Study population characteristics

Subjects included 50 ME/CFS cases and 50 healthy controls recruited at four sites across the USA (New York, NY; Salt Lake City, UT; Incline Village, NV; and Miami, FL) who met the 1994 CDC Fukuda [17] and the 2003 Canadian consensus criteria for ME/CFS [18]. Subject demographics are shown in Table 1. Cases included 41 female and 9 male ME/CFS patients (mean age 51.1 years; standard error of the mean (SEM) 1.6). Controls included 41 female and 9 male subjects (mean age 51.3 years; SEM 1.6). All case and control samples were collected between June 22, 2014, and October 27, 2014. IBS was diagnosed in 21 of 50 ME/CFS patients (42%) and none of 50 controls. Nine of 21 ME/CFS + IBS patients (43%) reported having IBS diagnosis prior to ME/CFS. No controls reported a diagnosis of IBS. Twenty-eight ME/CFS patients and 22 controls had a high body mass index (BMI) (>25 kg/m2).

Table 1 Characteristics of study cohort Full size table

TDA analysis of fecal microbiota, predicted bacterial metabolic pathways, plasma immune molecule profiles, and clinical features

Shotgun metagenomic sequencing of fecal samples was pursued to determine microbial composition (relative abundance of taxa) and infer bacterial metabolic pathways in the ME/CFS and control subjects. An average of 7 Gb of sequence per sample (from 100 bp, paired-end Illumina reads) was generated using high-throughput sequencing. Levels of plasma immune molecules were quantitated by immunoassay. We built a TDA network comprised of 100 samples (50 cases and 50 controls) and 1358 total variables. The variables consisted of the following elements: 574 representing the relative abundance of bacterial taxa; 586 representing metabolic pathways (131 superpathways and 455 individual metabolic pathways); 61 reflecting levels of each plasma immune molecule in the assay; 80 representing symptoms (health questionnaire items); and 57 co-morbidities and demographic variables.

Relationships among these datasets were analyzed using TDA (AYASDI software) to identify multidimensional networks and the individual factors (microbial, metabolic pathways, immune molecules, and clinical variables) that distinguish those networks.

The ME/CFS subjects formed separate topological networks from the control subjects in TDA (Fig. 1). IBS co-morbidity was the strongest driving factor in the separation of metagenomics in ME/CFS. TDA revealed differences in bacterial taxa and metabolic pathways between ME/CFS, ME/CFS + IBS, and ME/CFS without IBS vs. controls (Additional file 1: Table S1A). At the family level, the relative abundances of Lachnospiraceae and Porphyromonadaceae were lower in the ME/CFS (both with and without IBS) compared to the controls, whereas the relative abundance of the family Clostridiaceae was higher. At the genus level, the abundances of Dorea, Faecalibacterium, Coprococcus, Roseburia, and Odoribacter were lower in the ME/CFS compared to the controls, whereas abundances of Clostridium and Coprobacillus were higher. The 12 bacterial species driving the differences between the ME/CFS and control groups were Faecalibacterium prausnitzii, Faecalibacterium cf., Roseburia inulinivorans, Dorea longicatena, Dorea formicigenerans, Coprococcus catus, Odoribacter splanchnicus, Ruminococcus obeum, and Parabacteroides merdae (all decreased in ME/CFS) and Clostridium asparagiforme, Clostridium symbiosum, and Coprobacillus bacterium (all increased in ME/CFS).

