Implication of Gut Microbiome in Pathophysiology of Irritable Bowel Syndrome (IBS)

Abstract

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder, and highly prevalent among general public but its etiology is still unknow. A complex interaction between lifestyle, diet and intestinal microbiome play a role in inception and progression of IBS. Moreover, it is now clear that microbial factors play a key role in IBS pathophysiology. Acute gastric infection along with abnormal gut microbiota leads to IBS condition and studies proved changes in the gut microbiome in IBS patients. This study was designed to check epidemiological, hematological, and microbial diversity in IBS patients. For study of microbiome in IBS we calculated number of samples using WHO formula (http://www.who.int/chp/steps/resources/sampling/en/), which comes out to be 22, we choose 30 IBS patients to conduct our study. In the first phase of the study, role of anthropometrics parameters on progression of IBS was studied along with serum biochemistry analysis of IBS patients. Results concluded that IBS is disease of all ages having more prevalence in males then females (4:1). Diet particularly don’t have any vigorous effect in elevating IBS symptoms while reducing physical activity can elevate symptoms severity. IBS have three subtypes based upon symptoms i.e IBS-C (constipation),IBS-D (diarrhea) and IBS-M (mixed). In our study 56% of patients had IBS-Mixed symptoms while IBS-C and IBS-D reported to be 17 and 27% respectively. One of the important factors noted in triggering IBS was stress level, 50% of patients were having high-stress level when evaluated using a questionnaire. It was also noted that IBS symptoms were periodic and reoccur after certain time. IBS patients usually have long history of the disease, from our data we come to know that 27 % of patients were having symptoms from the last five years while 14 % had symptoms for more than half of their life. No drastic changes in hematological parameters of IBS patients were seen, a negative correlation was seen between IBS and vitamin D level, while that of liver enzymes, lipid profile, and complete blood picture remain the same in almost all patients. SCFAs are fermented by-products of bacterial metabolism, one of the important is butyrate production. As in IBS butyrate-producing, the bacterial level decreases so in general production of butyrate decrease in IBS patients. The second phase of study was gut microbiota changes in IBS patients using high DRSML QAU Page xxiv throughput sequencing technology. As fecal microbiota is representative of intestinal microflora so fecal sample was used to study intestinal dysbiosis in IBS patients. Five high prevalent bacterial phyla in IBS patients were: Firmicutes(94.9%), followed by Actinobacter, Bacteroidetes, Proteobacteria, and Tenericutes. Most of common genus present were ruminococcus and lactobacillus while that of Bifidobacterium and faecalibacterium were low. Another important order found was clostridia and bacilli which had an association of increasing gut dysbiosis, while those related to SCFAs production like Ruminococcaceae family were present in lower numbers. In the Family Enterobacteriaceae species of shigella/ salmonella were observed which showed association of diarrheal like symptoms in IBS patients. Gastroenteritis causing genus like Aeromonas, Weissella and Paenibacillus also increase in IBS patients. Comparing different IBS subtypes on basis of bacterial diversity we observe that IBS M have higher number of bacteriodetes and cynobacteria, while IBS-D have firmicutes and tenricutes. Fusobacteria was observed in higher number in IBS-C. Depression also changes gut microbial diversity among IBS patients, interesting interactions were found between many bacterial species and patients having high depression level, most prevalent phylum present in high level of depression patients was Enterobacteriaceae, Firmicutes phylum was also high in IBS patients having high level of depression and among this phyla genus Weissella, bromii, belonging to family Leuconostocaceae and Ruminococcaceae respectively ratio was more. Third phase was study of pathogenesis of culturable microbiota from IBS patients using long read sequencing approach. Main objective of study was to look into genetic diversity of culturable microorganism. Culturing, sanger sequencing and MinION techniques were used in this phase which revealed presence of important gram positive (Enterococcus) and pathogenic gram-negative species in IBS patients’ stool. One of important pathogen found was Klebsiella pneumonia, although K. pneumonia, present in normal healthy helps them to digest carbohydrates such as lactose, resistant starches, inulin, fructose, and mannose but this can cause infection in intestine when it became opportunistic and increase in numbers. E. coli which is mostly associated with travelers’ diarrhea and gut dysbiosis, its Strains were enriched in virulence genes associated with extraintestinal pathogenic E. coli (ExPEC) and/or DRSML QAU Page xxv adherent-invasive E. coli are related to IBS symptoms like dysbiosis were also found in our samples. Shigella flexneri which, altered bowel motility, visceral hypersensitivity, psychosocial distress, abnormal brain-gut interaction, enteric infection, gut-immune activation, low grade inflammation, intestinal permeability, and alterations in intestinal microflora were also present among IBS patients. Few strains of Proteus mirabilis causing bloating, abdominal discomfort and changes in stool form among IBS patients were also studied for their genetic diversity and virulence factor presence. Enterococcus now termed as an Emerging Pathogens were also in high abundant among IBS patients. Enterococcus (p=0.001) exhibited higher levels in IBS patients. Virulence and pathogenicity factors such as adhesins, invasions, pili, and hemolysin in Enterococcus an make them human pathogen, we found genes related to these in our Enterococcus isolates. In total we check 17 strains for whole genome all have varying amount of resistance genes. Concluding all results, we can summarize as 50% of IBS patients have symptoms of disease before they reach 35 years, and as age increases to 50 years or above IBS prevalence decreases to 50%. Highest rate of IBS was observed in age group between 31 years-40 years, and the lowest was between 71-80 years of age group. Duration of IBS was the time period patient is suffering from this disease, highest number of patients were recorded between age groups 1-5 i.e. 27. IBS can lead to reducing quality of life(QoL) while xercise can improve feelings and symptoms of fatigue, bloating and constipation. In our research we found that there was a relative high abundance of proinflammatory bacterial species like Enterobacteriaceae, while a reduced number of lactobacillus and bifidobacterium was observed. Moreover, Bifidobacterium families, all short chain fatty acids (SCFAs) producers were also observed to be lower in IBS patients due to which we observed low concentration of butyrate in IBS patients blood serum. Increased level of streptococcus species were present in IBS patients which shows assosiaction with inflammation . Veillonella in IBS patients and those with higher organic acid levels presented with worse gastrointestinal symptoms, quality of life, and negative emotions(depression). Although we could not successfully assemble the DRSML QAU Page xxvi genomes from metagenomic data at this stage, However, these results are showing very good diversity at species level. Although culturing reveals that gram negatives were present in all patients, but their quantity was low using culture independent techniques. Most of strains were resistance to common antibiotics like enterococcus strains were mostly resistance to vancomycin. All these resistance strains in IBS patients is an alarming situation for clinicians as they need to treat patients with narrow spectrum antibiotics instead of prescribing directly broad spectrum antibiotics.