Distinct gut microbial compositional and functional changes associated with impaired inhibitory control in patients with cirrhosis

Figures & data

Table 1. Details of Patients with Minimal Hepatic Encephalopathy on Psychometric Hepatic Encephalopathy Score (PHES) and on Inhibitory Control test (ICT) (N = 97)

	MHE on PHES			MHE on ICT
	No (n = 50)	Yes (n = 47)	P value	No (n = 21)	Yes (n = 76)	P value
Age	57.9 ± 6.9	62.3 ± 6.4	0.01	58.1 ± 8.2	60.6 ± 6.5	0.21
Gender	30 (60%)	42 (85%)	0.004	15 (71%)	57 (75%)	0.74
PPI	26 (52%)	34 (69%)	0.03	13 (62%)	47 (62%)	1.0
Diabetes	13 (26%)	18 (37%)	0.19	9 (43%)	22 (29%)	0.23
MELD score	9.6 ± 3.1	12.7 ± 3.5	<0.0001	10.8 ± 3.7	11.2 ± 3.6	0.66
Etiology (HCV/Alc/Both/ NASH &other)	15/13/15/7	14/26/4/3	0.02	10/8/5/2	19/31/14/8	0.41
Depression	9 (18%)	14 (29%)	0.14	5 (24%)	18 (24%)	0.53
Anxiety	3 (6%)	3 (4%)	0.9	1 (5%)	5 (6%)	1.0
SSRI	7 (14%)	9 (19%)	0.51	4 (20%)	12 (16%)	0.32
SNRI	2 (4%)	1 (2%)	0.58	2 (10%)	1 (1%)	0.10
Opioids	4 (8%)	2 (4%)	0.43	1 (5%)	5 (6%)	1.0
Benzodiazepines	4 (8%)	1 (2%)	0.16	1 (5%)	4 (5%)	1.0
Gabapentin	4 (8%)	3 (7%)	0.70	1 (5%)	6 (8%)	0.88
Prior HE	24 (48%)	36 (77%)	<0.0001	14 (67%)	46 (61%)	0.61
Lactulose	21 (42%)	36 (77%)	<0.0001	10 (48%)	49 (65%)	0.16
Rifaximin	12 (24%)	27 (57%)	0.001	8 (38%)	31 (41%)	0.81
Individual tests
PHES subtests
Number connection A (seconds)	31.6 ± 9.5	65.8 ± 21.9	<0.0001	40.6 ± 19.2	50.3 ± 24.8	0.06
Number connection B (seconds)	73.8 ± 19.7	193.0 ± 112.0	<0.0001	98.8 ± 76.6	141.0 ± 103.0	0.05
Digit Symbol (number)	59.2 ± 13.6	33.3 ± 10.7	<0.0001	54.8 ± 18.2	44.4 ± 17.2	0.03
Serial dotting (seconds)	56.6 ± 14.2	104.0 ± 44.2	<0.0001	64.5 ± 22.8	83.8 ± 42.9	0.008
Line tracing Errors (number)	33.1 ± 24.1	45.0 ± 31.7	0.04	32.2 ± 32.0	40.7 ± 27.5	0.28
Line tracing time (seconds)	79.6 ± 22.8	130.8 ± 68.5	<0.0001	98.8 ± 61.1	106.0 ± 55.3	0.63
Total PHES (low = good)	−0.6 ± 1.5	−9.1 ± 3.5	<0.0001	−2.7 ± 4.7	−5.3 ± 5.0	0.03
ICT subtest
ICT Lures (number out of 40)	12.5 ± 9.5	17.3 ± 9.9	0.02	3.1 ± 1.6	18.1 ± 8.7	<0.0001
ICT Targets (%)	96.2 ± 6.1	86.1 ± 14.8	<0.0001	97.7 ± 2.5	89.5 ± 13.2	<0.0001
ICT Weighted lures (number)	14.2 ± 11.7	27.9 ± 22.5	<0.0001	3.3 ± 1.8	25.6 ± 18.7	<0.0001

