Likewise, patient subgroups by IBS symptom-related variables were tested for differences in bacterial abundance. Because the mild IBS group contained only three patients, the mild and moderate IBS groups were combined and compared with severe IBS. The LEfSe analysis yielded three Bacteroidetes members (significant from phylum to order level), and three Firmicutes members associated with mild/moderate IBS. Postinfectious onset history of IBS was associated with an unclassified Erysipelotrichaceae genus. Patients with diarrhea-predominant IBS had higher Pseudobutyrivibrio genus, whereas IBS-mix patients had three elevated Propionibacterium taxa (members of Actinobacteria phylum, significant from order to genus level) (Figure 4, Table 6).
The complete list of signature bacteria, their taxonomic classification, model importance, and representative sequences are given in Table S7 (Supplemental Digital Content 7, http://links.lww.com/PSYMED/A511). The top 20 features were all unclassified species, as was confirmed by blast analyses of representative sequences (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Twelve of the top 20 features were members of the Lachnospiraceae family, and three were members of the Ruminococcaceae family (both members of Clostridiales order). These two families were also dominant in the whole set of 148 species, where taken together, they represented 66% of the signature OTUs. At the phylum level, most OTUs were members of Firmicutes (120 OTUs), followed by Bacteroidetes (20 OTUs), whereas Actinobacteria counted two OTUs, and Proteobacteria one, and five OTUs were unclassified up to phylum level (belonging to unknown bacteria).
This study assessed associations between psychological factors and gut microbiota in IBS by using several methods of microbial analysis, including tests of diversity and composition, correlational analyses, subgroup comparisons, and machine learning techniques.
Substantial limitations of this study are the lack of a control group and the small sample size. The results must therefore be interpreted with caution, and further studies are required to determine associations between psychological factors and gut microbiota in patients with IBS, and importantly, also in healthy individuals.
Furthermore, the outcomes of this study were highly dependent on self-report assessments with the HADS. The HADS has been subjected to criticism because of psychometric shortcomings (95), concerning the validity of the subscales anxiety and depression. Our results regarding anxiety and depression should therefore be interpreted carefully. A strength of the HADS however is its familiarity to many clinicians and its wide use in the field (63,66,68), which allows for comparisons across studies.
Another weakness can be seen in the specific OTU clustering technique used in this study, which is implemented in QIIME (88) and has been widely used but was found to inflate OTU counts under certain circumstances (96,97). Methodological advances have recently been made with regard to sequence clustering, and a possible transition from OTUs to higher resolution amplicon sequence variants has been proposed (98–100).
Microbial analyses are also afflicted with general limitations, for example, loss of information in beta-diversity analyses, or possibly misleading results by combination and analysis of bacteria at higher phylogenetic levels without sufficient resolution, the latter also related to the fact that many species are currently not even named. A detailed understanding of microbiome-body interactions cannot be reached until in-depth characterization of these unclassified bacteria has been achieved (e.g., their role in bile acid metabolism or production of neurotransmitter precursors (31,101)). Furthermore, it has been argued that functional properties can differ significantly even below the 97% genetic similarity criterion (102). In general, observing alterations of single bacteria can be misleading, because it remains unclear to which extent they are the results of microbe-microbe or microbe-host interactions. Taking into account comprehensive bacterial signatures, as proposed in this study, seem therefore reasonable. However, the machine learning was constrained by the small sample size, the lack of an external validation data set, and possible overfitting. The bacterial signature of psychological distress can therefore not claim generalization. It can only help orienting research toward relevant bacteria.
The bacterial diversity and the number of reads per sample in this study were high in comparison with other studies (62,63). Diversity was however not different between the subgroups, which may be due to the small sample size. Because several animal studies have reported decreased alpha diversity after exposure to stress (15,16,103), further investigations are required to assess the association between psychological variables and alpha diversity in humans. Microbiome composition was associated with psychological distress and depression, whereas other potentially confounding variables showed no association. This indicates systematic shifts in certain taxa in parallel with psychological burden. The cohort was separated in two clusters according to microbial composition. Psychological characteristics were equally distributed among these, with a slightly higher presence of psychological distress in the cluster with lower Firmicutes to Bacteroidetes ratio. This is similar to the results of Jeffery and colleagues (66), where psychological burden was higher in patients belonging to a cluster with microbiomes resembling those of healthy controls. The authors elegantly interpreted this discovery as a more “centrally triggered” IBS. In contrast to their work, our study lacks a control group for comparison with healthy microbiomes.
Irrespective of microbial analyses, the low correlation between IBS symptom burden and psychological distress was a surprising finding of this study. Previous studies reported mixed magnitudes of this relationship (104–106). In our opinion, the findings reflect that IBS is a heterogeneous disorder (43,45) and that association of psychological and IBS symptoms can occur in any configuration.
IBS symptom-related variables were also taken into consideration in the microbial subgroup analyses. The severity of IBS was correlated with Ruminococcus, which adds to previous findings (58,109). IBS-mix and IBS-C were characterized by elevated Propionibacterium. A possible role in slowing down intestinal transit has been previously observed (110) and warrants further investigation. Postinfectious history of IBS onset was associated with a significantly elevated Erysipelotrichaceae genus. Erysipelotrichaceae were previously found to flourish after treatment with broad-spectrum antibiotics and are rated as highly immunogenic (111). These properties might offer an explanation for subsequent bowel dysfunction after gastrointestinal infections. However, other previously described microbial characteristics of PI-IBS (67,109) were not replicated in our study.
This study assessed relationships between gut microbiota and psychological variables in a sample of patients with IBS. Notably, the study generated further evidence for a relationship between psyche and gut bacteria, underlining the importance of brain-gut alterations and the psychological dimension in IBS. Psychological distress was associated with gut microbiota composition, and a microbial signature corresponding with psychological distress was identified. In-depth characterization of these bacteria might lead to discovery of new biomarkers and therapeutics. The findings further emphasize the relevance of gut bacteria for stress reactivity in humans and for integrated approaches of clinical management of IBS. Future studies will also have to determine, if distress-associated microbial alterations are specific to IBS, a disease picture with both altered microbiome and stress reactivity characteristics, or if similar associations are also present in healthy individuals with varying levels of distress.
The authors thank Christoph Högenauer, Slave Trajanoski, Ingeborg Klymiuk, the Center for Medical Research Graz, Nina Rittershaus, Nicola Stephanou-Rieser, Stella Held, Sarah Russegger, Benjamin Block, and the participants for providing samples and information for the study.
Source of Funding and Conflicts of Interest: The study was supported by Österreichische Nationalbank Jubliäumsfonds (Grant Number 16506), Österreichische Gesellschaft für Gastroenterologie und Hepatologie M.T. has been on the Adisory Boards of Albireo, Gilead, Falk, Novartis, Intercept, MSD, and Phenex. He has received grants to the Medical University of Vienna from Albireo, Falk, Intercept, MSD, and Takeda. He has been on the speakers bureaus for Gilead, Falk, and MSD. He is holding patents for the use of nor-Ursodeoxycholic acid. He has received travel expenses from Gilead and Falk. J.P. has received travel expenses from Yakult. G.M. has been on the Advisory Boards of Allergan and Almirall, she has received grants to the Medical University of Vienna by AbbVie, Vifor, Almirall, Merck, Falk, Yakult, Sanova, Danone, and she has been on the speakers bureaus for Falk, Peri Consulting, Henrich Communication, Milton Erickson Institut Austria, Wirtschaftskammer Austria, and Gebro. She has received payments for development of educational presentations by Ärztekammer Austria and travel expenses by Gebro and Falk.
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