive S. mutans strains

Table 3 Comparison of the antibiotic resistance rates of Cnm + S. mutans strains and CBP gene-negative strains

Antibiotics	Susceptibility	Cnm mutation		P
		yes (%)	no (%)
PE	S I R	35.4 30.8 33.8	63.9 27.0 9.1	<0.001
CLI	S I R	43.1 33.8 23.1	78.4 15.4 6.3	<0.001
ERY	S I R	46.2 29.2 24.6	79.9 7.2 12.9	<0.001
DOX	S I R	38.5 30.8 30.8	57.4 36.7 6.0	<0.001
CEFT	S I R	46.2 35.4 18.5	76.2 16.3 7.5	<0.001
CEF	S I R	53.8 32.3 13.8	88.7 9.1 2.2	<0.001
TE	S I R	10.8 10.8 78.5	27.0 12.2 60.8	0.004
VAN	S I R	47.7 29.2 23.1	55.5 41.1 3.4	0.001

Discussion

Streptococcus mutans is one of the most important pathogens responsible for dental caries in humans. Dental caries develops over time as a result of a complex interplay between acidogenic bacteria, fermentable carbohydrates, oral hygiene, and various host-related factors such as teeth and saliva. This is a chronic disease that progresses slowly in most people worldwide [18]. Consistent toothbrushing and proper oral hygiene practices are among the most critical factors for prevention of dental caries. Elidrissi and Naidoo reported a lower incidence of dental caries in children who practiced proper toothbrushing compared to those without a regular toothbrushing habit [19]. In present study, 312 out of 384 patients (81.25%) were found to practice regular toothbrushing and oral hygiene habits, while 72 patients (18.75%) did not maintain proper oral hygiene during orthodontic treatment. Among those with inadequate oral hygiene, 26 (36.11%) had a history of dental caries during orthodontic treatment, specifically within the last 6 months. The questionnaire asking patients about any hereditary predisposition to caries revealed that 156/384 (40.62%) had at least one family member with a history of tooth decay in previous year.

Pathogenic bacteria often produce surface proteins that can bind to various elements of the mammalian extracellular matrix, such as collagen, laminin, fibronectin, and proteoglycans. S. mutans strains which harbor genes for collagen-binding proteins are known to be a major player in severe caries due to their affinity for dental structures. The extracellular matrix (ECM) is a macromolecular structure that becomes exposed when tissue integrity is damaged bytrauma or a lesion. These interactions between collagen-binding proteins and the ECM may occur through protein-protein binding, protein-carbohydrate recognition, or via a bridging mechanism involving a bivalent soluble host protein [20]. The initial step of invasion of host cells is binding to the ECM [21]. The Cnm and Cbm proteins of S. mutans strains exhibit a strong affinity for collagen [22,23], and it is well established that the presence of the Cbm and Cnm genes enhances collagen-binding capacity, thereby promoting the colonization of strains at the onset of dental caries. Moreover, several studies have shown that caries is strongly associated with sugar intake and feeding behavior. Oral intake of sugar-containing beverages or foods is known to cause caries in healthy people of any age [24]. It has been pointed that S. mutans strains are able to to use dietary sugar to assemble an insoluble polymeric matrix that provides mechanical stability and an acidic milieu, thus promoting the survival of other potentially acidogenic/aciduric microorganisms [25]. Lingström and co-workers have confirmed the contribution of liquid and solid/retentive sugars and starch/sugar combinations to severe early childhood caries [26]. In present study questionnaire about dietary sugar and feeding behavior revealed that many orthodontics patients (44.8%) had a sugar-rich diet. The prevalence of S. mutans strains lacking Cbm and/or Cnm genes was 82.55%, and these strains were isolated from 142 patients who had a high dietary sugar intake. Among them, 1.16% harbored Cbm + strains, 15.69% harbored Cnm + strains, and one (0.58%) harbored Cbm/Cnm + strains.

The prevalence of the Cnm gene in S. mutans strains is known to be approximately 10-20%, while the prevalence of the Cbm gene is around 3% [27]. For the present study, a small effect size was chosen to reflect the potential association between genetic variation and antibiotic resistance, and to ensure clinical significance. From a clinical relevance perspective, small effect sizes emphasize the importance of this study. Accordingly, although the prevalence of the strains examined was low, a small effect size of w = 0.15 was preferred in view of clinical significance.

