Archives of Oral Biology 67 (2016) 22–27

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Archives of Oral Biology

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Relationship between the IgA antibody response against Streptococcus

mutans GbpB and severity of dental caries in childhood
Natália Helena Colomboa , Jesse Augusto Pereiraa , Márjully Eduardo Rodrigues da Silvaa ,
Laís Fernanda Fonseca Ribasa , Thaís Manzano Parisottob ,
Renata de Oliveira Mattos-Granerc, Daniel J. Smithd, Cristiane Duquea,*
a
UNESP – Univ Estadual Paulista, Araçatuba Dental School, Department of Pediatric Dentistry and Public Health, Araçatuba, SP, Brazil
b
Sao Francisco University, Dental School, Laboratory of Microbiology and Molecular Biology, Bragança Paulista, SP, Brazil
c
UNICAMP – University of Campinas, Piracicaba Dental School, Department of Oral Diagnosis, Piracicaba, SP, Brazil
d
Forsyth Institute, Department of Immunology and Infectious Disease, Cambridge, MA, USA

A R T I C L E I N F O A B S T R A C T

Article history: Objective: Explore the associations between the severity of dental caries in childhood, mutans
Received 8 January 2016 streptococci (MS) levels and IgA antibody response against Streptococcus mutans GbpB. Moreover, other
Accepted 15 March 2016 caries-related etiological factors were also investigated.
Design: 36–60 month-old children were grouped into Caries-Free (CF, n = 19), Early Childhood Caries (ECC,
Keywords: n = 17) and Severe Early Childhood Caries (S-ECC, n = 21). Data from socio-economic-cultural status, oral
Early childhood caries hygiene habits and dietary patterns were obtained from a questionnaire and a food-frequency diary filled
Imunoglobulin A
out by parents. Saliva was collected from children for microbiological analysis and detection of salivary
S. mutans
Glucan binding proteins
IgA antibody reactive with S. mutans GbpB in western blot.
Results: S-ECC children had reduced family income compared to those with ECC and CF. There was
difference between CF and caries groups (ECC and S-ECC) in MS counts. Positive correlations between
salivary IgA antibody response against GbpB and MS counts were found when the entire population was
evaluated. When children with high MS counts were compared, S-ECC group showed significantly lower
IgA antibody levels to GbpB compared to CF group. This finding was not observed for the ECC group.
Conclusions: This study suggests that children with S-ECC have reduced salivary IgA immune responses to
S. mutans GbpB, potentially compromising their ability to modify MS infection and its cariogenic
potential. Furthermore, a reduced family income and high levels of MS were also associated with S-ECC.
ã 2016 Elsevier Ltd. All rights reserved.

1. Introduction three years of age, any sign of smooth-surface caries is indicative of

severe early childhood caries (S-ECC). From ages three through five,
Dental caries is an infectious disease that results from the one or more cavitated, missing (due to caries) or filled smooth
dissolution of tooth mineral by acids derived from bacterial surfaces in primary maxillary anterior teeth or a decayed, missing
fermentation of sucrose and other dietary carbohydrates (Loesche, or filled score of greater than or equal to four (age 3), five (age 4) or
1996). When occurring in young children, this disease is called six (age 5) surfaces also constitutes severe S-ECC. The disease can
early childhood caries (ECC). According to the American Academy progress and lead to destruction of the primary dentition affecting
of Pediatric Dentistry (2014) ECC is characterized by the presence negatively children’s physical and mental health, as well as
of one or more decayed (noncavitated or cavitated lesions), missing increasing the risk of new caries lesions in the permanent dentition
(due to caries), or filled tooth surfaces in any primary tooth of (Ng & Chase, 2013; Isaksson, Alm, Koch, Birkhed, & Wendt, 2013).
children under the age of six. However, in children younger than The mutans streptococci (MS) group, mainly Streptococcus
mutans, is most strongly associated with the pathogenic process of
ECC because of its high presence in the biofilms and saliva of the
affected children (Ge, Caufield, Fisch, & Li, 2008; Parisotto, Steiner-
* Correspondence at: UNESP – Univ Estadual Paulista, Araçatuba Dental School,
Department of Pediatric Dentistry and Public Health, R. José Bonifácio, 1193, CEP: Oliveira, Silva, Rodrigues, & Nobre-dos-Santos, 2010b). Three
16015-050, Araçatuba, SP, Brazil. important cell-associated antigens (Ags) are correlated directly
E-mail addresses: cristianeduque@yahoo.com.br, cduque@foa.unesp.br with the ability of S. mutans to adhere and accumulate in the tooth
(C. Duque).

