Fecal microbiota is associated with extraintestinal manifestations in inflammatory bowel disease

Figures & data

Table 1. Study participant characteristics.

	IBD (n = 131)		CD (n = 81)		UC (n = 50)
	IBD-EIM (n = 86)	IBD-C (n = 45)	CD-EIM (n = 60)	CD-C (n = 21)	UC-EIM (n = 26)	UC-C (n = 24)
Female (n/total (%))*	52/86 (60)*	18/45 (40)*	36/60 (60)	9/21 (43)	16/26 (62)	9/24 (38)
Age (mean (SD))	48 (±13)	44 (±14)	48 (±14)	44 (±13)	48 (±11)	44 (±14)
BMI (kg/m^2) (mean (SD))*	28.1 (±5.3)*	25.6 (±5.3)*	28.4 (±5.4)*	24.8 (±3.9)*	27.4 (±5.2)	26.4 (±6.3)
BMI (n/total (%))*	*	*	*	*
Underweight (<18.5)	0/86 (0)	1/45 (2)	0/60 (0)	1/21 (5)	0/26 (0)	0/24 (0)
Normal (18.5–24.9)	26/86 (30)	27/45 (60)	17/60 (28)	12/21 (57)	15/26 (58)	9/24 (38)
Overweight (25–29.9)	35/86 (41)	11/45 (25)	24/60 (40)	6/21 (29)	5/26 (19)	11/24 (46)
Obese (>30)	25/86 (29)	6/45 (13)	19/60 (32)	2/21 (9)	6/26 (23)	4/24 (16)
Years since IBD diagnosis (mean (SD))	15 (±9)	13 (±8)	16 (±9)	12 (±6)	14 (±8)	14 (±9)
Family disposition to IBD (n/total (%))	28/84 (33)	14/29 (33)	34/58 (40)	4/20 (20)	5/26 (20)	10/23 (43)
IBD related hospitalization (n/total (%))*	65/85 (76)*	20/45 (44)*	49/59 (83)*	11/21 (52)*	16/26 (62)	9/24 (38)
IBD related surgery (n/total (%))	32/86 (37)	12/45 (27)	29/60 (48)	12/21 (57)	3/26 (12)	0/24 (0)
Montreal classification
A: age of diagnosis (n/total (%))
A1: below 16 years	5/86 (6)	6/45 (13)	5/60 (8)	1/21 (5)	0/26 (0)	5/24 (21)
A2: between 17 and 40 years	67/86 (78)	29/45 (65)	48/60 (80)	14/21 (66)	19/26 (73)	15/24 (63)
A3: above 40 years	14/86 (16)	10/45 (22)	7/60 (12)	6/21 (29)	7/26 (27)	4/24 (16)
L: disease location in CD (n/total (%))
L1: ileal			7/58 (12)	2/21 (10)
L2: colonic			28/58 (48)	12/21 (57)
L3: ileocolonic			14/58 (26)	3/21 (14)
L4: upper GI involvement			9/58 (16)	4/21 (19)
B: behaviour in CD (n/total (%))
B1: non-stricturing, non-penetrating			47/58 (81)	14/21 (67)
B2: stricturing			6/58 (10)	3/21 (14)
B3: penetrating			5/58 (9)	4/21 (19)
E: disease extent in UC (n (%))*					*	*
E1: ulcerative proctitis					3/26 (11)	6/24 (25)
E2: left sided colitis					7/26 (27)	12/24 (50)
E3: extensive colitis					16/26 (62)	6/24 (25)
Treatment
No treatment (n/total (%))	5/86 (6)	6/45 (13)	3/60 (5)	4/21 (19)	2/26 (8)	2/24 (8)
Standard treatment (n/total (%))*	37/86 (43)*	29 (64)*	17/60 (28)	9/21 (43)	20/26 (77)	20 (83)
5-ASA (topical/systemic)*	22/86 (26)*	20/45 (44)*	4/60 (7)	0/21 (0)	18/26 (70)	19/24 (79)
Corticosteroids (topical/systemic)	3/60 (5)	1/45 (2)	2/60 (3)	0/21 (0)	1/26 (4)	1/24 (4)
Azathioprine*	11/75 (13)*	24/45 (29)*	10/60 (17)	7/21 (33)	1/26 (4)*	6/24 (24)*
Mercaptopurine	2/86 (2)	1/45 (2)	1/60 (2)	1/21 (5)	1/26 (4)	1/24 (7)
Methotrexate	5/86 (6)	0/45 (0)	2/60 (3)	0/21 (0)	3/26 (12)	0/24 (0)
Biologics and targeted small molecule drugs (n/total (%))*	70/86 (81)*	23/45 (51)*	56/60 (93)	14/21 (67)	14/26 (54)	9/24 (38)
TNF-α inhibitors*	55/86 (64)	26/45 (58)	41/60 (68)	13/21 (62)	14/26 (54)*	6/24 (25)*
Vedolizumab	11/86 (13)	2/45 (4)	11/60 (18)	1/21 (5)	1/26 (4)	0/24 (0)
Ustekinumab	4/86 (5)	0/45 (0)	4/60 (7)	0/21 (0)	0/26 (0)	0/24 (0)
Other	2/86 (2)	0/45 (0)	1/60 (2)	0/21 (0)	0/26 (0)	1/24 (4)
Quality of life/disease activity
Short Health Score*	144 (±92)*	68 (±73)*	153 (±95)*	77 (±96)*	123 (±85)*	60 (±44)*
Harvey–Bradshaw Index (HBI)*			*	*
Remission (<5)			30/59 (51)	17/21 (81)
Mild activity (5–7)			15/59 (25)	4/21 (19)
Moderate activity (8–16)			14/59 (24)	0/21 (0)
Severe activity (>16)			0/59 (0)	0/21 (0)
Simple Clinical Colitis Activity Index (SCCAI)
Remission (≤2)					16/26 (62)	18/24 (75)
Mild (3–5)					7/26 (27)	6/24 (25)
Moderate (6–11)					4/26 (15)	0/24 (0)
Severe (≥12)					0/26 (0)	0/24 (0)