Figure 1. Bacterial species comparison between patients with MHEPHES (n = 47) versus not (n = 50)1A: Alpha diversity analyses did not show any differences between groups 1B: Cleveland plot derived from DESeq2 comparison 1 C: PCoA showing no significant separation between groups (PERMANOVA not significant)

Table 2. MAAsLin2 Top 30 Bacterial Species and Clinical Variables associated with MHE on Individual tests

MHE on PHES	coefficient	P-value	Q-value	MHE on ICT	Coefficient	P-value	Q-value
Clostridium_aerotolerans	−7.3860288	1.14E-07	4.82E-05	Pseudomonas_stutzeri	−11.076275	6.59E-08	3.79E-06
Ruminococcaceae_bacteriumCPB6	−7.1714167	1.17E-06	0.00016485	Clostridium_aerotolerans	−8.5224458	6.89E-18	8.31E-15
Hafnia_alvei	−6.825657	6.92E-07	0.00012514	Ruminococcaceae_bacterium_CPB6	−8.1487537	2.94E-16	1.78E-13
Desulfovibrio_fairfieldensis	−6.559384	1.67E-06	0.00021139	Enterococcus_sp._HMSC064A12	−7.351274	8.60E-16	3.46E-13
Desulfovibrio_sp._6_1_46AFAA	−6.4232119	5.51E-06	0.0002792	Enterococcus_sp._HMSC070F12	−6.9835319	7.14E-12	1.08E-09
Ethanoligenens_harbinense	−6.4218669	9.70E-06	0.00045469	Enterococcus_sp._HMSC076D08	−6.8696824	5.83E-14	1.17E-11
Hafnia_sp._HMSC23F03	−6.4054216	5.42E-06	0.0002792	Enterococcus_sp._HMSC035B04	−6.7896399	1.10E-14	3.31E-12
Prevotella_ruminicola	−5.4842961	1.87E-07	5.93E-05	Enterococcus_sp._HMSC077E07	−6.492291	3.67E-09	2.95E-07
Corynebacterium_argentoratense	−5.3129369	3.40E-06	0.00027784	Ethanoligenens_harbinense	−6.4231516	7.25E-10	7.29E-08
Bacteroides_coprocola	−2.898706679	1.38E-05	0.000581536	Veillonella_seminalis	−6.3948881	1.20E-10	1.45E-08
Prevotellamassilia_timonensis	−2.8492228	3.13E-06	0.00027784	Desulfovibrio_sp._6_1_46AFAA	−6.2707191	5.57E-07	2.50E-05
MELD score	0.10135968	4.62E-06	0.0002792	Enterococcus_sp._HMSC060D09	−6.245521	1.09E-08	7.72E-07
Lactobacillus_zeae	4.15539892	5.05E-06	0.0002792	Clostridium_sp._Marseille-P2415	−6.1345706	1.49E-12	2.58E-10
Prevotella_oris	4.20943187	4.17E-06	0.00027784	Enterococcus_sp._HMSC073E08	−6.045244	2.71E-10	2.98E-08
Klebsiella_aerogenes	4.27465841	3.71E-07	7.83E-05	Enterococcus_sp._HMSC077E04	−5.9138986	1.12E-10	1.45E-08
Synergistes_sp._3_1_syn1	4.53235446	5.18E-06	0.0002792	Atopobium_minutum	−5.7353621	2.91E-07	1.40E-05
Cloacibacillus_evryensis	4.8373667	5.82E-06	0.00028338	Enterococcus_sp._HMSC065H03	−5.6796115	2.68E-09	2.31E-07
Lactobacillus_acidophilus	4.96799149	3.84E-08	4.23E-05	Enterococcus_sp._HMSC060E05	−5.673559	9.69E-09	7.31E-07
Lactobacillus_saerimneri	5.035620618	1.26E-05	0.000549872	Enterococcus_sp._HMSC056C08	−5.5581075	7.27E-08	3.99E-06
Bifidobacterium_mongoliense	5.05623943	2.42E-06	0.00027784	Turicibacter_sp._H121	−5.3751102	7.76E-07	3.23E-05
Lactobacillus_animalis	6.40954148	3.53E-06	0.00027784	Lactobacillus_mucosae	−5.0214959	1.62E-09	1.51E-07
Campylobacter_helveticus	6.5153248	5.10E-06	0.0002792	Enterococcus_faecium	−4.9810595	1.60E-14	3.87E-12
Kluyvera_ascorbata	6.6757588	8.73E-07	0.00013808	Enterococcus_sp._HMSC034B11	−4.9491816	2.86E-07	1.40E-05
Lactobacillus_perolens	6.7806971	1.05E-05	0.00047562	Pseudopropionibacterium_propionicum	−4.5684879	6.28E-08	3.79E-06
Lactobacillus_harbinensis	6.89835971	6.69E-08	4.23E-05	Dysgonomonas_capnocytophagoides	−4.2552989	5.70E-08	3.62E-06
Pectobacterium_polaris	7.31494204	2.90E-06	0.00027784	Enterococcus gallinarum	−3.8421406	1.99E-08	1.33E-06
Providencia_rettgeri	7.87775259	4.01E-06	0.00027784	Parvimonas micra	−3.8132837	1.08E-07	5.67E-06
Atopobium_minutum	12.7125871	3.16E-06	0.00027784	Lactobacillus sakei	−3.6981035	6.35E-07	2.74E-05
Allofustis_seminis	13.4373102	4.02E-06	0.00027784	Clostridium hylemonae	−3.2696316	1.62E-06	6.53E-05
Clostridium_sp. BNL1100	13.6372933	2.53E-07	6.41E-05	Dakarella massiliensis	6.08660083	5.60E-07	2.50E-05

