The intra- and extraluminal appendiceal microbiome in pediatric patients: A comparative study

Abstract

Intestinal microbiota is involved in metabolic processes and the pathophysiology of various gastrointestinal disorders. We aimed to characterize the microbiome of the appendix in acute pediatric appendicitis comparing extraluminal and intraluminal samples.Between January and June 2015, 29 children (3-17 years, mean age 10.7 ± 3.4 years, sex M:F = 2.6:1) undergoing laparoscopic appendectomy for acute appendicitis were prospectively included in the study. Samples for bacterial cultures (n = 29) and 16S ribosomal desoxyribonucleic acid (rDNA) sequencing (randomly chosen n = 16/29) were taken intracorporeally from the appendiceal surface before preparation ("extraluminal") and from the appendiceal lumen after removal ("intraluminal"). The degree of inflammation was histologically classified into catarrhal, phlegmonous, and gangrenous appendicitis.Seventeen bacterial species were cultivated in 28 of 29 intraluminal samples and 4 species were cultivated in 2 of 29 extraluminal samples. Using 16S rDNA sequencing, 267 species were detected in intraluminal but none in extraluminal samples. Abundance and diversity of detected species differed significantly between histological groups of acute appendicitis in bacterial cultures (P = .001), but not after 16S rDNA sequencing.The appendiceal microbiome showed a high diversity in acute pediatric appendicitis. The intraluminal microbial composition differed significantly depending on the degree of inflammation. As bacteria were rarely found extraluminally by culture and not at all by sequencing, the inflammation in acute appendicitis may start inside the appendix and spread transmurally.

Figure 1

Histological differentiation into (A) catarrhal, (B) phlegmonous, and (C) gangrenous appendicitis using hematoxylin–eosin staining of paraffin-embedded tissue sections. (A) Minimal appendicitis with focal erosion of mucosa and an inflammatory infiltrate only in the submucosal layer. (B) Acute appendicitis with focal ulceration of mucosa, hemorrhage, and an inflammatory infiltrate in the submucosal, muscular, and serosal layer. (C) Extensive ulceration of the mucosa with a loss of mucosa, a massive inflammation of all layers of the wall next to necrotic areas. Magnification: ×25.

Figure 2

Microbiome analyses at phylum level in acute appendicitis. Four different phyla were detected by cultivation and 9 different phyla by 16S rDNA sequencing. No significant differences in abundance were found between the different groups for both methods. DNA reads that could not be assigned to a phylum are represented as “not assigned.”

Figure 3

Microbiome analyses at genus level in acute appendicitis. Eleven genera could be identified by cultivation with the highest abundance of Escherichia in all 3 stages of inflammation, which differed significantly from each other in their compositions (P < .05); 109 genera were found by 16S rDNA sequencing, with highest abundance of Fusobacterium. No significant differences could be detected between the different stages after sequencing. Nine of 11 cultivated genera were also found by 16S rDNA sequencing. Bacterial genera with an abundance ≥2% in at least 1 group were included in the figure. Bacterial genera showing abundance <2% are summarized in “other.” Nonallocable sequences as well as bacterial genera showing an abundance <0.1% are indicated “not assigned.”

Figure 4

Microbiome analyses at species level in acute appendicitis. Seventeen species were found by bacterial culturing with the highest abundance of Escherichia coli in all 3 stages of inflammation, which differed significantly from each other in their composition (P < .05). By 16S rDNA sequencing, 267 species were identified, which did not differ between the histological groups. Species with an abundance of ≥2% in at least 1 group were included in the figure. Bacterial species showing an abundance <2% or detected only once in bacterial culture are summarized in “other.” Nonallocable sequences are indicated “not assigned.”