Many previous studies have investigated the frequency of the Cbm and Cnm genes in S. mutans strains in relation to odontogenic infections including caries. Momenni and co-workers reported that the prevalence rates of the Cnm and Cbm genes in S. mutans strains were 7% and 11%, respectively [17]. One study revealed 15% Cnm gene positivity in S. mutans strains and [28], and this was supported by other two studies [29,30]. Two studies performed between 2012 and 2014 by Nomura et al. reported Cbm gene positivity in less than 3% of the strains examined [12,31]. In the S. mutans strains isolated from the present cohort, the prevalence of Cbm was2.08% (8/384), that of Cnm was 16.14% (62/384), and that of Cbm/Cnm was 0.78% (3/384).

The overall in vitro antibiotic resistance rates of these isolates were 30.6% (19/62) for PE, 22.5% (14/62) for CLIN, 24.2% (15/62) for ERY, 29.03% (18/62) for DOX, 17.7% (11/62) for CEFT, 14.5% (9/62) for CEF, 77.4% (48/62) for TE, and 24.2% (15/62) for VAN. Three isolates those harbored both Cbm and Cnm were resistant to PE (100%), CLIN (33.3%), DOX (66.6%), CEFT (33.3%), and TE (100%). Similar results were found in a study by Kiros and co-workers (6.6% for CLIN, 20.4% for ERY, 10.2% for DOX, 11.2% for CEFT, 3.1% for CEF, and 63.7% for TE) [32]. Another study by Al-Shami and co-workers found resistance rates of 14.9% for PE, 24.1% for ERY, 8% for CLIN, and 2.3% for VAN [33]. In present study, antibiogram tests of the 311 isolates those harbored neither Cbm nor Cnm revealed resistance rates of 8.3% (26/311) for PE, 5.4% (17/311) for CLIN, 11.9% (37/311) for ERY, 5.4% (17/311) for DOX, 7.4% (23/311) for CEFT, 1.6% (5/311) CEF, 60.7% (189/311) for TE, and 3.5% (11/311) for VAN. The resistance rates of Cbm + S. mutans strains were 37.5% (3/8) for PE, 37.5% (3/8) for CLIN, 50% (4/8) for ERY, 25% (2/8) for DOX, 12.5% (1/8) for CEFT, and 62.5% (5/8) for TE.

S. mutans strains harboring the Cbm and Cnm genes possess collagen-binding proteins (Cbm and Cnm) on their cell surface, which enhance their ability to adhere to dentin. This increased adhesion capacity promotes the persistence of the bacterium on tooth surfaces and oral devices such as dental arches and brackets, thereby significantly raising the risk of dental caries. Moreover, studies have shown that strains harboring these genes tend to exhibit higher resistance to antibiotics. This not only strengthens their role in the development of dental caries but also complicates treatment strategies. In addition, lack of toothbrushing and oral hygiene directly contributes to caries in orthodontic patients. Several factors such as salivary pH and composition feeding behavior, sugar intake, trauma to teeth, lack of oral hygiene and foreign dental devices such as dental arches and brackets can facilitate the formation of caries through bacterial affinity and colonization. Such conditions may favor S. mutans strains harboring Cbm and Cnm since they are more resistant to antibiotics. Location, and demographic and sociocultural features of patient groups may have a role in determining differences in Cbm and Cnm gene frequency. On the basis of the present findings, it can be concluded that S. mutans strains harboring the Cbm, Cnm, and Cbm/Cnm genes are more resistant to the antibiotics tested than other strains, in accordance with the literature. The present data are important for raising awareness in patients receiving orthodontic treatment. Larger future studies, will be needed to reveal the other contributory factors to caries associated with these strains.

Abbreviations

CBPs: collagen binding proteins; CEF: cefoxitin; CEFT: ceftriaxone; CLIN: clindamycin; CLSI: The Clinical and Laboratory Standards Institute; DOX: doxycycline; ECM: extracellular matrix; ERY: erythromycin; EUCAST: The European Committee on Antimicrobial Susceptibility Testing; I: susceptible, increased exposure; PCR: polymerase chain reaction; PE: penicillin; R: resistant; S: susceptible; TE: tetracycline; VAN: vancomycin

Ethical Statements

This study was approved by decision number 04 of the Clinical Ethics Committee at its meeting dated 25.02.2021.

Conflicts of Interest

None of the authors have any conflicts of interest or competing financial interests to report.

Funding

This study was supported financially by the Scientific Research Projects Coordinator (number 21.GAP.029).

Author Contributions

HSB: study concept and design, data acquisition and interpretation, preparation of the manuscript, and approval of the final version of the manuscript; HY: study concept, critical review of the manuscript, and approval of the final version of the manuscript. İY: data acquisition, FD: data acquisition. All authors read and approved the final version of the manuscript.

ORCID iD

¹⁾HSB*: [e-mail], https://orcid.org/0000-0003-3400-9292

²⁾HY: [e-mail], https://orcid.org/0000-0002-4666-8247

³⁾İY: [e-mail], https://orcid.org/0009-0004-8834-8155

²⁾FD: [e-mail], https://orcid.org/0009-0009-8412-509x

Data Availability Statements

Data related to this article are available from the corresponding author upon request.