Table 3. MAAsLin2 Top 30 Bacterial Species and Clinical Variables

MHEICT only (n = 35) versus MHEPHES only (n = 6)			MHEPHES only (n = 6) versus No MHEPHES (n = 50)			MHEICT only (n = 35) versuss NoMHEICT (n = 21)
Feature	Higher in	P-value	Feature	Higher in	P-value	Feature	Higher in	P-value
Turicibacter_sp. H121	PHESMHE-NoICT	1.25E-16	Dysgonomonas capnocytophagoides	MHEPHES	9.75E-04	Clostridium sp. ASF502	NoMHEICT	2.98E-04
Enterococcus_sp. HMSC064A12	PHESMHE-NoICT	1.29E-13	Lautropia mirabilis	MHEPHES	0.001	Clostridium sp. D5	NoMHEICT	8.96E-04
Enterococcus_sp. HMSC035B04	PHESMHE-NoICT	2.61E-13	Fusicatenibacter_sp. 2789STDY5834925	MHEPHES	0.002	Lachnoclostridium sp._YL32	NoMHEICT	0.003
Enterococcus_sp. HMSC14A10	PHESMHE-NoICT	1.58E-11	Rothia_sp. HMSC072B03	MHEPHES	0.003	Blautia schinkii	NoMHEICT	0.008
Citrobacter_freundii complex_sp._CFNIH2	PHESMHE-NoICT	1.53E-10	Absielladolichum	MHEPHES	0.005	Dakarella massiliensis	MHEICT	0.008
Enterococcus_sp HMSC073E08	PHESMHE-NoICT	7.24E-10	MELD score	MHEPHES	0.005	Erysipelotrichaceae_bacterium_2_2_44A	NoMHEICT	0.009
Proteus_mirabilis	PHESMHE-NoICT	2.31E-09	Enterococcus sp. HMSC069A01	MHEPHES	0.005	Lactobacillus timonensis	NoMHEICT	0.014
Enterococcus_sp. HMSC065H03	PHESMHE-NoICT	2.33E-09	Rothia_mucilaginosa	MHEPHES	0.006	Streptococcus infantarius	NoMHEICT	0.015
Enterococcus_sp. HMSC076D08	PHESMHE-NoICT	2.83E-09	Rifaximin use	MHEPHES	0.008	Prevotella intermedia	MHEICT	0.020
Enterococcus_sp. HMSC070F12	PHESMHE-NoICT	3.42E-09	Lactococcus_piscium	MHEPHES	0.010	Lactobacillus reuteri	NoMHEICT	0.023
Enterococcus_sp. HMSC077E07	PHESMHE-NoICT	4.00E-09	Eisenbergiella_tayi	MHEPHES	0.010	Dysgonomonas capnocytophagoides	NoMHEICT	0.024
Enterococcus_sp. HMSC060E05	PHESMHE-NoICT	1.27E-08	Enterococcus sp. HMSC066C04	MHEPHES	0.011	Cellulomonas carbonis	MHEICT	0.025
Clostridium sp. BNL1100	PHESMHE-NoICT	2.54E-08	Lachnospiraceae bacterium 3_1_57FAA_CT1	MHEPHES	0.011	Bifidobacterium animalis	NoMHEICT	0.029
Enterococcus_sp. HMSC056C08	PHESMHE-NoICT	2.69E-08	Enterococcus sp. HMSC061C05	MHEPHES	0.012	Prevotella_bergensis	MHEICT	0.032
Enterococcus_sp. HMSC034B11	PHESMHE-NoICT	4.17E-08	Coprobacillus sp. 29 1	MHEPHES	0.012	Enterococcus_sp._HMSC035C10	NoMHEICT	0.033