http://dx.doi.org/10.1016/j.archoralbio.2016.03.006
0003-9969/ ã 2016 Elsevier Ltd. All rights reserved.
 N.H. Colombo et al. / Archives of Oral Biology 67 (2016) 22–27 23

surfaces forming dental biofilm: antigen I/II (AgI/II); glucosyl- 0.75) using mouth mirrors and probes under natural light.
transferases (GtfB, GtfC, GtfD) and glucan-binding proteins (GbpA, Decayed, missing and filled surfaces in deciduous teeth (dmfs)
GbpB, GbpC, GbpD) (Smith, 2002). Ag I/II promotes interaction of were counted in each child. The exclusion criteria for this study
bacterial proteins and host-derived components; Gtf catalyzes were: children suffering from systemic disease or using long-term
glucan synthesis and Gbps increase the binding of S. mutans to each medications or antibiotics less than one month before the
other and to glucans deposited on tooth surfaces, contributing to examination and children with mucosal lesions. Children with
the sucrose-dependent adherence to teeth (Smith, 2002). Several only restored or missing teeth were also excluded. Preschoolers
studies have demonstrated that induction of specific antibodies who had white chalky spot lesions were excluded from caries free
against these antigens, mainly GtfB/C and GbpB, can prevent the group. All children were encouraged and instructed on dental
development of dental caries in animal models (Jespersgaard et al., hygiene and received all other necessary oral care. The inclusion
1999; Koga, Oho, Shimazaki, & Nakano, 2002; Smith & Taubman, criteria of this study comprise oral/systemic health children from
1996) and modify infection in humans (Smith & Taubman, 1987). both genders, 36–60 months of age. Initially sixty children were
Salivary immunoglobulins, particularly secretory IgA have selected. Three of them were absent from schools when saliva was
major importance in the resistance of the mucosa to oral collected. So, fifty-seven children were divided into three groups
infections. The secretory IgA immune response represents the according to oral health status: caries-free group (CF) (n = 19), early
first line of adaptive immune defense against mutans streptococci, childhood caries group (ECC) (n = 17) and severe early childhood
blocking microbial adhesins and potentially reducing oral coloni- caries group (S-ECC) (n = 21). ECC was defined for this study as the
zation with this cariogenic microflora. Salivary IgA has also been presence of 1 through 3 decayed tooth surface (cavitated lesions) in
shown to enhance the activity of several enzymes such as primary teeth, S-ECC was defined as the presence of decayed
lactoferrins and lysozymes (Law, Seow, & Townsend, 2007). A surfaces score of 4 (age 3 years), 5 (age 4 years), with at least
clinical study showed that the IgA antibody response against S. one smooth-surface carious lesion (American Academy of Pediatric
mutans GbpB was predominant in the first year of life and Dentistry, 2014).
frequently correlated with the delay in the oral infection with S.
mutans (Nogueira, Alves, Napimoga, Smith, & Mattos-Graner, 2.2. Saliva samples
2005). The intensity of IgA patterns against antigens of S. mutans
was previously studied and lower levels of salivary IgA against About 2 ml of unstimulated saliva was collected from children
GbpB were associated with higher caries risk (Parisotto et al., at afternoon and at least 1 h after feeding. Tubes were transported
2011). However, the relationship between IgA antibody levels on ice to the laboratory and processed within 1 h. After agitation,
against this antigen and different levels of caries has not yet been one hundred microliters of saliva were separated for microbiolog-
evaluated. The objective of this study was to explore the ical procedures. The remaining saliva was clarified by centrifuga-
associations among the severity of dental caries in childhood, tion at 10000 rpm at 4  C for 10 min. The supernatants were
mutans streptococci levels and IgA response against S. mutans collected and 250 mM EDTA was added to minimize salivary IgA
GbpB. Moreover, caries-related etiological factors were also aggregation. Aliquots of 50 ml of each saliva samples were frozen at
investigated. The null hypotheses formulated was that immune 70  C until immunological analysis (Nogueira et al., 2005).
response, MS levels, socio-economic-cultural aspects, hygiene and
dietary habits presented by ECC and S-ECC are not different from 2.3. Microbiological procedures
that exhibited by CF children.
Aliquots of saliva were homogenized by vortexing for 1 min and
2. Methods the suspensions were serially diluted in 0.9% NaCl solution. Each
dilution was cultivated in triplicate on Mitis Salivaris Agar (Difco
2.1. Subjects Laboratories, Detroit, MI, USA) with 0.2 U/mL bacitracin for
isolation of mutans streptococci (MS). All plates were incubated
The study population comprised 36- to 60-month-old children at 37  C for 48 h in a 5% CO2 atmosphere. After 48 h, the total
who attended the four public nursery schools in the city of number of colony-forming units (CFU) was counted using a
Araçatuba, São Paulo, Brazil. The city’s population has access to stereoscopic microscope. Results were expressed as CFU/ml.
public water supply with fluoride level of 0.7 ppm. Children’s
parents as well as administrators at the preschools involved 2.4. Total salivary IgA level measurement
granted written permission for the study which was previously
approved by the Research Ethics Committee of Araçatuba Dental The concentration of total IgA in saliva samples were
School—Univ. Estadual Paulista (UNESP), Brazil (CAAE # determined by an enzyme-linked immunosorbent assay (ELISA)
13079213.4.0000.5420). kit using a commercially available analysis kit (Mabtech Inc.,
Data from socio-economic-cultural status, including family Cincinnati OH, USA) and following the manufacturer’s instructions.
income and mother’s education level and from dietary habits were
obtained from a questionnaire and a food-frequency diary filled 2.5. Western blot analysis of salivary antibody to mutans GbpB
out by parents. The diet reflected food consumption of three
consecutive days during the workweek, because in the weekend In order to analyze the influence of patterns of specificity of IgA
the diet can be highly variable. In addition to the diet data reported response to S. mutans GbpB, levels of MS infection and caries status,
by parents, the preschool staff provided information about infant Western blot assays were performed using saliva samples from
feeding in the school period, as many of the children were in school children and tested against Ags extracted from a standard S. mutans
full time. This diet chart included the time of day that the children strain (ATCC 25175). For Ags preparation, colonies of S. mutans
ate and drank anything and the content of all meals and snacks. The from fresh Brain Heart Infusion Agar (BHI, Difco) were inoculated
daily frequencies of total sugar exposure and consumption from in 5 ml BHI broth and incubated for 18 h. Bacterial cells were then
baby bottles was calculated from chart data (Parisotto et al., harvested from 1 ml of cultures previously adjusted to an
2010a). absorbance of 1.0 (A550 nm). Cells were ressuspended in TE
Clinical examinations were performed at the school by a single containing 100-mm-diameter zirconia/silica beads and mechani-
calibrated examiner (NHC) previously training (Kappa value of cally disrupted using a Mini-BeadBeater (BioSpec) at maximum
24 N.H. Colombo et al. / Archives of Oral Biology 67 (2016) 22–27

