The human oral cavity refers to a complex microbial community consisting of bacteria, fungi, viruses, archaea, and protozoa, and the microorganisms constantly interact with each other (Lamont et al., 2018). Eubiosis of the oral microbiome is characterized by the “healthy oral microbiota” with specific microbial patterns, and the oral microbiome lives in homeostasis with the host. Moreover, changes in the balance of the oral microbiome cause dysbiosis, which leads to a pathogenic state and the occurrence and progression of oral diseases (Jiao et al., 2014). Microbial structure in the oral cavity is dependent on niche and age (Xu et al., 2015, 2022). Oral microbial maturation is triggered by age-associated biological alterations (Crielaard et al., 2011), whereas oral microbial communities formed during early life can affect the long-term systemic and oral health of a child (Dzidic et al., 2018).

The mother plays a crucial role in shaping the oral environment in early life (Sampaio-Maia and Monteiro-Silva, 2014; Teng et al., 2015; Dzidic et al., 2018), which subsequently develops into a complicated and mature microbial ecosystem due to parental vertical transmission and the external environment (Hesselmar et al., 2013; Sampaio-Maia and Monteiro-Silva, 2014). Almost all previous studies have classified diverse oral microbial developmental stages on the basis of age in months while simultaneously ignoring the effect brought by tooth eruption state. Typically, the eruption of the primary incisor starts at approximately 6 months after birth. It is of great importance to evaluate the influence of tooth eruption on the course of microbial colonization.

Preterm birth has been a major factor resulting in neonatal death worldwide and leading to higher risks for poor neurobehavioral development, metabolic disorders, and respiratory distress in neonates and mothers. Low birth weight in preterm infants has been associated with pathogenic bacteria in the oral cavity (Han and Wang, 2013; Cobb et al., 2017; Ye et al., 2022). Thus, higher detection frequencies of periodontal-associated microbial species, including Porphyromonas gingivalis, Treponema denticola, Fusobacterium nucleatum, Prevotella intermedia, and Tannerella forsythia, in saliva or dental plaque samples have recently been shown to be strongly associated with preterm low birth weight (Swati et al., 2012; Ye et al., 2022). Moreover, P. gingivalis infection-induced preterm birth as well as low birth weight in pregnant mice (Ao et al., 2015). In addition, it was considered that dysbiosis in the placental microbiome showed a relationship to preterm birth. To date, the impact of preterm birth on the development of the early oral microbiota has not been thoroughly investigated in longitudinal studies.

There are more and more studies on the interactions between the oral microbiota of mother and child and on the progressive development of the intestinal microbiota of infants (Backhed et al., 2015; Gomez and Nelson, 2017; Hill et al., 2017; Mason et al., 2018; Yang et al., 2020; Kaan et al., 2021). However, the changes observed in the oral microbiome and the progressive development of the infants’ microbiome during teeth eruption are not well understood. To date, there is no consensus on the characteristics of the oral microbiome colonization of preterm infants after birth.

On the basis of this, we aimed to characterize the dynamic shift in microbial structure and external factors associated with the microbiome of preterm infants during teeth eruption. In this longitudinal study, we collected saliva samples from normally delivered and preterm infants during the eruption of deciduous teeth and examined the dynamic microbial changes of the newborns at two time points using Illumina MiSeq sequencing of the 16S rRNA gene.

Microbiota colonization dynamics in infancy remains an interesting topic in microbial ecology and human health (Derrien et al., 2019). We know little about the growth of the infant oral microbial ecosystem (Del Chierico et al., 2015). The current work examined the effects of preterm birth on flora during a dental eruption. This study matched the month age of the two groups at baseline, which is a critical point and helps us understand the variations in the oral microbiome during tooth eruption.

