Fluctuations in butyrate-producing bacteria in ulcerative colitis patients of North India

Table 1 Clinical and demographic features of ulcerative colitis patients and controls n (%)

Feature	UC (n = 26)	Control (n = 14)
Sex (F/M)	11 (42.30)/15 (57.69)	7 (50)/7(50)
Age at diagnosis (yr), mean ± SD	38.35 ± 11.49	35.00 ± 14.14
15-40	18 (69.23)	8 (57.14)
> 40	8 (30.76)	6 (42.85)
Disease behavior
Severe	12 (46.15)	-
Moderate	6 (23.07)	-
Remission	8 (30.76)	-
Disease extent
Proctitis	4 (15.38)	-
Left sided colitis	6 (23.00)	-
Pancolitis	6 (23.00)	-
None of the above	10 (38.46)	-
Smoking history
Yes	3 (11.53)	2 (14.28)
No	23 (88.46)	12 (85.70)
Treatment history
Immunosuppressant	16 (61.53)	-
Steroids	14 (53.84)	-
Appendectomy Y/N	4 (15.38)/22 (84.61)	0/14
Family history Y/N	2 (7.69)/24 (92.30)	0/14

UC: Ulcerative colitis; F: Female; M: Male; Y: Yes; N: No.

Table 2 Probes and oligonucleotides employed in the study

Primer or probe	Target (phylogenetic group)	Sequence (5’-3’) from 16S rRNA gene	Used in FISH-flow cytometry or qPCR	Ref.
NON338	No bacteria	ACATCCTACGGGAGGC	Probe1	[21]
EUB338	Most bacteria	GCTGCCTCCCGTAGGAGT	Probe1	[20]
Erec 482	C. coccoides/E.rectale cluster	GCTTCTTAGTCARGTACCG	Probe1	[34]
Clep 1156	Clostridium leptum subgroup	GTTTTRTCAACGGCAGTC	Probe1	[35]
Fpra-655	F. prausnitzii	CGCCTACCTCTGCACTAC	Probe1	[16]
Rint-623	R. intestinalis subcluster	TTCCAATGCAGTACCGGG	Probe1	[16]
Ehal-057	E. halliii L2-7/E. halliii	TTGCACTGCCACCTACGC	Probe1	[16]
Cp1	Competitor 1	GRTTTRTCAYCGGCAGTC	Competitor1	[23]
Cp2	Competitor 1	GTVTTRTCBACGGCAGTC	Competitor1	[23]
H1174	Helper oligonucleotide	TTGACGTCRTCCCCACCTTCCTCC	Helper1	[23]
H1129	Helper oligonucleotide	TAGAGTGMTCTTGCGTA	Helper1	[23]
H1090	Helper oligonucleotide	GGTTGCGCTCGTTGCGGGACTTAA	Helper1	[23]
H750	Helper oligonucleotide	TCGHGCCTCAGCGTCAG	Helper1	[23]
H122	Helper oligonucleotide	GAAGGCAGGTTACTCACGC	Helper1	[23]
Clep FP	C. leptum subgroup	CGTCAGCTCGTGTCGTGAGAT	Primer set2	[36]
Clep RP		CGTCATCCCCACCTTCCTCC
C.cocci FP	C. coccoides subgroup	GCCACATTGGGACTGAGA	Primer set2	[36]
C.cocci RP		GCTTCTTAGTCAGGTACCG
Fpraus FP	F. prausnitzii	GATGGCCTCGCGTCCGATTAG	Primer set2	[37]
Fpraus RP		CCGAAGACCTTCTTCCTC
Rint FP	Roseburia/E. rectale cluster	CKGCAAGTCTGATGTGAAAG	Primer set2	This study
Rint RP		GCGGGTCCCCGTCAATTCC
Ehal FP	E. hallii L2-7/E. hallii members	GCGTAGGTGGCAGTGCAA	Primer set2	[38]
Ehal RP		GCACCGRAGCCTATACGG

¹Respective probe or oligonucleotide was used in fluorescent in situ hybridization (FISH)-flow cytometry;

²Respective primer was used in quantitative polymerase chain reaction (qPCR). FP: Forward primer; RP: Reverse primer; R. intestinalis: Roseburia intestinalis; E. hallii: Eubacterium hallii; F. prausnitzii: Fecalibacterium prausnitzii; C. coccoides: Clostridium coccoides; C. leptum: Clostridium leptum; E. rectale: Eubacterium rectale.

Table 3 Accession number of reference strain used in the study

Bacteria	Source	Accession No.
C. leptum	Healthy human fecal sample	AM042697
F. prausnitzii	Healthy human fecal sample	JX556686
R. intestinalis	Healthy human fecal sample	JX556688
E. hallii	Healthy human fecal sample	JX556687

C. leptum:Clostridium leptum;F. prausnitzii:Fecalibacterium prausnitzii;R. intestinalis:Roseburia intestinalis; E. hallii:Eubacterium hallii.

Table 4 Enumeration of butyrate producers in fecal samples through fluorescent in situ hybridization-flow cytometry (mean ± SE)

Cluster or species	Out of total microbiota	Species	Out of respective cluster
Control fecal samples
C. coccoides cluster	25.69% ± 1.62%	R. intestinalis	14.48% ± 1.52%
		E. hallii	5.93% ± 0.54%
C. leptum cluster	13.74% ± 1.05%	F. prausnitzii	11.66% ± 1.55%
R .intestinalis1	3.71% ± 0.39%
E. hallii1	1.52% ± 0.13%
F. prausnitzii1	1.60% ± 0.21%
UC fecal samples (moderate)
C. coccoides cluster	12.70% ± 4.60%	R. intestinalis	10.00% ± 2.25%
		E. hallii	4.03% ± 0.3%
C. leptum cluster	6.40% ± 3.40%	F. prausnitzii	7.5% ± 2.3%
R .intestinalis1	1.27% ± 0.10%
E. hallii1	0.51% ± 0.01%
F. prausnitzii1	0.48% ± 0.14%
UC fecal samples (severe)
C. coccoides cluster	9.80% ± 2.40%	R. intestinalis	9% ± 1.83%
		E. hallii	4.2% ± 0.2%
C. leptum cluster	6.20% ± 1.80%	F. prausnitzii	6.01% ± 1.6%
R .intestinalis1	0.82% ± 0.04%
E. hallii1	0.41% ± 0.01%
F. prausnitzii1	0.37% ± 0.02%
UC fecal samples (remission)
C. coccoides cluster	12.99% ± 2.65%	R. intestinalis	11.40% ± 1.83%
		E. hallii	5.07% ± 0.6%
C. leptum cluster	7.89% ± 1.50%	F. prausnitzii	7.40% ± 2.32%
R .intestinalis1	1.48% ± 0.04%
E. hallii1	0.65% ± 0.01%
F. prausnitzii1	0.58% ± 0.03%

¹Abundance of Roseburia intestinalis (R. intestinalis), Eubacterium hallii (E. hallii) and Fecalibacterium prausnitzii (F. prausnitzii) were calculated out of total microbiota. Clostridium coccoides (C. coccoides) and Clostridium leptum (C. leptum) were enumerated as proportion of total bacterial cell hybridized with group-specific probe. R. intestinalis, E. hallii and F. prausnitzii were enumerated as proportion of cell hybridized with species-specific probe. UC: Ulcerative colitis.