speed (2800 rpm) for 1-min pulses, three times, with a 30-s rest on the sample showed zero CFUs. Pearson correlation tests were
ice between pulses. Cell pellets were then boiled in Laemmli buffer conducted to compare IgA levels against S. mutans GbpB and
for 5 min, and protein extracts were separated by centrifugation at bacterial counts for the entire population. The reactivities of
4  C (10000 rpm for 4 min). Protein concentrations were deter- salivary IgA antibody with S. mutans GbpB were compared with
mined by the method of Bradford and a total of 16 mg of protein respect to caries severity (CF, ECC and S-ECC) and mutans
extract was used for Western blot analysis (Nogueira et al., 2005). streptococci levels (low mutans streptococci LMS and high
Ags extracts were loaded per lane, separated by sodium dodecyl mutans streptococci—HMS) using Mann-Whitney tests.
sulfate–6% polyacrylamide gel electrophoresis, and transferred to
nitrocellulose membranes. After transference, membranes were 3. Results
washed and blocked overnight at 4  C (in Tris-buffered saline–
Tween, pH 7.5, 5% nonfat milk). Incubations with saliva samples Means/standard deviations obtained for caries levels (dmfs)
diluted 1:100 were performed at room temperature for 2 h. As were: CF (0), ECC (2  1.06) and S-ECC (23.43  17.17). Considering
negative controls, membranes were incubated only with blocking white spots lesions, dmfs were: CF (0), ECC (2.94  1.67) and S-ECC
buffer, and as positive controls, membranes were incubated with a (28.28  18.76). There were no statistical differences among the
standard saliva sample obtained from an adult subject whose groups in relation to age, gender, mother’s education level, diet
pattern of reaction with S. mutans antigen extracts had been habits (artificial feeding and sugar intake) and total IgA levels
previously measured. The secondary antibody was HRP-Goat Anti- (Table 1). Families of S-ECC children had reduced income
Human IgA (1:4000 dilution) (Invitrogen, Life Technologies, USA). compared to families of ECC and CF children showing a
Immunoreactive bands were detected by autoradiography using relationship between high scores of caries and economic condition
ECL chemiluminescent substrate reagent kit (Invitrogen, Life of family. Statistical differences between CF  ECC and CF  S-ECC
Technologies, USA) according to the manufacturer’s instructions. children were observed for mutans streptococci (MS) counts. S-ECC
X-ray films were scanned in a transluminator using a White Light children were heavily colonized by MS. There was a gradual
Converter Plate (UVP, LLC, Upland, CA, USA) and the patterns of
antigen recognition, including the number and intensity of reactive
bands were analyzed with UVP Image software. The molecular
weight of S. mutans GbpB was about 60 kDa. Migration position of
GbpB were determined in parallel western blot assays performed
with specific polyclonal rat antiserum to GbpB (Smith & Taubman,
1996).

