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Published in final edited form as: Am J Perinatol. 2023 Oct 31;41(Suppl 1):e2990–e3002. doi: 10.1055/s-0043-1776344

A Scoping Review of the Oral Microbiome in Preterm Infants

Rebecca Koerner 1, Marion M Bendixen 2, Angela Monk 1, Monica FT Lamberti 3, Graciela L Lorca 3, Josef Neu 4, Leslie A Parker 1
PMCID: PMC11058105  NIHMSID: NIHMS1961774  PMID: 37907200

Abstract

The purpose of this scoping review was to examine the oral microbiome composition in preterm infants, sampling and collection methods, as well as exposures associated with oral microbiome composition and health implications. We conducted a scoping review of the literature using the Arskey and O’Malley framework. We identified a total of 13 articles which met our inclusion criteria and purpose of this scoping review. Articles included in this review compared the oral microbiome in preterm infants to term infants, examined alterations to the oral microbiome over time, compared the oral microbiome to different body site microbiomes, and explored associations with clinically relevant covariates and outcomes. Exposures associated with the diversity and composition of the oral microbiome in preterm infants included delivery mode, oral feeding, oropharyngeal care, skin-to-skin care, and antibiotics. Day of life and birth weight were also associated with oral microbiome composition. The oral microbiome may be associated with the composition of the tracheal and gut microbiomes, likely due to their proximity. Alpha and beta diversity findings varied across studies as well as the relative abundance of taxa. This is likely due to the different sampling techniques and timing of collection, as well as the wide range of infant clinical characteristics. Multiple factors may influence the composition of the oral microbiome in preterm infants. However, given the heterogeneity of sampling techniques and results within this review, the evidence is not conclusive on the development as well as short- and long-term implications of the oral microbiome in preterm infants and needs to be explored in future research studies.

Keywords: preterm, infant, oral, microbiome

The first 1,000 days of an infant’s life are hypothesized to have tremendous impact on lifelong health outcomes.1 Early development of the microbiome has lasting implications, as the microbiome plays a critical role in health and disease.1 The oral microbiome, which is associated with oral health, is linked to systemic health conditions across the lifespan including cardiovascular disease, diabetes, and cancer.2

The oral microbiome develops throughout the first several years of life, with microbial diversity increasing as the child ages.3 Early colonizers of the term infant oral microbiome are consistently Streptococcus and Staphylococcus, which are known to easily adhere to the mucosal surface, are prominent in mother’s own milk (MOM), and are common breast epithelium microbes.3 Early colonization of the term infant oral microbiome is affected by delivery mode, antibiotic exposure, and feeding method.3

In contrast to healthy term infants, preterm infants are often prescribed antibiotics soon after birth and intubated due to respiratory distress. In addition, preterm infants are fed via orogastric/nasogastric feeding tubes due to being too immature to orally feed, thus preventing or delaying the contact with important colonizers obtained from breastfeeding. As a result, there is an increased risk for pathogenic bacterial colonization and a dysbiotic oral microbiome, which may be especially important in preterm infants due to their immature immune development.4–6

The neonatal intensive care unit (NICU) harbors opportunistic pathogens associated with nosocomial diseases including Staphylococcus aureus, Escherichia coli, and Klebsiella.7 In preterm infants, pathogenic bacteria colonization and/or microbiome dysbiosis is associated with development of potentially serious complications including necrotizing enterocolitis (NEC),8 bronchopulmonary dysplasia,9 and late-onset sepsis10 which significantly increase morbidity and mortality.11 The oral microbiome may be responsible for seeding the lung and gut microbiome due to their proximity,12–14 and thus early colonization of the oral cavity can have systemic health implications.

Given the long-term health implications of preterm birth, it is imperative to understand the role of the oral microbiome in health and disease. Specifically, understanding how the NICU milieu and medical interventions shape the oral microbiome may guide future interventions to improve oral health in this population. The aim of this scoping review was to (1) examine oral microbiome composition in preterm infants,(2) identify factors associated with alterations to the oral microbiome in preterm infants, (3) explore methods of collecting specimens for oral microbiome analysis in preterm infants, and (4) explore associations between the oral microbiome and health outcomes in preterm infants.

Materials and Methods

This scoping review was guided by the Arskey and O’Malley framework and followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses-scoping review guidelines.15 The Arskey and O’Malley framework encompasses a five step process—(1) develop a research question, (2) examine the extant literature, (3) include relevant studies based on specified inclusion/exclusion criteria, (4) chart the data, and (5) appraise the evidence, synthesize, and report the findings.15 We chose to utilize a scoping review format to examine the current literature, evaluate research methods for examining the oral microbiome in preterm infants, and identify gaps in knowledge to guide future research studies.

