Methodological issues in the study of intestinal microbiota in irritable bowel syndrome

Table 1 Outcomes of the selected original research studies (see text for details), which have been carried out to characterize the intestinal microbiota composition in irritable bowel syndrome

Participants	Rome criteria	Results	Sample	Technique	Ref.
27 IBS (20 F/7 M)	II		Fecal	qPCR (SYBR Green)	[16]
12 IBS-D		↑Ruminococcus productus - Clostridium coccoides
9 IBS-C		↓Lactobacillus (IBS-D vs IBS-C)
6 IBS-A		↓Bifidobacterium (IBS-D vs HC, IBD-C, IBS-A)
Age: 20-65		↓Desulfovibrio (IBS-D vs HC, IBD-C, IBS-A)
22 HCs (15 F/7 M)		↑Veillonella (IBS-C vs HC)
Age: 25-64
26 IBS (19 F/7 M)	II		Feces	DGGE	[23]
12 IBS-D		More temporal instability in predominant bacterial population in IBS subjects
9 IBS-C		Slight increase of coliforms in IBS and higher aerobe/anaerobe ratio in IBS		Cloning and sequencing of 5 IBS and 4 HC samples (29 IBS and 16 HC clones); ABI PRISM BigDye terminator Cycle sequencing kit v. 3.0
5 IBS-A		↑Clostridium spp.
Age: 20-65		↓Eubacterium spp.
25 HCs (18 F/7 M)
Age: 23-63				Culture method
20 IBS (14 F/6 M)	II		Ileal and	FISH	[33]
Mean age: 47.8		Mucosal bacteria concentration higher than 109 cells/mL in 65% of IBS subjects (35% in HC)	colonic biopsies
20 HCs (13 F/7 M)		Prevalence of Eubacterium rectale-Clostridium coccoides in IBS biofilm
Mean age: 46.2
16 IBS (11 F/5 M)	II	More temporal instability of predominant microbiota only in RNA-DGGE profiles in IBS vs HCs (not in DNA-DGGE)	Feces	DGGE	[49]
7 IBS-D
6 IBS-C
3 IBS-A		↓C. coccoides - E. rectale in IBS-C vs HC No differences in Clostridium population stability between IBS and HC		Multiplexed quantification of clostridial 16S rRNA through multiple transcript analysis with the aid of affinity capture (TRAC)
Age: 24-64
16 HCs (12 F/4 M)
Age: 26-63
24 IBS	II	Significant differences in microbiota composition in different IBS subcategories pooled in 3 groups on the basis of %GC (7-10-13 fractions)	Feces	16S rRNA gene cloning and sequencing of 3753 clones	[37]
10 IBS-D
8 IBS-C
		In fraction group 7:
6 IBS-A		↓Lactobacillus in all IBS subgroups vs HC
Age: 21-65		↑Ruminococcus in IBS-C and IBS-A
23 HCs (16 F/7 M)		↑Streptococcus in IBS-D
Age: 26-64
		In fraction group 13:
		↓Collinsella in IBS-C and IBS-D
20 IBS	II	↑Clostridium thermosuccinogenes (IBS-A vs IBS-D)	Feces	qPCR (SYBR Green)	[46]
8 IBS-D		↑Ruminococcus torques 94% phylotype (IBS-D vs HCs and IBS-A)
8 IBS-C		↑Ruminococcus bromii-like phylotype (IBS-C vs HCs)
4 IBS-A		↑Bacteroides intestinalis-like and C. cocleatum (IBS-A and HCs vs IBS-D)
Age: 24-64		↓Clostridium aerofaciens-like (IBS-D vs other groups)
15 HCs
Age: 25-64
41 IBS (29 F/12 M)		↓Bifidobacterium	Feces	FISH	[31]
14 IBS-D		↓B. catenulatum	Feces and duodenal brushes	qPCR (Taqman)
11 IBS-C
16 IBS-A
Mean age: 42
