Orofacial clefts alter early life oral microbiome maturation towards higher levels of potentially pathogenic species: A prospective observational study

Abstract

Orofacial clefts (OFC) present different phenotypes with a postnatal challenge for oral microbiota development. In order to investigate the impact of OFC on oral microbiota, smear samples from 15 neonates with OFC and 17 neonates without OFC were collected from two oral niches (tongue, cheek) at two time points, i.e. after birth (T0: Ø3d OFC group; Ø2d control group) and 4-5 weeks later (T1: Ø32d OFC group; Ø31d control group). Subsequently, the samples were analyzed using next-generation sequencing. We detected a significant increase of alpha diversity and anaerobic and Gram-negative species from T0 to T1 in both groups. Further, we found that at T1 OFC neonates presented a significantly lower alpha diversity (lowest values for high cleft severity) and significantly higher levels of Enterobacteriaceae (Citrobacter, Enterobacter, Escherichia-Shigella, Klebsiella), Enterococcus, Bifidobacterium, Corynebacterium, Lactocaseibacillus, Staphylococcus, Acinetobacter and Lawsonella compared to controls. Notably, neonates with unilateral and bilateral cleft lip and palate (UCLP/BCLP) presented similarities in beta diversity and a mixture with skin microbiota. However, significant differences were seen in neonates with cleft palate only compared to UCLP/BCLP with higher levels of anaerobic species. Our findings revealed an influence of OFC as well as cleft phenotype and severity on postnatal oral microbiota maturation.

Keywords: Orofacial clefts; early life dysbiosis; microbiome maturation; neonates; oral microbiome.

Figure 1.

Flow of study participants.

Figure 2.

Dendrogram and genus frequencies plot. A dendrogram based on generalized UniFrac distances is given for each individual sample (excluded samples see Material & Methods) collected from two groups (red: cleft lip palate (‘LKG’/CLP) group; blue: control (‘LKGc’) group), two niches (orange: tongue T; turquoise: cheek C) and two time points (purple: T0, green: T1). A heatmap representing relative abundances (black = no abundance; red = highest abundance) of microbial genera found in individual samples. Samples are ordered vertically according to the dendrogram, while microbial genera are presented horizontally with left to right decreasing mean relative abundance in all samples. The Simpson diversity index representing alpha diversity (Alpha) is shown on the right side for each sample with sample identifier next to it.

Figure 3.

Alpha Diversity. Box and scatter plots of Simpson diversity is given for both time points T0 and T1 (orange = T0; turquoise = T1) for (a) cleft lip palate (CLP) vs. control (CTRL), tongue (T) vs. cheek (C); (b) CLP vs. CTRL; (c) CTRL vs. CLP with low Severity score 2–7 (CLPlow) vs. CLP with high Severity score 8–14 (CLPhigh); (d) CLP vs. control CTRL considering different type of birth vaginal (v) vs. caesarian birth (c). T1). Simpson diversity of individual samples was calculated based on OTU counts (dots). Boxes show 25^th percentile, median and 75^th percentile. Wilcox tests were used to calculate pairwise comparison statistics as indicated. Differences were considered statistically significant for p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

Figure 4.

Beta Diversity. Multidimensional scaling (MDS) plots based on generalized UniFrac distances of log-transformed genus counts are shown for (a) cleft lip palate (CLP) vs. control (Ctrl), tongue (T) vs. cheek (C) and time points T0 vs. T1; (b) Cleft Lip only (CLo) vs. Cleft Palate only (CPo), unilateral cleft lip palate (UCLP), bilateral cleft lip palate (BCLP) and T0 vs. T1 and (c) CLP vs. Ctrl and T0 vs. T1. In (d) a Canonical Correspondence Analysis (CCA) of log-transformed genus counts constrained to group (CLP, Ctrl) and time point (T0, T1) is depicted.

Figure 5.

High level phenotype analysis. Relative abundance is given for cleft lip palate (CLP) vs. control (CTRL) and time points T0 (orange colour) vs. T1 (turquoise colour) regarding (a) aerobic, (b) facultative anaerobic, (c) anaerobic, (c) Gram-negative, (d) Gram-positive, (e) biofilm forming and (f) stress tolerant species. For the significant different groups in (f) median relative abundances are given to aid readability. Wilcox tests were used to calculate pairwise comparison statistics as indicated. Differences were considered statistically significant for p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

Figure 6.

High level phenotype analysis regarding cleft phenotype and severity. Relative abundance is given for different cleft phenotypes Cleft Lip only (CLo), Cleft Palate only (CPo), unilateral cleft lip palate (UCLP), bilateral cleft lip palate (BCLP) and time points T0 (orange colour) vs. T1 (turquoise colour) regarding (a) aerobic, (b) anaerobic, (c) facultatively anaerobic, (d) Gram-negative, (e) Gram-positive species. Relative abundance is given for CLP with low Severity score 2–7 (CLPlow) vs. CLP with high Severity score 8–14 (CLPhigh) and time points T0 (orange colour) vs. T1 (turquoise colour) regarding (f) aerobic, (g) anaerobic, (h) facultatively anaerobic, (i) Gram-negative, (j) Gram-positive species. Wilcox tests were used to calculate pairwise comparison statistics as indicated. Differences were considered statistically significant for p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

Figure 7.

Differential abundance analyses of genera. Volcano plots on genus level is given for a) the cleft lip palate (CLP) vs. control (CTRL) group at T0 and (b) CLP vs. CTRL at T1; d) time point T0 vs. T1 for CTRL group and d) T0 vs. T1 for CLP group. Genus names are given next to colored dots for significant different genera. Dots are colored according to genus phylum as indicated and dashed lines indicate thresholds of significance (>2-fold, adj. p (q) < 0.05).

Figure 8.

Differential abundance analyses of OTUs. Volcano plots on operational taxonomic (OTU) level is given for a) bilateral cleft lip palate (BCLP) vs. unilateral cleft lip palate (UCLP) at T0; b) BCLP vs. UCLP at T1; c) BCLP vs. CPo at T0; d) BCLP vs. CPo at T1; e) UCLP vs. CPo at T0; f) UCLP vs. CPo group at T1. OTU designations are given next to colored dots for significant different OTUs. Dots are colored according to OTU phylum as indicated and dashed lines indicate thresholds of significance (>2-fold, adj. p (q) < 0.05) between the comparators.