Eligibility Criteria

We included peer-reviewed, original research studies that examined the oral microbiome using 16S ribosomal RNA (rRNA) sequencing in preterm infants born at 23 to 36 weeks’ gestation and were written in English. We excluded articles that were not peer-reviewed, animal studies, were literature reviews, or did not conduct 16S rRNA sequencing on the oral microbiome. We did not set a date limit on articles included, so we could gather all relevant information regarding our topic, though all articles were published within the last 10 years. One author (R.K.) screened articles based on title and abstract, using Covidence, a systematic review software, and then two authors (R.K. and L.A.P.) reviewed the remaining full-text articles for inclusion. For any conflicts, the authors discussed the reasoning for inclusion or exclusion and came to a mutual agreement.

Information Sources and Search Strategy

One author (R.K.) searched four electronic databases— PubMed, CINAHL, Scopus, and Web of Science—to conduct our literatre search. The search was conducted in November 2022. A medical librarian assisted in developing search terms for our search strategy and we included terms related to the microbiome, oral cavity, and preterm infants. In PubMed, we used appropriate Medical Subject Headings (MeSH) and identified appropriate major headings in CINAHL. Our database searches can be found in Supplementary Table S1, available in the online version.

Data Charting Process and Data Items

One author (R.K.) extracted data from original research articles and their supplementary materials. Then, data were charted using an Excel spreadsheet. The following headings were used to guide our data charting: author, study aim, study design, sample size and population, sample collection methods and analysis, study inclusion/exclusion criteria, covariates included in analysis (delivery mode, feeding method, antibiotic exposure), primary and secondary outcome results, and limitations.

Critical Appraisal of Evidence and Synthesis of Results

One author (M.M.B.) critically appraised the evidence using the Newcastle–Ottawa Quality Assessment Scale for cohort studies and the Cochrane Risk of Bias Tool for randomized controlled trials. Results were synthesized based on characteristics of studies, participants, and outcome measures (oral microbiome diversity and abundance, associations with clinical covariates and other microbiomes, oropharyngeal care, and health outcomes).

Results

We identified 775 studies in our literature search (Fig. 1). A total of 399 duplicate articles were identified, 376 studies were reviewed based on title and abstract, and 16 studies were assessed with full-text review. We excluded three studies as they did not include 16S rRNA sequencing of the oral microbiome, only conducted whole-genome sequencing, or only evaluated the microbiome. A total of 13 studies are included in our review and are presented in Table 1. We critically appraised our evidence and findings, which are presented in Tables 2 and 3.

Fig. 1.

Fig. 1

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Preferred Reporting Items for Systematic Reviews and Meta-analyses flow diagram.

Table 1.

