Diarrhoea-predominant irritable bowel syndrome distinguishable by 16S rRNA gene phylotype quantification

Table 1 Characteristics of IBS patients and control subjects

	IBS-D	IBS-C	IBS-M	Controls
n	8	8	4	15
Age (yr): mean (range)	43.6 (26-60)	48.6 (24-64)	50.8 (31-62)	47 (25-64)
Gender (female/male)	4/4	9/1	3/1	10/5
Predominant bowel habit	Diarrhoea	Constipation	Mixed-type	-

IBS: Irritable bowel syndrome; IBS-D: Diarrhoea-predominant IBS; IBS-C: Constipation-predominant IBS; IBS-M: Mixed symptom-subtype IBS.

Table 2 Phylum level classification, primers and assay conditions of qPCR assays

qPCR assay (Phylum)	Primers (5’→3’)	Standard	Target size (bp)	MgCl2 (mmol/L)	Annealing T (°C)	Detection T (°C)	Average PCR efficiency ± SD
Bacteroides intestinalis-like (Bacteroidetes)	F: AGCATGACCTAGCAATAGGTT	AM277809	124	3	63	83	87 ± 6
	R: CCTTCTCGTTATACTATCCGGTAT
Bifidobacterium catenulatum/Bifidobacterium pseudocatenulatum-like[11] (Actinobacteria)	F: ACTCCTCGCATGGGGTGTC	AM277149	275	3	68	87	90 ± 8
	R: CCGAAGGCTTGCTCCCGAT
Butyrivibrio crossotus-like (Firmicutes)	F: TGCTAATACCGCATAAAACAGCAGA	AM275497	232	4	63	85	82 ± 5
	R: CGCTGGATCAGGCTTTCG
Clostridium cocleatum 88%[11] (Firmicutes)	F: AATACATAAGTAACCTGGCRTC	AM276544	104	4	60	80	88 ± 10
	R: CGTAGCACTTTTCATATAGAGTT
Clostridium thermosuccinogenes 85% (Firmicutes)	F: ACATGCAAGTCGAACGGAAGTC	AM275406	373	2	62	81	88 ± 8
	R: TGCGTCAGAGTTTCCTCCATTG
Collinsella aerofacien-like[11] (Actinobacteria)	F: CCCGACGGGAGGGGAT	AM276090	260	4	67	89	75 ± 3
	R: CTTCTGCAGGTACAGTCTTGAC
Coprobacillus catenaformis 91% (Firmicutes)	F: CGGACGCGATGCTTCT(A/G)GC	AM275478	133	4	62	82	93 ± 8
	R: AACATATCTCCCATGCGGTTG
Coprococcus eutactus 97%[11] (Firmicutes)	F: AGCTTGCTCCGGCYGATTTA	AM275825	97	2	63	83	73 ± 5
	R: CGGTTTTACCAGTCGTTTCCAA
Ruminococcus bromii-like (Firmicutes)	F: CGAACGGAACTGTTTTGAAAGA	AM275413	156	4	62	81	97 ± 6
	R: CAAAACCATGTGGTTCCGATAT
Ruminococcus torques 91%[11] (Firmicutes)	F: TGCTTAACTGATCTTCTTCGGA	AM276624	119	5	62	82	88 ± 5
	R: CGGTATTAGCAGTCATTTCTG
R. torques 93%1(Firmicutes)	F: GACTGCTTTTGAAACTGTCA	AM275798	396	4	61	83	85 ± 4
	R: AGGTCCGGTTAAGGA
R. torques 94%[11] (Firmicutes)	F: AATCTTCGGAGGAAGAGGACA	AM275522	137	2	65	85	81 ± 6
	R: ACACTACACCATGCGGTCCT
Slackia faecicanis 91% (Actinobacteria)	F: GAGTAACGCGTGACCGACCTT	AM276086	75	4	64	86	90 ± 5
	R: CCCGGAGTACCCGGTATCA
Spiroplasma chinense 84% (Firmicutes)	F: ATGGCCCAGTGAAGGTTG	AM275518	101	4	66	83	79 ± 4
	R: CCCAACGAAAAGGTAGGTCA
Universal[61]	F: TCCTACGGGAGGCAGCAGT	B. longum2	466	3	50	80	92 ± 6
	R: GGACTACCAGGGTATCTAATCCTGTT

