Association of symptoms with gastrointestinal microbiota in irritable bowel syndrome

Table 1 Characteristics of the study subjects

Characteristic	mean ± SD	Median (range)
Age (yr)	48.4 ± 11.9	49.0 (20-72)
Body mass index (kg/m2)	26.3 ± 5.1	26.0 (19.4-45.6)
Systolic blood pressure (mmHg)	129.2 ± 19.1	129.5 (95-175)
Diastolic blood pressure (mmHg)	83.0 ± 10.2	83.5 (61-100)
Hemoglobin (g/L)	139.0 ± 12.0	139.0 (108-165)
B-GHba1c (%)	5.6 ± 0.38	5.6 (4.8-6.4)
Total cholesterol (mmol/L)	5.1 ± 0.73	5.0 (3.8-7.3)
Sedimentation rate (mm/h)	7.4 ± 7.1	5.0 (2-43)

Table 2 Targets, assay conditions and primers of quantitative real-time polymerase chain reaction assays

Target bacterial group/phylotype	Positive control strain or clone	MgCl2 (mmol/L)	Detection (°C)	Annealing (°C)	Primer sequences (F: forward, R: reverse)
Bifidobacterium catenulatum/Pseudocatenulatum-like[4]	AM277302	3	87	68	F: 5'-ACTCCTCGCATGGGGTGTC-3'
					R: 5'-CCGAAGGCTTGCTCCCGAT-3'
Bifidobacterium spp.[30]	Bifidobacterium longum	3	85	58	F: 5'-TCGCGTC(C/T)GGTGTGAAAG-3'
	DSM20219				R: 5'-CCACATCCAGC(A/G)TCCAC-3'
Clostridium coccoides/Eubacterium rectale-group[30]	Ruminococcus productus	4	85	55	F: 5'-CGGTACCTGACTAAGAAG-3'
					R: 5'-AGTTT(C/T)ATTCTTGCGAAC-3'
	DSM2950
Clostridium cocleatum 88%[4]	AM275477	4	80	60	F: 5'-AATACATAAGTAACCTGGCRTC-3'
					R: 5'-CGTAGCACTTTTCATATAGAGTT-3'
Collinsella aerofaciens-like[4]	AM276364	4	89	67	F: 5'-CCCGACGGGAGGGGAT-3'
					R: 5'-CTTCTGCAGGTACAGTCTTGAC-3'
Coprococcus eutactus 97%[4]	AM278899	2	83	63	F: 5'-AGCTTGCTCCGGCYGATTTA-3'
					R: 5'-CGGTTTTACCAGTCGTTTCCAA-3'
Desulfovibrio desulfuricans-group[30]	Desulfovibrio desulfuricans	4	85	58	F: 5'-GGTACCTTCAAAGGAAGCAC-3'
	ATCC7757				R: 5'-GGGATTTCACCCCTGACTTA-3'
Eubacterial 16S[31]	Bifidobacterium longum	3	80	50	F: 5'-TCCTACGGGAGGCAGCAGT-3'
	DSM20219				R: 5'-GGACTACCAGGGTATCTAATCCTGTT-3'
Lactobacillus-group[32,33]	Lactobacillus acidophilus	2	85	58	F: 5'-AGCAGTAGGGAATCTTCCA-3'
	ATCC4356				R: 5'-CACCGCTACACATGGAG-3'
Ruminococcus torques 91%[4]	AM276624	5	82	62	F: 5'-TGCTTAACTGATCTTCTTCGGA-3'
					R: 5'-CGGTATTAGCAGTCATTTCTG-3'
Ruminococcus torques 94%[4]	AM277929	2	85	65	F: 5'-AATCTTCGGAGGAAGAGGACA-3'
					R: 5'-ACACTACACCATGCGGTCCT-3'
Streptococcus bovis-like[4]	AM276479	5	80	60	F: 5'-TTAGCTTGCTAAAGTTGGAA-3'
					R: 5'-ATCTACTAGTGAAGCAATTGCT-3'
Veillonella spp.[30]	Veillonella parvula ATCC10790	3	85	62	F: 5'-A(C/T)CAACCTGCCCTTCAGA-3'
					R: 5'-CGTCCCGATTAACAGAGCTT-3'