Sampling methods and results of included studies

Study (year) Study design and purpose Sample size and population Methods Oral sampling technique Main study findings
Biagi et al13 (2018) Prospective cohort study examining the oral and gut microbiomes and associations with MOM microbiome before and after breastfeeding, for up to 210 days after birth 21 infants, 32 to 34 weeks’ GA and 16 mothers Oral, stool, and MOM samples were collected at birth, 4, 7, 14, 21, and 30 days after birth. Additional samples from these sites were also collected up to 210 days after birth when possible. The V3-V4 region was amplified for microbiome sequencing. Alpha diversity was measured using observed species and Shannon indices. Beta diversity was measured using Bray–Curtis distances Swab of inside of infant’s cheek using a sterile cotton-tipped applicator Breastfeeding significantly impacted oral microbiome composition and MOM microbiome (p = 0.001). Streptococcus and Rothia were highly abundant in the oral microbiome (p < 0.001) and were present in the milk microbiome once infants began breastfeeding
Brewer et al12 (2021) Prospective cohort study comparing the oral and tracheal microbiome during the first week of life 42 intubated infants, ≤32 weeks GA and <2,000 g Oral samples were collected 3 days after birth and tracheal samples were collected 3 and 7 days after birth. The V3-V6 region was amplified for microbiome sequencing. Alpha diversity was measured using the Shannon index and Beta diversity was measured using the Bray–Curtis similarity and analysis of similarities Sterile suction connected to a trap, cleared with 1 mL of sterile saline Tracheal alpha diversity (Shannon index) was higher than oral alpha diversity DOL 3 (p = 0.02). The tracheal microbiome on DOL 7 was not significantly different than the oral microbiome on DOL 3 (p = 0.92). No difference in beta diversity between the oral microbiome on DOL 3 and tracheal microbiome on DOL 7
Cortez et al17 (2021) Prospective, quasi-experimental study examining the effect of OPC on the oral microbiome 20 infants, 28 to 35 weeks’ GA Oral samples were collected within 24 hours, 7, 14, and 21 days after birth. The V3-V4 region was amplified for microbiome sequencing. Alpha diversity was measured by the Chao 1, Shannon, Simpson, and Faith’s phylogenetic diversity indices. Beta diversity was measured by weighted, unweighted, and generalized UniFrac distances as well as Bray–Curtis dissimilarity. Confounding variables including delivery mode, GA, and antibiotic exposure were included in the analysis. OPC (MOM or donor human was initiated 24 to 48 hours after birth, continued for ≥3 days, and was administered every 3 hours Swab sample was collected from cheek and tongue for 30 seconds Significant decreases in Chao 1 richness (p = 0.001), Faith’s phylogenetic diversity index, Shannon and Simpson indices (p < 0.001) in both groups. The Shannon index was higher in the standard care group at 21 days (p = 0.015). No significant differences in beta diversity between the groups. DOL significantly affected beta diversity (p = 0.001)
Costello et al16 (2013) Prospective cohort study evaluating the oral, fecal, and skin microbiome in low birth weight infants 6 infants, <2,500 g at birth Oral, stool, and skin samples were collected 8, 10, 12, 15, 18, and 21 days after birth. The V3-V5 region was amplified for microbiome sequencing. Alpha diversity was measured using observed taxonomic unit richness. Beta diversity was measured using unweighted UniFrac distances Swab sample of the dorsum of the tongue was collected with either a sterile nylon or cotton swab Body site is the most important factor regarding microbiome composition (p < 0.001). The oral microbiome was stable over the first 3 weeks of life (p = 0.935). Compositional differences between oral and gut microbiomes increased over time (p = 0.0359)
Hendricks-Munoz et al18 (2015) Prospective cohort study examining the effect of STS on the oral microbiome 42 infants, <1,500 g and <32 weeks’ GA Oral samples were collected at either 1 month after birth or at discharge. Participants were then grouped by corrected gestational age. The V4-V6 region was amplified for microbiome sequencing. Alpha diversity was measured using the reverse Simpson index and Beta diversity was measured using the Bray–Curtis dissimilarity index Sterile dry soft cotton swab rolled along the mouth, cheeks, and tongue until saturated with saliva. Taken prior to feeding No significant differences in alpha diversity. Infants ≤32 weeks without STS care had increased beta diversity. In infants without STS, greater relative abundance of Pseudomonas (p < 0.019) and Corynebacterium (p < 0.023), and decreased abundance of Streptococcus (p< 0.024). Length of hospitalization was significantly decreased in infants exposed to STS (p < 0.0095).
Li et al25 (2021) Cross-sectional study examining the microbiome between mothers, term infants, and preterm infants 15 infants, 28 to 366/7 weeks’ GA, 11 infants 37 to 42 weeks’ GA, and 26 mothers Infant oral and rectal swabs as well as maternal vaginal and rectal swabs were collected within 24 hours after birth. The V3-V4 region was amplified for microbiome sequencing. Alpha diversity was measured using the Shannon, phylogenetic diversity, and abundance-based coverage estimator indices. Beta diversity was measured using unweighted UniFrac distances Buccal mucosa swab No differences in Shannon index, or beta diversity measures between pre-term infants and term infants. There was an increased relative abundance of Ralstonia, Ureaplasma, and Streptococcus in the preterm group
Li et al19 (2020) Cross-sectional study examining how delivery mode affects the oral microbiome in very low birth weight infants 23 infants, <1,500 g and <32 weeks’ GA Oral samples were collected immediately after delivery. The V3-V4 region was amplified for microbiome sequencing. Alpha diversity was measured using Shannon and Simpson indices. Beta diversity was assessed using principal coordinates analysis Swab of oral mucosa Delivery mode was not associated with significant differences in Shannon (p = 0.058) or Simpson (p = 0.3) indices or principal coordinates analysis. Infants born via vaginal birth were more likely to have a higher abundance of Ureaplasma and Pantoea in comparison to Corynebacterium and Methylobacterium in infants born via C-section
