The oral microbiome plays an essential role in both oral and systemic health (Jia et al., 2018; Nardi et al., 2021). In the first two years of life, the oral microbiome undergoes significant changes as infants progress from a predominantly milk-based diet to the introduction of solid foods (Oba et al., 2020). This window of time is characterized by rapid microbial succession and is influenced by various factors. Current evidence suggests that, the infant oral microbiome is predominantly composed of Streptococcus (Drell et al., 2017; Verma et al., 2018) with Gemella, Rothia, Haemophilus, and Lactobacillus also prevalent (Blum et al., 2022). However, the temporal development of the oral microbiome during early life and the influence of various maternal, infant, and environmental covariates remains underexplored. Previous studies have highlighted that pacifier use (Plonka et al., 2012), intrapartum antibiotic prophylaxis (Gomez-Arango et al., 2017; Li et al., 2019), and mode of delivery (Li et al., 2005, 2018; Holgerson et al., 2011; Nelun Barfod et al., 2011; Thakur et al., 2012; Dominguez-Bello et al., 2016; Hurley et al., 2019) are associated with the composition and diversity of the infant oral microbiome. However, comprehensive longitudinal data capturing these influences over the early years of life are limited.

Solid food introduction also represents a crucial milestone during infancy, influencing microbial colonization and diversity in the oral cavity with demonstrated shifts in oral microbiome composition (Sulyanto et al., 2019; Oba et al., 2020). For instance, one study of nine infants reported an increase in bacterial diversity and richness and decreased relative abundance of Streptococcus mitis with food introduction (Sulyanto et al., 2019). Another study of 12 infants found a rise in the relative abundance of Gemella, Neisseria, Veillonella, and Fusobacterium following solid food introduction (Oba et al., 2020). Unfortunately, both studies have limitations related to small sample sizes and infrequent sampling. The small sample sizes of these studies were further complicated by the inclusion of a mix of breastfed, mixed fed, and formula fed infants. Given the significant impact of breastfeeding on the oral microbiome (Sulyanto et al., 2019; Oba et al., 2020), different milk-feeding styles should not be grouped for such an analysis.

Cessation of breastfeeding may represent another inflection point in infant oral microbiome development; however, no studies have investigated this. Nevertheless, infants partially breastfed for at least 12 months have been shown to harbor different oral microbiota compared to those who ceased breastfeeding by 12 months (n=90) (Dzidic et al., 2018). These differences persisted up to 7 years of age, with the group that continued breastfeeding showing higher relative abundance of Streptococcus at 12 months and Veillonella at 7 years of age. However, distinguishing the effects of breastfeeding cessation from those of formula supplementation remains challenging.

The aim of this study was to comprehensively characterize the temporal development of the oral microbiome in exclusively and predominantly breastfed infants over the first two years of life. In addition, we aimed to identify maternal, infant, and environmental determinants of early oral microbiome development.

To investigate the temporal development of the oral microbiome during early life, we performed full-length 16S rRNA gene sequencing on 667 oral samples collected from 84 infants in the BLOSOM cohort. Maternal and infant characteristics are detailed in Table 1 . All infants were born at term without major health complications and were exclusively or predominantly breastfed prior to the introduction of solid foods. Samples were collected at ten time points across the first two years of life ( Figure 1 ).

Our longitudinal analysis of the infant oral microbiome in predominantly breastfed infants showed dynamic shifts in microbial diversity and abundance over the first two years of life. The oral microbiome was predominantly composed of taxa such as S. mitis, G. haemolysans, and R. mucilaginosa, aligning with previous studies (Cephas et al., 2011; Dzidic et al., 2018; Kennedy et al., 2019; Sulyanto et al., 2019; Li et al., 2023) and was impacted by various maternal, infant, and environmental factors.

The introduction of solid foods represents a pivotal milestone in infant development as this dietary shift not only provides new substrates for bacterial metabolism but also gradually reduces the intake of human milk, potentially altering the composition of the oral microbiome (Oba et al., 2020). This dietary change increases the availability of polysaccharides, favoring the growth of taxa that efficiently utilize these complex carbohydrates (Takahashi, 2015). The significant shift in nutrient sources that occurs with the introduction of solid foods is expected to impact the diversity and composition of the oral microbiome (Dzidic et al., 2018). Our study revealed significant shifts in the abundances of S. mitis, R. mucilaginosa, N. subflava, S. lugdunensis, B. longum, L. gasseri, and G. elegans after the introduction of food. Our results match those previously reported in two small studies (n=9 and n=12), both of which identified a decrease in S. mitis following the first intake of solids foods (Sulyanto et al., 2019; Oba et al., 2020). These studies also reported an increase in Shannon diversity and richness following the introduction of solid foods (Sulyanto et al., 2019), as well as an increase in Gemella, Neisseria, Veillonella, and Fusobacterium (Oba et al., 2020). Another study examining the oral microbiome of infants and their mothers found that incorporation of solid foods into the infant diet was associated with a compositional shifts in the oral microbiome, resulting in clustering patterns that more closely resemble those of their mothers rather than those of other infants (Cephas et al., 2011). These findings highlight the critical role of dietary factors in shaping the development of the oral microbial community. Given that early colonization shapes later waves of succession in microbial communities, more research attention should be given to the study of the effect of diet on the early oral microbiome.

