2.1

Sampling design

The sample size calculation was based on the estimated proportion of 78 % that the children and mothers shared similar C . albicans strains in our pilot study and on the proportion of 14–61 % in the general population reported in the previous studies [ , , ]. The mean of the reported proportions was employed as the null proportion (38 %) and used in a one-sided alternative z-test at alpha = 0.05. The z-test was performed by using analysis software “R” (version 4.0.2). The sample size required to achieve 90 % power was 13 child-mother pairs.

2.2

Subjects

A total of 80 mother-child pairs who visited the Department of Pediatric Dentistry, Nihon University School of Dentistry at Matsudo, Japan between June 2021 and April 2023 were enrolled. Healthy children aged 3–10 years were eligible. Children and their mothers who had been treated with antibiotics or antifungal agents within the past 3 months were excluded. The participants were classified into the Caries group (i.e., children with decayed, missing, and filled primary teeth [dmft] ≥ 1 and their mothers), and the caries-free (CF) group (i.e., children with dmft = 0 and their mothers).

This study was approved by the ethics committee of Nihon University School of Dentistry at Matsudo (EC20-28) and was conducted according to the tenets of the Declaration of Helsinki. Written informed consent was obtained from the mothers before study participation.

2.3

Questionnaire survey and DMFT records

The children’s background (age, sex, tooth brushing times/day, antibiotic use >3 months, low birth weight), what to eat for snacking, frequency of snacking and daily juice consumption were investigated using a questionnaire. Caries status was assessed as the number of decayed teeth, missing teeth due to caries, and filled teeth (dmft/DMFT) for the deciduous and permanent dentitions of children and their mothers.

2.4

Dental plaque collection

Dental plaque samples were collected from all tooth surfaces using a sterile spoon excavator and sterile probe and suspended in sterile tubes containing 1 ml 0.89 % physiological saline. Children in the Caries group had dental plaque samples containing carious lesions. The samples were vortexed and sonicated (sonication at 20 W for 10 s and resting on ice for 30 s, repeated three times) before plating and then prepared in saline to 10 ¹ –10 ⁻⁵ and plated within 2 h, based on the method described by Xiao et al. [ ]. The plaque samples were prepared at 10-fold condensation (10 ¹ ) because, in our preliminary experiments, C . albicans was sometimes below the detection limit when it was not condensed.

2.5

Isolation of C . albicans and S . mutans

Dental plaque samples prepared to 10 ¹ –10 ⁻¹ were plated at 50 μl each on CHROMagar™ Candida medium (Kanto-kagaku, Japan) and incubated at 37 °C for 48 h to isolate C . albicans . Green colonies on CHROMagar™ Candida medium were used as candidates for C . albicans based on the report by Odds et al. [ ]. Meanwhile, for the isolation of S . mutans , dental plaque samples prepared to 10 ⁻² –10 ⁻⁵ were plated at 50 μl each on Mitis Salivarius with Bacitracin selective medium (MSB medium) and incubated at 37 °C for 48 h to isolate S . mutans . Rough-type colonies grown on MSB medium were designated as S . mutans .

2.6

Identification and genotyping of C . albicans

Primers capable of identifying three species of Candida whose colors were difficult to distinguish in the culture medium, namely, C . albicans (light green), Candida dubliniensis (dark green) [ ], and Candida tropicalis (dark blue), were created according to the method by Fukatsu et al. [ ] ( Table 1 ). C . albicans SC5314, C . dubliniensis IFM54605, and C . tropicalis JCM1541 strains were used as controls for polymerase chain reaction (PCR). The conditions for PCR identification of C . albicans were as follows: initial denaturation at 98 °C for 2 min and additional 30 cycles consisting of 10 min at 98 °C and 1 min at 68 °C for chain elongation. All PCR products were analyzed by electrophoresis on 2.0 % agarose gels run at 100V for 40 min. We used 100 bp DNA Ladder Dye Plus (TAKARA BIO, Japan) as a molecular weight marker. The amplified fragment lengths were used to identify C . albicans ( Fig. 1 ). Genotyping was performed using the primers described by McCullough et al. [ ], and the PCR conditions were the same as those described above ( Table S1 , Fig. S1 ).

PCR amplification product of Multiplex PCR.

The PCR product size is approximately 624 bp, and bands 1–8 correspond to C . albicans . Molecular size markers are shown in lanes marked as M, and the corresponding sizes are shown on the right.

2.7

Creation of a dendrogram and evaluation of genetic relatedness of C . albicans

DNA from 118 C . albicans (types A-D) isolates from the mother and child were extracted using the gram-positive bacteria protocol of the DNeasy Blood & Tissue Kit (QIAGEN, Netherlands). PCR was performed using 5‘-CCGGCGGCG-3’ primers. Briefly, we prepared 10 μL PCR solution consisting the following: 3 μL Premix Taq (TaKaRa Taq μVersion 2.0), 5 ng DNA, and 5 μM primers, scaled to 10 μL with PCR-grade water. PCR was performed as previously reported [ ]. PCR products were confirmed using the capillary electrophoresis system MCE-202 MultiNA (Shimadzu, Kyoto, Japan). Genotypes were scored as 0 or 1 according to the absence or presence of bands, respectively. Phylogenetic trees were constructed using the statistical analysis software “R” (version 3.4.3) based on the unweighted pair group method with arithmetic mean (UPGMA method). The dendrogram was divided into five clusters using 0.2 (i.e., the divergence between C . albicans and C . dubliniensis ) as a guide, and a table was created ( Fig. S2 ). In our criteria, strains of Candida species classified in the same cluster to be genetically “similar”.

2.8

Statistical analysis

Statistical analysis was performed by using analysis software “R” (version 4.0.2). P values were calculated using Welch’s t -test. The correlation between S . mutans and C . albicans in children in the Caries group was also analyzed. To narrow down the dietary factors that could influence caries severity (dmft), a multiple regression analysis was performed using dmft scores as the objective variable, and sugar-containing sweets (chocolate, gummy candy, drop/lollipop, and cookie), snacking frequency, and daily juice consumption as explanatory variables. A logistic regression analysis was further conducted to evaluate the impact of sugar-containing diet on the carriage of oral C . albicans in children. Statistical significance was set at p < 0.05.