Orchanian et al20 (2022) Prospective cohort study examining the oral, skin, and gut microbiomes and metabolome of preterm infants 57 infants, 34 to 366/7 weeks’ GA; 18 infants, 23 to 34 weeks’ GA and 500 to 1,500 g at birth Oral, skin, and stool samples were collected daily from birth to 21 days after birth or discharge, whichever came first. The V4 region was amplified for microbiome sequencing. Alpha diversity was measured using the Shannon index. Beta diversity was measured using the Aitchison distance Double tip swab inside of the cheek, one side for 16S rRNA sequencing and other side for metabolomics analysis Antibiotic exposure had the greatest effect on oral microbiome community composition (p = 0.002) followed by chronologic age, birth weight, and delivery mode. No difference in oral microbiome alpha diversity over the first week of life. The composition of the oral microbiome changed over time, regardless of delivery mode or infant age
Romano-Keeler et al23 (2017) Randomized controlled trial examining the effects of OPC on the oral microbiome and salivary immune peptides 99 infants, <32 weeks’ GA Oral samples were collected within the first 24 to 48 hours, 8 to 9 days, and 30 days after birth. The V1-V2 region was amplified for microbiome sequencing. Alpha diversity was measured using the Shannon index and beta diversity was measured using the unweighted UniFrac distance. MOM (0.2 mL) was administered into the oral cavity every 6 hours, starting within the first 48 hours after birth and continued for 5 days A wicking sterile sponge was inserted into the mouth for a minimum of 60 seconds. If there was not adequate saliva, 100 μL of sterile saline was used to irrigate the mouth before a second wicking sponge was used There were no significant differences in Shannon index or unweighted UniFrac distance based on whether infants were exposed to OPC. DOL significantly impacted oral microbiome composition for both groups (unweighted UniFrac distance; p < 0.001) with significant increases in Streptococcus and Staphylococcus at DOL 8 to 9 (p < 0.05)
Sohn et al21 (2016) Randomized controlled trial examining the effect of OPC on the oral microbiome in preterm infants 12 infants, <1,500 g, <7 days old, and intubated within 48 hours of birth Oral samples were collected before initiation of OPC as well as 2 and 50 hours after completion of the intervention. OPC using 0.2 mL of MOM was initiated when MOM was available every 2 hours for 46 hours. The V4 region was amplified for microbiome sequencing. Linear discriminate analysis effect size was used to compare microbiome communities Sterile cotton tipped applicators that were held in the lower buccal pouch for 5 seconds and then the inside of the cheek for 5 seconds, then repeated on the other side Infants exposed to OPC had significantly lower relative abundance of Moraxellaceae (p = 0.03), a lower relative abundance of Staphylococcus at 96 hours (p = 0.01), and a greater relative abundance of Planococcus (p = 0.009). For all infants, Proteobacteria and Actinobacteria were increased on DOL 0 and Firmicutes was greater at 96 hours
Younge et al24 (2018) Cross-sectional study examining and comparing the skin, oral, and stool microbiomes of preterm and term infants and their relationship to the NICU environment 40 infants, <37 weeks’ GA; 89 infants, >37 weeks’ GA Oral and skin samples were collected at time of recruitment. Stool samples were collected only if a fresh specimen was available. The V4 region was amplified for microbiome sequencing. Alpha diversity was measured using the Shannon index. Beta diversity was measured using generalized UniFrac and Bray–Curtis distances Sterile swab of oral cavity No statistically significant differences in oral microbiome alpha diversity between preterm and term infants. There was a greater relative abundance of Stenotrophomonas, Lactococcus, and Enterobacter in preterm infants. Microbiome composition was significantly different dependent on body site between and within subjects (p = 0.001)
Young et al14 (2020) Prospective cohort study examining development of the oral, tracheal, stool microbiome of extremely preterm infants and MOM microbiome of their mothers 7 infants, <26 weeks’ GA Samples were collected over the first 60 days of life, with oral and endotracheal samples collected only when suctioning was clinically indicated. MOM samples were collected from extra volume left in nasogastric tubes, bottles, or syringes. The V4 region was amplified for microbiome sequencing. Alpha diversity was measured using the Shannon index and Beta diversity was measured using the weighted Bray–Curtis dissimilarity index Sterile catheter via routine suction of oral cavity when clinically indicated Significant differences in microbiome diversity by body site by the seventh and eighth week of life (p = 0.05). The oral microbiome was most similar to stool with a 26% similarity in composition. Bray–Curtis dissimilarity indicated that DOL (p = 0.02) and sampling sites (p = 0.001) were the most important factors affecting oral microbiome composition
Zioutis et al22 (2022) Prospective cohort study examining the skin, gut, and oral microbiomes in extremely low birth weight infants and the relationship between communities at these sites 15 infants, <1,000 g at birth Oral, skin, and stool samples were collected 1, 3 to 4, 7 to 8, and 14 to 16 days after birth. The V3-V4 region was amplified for microbiome sequencing. Alpha diversity was measured using the Shannon index, evenness, and ASV richness. Beta diversity was measured using the Bray–Curtis dissimilarity index Swab of oral cavity using ESwab Similar composition at all body sites but only body site explained the variation within the sample population (p = 0.001). Delivery mode and gestational age explained little of the microbiome composition variability. No significant differences in oral microbiome alpha or beta diversity over the first 2 weeks of life
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Abbreviations: ASV, amplicon sequence variant; C-section, cesarean section; DOL, day of life; GA, gestational age;MOM,mother’s own milk; NICU, neonatal intensive care unit; OPC, oropharyngeal colostrum care; rRNA, ribosomal RNA; STS, skin-to-skin.