The complete cessation of breastfeeding represents another dietary transition for the infant/child. In our study, the duration of breastfeeding was relatively long; however, infants who ceased breastfeeding by 24 months exhibited an increase in S. mitis and S. lugdunensis, while showing a reduction in N. subflava compared to those who were still breastfeeding. Dzidic et al (Dzidic et al., 2018). observed that infants breastfed for at least 12 months showed distinct oral microbiome profiles compared to those who ceased earlier, with persistent differences detected up to 7 years of age (Dzidic et al., 2018). These findings, although not directly comparable, highlight the role of human milk in shaping the infant oral microbiome beyond the first year of life. Interestingly, S. mitis abundance decreases following the introduction of solid foods, suggesting a preference for a breastfed-exclusive environment. However, by 24 months, S. mitis is slightly less abundant in breastfed infants compared to those who ceased infants. This pattern may reflect shifts in microbial community dynamics, competition with other taxa, or changes in oral environmental factors such as pH, salivary composition, and dietary substrates (Sedghi et al., 2021). While the underlying mechanisms remain unclear, these findings highlight the complex interactions between diet and the oral microbiome throughout early life and warrant further investigation.

Pacifiers are commonly employed to soothe infants and are often introduced early in life; however, emerging research suggests associations with alterations in oral microbial composition. Our study revealed a higher abundance of S. salivarius group 1 and S. parasanguinis in the oral cavities of infants who used pacifiers in the first week of life. This increase may be linked to the mechanical interaction between the pacifier, saliva, and the oral microbiome, which could create favorable conditions for biofilm formation (Brook and Gober, 1997; Comina et al., 2006). Notably, Streptococcus species preferentially form biofilms, as they account for 80% of the early colonizers during the initial adhesion phase, relying primarily on oral glycoproteins and salivary mucins (Radaic and Kapila, 2021). Additionally, the act of sucking on a pacifier can enhance saliva production and alter its flow and distribution, thereby creating an environment for bacteria that thrive in saliva-rich conditions (Comina et al., 2006). Two studies have identified a significant positive association between pacifier use and the abundance of lactobacilli and Candida spp. in the oral microbiome of breastfed infants (Ollila et al., 1997; Ersin et al., 2006). However, it is important to note that pacifier use was only recorded for the first week of life in our cohort. Whilst it is likely that use of the pacifier in these infants was continued, we do not have data to support this, thus our findings should be regarded as preliminary and should be verified in other cohorts.

Intrapartum antibiotic prophylaxis is administered for caesarean section delivery, to prevent Group B Streptococcus (GBS) infection, and for prolonged rupture of membranes (PROM). Approximately 20% of women test positive for GBS in the third trimester of pregnancy; however, not all of them consent to intrapartum antibiotic prophylaxis (Le Doare et al., 2017). Despite its widespread use, there has been limited comprehensive investigation into the immediate and long-term effects of perinatal antibiotic administration on the infant oral microbiome (Kuperman and Koren, 2016). Our findings revealed that infants born to antibiotic-exposed mothers, harbored oral microbiomes with reduced abundance of S. lugdunensis compared to infants born to non-exposed mothers. Interestingly this differs from two other studies that analyzed the microbiome at the family level showing a higher abundance of Proteobacteria in exposed infants, while unexposed displayed a dominance of Streptococcaceae, Gemellaceae, and Lactobacillales (Gomez-Arango et al., 2017). In another study, unexposed infants having elevated levels of Firmicutes and decreased levels of Actinobacteria, Bacteroidetes, and Proteobacteria compared to the exposed group (Li et al., 2019). Further large studies are required to unravel the effects of intrapartum antibiotic use in breastfed infants, with a focus on the class of antibiotic used.

Delivery mode is considered a major covariate in the establishment of oral bacteria in infants and has been explored in various studies (Li et al., 2005, 2018; Holgerson et al., 2011; Nelun Barfod et al., 2011; Thakur et al., 2012; Dominguez-Bello et al., 2016; Hurley et al., 2019); however, the differential effects of planned and intrapartum caesareans remain under explored. We observed lower Shannon diversity and richness in the oral cavities of infants delivered via planned, but not intrapartum, caesarean section. Further, whilst previous studies have reported compositional differences in the oral microbiome between vaginally born infants and those delivered by caesarean section (Li et al., 2005, 2018; Holgerson et al., 2011; Nelun Barfod et al., 2011; Thakur et al., 2012; Dominguez-Bello et al., 2016; Hurley et al., 2019), we found no such differences when conducting full-length sequencing of the 16S rRNA gene.

