Evaluation of Physiological Parameters of Intestinal Sulfate-Reducing Bacteria Isolated from Patients Suffering from IBD and Healthy People.
ABSTRACT: BACKGROUND:Inflammatory bowel diseases (IBDs) are multifactorial illnesses of the intestine, to which microorganisms are contributing. Among the contributing microorganisms, sulfate-reducing bacteria (SRB) are suggested to be involved in the process of bowel inflammation due to the production of hydrogen sulfide (H2S) by dissimilatory sulfate reduction. The aims of our research were to physiologically examine SRB in fecal samples of patients with IBD and a control group, their identification, the study of the process of dissimilatory sulfate reduction (sulfate consumption and H2S production) and biomass accumulation. Determination of biogenic elements of the SRB and evaluation of obtained parameters by using statistical methods were also included in the research. The material for the research consisted of 14 fecal samples, which was obtained from patients and control subjects. METHODS:Microscopic techniques, microbiological, biochemical, biophysical methods and statistical analysis were included. RESULTS:Colonies of SRB were isolated from all the fecal samples, and subsequently, 35 strains were obtained. Vibrio-shaped cells stained Gram-negative were dominant in all purified studied strains. All strains had a high percentage of similarity by the 16S rRNA gene with deposited sequences in GenBank of Desulfovibrio vulgaris. Cluster analysis of sulfate reduction parameters allowed the grouping of SRB strains. Significant (p < 0.05) differences were not observed between healthy individuals and patients with IBD with regard to sulfate reduction parameters (sulfate consumption, H2S and biomass accumulation). Moreover, we found that manganese and iron contents in the cell extracts are higher among healthy individuals in comparison to unhealthy individuals that have an intestinal bowel disease, especially ulcerative colitis. CONCLUSIONS:The observations obtained from studying SRB emphasize differences in the intestinal microbial processes of healthy and unhealthy people.
Project description:A comparative study of the kinetic characteristics (specific activity, initial and maximum rate, and affinity for substrates) of key enzymes of assimilatory sulfate reduction (APS reductase and dissimilatory sulfite reductase) in cell-free extracts of sulphate-reducing bacteria (SRB) from various biotopes was performed. The material for the study represented different strains of SRB from various ecotopes. Microbiological (isolation and cultivation), biochemical (free cell extract preparation) and chemical (enzyme activity determination) methods served in defining kinetic characteristics of SRB enzymes. The determined affinity data for substrates (i.e., sulfite) were 10 times higher for SRB strains isolated from environmental (soil) ecotopes than for strains from the human intestine. The maximum rate of APS reductase reached 0.282-0.862 µmol/min×mg-1 of protein that is only 10 to 28% higher than similar initial values. The maximum rate of sulfite reductase for corrosive relevant collection strains and SRB strains isolated from heating systems were increased by 3 to 10 times. A completely different picture was found for the intestinal SRB Vmax in the strains Desulfovibrio piger Vib-7 (0.67 µmol/min × mg-1 protein) and Desulfomicrobium orale Rod-9 (0.45 µmol/min × mg-1 protein). The determinant in the cluster distribution of SRB strains is the activity of the terminal enzyme of dissimilatory sulfate reduction-sulfite reductase, but not APS reductase. The data obtained from the activity of sulfate reduction enzymes indicated the adaptive plasticity of SRB strains that is manifested in the change in enzymatic activity.
Project description:Sulfate is present in foods, beverages, and drinking water. Its reduction and concentration in the gut depend on the intestinal microbiome activity, especially sulfate-reducing bacteria (SRB), which can be involved in inflammatory bowel disease (IBD). Assimilatory sulfate reduction (ASR) is present in all living organisms. In this process, sulfate is reduced to hydrogen sulfide and then included in cysteine and methionine biosynthesis. In contrast to assimilatory sulfate reduction, the dissimilatory process is typical for SRB. A terminal product of this metabolism pathway is hydrogen sulfide, which can be involved in gut inflammation and also causes problems in industries (due to corrosion effects). The aim of the review was to compare assimilatory and dissimilatory sulfate reduction (DSR). These processes occur in some species of intestinal bacteria (e.g., Escherichia and Desulfovibrio genera). The main attention was focused on the description of genes and their location in selected strains. Their coding expression of the enzymes is associated with anabolic processes in various intestinal bacteria. These analyzed recent advances can be important factors for proposing possibilities of metabolic pathway extension from hydrogen sulfide to cysteine in intestinal SRB. The switch from the DSR metabolic pathway to the ASR metabolic pathway is important since toxic sulfide is not produced as a final product.