Table 2.

Critical appraisal of included cohort studies

Study (year) Selection Comparability Outcome Total
Biagi et al13 (2018) *** * ** 6 stars
Brewer et al12 (2021) *** ** * 6 stars
Cortez et al17 (2021) **** * ** 7 stars
Costello et al16 (2013) * * ** 4 stars
Hendricks-Munoz et al18 (2015) *** * ** 6 stars
Li et al25 (2021) **** ** ** 8 stars
Li et al19 (2020) *** * ** 6 stars
Orchanian et al20 (2022) **** ** *** 9 stars
Younge et al24 (2018) ** ** * 5 stars
Young et al14 (2020) *** ** *** 8 stars
Zioutis et al22 (2022) **** ** *** 9 stars
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Good quality: three or four stars in selection domain AND one or two stars in comparability domain AND two or three stars in outcome/exposure domain.

Fair quality: two stars in selection domain AND one or two stars in comparability domain AND two or three stars in outcome/exposure domain.

Poor quality: zero or one star in selection domain OR zero star in comparability domain OR zero or one star in outcome/exposure domain.

Table 3.

Critical appraisal of included randomized controlled trials

Domain Study (year)
Romano-Keeler et al23 (2017)		 Sohn et al21 (2016)
Risk of bias arising from the randomization process Low Low
Risk of bias due to deviations from the intended interventions (effect of assignment to intervention) Some concerns Some concerns
Risk of bias due to deviations from the intended interventions (effect of adhering to intervention) Some concerns Some concerns
Missing outcome data Some concerns Low
Risk of bias in measurement of the outcome Some concerns Low
Risk of bias in selection of the reported result Some concerns Low
Overall Some concerns Low
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Characteristics of Included Studies and Participants

A total of 526 preterm infants are included in this scoping review. Sample sizes of studies ranged from 6 to 129. One study included a low birth weight infant who was born full term.16 We chose to include this study as most infants were born preterm and a novel operational taxonomic unit (OTU) was identified in the most preterm infant with significant health implications. Seven studies excluded participants if they had congenital anomalies.12,17–22 Study designs are presented in Table 1, with nine conducting longitudinal, prospective cohort studies. Studies took place in the United States,12,16,18,20,21,23,24 England,14 Vienna,22 China,19,25 Italy,13 and Brazil.17

Samples were collected at various intervals throughout the first 7 months of life. A total of five studies collected samples within the first 24 hours after birth,13,17,19,20,25 whereas the remainder collected samples after the first 24 hours. All longitudinal studies collected oral samples at varying intervals. Collection methods of oral samples varied among studies and are reviewed in Table 1. In addition, studies differed on variable regions sequenced. A total of five studies amplified the V3–V4 region,13,17,19,22,25 four studies amplified the V4 region,14,20,21,24 and the remaining studies amplified the V3–V6 region,12 the V3–V5 region,16 the V4–V6 region,18 and V1–V3 region.23

The Oral Microbiome in Preterm Infants

Two studies compared the oral microbiome between preterm and term infants and reported no statistically significant differences in alpha diversity between the groups.24,25 Li et al25 reported no differences in beta diversity, whereas Younge et al24 reported UniFrac distance was less in preterm infants compared to term infants across all body sites (p < 0.01). The relative abundance of oral taxa was evaluated in eight studies, with no clear consensus of a core oral microbiome.13,14,16–19,21,25 Five longitudinal studies evaluated temporal development of the oral microbiome using alpha diversity14,17,20,22,23 and beta diversity measures17,20,22,23 and found day of life was a key factor related to oral microbiome development.