Fig. 1 Topological data analysis (TDA) reveals altered metagenomic profiles in ME/CFS. Metagenomic data including bacterial composition predicted bacterial metabolic pathways, plasma immune profiles and symptom severity scores were analyzed by using TDA (AYASDI software) to define multidimensional subgroups. TDA of variance-normalized Euclidean distance metric with four lenses [neighborhood lenses (NL1 and NL2), ME/CFS, and IBS diagnosis] revealed that ME/CFS samples formed distinct networks separately from controls. The controls grouped more tightly than the ME/CFS patients. IBS co-morbidity was identified as the strongest driving factor in the separation of metagenomic and immune profile of ME/CFS individuals. Dots that are not connected to the networks represent outliers. Metagenomic data including bacterial composition and inferred metabolic pathways, plasma immune profiles, and health symptom severity scores were integrated for topological data analysis (TDA) using the AYASDI platform (Ayasdi, Menlo Park, California). AYASDI represents high-dimensional, complex biological data sets as a structured 3-dimensional network [56]. Each node in the network comprises one or more subject(s) who share variables in multiple dimensions. Lines connect network nodes that contain shared data points. Unlike traditional network models where a single sample makes a single node, the size of a node in the topological network was proportional to the number of variables with a similar profile. We built a network comprised of 100 samples and 1358 variables (574 variables representing bacterial relative abundance at different taxonomic levels, 61 variables reflecting levels of each immune molecule in the assay, 586 variables representing metabolic pathways, 80 variables representing different ME/CFS fatigue, and other symptom score/health questionnaire items and information on co-morbidities, and demographic variables). All variables were weighted equally. Variance-normalized Euclidean distance method was used as the distance metric; a range of filter lenses (neighborhood lens 1 and 2, ME/CFS, and IBS diagnosis) was used to identify networks Full size image

Intragroup variability in the control group was smaller than in the ME/CFS group (Fig. 1). The TDA-based microbial taxa and metabolic pathways distinguishing between ME/CFS + IBS and ME/CFS without IBS vs. control are shown in Additional file 1: Table S1A. ME/CFS + IBS had decreased representation from the Proteobacteria phylum, C. catus, and F. prausnitzii species and increased representation from the Clostridiaceae family compared to the controls. Superpathway analysis showed alteration in heme biosynthesis, carboxylates, amino acid (AA), and polyamine metabolism. In the ME/CFS without the IBS group, the difference was driven by the increased abundance of members of the Clostridiaceae family, the Clostridium, and Pseudoflavonifractor genera, and the decreased abundance of members of the Porphyromonadaceae family and Odoribacter and Parabacteroides genera. The bacterial species driving the differences between the ME/CFS without IBS and control groups were D. formicigenerans, C. catus, Blautia hansenii, and Parabacteroides distasonis (all decreased in the ME/CFS without IBS) and unclassified Bacteroides, D. longicatena, Ruminococcus gnavus, C. symbiosum, Eggerthella lenta, Pseudoflavonifractor capillosus, C. bacterium, Clostridium cf. and scindens (all increased in the ME/CFS without IBS). The ME/CFS subgroup comparison between the ME/CFS + IBS and ME/CFS without IBS is shown in Additional file 1: Table S1A. Higher KS (Kolmogorov-Smirnov) scores were found for bacterial taxa and metabolic pathways distinguishing the ME/CFS + IBS group from controls. Comparisons involving the ME/CFS + IBS subgroup (vs. controls and vs. ME/CFS without IBS) showed stronger associations with bacterial taxa and metabolic pathways than any of the other comparisons.

Four networks defined by IBS co-morbidity and BMI (Additional file 2: Figure S1, Additional file 1: Table S1B) were associated with a distinct metagenomic and immune profile.

ME/CFS and ME/CFS subgroups are associated with an altered microbial composition

Compositional taxonomic analysis based on metagenomic sequencing indicated that the two dominant phyla in both ME/CFS and control individuals were Bacteroidetes (64.9 and 63.4%, respectively) and Firmicutes (26.6 and 29.7%, respectively) (Fig. 2a). Combined, Bacteroidetes and Firmicutes accounted for a mean relative abundance of 91.5% in ME/CFS cases and 93.1% in controls. The other phyla (Actinobacteria, Proteobacteria, Verrucomicrobia, Euryarchaeota, Lentisphaerae, and Fusobacteria) were represented at low relative abundance (mean relative abundance <5%) in samples.

