Dynamic changes of intestinal flora in patients with irritable bowel syndrome combined with anxiety and depression after oral administration of enterobacteria capsules

ABSTRACT This study investigated the clinical characteristics and dynamic changes of intestinal bacterial community to evaluate the curative effect of fecal microbiota transplantation (FMT) on irritable bowel syndrome with predominant diarrhea (IBS-D) comorbid with anxiety and depression. Total two treatments were designed in randomize-controlled trial includes oral FMT capsules with 1 week (A1), 8 weeks (A2), and 12 weeks (A3), as well as oral empty capsules with 1 week (B1), 8 weeks (B2), and 12 weeks (B3) as control for comparison. The positive therapeutic effects occurred in FMT colonized patient with IBS-D comorbid psychological disorder, demonstrated at alleviated IBS-D severity (IBS-SSS score from 291.11 reduced to 144.44), altered stool type (from 6 changed to 4), reduced anxiety and depression scores (from 18.33 to 8.39 and from 22.33 to 17.78) after FMT-treated 12 weeks. The FMT therapy improved bacterial alpha diversity and the majority bacterial community predominant by Bacteroidetes and Firmicutes, and the relative abundance (RA) was higher after FMT-treated 12 weeks (50.61% and 45.52%) than control (47.62% and 38.96%). In short, FMT therapy has great potential for IBS-D patients combined with anxiety and depression by alleviated clinical symptoms and restore the intestinal micro-ecology.


Introduction
Irritable bowel syndrome (IBS) is a persistent or intermittent functional-gastrointestinal disorder and characterized by altered bowel habits as well as abdominal pain. According to statistics, the global IBS prevalence rate ranged from 7% to 21%, and approximately one third of patients are dominant by constipation, and one third suffer from diarrhea, and the rest patients are mixed or unclassified bowel pattern [1,2]. Previous study has recognized the gastrointestinal diseases usually overlap with other dysfunctions and psychiatric diseases, as estimated that 60-85% of clinical gastrointestinal patients suffer from mental disorders. There seems to be bidirectional between IBS and psychiatric diseases, and it is reported that the prevalence of depression in IBS patients is as high as 84% and the prevalence of anxiety is 44%; meanwhile, 25-30% of depression and 10-45% of anxiety patients will be developed into IBS [3]. Since the depression contributed as the major factor of disability worldwide, anxiety is also ranked the sixth largest actor to the non-fatal burden. Therefore, the co-occurrence of chronic diseases and psychiatric disorders is a huge burden on patients.
The pathogenesis of IBS is still unclear and the destruction of symbiosis between human hosts and microbial communities, and the comprehensive interaction or communication between microbial communities and autonomic nervous system is considered to be the core factor for the persistence of IBS symptoms [4,5]. Importantly, the intestinal microbiota is known as second brain of humans and essential in regulating the central nervous system [6,7]. Recent studies have hypothesized that the ecological disorders and immune responses observed in IBS may drive and maintain the IBS gastrointestinal symptoms [4]. As reported, the fecal microbial characteristics are similar between irritable bowel syndrome with predominant diarrhea (IBS-D) and depression patients, while lower microbial diversity observed compare with healthy donors and speculated to be the mucosal immune barrier dysfunction derived by microbial disorders [6][7][8]. Pittayanon et al. found higher abundance of Proteobacteria and Bacteroides in IBS with psychiatric disorders patients, while lower alpha diversity and abundance of Bacteroidetes, Actinobacteria, and Faecalibacterium [9,10]. Therefore, to balance the intestinal microbial ecosystem, researchers keep positively and constantly exploring pharmacological and non-pharmacological treatments comprised diet change, antispasmodics, anti-diarrhoeals, antidepressants, probiotics, antibiotics, serotonin agonists, hypnotherapy, and guanylate cyclase C agonists intervention [11][12][13].
Among them, the technique of fecal microbiota transplantation (FMT) has been proposed through transferring the healthy donors gut microbiota to recipient, aim to restore the imbalanced microbial communities, and establish a complex and stable microbial community system [14,15]. In current clinical practice, FMT has been proved to be very successful to treat Clostridium difficile infection with 90% cure rate [16,17]. However, the efficiency of FMT to treat IBS is still controversial, Kurokawa et al. suggested that FMT may effectively alleviate certain mental disorders after 8 weeks oral microbial transplantation in autistic children [3]. While Aroniadis et al. pointed out that hadn't relief symptoms after 12 weeks FMT treated on IBS-D through a randomized, placebo-controlled and double-blind trial [18]. However, a cohort trial observed the improvement in IBS-SSS-based symptoms after 3 months of FMT treatment [19][20][21][22].
Although previous study confirmed that oral FMT have been used for IBS therapy, while there is no specific treatment for IBS-D comorbid with anxiety and depression, as well as limited information in intestinal bacterial community dynamics response to FMT treatment. Therefore, the present study investigates clinical symptoms and detected the bacterial community dynamics through 16S rRNA Miseq_PE300 sequencing and bioinformatics analysis on fecal samples of FMT-treated IBS-D patients compared with control, aimed at evaluating the curative effect of FMT on alleviated clinical symptoms and reconstruction of intestinal micro-ecology in IBS-D and psychiatric comorbidities patients.