Oral Microbiome and Clinical Covariates

Clinical covariates including birth weight, feeding method, delivery mode, antibiotic exposure, days of mechanical ventilation, days on oxygen supplementation, and skin-to-skin (STS) care were examined for their associations with the oral microbiome in preterm infants. Only one study examined associations between birth weight and the oral microbiome, reporting birth weight affecting oral microbiome composition (p = 0.002).20 While feeding method (oral vs. via a feeding tube) may impact oral microbiome development, only one study evaluated feeding method, reporting breastfeeding had a significant impact on oral microbiome composition (p = 0.001).13

The effect of delivery mode on the oral microbiome was examined in three studies.19,20,25 Orchanian et al20 reported delivery mode had a significant effect on oral microbiome composition (p = 0.024). In contrast, Li et al25 reported the infant oral microbiome was correlated with the maternal vaginal microbiome, regardless of delivery mode and Li et al19 reported no differences in Shannon (p = 0.058) or Simpson (p = 0.3) diversity indices related to delivery mode.

Four studies evaluated the effect of antibiotic exposure with mixed findings.13,16,20,22 One study reported antibiotic exposure had the strongest effect on oral microbiome composition compared to delivery mode, birth weight, or age (p = 0.002),20 while Biagi et al13 reported no effect of antibiotic exposure. Zioutis et al22 reported that antibiotic exposure was a key factor in influencing oral microbiome composition, though it only explained a small amount of community composition variation, in addition to other factors (elevated interleukin-6, day of life, days of mechanical ventilation, and days of oxygen supplementation). However, one study reported antibiotic exposure coincided with expansion of a previously uncultivated Mycoplasma species.16

Only one study examined STS care, defined as holding an infant, clad only in a diaper for a minimum of 1 hour, at least once during the first 21 days of life, and its association with the oral microbiome.18 Infants were categorized as being ≤ or >32 weeks at the time of sampling, though all were born prior to 32 weeks for study inclusion criteria.18 Infants who received STS care were more likely to have an increased relative abundance of Streptococcus (p < 0.024) but there were no significant differences in alpha diversity (reverse Simpson’s index). Infants ≤32 weeks at the time of sampling without STS exposure had increased beta diversity yet this was not found in those >32 weeks.

Oral Microbiome and Associations with the Lung, Gut, and Skin Microbiome

Seven studies evaluated development of the microbiome and associations among body sites. Comparisons were made among the oral, tracheal,12,14 skin,16,22,24 and gut microbiome,13,14,16,22–25 with five studies reporting sampling site was a key factor of microbiome composition.14,16,20,22,24 Brewer et al12 reported that the tracheal microbiome demonstrated a higher alpha diversity than the oral microbiome at 3 days of life (p = 0.02) but at 7 days of life, neither alpha nor beta diversity were significantly different (alpha diversity, Shannon index, p = 0.92). Young et al14 reported the oral and tracheal microbiomes shared approximately a 10% site similarity. Four studies collected both skin and oral samples but only three compared microbiome differences between sites with all finding significant differences.16,20,22,24

Diversity and compositional similarity between oral and gut microbiomes varied among studies with two reporting significant changes over time14,16 and one reporting more similarity as time progressed.13 Furthermore, alpha diversity differed between sites,24 yet microbial composition was reported to be similar.25 Likewise, one study reported the oral microbiome was more similar to the gut14 and one study found amplicon sequencing variants (ASVs) to be more similar in the oral cavity and gut in comparison to other body sites, indicating site similarity due to proximity and/or environmental factors.22