2.6. Statistical analysis

The statistical analysis was performed considering caries

status: CF, ECC and S-ECC as the dependent variables. The
comparisons among the groups were performed according to
data distribution, considering Kolmogorov-Smirnov Z tests.
ANOVA and Tukey tests were applied for age and sugar intake.
Kruskal-Wallis and Mann-Whitney tests were applied for gender
comparison, family income, mother’s education level, artificial
(bottle) feeding, mutans streptococci/lactobacilli counts and total Fig. 1. Scatter plot showing correlation between levels of IgA antibody reactive with
GbpB and mutans streptococci (MS). The analysis was carried out for the total
IgA levels. Medians and ranges of bacterial counts were expressed
population, regardless the caries status. Positive Pearson correlation (R = 0.583,
as log (CFU +1) and the constant 1 was added to CFU counts, when p = 0.00) was observed for MS counts.

Table 1
Comparative analysis between the severity of early childhood caries and related etiological factors.

CF ECC S-ECC p value

Dmfs (mean  SD) 0a 2  1.06b 23.43  17.17c 0.00
Age (months) Mean  SD 46.26  5.05a 45.94  9.83a 48.10  8.59a 0.664
Gender (%) Female 47.37A 35.29A 52.38A 0.571
Male 52.63 64.71 47.62

Family income per month R$ 1448.00* 41.1A 25B 64.71C 0.05
Mother’s education (%) Up to 8 years 37.50A 35.71A 47.62A 0.737
Mother’s help with tooth brushing (%) 82.35A 80.0A 65.0A 0.961
Bottle feeding (%) 82.36A 66.67A 85A 0.692
Sugar intake liquid 4.21  1.37a 4.02  1.18a 4.56  1.38a 0.465
(mean  SD) solid 2.70  0.90a 3.17  1.43a 2.97  1.40a 0.565
total 6.88  1.62a 7.19  1.66a 7.53  2.32a 0.579

