**2. Experimental Section**

#### *2.1. Manipulation with Animals*

Male C57Bl/6 mice (20 g ± 2 g) were obtained from the Animal Breeding Facility of Masaryk University (Brno, Czech Republic). They were kept under standard conditions (22 ± 2 ◦C, 50 ± 10% relative humidity) and alternating 12 h light/dark cycles. The animals had access to a standard diet and drinking water ad libitum. Manipulations with the animals were carried out according to the bioethical rules as per the principles of the "European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes" adopted in Strasbourg in 1986. The study was also approved by the "Commission for the Protection of Animals against Cruelty" and the Ethics Committee of the University of Veterinary and Pharmaceutical Sciences in Brno, Czech Republic. In total, six animals in two groups (4 + 2 animals in the first and second group, respectively) were randomly separated and used in this experiment. In the dextran sulfate sodium (DSS) group (*n* = 4), colitis was induced by administering 5% (*w*/*v*) DSS (MP Biomedicals, Illkirch-Graffenstaden, France, MW 36,000–50,000 Da) in drinking water for 7 days. The mice in the intact group (*n* = 2) received drinking water only. On the last day of the experiment, the animals were killed by decapitation under isoflurane anesthesia. The isolated distal colonic segments were selected for the analysis of the qualitative and quantitative composition of intestinal microflora of both groups of the animals.

#### *2.2. Bacterial Mixed Cultures*

The material used for the study consisted out of mixed sulfate-reducing bacteria cultures that were isolated from feces of healthy and with ulcerative colitis mice. After the autopsy, the samples were placed in the tubes. The bacteria were studied as mixed cultures because the aim of the study was not the purification of SRB. Mixed cultures were kept at the Laboratory of Anaerobic Microorganisms of the Department of Experimental Biology at Masaryk University (Brno, Czech Republic).

#### *2.3. Cultivation of SRB Cultures*

SRB cultures were cultivated according to Kovac and Kushkevych (2017) [30] and Postgate (1984) in a modified Postgate C medium [23]. Mohr's salt (ammonium iron sulfate hexahydrate, Sigma-Aldrich, Prague, Czech Republic) was used as a simple growth detection. Ferrous salt forms reacted with sulfide produced by SRB (dark black precipitate of FeS) and indicated the presence of SRB (the presence of dissimilatory sulfate reduction). Due to the method, it was possible to optically determine the presence of metabolic activity qualitatively and quantitatively.

The cultures were kept in medium with Mohr's salt and without is since color changes are not desirable for spectrophotometric and turbidimetric methods. In cultures kept in medium without Mohr's salt, the SRB can be detected by the sharp smell of hydrogen sulfide same as by optical turbidity. The medium was sterilized (pH 7.5–7.7, Eh = −100 mV). Redox potential was adjusted by Na2S (Sigma-Aldrich, Prague, Czech Republic) and ascorbic acid (Sigma-Aldrich, Prague, Czech Republic). The anoxic atmosphere was ensured by the nitrogen gas addition, inhibiting oxygen from the air to di ffuse into the medium. The oxygen proof layer was secured by the addition of para ffin (Sigma-Aldrich, Prague, Czech Republic) drops to each cultivation tube. The strains were able to grow 10 days under these conditions.

The long storage (up to one month) conditions for cultures were provided by Postgate B medium with the addition of Mohr's salt. In this medium there is always tending of bacteria to descend to the bottom of the tube due to the presence of the precipitate. Bacteria usually stick to the walls of the tube when is used modified Postgate C medium.

#### *2.4. Description of Morphology*

Microscope Olympus BX50 (lympus, Japan) was used for the observation of cells.

Phase-contrast microscopy is a technique that allows images of transparent specimens (living cells). The advantage of this technique is the possibility to do the measuring without cell killing since cells can be monitored with real-time motility. The bacterial suspension (a drop) was placed on a glass slide. The slide (cover glass added to the top of bacterial suspension) was analyzed immediately after immersion and with 100× objective.

The Gram staining method provides observation of gram-positive and gram-negative bacteria by di fferential staining with the use of crystal violet-iodine complex and a safranin counterstain. Gram-positive bacteria appear purple after treatment with alcohol while gram-negative bacteria appear pink. After drying samples were microscopically observed, including oil immersion 100× objective.

Capsule staining. Acidic and basic stains cannot be used for bacterial capsules. Therefore, the best way to visualize them is to stain the background using an acidic dye (e.g., nigrosine, Congo red) and to stain the cell itself using a basic stain (e.g., crystal violet, safranin, methylene blue). One drop of Congo red dye was mixed with one drop of bacterial suspension on a glass slide. After spreading throughout the slide and letting dry, it was immersed in hydrochloric acid (4 mol/L) and after a few seconds, it was let dry again. Subsequently, methylene blue dye was added on the slide and it was let standing for three minutes. After three minutes, the slide was washed with deionized water, dried, and observed with immersion oil and 100× objective. The cells were stained blue and their capsules remained white and visible on a dark background.