Fig. 2 The altered microbial profile of ME/CFS compared to controls. a Heatmap representing the relative abundance of phyla in ME/CFS and control subjects. Bacteroidetes and Firmicutes were the two dominant phyla in both ME/CFS and control individuals. The heatmap represents the individual values (relative abundances) of bacterial phyla as colors where blue is the minimum percentage (0%) and red is the maximum percentage (100%). b Principal coordinate analysis (PCoA) based on the Bray-Curtis dissimilarity among species-level relative abundance distributions showed overlap between ME/CFS and control subject microbiota. c Bar charts showing significant separation of ME/CFS and controls along the first two PCs (PC1 explaining 12.67% of the variance, ***p < 0.001; PC2 explaining 10.69% of the variance, *p = 0.030). PCoA coordinates were compared as continuous variables with nonparametric Mann-Whitney U test. d Bar chart showing the BC dissimilarity within control subjects, within ME/CFS subjects, and between ME/CFS vs. control subjects. BC dissimilarity values were compared as continuous variables with nonparametric Mann-Whitney U test; error bars show the mean with SEM (standard error of the mean). *p < 0.05; ***p < 0.001. ns not significant Full size image

Principal coordinate analysis (PCoA) based on the species-level Bray-Curtis [19] dissimilarity revealed overlap between the ME/CFS and control subjects (Fig. 2b). However, the ME/CFS subjects overall varied from the controls in the first two principal coordinates [20], accounting for 25% of the total variance (Fig. 2c: PC1 12.67%, p < 0.001; PC2 10.69%, p = 0.03). Within control subjects, BC dissimilarity was significantly lower than within ME/CFS subjects, consistent with our findings based on TDA analysis (Fig. 1) and suggests greater variability in the ME/CFS microbiota (Fig. 2d). The between-group (ME/CFS vs. control) BC dissimilarity comparisons were higher than the within-control comparisons (p < 0.001) but was not higher than the within ME/CFS comparisons (Fig. 2d). Together, these data provide evidence of greater variability in the microbiota of ME/CFS patients.

Metagenomic biomarker discovery (linear discriminant analysis effect size (LEfSe)) identified 22 bacterial taxa enriched in ME/CFS and 27 enriched in controls (Fig. 3a). Based on nonparametric Mann-Whitney U test with Benjamini-Hochberg correction (p < 0.05 and adjusted p < 0.2), 41 bacterial species, genera, families, or orders differed between the ME/CFS and control groups (Additional file 1: Table S2A). Thirty-seven bacterial taxa differentiated ME/CFS from controls by both statistical methods. At the bacterial order and of family levels, the relative abundances of members of the order Pasteurellales and of the families of Lachnospiraceae, unclassified Bacillales and Pasteurellaceae were lower in the ME/CFS patients than in the controls, whereas the relative abundance of members of the family Clostridiaceae was higher in the ME/CFS. At the genus levels, the abundances of members of the genera Faecalibacterium, Roseburia, Coprococcus, Gemella, Dorea, and Haemophilus were lower in the ME/CFS, whereas the abundances of the genera Clostridium, Pseudoflavonifractor, Anaerostipes and Coprobacillus were higher in the ME/CFS. The bacterial species driving the differences between the ME/CFS and control groups were F. prausnitzii, Alistipes putredinis, Faecalibacterium cf., R. inulinivorans, D. longicatena, D. formicigenerans, Eubacterium ventriosum, Eubacterium hallii, Haemophilus parainfluenzae, P. distasonis, R. obeum and C. catus (all decreased in ME/CFS) and unclassified Bacteroides, unclassified Alistipes, P. capillosus, Clostridium bolteae, R. gnavus, C. asparagiforme, Anaerostipes caccae, C. bacterium, C. symbiosum, and C. scindens (all increased in ME/CFS). Species in Faecalibacterium, Roseburia, Dorea, Coprococcus, Clostridium, Ruminococcus, and Coprobacillus species were significantly different in the ME/CFS patients compared to the controls by TDA, LEfSe, and nonparametric testing (Fig. 3a, Additional file 1: Table S1A and Additional file 1: Table S2A).