Oral Microbiome and Associations with Oropharyngeal Colostrum Care

Three studies examined the effects of oropharyngeal colostrum care (OPC; placement of human milk onto the buccal mucosa).17,21,23 All three studies used a dose of 0.2 mL,17,21,23 but studies varied regarding frequency of administration (every 2,21 3,17 and 6 h23), time of initiation (within 48 h after birth,17,23 or between 32 and 87 h after birth21), and length of administration (46 h,21 3 d,17 and 5 d23). Studies differed on whether OPC affected microbial alpha or beta diversity.17,23 Cortez et al17 reported signifi-cant differences in alpha diversity measures throughout the course of the intervention with a significant increase in Shannon index for infants who did not receive OPC (p = 0.015) but found no differences in beta diversity. However, Romano-Keeler et al23 found no differences in alpha diversity and similarly, found no differences in beta diversity when OPC was administered. In terms of relative abundance of taxa, studies reported significantly lower Streptococcus (p < 0.05),23 Moraxellaceae (p = 0.03), and Staphylococcacae (p = 0.01);21 Gemella (p < 0.001), Agathobacter (p = 0.041), and Blautia (p = 0.013);17 and increased Haemophilus and Bifidobacterium (p < 0.001)17 in infants who received OPC.

Oral Microbiome and Associations with Health Outcomes

Two studies evaluated whether oral microbiome composition was associated with infant health outcomes.14,22 Zioutis et al22 reported oral microbiome composition was significantly associated with intraventricular hemorrhage (IVH), but no associated ASVs were identified. Young et al14 found no association between oral microbiome composition and either NEC or late-onset sepsis. However, in one study, one very low birth weight infant with presumed but culture-negative sepsis had an increased relative abundance of Pseudomonas aeruginosa and a novel OTU belonging to the Mycoplasma genus.16

Discussion

The purpose of this scoping review was to explore the current methodology and evidence regarding the oral microbiome in preterm infants, including identification of factors associated with oral microbiome development, and associations with health outcomes. We identified a variety of sampling techniques utilized to explore the oral microbiome in preterm infants, found day of life to be important in oral microbiome development, and several exposures which may contribute to oral microbiome composition. However, most studies had small sample sizes and were heterogenous in their methodology making it difficult to draw conclusions regarding development and colonization patterns of the preterm infant’s oral microbiome.

Specifically, sampling techniques among studies varied as did their findings of the relative abundance of certain taxa. Sampling from different oral niches such as subgingival or supragingival plaque in comparison to the tongue or saliva, can harbor different bacteria due to the unique properties of each niche.26 However, we found no evidence-based sampling protocols or recommendations for different sampling sites in predentate infants. Sampling from multiple sites within the oral cavity, including the buccal mucosa, tongue, and saliva, using a sterile swab in predentate infants may help provide insight into the overall composition of the oral microbiome. Given variations in sampling methodologies, it is difficult to draw conclusions and develop frameworks for biologic pathways in which changes to the preterm infant oral microbiome may be associated with health outcomes. As such, standardization of sampling techniques in this population is an important area for future research, as different sampling sites may be associated with different findings.

Day of life was found to be important in oral microbiome development. While sampling times varied, most longitudinal studies reported oral microbiome composition changed over time.14,17,20,22,23 As such, cross-sectional studies miss important temporal dynamics, which may be associated with short- and long-term health outcomes. When making comparisons among studies with oral samples, timing and day of life are critical to examine, as time significantly contributes to alterations in diversity and relative abundance.14,16,17,20,22–24

Several key characteristics relevant to preterm infants including birth weight, delivery mode, exposures to oral feeding, OPC, STS care, and antibiotics may be integral components to oral microbiome development. However, most studies did not evaluate associations between oral microbiome and clinical characteristics nor did they control for these in their analyses. As such, future research should examine how these exposures may interact and influence oral microbiome development in preterm infants.

Only two studies examined associations between the oral microbiome and health outcomes, indicating a dire need for additional research in this area. One study reported an association with IVH, but only 15 infants were included in their analysis.22 No specific ASVs were identified, though a larger sample size may help identify pathogens associated with the oral microbiome and IVH.22 The gut–brain axis may be associated with neurodevelopmental outcomes in preterm infants as certain gram-negative bacteria, may alter immunologic responses which result in neuroinflammation, and thus, may worsen neurological outcomes.27 Seki et al27 found preterm infants with severe brain injury who were more likely to have Klebsiella overgrowth in the gastrointestinal tract, which was associated with increased IL-17a and Vascular Endothelial Growth Factor (VEGF)-A, and thus likely contributed to neuroinflammation, in comparison to preterm infants without brain injury. In addition, Young et al14 reported no associations with NEC or late-onset sepsis and the oral microbiome, although only seven infants were included in this sample. Both research studies highlight the need for larger sample sizes to evaluate associations with the microbiome and health outcomes. Preterm infants often require endotracheal intubation and placement of naso/orogastric feeding tubes which may contribute to the development of pathogenic biofilms in the oral cavity,5,28,29 and unlike adult or pediatric intensive care units, there are no standardized oral care interventions in the NICU, which may further promote oral microbiome dysbiosis.30 As the oral microbiome may play a role in colonizing the gut microbiome, oral microbiome dysbiosis may be a mechanism associated with the gut–brain axis and gastrointestinal disease processes, and exploration of the full gastrointestinal tract should be incorporated into future research studies. We hypothesize that oral microbiome dysbiosis may allow for pathogenic bacteria to colonize in the gut microbiome and/or induce a local or systemic inflammatory response, and thus, contribute to neuroinflammation and/or inflammatory gastrointestinal disease processes, such as NEC. However, the oral–gut–brain axis has been relatively unexplored in preterm infants. As such, future research determining mechanisms associated with the oral microbiome and health outcomes, with larger sample populations, and incorporation of potentially confounding variables is needed.