DAPI (4,6-diamidino-2-phenylindole) staining is a fluorescent dye, binding by preference to the AT-rich regions of DNA [31]. Microorganisms with thick cell walls can be stained with DAPI after permeabilization of the cell wall by ethanol. For this type of microscopy, using a 48-hour old culture was found most suitable. A 48-h-old cell suspension of a volume 25 μL to 100 μL was diluted in several ml of MiliQ deionized water and washed by vacuum filtration. After washing, the filtration paper with cells was let dry. Consequently, 20 μL of DAPI stain (Sigma-Aldrich, Prague, Czech Republic) was applied and the filtration paper with cells was kept in the dark in a refrigerator for 10 min. After that, the filtration paper was washed in water, ethanol, and water, respectively, and let dry. Next, it was put on a glass slide with immersion oil applied both under and over the filtration paper with cells, and the slide was observed in a microscope, using WU filter (Sigma-Aldrich, Prague, Czech Republic) and 100× objective.

#### *2.5. pH Tolerance and Temperature Range Test*

As measured before, the optimal pH for the cultivation of intestinal SRB is from 7 to 8 [15]. The measuring was done by performing a simple pH test. The modified Postgate C medium was prepared by adjusting various pH values, performed by adding drops of sodium hydroxide (aqueous solution) and hydrochloric acid (aqueous solution), respectively. CyberScan 510 pH-meter (PreSens, Regensburg, Germany) was used to measure the exact pH values (pH ranged from 4 to 12). Media were heated to 37 ◦C in Wasserman tubes inoculums (obtained from healthy and not healthy mice) of cultures. Paraffin oil (500 μL) was added on the top of the medium to provide an oxygen-proof layer. The optical density of the suspension was measured at 430 nm using spectrophotometer Spectronics Genesys 5 (Thermo Fisher Scientific, Prague, Czech Republic). Blank samples were media without inoculum. Optical density was measured after 24 h of cultivation again. Bacteria were added in Eppendorf tubes and placed in thermostats (1-CUBE, Havlickuv Brod, Czech Republic) set at 5, 25, 35, 45, 50, and 60 ◦C. Optical density was measured at 430 nm using Spectronic Genesys 5, after 72 h of cultivation.

#### *2.6. Production of Hydrogen Sulfide*

Spectrophotometrical methylene blue method was used for measuring the presence of hydrogen sulfide in solution [32]. The bacterial suspension (1 mL) was pipetted to 5 mL of aqueous zinc acetate (5 g/L). 2 mL of p-aminodimethylaniline (Sigma-Aldrich, Prague, Czech Republic) solution (0.75 g/<sup>L</sup> in 2 M sulfuric acid) was added immediately and the solution was let stand at room temperature for 5 min. 0.5 mL of ferric chloride (FeCl3) (12 g/<sup>L</sup> in 0.015 M sulfuric acid) solution was consequently added. The solution was centrifuged at 2200 RPM (10 ◦C for 5 min). After centrifuging, the samples lost the original light pink color and had a blue color. The absorbance was measured at 665 nm by Spectronic Genesys 5 spectrophotometer. The procedure for blank sample preparation included preparation that a clear cultivation medium was added in step 1. The concentrations used for calibration solutions ranged from 6 μmol/L to 100 μmol/L (Figure 1).

**Figure 1.** The calibration used for the determination of sulfide concentrations.

## *2.7. Statistical Analysis*

Using the experimental data, the basic statistical parameters (M—mean, m—standard error, M ± m) were calculated. The accurate approximation was when *p* ≤ 0.0533 [33]. Statistical analysis was done by SPSS 20 statistical software (IBM Corporation, Armonk, NY, USA). Plots were built by software package Origin 7.0 (Northampton, MA, USA).

Meta-analysis consisted of studies found on the WEB OF KNOWLEDGE database. The database found 38 studies, from the year 1945 to 2019.considering sulfate-reducing bacteria. Only six studies were included in the meta-analysis since other studies did not satisfy the specific hypothesis of the study. The Review Manager Software (Cochrane, Brno, Czech-Republic) (number 5.3 developed by Cochrane Collaboration) was used. In the included studies the data consisted of the number of participants with the positive occurrence of the SRB bacteria in the group of healthy people and people with ulcerative colitis. In other studies, the data consisted of the mean, standard deviation and the number of the measurements. Heterogeneity was expressed by the *I 2* test, where the higher *I 2* represented a higher heterogeneity.