Participant's recruitment
Present study was carried out at Ping Xiang People's Hospital in Jiangxi, China. Patients diagnosed of IBS-D as well as Hamilton anxiety and depression score within 14-28 and 20-34 points were recruited for this trial. The exclusion criteria were as follows: had any abdominal surgery, human immunodeficiency virus infection, kidney disease, psychosis (mania and schizophrenia), pregnancy, active infection, abnormal thyroid function, abnormal liver function, use of probiotics, prebiotics, and antibiotics in the last 2 weeks, as well as participate in other clinical trials within 3 months were excluded. Total 18 patients were included to evaluate the response of fecal bacteria transplantation to clinical symptoms and dynamic changes of intestinal bacteria after FMT treatment. This study protocol was registered in China Clinical Trial Registry Center (ChiCTR1900024924) and approved by the Ethics Committee of Pingxiang People's Hospital (2019R001-F04). The informed consent and voluntarily accept fecal bacteria transplantation of participants was obtained before enrollment.

Trial design as well as feces collection
Present study belongs to randomized-controlled observational clinical trial, and 18 patients diagnosed as IBS-D comorbid with anxiety and depression were randomly divided into two group of FMT therapy and control (9 patients in each group). The FMT treatment was intervened by oral enteric capsules for 3 times (every 2 days one time and 30 capsules each time) and recorded the symptoms after therapeutic 1 week (A1), 8 weeks (A2), and 12 weeks (A3). At the same time, oral empty capsules as control and record the symptoms after 1 week (B1), 8 weeks (B2), and 12 weeks (B3). Enterobacter FMT capsules and empty capsules were technical supported by Chengge Biotechnology Co., Ltd. (Xiamen, China). The feces collection was carried out by using the feces collection box provided from Allwegene Technology Co., Ltd (Beijing, China).

16S rRNA Miseq_PE300 sequencing
After completing the genomic DNA extraction from collected feces samples, quality was determined by using 1% agarose gel electrophoresis. The specific primers with barcode were synthesized according to the target sequencing region V3-4, and the sizes of the amplified bands of polymerase chain reaction products were detected by 1% agarose gel electrophoresis and purified by Agencourt AMPure XP nucleic acid purification kit. Furthermore, Miseq library construction and computer sequencing was by Allwegene Technology Co., Ltd (Beijing, China).

Bioinformatics and statistical analysis
The obtained sequencing from Miseq sequencing was Pair-End (PE) double-ended sequence data; the measured Fastq data perform quality control by using Trimmomatic (v0.36) and Pear (v0.9.6), and then merged the two ends of the sequence according to the overlap relationship of PE by using Flash (v1.20) and Pear (v0.9.6), remove the chimera of the Fasta sequence and short sequence, finally obtained high quality Fasta data. The valid data from Raw PE remove barcode and primer obtained raw tags further remove chimera and short sequences get clean tags. Clustering all clean tags and classified Operational Taxonomic Units (OTUs) according to different similarity levels, then perform biological information statistical analysis by Qiime (Version 1.8.0 http://www. qiime.org/) and Vsearch (2.7.1) at 97% similarity level.
Bacterial annotation taxonomy analysis obtained from blast or RDP Classifier compare the representative sequences of OTUs and the annotated at phylum, class, order, family, and genus levels. The heatmap was obtained from distance calculation and clustering analysis by R language (vegan packages), vegdist, and hclust. Alpha diversity comprised of Chao1, Simpson, Observed species, PD whole tree, and Shannon were calculated by qiime (v.18.0). Beta diversity based on unifrac and Bray-Curtis of principal coordinates and component analysis as well as nonmetric multidimensional scaling carried out by R language. The network analysis was performed during top 20 genera by using spearman method in R language.
For clinical indicators, the severity of abdominal symptoms was evaluated by irritable bowel syndrome scoring system (IBS-SSS) and classified as mild, moderate, and severe (range of 75-175, 175-300, 300-500) [22,23]. The psychiatric symptoms of patients were evaluated by trained psychiatrists using Hamilton Anxiety and Depression Rating Scale (HAM-A and HAM-D). Based on American Psychiatric Association guidelines, the normal range of anxiety and depression were HAM-A < 14 and HAM-D < 8 [24]. Additionally, Bristol Stool Form Scale was used as feces shape classification (7 = liquid, 3 and 4 represent ideal shape, 1 = very hard), and quality of life was assessed by irritable bowel syndrome quality of life Scale (IBS-QOL) [25][26][27].