* p Value < .05. TNF-α inhibitors: Tumor Necrosis Factor alpha inhibitor; 5-ASA: 5-aminosalicylic acid.

Table 2. Extraintestinal manifestations.

	EIM (n = 86)	CD-EIM (n = 60)	UC-EIM (n = 26)
Number of EIMs
1	60	39	21
2	22	18	4
≥3	4	2	1
EIM at incl.	70	49	21
Types of EIMs
Arthropathies	64	45	19
Spondyloarthritis (SpA)	34	27	7
Arthropathy	30	18	12
Ocular manifestations	25	19	6
Uveitis*	19	15	4
Other	6	4	2
Hepatobiliary manifestations	14	6	8
PSC	10	4	6
Autoimmune hepatitis	4	2	2
Skin manifestations	12	12	0
Erythema nodosum	12	12	0
Other	28	26	2

*Incl. anterior uveitis, iridocyclitis, posterior uveitis.

Figure 1. Blood monocyte concentration is higher in IBD patients with EIMs. (A) Monocyte concentration (×10⁹/L) between IBD-EIM and IBD-C. Values above the redline are above reference levels and represents monocytosis (>0.70 × 10⁹/L). (B) Monocyte concentration by EIM count (0, 1, ≥2). (C) Monocyte concentration between CD-EIM and CD-C. (D) Monocyte concentration between UC-EIM and UC-C. (E) Monocytosis in IBD-EIM vs. IBD-C. (F) Monocytosis by EIM count. (G) Monocytosis between CD-EIM and CD-C. (H) Monocytosis between UC-EIM and UC-C. MW: Mann–Whitney’s test; KW: Kruskal–Wallis test; FE: Fisher’s exact test. Monocytes were not available for all participants, leading to reduced group sizes for this analysis: IBD-EIM (n = 85), IBD-C (n = 40), CD-EIM (n = 59), CD-C (n = 18), UC-EIM (n = 26) and UC-C (n = 22). p Values < .05 were considered significant.

Figure 2. Fecal microbiota amplicon sequence variants (ASVs) differ between inflammatory bowel diseases patients with (IBD-EIM) or without (IBD-C) extraintestinal manifestations (EIM). (A) Alpha-diversity indices between IBD-EIM (red) and IBD-C (blue) (Pielou’s evenness, p = .01, Shannon’s diversity, p = .02). (B) Principal coordinate analysis plot depicts beta-diversity distance matrices between IBD-EIM (red) and IBD-C (blue) patients (weighted UniFrac, PERMANOVA; R² = 0.02, p = .01). (C) Volcano plot of significant ASVs between IBD-EIM and IBD-C. Significant ASVs (p.fdr < .05, LogFC > 1) are coloured by genus or the highest taxonomic level identified. Significantly enriched ASVs in IBD-EIM (n = 5) are depicted on the right axis and ASVs depleted in IBD-EIM (n = 19) are depicted on the left axis.