Enterococcus gallinarum	PHESMHE-NoICT	1.34E-07	Enterococcus sp. 10F3_DIV0382	MHEPHES	0.013	Prevotella lascolaii	MHEICT	0.036
Enterococcus_sp. HMSC077E04	PHESMHE-NoICT	2.41E-07	Lactulose use	MHEPHES	0.014	Streptococcus equinus	NoMHEICT	0.037
Enterococcus asini	PHESMHE-NoICT	5.91E-07	Enterococcus sp. HMSC058D07	MHEPHES	0.015	Lactobacillus rogosae	MHEICT	0.039
Streptococcus_sp. HMSC072D07	PHESMHE-NoICT	6.32E-07	Candidatus_Saccharibacteria_oral_taxon_TM7x	MHEPHES	0.019	Clostridium hylemonae	NoMHEICT	0.043
Eisenbergiella tayi	PHESMHE-NoICT	1.56E-06	Rothia_sp. HMSC062F03	MHEPHES	0.021	Megamonas_sp._Calf98_2	NoMHEICT	0.044
Prevotella copri	ICTMHE-NoPHES	1.64E-06	Clostridium sp. D5	MHEPHES	0.021	Microvirgula aerodenitrificans	NoMHEICT	0.047
Clostridium_sp._ASF502	PHESMHE-NoICT	1.73E-06	Enterococcus sp. 5B3_DIV0040	MHEPHES	0.021	Hungatella hathewayi	NoMHEICT	0.047
Streptococcus_sp. HMSC076C08	PHESMHE-NoICT	1.23E-05	Enterococcus gilvus	MHEPHES	0.022	Bacteroides bouchesdurhonensis	MHEICT	0.048
Dysgonomonas_capnocytophagoides	PHESMHE-NoICT	1.67E-05	Streptococcus sp. UMB0029	MHEPHES	0.023	Prevotella albensis	MHEICT	0.048
Prevotella_sp._Marseille_P4119	PHESMHE-NoICT	2.27E-05	Streptococcus sp. HPH0090	MHEPHES	0.023	Prevotella buccalis	MHEICT	0.049
Enterococcus_sp. HMSC072F02	PHESMHE-NoICT	2.73E-05	Bacteroides paurosaccharolyticus	MHEPHES	0.023	Desulfovibrio desulfuricans	MHEICT	0.049
Megasphaera massiliensis	ICTMHE-NoPHES	3.14E-05	Enterococcus sp. HMSC035B04	MHEPHES	0.025	Lactobacillus gastricus	NoMHEICT	0.050
Eggerthella_sp. YY7918	ICTMHE-NoPHES	3.58E-05	Prior HE	MHEPHES	0.025	Lachnospiraceae_bacterium_3_1	NoMHEICT	0.050
Alistipes ihumii	ICTMHE-NoPHES	3.83E-05	Streptococcus_sp._NPS_308	MHEPHES	0.026	Prevotella copri	MHEICT	0.050
Prevotella oralis	PHESMHE-NoICT	4.50E-05	Enterococcus sp. HMSC070F12	MHEPHES	0.028	Enterococcus faecium	NoMHEICT	0.050
Variables that are positively linked with MHE on ICT not PHES are in bold font			All variables were higher in those with MHEPHES only versus the rest			Variables that are positively linked without MHE on ICT are in bold font