Clinical response of IBS-D patients to fecal microbiota transplantation therapy
To evaluate the curative effect of FMT on clinical symptoms relief of IBS-D severity and anxiety/ depression, the distribution of clinically characters is demonstrated in Tables 1 and 2. A total of 18 patients were enrolled and compared 9 FMT treated and 9 without FMT-treated IBS-D patients in randomized controlled trials. The stool samples were collected from all of the patients in the FMT group or placebo group. Moreover, the FMT group consisted of 5 males and 4 females with a mean age of 44 years, while 5 males and 4 females with an average age of 50 years in the control group. The positive clinical response occurred in FMT group, while the clinical symptom was similarly between two groups at the initially stage. Specifically, the mean score of IBS-SSS and stool type were 284. 44 Table 2).
The primary clinical response of FMT-treated IBS-D patients was IBS-SSS score declined by 113 points  Week 8 Week 12     after 12 weeks, simultaneously the anxiety and depression scores decreased 9 and 4 points. In contrast, the control basically remained stable or slightly increased among all indexes. Schmulson [10]. From the clinical response of IBS-D patients to FMT treatment, stool characteristics were improved and alleviated the severity of IBS-D combined with anxiety and depression.

Alteration of alpha and beta diversity after fecal microbiota transplantation therapy IBS-D patients
The intestinal microbial community maintains dynamic balance to form a stable intestinal microecosystem, which participates in the differentiation and proliferation of intestinal mucosal epithelial cells and promotes the development of the mucosal immune system [34]. While intestinal microbial disordered and relative abundance of bacterial disbalance in proportions, such as reduced probiotics and increased pathogenic bacteria including Escherichia coli and Bacillus, which triggered various symptoms [35,36]. Several studies proposed that the increased alpha diversity and the proximity of beta diversity to the donor microbiota were predictors of successful FMT therapy [37,38].   (Figure 3). On comparison, the alpha diversity index between FMT-treated group and control concluded that the FMT therapy elevated the bacterial diversity of the intestinal microbiota and enriched with therapy duration compare with control. In addition, as demonstrated in beta diversity, the distribution of intestinal bacterial community during FMT was dispersed obviously, closely cluster observed in FMTtreated 8 weeks and 12 weeks (A2 and A3), while the control microbial distribution was concentrated among FMT-treated 1, 8, and 12 weeks (Figure 4), that indicated the altered bacterial community contributed by FMT therapy and similar result also identified in previous studies [39,40] [19]. However, this study cannot identified the accurately bacterial species associated with clinical condition of IBS-D combined with anxiety and depression due to limited samples, and further study need to expand the sample range and clarify the correlation between specific bacteria and clinical indicators. Additionally, Johannes et al. reported that the high abundance of Proteobacteria was recorded in depressive disorder and contributed as a feature for IBS, and Prevotellaceae was related with IBS severity [24], while the reduced abundance in this study was supported the FMT positive therapeutic effect. Antushevich et al. proposed the positive effect of FMT on IBS by adjusted clinical symptoms and decreased relative abundance of Proteobacteria and increased in Bacteroides and Firmicutes, meanwhile fecal characteristics changes were related to increased Firmicutes and Clostridia and decreased Bacteroidetes [41][42][43][44][45]. Overall, FMT therapy increased bacterial diversity and adjust the bacterial community distribution, which further influence clinical and psychiatric symptoms, and FMT could be considered as potential and promising therapy for IBS-D combined with anxiety and depression to restore the intestinal microecology.

Conclusion
The positive therapeutic effect of oral FMT alleviated the IBS-D severity and psychological depression/anxiety and altered bacterial community. The FMT therapy relieved the clinical symptoms of IBS-D patients by decreased the IBS-SSS score (113 points) and mitigated anxiety and depression score (9 and 4 points). Introduced healthy microbiota restores the gut ecological imbalance by enriched bacterial diversity, adjusted the community distribution, including improved the beneficial Bacteroidetes and Firmicutes and suppressed the toxic releaser Enterobacteriaceae, Bacteroides and Escherichia −Shigella. In short, FMT therapy has great potential for IBS-D patients combined with anxiety and depression to restore the intestinal micro-ecology and alleviate the clinical symptom.

Disclosure statement
No potential conflict of interest was reported by the author(s).