Fig. 3 Bacterial taxa differentiate ME/CFS and ME/CFS subgroups (ME/CFS + IBS and ME/CFS without IBS) compared to controls. Histogram showing the log-transformed LDA scores computed with LEfSe for bacterial taxa differentially abundant between ME/CFS groups and controls. Positive LDA score indicates enrichment in a ME/CFS, b ME/CFS + IBS, and c ME/CFS without IBS vs. controls. Negative LDA indicates enrichment in control subjects (reduced in ME/CFS subgroups). The LDA score indicates the effect size and ranking of each bacterial taxon. Names of different taxonomic categories (genus, family, or order) are indicated with shades of blue; species names are written in black. An alpha value of 0.05 for the Kruskal-Wallis test and a log-transformed LDA score of 2.0 were used as thresholds for significance in LEfSe analyses. Asterisks next to taxa names indicate significant differences that were also found for a given taxa based on nonparametric Mann-Whitney U test with Benjamini correction (adjusted p < 0.2) Full size image

Evidence from TDA that IBS was linked to differences in disease severity, microbiota, and immune profiles led us to stratify the patient cohort into ME/CFS + IBS and ME/CFS without IBS and test for group-specific differences in the microbiota using linear statistical models.

In comparisons of the ME/CFS + IBS and control groups, LEfSe identified 12 bacterial species, genera, or families enriched in the ME/CFS + IBS and 26 bacterial taxa enriched in the controls (Fig. 3b). Based on nonparametric Mann-Whitney U test with Benjamini-Hochberg correction (adjusted p < 0.2), 21 bacterial taxa differed between the ME/CFS + IBS and control groups (Additional file 1: Table S2B). The two statistical methods yielded results with overlap in all 21 bacterial taxa. Differences were driven by increases in representatives of the Clostridiaceae family, Clostridium and Anaerostipes genera; and decreases in representatives of the Lachnospiraceae family, Faecalibacterium, Roseburia, Coprococcus, and Dorea genera. The 13 bacterial species driving the differences between ME/CFS + IBS and controls were F. prausnitzii, F. cf., unclassified Faecalibacterium, R. inulinivorans, C. comes, D. longicatena, E. hallii, D. formicigenerans, R. obeum, and C. catus (each of which was decreased in the ME/CFS + IBS) and unclassified Alistipes, C. bolteae, and A. caccae (which were increased in the ME/CFS + IBS).

In the ME/CFS without IBS, LEfSe identified 29 bacterial species, genera or families enriched in ME/CFS without IBS and 16 bacterial taxa enriched in controls (Fig. 3c). Based on nonparametric Mann-Whitney U test with Benjamini-Hochberg correction (adjusted p < 0.2), 22 bacterial taxa differed between the ME/CFS without IBS and control groups (Additional file 1: Table S2B). Twenty-one bacterial taxa showed overlap between the two statistical methods. The differences in ME/CFS without IBS were driven by the increased abundance of members of Pseudomonadales order, the Clostridiaceae and Pseudomonadaceae family, and the Clostridium, Pseudomonas, Pseudoflavonifractor, Eggerthella, and Coprobacillus genera and the decreased abundance of members of the Dorea genus. The 13 bacterial species driving the differences between the ME/CFS without IBS and control groups were D. formicigenerans, C. catus, and P. distasonis, (all decreased in ME/CFS without IBS) and unclassified Bacteroides, R. gnavus, D. longicatena, P. capillosus, E. lenta, C. symbiosum and scindens, C. bacterium, and Clostridium cf. (all increased in ME/CFS without IBS).