The investigators reported an increase not only in bacterial diversity (Shannon) but also in richness (Chao) in the upbringing of both groups. The teeth eruption into the infant oral cavity starts at around 6 months of age in healthy infants and significantly later in preterm babies; thus, explaining a higher Chao diversity in PT1 than CT1 infants in this study. In the PT1 group, community development was more advanced. Mason et al. (2018) elucidated that oral microbiome in predentation infants is very similar to the maternal oral microbiome, and it drastically changes at the age of tooth eruption. The microbiome is also affected by the food diversity introduced along with milk (Mason et al., 2018). The CT0 and PT0 groups had distinctive microbial communities differing in the microbial structure at baseline and in shift patterns along with the teeth eruption period. PT0 showed irregular variations in microbiome structures. Tooth emergence can be related to ecological change. In addition, the tooth surface, gingival sulci, and interproximal spaces between teeth provide a wide variety of niches to develop highly complex microbiota and form a higher diversity of environments for microbial colonization and growth in the oral cavity (Jakubovics, 2015; Mark Welch et al., 2020).

In this study, the composition and displacement trend, as well as tooth eruption of oral nuclear flora differed in the full-term and preterm groups. In addition, an oral core microbiota composed mainly of Streptococcus spp. salivarius, Pseudomonas spp., R. mucilaginosa, V. atypica, V. dispar, Gemella spp. and Neisseria spp. (approximately more than 80.0% of the total oral microbiota) can be identified in the preterm group. While in the healthy birth group, Streptococcus, especially S. salivarius, V. atypica, Gemella spp. and Staphylococcus spp., dominated the infant oral microbiota. Streptococcus accounted for more than 70.0% of the total microbiota at the predental stage and then decreased to 44.9% at the CT1 stage. The obtained result is consistent with other studies (Dashper et al., 2019; Lif Holgerson et al., 2020). Cheema et al. (2022) have reported specific taxa containing Streptococcus mitis, R. mucilaginosa, Veillonella parvula, S. salivarius, Gemella haemolysans, and Veillonella HB016, which formed the infant core oral microbiota at the first 3 months of life. Dzidic et al. (2018) indicated Streptococcus, Veillonella, and Lactobacillus spp. to be “early colonizers” in saliva in the oral cavity. Mason et al. (2018) found that tooth eruption provides habitats for Rothia, Fusobacterium, and Neisseria, resulting in shifts in the core microbiome, and that the tooth environment could strongly influence the composition of the oral microbiome at different life stages relative to other factors. In addition, Neisseria spp. and R. mucilaginosa were more abundantly found in the healthy group because of teeth eruption. In preterm infants, Neisseria spp. increased notably, and R. mucilaginosa markedly decreased along with two lower primary central incisors teeth eruption. Gram-positive aerobic coccus R. mucilaginosa has been considered a normal oral microbiome, and it can play the role of the opportunistic pathogen leading to impairment, including septicemia and endocarditis under certain conditions (Maraki and Papadakis, 2015). The onset of tooth eruption may create new ecological niches in the oral cavity, leading to new surfaces of adhesion and thus favoring colonization by different flora. Whether the core microbiota shifts predominantly due to tooth eruption in preterm infants is still controversial.

The oral Streptococcus genus is the most comprehensively investigated genera of oral bacteria and controls the oral microbiome from birth until at least 7 years old (Sampaio-Maia and Monteiro-Silva, 2014; Dzidic et al., 2018). Because of the capability of adhering to and colonizing the mucosal surface lining, Streptococcus is especially abundant within the first months after birth and reduces with age (Sampaio-Maia and Monteiro-Silva, 2014; Dzidic et al., 2018). Xu et al. (2022) conducted a longitudinal study of the gradual eruption of deciduous teeth in infants and observed that Streptococcus accounted for more than 60% of the total microbiota and subsequently declined dramatically to 26.1% after teeth eruption, which is inconsistent with our results in both groups. S. mitis and S. salivarius species occupied the majority of oral streptococci (Dashper et al., 2019; Lif Holgerson et al., 2020). We observed that Streptococcus species were more abundant in the healthy controls than the preterm babies at early stages. Moreover, various findings demonstrated the reduction in Streptococcus abundance was accompanied by enhanced microbial diversity (Blum et al., 2022). In addition, this decreasing phenomenon of Streptococcus was observed in our study, which may be because of bacterial community development.

