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. 2015 Dec;21(12):2797-805.
doi: 10.1097/MIB.0000000000000557.

Alterations of the Subgingival Microbiota in Pediatric Crohn's Disease Studied Longitudinally in Discovery and Validation Cohorts

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Alterations of the Subgingival Microbiota in Pediatric Crohn's Disease Studied Longitudinally in Discovery and Validation Cohorts

Judith Kelsen et al. Inflamm Bowel Dis. 2015 Dec.

Abstract

Background: Oral manifestations are common in Crohn's disease (CD). Here we characterized the subgingival microbiota in pediatric patients with CD initiating therapy and after 8 weeks to identify microbial community features associated with CD and therapy.

Methods: Pediatric patients with CD were recruited from The Children's Hospital of Pennsylvania. Healthy control subjects were recruited from primary care or orthopedics clinic. Subgingival plaque samples were collected at initiation of therapy and after 8 weeks. Treatment exposures included 5-ASAs, immunomodulators, steroids, and infliximab. The microbiota was characterized by 16S rRNA gene sequencing. The study was repeated in separate discovery (35 CD, 43 healthy) and validation cohorts (43 CD, 31 healthy).

Results: Most subjects in both cohorts demonstrated clinical response after 8 weeks of therapy (discovery cohort 88%, validation cohort 79%). At week 0, both antibiotic exposure and disease state were associated with differences in bacterial community composition. Seventeen genera were identified in the discovery cohort as candidate biomarkers, of which 11 were confirmed in the validation cohort. Capnocytophaga, Rothia, and TM7 were more abundant in CD relative to healthy controls. Other bacteria were reduced in abundance with antibiotic exposure among CD subjects. CD-associated genera were not enriched compared with healthy controls after 8 weeks of therapy.

Conclusions: Subgingival microbial community structure differed with CD and antibiotic use. Results in the discovery cohort were replicated in a separate validation cohort. Several potentially pathogenic bacterial lineages were associated with CD but were not diminished in abundance by antibiotic treatment, suggesting targets for additional surveillance.

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Conflict of interest statement

The authors have no potential conflicts to disclose.

Figures

Figure 1
Figure 1. Summary of bacterial phyla observed in the discovery and validation cohorts
Proportions of the five dominant bacterial phyla identified in 16S tag sequencing results from subgingival brushing samples. Patients with CD were sampled prior to initiation of therapy and again after 8 weeks. CD subjects currently receiving antibiotics (CD Abx+) are summarized separately from other CD patients (CD Abx-). Healthy control subjects (Control Abx-) were sampled at the same time interval and were not exposed to antibiotics during the study or 2 months prior. The study was repeated using separate (A) discovery and (B) validation cohorts.
Figure 2
Figure 2. Analysis of bacterial community composition in the discovery and validation cohorts
Communities were compared to determine differential membership using unweighted UniFrac distance (A, B) or community proportional abundance using weighted UniFrac distance (C, D). Samples were ordinated using principal coordinate analysis. Open triangles represent the group centroid position for healthy control subjects (blue), CD patients not on antibiotics (red), and CD patients on antibiotics (green). The PERMANOVA test was used to assess association of sample-sample distance with disease state (CD) and current antibiotic usage (Abx).
Figure 3
Figure 3. Genera identified as candidate biomarkers in the discovery cohort and confirmed in the validation cohort
(A) Candidate genera differing between healthy control subjects (Control Abx-), CD subjects not on antibiotics (CD Abx-), and CD subjects currently on antibiotics (CD Abx+) in the discovery cohort at week 0 (* P<0.05, ** P<0.01, *** P<0.001). (B) Genera confirmed to be differing in the validation cohort at week 0. Confirmed genera are color-coded according to phylum.
Figure 4
Figure 4. Antibiotic use is associated with lower bacterial diversity
(A) Rarefaction curves representing the expected number of Operational Taxonomic Units (OTUs) observed in 16S sequencing results from healthy controls (Control Abx-), CD patients not on antibiotics (CD Abx-), and CD patients receiving antibiotics (CD Abx+) at week 0 in the discovery cohort. (B) Boxplots showing expected number of OTUs at 1000 reads per sample in the discovery cohort at week 0. (C) Rarefaction curves and (D) boxplots for the validation cohort at week 0. In both cohorts, CD patients on antibiotics have fewer OTUs per sample than CD patients not on antibiotics or healthy controls (Mann-Whitney test, ** P<0.01, *** P<0.001).
Figure 5
Figure 5. A random forest classifier trained on Discovery Cohort samples
(A) Probability of CD assignment for samples in the validation cohort at week 0 based on a random forest classifier trained on the week 0 discovery cohort data. (B) The receiver operating characteristic curve for the classifier, which plots the true positive rate against the false positive rate. A perfect classifier would have an “area under the curve” of 1, and a random classifier would score 0.5. (C) The 20 most important OTUs for classification are shown, ordered by an out-of-bag estimate for mean decrease in accuracy when that OTU is removed. The fold difference in OTU proportion is shown to the right: OTUs that are more abundant in CD samples appear to the right of the dashed line.

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