When comparing the oral microbiome to other body sites and their microbiomes, associations between body sites varied. Based on the findings from this review, the oral microbiome may play a role in seeding the tracheal microbiome and is similar to the gut microbiome, but the mechanisms of these relationships need exploration. We were only able to find seven studies that examined associations among oral and gut/tracheal/skin microbiomes, and sample sizes were small. As the oral microbiome may seed the tracheal and gut microbiomes, the oral cavity may be a critical niche for future interventions to help prevent lung and gut dysbiosis.

The use of OPC is associated with reduced length of hospital stay,23 decreased risk of ventilator-associated pneumonia,31 and faster time to full feeds.32 Within the studies included in this review, frequency and length of administration varied which may lead to inconsistent effects to the oral microbiome. Additionally, it is unclear whether colostrum or more mature MOM was administered, which may be important due to compositional differences between colostrum and more mature milk including increased immunological components in colostrum33 and more diverse microbiome.34 One study also used donor human milk for OPC, which is devoid of live bacteria.17

How OPC effects oral microbiome colonization is yet to be elucidated. Studies reported variations in the relative abundances of taxa, but only two studies evaluated alpha and beta diversity.17,23 As the longest duration of OPC was 5 days,23 no studies examined OPC for preterm infants beyond the first week of life. Since the milk microbiome changes over time and varies among women, this may also affect the infant’s oral microbiome composition after OPC administration.34 No study examined oral care interventions, which are rarely implemented in the NICU. As many preterm infants require long-term intubation due to respiratory immaturity, oral care interventions similar to those practiced in pediatric and adult intensive care units should be considered in clinical practice and evaluated for their effect on the oral microbiome.

Limitations

There are several limitations to the methodology of this review and the included studies. First, this was a scoping review, and thus, our search was broad and only conducted once. Second, many of these studies had small sample sizes, especially those who examined very preterm infants. Thirdly, in terms of sampling sites and timing, the studies included were heterogenous in their methodology. Additionally, studies differed on which hypervariable region of the 16S rRNA gene they amplified and did not evaluate the contribution of DNA or RNA viruses or fungal organisms. Lastly, while 16S rRNA sequencing can identify both culturable and unculturable bacteria, it was unable to differentiate between live and inactive microbes. Despite these limitations, this is the first review to our knowledge to examine the literature regarding the oral microbiome in preterm infants.

Conclusion

We identified a total of 13 articles which met our inclusion criteria and purpose of this scoping review. Within our review, we identified articles that compared preterm infants to term infants, changes to the oral microbiome over time, associations with other body site microbiomes, and factors associated with oral microbiome development. We found that within these studies, findings varied among alpha and beta diversity measures as well as the relative abundance of taxa. This is likely due to the different sampling techniques and timing of collection, as well as the wide range of infant clinical characteristics. With this heterogeneity of methods and results, the evidence is not conclusive on the development as well as short- and long-term implications of the oral microbiome in preterm infants, though the oral microbiome may be associated with adverse outcomes such as IVH. However, this review recognizes the need for future research to identify a standardized method for collecting oral samples, and determining how various early life exposures associated with preterm birth may relate to short- and long-term health outcomes in the preterm infant.

Supplementary Material

Supplemental Material

Key Points.

  • Day of life is a critical factor in oral microbiome development in preterm infants.

  • The oral microbiome may be associated with tracheal and gut microbiome colonization.

  • Future research should examine sampling methodology for examining the oral microbiome.

  • Future research should explore associations with the oral microbiome and adverse health outcomes.

Footnotes

Conflict of Interest

None declared.

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Supplemental Material
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