PCoA analysis based on the BC dissimilarity with ME/CFS stratified by IBS co-morbidity again showed overlap in controls and ME/CFS (Fig. 4a). Both the ME/CFS + IBS and ME/CFS without IBS individuals separated along PC1 compared to the controls (Fig. 4b: PC1 12.67%, ME/CFS + IBS vs. control p < 0.05, ME/CFS without IBS vs. control p < 0.001); however, only ME/CFS without IBS showed separation along PC2 (PC2 10.69%, ME/CFS without IBS vs. control p = 0.030). While controls had significantly lower within-group dissimilarity compared to within-group dissimilarities of both the ME/CFS without IBS and ME/CFS + IBS, ME/CFS + IBS had the highest within-group dissimilarity (Fig. 4c). Between-group comparisons demonstrated that there was lower dissimilarity between the controls and the ME/CFS without IBS group than there was between the controls and the ME/CFS group with IBS. Comparison between ME/CFS without IBS vs. ME/CFS with IBS showed higher dissimilarity than control vs. ME/CFS without IBS but similar dissimilarity as between control vs. ME/CFS with IBS. Thus, there was as much dissimilarity between the two subgroups of ME/CFS defined by IBS co-morbidity as between control and ME/CFS with IBS and even greater dissimilarity between the subgroups of IBS. Together, these results suggest that the ME/CFS subjects have greater variation between their microbiota than the control subjects and that ME/CFS subjects with IBS have the greatest within group variation.

Fig. 4 Distinct microbial profiles of ME/CFS + IBS and ME/CFS without IBS compared to controls. a Principal coordinate analysis (PCoA) analysis based on the BC dissimilarity with ME/CFS stratified by IBS co-morbidity. b Bar charts showing separation of ME/CFS with and without IBS compared to controls along PC1 (top panel); only ME/CFS without IBS showed significant separation along PC2 (bottom panel). c Bar chart showing the mean BC dissimilarity within control subjects, within ME/CFS without IBS and within ME/CFS with IBS subjects, as well as, between control vs. ME/CFS without IBS, between control vs. ME/CFS with IBS, and between ME/CFS without IBS vs. ME/CFS with IBS subjects. PCoA coordinates and BC dissimilarity values were compared as continuous variables with nonparametric Mann-Whitney U test; error bars show the SEM (standard error of the mean). *p < 0.05; ***p < 0.001. ns not significant. d, e Unique and overlapping bacterial species differentiate ME/CFS and ME/CFS IBS subgroups from control subjects. d Proportional Venn diagram showing the number of unique and overlapping bacterial species that differentiate ME/CFS groups from control subjects. e Circular visualization (Circos) showing the individual bacterial species and their unique or overlapping relationship between the total ME/CFS group, the ME/CFS without IBS group, and the ME/CFS with IBS group. Unique to total ME/CFS: Gemella unclassified (1), Streptococcus peroris (12), Eubacterium rectale (23), and Ruminococcus torques (34). Unique to ME/CFS without IBS: Bacteroides caccae (2), Bacteroides ovatus [64], Bacteroides vulgatus (4), Lactobacillus gasseri (5), Streptococcus infantis (6), Solobacterium moorei (7), Pseudomonas aeruginosa (8), and Eggerthella lenta (46). Unique to ME/CFS + IBS: Butyrivibrio unclassified (42), Coprococcus comes (43), Roseburia intestinalis (44), and Desulfovibrio desulfuricans (45). Overlapping between all three comparisons (total ME/CFS, ME/CFS without IBS and ME/CFS + IBS groups vs. control): Alistipes unclassified (9), Clostridium asparagiforme (10), Clostridium bolteae (11), Eubacterium ventriosum (13), Coprococcus catus (14), Dorea formicigenerans (15), Dorea longicatena (16), Marvinbryantia formatexigens (17), Roseburia inulinivorans (18), Ruminococcus gnavus (19), Subdoligranulum variabile (20),and Coprobacillus bacterium (21). Overlapping between total ME/CFS and ME/CFS + IBS groups compared to controls: Alistipes putredinis (22), Eubacterium hallii (24), Anaerostipes caccae (25), Faecalibacterium cf. (26), Faecalibacterium prausnitzii (27), Faecalibacterium unclassified (28), and Ruminococcus obeum (29). Overlapping between ME/CFS without IBS and ME/CFS + IBS groups compared to controls: Bacteroides unclassified (30), Odoribacter splanchnicus (31), Parabacteroides distasonis (32), Parabacteroides merdae (33), Clostridium cf. (35), Clostridium scindens (36), Clostridium symbiosum (37), Pseudoflavonifractor capillosus (38), Blautia hansenii (39), Dorea unclassified (40), and Haemophilus parainfluenzae (41) Full size image

