The composition of the oral microbiota varies with the age of the host. Age-related changes in the oral cavity result from tooth eruption, dietary changes, hormonal fluctuations and salivary flow. Infants are especially susceptible to microbial colonization, as specific antibodies, such a secretory immunoglobulin A, are present at relatively low levels during infancy. At birth, the oral cavity is usually void of microbes, except in cases of chorioamnionitis . Rapid contamination of external and internal surfaces occurs when the fetus is exposed to the flora of the birth canal and to the external environment, especially through contact with the mother and with contaminated milk and water. The rate and extent of neonatal oral cavity colonization depends on various perinatal and neonatal factors, such as the mucosal immune system, gestational age, mode of delivery, hospitalization in an intensive care unit, type and mode of feeding and antibiotic treatment. The mucosal immune system represents the first line immune response against oral infection. Secretory IgA present in the saliva may control the oral microbes by preventing their adherence to the oral mucosa and the teeth . Salivary IgA concentrations and IgA antibody specificities appear to be influenced by gestational age, which may reflect the level of the mucosal immune system . Because neonates are immunologically immature, they are at risk of developing infections. Premature or low-birthweight neonates are thus at a particularly high risk of infection . However, the colonization of the oral cavity after birth could be delayed by Caesarean section delivery or total parenteral nutrition [14, 15].
Cariogenic microbe colonization, especially with the major pathogenic microorganism associated with dental caries (S. mutans), was found to occur during a window of infectivity between 19 and 31 months of age. This so-called “window of infectivity” theory of cariogenic microbe acquisition should be reevaluated using modern molecular techniques for microbial detection. Real-time PCR is a clinical tool for detecting and quantifying the presence of bacterial pathogens. Compared to traditional culturing methods, real-time PCR is fast and cost effective, but it is limited by the quality of the primers and probes chosen. These primers and probes must be sensitive enough to detect all of the target organisms yet specific enough to exclude all others . Cephas et al.  demonstrated that 62.2% of the edentulous infants had Streptococcus genera in their salivary samples. Milgrom et al.  studied the occurrence of S. mutans in infants between 6 and 12 months of age and found these microbes in 48.3% of the plaque samples and in 58.3% of the saliva samples collected from their tongues. In our study, S. mutans was detected in all full-term one-year-old infants.
It has been speculated that birth by Caesarean section would decrease an infant’s early colonization by S. mutans because the immune defense system of neonates can avoid the extensive exposure to maternal microorganisms that occurs during vaginal birth . Our results did not confirm speculations about the relationship of the delivery mode and the early colonization of the oral cavity with S. mutans. In our study, most premature infants (75%) were born via Caesarean section and still had minimal S. mutans colonization of the oral cavity.
The presence of Lactobacillus species is correlated with both active caries and an increased predisposition for future caries, as these bacteria interact with other microorganisms during their colonization. We found very high positive levels of Lactobacillus species (98.7%) in the saliva samples of the one-year-old infants. However, Könönen et al.  detected Lactobacillus species in only 2% of 12-month-old infants.
Actinomyces species play an important role in the initiation and progression of caries due to their aciduricity and ability to co-aggregate with S. mutans. Actinomyces species were present in 83% of the caries-free infants in our study, which is consistent with the reported 91% prevalence of this species in one-year-old infants described by Könönen et al. .
Anaerobic bacterial species associated with the onset of periodontal diseases constitute an important part of the bacterial community of the mouth. The timing and type of the initial oral colonization of infants is of great importance, as it lays the groundwork for all further colonization. According to some previous studies [22, 23], the colonization of the oral cavity in infants by periodontal pathogens is thought to be rare, occurring most commonly when the mother has periodontitis. Improved methods based on the PCR technique suggest that the acquisition of some microbes associated with periodontal disease occurs in the oral cavity earlier than was previously supposed. Obligate anaerobes can and do begin to colonize the oral cavities of infants prior to tooth eruption . Some authors [25–27] have detected periodontal pathogens in young children, although the prevalence of these microbes has been relatively low. In our earlier study , periodontal pathogens (Aggregatibacter actinomycetemcomitans, Prevotella intermedia and F. nucleatum) were found in the saliva of 2.9% of newborns, 63.8% of six-month-old predentate infants and 97.8% of 12- month-old infants. Yang et al.  detected periodontal pathogens in 71% of children with an average age of 32.4 ± 7.5 months. In our recent study, we found that 91% of full-term one-year-old infants had periodontal pathogens. Thus, the reported prevalence of periodontal pathogens in the oral cavity varies considerably across different studies [24–27]. One of the most important periodontal pathogens is Aggregatibacter actinomycetemcomitans. This microbe was the most frequently detected periodontal pathogen found in our study and was present in the saliva of 83% of the very-low birthweight infants and in 96% of the full-term infants. Lamell et al.  detected Aggregatibacter actinomycetemcomitans in only 25% of infants under one year of age and, according to their work, Aggregatibacter actinomycetemcomitans is usually a transient colonizer of a child’s oral cavity.