Microbial correlation analysis showed positive relationships between Streptococcus spp. and Gemella spp. and between S. salivarius and R. mucilaginosa at the PT1 stage. Members of Streptococcus can bind to other bacteria via surface adhesins, which can promote the development of certain bacterial communities, and occur together with G. haemolysans, and R. mucilaginosa (Kolenbrander et al., 2006; Nobbs et al., 2011; Sulyanto et al., 2019). In addition, the facultative anaerobic, Gram-positive diplococcus G. haemolysans primarily mucosal in mammals, particularly in the upper gastrointestinal tract with the oral cavity, and ferments free sugars into a mixture of acidic end products (Blum et al., 2022). This was associated with allergies in the oral microbiome of children (Dzidic et al., 2018), which were not found in our questionnaire analysis. Moreover, unlike the downward trend in the healthy group, Gemella spp. showed an upward trend along with two lower primary central incisors teeth eruption in preterm infants. As Gemella colonizes early, it helps in oral microbiome development relatively later in premature babies.

Correlation networks of saliva microbiota in preterm infants showed that S. salivarius had a strong positive correlation with V. atypica and V. dispar in the PT1 stage. Production of lactate from fermentation of free sugars by oral Streptococcus may be the essential nutrient for Veillonella spp. demonstrating synergistic interactions between the two species (Jakubovics, 2015). Other key bacteria, Veillonella spp. have also been considered early colonizers. The general uptrend of Veillonella spp. abundance was shown in several studies in healthy and preterm infants (Dzidic et al., 2018; Yin et al., 2021; Xu et al., 2022). In this study, the relative abundance of V. dispar also increased while growing up in both groups; however, V. atypica did not show this trend, which was stable in the healthy controls from CT0 to CT1 stage and decreased markedly in the preterm infants along with two lower primary central incisors teeth eruption. Metabolites produced by Streptococcus, including lactic acid, can pave the way for other microorganisms to colonize saliva, such as Veillonella (Gomez and Nelson, 2017). Early symbiosis in saliva may have ecological advantages over later symbiosis, promoting environmental changes through their metabolic products that are conducive to the growth of additional oral bacterial communities. An important consequence of the healthy development of the oral microbiome is the prevention of the most common childhood oral disease, dental caries. The early presence of oral pathogenic microorganisms in edentulous children may use saliva as a reservoir. However, the longitudinal impact of these initial colonizers on infant oral and systemic health is largely unknown and should be further investigated to provide useful information for active and passive immunization strategies against childhood oral diseases.

It is noteworthy that some species were detected only in the preterm group and were hardly present in the control groups, such as Pseudomonas spp. There was a high abundance of Pseudomonas spp. at baseline that decreased afterward, which may be because of hospitalization and antibiotic use of premature infants (Henares et al., 2021; Warniment et al., 2021). Common nosocomial genera, including Staphylococcus, Acinetobacter, and Pseudomonas, developed in infants subjected to antibiotics (Henares et al., 2021). A higher abundance of Pseudomonas spp. in the PT0 and PT1 groups indicated that preterm infants were more susceptible to oral or systemic infections.

