Spatial organization of bacterial flora in normal and inflamed intestine: A fluorescence in situ hybridization study in mice

Table 1 FISH probes.

Name	Target	Reference
Eub338	Virtually all Bacteria, Kingdom Eubacteria	6
Alf1b	Alpha group of Proteobacteria: Rhodobacter, Acetobacter, Paracoccus	7
Beta42a	Beta subclass of Proteobacteria: Rhodocyclus, Bordetella, Neisseria,Thiobacillus, Alcaligenes and other	7
Gam42a	Gamma subclass of Proteobacteria: Enterobacteriaceae, Proteus, Legionella, Azotobacter	7
Ebac	Enterobacteriaceae	8
Ec1531	Escherichia coli	9
Y16s-69	Yersinia species	10
Srb385	Sulfate reducing bacteria	11
Sgd	Desulfotomaculum subgroup	12
Hpy-1	Helicobacter pylori epsilon subclass of Proteobacteria	13
Arc1430	Arcobacter ssp. epsilon subclass of Proteobacteria	14
HGC	Gram-positive bacteria with high G+C content:Actinobacteria	15
LGC	Gram-positive bacteria with low G+C content: Firmicutes	16
Sfb	Segmented filamentous bacteria	17
Erec	Clostridium coccoides - Eubacterium rectale group	18
Lach	Subgroup of Erec	19
Ehal	Subgroup of Erec	19
Chis150	Clostridium histolyticum group (Incl. Clostridiumperfringens and Clostridium botulinum)	18
Clit135	Clostridium lituseburense group (incl. Clostridium difficile)	18
Lab158	Lactobacillus and Enterococcus group	20
Strc493	Streptococcus group	18
Enc131	Enterococcus spp and other	21
Efaec	Enterococcus faecalis, Enterococcus sulfuricus	22
Ato291	Atopobium, Coriobacterium, Eggerthella and Collinsella spp	23
Cor653	Coriobacterium group	23
Ecyl	Eubacterium cylindroides, Clostridium innocuum, Eubacterium biforme, Eubacterium tortuosum and other	19
Phasco	Phascolarctobacterium faecium, Acidaminococcus fermentans, and Succiniclasticum ruminis	19
Veil	Veillonella group	19
Rbro, Rfla	Clostridium sporosphaeroides, Ruminococcus bromii, andClostridium leptum, Ruminococcus albus and Ruminococcus flavefaciens	19
UroA, UroB	Ruminococcus obeum-like bacteria (subgroup of Erec)	24
Ser1410	Genus Brachyspira	25
Bif164	Bifidobacterium	26
CF319a	Cytophaga-Flavobacteria group	27
Bac303	Bacteroides/Prevotella group	27
Bfra602	Bacteroides fragilis group	18
Bdis656	Bacteroides distasonis group	18
Fprau	Fusobacterium prausnitzii group	28
Dss658	Sulfate reducing bacteria subgroup of delta proteobacteria	29
Arch915	Archaea	30
Non338	Nonsense probe used to test for nonspecific binding	7

Table 2 Spatial organization of intestinal bacteria based on FISH with Eub338 universal bacterial probe.

	Group	Ileum	Cecum	Proximal	Middle	Distal	Rectum
Mean lumen width in	WT	Narrow 0.2	Broad 6.4	Narrowing 2.9	Narrow <0.1	Widening 2.4	Broad 2.5
microscopic fields	DSS	Narrow 0.1	Broad 3.0	Irregular 0.9	Irregular 0.8	Irregular 0.6	Irregular 0.6
(×1000) between	IL-10 C-	Narrow 0.1	Broad 2.1	Irregular 0.6	Irregular 0.5	Irregular 0.7	Irregular 1.2
opposite walls	IL-10 C+	Narrow 0.3	Broad 2.0	Irregular 0.6	Irregular 0.5	Irregular 0.5	Narrow 0.2
Min -max	WT	0.2-2.5×107	>1012	>1012	-	>1012	>1012
concentrations of	DSS	<107	1.3-6.7×109	1.2-4.0×109	2.1-6.6×109	1.8-2.2×109	1.1-2.2×109
bacteria per mL	IL-10 C-	<107	1.0-5.3×1010	1.0-5.2×1010	0.6-3.4×1010	1.2-1.9×1010	1.4-2.3×1010
of feces	IL-10 C+	<107	1.7-5.0×109	0.7-4.2×109	0.7-4.2×109	4.7-7.1×109	4.7-9.2×109
Min -max. length	WT	None	None	8-10%	80-90%	Complete	Complete
of mucus gap	DSS	None	8-20%	40-50%	Complete	Complete	Exudate
separating feces	IL-10 C-	None	5-10%	70-80%	Complete	Complete	Complete
from epithelium	IL-10 C+	None	10-20%	50-80%	Complete	Complete	Exudate
Min-max width	WT	-	0	0- 5	0-20	20-50	30-100
of the mucus in	DSS	-	0- 5	0-10	10-50	40-75	60-150
µm between	IL-10 C-	-	0-10	0-10	15-50	40-100	60-200
epithelium and feces	IL-10 C+	-	0-20	0-20	5-50	60-200	Infiltrate
Mean percent of	WT	-	10% (3-12)	16% (6-16)	5% (single)	<1% (single)	0
crypts with bacteria	DSS	-	22% (4-25)	44% (9-30)	27% (4-15)	16% (2-9)	<1%
(mean-max number of	IL-10 C-	-	<1% (single)	5% (1-3)	2% (single)	1% (single)	No
bacteria within each)	IL-10 C+		28% ( 3-20)	34% (8-34)	27% (2-20)	16% (3-12)	<1%
Min-max thickness	WT	-	0	5-25	0-10	0	0
of the interlaced	DSS	-	0-100	80-400	8-40	0-2	0-2
layer (µm)	IL-10 C-	-	2-10	<5	0	0-2	0
	IL-10 C+		0-120	200-500	80-150	0-15	Infiltrate

Mean number of bacteria within crypts were counted in two different microscopic fields. Only crypts with bacteria were evaluated when mean number of bacteria within crypts was determined.

Table 3 Bacterial groups identified within defined compartments.

FISH Probe	Feces	Interlaced	Crypt
Erec	+	+	+
Alf1b	+	+	+
Lach	+	+	+
Phasco	+	+	+
Cor653	+	+	+
Lab158	+	+/-1	+
Rbro	+	+	-
Rfla	+	+	-
Ehal	+	+/-1	-
Bdis	+	+/-1	-
Bac303	+	-	-
LGC	+	-	-
lit1C35	+	-	-
Chis150	+	-	-
Ecyl	+	-	-
Bif164	+	-	-
Veil	+	-	-
UroB	+	-	-
UroA	+	-	-
HGC	+	-	-
Strc493	+	-	-
Ebac	+	-	-
Arc1430	+	-	-
Enc131	+	-	-
Ato291	+	-	-

¹Composed of different morphotypes one of which could be found within and one outside of the interlaced layer.

Table 4 Mean concentrations of selected bacterial groups comprising more than 10% of the population within the fecal compartment of cecum and rectum.

Probe	Group	Cecum	Rectum
Erec	WT	80×109	0.8×109
	DSS	13×108	0.3×108
	IL-10 C-	15×108	25×108
	IL-10 C+	8×108	12×108
Bac	WT	2.8×108	2.1×108
	DSS	1×108	0.5×108
	IL-10 C-	5×108	2×108
	IL-10 C+ 28 wk	11×108	5×108