Proportional Venn and circular visualization diagrams were used to display the overlapping and differentiating bacterial taxa between the controls and each of the ME/CFS groups (total ME/CFS, ME/CFS + IBS, and ME/CFS without IBS) (Fig. 4d, e). The proportional Venn diagram (Fig. 4d) showed that the relative abundance of 11 bacterial species distinguished both ME/CFS + IBS and ME/CFS without IBS from the controls. The relative abundance of 19 bacterial species distinguished the ME/CFS subjects without IBS from the controls, but did not help to differentiate the total ME/CFS group from the control group. Circular visualization (Fig. 4e) showed both the distinct and overlapping bacterial taxa whose abundance differentiated the total ME/CFS group, the ME/CFS + IBS group, and the ME/CFS group without IBS from the control group.

We compared the number of bacterial species among the ME/CFS diagnostic groups and the controls. A total of 363 bacterial species were identified in the entire cohort. The number of species identified was similar in ME/CFS patients and controls (average number of bacterial species ± the standard error of the mean: ME/CFS 74.24 ± 1.67; control 77.5 ± 2.07). We also did not find differences in the number of bacterial species when comparing subgroups based on IBS status (average number of bacterial species ± the standard error of the mean: ME/CFS + IBS 71.62 ± 2.26; ME/CFS without IBS 76.14 ± 2.34; control 77.5 ± 2.07).

Bacterial species distinguish ME/CFS and ME/CFS with IBS from healthy control subjects

We used three dimensionality reduction methods to further analyze the microbial communities that distinguish ME/CFS subgroups and controls: least absolute shrinkage and selection operation (LASSO), random forest (RF) and partial least squares (PLS) (Table 2). Bacterial species were selected for the predictive logistic regression model if they met criteria in the dimensionality reduction models. Species from the Firmicutes phylum were the chief determinants of ME/CFS group status. The relative abundance of four bacterial species (C. catus, P. capillosus, D. formicigenerans, and F. prausnitzii) distinguished the ME/CFS patients from the controls (ROC AUC = 0.831 and cross-validated AUC = 0.684). The addition of four more bacterial species (C. asparigiforme, Sutterella wadsworthensis, A. putredinis, and Anaerotruncus colihominis) improved predictive performance (ROC AUC = 0.893, cross-validated AUC = 0.745). This predictive selection model corresponds to our TDA, LEfSe, and nonparametric statistical findings of differences in Coprococcus, Dorea, Faecalibacterium, and Clostridium spp. in total ME/CFS and control group comparisons.

Table 2 Predictive selection model defined different bacterial composition in ME/CFS diagnostic groups Full size table

The relative abundance of distinct bacterial taxa defined ME/CFS patients with IBS. The relative abundance of just two bacterial species (Faecalibacterium cf. and Bacteroides vulgatus) distinguished the ME/CFS + IBS from the control subjects with a moderate degree of accuracy (ROC AUC = 0.771; cross-validated AUC = 0.571). Accuracy of the outcome prediction improved when nine additional bacterial species: F. cf., F. prausnitzii, B. vulgatus, A. putredinis, C. catus, A. caccae, D. formicigenerans, A. colihominis, and C. asparagiforme were added to the model (ROC AUC = 1, cross-validated AUC = 0.791).

