Characteristics of the gut microbiota and the content of short-chain fatty acids in patients with irritable bowel syndrome

The article presents the issues related to gut microbiota and short-chain fatty acids in patients with irritable bowel syndrome.  It was found that  the  faecal concentration of acetic, propionic, butyric acids  is higher in patients with  irritable bowel syndrome with diarrhea, compared with  the patients without diarrhea syndrome. 83.3–88.9% of patients with various forms of irritable bowel syndrome presented with dysbiotic changes. The reduced concentration of Bifidobacterium species, mainly lactic acid bacteria, is more often registered in patients with diarrheal form. There is a certain correlation between the concentration of short-chain fatty acids and the content of Bifidobacterium, Lactobacillus, Candida and opportunistic bacteria.


Introduction
Many studies have found that impaired gut microbiota is an important component of the development of irritable bowel syndrome (IBS). Throughout the gut, microbiota plays an important role in the normal functioning of the gut. Molecular technologies have established the dominance of four classes of microorganisms: Firmicutes (64 %), Bacteroidetes (23 %), Proteobacteria (8 %), Actinobacteria (3 %) [1].
In recent decades, a large number of studies have been conducted to investigate the incidence of dysbiosis in patients with IBS, as well as the nature of changes in the individual composition of the gut microbiota. In a famous study by Casén C. et al. (2015), which was conducted in Sweden, Norway, Denmark and Spain, intestinal dysbiosis was detected by genetic methods in 73 % of IBS patients and 16 % of healthy individuals [2].
The studies examining the species composition of microbiota in IBS have shown varied results, given the different techniques used in these studies. However, the general trend is a decrease in Firmicutes and Bacteroidetes with different ratios depending on the form of IBS. Among these classes of bacteria, the content of Lactobacillus and Bifidobacterium is most commonly decreased, especially in the diarrheal form of IBS [1].
In contrast, many studies have shown an increase of other microorganisms in IBS, including Pseudomonas aeruginosa, Ruminococcus spp., Escherichia, Clostridium spp., Streptococcus spp. and others. This category of patients demonstrated the increased levels of C-reactive protein, proinflammatory cytokines (IL-6 and IL-8), bacterial lipopolysaccharides [3].
The question is, how do the altered gut microbiota impact IBS development?
Microbiota performs several important functions. First of all, it concerns the prevention of infectious factors. In the intestine, there is a constant fight or "competition" between the beneficial commensals (symbionts) to which Firmicutes and Bacteroidetes belong, with pathogenic or potentially www.gastro.org.ua, http://gastro.zaslavsky.com.ua Патологія кишечника / Bowel Pathology pathogenic pathobionts. This "competition" is called colonization resistance and is an important guarantee of the normal functioning of the gastrointestinal system. Certain important molecules, which may include short-chain fatty acids (SCFA) and bacteriocins, are implicated in the course of colonization resistance [4].
Researchers' interest in SCFA has been around for decades. Back in 1980, Roediger W.E. found that SCFA produced by anaerobic bacteria are an important source of energy for colonocytes [5].
Since that time there were performed many studies which revealed important diverse functions of SCFA, including the effect on intestinal motor activity, stimulation of the immune system, blocking the activation of pathogenic flora, impact on metabolic processes. SCFA lowers the pH around the intestinal epithelial cells that has a protective effect on them. Besides, SCFA provide antibacterial protection against pathogenic bacteria by attracting neutrophils and cytokines, with immune tolerance to commensal flora remaining [4]. It is believed that different SCFA can have various effects, in particular, butyrate is more present in the intestine, propionate -in the enterohepatic circulation, acetate -in the systemic circulation [4,6].
The article reviews the results of some recent studies. The experimental research by Wang H.-B. et al. studied the anti-inflammatory properties of intestinal bacteria, in particular in acute systemic inflammation due to septic shock. Butyrate (butyric acid) has been found to reduce the plasma levels of proinflammatory compounds TNF-α, IL-6 and IL-1β in the experimental animals; however, butyrate significantly increases the anti-inflammatory IL-10 [7]. The study by Tedelind S. et al. (2007) found anti-inflammatory effects of other SCFA: acetate, propionate, butyrate reduced TNF-α release stimulated by lipopolysaccharide [8].
Butyrate also stabilizes the intestinal barrier function, in particular by influencing hypoxia-inducible factor [9]. In a Chinese study, Feng Y. et al. (2018) found that SCFA stimulate the formation of the intestinal epithelial barrier and protect it against damage by lipopolysaccharides, in particular through inhibition of NLRP3 inflammasome and autophagy [10].
The famous Spanish study of Pozuelo M. et al. (2015) investigated the microbiome and SCFA in 113 patients with IBS and 66 healthy individuals. With the use of 16S rRNA genetic research methods, it was found that IBS was associated with a reduced heterogeneity of microorganisms, as well as a decrease in butyrate-producing bacteria, especially in patients with diarrheal and mixed forms of IBS [11]. A decrease in butyrate production can augment intestinal permeability, enhance nociceptive sensory response, and exacerbate IBS symptoms [1].
Given the multifactorial nature of the mechanisms of IBS development, the diverse action of SCFA may be important in IBS. SCFA is believed to be an important component of maintaining intestinal and immune homeostasis [12]. However, many questions about the SCFA and the development of IBS remain unclear.
Purpose of the study was to investigate the features of SCFA faecal content in IBS patients depending on dysbiotic changes in the gut microbiome.