In this study, birth weight and breastfeeding durations in preterm babies influenced the saliva microbiome at the predentate stage. Subsequently, birth weight and gestational weeks at birth presented a significant association with the bacterial community. Protective factors of breast milk, such as maternal sIgA, may stimulate colonization with commensal bacteria, which perform an essential function in fulfilling the critical tasks required to build a microbial community to establish a healthy microbiota profile in the oral cavity (Berger et al., 2020; Blum et al., 2022). Cheema et al. (2022) indicated that the ingestion of oligosaccharides from human milk was associated with infant oral and fecal taxa, while their direct clinical relevance was unknown. Streptococcus spp. was associated with breastfeeding duration in PT0. Streptococcus is one of the leading bacterial groups in human breast milk (Rodriguez, 2014; Boix-Amoros et al., 2016). In addition, the metabolic products (including lactic acid) that originated from Streptococcus species from the dietary oligosaccharides in breast milk can help in establishing other oral microorganisms, including Veillonella (Wernersson et al., 2006; Gomez and Nelson, 2017). The transmission of bacteria from breast milk, as well as the nutrients found at the vital time point in the growth of babies, might influence the colonization window of certain bacterial genera (Dzidic et al., 2018). According to our results, the duration of breastfeeding may induce variations in oral microbial succession patterns that persist over time. Consideration of neonatal treatment needs, including antibiotic use, is necessary depending on the physical condition, birth weight, and gestational weeks of preterm infants to exert a long-term influence on the oral bacterial composition.

Nevertheless, this work still has a few limitations. At first, the sample size was limited, particularly in the healthy control group. In this study, the enrollment and follow-up of the healthy babies were more challenging than the preterm babies. This may be because parents of babies without disease are less interested in participating in research projects, and parents of healthy babies often do not attend appointments; parents of premature babies, on the other hand, prefer follow-up appointments. Moreover, this study was conducted at only two childcare institutions, preventing us from obtaining a larger sample size. Second, this study followed up only two lower incisors’ eruption, which may limit the overall results of the study. Larger and longer longitudinal studies are required to explore the effect of oral microbial change because of the complicated oral microbial structures during teeth eruption. Third, because tooth eruption and the introduction of food in addition to milk can occur simultaneously [45], it is difficult to distinguish the effects of dietary habits and tooth eruption on the development of the oral microbial community. At last, the mode of birth delivery was not included in the bioinformatics analysis. Various researchers have compared colonization patterns of microbial species between infants delivered vaginally and those delivered by cesarean section. Hurley et al. (2019) reported that the effects of birth mode on infant oral microbiome development are short-lived. Infants delivered by cesarean section showed an obviously more diverse oral microbiota when compared with infants delivered vaginally at 1 week of age, whereas the existing difference did not persist after 4 weeks of birth. This study included infants at least 1 month of age, so there are no subgroups by mode of delivery. Considering that antibiotics are typically administered prophylactically during intrapartum care to reduce the risk of infectious complications during cesarean deliveries, which may confound microbiome results (Smaill and Grivell, 2014), a thorough investigation is needed to determine the effects of mode of delivery on the oral microbiome and whether they may persist over time.

To conclude, this study provides certain knowledge on how the oral microbiota changes during teeth eruption in preterm newborns. Compared with full-term infants, the composition and shifting pattern, along with two lower primary central incisors teeth eruption, of core oral flora differed in preterm groups. Several microorganisms that populate the oral microbiome develop relatively late in preterm infants, such as Gemella. In addition to tooth eruption, the results of this study confirmed that breastfeeding duration and birth weight also influence the growth of the salivary microbiome in preterm infants.

The data presented in the study are deposited in the SRA repository, accession number PRJNA876796.

The studies involving human participants were reviewed and approved by the Ethics Committee at the Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

YZ and XC contributed to the study design, sample collection, clinical examination, data analysis, statistics, and interpretation, and drafting and critically revising the manuscript. G-ZC, Y-PW, and VF contributed to sample collection and drafting and critically revising the manuscript. X-PF contributed to the study design and drafting and critically revising the manuscript. All authors give final approval and agree to be accountable for all aspects of the work.

This study was funded by the National Natural Science Foundation of China (No. 81800967), Shanghai Commission of Science and Technology (No. 22Y11903200), and Shanghai Municipal Health Commission (Nos. 2019SY027 and 2020YJZX0114).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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