Metabolic Activity of Sulfate-Reducing Bacteria from Rodents with Colitis

Open Med. 2018; 13: 344-349

Research Article

Jozef Kováč, Monika Vítězová, Ivan Kushkevych* Metabolic activity of sulfate-reducing bacteria from rodents with colitis https://doi.org/10.1515/med-2018-0052 The dissimilatory sulfate reduction is a multistage and received April 5, 2018; accepted July 26, 2018 complex process which provides SRB cell’s energy with the formation of ATP. SRB consume sulfate as a terminal Abstract: Sulfate-reducing bacteria (SRB) are anaerobic electron acceptor and due to the oxidation of organic microorganisms, which use sulfate as an electron accep- compounds and hydrogen obtain energy for their growth tor in the process of dissimilatory sulfate reduction. The [4,5]. Hydrogen sulfide (H2S) is the final product of sulfate final metabolic product of these anaerobic microorgan- reduction [6]. isms is hydrogen sulfide, which is known as toxic and can Sulfate in food may be important in human metabo- lead to damage to epithelial cells of the large intestine at lism. Free sulfate ions affect large bowel metabolism where high concentrations. Different genera of SRB are detected it is reduced to hydrogen sulfide, a substance potentially in the large intestine of healthy human and animals, and toxic to the colonic epithelium [7]. Sulfate concentrations with diseases like Crohn’s disease and ulcerative colitis. have therefore been measured in more than 200 individual SRB isolated from rodents with ulcerative colitis have pro- foods and beverages [8]. High-sulfate foods (>10 μmol/g or duced 1.14 (mice) and 1.03 (rats) times more sulfide ions 1 mg/g) include some breads, soya flour, some dried fruits, than healthy rodents. The species of Desulfovibrio genus some brassicas, and some sausages. High-sulfate bever- are the most widespread among all SRB in the intestine. ages (>2.5 μmol/ml or 0.25 mg/ml)) include some beers, The object of our research was to observe and compare ciders, and wines. The sulfate content of beer is discussed the difference of production of sulfide and reduction of with particular relation to epidemiological observations sulfate in intestinal SRB isolated from healthy rodents which link ingestion of beer with colorectal cancer [8]. and rodents with ulcerative colitis. Inflammatory bowel disease (IBD) including ulcerative colitis (UC) or Crohn’s disease is characterized by

chronic inflammation of the large intestine in genetically Keywords: Sulfate-reducing bacteria; Desulfovibrio susceptible individuals of unknown etiology [9,10]. One of genera; Hydrogen sulfide; Ulcerative colitis the hypotheses is that UC is caused by the toxic molecule

of H2S. In persons with ankylosing spondylitis and, with rheu- matic diseases, etc. are often found SRB [4,11-13], which in 1 Introduction large amounts can cause intense process of dissimilatory sulfate reduction in the gut leading to inflammatory bowel Sulfate-reducing bacteria (SRB) are anaerobic microor- diseases [1,7,14]. On the other hand, the increased number ganisms, which use sulfate as an electron acceptor in the of SRB was found in feces from people with ulcerative process of dissimilatory sulfate reduction. SRB are spread- colitis compared with healthy individuals [15-19]. wide not only in the environmental sources, but are also SRB, especially Desulfovibrio genus, has been studied present in the digestive tract of humans and animals [1-3]. for over a century because of their ubiquity and their important roles in chemical processes and elemental cycles [20]. Also, Desulfovibrio genus is the most common *Corresponding author: Ivan Kushkevych, Department of Expe- species of SRB and its species are most often isolated from rimental Biology (Section of Microbiology), Faculty of Science, the large intestine of human and animals [15,21,22]. Masaryk University, Kamenice 753/5, 62500 Brno, Czech Republic; Due to lack of information about dissimilatory sulfate E-mail: [email protected] reduction of intestinal SRB and its comparison between Jozef Kováč, Monika Vítězová, Department of Experimental Biology (Section of Microbiology), Faculty of Science, Masaryk University, healthy and samples from ulcerative colitis, more obser- Kamenice 753/5, 625 00 Brno, Czech Republic. vation was needed. In our research, we have been focused

Open Access. © 2018 Jozef Kováč et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution-NonCom- mercial-NoDerivatives 4.0 License. Metabolic activity of sulfate-reducing bacteria 345 on production of hydrogen sulfide and decrease of sulfate 2.4 Sulfate ions detection ions during sulfate respiration in the samples from rodents with and without ulcerative colitis. The content of sulfate in the medium was determined by turbidimetric method right after seeding and after 24 hours cultivation. The calibration curve was constructed with the same process. Calibration solutions were pre- 2 Materials and methods pared in distilled water at concentrations of 2, 4, 8, 16, 24, 32, 40 and 48 µM sodium sulfate. A suspension of

