Background
Respiratory complications are a common extra-intestinal manifestation of esophageal atresia (EA). The underlying pathophysiology behind these complications is yet to be understood. EA patients’ increased predisposition to esophageal dysmotility, severe gastroesophageal reflux disease and reflux aspiration is suspected to contribute to these complications, potentially leading to airway dysbiosis and inflammation. This study aims to assess the degree of airway dysbiosis and inflammation present in children with EA compared to healthy controls (HC) and explore its association with reflux and respiratory outcomes.

Methods
As a part of the Evaluating the Alimentary and Respiratory Tracts in Health and disease (EARTH) research program, this prospective, cross-sectional, controlled, observational study involved the collection of clinical data and oropharyngeal swabs from children with EA and HC, aged 0 to 17 years. 16S rRNA gene sequencing (V4 region) was performed. Oropharyngeal S100A12 and pepsin levels were measured using enzyme linked immunosorbent assays. Targeted liquid chromatography mass spectrometry was utilized to measure bile acids. Associations between airway microbiota, inflammation, markers of reflux and respiratory outcomes were analyzed.

Results
Airway microbiome analysis of EA (n=44) and age matched HC (n=44) samples indicated children with EA had a lower mean bacterial richness of 96.7(SD 29.3) compared to the HC cohort, 121.3(SD 29.7) (p=2.8 × 10^-7). The Shannon diversity index was also significantly lower in the EA cohort, 2.47(SD 0.52), compared to the HC cohort, 2.80(SD 0.45) (p=0.001). Beta diversity analysis indicated significant differences in bacterial composition between the HC and EA cohorts. Children with EA who experienced a chronic cough, had decreased bacterial richness, Shannon diversity and significant differences in beta diversity compared to patients without a cough. Oropharyngeal S100A12 levels were higher in patients with EA (n=30) compared to HC (n=30), 50.7 ng/ml (IQR 14.0 – 100.8) vs 14.7 ng/ml (IQR 7.8 - 43.2), respectively (p=0.02). There was a positive correlation between pepsin and S100A12 levels in the EA cohort (r=0.38 p=0.04). Several bile acid concentrations including cholic, taurocholic, glycocholic, taurodeoxycholic and taurochenodeoxycholic acid, were significantly higher in children with EA (n=12) compared to HC (n=12). However, bile acid concentrations did not significantly correlate with pepsin or S100A12 levels.

Conclusion
Children with EA have an altered oropharyngeal microbiota and higher levels of inflammation compared to HC. Positive correlations of pepsin and S100A12 levels suggest a potential role of reflux in the development of airway inflammation in EA.

<b>Figure 1.</b> <b>Alpha Diversity in Esophageal Atresia and Healthy Control Cohorts </b><br /> Boxplots of EA and HC cohorts comparing sample richness (number of zOTUS) (p=2.8 × 10<sup>-7</sup>) (<b>A</b>) and Shannon index (p=0.001) (<b>B</b>). Scatterplots of EA and HC sample richness (number of zOTUs) against age (p=0.0002) (<b>C</b>) and Shannon Index (p=0.004) (<b>D</b>). The grey shaded regions represent the 95% confidence intervals constructed from generalized linear models, each line representing the mean.<br /> Abbreviations: EA, Esophageal Atresia; HC, Healthy Controls; zOTU, zero-distance operational taxonomic unit

Figure 1. Alpha Diversity in Esophageal Atresia and Healthy Control Cohorts
Boxplots of EA and HC cohorts comparing sample richness (number of zOTUS) (p=2.8 × 10^-7) (A) and Shannon index (p=0.001) (B). Scatterplots of EA and HC sample richness (number of zOTUs) against age (p=0.0002) (C) and Shannon Index (p=0.004) (D). The grey shaded regions represent the 95% confidence intervals constructed from generalized linear models, each line representing the mean.
Abbreviations: EA, Esophageal Atresia; HC, Healthy Controls; zOTU, zero-distance operational taxonomic unit

<b>Figure 2. Oropharyngeal</b> <b>S100A12 levels between</b> <b>Esophageal Atresia and Healthy Control Cohorts </b><br /> Scatterplot of oropharyngeal S100A12 levels against age in the EA and HC cohorts (p=0.02)<b>.</b> The grey shaded regions represent the 95% confidence intervals constructed from generalized linear models, each line representing the mean.<br /> Abbreviations: EA, Esophageal Atresia; HC, Healthy Controls

Figure 2. Oropharyngeal S100A12 levels between Esophageal Atresia and Healthy Control Cohorts
Scatterplot of oropharyngeal S100A12 levels against age in the EA and HC cohorts (p=0.02). The grey shaded regions represent the 95% confidence intervals constructed from generalized linear models, each line representing the mean.
Abbreviations: EA, Esophageal Atresia; HC, Healthy Controls

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