Table 3. Significant ASVs between IBD-EIM and IBD-C.

ASV	p.fdr	Log2 FC	Mean diff.	Phylum	Family	Genus	Species
ASV_20	.0344	−9.14	−563	Firmicutes	Lachnospiraceae	Blautia	obeum
ASV_36	.0112	−8.94	−490	Firmicutes	Lachnospiraceae	Agathobacter	NA
ASV_48	.0292	−8.34	−324	Bacteroidota	Bacteroidaceae	Bacteroides	vulgatus
ASV_91	.0002	−8.21	−296	Firmicutes	Ruminococcaceae	Faecalibacterium	NA
ASV_94	.0221	−6.94	−123	Bacteroidota	Bacteroidaceae	Bacteroides	uniformis
ASV_204	.0016	−6.87	−117	Bacteroidota	Bacteroidaceae	Bacteroides	fragilis/ koreensis/ kribbi/ovatus
ASV_241	.0001	−6.52	−92	Bacteroidota	Tannerellaceae	Parabacteroides	distasonis
ASV_233	.0221	−6.49	−90	Firmicutes	Lachnospiraceae	Lachnoclostridium	NA
ASV_50	.0368	−6.37	−83	Bacteroidota	Prevotellaceae	Prevotella	copri
ASV_158	.0004	−6.33	−81	Firmicutes	Lachnospiraceae	Agathobacter	NA
ASV_108	.0007	−6.33	−80	Bacteroidota	Rikenellaceae	Alistipes	putredinis
ASV_272	.0028	−6.28	−78	Firmicutes	Ruminococcaceae	NA	NA
ASV_192	.0020	−6.11	−69	Firmicutes	Lachnospiraceae	Roseburia	NA
ASV_307	.0005	−5.70	−52	Firmicutes	Lachnospiraceae	Lachnoclostridium	edouardi
ASV_202	.0344	−5.26	−38	Firmicutes	Ruminococcaceae	Faecalibacterium	NA
ASV_271	.0004	−5.13	−35	Firmicutes	Lachnospiraceae	NA	NA
ASV_342	.0003	−4.82	−28	Proteobacteria	Sutterellaceae	Sutterella	massiliensis/ stercoricanis/ wadsworthensis
ASV_284	.0170	−4.54	−23	Firmicutes	Lachnospiraceae	Agathobacter	NA
ASV_267	.0368	−4.13	−18	Firmicutes	Lachnospiraceae	Agathobacter	NA
ASV_242	.0001	4.45	22	Firmicutes	NA	NA	NA
ASV_217	.0450	4.46	22	Firmicutes	Anaerovoracaceae	Family_XIII_AD3011_group	NA
ASV_296	.0221	4.67	25	Firmicutes	Ruminococcaceae	NA	NA
ASV_93	.0002	6.84	115	Firmicutes	Lachnospiraceae	NA	NA
ASV_83	.0074	7.10	137	Actinobacteriota	Eggerthellaceae	Eggerthella	lenta

Figure 3. Fecal microbiota bacterial networks differ between IBD-EIM and IBD-C. Twenty-seven bacterial networks were identified using sparse correlations for compositional data (SparCC) on amplicon sequence variants (ASVs) with a correlation cutoff of 0.5. Three bacterial networks (module 7 (n = 16), module 8 (n = 6) and module 23 (n = 2)) were significantly different between IBD-EIM and IBD-C (logistic regression, p < .05), and all were negatively correlated with IBD-EIM. Significant modules are marked in dark red, and non-significant modules are marked in grey.

Table 4. Significant bacterial network modules with correlation ≥0.5.

Module	Module size	p Value	ASV members
Module 7	16	.004	Blautia obeum Blautia faecis Dorea longicatena Erysipelotrichaceae_UCG-003 bacterium Erysipelotrichaceae_UCG-003 NA Faecalibacterium cf./prausnitzii Faecalibacterium prausnitzii Faecalibacterium prausnitzii Faecalibacterium NA Faecalibacterium NA Faecalibacterium NA Faecalibacterium NA Faecalibacterium NA Monoglobus NA Ruminococcaceae NA Ruminococcaceae NA
Module 8	6	.008	Agathobacter NA Agathobacter NA Agathobacter NA Agathobacter NA Agathobacter NA Fusicatenibacter saccharivorans
Module 25	2	.025	Bacteroides vulgatus Bacteroides NA

Data availability statement

The data supporting the findings of this study are available from the corresponding author [S.H.] upon reasonable request.