2.1 Bacterial cultures 40 mg/L BaCl2 was prepared in 0.12 M HCl. The resulting solution was mixed with glycerol in a 1:1 ratio. To the 1 mL Crude cultures of intestinal SRB have been isolated from of sample supernatant after centrifugation at 5000 × g at the intestine of rats and mice. The cultures have been kept 23˚C was added 10 mL of prepared BaCl2:glycerol solution in the collection of microorganisms at the Laboratory of and carefully stirred. The mixture was allowed to stand for Anaerobic Microorganisms of Department of Experimen- 10 minutes and right after that the absorbance was meas- tal Biology at Masaryk University (Brno, Czech Republic). ured at 520 nm (Spectrosonic Genesis 5). As a control, the measurement was repeated in the same manner using a cultivation medium [26]. 2.2 Bacterial growth and cultivation

Bacteria were grown in a nutrition-modified Postgate’s 2.5 DAPI liquid medium “C” with the following composition (g/L)

[23]: Na2SO4 (4.0), (NH4)2SO4 (0.2), MgSO4 × 7H2O (1.0), Morphological characteristics of intestinal SRB were eval-

K2HPO4 (0.5), KH2PO4 (0.3), CaCl2 × 6H2O (0.06), NH4Cl uated by fluorescent DAPI (4’, 6-diamidino-2-fenylindol)

(1.0), lactate (6 mL), yeast extract (1.0), sodium citrate × straining. DAPI was used as cytochemical detection for

2H2O (0.3). The final optimal pH 8 for cultivation of intes- bacterial DNA. This DNA-DAPI connection is visible in 365 tine SRB was provided by a sterile 1 M solution of NaOH nm [27]. (0.9 ml/l). The bacteria were grown for 72 hours at 37°C under anaerobic conditions [24]. 2.6 Statistics

2.3 Hydrogen sulfide assay Statistical calculations of the results were carried out using the software MS Office, Origin and Statistica12 com- Hydrogen sulfide produced by intestinal SRB has been puter programs. Using the experimental data, the basic measured spectrophotometrically right after seeding and statistical parameters (mean: M, standard error: m, M ± after 24 hours cultivation. Calibration solutions were pre- m) were calculated. The research results were treated by pared in distilled water at concentrations of 12.5, 25, 50, methods of variation statistics using Student’s t-test. The and 100 µM of sodium sulfide. The calibration curve was significance of the calculated indicators was tested by constructed with the same process. A sample of volume 1 Fisher’s F-test. The accurate approximation was when P≤ mL was added to 10 mL of a 5 g/L aqueous solution of zinc 0.05 [28]. acetate. Right after, 2 mL of 0.75 g/mL p-aminodimethy- laniline in a solution of sulfuric acid (2 M) was added. The mixture was left to stand for 5 minutes at room temperature. After that, 0.5 mL of 12 g/L solution of ferric chloride 3 Results dissolved in 15 mM sulfuric acid was added. After stand- Crude samples of intestinal SRB were cultivated in the ing another 5 minutes at room temperature, the mixture selective modified medium and after cultivation, vibrio was centrifuged at 5000 × g at 23˚C. The absorbance of like colonies was observed (Fig. 1). Therefore, these mixture was determined to measure hydrogen sulfide at a samples were subjected to measurement of metabolites of wavelength of 665 nm by spectrophotometer Spectrosonic their dissimilatory sulfate reduction. Genesis 5. As a control, the measurement was repeated in Detection and monitoring of sulfate reduction and the same manner using a cultivation medium [25]. production of sulfide in samples obtained from healthy 346 Jozef Kováč et al.

rodents and rodents with ulcerative colitis for 24 hours cultivation were obtained. The percentage ratio of these data was calculated in every sample (Table 1.). Sulfide production was increased in samples from mice and rats with UC, 4.06±0.34 and 4.10±0.46 mM, in comparison with healthy samples, 3.55±0.29 and 3.99±0.28 mM, respectively. However, sulfate reduction was increased in the same way, 7.15±0.75, 9.72±0.53 mM (UC) and 6.20±0.23, 9.40±0.36 mM (healthy). In intestinal SRB suspension from rats sulfate reduction was significantly increased with 9.40±0.36 and 9.72±0.53 mM compared with the suspension from mice at 6.20±0.23 and 7.15±0.75 mM. On the other hand, the production of sulfide is not as different as sulfate reduction. Based on the percentage ratio calcu- Figure 1: The crude culture of intestinal SRB isolated from a mouse lations for metabolites of dissimilatory sulfate reduction with ulcerative colitis (magnified 10,000 ×). mentioned above, two graphs were drawn (Fig. 2).