26 HCs (18 F/8 M)
Mean age:32
10 IBS-D (6 F/4 M)	II	Decreased diversity in the intestinal microbiota of IBS-D vs HCs ↑ Proteobacteria and Firmicutes ↑ Lachnospiraceae ↓ Actinobacteria and Bacteroidetes	Feces	Genomic DNA fractioning on the basis of %GC (35%-40%/40%-45%/50%-55%/55%-60%/60-65/65%-70%/70%-75%); amplification of 16S rRNA gene; sequencing of 3267 clones for IBS subjects	[47]
Age average: 46.5
23 HCs
Age average: 45
12 IBS-D (7 F/5 M)		No significant differences in Enterobacteriaceae and Eggerthella lenta-type (Atopobium) phylotype between IBS-D and HCs		qPCR (SYBR Green)
Age average: 46.5
22 HCs
Age average: 45
47 IBS (47 F)	II	Significant difference in DGGE profile between IBS and HC, less microbial variation in IBS	Feces	DGGE of V1-V3 region of the 16S rRNA	[70]
Age: 24-66
33 HCs		No significant intra and inter-differences in IBS subjects between luminal and mucosal microbiota. IBS impacts equally on both communities	Feces and colonic biopsies	DGGE of V6-V8 Region of the16S rRNA
Age: 21-38
26 IBS (13 F/13 M)		↑Veillonella	Feces	qPCR (SYBR Green)	[12]
8 IBS-D
11 IBS-C		↑Lactobacillus spp.		Culture method
7 IBS-A
Age: 21.7 ± 2.0
26 HCs
Age: 21.9 ± 2.9
10 IBS-D (8 F/2 M)	III	↓ Aerobic counts in fecal samples of IBS-D	Feces samples and colonic biopsy	Culture method	[27]
Age: 23-50		No difference in mucosal samples between IBS-D and HC
10 HCs (6 F/4 M)		↑Lactobacillus spp. in fecal samples of IBS-D vs HC
Age: 21-54		No difference in mucosal samples between IBS-D and HC		qPCR (SYBR Green)
11 IBS (7 F/4 M)	II	Reduced biodiversity in IBS subjects	Feces	DGGE on universal and specific primers for Bacteroides Sequencing of V3 region of the 16S rRNA genes	[69]
Age: 25-64		Significant differences in profiles between IBS and HC subjects
22 HCs (17 F/5 M)		↓B. vulgatus, B. ovatus, B. uniformis, Parabacteroides
Age: 21-61		sp. in IBS vs HC
22 IBS (8 F/14 M)	Pediatric Rome III	No differences in total bacterial load between IBS and HCs	Feces	16S Metagenomics 454 Pyrosequencing (V1-V3 and V3-V5 regions of 16S rRNA)	[35]
1 IBS-D		Profile differences in IBS subtypes among each other, and between IBS and HCs
13 IBS-C		In IBS:
8 IBS-A		↑ Proteobacteria
Age: 7-12		↑γ-Proteobacteria
22 HCs (11 F/11 M)		↑Haemophilus parainfluenzae
		↑Veillonella		PhyloChip Microarray Hybridization on purified 27F and 1492R regions of 16S rRNA (on 28 IBS and 27 HC)
		↑Dorea
		↓Eubacterium
		↓Anaerovorax
		↓Bacteroides vulgatus
62 IBS (57 F/5 M)	II	↑ Firmicutes/Bacteroidetes ratio	Feces	HITChip phylogenetic microarray	[36]
25 IBS-D		↑Bacillus
19 IBS-C		↑Steptococcus
19 IBS-A		↑Dorea
Age: 22-66		↑Ruminococcus
46 HCs (34 F/12 M)		↑R. gnavus
Age: 23-58		↑Blautia
		↑Clostridium
		↓Faecalibacterium
		↓Bacteroides
		↓B. vulgatus
		↓Prevotella
		↓Bifidobacterium
		↓B. gallicum
		↓B. pseudocatenulatum