Table 3 Symptom questionnaire results

Symptom groups	Median (interquartile range)	Minimum value	Maximum value
Bowel symptoms
Increased frequency of defecation	3 (2)	1	6
Abdominal cramps	3 (1)	1	5
Passing gas	4 (2)	1	7
Abdominal bloating	5 (2)	1	7
Feeling a need to defecate	3 (2)	1	7
Soiling	1 (1.25)	1	5
Stomach pain	3 (1)	1	6
Systemic symptoms
Tired and worn out	3.5 (1)	1	6
Nausea	3 (2)	1	6
Generally unwell	3 (1.25)	1	5
Sleep disturbance	4 (2)	1	7
Weight problems	3 (2.25)	1	7
Social function
Work/school activities	1 (0)	1	7
Cancel social engagements	1 (1)	1	7
Leisure/sports activity	3 (3)	1	7
Avoid events lacking toilet	1 (1)	1	4
Sexual activity	2 (2)	1	7
Emotional function
Frustrated/impatient	3 (1)	1	6
Energy	3 (2)	2	6
Fear of not finding a toilet	2 (2)	1	4
Depressed/discouraged	3 (1)	1	5
Worried about illness	3 (1.25)	1	7
Relaxed/free from worries	5 (1)	2	7
Tearful or upset	2 (1.25)	1	5
Angry	3 (2)	1	7
Irritable	3 (2)	1	5
Lack of understanding by others	2 (1)	1	6
Satisfaction with personal life	4 (2)	1	6

Table 4 Mean values and Spearman correlations (r_s) of symptom groups

Variable	Mean (SD)	Bowel symptoms rs	Systemic symptoms rs	Social symptoms rs
Bowel symptoms	24.0 (6.5)	-	-	-
Systemic symptoms	16.4 (8.1)	0.58b	-	-
Social function	9.3 (4.3)	0.60b	0.34a	-
Emotional function	39.9 (8.1)	0.71b	0.76b	0.58b

^aP < 0.05,

^bP < 0.001.

Table 5 Number of 16S rRNA gene copies detected in 50 ng of fecal DNA

qPCR assay	All (n = 44)	BMI < 251 (n = 17)	BMI > 25 (n = 22)	P2
Bifidobacterium catenulatum/Pseudocatenulatum-like	4.8 (1.6)3	5.4 (0.7)	4.3 (1.8)	0.366
Bifidobacterium spp.	5.8 (1.0)	5.5 (1.0)	6.2 (0.9)	0.009
Clostridium coccoides/Eubacterium rectale-group	7.3 (0.2)	7.3 (0.1)	7.3 (0.2)	0.630
Clostridium cocleatum 88%	5.6 (1.1)	5.5 (1.4)	5.6 (1.0)	0.483
Collinsella aerofaciens-like	5.6 (1.1)	5.3 (1.2)	5.8 (0.9)	0.294
Coprococcus eutactus 97%	5.3 (1.5)	5.5 (1.7)	5.4 (1.3)	0.639
Desulfovibrio desulfuricans-group	3.9 (0.8)	3.8 (0.6)	4.0 (0.9)	0.403
Eubacterial 16S	6.0 (0.3)	5.9 (0.4)	6.0 (0.3)	0.630
Lactobacillus-group	3.9 (0.9)	4.2 (0.7)	3.7 (0.9)	0.060
Ruminococcus torques 91%	4.6 (0.8)	4.4 (0.8)	4.8 (0.7)	0.461
Ruminococcus torques 94%	3.8 (1.0)	4.0 (1.3)	3.6 (0.8)	0.409
Streptococcus bovis-like	2.7 (1.5)	3.2 (2.1)	2.5 (1.2)	0.732
Veillonella spp.	3.4 (1.0)	3.5 (1.1)	3.4 (1.1)	0.745

¹Body mass index (BMI) data missing for 5 subjects;

²Calculated with Mann-Whitney U-test;

³Values are presented as log10 averages with standard deviation in parentheses. qPCR: Quantitative real-time polymerase chain reaction.