Table 4. Comparison of Gut-Brain Modules Different in Patients According to Cognitive Strategy used

PHES GBM Lineage	LOG2FC	Direction	P-value
MGB056_Propionate_degradation_I	−1.06	MHE PHES	0.001806
MGB022_GABA_synthesis_III	−0.75	MHE PHES	0.03326
MGB021_GABA_synthesis_II	−0.58	MHE PHES	0.029268
MGB020_GABA_synthesis_I	−0.51	MHE PHES	0.038805
MGB047_Acetate_degradation	−0.47	MHE PHES	0.015038
MGB006_Glutamate_synthesis_I	−0.02	MHE PHES	0.011326
MGB027_Nitric_oxide_degradation_I_NO_dioxygenase	0	MHE PHES	0.012334
MGB029_ClpB_ATP_dependent_chaperone_protein	0.03	No MHE PHES	0.023788
MGB050_Glutamate_degradation_I	0.04	No MHE PHES	0.007327
MGB034_Isovaleric_acid_synthesis_I_KADH_pathway	0.11	No MHE PHES	0.032533
MGB040_Menaquinone_synthesis_vitamin_K2_I	0.12	No MHE PHES	0.026581
MGB038_Inositol_degradation	0.13	No MHE PHES	0.040531
MGB033_Quinolinic_acid_degradation	0.23	No MHE PHES	0.022053
MGB052_Butyrate_synthesis_I	0.27	No MHE PHES	0.005594
MGB041_Menaquinone_synthesis_vitamin_K2_II_alternative_pathway_futalosine_pathway	0.56	No MHE PHES	0.038444
MGB028_Nitric_oxide_degradation_II_NO_reductase	0.81	No MHE PHES	0.044083
MGB024_DOPAC_synthesis	1	No MHE PHES	0.023769
ICT GBM Lineage
MGB032_Quinolinic_acid_synthesis	−5.1	MHE ICT	0.001
MGB043_Acetate_synthesis_I	−4.69	MHE ICT	0.005995
MGB047_Acetate_degradation	−3.97	MHE ICT	0.045925
MGB041_Menaquinone_synthesis_vitamin_K2_II_alternative_pathway_futalosine_pathway	−3.72	MHE ICT	0.02515
MGB033_Quinolinic_acid_degradation	−3.72	MHE ICT	0.0322
MGB040_Menaquinone_synthesis_vitamin_K2_I	−3.7	MHE ICT	0.00621
MGB034_Isovaleric_acid_synthesis_I_KADH_pathway	−3.54	MHE ICT	0.021862
MGB029_ClpB_ATP_dependent_chaperone_protein	−3.54	MHE ICT	0.029987
MGB006_Glutamate_synthesis_I	−3.37	MHE ICT	0.014197
MGB038_Inositol_degradation	−3.29	MHE ICT	3.97E-04
MGB053_Butyrate_synthesis_II	−3.25	MHE ICT	0.008637
MGB020_GABA_synthesis_I	−3.17	MHE ICT	0.008621
MGB024_DOPAC_synthesis	−3.17	MHE ICT	0.023021
MGB052_Butyrate_synthesis_I	−3.11	MHE ICT	0.007033
MGB050_Glutamate_degradation_I	−2.96	MHE ICT	0.042206
MGB021_GABA_synthesis_II	−2.81	MHE ICT	0.015873
MGB027_Nitric_oxide_degradation_I_NO_dioxygenase	−2.58	MHE ICT	0.002261
MGB056_Propionate_degradation_I	−2.21	MHE ICT	0.034031
MGB028_Nitric_oxide_degradation_II_NO_reductase	−1	MHE ICT	2.74E-05
Discordant GBM Lineage
MGB005_Tryptophan_synthesis	6.15	ICTMHE-NoPHES	8.40E-05
MGB032_Quinolinic_acid_synthesis	5	ICTMHE-NoPHES	0.003334
MGB040_Menaquinone_synthesis_vitamin_K2_I	4.49	ICTMHE-NoPHES	0.009683
MGB033_Quinolinic_acid_degradation	4.43	ICTMHE-NoPHES	0.012096
MGB041_Menaquinone_synthesis_vitamin_K2_II_alternative_pathway_futalosine_pathway	4	ICTMHE-NoPHES	0.005421
MGB029_ClpB_ATP_dependent_chaperone_protein	4	ICTMHE-NoPHES	0.023231
MGB034_Isovaleric_acid_synthesis_I_KADH_pathway	3.91	ICTMHE-NoPHES	0.049365
MGB053_Butyrate_synthesis_II	3.81	ICTMHE-NoPHES	0.047532
MGB047_Acetate_degradation	3.7	ICTMHE-NoPHES	0.031842
MGB038_Inositol_degradation	3.17	ICTMHE-NoPHES	0.023127
MGB052_Butyrate_synthesis_I	3.09	ICTMHE-NoPHES	0.034837
MGB020_GABA_synthesis_I	2.81	ICTMHE-NoPHES	0.017337
MGB050_Glutamate_degradation_I	2.74	ICTMHE-NoPHES	0.017733
MGB044_Acetate_synthesis_II	2.68	ICTMHE-NoPHES	0.031584
MGB006_Glutamate_synthesis_I	2.66	ICTMHE-NoPHES	0.001651
MGB024_DOPAC_synthesis	1.58	ICTMHE-NoPHES	0.041681