Table 1: Concentrations of sulfate and sulfide in media with intestinal SRB from mice and rats for 24 hours cycle.

SRB suspension from mice

SO 2- at the begin- SO 2- after 24 hours SO 2- decreased S2- after 24 hours S2- produced Number of samples 4 4 4 ning [mM] cultivation [mM] (%) cultivation [mM] (%)

Healthy

1 20.49±1.64 13.19±0.62 35.63 7.31±0.12 40.49

2 17.28±1.63 14.19±0.79 17.88 8.22±0.63 53.04

3 19.82±1.41 14.30±0.44 27.85 7.12±0.15 41.85

4 21.70±1.38 14.43±0.81 33.50 7.83±0.83 41.76

5 21.52±1.37 16.06±1.14 25.37 6.77±0.31 59.38

6 20.91±2.10 15.42±0.71 26.26 8.96±0.48 48.10

7 19.68±1.44 12.27±0.89 37.65 7.99±0.45 37.42

Average 20.27±1.56 14.06±0.77 29.38 7.74±0.30 45.92

Ulcerative colitis

1 20.77±2.32 13.50±1.45 35.00 8.21±0.98 52.50

2 20.23±1.72 13.21±1.42 34.70 8.44±0.65 45.26

Average 20.50±2.02 13.35±1.44 34.85 8.32±0.82 48.83

SRB suspension from rats

Healthy

1 24.31±1.21 13.48±0.74 44.55 9.17±0.59 52.02

2 21.97±0.82 14.01±0.66 36,23 7.57±0.12 42.54

Average 23.14±1.02 13.75±0.70 40.60 8.37±0.36 47.73

Ulcerative colitis

1 24.40±2.39 14.68±1.45 39.84 7.46±0.83 54.96 Metabolic activity of sulfate-reducing bacteria 347

Figure 2: Percentage ratio of sulfate reduction ions (A), the percentage of starting and final metabolites during sulfate-reduction (B) from healthy samples and samples with UC.

During sulfate reduction in samples from mice, intermediate compounds were 24.7 and 16.32 % of total compounds compared to samples from rats (11.67, 5.2 %) (Fig. 2A). Additionally, the differential percentage ratio between samples with colitis and healthy control is visible in samples from mice for sulfate states 54.63:45.37 % and for sulfide ions 49.15:50.85 % in comparison to samples from rats, 53.34:46.66%; 50.65:49.35% respectively (Fig. 2B). The diagram of cluster analysis shows that sulfate ions from healthy rodents are not as related to rodents with colitis as are sulfide ions (Fig. 3). However, samples with UC from both rodents have shorter linkage distance. Healthy control of sulfide ions is also incorporated into the same cluster as are with colitis.

4 Discussion Figure 3: Cluster analysis of consuming sulfate and produced sulfide The presence of intestinal SRB isolated from healthy after 24 hours. human feces was previously presented by Moore et al. nificant, which indicates that sulfate is evenly transported [21], Beerens and Romond [29], and by Gibson and his to the bacterial cell. colleagues [15,16], who also measured the rate of pro- The concentration of sulfate in the intestine depends ducing H S. The lower percentage ratio of intermediate 2 on the food introduced. Oxidized forms of sulfur includ- compounds in samples from rodents with colitis indi- ing sulfite and sulfate are present in food such as com- cates that they faster convert incorporated sulfate ions to mercial bread, nuts, dried fruits and vegetables, brassica sulfide. This faster reduction of sulfate can be involved vegetable and fermented beverages. The sulfate mainly with enzymes of dissimilatory sulfate-reduction (adeno- is in the free anionic form. About 2±15 mM of sulfate is sine-5’-phosphosulfate reductase and sulfite reductase) passes through the human gastrointestinal tract every or even by other enzymes such as pyruvate ferredoxin day by food. However, the concentration of sulfate ions oxidoreductase, Na+/K+-activated Mg2+-dependent ATP-hy- in the feces is much lower and is it about 0.26 mM/day. drolase, acetate kinase, and phosphotransacetylase It was also observed that 95% of sulfate is absorbed in [5,30-32]. However, the difference in sulfate consumption the gastrointestinal tract and only 5% remains detected between samples with UC and healthy control is not sig- in the feces [8]. Other researchers have also reported that 348 Jozef Kováč et al.