¹For the R. torques 93% assay the sequence AY305319[62] was used for primer design;

²The Bifidobacterium longum DSM 20219T 16S rRNA gene was used as standard in the universal qPCR assay. qPCR: Quantitative real-time polymerase chain reaction.

Table 3 The average relative log₁₀ amount of the 16S rRNA gene copies detected with qPCR assays in proportion to the universal qPCR results

qPCR assay	Control (n = 15)	IBS-C (n = 8)	IBS-D (n = 8)	IBS-M (n = 4)
Bacteroides intestinalis-like	-4.85 ± 1.521 (12)	-4.71 ± 1.42 (5)	-5.8 ± 1.32 (5)	-3.46 ± 1.26 (4)
Bifidobacterium catenulatum/Bifidobacterium pseudocatenulatum-like	-4.1 ± 2.22 (14)	-5.63 ± 2.52 (7)	-5.42 ± 2.63 (5)	-4.4 ± 2.54 (4)
Butyrivibrio crossotus-like	-6.2 ± 2.03 (8)	-6.5 ± 1.97 (3)	-7.34 ± 1.58 (0)	-6.04 ± 2.19 (2)
Clostridium cocleatum 88%	-1.7 ± 1.32 (15)	-2.36 ± 2.35 (8)	-2.69 ± 2.33 (8)	-0.72 ± 0.98 (4)
Clostridium thermosuccinogenes 85%	-3.33 ± 1.16a (15)	-3.7 ± 0.84 (8)	-4.08 ± 0.90ab (8)	-3.08 ± 1.38b (4)
Collinsella aerofaciens-like	-2.45 ± 1.16 (15)	-2.9 ± 2.33 (7)	-4.63 ± 2.35 (7)	-1.73 ± 2.61 (4)
Coprobacillus catenaformis 91%	-4.72 ± 0.77 (15)	-4.41 ± 0.67 (8)	-4.79 ± 0.61 (8)	-4.71 ± 0.25 (4)
Coprococcus eutactus 97%	-5.44 ± 2.53 (9)	-5.91 ± 2.61 (3)	-6.55 ± 2.28 (2)	-4.09 ± 2.69 (3)
Ruminococcus bromii-like	-3.69 ± 2.42c (15)	-1.61 ± 1.83c (8)	-3.4 ± 2.49 (8)	-2.08 ± 1.56 (4)
Ruminococcus torques 91%	-3.13 ± 0.77 (15)	-2.87 ± 1.10 (8)	-2.58 ± 1.09 (8)	-2.83 ± 1.26 (4)
R. torques 93%	-2.41 ± 0.53d (15)	-2.61 ± 0.72 (8)	-2.65 ± 0.59 (8)	-2.92 ± 0.56d (4)
R. torques 94%	-4.02 ± 1.63c (14)	-3.39 ± 1.40 (8)	-2.43 ± 1.49c (8)	-3.82 ± 2.16 (3)
Slackia faecicanis 91%	-5.53 ± 2.26 (8)	-5.6 ± 2.33 (4)	-6.22 ± 2.16 (3)	-4.01 ± 2.28 (4)
Spiroplasma chinense 84%	-5.62 ± 2.04 (9)	-5.36 ± 2.20 (5)	-6.51 ± 1.98 (2)	-5.7 ± 2.22 (2)

The number of subjects with target 16S rRNA gene copies detected above the calculated threshold value in any of the three samples analysed are given in parentheses.

¹Values are presented as averages of log₁₀-values ± SD from three time-points (0, 3 and 6 mo).

^aP = 0.04,

^bP = 0.05,

^cP = 0.01,

^dP = 0.00.