Fig 1.
Intestinal tissues examined in present study.
Mucosal tissues examined by other methods, such as snip biopsy (A), predominately examine microbes of the mucosal surface; Mucosal (B) and Submucosal (C) tissues examined by the present study examined bacterial populations deep within the diseased tissues.
Table 1.
Differences in the number of OTU’s detected between mucosal and submucosal DNA samples suggesting that inadvertent influence of the submucosa with microbes from the mucosa was minimal.*
Fig 2.
Influence and effect of rRNA gene copy number on the relative abundance of bacterial populations.
Conversion of data to relative percent of population based on the number of 16s rRNA operons in each organism, thereby representing the number of bacteria or genomes, had a major effect on the composition of the bacterial communities within the mucosa and submucosa. Certain bacteria are either over or under represented when a 1:1 ratio is assumed per normal bioinformatics. Similar disparities were found at all bacterial divisions. Legend: Fasta Count: Relative abundance based on the assumption that each rRNA sequence alignment is equivalent to a single bacterial genome/cell as used in most bioinformatics analyses. rrn Operon Count: relative bacterial abundance based on the average number of rRNA operons in each bacterial cell (fasta count/rrn operons) thereby more accurately reflecting bacterial prevalence.
Fig 3.
Submucosal and mucosal microbiota in nIBD controls.
Major bacterial populations and average relative frequencies found in deep mucosal and submucosal ileal tissues from non-inflammatory bowel disease controls accounting for > 95% of the total microbiome. There was no statistically significant increase in bacteria within the submucosa as compared to the mucosa. Percent normalized to whole bacterial genomes based on rrn operon counts.
Fig 4.
Differences in the relative abundance of bacterial Orders (A) and Families (B) in the mucosa of patients with ileal Crohn's disease compared to the ileal mucosa in nIBD controls.
Except for an increase in bacteria of the Order Bacteroidales and Enterobacteriales, mucosa from ileal Crohn's disease patients had a reduced abundance of most other bacteria. There were major differences in most bacterial Families between Crohn's disease and controls. Illustrated are only bacteria comprising > 1% of the total bacterial population and present in >50% of patients.
Fig 5.
Bacterial populations of the diseased submucosa and mucosa at the center of the diseased tissues in Crohn's disease.
The values in parentheses represent the average relative frequency in mucosa/submucosa. An asterisk (*) denotes that the difference between the mucosa and submucosa was statistically significant (p = <0.05) while a plus (+) represents a trend toward statistical significance (p = >0.05 but <0.065).
Fig 6.
Certain bacterial families have a greater tendency to transgress mucosal barriers and colonize submucosal tissues.
Relative fold increase/decrease in the abundance of bacterial Families (A) and Genera (B) within the subjacent ileal submucosa as compared to the parallel superjacent mucosa from tissues in the center of the disease lesion suggesting the ability of certain bacteria to invade and colonize the diseased submucosal tissues. Four families and 8 Genera had a predilection to penetrate mucosal barriers and colonize the submucosa while 4 Families and 3 Genera had a diminished capacity to invade submucosal tissues. Parasutterella spp. (dark blue, panel B) had a particular predilection to penetrate mucosal barriers. Legend: Bacteria present in less than 50% of patients are excluded. All increases/decreases were statistically significant (p = <0.05) except as noted with an asterisk (*) in which p = >0.05 but <0.065 suggesting a trend toward statistical significance.
Fig 7.
Comparison of the relative abundance of bacterial Orders within the submucosa of the diseased ileum in Crohn's disease as compared to the ileal submucosa in nIBD controls.
Various Orders, such as Selenomonadales and Rhizobiales, are absent or poorly represented with the mucosa of Crohn's disease tissue while other orders such as Desulfovibrionales and Coriobacteriales are absent or poorly represented in nIBD controls. Panel A: Relative abundance within submucosal tissues of each bacterial Order in each disease group. Panel B: Relative comparison and contribution of each Order to the total. Legend: Bacteria present in less than 50% of patients are excluded. All data presented are statistically significant (p = <0.05).
Fig 8.
Comparison of the relative abundance of bacterial Families (A) and Genera (B) within the submucosa of the diseased ileum in Crohn's disease as compared to the ileal submucosa in nIBD controls.
There were major changes in 12 families and 20 genera. Legend: Bacteria present in less than 50% of patients are excluded. All data presented are statistically significant (p = <0.05).
Table 2.
Comparison of changes in the mucosal and submucosal microbiome in Crohn's disease versus non-inflammatory bowel disease controls as reported in the present study and in treatment-naïve new-onset patients.