[9] Podolsky D.K., Inflammatory Bowel Disease. New England absorption of sulfate by the human gastrointestinal tract Journal of Medicine, 2002, 347(6), 417-429 is believed to be bad [33,34]. [10] Schirbel A., Fiocchi C., Inflammatory bowel disease: Due to the possible connection of the intestinal SRB Established and evolving considerations on its etiopatho- to the development of UC various researches have studied genesis and therapy. Journal of Digestive Diseases, 2010, synthesized organic compounds on their inhibition [35-37]. 11(5), 266-276 To conclude our research, differences in production of [11] Cummings J., Macfarlane G., Macfarlane S., Intestinal bacteria and ulcerative colitis. Current issues in intestinal sulfide and reduction of sulfate in suspension of SRB from microbiology, 2003, 4(1), 9-20 rodents with and without ulcerative colitis was observed. [12] Sekirov I., Russell S.L., Antunes L.C., Finlay B.B., Gut

Differences in produced H2S are visible between samples Microbiota in Health and Disease. Physiological Reviews, with UC and healthy controls. The consumption of sulfate 2010, 90(3), 859-904 ions was not significant. The observed difference can lead [13] Kushkevych I., Kollar P., Ferreira A.L., Palma D., Duarte A., Lopes M.M., Jampilek J., Antimicrobial effect of salicylamide to a better understanding of the etiology of the UC and derivatives against intestinal sulfate-reducing bacteria. association of SRB in its development. Journal of Applied Biomedicine, 2016, 14(2), 125-130 [14] Macfarlane S., Dillon J., Microbial biofilms in the human Acknowledgements: This study was supported by Grant gastrointestinal tract. Journal of Applied Microbiology, 2007, Agency of the Masaryk University (MUNI/A/0906/2017) 102(5), 1187-1196 [15] Gibson G.R., Cummings J.H., Macfarlane G.T., Growth and activities of sulphate-reducing bacteria in gut contents of Conflict of interest statement: Authors state no conflict healthy subjects and patients with ulcerative colitis. FEMS of interest. Microbiology Ecology, 1991, 9(2), 103-111 [16] Gibson G.R., Macfarlane G.T., Cummings J.H., Sulphate reducing bacteria and hydrogen metabolism in the human large intestine. Gut, 1993, 34(4), 437-439 References [17] Levitt M.D., Furne J., Springfield J., Suarez F., DeMaster E., Detoxification of hydrogen sulfide and methanethiol in the [1] Loubinoux J., Bronowicki J.-P., Pereira I.A., Mougenel, J.-L., cecal mucosa. Journal of Clinical Investigation, 1999, 104(8), Faou A.E., Sulfate-reducing bacteria in human feces and 1107-1114 their association with inflammatory bowel diseases. FEMS [18] Macfarlane, G.T., McBain A.J., The Human Colonic Microbiota. Microbiology Ecology, 2002, 40(2), 107-112 In G. R. Gibson, M. B. Roberfroid, Colonic Microbiota, [2] Kushkevych I., Vítězová M., Vítěz T., Bartoš M., Production Nutrition and Health (1 ed., pp. 1-25). Dordrecht: Springer of biogas relationship between methanogenic and Netherlands. 1999 sulfate-reducing microorganisms: relationship between [19] Zinkevich V., Beech I.B., Screening of sulfate-reducing methanogenic and sulfate-reducing microorganisms. Open bacteria in colonoscopy samples from healthy and colitic Life Sciences, 2017, 12(1), 82-91 human gut mucosa. FEMS Microbiology Ecology, 2000, 34(2), [3] Kushkevych I., Kováč J., Vítězová M., Vítěz T., Bartoš M., 147-155 The diversity of sulfate-reducing bacteria in the seven [20] Voordouw G., Carbon Monoxide Cycling by Desulfovibrio bioreactors. Archives of Microbiology, 2018, 200(6), 945-950 vulgaris Hildenborough. Journal of Bacteriology, 2002, [4] Barton L.L., Hamilton A.W., Sulphate-Reducing Bacteria 184(21), 5903-5911 Environmental and Engineered Systems. Cambridge: [21] Moore W.E., Johnson J.L., Holdeman L.V., Emendation Cambridge University Press, 2007 of Bacteroidaceae and Butyrivibrio and Descriptions of [5] Kushkevych I., Fafula R., Parák T., Bartoš M., Activity of Na+ / Desulfomonas gen. nov. and Ten New Species in the Genera K+ -activated Mg2- -dependent ATP-hydrolase in the cell-free Desulfomonas, Butyrivibrio, Eubacterium, Clostridium, extracts of the sulfate-reducing bacteria Desulfovibrio piger and Ruminococcus. International Journal of Systematic Vib-7 and Desulfomicrobium sp. Rod-9. Acta Veterinaria Brno, Bacteriology, 1976, 26(2), 238-252 2015, 84(1), 3-12 [22] Willis C.L., Cummings J.H., Neale G., Gibson G.R., Nutritional [6] Kushkevych I., Vítězová M., Fedrová M., Vochyanová Z., Aspects of Dissimilatory Sulfate Reduction in the Human Paráková L., Hošek J., Kinetic properties of growth of Large Intestine. Current Microbiology, 1997, 35(5), 294-298 intestinal sulphate-reducing bacteria isolated from healthy [23] Postgate J.R. The sulphate-reducing bacteria. New York: mice and mice with ulcerative colitis. Acta Veterinaria Brno, Cambridge University Press. 1979 2017, 86, 405-411 [24] Kováč J., Kushkevych I., New modification of cultivation [7] Pitcher M., Beatty E., Gibson G., Cummings J., Hydrogen medium for isolation and growth of intestinal sulfate- Sulphide Production by Colonic Sulphate-Reducing Bacteria: reducing bacteria. In MendelNet 2017 Proceedings of 24th A Luminal Toxin in Ulcerative Colitis? Clinical Science, 1995, International PhD Students Conference, Mendel University in 89(33), 26P.2-26P Brno, 2017, 702-707 [8] Florin T.H., Neale G., Goretski S., Cummings J. H., Sulfate [25] Bailey T.S., Pluth M.D., Chemiluminescent Detection of in food and beverages. Journal of Food Composition and Enzymatically Produced Hydrogen Sulfide. Journal of the Analysis, 1993, 6(2), 140-151 American Chemical Society, 2013, 135(44), 16697-16704 Metabolic activity of sulfate-reducing bacteria 349