Materials and methods
The study was conducted at the State Institution "Institute of Gastroenterology of the National Academy of Medical Sciences of Ukraine". The study involved 15 IBS patients. The diagnosis of irritable bowel syndrome was established after a thorough clinical and anamnestic examination, taking into account compliance with the Rome IV criteria (2016) with the exclusion of anxiety symptoms. All patients experienced intestinal pain. Other symptoms include bloating and abdominal distension; some patients presented with anxiety. Attention was first of all paid to the nature of the emptying. According to the Bristol Stool Chart, patients had diarrheal IBS (9 patients) and non-diarrheal forms (with or without constipation) (6 patients).
All patients enrolled in the study were evaluated for SCFA content. The SCFA level was measured by the chromatographic method using the hardware-software complex for medical research based on the gas chromatograph "Chromatec-Crystal 5000" by the method of Guohua Zhao [13]. The quantitative identification of the SCFA fractions (µg/mg) of acetic (C2), propionic (C3), butyric (C4) acids, column calibration, and chromatogram calculation were performed by the method of normalization of peak areas and their fractions according to the standards of "Sigma-Aldrich Acids" (USA).
Besides, all patients underwent bacteriological (cultural) study of feces with the determination of the gut microbiota composition (the content of Bifidobacteria, Lactobacilli, Escherichia, Enterococci, potentially pathogenic and Candida flora). Investigation of the species and quantitative composition of the colonic microbiota was performed using ten-fold dilutions (10 -1 -10 -9 ) on a standard set of elective and differential diagnostic nutrient media for isolation of aerobic and anaerobic microorganisms.

Results ans discussion
According to the results of the determination of faecal SCFA content, the level of acetic acid (C2) in patients with diarrheal IBS varied within a range of 0-0.461 µg/mg; the average level was (0.236 ± 0.044) µg/mg. The content of acetic acid (C2) in IBS patients with no diarrhea ranged 0.034-0.251 µg/mg; the average value was (0.120 ± 0.041) µg/mg (p = 0.039).
Thus, the concentration of all SCFA in IBS patients is higher in the presence of diarrhea compared with the patients without diarrhea (Fig. 1). It is also noticeable that the acetic acid content is the highest. Gastroenterologìa, ISSN 2308-2097 (print), ISSN 2518-7880 (online)

Патологія кишечника / Bowel Pathology
The bacteriological examination found the signs of intestinal dysbiosis in 88.9 % patients with diarrheal IBS and 83.3 % patients with non-diarrheal IBS. The average content of Bifidobacteria (logarithm) did not differ for diarrheal and non-diarrheal forms of IBS and accounted for (8.67 ± 0.24) and (8.67 ± 0.33), respectively. The Lactobacilli content (logarithm) was lower in diarrheal form (4.67 ± 0.37) compared with non-diarrheal (5.33 ± 0.21). The content of E.coli with normal properties between the groups differed slightly: (6.44 ± 0.24) for diarrhea, (6.67 ± 0.67) for non-diarrheal form. The Enterococci content was slightly lower in diarrheal form (6.33 ± 0.29) than in the absence of diarrhea (7.17 ± 0.31) (Fig. 2).
In patients with diarrhea, a decrease in Bifidobacteria content was observed in 22.2 % of cases, and a decrease in Lactobacilli content in 66.7 % people. In 16.7 % patients without diarrhea, the Bifidobacteria content was reduced; there was no reduction in Lactobacilli concentration. In 55.6 % of patients with diarrheal syndrome and 50.0 % of diarrhea-free patients, an increase in the concentration of Candida was observed; 66.7 % people with diarrhea and 100 % diarrhea-free patients presented with an increase in potentially pathogenic flora.
The selection of SCFA was estimated depending on the disturbances of the gut microbiota.
Thus, in patients with IBS, there is a certain interaction between the state of gut microbiota and the faecal content of SCFA: the increase in the concentration of SCFA is slightly higher with a decrease in Bifidobacteria and Lactobacilli and increase of the Candida flora; on the contrary, the decrease in the concentration of SCFA may be established in case of increased potentially pathogenic flora. Further study of the relationship between the state of the gut microbiota, the nature of dysbiotic changes, and the release of SCFA is needed.

Conclusions
1. The faecal concentration of SCFA is higher in IBS patients with diarrhea compared with the patients without diarrheal syndrome.
2. 83.3-88.9 % of patients with various forms of IBS presented with gut dysbiosis; the patients with diarrhea are more likely to have a reduced content of Bifidobacteria and, in particular, Lactobacilli.
3. There is a certain relationship between the SCFA concentration and the content of Bifidobacteria, Lactobacilli, Candida and potentially pathogenic flora, which need to be evaluated in further investigations.