Culture-independent evaluation of the appendix and rectum microbiomes in children with and without appendicitis

Abstract

Purpose: The function of the appendix is largely unknown, but its microbiota likely contributes to function. Alterations in microbiota may contribute to appendicitis, but conventional culture studies have not yielded conclusive information. We conducted a pilot, culture-independent 16S rRNA-based microbiota study of paired appendix and rectal samples.

Methods: We collected appendix and rectal swabs from 21 children undergoing appendectomy, six with normal appendices and fifteen with appendicitis (nine perforated). After DNA extraction, we amplified and sequenced 16S rRNA genes and analyzed sequences using CLoVR. We identified organisms differing in relative abundance using ANOVA (p<0.05) by location (appendix vs. rectum), disease (appendicitis vs. normal), and disease severity (perforated vs. non-perforated).

Results: We identified 290 taxa in the study's samples. Three taxa were significantly increased in normal appendices vs. normal rectal samples: Fusibacter (p = 0.009), Selenomonas (p = 0.026), and Peptostreptococcus (p = 0.049). Five taxa were increased in abundance in normal vs. diseased appendices: Paenibacillaceae (p = 0.005), Acidobacteriaceae GP4 (p = 0.019), Pseudonocardinae (p = 0.019), Bergeyella (p = 0.019) and Rhizobium (p = 0.045). Twelve taxa were increased in the appendices of appendicitis patients vs. normal appendix: Peptostreptococcus (p = 0.0003), Bilophila (p = 0.0004), Bulleidia (p = 0.012), Fusobacterium (p = 0.018), Parvimonas (p = 0.003), Mogibacterium (p = 0.012), Aminobacterium (p = 0.019), Proteus (p = 0.028), Actinomycineae (p = 0.028), Anaerovorax (p = 0.041), Anaerofilum (p = 0.045), Porphyromonas (p = 0.010). Five taxa were increased in appendices in patients with perforated vs. nonperforated appendicitis: Bulleidia (p = 0.004), Fusibacter (p = 0.005), Prevotella (p = 0.021), Porphyromonas (p = 0.030), Dialister (p = 0.035). Three taxa were increased in rectum samples of patients with appendicitis compared to the normal patients: Bulleidia (p = 0.034), Dialister (p = 0.003), and Porphyromonas (p = 0.026).

Conclusion: Specific taxa are more abundant in normal appendices compared to the rectum, suggesting that a distinctive appendix microbiota exists. Taxa with altered abundance in diseased and severely diseased (perforated) samples may contribute to appendicitis pathogenesis, and may provide microbial signatures in the rectum useful for guiding both treatment and diagnosis of appendicitis.

Figure 1. Participant demographics and clinical data.

Of note, in those patients where an upper GI or no diagnostic imaging was performed, appendicitis was not the preoperative diagnosis and the appendectomy was incidental.

Figure 2. Bray-Curtis Cluster Dendogram.

This abundance-weighted measures how similar two communities are in terms of their genus composition using the Bray-Curtis metric , . 37 samples were analyzed (16 rectal samples and 20 appendix samples). 13 appendix samples clustered together (green box). A group of 10 rectal samples (blue box) clustered separately from the appendix samples, suggesting that the microbiome of the rectum differs from the microbiome of the appendix. Only one pair of rectal-appendix samples from the same subject (subject 10; red arrows) clustered together. The appendix cluster (green box) was composed almost entirely (12 out of 13 samples) of appendicitis samples, both non-perforated and perforated, suggesting that the appendix microbiome associated with appendicitis differs from the microbiome of the normal appendix. For the rectal sample cluster, 8 samples out of 10 samples from patients with appendicitis, both non-perforating and perforating, clustered together, suggesting that the microbiome of the rectum in patients with appendicitis is distinct from the microbiome of the rectum from patients without appendicitis. Samples are listed by ID number, SnX/Y, where n is the subject identification number, X describes the body site (A for appendix, R for rectum), and Y describes the patient's diagnosis (N for normal appendix, NP for an appendix with non-perforating appendicitis, and P for an appendix with perforating appendicitis).

Figure 3. Schematic of Bacterial Genera with Significant Differences in Abundance in Appendix and Rectum, in Patients with and without Appendicitis.

Top row, left to right: bacteria with elevated abundance in the normal appendix compared to the rectum, elevated abundance in appendicitis and elevated abundance in the normal appendix compared to the diseased appendix (appendicitis); Bottom row, left to right: Elevated abundance in the rectum of patient's with appendicitis compared to those with normal rectum samples, elevated abundance in perforated appendicitis compared to non perforated appendicitis and elevated abundance in the normal rectum compared to the normal appendix. The figure lists the taxa in each category; the numbers in parentheses for each heading lists the number of genera in that category.