		↓Methanobrevibacter in IBS vs HC, particularly in IBS-C subgroup
11 IBS	II	Greater biological variability of predominant bacteria among IBS subjects vs HC and higher microbial diversity (especially Bacteroides and lactobacilli) in IBS vs HC	Feces	qPCR (SYBR Green)	[63]
(5 F/6 M)		In IBS, Exclusive detection of Eubacterium biforme (absent in HC)		DGGE on V3-V5 region of 16S rRNA gene
8 HCs (2 F/6 M)		↑Bacteroidetes		qPCR (SYBR Green)
Age: 18-74		↑Lactobacillus
		↓Bifidobacterium
		↓C. coccoides
37 IBS (26 F/11 M)	II	No evident difference in predominant microbiota from profiles of both sample sites between IBS and HC	Duodenal brushes and feces	DGGE on V6-V8 region of 16S rRNA gene, generation of 51 clones and sequencing	[57]
13 IBS-D
13 IBS-C
13 IBS-A		↑P. aeruginosa in all subgroups if IBS and in both		qPCR (Taqman)
Age: 21.7 ± 2.0		body niche samples
20 HC (15 F/5 M)
Age: 21.7 ± 2.0
16 IBS-D (11 F/5 M)	III	Lower biodiversity in IBS-D vs HCs in fecal samples, no biodiversity differences in mucosal samples	Feces samples and colonic biopsy	T-RFLP	[71]
Age: 23-52		↓ Clostridiales
21 HCs (17 F/4 M)		↓ Planctomycetaceae			[62]
Age: 21-60
81 IBS (69 F/27 M)	I and II	Staphylococcus aureus detected only in IBS subjects, with higher prevalence in IBS-C	Feces
15 IBS-C		Enterotoxin-encoding gene of C. perfringens detected		qPCR (SYBR Green)
Age: 20-73		only in IBS subjects
23 HCs (16 F/7 M)		Helicobacter pylori detected in 3 IBS subjects, none in		Sequencing of S. aureus amplicons
Age: 26-64		HCs
23IBS-D (17 F/6 M)	III	Lower microbial richness in IBS-D	Feces	454 Pyrosequencing of the V1-V3 and V6 regions of 16S rRNA gene	[54]
Age: 23-70		Structural changes in IBS-D vs HC, from phylum to genus
23 HCs (18/5 M)		↑ Proteobacteria
Age: 21-58		↑γ-proteobacteria
		↑Enterobacteriales
		↑Enterobacteriaceae
		↓Faecalibacterium
		↓F. prausnitzii		qPCR (SYBR Green)
37 IBS (26 F/11 M)	II	Clustering by microbiota composition revealed subgroups of IBS patients: (1) a group (n = 15) with normal-like microbiota composition compared with HCs; and (2) a group (n = 22) with large microbiota-wide changes characterized by an increase of Firmicutes (mainly clostridia/Clostridiales) and a depletion of Bacteroidetes	Feces	Pyrosequencing of the V4 region of 16S rRNA gene	[77]
15 IBS-D		↓Bacteroides
10 IBS-C		↓Alistipes
12 IBS-A		↑Lachnospiraceae incertae sedis
Age: 37 ± 12		↑ Butyrate-producing Eubacterium halli and
20 HCs (13 F/7 M)		desmolans
Age: 39 ± 9		↑B. adolescentis
47 IBS	III	Higher number of mucosa-associated bacteria in	Rectal	FISH	[32]
27 IBS-D		IBS	biopsies
20 IBS-C
Age average: 34.3		↑Bacteroides
26 HCs		↑Eubacterium rectale-C. coccoides
Age average: 46.1		↓Bifidobacterium in IBS-D than in IBS-C
75 rural IBS	III	↓Enterobacter	Feces	Culture-based analysis	[29]
Age: 4-18		↓Enterococcus
20 Hurban HCs		↓Lactobacillus
Age: 5-15		↓Bifidobacterium