Membership in the ME/CFS without IBS subgroup as compared with the control group was predicted by the relative abundance of Bacteroides caccae, P. capillosus, P. distasonis, Bacteroides fragilis, Prevotella buccalis, Bacteroides xylanisolvens, and D. formicigenerans (ROC AUC = 0.948, cross-validated AUC = 0.754). TDA-selected bacterial species of Bacteroides, Clostridium, Pseudoflavonifractor, and Parabacteroides were found in all statistical tests (LEfSe, nonparametric and reduction model tests) in the ME/CFS without IBS group compared to the controls. The decreased relative abundance of Bacteroides vulgatus distinguished the ME/CFS without IBS group from the ME/CFS + IBS group based on all statistical tests (TDA, LEfSe, nonparametric and reduction model tests).

ME/CFS patients with IBS were distinguished from ME/CFS patients without IBS by the relative abundance of eleven bacterial species (ROC AUC = 0.956, cross-validated AUC = 0.604).

Distinct bacterial metabolic pathways in ME/CFS

Bacterial metagenomic data were used to predict differences in functional metabolic pathways in the ME/CFS subgroups. Altogether, 455 individual bacterial metabolic pathways were identified and analyzed. In superpathway analyses (total 131 superpathways), LEfSe revealed that bacterial vitamin B6 biosynthesis and salvage, pyrimidine ribonucleoside degradation, and atrazine degradation were significantly enriched while bacterial pathways for the biosynthesis of arginine, polyamine, unsaturated fatty acid (FA), and mycolate were significantly reduced in the ME/CFS compared to the controls (Fig. 5a). The ME/CFS + IBS group had predicted enrichment in bacterial pathways for fucose, rhamnose, atrazine degradation and L-threonine biosynthesis, reduced heme, AA and polyamine biosynthesis, and reduced purine, pyrimidine, and unsaturated FA metabolism compared to the controls (Fig. 5b). In the ME/CFS without the IBS group, predicted bacterial pathways of vitamin B6 biosynthesis and salvage, pyrimidine ribonucleosides, atrazine, glycerol and sulfolactate degradation were increased, whereas unsaturated FA and mycolate biosynthesis were decreased compared to the controls (Fig. 5c). Nonparametric Mann-Whitney U test with Benjamini-Hochberg correction (adjusted p < 0.2) further supported findings from LEfSe showing enrichment in the pathway of atrazine degradation in both the ME/CFS and ME/CFS + IBS groups compared to the controls; predicted bacterial pathways of arginine, polyamine biosynthesis, and pyrimidine ribonucleoside degradation were reduced in the ME/CFS + IBS (Additional file 1: Table S3). Based on nonparametric Mann-Whitney U test with Benjamini-Hochberg correction (adjusted p < 0.2), ME/CFS showed altered representation of individual bacterial metabolic pathways linked to the tricarboxylic acid [21] cycle, alcohol and aromatic compound degradation, and FA/lipid metabolism (Additional file 1: Table S3). The ME/CFS + IBS group was associated with altered bacterial pathways for FA/lipid metabolism, aromatic compounds biosynthesis, and carbohydrate (CHO)/carboxylate degradation (Additional file 1: Table S3).