[26] Kolmert Å., Wikström P., Hallberg K.B. A fast and simple Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9. turbidimetric method for the determination of sulfate in Polish J Microbiol, 2015, 64, 107-114 sulfate-reducing bacterial cultures. Journal of Microbiological [33] Wilson T., Intestinal absorption. Saunders, 1962 Methods, 2000, 41(3), 179-184 [34] Goodman L.S., Gilman A., The Pharmacological basis of [27] Porter K.G., Feig Y.S., The use of DAPI for identifying and therapeutics (5th Ed.). New York: Macmillan., 1975 counting aquatic microflora1. Limnology and Oceanography, [35] Kushkevych I., Kollar P., Suchy P., Parak K., Pauk K., 1980, 25(5), 943-948 Imramovsky A., Activity of selected salicylamides against [28] Bailey N.T.J. Statistical Methods in Biology. Cambridge intestinal sulfate-reducing bacteria. Neuroendocrinol Lett, University Press, Cambridge, 1995, 252 p 2015, 36, 106-113 [29] Beerens H., Romond C., Sulfate-reducing anaerobic bacteria [36] Kushkevych I., Kos J., Kollar P., Kralova K., Jampilek J., Activity in human feces. The American Journal of Clinical Nutrition, of ring-substituted 8-hydroxyquinoline-2-carboxanilides 1977, 30(11), 1770-1776 against intestinal sulfate-reducing bacteria Desulfovibrio [30] Kushkevych I., Acetate kinase Activity and Kinetic Properties piger. Medicinal Chemistry Research, 2018, 27(1), 278-284 of the Enzyme in Desulfovibrio piger Vib-7 and Desulfomi- [37] Kushkevych I., Vítězová M., Kos J., Kollár P., Jampílek J., Effect crobium sp. Rod-9 Intestinal Bacterial Strains. The Open of selected 8-hydroxyquinoline-2-carboxanilides on viability Microbiology Journal, 2014, 8(1), 138-143 and sulfate metabolism of Desulfovibrio piger. Journal of [31] Kushkevych I.V., Activity and kinetic properties of phospho- Applied Biomedicine, 2018, 16(3), 241-246 transacetylase from intestinal sulfate-reducing bacteria. Acta Biochimica Polonica, 2015, 62(1), 103-108 [32] Kushkevych I.V., Kinetic Properties of Pyruvate Ferredoxin Oxidoreductase of Intestinal Sulfate-Reducing Bacteria