Figure 2. Gut brain module comparison between patients with MHEPHES (n = 47) versus not (n = 50) 1A: Alpha diversity analyses did not show any differences between groups 1B: Cleveland plot derived from Metastats comparison 1 C: PCoA showing no significant separation between groups (PERMANOVA not significant)

Figure 3. Bacterial species comparison between patients with MHEICT (n = 76) versus not (n = 21) 1A: Alpha diversity analyses did not show any differences between groups 1B: Cleveland plot derived from DESeq2 comparison 1 C: PCoA showing trend toward a significant separation between groups (PERMANOVA p = .08)

Figure 4. Gut brain module comparison between patients with MHEICT (n = 76) versus not (n = 21)1A: Alpha diversity analyses showed significantly higher diversity in the MHEICT group compared to no-MHE 1B: Cleveland plot derived from Metastats comparison 1 C: PCoA showing a significant separation between groups

Figure 5. Bacterial species comparison between patients with MHEICT-only (n = 35) versus MHEPHES only (n = 6)1A: Alpha diversity analyses did not show any differences between groups 1B: Cleveland plot derived from DESeq2 comparison 1 C: PCoA showing no significant separation between groups

Figure 6. Gut brain module comparison between patients with MHEICT-only (n = 35) versus MHEPHES only (n = 6) 1A: Alpha diversity analyses showed significantly higher diversity in the MHEICT only group compared to MHEPHES only group 1B: Cleveland plot derived from Metastats comparison 1 C: PCoA showing a significant separation between groups