22 IBS-D	II	Higher variability among IBS subjects	Feces	Microbiota array	[40]
Age: 8-18		No difference between IBS-D and HCs at phylum level. No difference for Clostridium and Faecalibacterium
22 HCs		↑Veillonella		Pyrosequencing (V1-V2-V3 region of 16S
Age: 11-18		↑Prevotella		rRNA)
		↑Enterobacter
		↑Lactobacillus
		↓Bifidobacterium
		↓Verrucomicrobium
		Difference at species level in the genus Bacteroides:
		↓B. fragilis
		↓B. thetaiotaomicron
		↑B. ovatus
		↑B. salyersiae
		Positive abundance correlation between Veillonella-Haemophilus and Streptococcus; negative for Ruminococcus
		Confermation of data on Clostridia, Bacteroidetes, Bifidobacterium		FISH
		Confermation of data on Bifidobacterium, Prevotella, Faecalibacterium		qPCR
14 IBS-C (14 F)	II	No differences in total strict and facultative anaerobes between IBS-C and HCs	Feces	Culture-based analysis	[28]
12 HCs (14 F)		No difference in hydrolytic bacterial communities
Age: 20-59		↑ Lactate utilizing sulphate-reducing bacteria (SRB)
		↓ Lactate non SRB (butyrate-producing)
		↑ H2-utilizing SRB
		↓ H2-utilizing non SRB (acetogenic, methanogens)
		↑Enterobacteriaceae
		↓Bifidobacterium
		↓Lactobacillus
		↓Bifidobacterium		FISH
		↓Roseburia-E. rectale
19 IBS	III	↑ Bifidobacteriaceae	Feces	Microbiota Array	[42]
24 HCs		↑ Lactobacillaceae
Age: 33.6 ± 9.1		↑Clostridium cluster IX
		↑Eubacterium rectale
		↑Enterococcus faecium
		↑Clostridium difficile
		↑Bacillus cereus and B. clausii
		↑Campilobacter spp.
		↓Bacteroides/Prevotella
		↓Veillonella
14 IBS-D(3 F/11 M)	III	↑E. coli	Feces	qPCR (SYBR Green)	[58]
18 HCs (7 F/11 M)		↓Clostridium leptum
Age: 18-65		↓Bifidobacterium
16 IBS		Reduced microbial diversity in IBS	Colonic biopsies and feces	Pyrosequencing (V1-V2 regions of 16S rRNA)	[48]
9 HCs		In mucosal samples:
		↑ Bacteroidaceae
		In fecal samples:
		↑ Rikenellaceae
		↑ Porphyromonadaceae
		↓ Ruminococcaceae
		IBS-D:
		↑Acinetobacter, Leuconostoc, Butyricimonas, Odoribacter (fecal)
		↓Desulfovibrio, Oribacterium (biopsies)
		IBS-C:
		↑Alistipes, Butyricimonas (feces) and Bacteroides (biopsies)
		↓Fusobacterium, Eubacterium, Coprococcus, Eubacterium, Haemophilus, Neisseria, Streptococcus, Veillonella
2 IBS-D	III	↑Alphaproteobacteria	Feces	Pyrosequencing (16S rRNA gene)	[89]
1 HCs		↑ Facultative anaerobe (Proteobacteria,
Several sampling over 6-8 wk		Streptococcus) in days of acute diarrhea

qPCR: Real time quantitative polymerase chain reaction; DGGE: Denaturing gradient gel electrophoresis; T-RFLP: Terminal restriction fragment length polymorphism; FISH: Fluorescence in situ hybridization; Ref.: Reference; IBS: Irritable bowel syndrome; IBS-D: Diarrhea-associated IBS; IBS-C: Constipation-associated IBS; IBS-A: Alternating symptoms IBS; HCs: Healthy controls. ↑: Increased presence in IBS; ↓: Reduced presence in IBS.