Fusobacterium nucleatum is a quantitatively prominent component of the dental plaque and is one of the first Gram-negative species to become established in the plaque biofilm. Fusobacterium nucleatum is the most frequent strictly anaerobic species that exists in the oral cavity at one year of age. This microbe has been found to be present in the oral cavities of 91% of one-year-old infants and of 60% of younger infants with a mean age of 3 months . Here, we found the incidence of F. nucleatum to be low in both groups of infants.
Cortelli et al.  described the initial colonization by Prevotella intermedia and Tannerella forsythia in an age group of 6 to 12 years, and P. gingivalis was first detected in much older individuals, aged 19 to 44 years. Cortelli et al.  suggested that alterations in the oral microenvironment accompanied by the eruption of teeth and the formation of the gingival sulcus provide environmental niches that are favorable for the growth of Tannerella forsythia, Prevotella intermedia and Prophyromonas gingivalis. Könönen et al.  detected these microbes, including T. denticola and Peptostreptococcus micros, in the oral cavity of 12-month-old infants, but their incidence was very low. Our findings regarding the oral colonization of these periodontal pathogens in the same age group are consistent with those of the described studies. Treponema denticola was not detected in the children we examined.
Oral colonization with cariogenic and periodontal pathogens in pre-term infants has not been adequately investigated. The authors [30, 31] focused only on a comparison of the S. mutans colonization of the oral cavity in pre-term and full-term infants. Wan et al.  used selective tryptone-yeast-cysteine-sucrose-bacitracin agar for S. mutans isolation and detected S. mutans in 60% of full-term six-month-old infants and in over 50% of pre-term six-month-old infants. In their longitudinal study, Wan et al.  found that S. mutans colonization increased with infant age. At 12 months of age, 37% of infants harbored S. mutans, and there was a higher prevalence of these microbes in full-term infants than in pre-term infants, although this difference was not statistically significant. After 12 months of age, a higher prevalence of S. mutans was reported in the pre-term infants, but again, this difference was not statistically significant. Factors associated with S. mutans colonization included sweet liquids being taken to bed, frequent sugar exposure, snacking, sharing of adult food and high maternal S. mutans levels. In contrast, a lack of S. mutans colonization was connected with tooth brushing and multiple courses of antibiotics.
Seow et al.  studied the presence of S. mutans in 12-month-old infants and the differences between pre-term and full-term infants using quantitative real-time PCR techniques. They found no significant differences between these groups of infants. This may be related to the similar dietary and oral hygiene habits in both groups. The results of this research contradict the findings of our own study. In our study, we found a significant difference in the presence of oral S. mutans between pre-term and the full-term infants.
A number of factors could help explain the low observed occurrence of S. mutans in the oral cavities of the pre-term infants. Late transmission and colonization with S. mutans can be considered because these infants were not in frequent contact with their mothers from birth. Pre-term infants are also repeatedly treated with antibiotics, which impacts the composition of their oral microbial flora. Wan et al.  found that non-colonization by S. mutans was associated with multiple courses of antibiotics. In addition to antibiotic dosing, frequent parenteral nutrition, differences in mucosal immunity, and repeated endotracheal intubations and laryngoscopies may all affect the bacterial colonization of the oral cavities of pre-term infants. Lengthy hospitalizations of pre-term infants in intensive care units may also influence bacterial colonization. These factors may not affect the periodontal pathogens, which are much later colonizers of the oral cavity. This was confirmed in our study, as we found that the incidence of the major periodontal pathogens was similar in both study groups.
The authors are aware of certain limitations of this study. The infants’ dietary and tooth brushing histories were not obtained, a relatively small number of infants were included in the research group, and only one sample was taken from each infant for microbial detection.