References

• 1.  Zhang M, McGrath C, Hägg U (2006) The impact of malocclusion and its treatment on quality of life: A literature review. Int J Paediatr Dent 16, 381-387.
• 2.  Johal A, Alyaqoobi I, Patel R, Cox S (2015) The impact of orthodontic treatment on quality of life and selfesteem in adult patients. Eur J Orthod 37, 233-237.
• 3.  Tornos P, Gonzalez-Alujas T, Thuny F, Habib G (2011) Infective endocarditis: the European viewpoint.’’ Curr Probl Cardiol 36, 175-222. doi: 10.1016/j.cpcardiol.2011.03.004
• 4.  Höchli D, Hersberger-Zurfluh M, Papageorgiou SN, Eliades T (2017). Interventions for orthodontically induced white spot lesions: a systematic review and meta-analysis. Eur J Orthod 39, 122-133. doi: 10.1093/ejo/cjw065
• 5.  Miura KK, Ito IY, Enoki C, Elias AM, Matsumoto MAN (2007) Anticariogenic effect of fluoride-releasing elastomers in orthodontic patients. Braz Oral Res 21, 228-233. doi: 10.1590/s1806-83242007000300007
• 6.  Boersma J, van der Veen M, Lagerweij M, Bokhout B, Prahl-Andersen B (2005) Caries prevalence measured with QLF after treatment with fixed orthodontic appliances: influencing factors. Caries Res 39, 41-47. doi: 10.1159/000081655
• 7.   Kanthathas K, Willmot DR, Benson PE (2005) Differentiation of developmental and post-orthodontic white lesions using image analysis. Eur J Orthod 27, 167-172. doi: 10.1093/ejo/cjh084
• 8.  Chen W, Zhou Y (2015) Caries outcomes after orthodontic treatment with fixed appliances: a longitudinal prospective study. Int J Clin Exp Med. 8, 2815-2822.
• 9.  Nakano K, Hokamura K, Taniguchi N, Wada K, Kudo C, Nomura R et al. (2011) The collagen-binding protein of Streptococcus mutans is involved in haemorrhagic stroke. Nat Commun 2, 485. doi: 10.1038/ncomms1491
• 10.  Aviles-Reyes A, Miller JH, Simpson-Haidaris PJ, Lemos JA, Abranches J (2014) Cnm is a major virulence factor of invasive Streptococcus mutans and part of a conserved three-gene locus. Mol Oral Microbiol 29, 11-23. doi: 10.1111/mom.12041
• 11.  Nomura R, Nakano K, Taniguchi N, Lapirattanakul J, Nemoto H, Grönroos L et al. (2009) Molecular and clinical analyses of the gene encoding the collagen-binding adhesin of Streptococcus mutans. J Med Microbiol 58, 469-475. doi: 10.1099/jmm.0.007559-0
• 12.  Nomura R, Nakano K, Naka S, Nemoto H, Masuda K, Lapirattanakul J et al. (2012) Identification and characterization of a collagen-binding protein, Cbm, in Streptococcus mutans. Mol Oral Microbiol 27, 308-323. doi: 10.1111/j.2041-1014.2012.00649.x
• 13.  Avilés-Reyes A, Miller JH, Lemos JA, Abranches J (2017) Collagen-binding proteins of Streptococcus mutans and related streptococci. Mol Oral Microbiol 32, 89-106. doi:101111/o mi.12158
• 14.  Gwinnett AJ, Ceen RF (1979) Plaque distribution on bonded brackets: a scanning microscope study. Am J Orthod. 75, 667-677. doi: 10.1016/0002-9416(79)90098-8
• 15.  Mitchell L (1992) Decalcification during orthodontic treatment with fixed appliance—an overview. Br J Orthod 19, 199-205. doi: 10.1179/bjo.19.3.199
• 16.  Ahn SJ, Lim BS, Yang HC, Chang YI (2005) Quantitative analysis of the adhesion of cariogenic streptococci to orthodontic metal brackets. Angle Orthod 75, 666-671. doi: 10.1043/0003-3219(2005)75[666:QAOTAO]2.0.CO;2
• 17.  Momeni SS, Ghazal T, Grenett H, Whiddon J, Moser SA, Childers NK (2019) Streptococcus mutans serotypes and collagen-binding proteins Cnm/Cbm in children with caries analysed by PCR. Mol Oral Microbiol 34, 64-73. doi: 10.1111/omi.12254. Epub 2019 Feb 26
• 18.  Selwitz RH, Ismail AI, Pitts NB (2007) Dental caries. Lancet 369, 51-59. doi: 10.1016/S0140-6736(07)60031-2