mutans streptococci (log UFC + 1) 3.35 (0–7.29)A 3.74 (0–6.66)A 5.63 (3.15–8.3)B 0.012
median (range)
A A A
total lactobacilli (log UFC + 1) 0 (0–6.79) 3.30 (0–6.47) 1.97 (0–6.72) 0.447
median(range)
A A A
Total IgA (ng/ml) Median (range) 99.15(24.52–114.24) 83.24 (32.27–114.56) 88.04 (18.17–109.31) 0.125
a,b,c
Different lower case letters show statistical difference among the groups, according to ANOVA and Tukey tests.
A,B,C
Different upper case letters show statistical difference among the groups, according to Kruskal-Wallis and Mann-Whitney tests.
CF—caries free, ECC—early childhood caries, S-ECC—severe early childhood caries.
*
R$—Brazilian real. 1 US$  R$ 3.50 (2015, August).
Bold represents values of p < 0.05.
 N.H. Colombo et al. / Archives of Oral Biology 67 (2016) 22–27 25

Table 2
Medians (range) of total salivary IgA, distributed according to mutans streptococci
levels.

MS levelsa,b

LMS HMS p value

CF 101.9 (24.52–114.24) 91.05 (60.04–107.43) 0.375
ECC 81.93 (32.27–107.43) 84.55 (48.02–114.56) 0.341
S-ECC 98.92 (18.17–104.14) 84.10 (26.01–109.31) 0.357
p value 0.380 0.243
a
There was no statistical difference among groups of children (columns),
considering each MS levels separately (LMS or HMS), using Kruskal-Wallis tests.
b
There was no statistical difference inside each group of children (rows),
comparing MS levels (LMS x HMS), using Mann-Whitney tests.