Interestingly, our findings suggest that the type of caesarean delivery is important, with differences seen in infants delivered via planned, but not intrapartum, caesarean section. Although this distinction has not been investigated in the context of the infant oral microbiome previously, our findings do match up with observations in the infant gut microbiome, where infants born via intrapartum caesarean more closely resemble those born vaginally (Penders et al., 2006; Stokholm et al., 2016; Liu et al., 2023; Weng et al., 2023; Yu et al., 2024). These findings underscore the necessity of analyzing delivery mode as a three-class variable for infant microbiome studies.

Maternal BMI has been identified as a potential determinant of the infant oral microbiome. In our study, we found significantly higher relative abundances of N. subflava and Prevotella sp. in infants born to mother with underweight/normal-weight compared to those born to mothers with overweight or obesity. While the reason for this association is unknown, previous studies have found that the human milk microbiome differs based on maternal BMI (Lundgren et al., 2019; LeMay-Nedjelski et al., 2020; Cortés-Macías et al., 2021). Additionally, the infant oral microbiome is likely influenced by bioactive components, such as immune proteins, in milk, which may also be impacted by maternal BMI (Cortés-Macías et al., 2021). More in depth studies of multiple milk components may aid in interpreting the impact of maternal body composition on infant microbiome colonization.

Our investigation also explored, for the first time, the potential influence of household pets on the infant oral microbiome. Dogs and cats, known to harbor diverse microbial communities, have been associated with changes in the human gut microbiome (Azad et al., 2013; Tun et al., 2017). Our results revealed a lower abundance of Prevotella spp. in the oral microbiome of infants living with pets compared to those in pet-free homes. Although this finding does not fully align with previous studies on the infant gut microbiome, where exposure to furry pets has been linked to increased microbial diversity (Azad et al., 2013; Tun et al., 2017; Panzer et al., 2023), it suggests that similar mechanisms may influence the oral microbiome. Pets can harbor and transmit various microbes. Microbial transmission to infants may occur through direct contact with pets or via intermediate reservoirs in the home, such as house dust or the maternal microbiome (Azad et al., 2013; Konya et al., 2014) highlighting the need for further investigation.

Birth season has also been recognized as a potential determinant in shaping the development of the oral microbiome during infancy however no studies have investigated this. We found that infants born in winter or autumn exhibited a lower relative abundance of S. parasanguinis. In contrast, a study of the infant nasopharyngeal microbiome found that season of birth was linked to the diversity of bacteria in infants’ nasopharynx at one month of age, with greater richness and altered taxonomic composition observed among those born in summer (Schoos et al., 2020). These findings suggest that birth season may influence microbial colonization in different body sites, indicating a broader seasonal impact on microbial communities. Further research is needed to explore these seasonal variations in the oral microbiome and their potential implications for infant health, particularly in light of association between birth season and infant allergic disease (Kuwabara et al., 2020; Tsuchida et al., 2023).

The key strengths of this study are its longitudinal nature, allowing identification of temporal patterns over 24 months, and the relatively large sample size compared to previous similar studies. In addition, the use of full-length 16S rRNA gene sequencing, offers advantages over short-read sequencing techniques, including improved taxonomic resolution and accuracy in microbial identification.

Several limitations, however, must be acknowledged. Firstly, the precise timing of solid food introduction was not recorded, nor were the types and quantities of food to which infants were exposed to. However, our study partly addressed this limitation by analyzing data at two subsequent time points after the introduction of solid foods. Secondly, the timing of tooth eruption was not documented. Tooth eruption and the introduction of solid foods often occur around the same time and may influence the composition and diversity of the oral microbiome (Sulyanto et al., 2019). Additionally, daycare attendance data were collected only at 12 months as a binary variable (yes/no), with substantial missing data and no information on attendance frequency or location. Due to these limitations, it was not included in our modelling; however, it should be considered in future studies.

In summary, this study represents one of the most comprehensive examinations of the infant oral microbiome with respect to breastfeeding conducted to date. Our findings illustrate the dynamic nature of the oral microbiome over the first two years of life, with significant associations identified with multiple factors, including pacifier use, intrapartum antibiotic prophylaxis, maternal allergy, pre-pregnancy maternal BMI, delivery mode, maternal age, presence of pets at home, siblings, and birth season. We demonstrated that the introduction of solid foods is a significant milestone in the development of the oral microbiome along with cessation of breastfeeding. The impact of these changes on infant health and development requires further investigation.