• 19.  Elidrissi SM, Naidoo S (2016) Prevalence of dental caries and toothbrushing habits among preschool children in Khartoum State, Sudan. Int Dent J 66, 215-220. doi: 10.1111/idj.12223
• 20.  Westerlund B, Korhonen TK (1993) Bacterial proteins binding to the mammalian extracellular matrix. Mol Microbiol 9, 687-694. doi: 10.1111/j.1365-2958.1993.tb01729.x
• 21.  Tamura GS, Kuypers JM, Smith S, Raff H, Rubens CE (1994) Adherence of group B streptococci to cultured epithelial cells: roles of environmental factors and bacterial surface components. Infect Immun 62, 2450-2458. doi: 10.1128/iai.62.6.2450-2458.1994
• 22.  Sato Y, Okamoto K, Kagami A, Yamamoto Y, Igarashi T, Kizaki H (2004) Streptococcus mutans strains harboring collagen-binding adhesin. J Dent Res 83, 534-539. doi: 10.1177/154405910408300705
• 23.  Lapirattanakul J, Nakano K, Nomura R, Leelataweewud P, Chalermsarp N, Klaophimai A et al. (2011) Multilocus sequence typing analysis of Streptococcus mutans strains with the Cnm gene encoding collagen-binding adhesin. J Med Microbiol 60, 1677-1684. doi: 10.1099/jmm.0.033415-0. Epub 2011 Jun 16.
• 24.  Grindefjord M, Dahllöf G, Nilsson B, Modéer T (1996) Stepwise prediction of dental caries in children up to 3.5 years of age. Caries Res 30, 256-266. doi: 10.1159/000262333
• 25.  Bowen WH, Burne RA, Wu H, Koo H (2018) Oral biofilms: pathogens, matrix, and polymicrobial interactions in microenvironments. Trends Microbiol 26, 229-242. doi: 10.1016/j.tim.2017.09.008. Epub 2017 Oct 30.
• 26.  Lingström P, van Houte J, Kashket S. (2000) Food starches and dental caries. Crit Rev Oral Biol Med 11, 366-380. doi: 10.1177/10454411000110030601
• 27.  Regis WFM, Mustafa Gomes Muniz F, Rocha FR, Sales EMA, Vieira PHAGP, Barros MMAF et al. (2025) Streptococcus mutans serotyping, collagen-binding genes and Candida albicans in dentin carious lesions: a molecular approach. Clin Oral Invest 29, 52. doi: 10.1007/s00784-024-06140-0
• 28.  Cornejo OE, Lefebure T, Bitar PD, Lang P, Richards VP, Eilertson K et al. (2013) Evolutionary and population genomics of the cavity causing bacteria Streptococcus mutans. Mol Biol Evol 30, 881-893. doi: 10.1093/molbev/mss278
• 29.  Nakano K, Nomura R, Taniguchi N, Lapirattanakul J, Kojima A, Naka S et al. (2010) Molecular characterization of Streptococcus mutans strains containing the Cnm gene encoding a collagen-binding adhesin. Arch Oral Biol 55, 34-39. doi: 10.1016/j.archoralbio.2009.11.008
• 30.  Abranches J, Miller JH, Martinez AR, Simpson-Haidaris PJ, Burne RA, Lemos JA (2011) The collagen binding protein Cnm is required for Streptococcus mutans adherence to and intracellular invasion of human coronary artery endothelial cells. Infect Immun 79, 2277-2284. doi: 10.1128/IAI.00767-10
• 31.  Nomura R, Otsugu M, Naka S, Teramoto N, Kojima A, Muranaka Y et al. (2014) Contribution of the interaction of Streptococcus mutans serotype k strains with fibrinogen to the pathogenicity of infective endocarditis Infect Immun 82, 5223-5234. doi: 10.1128/IAI.02164-14
• 32.  Kiros A, Saravanan M, Niguse S, Gebregziabher D, Kahsay G, Dhandapani R et al. (2022) Bacterial profile, antimicrobial susceptibility pattern, and associated factors among dental caries suspected patients attending the ayder comprehensive specialized hospital and private dental clinic in Mekelle, northern Ethiopia. Biomed Res Int 17, 2022:3463472. doi: 10.1155/2022/3463472
• 33.  Al-Shami IZ, Al-Hamzi MA, Al-Shamahy HA, Abdul Majeed ALA (2019) Efficacy of some antibiotics against Streptococcus mutans associated with tooth decay in children and their mothers. On J Dent & Oral Health 2. doi: 10.33552/ojdoh.2019.02.000530