Fig. 2. Box plots of the western blot reactivity of salivary IgA antibody with S.
increase in the MS count with respect to the severity of the disease mutans GbpB among the groups of children, distributed according to caries status
(CF, ECC and S-ECC) and mutans streptococci levels (LMS and HMS). Bars indicate
(Table 1). Considering the total population, positive correlations
minimum and maximum values. Black and white boxes indicate lower and upper
between salivary IgA levels to GbpB and MS counts were found quartiles, respectively. Line in the middle of boxes is median.
(Fig. 1). For these reasons, children were paired according to levels A—Different upper case letters show statistical difference among groups of children
of MS within each group. Considering the mean of MS counts of the considering each MS level separately (LMS or HMS), according to Mann-Whitney
population, the groups of children (CF, ECC and S-ECC) were tests. For example: GbpB CF (LMS) x GbpB ECC (LMS).
a—Different lower case letters show statistical difference inside each group of
subdivided as follows: LMS low mutans streptococci count (with children, comparing MS level (LMS x HMS), according to Mann-Whitney tests. For
MS counts log 4 CFU/ml) and HMS high mutans streptococci example: GbpB CF (LMS) x GbpB CF (HMS).
count (with MS counts log 5 CFU/ml). Comparing LMS and HMS, CF—caries free, ECC—early childhood caries, S-ECC—severe early childhood caries.
there was no significant difference among the groups in relation to LMS- Low mutans streptococcus levels.
HMS High mutans streptococcus levels.
total IgA levels (Table 2). The levels of IgA antibody reactive with
GbpB were lower in S-ECC compared to ECC and CF children, only
for HMS (Fig. 2). IgA antibody levels to GbpB increased for all
groups, when compared with the same groups of children (CF, ECC et al., 2000; Ohlund et al., 2007). Our results showed that S-ECC
and S-ECC) with LMS and HMS counts (Fig. 2). However, S-ECC children were highly colonized by mutans streptococci compared
children with high mutans streptococci counts showed signifi- with CF and ECC children. This information confirmed data from
cantly lower IgA antibody levels to GbpB compared to the CF group literature which have demonstrated that children with high S.
with HMS. No statistically significant differences were observed mutans counts have more risk to develop dental caries (Ge et al.,
comparing ECC with the other groups with HMS (Fig. 2). 2008; Milgrom et al., 2000; Ohlund et al., 2007). A recent
systematic review and meta-analysis showed that there is scientific
4. Discussion evidence of S. mutans transmission from mother to child especially
when the mother is the primary caregiver (da Silva Bastos et al.,
Early childhood caries has a complex etiology with biological, 2015), although 18% of bacterial strains were from an unknown
behavioral, and socioeconomic influences (Arora, Schwarz, & source (Kozai et al., 1999). The American Academy of Pediatric
Blinkhorn, 2011; Ng & Chase, 2013). For this reason, in this study, Dentistry (2014) recommended the reduction of mutans strepto-
some factors determining oral health were concomitantly ana- cocci levels of mothers, primary caregivers and sibling(s),
lyzed. S-ECC children had a reduced family income compared to CF educating them on avoiding saliva-sharing behaviors (e.g., sharing
and ECC groups, however, no difference among the groups were spoons, cups and other utensils, cleaning a dropped pacifier or toy
found in relation to mother's education level. High prevalence of with their mouth), to reduce the child’s risk of ECC.
dental caries in children with adverse socio-economic conditions The literature has shown that a better immune response to oral
was previously confirmed by another studies (Oliveira, Sheiham, & microorganisms, mainly S. mutans, may be a protective factor
Bönecker, 2008; Parisotto et al., 2010a). However, discrepancy in against the development of dental caries (Davidopoulou, Diza,
relation to level of mother’s education was found in the literature, Menexes, & Kalfas, 2012; Nogueira et al., 2005; Tao et al., 2005).
with children whose mothers had less than 8 years of education This observation may be valid for both innate and adaptive
with high caries levels (Oliveira et al., 2008) and absence of immune responses. Reduced levels of some antimicrobial peptides,
difference between caries-free and caries group (Parisotto et al., components of innate immune response, are associated with caries
2010a), in agreement with our study. This lack of difference in childhood (Davidopoulou et al., 2012; Tao et al., 2005). The
probably occurred because children were selected from the schools action of salivary IgA against specific surface proteins of cariogenic
with similar social characteristics. A strong association between bacteria such as S. mutans has been the subject of many studies
high frequency of sugar exposure and occurrence of dental caries (Nogueira et al., 2007, 2005; Parisotto et al., 2011), with focus on
has been established yet (Kalsbeek & Verrips, 1994; Milgrom et al., GbpB and Gtf expression. These proteins may be targets in the
2000; Parisotto et al., 2010a). However, the present study did not development of vaccines against dental caries (Kim et al., 2011;
find a significant difference in sugar exposure among the groups, Smith & Taubman, 1996). The immunization with GbpB induced an
similar to found in another study (Ohlund et al., 2007) which it was immune response that interfered with the accumulation of S.
not correlated caries status with sugar intake frequency. One mutans and reduced the levels of dental caries in rats (Smith &
possible explanation for this finding is that the responses from Taubman, 1996). The caries protection resulting from immuniza-
questionnaires may have reflected present, not historical experi- tion of rats with Gtf was lower than observed after immunization
ence, which would have better revealed habits during the period with S. mutans GbpB. The same study showed that saliva of sham-
when caries had started. immunized/S. mutans infected rats contained antibody to GbpB in
Several previous studies found mutans streptococcus is a saliva at the end of the experiment, indicating that infection with S.
significant factor for the presence of ECC (Ge et al., 2008; Milgrom mutans can induce an immune response to this antigen.
26 N.H. Colombo et al. / Archives of Oral Biology 67 (2016) 22–27

Preschoolers with a lower baseline level of salivary IgA antibody Acknowledgements

reactive with GbpB had 7.5 higher risk to develop caries, but any
differences between CF and caries group (ECC mixed with S-ECC) in This study was supported by grants (2012/19235-5; 2013/
relation to salivary IgA against S. mutans antigens (GbpB, Gtf) was 12167-7) from São Paulo Research Foundation (FAPESP), São Paulo,
found (Parisotto et al., 2011). The results of the current study are SP, Brazil and Coordination for the Improvement of Higher
partially in accordance with this mentioned study. We did not find Education Personnel (CAPES).
a difference in IgA antibody levels against GbpB between CF and
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prevalence country. Caries Research, 41, 26–33.
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Conflict of interest dental caries with social factors and nutritional status in Brazilian preschool
children. European Journal of Oral Sciences, 116, 37–43.
The authors declare that they have no conflict of interest.
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