Fig. 5 Altered bacterial metabolic pathways define ME/CFS and ME/CFS subgroup with IBS co-morbidity. Histogram of the log-transformed LDA scores computed with LEfSe for bacterial metabolic pathways found to be differentially abundant between ME/CFS groups and controls. Positive LDA score indicates enrichment of pathways in a ME/CFS, b ME/CFS + IBS, and c ME/CFS without IBS vs. controls. Negative LDA indicates enrichment of pathways in control subjects (reduced in ME/CFS groups). The LDA score indicates the effect size and ranking of each superpathway. An alpha value of 0.05 for the Kruskal-Wallis test and a log-transformed LDA score of 2.0 were used as thresholds for significance in LEfSe analyses. Asterisks next to pathways indicate significant differences were also found for a given pathway based on nonparametric Mann-Whitney U test with Benjamini-Hochberg correction (adjusted p < 0.2) Full size image

Plasma cytokine concentrations do not distinguish ME/CFS groups

Immune profiling (Additional file 1: Table S4A) was performed to test for alterations in the ME/CFS and IBS subgroups. No significant findings were obtained after adjusting for multiple comparisons of all 61 cytokines. However, prior to adjustment, TNF-α was increased in the ME/CFS cases compared to the controls (Additional file 1: Table S4B) and plasma levels of leptin, CSF-2, CXCL-8, and TNF-α were higher in the ME/CFS + IBS patients than the controls (Additional file 1: Table S4B). The ME/CFS patients without IBS had a pre-adjustment trend toward increased TNF-α compared to controls.

Unsupervised hierarchical clustering was used to visualize the variation in plasma cytokine levels between the total ME/CFS, ME/CFS + IBS, ME/CFS without IBS, and controls. Although the clusters did distinguish a range of cytokine profiles in individuals (ranging from high to low cytokine profiles), there was no distinct clustering observed between disease groups (Additional file 2: Figure S2).

A predictive logistic regression model restricted solely to immune data showed little accuracy in distinguishing between the ME/CFS diagnostic groups and controls (total ME/CFS, ME/CFS + IBS, or ME/CFS without IBS vs. controls; data not shown).

Correlations of symptom severity scores with bacterial species abundance and predicted bacterial metabolic pathways in ME/CFS and ME/CFS subgroups

We investigated whether the relative abundance of individual bacterial species, defined by their association with ME/CFS and ME/CFS subgroups (Fig. 3, Additional file 1: Table S2), correlated with Short Form 36 Health Survey (SF-36) and the Multidimensional Fatigue Inventory (MFI). Additional file 1: Table S5 shows the significant correlations of bacterial species with disease severity scores in all ME/CFS, ME/CFS + IBS, or ME/CFS without IBS cases.

Among all ME/CFS cases, the increased relative abundances of R. gnavus, C. bacterium, C. bolteae, and C. asparagiforme were associated with better vitality, health change, and motivation scores. Decreased relative abundances of F. prausnitzii and C. catus were associated with worse emotional wellbeing scores, while decreased abundances of R. inulinivorans and D. formicigenerans were associated with improved motivation scores.

In the ME/CFS + IBS cases, decreased relative abundance of unclassified Alistipes, D. longicatena, and R. inulinivorans were associated with improved vitality, health change, and fatigue scores. Decreased relative abundance of C. comes and Faecalibacterium species were associated with worse fatigue scores and worse pain scores, respectively.

In ME/CFS without IBS cases, the increased relative abundance of P. capillosus was associated with worse vitality, emotional wellbeing, health changes and motivation scores. The relative abundances of D. formicigenerans and C. scindens were associated with improved motivation scores, similar to patterns observed in total ME/CFS.

Metabolic pathways predicted from bacterial metagenomic gene content revealed correlations between activity in specific pathways and clinical features. Decreased polyamine biosynthesis in both ME/CFS and ME/CFS + IBS cases was associated with worse physical function scores and increased fatigue. In ME/CFS + IBS cases, increased fucose and rhamnose degradation and increased threonin biosynthesis were associated with worse general wellbeing and pain scores, decreased phenylalanine and tyrosine biosynthesis, and decreased pyrimidine deoxyribonucleoside degradation were associated with worse general wellbeing, mental fatigue and pain scores. Increased sulfolactate degradation in ME/CFS without IBS was associated with better pain scores.