Table 6 Prevalence of target 16S rRNA genes detected for each quantitative real-time polymerase chain reaction assay

qPCR assay	All (n = 44)	BMI < 251 (n = 17)	BMI > 25 (n = 22)	P2
Bifidobacterium catenulatum/Pseudocatenulatum	12	3	7	0.315
Bifidobacterium spp.	44	17	22	ND
Clostridium coccoides/Eubacterium rectale-group	44	17	22	ND
Clostridium cocleatum 88%	33	13	15	0.568
Collinsella aerofaciens-like	29	15	12	0.024
Coprococcus eutactus 97%	16	7	8	0.759
Desulfovibrio desulfuricans-group	29	11	15	0.819
Eubacterial 16S	44	17	22	ND
Lactobacillus-group	44	17	22	ND
Ruminococcus torques 91%	38	15	18	0.582
Ruminococcus torques 94%	29	9	16	0.202
Streptococcus bovi-like	33	11	18	0.225
Veillonella spp.	41	16	20	0.709

¹Body mass index (BMI) data missing for 5 subjects;

²Calculated with pearson χ² test. ND: Not determined; qPCR: Quantitative real-time polymerase chain reaction.

Table 7 Association of Ruminococcus torques 94% phylotype with bowel symptoms and other fecal bacterial phylotypes

Variable	All samples	Ruminococcus torques 94%
		Detected	Undetected
Symptom sums1
All symptoms	83.6 (20.0)	87.6 (19.6)	75.9 (19.1)
Bowel symptoms	24.1 (6.5)	25.4 (6.5)	21.5 (5.8)23
Systemic symptoms	16.4 (4.8)	17.0 (4.7)	15.2 (4.8)
Social function	9.3 (4.3)	10.0 (4.5)	7.8 (3.6)
Emotional function	33.9 (8.1)	35.2 (8.0)	31.5 (7.9)
qPCR assays4
Bifidobacterium catenulatum/Pseudocatenulatum	2.5 (1.7)	2.5 (1.7)	2.5 (1.6)
Bifidobacterium spp.	5.8 (1.0)	5.8 (1.0)	5.9 (1.1)
Clostridium coccoides /Eubacterium rectale-group	7.3 (0.2)	7.3 (0.2)	7.3 (0.2)
Clostridium cocleatum 88%	3.9 (2.8)	3.1 (3.0)	5.4 (1.7)5
Collinsella aerofaciens-like	3.0 (3.8)	1.6 (4.0)	5.6 (0.7)5
Coprococcus eutactus 97%	1.9 (2.8)	0.9 (2.2)	3.7 (3.0)6
Desulfovibrio desulfuricans-group	1.9 (3.0)	1.4 (3.1)	2.7 (2.6)
Eubacterial 16S	6.0 (0.3)	5.9 (0.3)	6.0 (0.4)
Lactobacillus-group	3.9 (0.9)	3.8 (0.8)	4.1 (1.0)
Ruminococcus torques 91%	4.1 (1.5)	4.1 (1.6)	3.9 (1.3)
Ruminococcus torques 94%	2.7 (1.8)	3.8 (1.0)	-
Streptococcus bovis-like	1.9 (1.9)	1.6 (1.6)	2.4 (2.3)
Veillonella spp.	3.0 (1.7)	3.2 (1.7)	3.0 (1.8)

¹Values are presented as averages with standard deviation in parentheses;

²All statistical comparisons made between samples with Ruminococcus torques 94% detected and undetected;

³Significant difference (P < 0.05) using analysis of variance (ANOVA) test with continuous data;

⁴The values are presented as log10 averages with standard deviation in parentheses;

⁵Significant difference (P < 0.05) using ANOVA test with binary data;

⁶Significant difference (P < 0.01) using ANOVA test with binary data.