Project description:<h4>Introduction</h4>Increased numbers of sulfate-reducing bacteria (SRB) are often found in the feces of people and animals with inflammatory bowel disease. The final products of their metabolism are hydrogen sulfide and acetate, which are produced during dissimilatory sulfate reduction process.<h4>Objectives</h4>The aim of the study was to monitor processes concerning sulfate reduction microbial metabolisms, including: the main microbial genera monitoring and their hydrogen sulfide production in the intestines of healthy and not healthy individuals, phylogenetic analysis of SRB isolates, cluster analysis of SRB physiological and biochemical parameters, SRB growth kinetic parameters calculation, same as the application of the two-factor dispersion analysis for finding relationship between SRB biomass accumulation, temperature and pH. Feces samples from healthy people and patients with colitis were used for isolation of sulfate-reducing microbial communities.<h4>Methods</h4>Microbiological, biochemical, biophysical, molecular biology methods, and statistical processing of the results have been used for making an evaluation of gained results.<h4>Results</h4>Two dominant SRB morphotypes differed in colony size and quantitative ratio in the feces of healthy and colitis patients were observed and identified. In the feces of healthy people, 93% of SRB of morphotype I prevailed <i>(Desulfovibrio)</i> while morphotype II made only 7% <i>(Desulfomicrobium)</i>; in the feces of patients with colitis, the ratio of these morphotypes was 99:1, respectively. Hydrogen sulfide concentrations are also higher in the feces of people with colitis and certain synergy effects exist among acetate produced by SRB.<h4>Conclusions</h4>The study results brought important findings concerning colony environments with developed colitis and these findings can lead to the development of possible risk indicators of ulcerative colitis prevalence.
Project description:Sulfate-reducing bacteria (SRB) colonize the guts of ~50% of humans and produce H2S, a signaling molecule with numerous host effects. We used genome-wide transposon mutagenesis and insertion-site sequencing (INSeq), RNA-Seq, plus mass spectrometry to characterize genetic and environmental factors that impact the niche of Desulfovibrio piger, the most common SRB in a surveyed cohort of healthy USA adults. Gnotobiotic mice were colonized with an assemblage of sequenced human gut bacterial species with or without D. piger and fed diets with different levels and types of carbohydrates and sulfur sources. Diet was a major determinant of functions expressed by this artificial 9-member community and of the genes that impact D. piger fitness; the latter includes high- and low-affinity systems for utilizing ammonia, a limiting resource for D. piger in mice consuming a polysaccharide-rich diet. While genes involved in hydrogen consumption and sulfate reduction are necessary for its colonization, varying dietary free sulfate levels did not significantly alter levels of D. piger, which can obtain sulfate from the host in part via cross-feeding mediated by Bacteroides-encoded sulfatases. Chondroitin sulfate, a common dietary supplement, increased D. piger and H2S levels without compromising gut barrier integrity. A chondroitin sulfate-supplemented diet together with D. piger impacted the assemblage’s substrate utilization preferences, allowing consumption of more reduced carbon sources, and increasing the abundance of the H2-producing Actinobacterium, Collinsella aerofaciens. Our findings provide genetic and metabolic details of how this H2-consuming SRB shapes the responses of a microbiota to diet ingredients, and a framework for examining how individuals lacking D. piger differ from those that harbor it. Overall design: mRNA profiles of fecal contents from gnotobiotic mice colonized with defined consortia of human gut associated microbes
Project description:Lower intraluminal colonic pH is an indication for the development of inflammatory bowel disease including active ulcerative colitis. Involvement of intestinal sulfate-reducing bacteria in decreasing bowel pH by the production of H2S and acetate as well as their sensitivity has never been reported before. The study of the relative pH and survival of Desulfovibrio piger Vib-7 by monitoring sulfate reduction parameters was the aim of this work. Monitoring was done through the measurement of bacterial growth (biomass), dissimilatory sulfate reduction parameters: sulfate consumption, lactate oxidation, hydrogen sulfide and acetate production. According to our results, we observed that lower pH (<5) significantly inhibited D. piger Vib-7 growth. This inhibition was also noticed when alkaline media (>9 pH) was used, though the reduction was not at the rate as in media with pH of 4. The research indicates that the growth of D. piger Vib-7 is inhibited at pH of 4 which is not as low as the pH found in people with severely developed inflammatory bowel diseases such as ulcerative colitis. Certainly the interaction (synergistic effect) between both hydrogen sulfide and acetate accumulation can also play an important etiological role in the development of bowel inflammation in humans and animals.
Project description:Abundance and seasonal dynamics of sulfate-reducing bacteria (SRB), in general, and of extreme halophilic SRB (belonging to Desulfocella halophila) in particular, were examined in highly saline industrial wastewater evaporation ponds over a forty one month period. Industrial wastewater was sampled and the presence of SRB was determined by quantitative real-time PCR (qPCR) with a set of primers designed to amplify the dissimilatory sulfite reductase (dsrA) gene. SRB displayed higher abundance during the summer (10(6)-10(8) targets ml(-1)) and lower abundance from the autumn-spring (10(3)-10(5) targets ml(-1)). However, addition of concentrated dissolved organic matter into the evaporation ponds during winter immediately resulted in a proliferation of SRB, despite the lower wastewater temperature (12-14 degrees C). These results indicate that the qPCR approach can be used for rapid measurement of SRB to provide valuable information about the abundance of SRB in harsh environments, such as highly saline industrial wastewaters. Low level of H2S has been maintained over five years, which indicates a possible inhibition of SRB activity, following artificial salination (approximately 16% w/v of NaCl) of wastewater evaporation ponds, despite SRB reproduction being detected by qPCR.
Project description:Background:Among the gut microbiota, sulfate-reducing bacteria (SRB) is a kind of hydrogen-utilizing functional bacteria that plays an important role in intestinal hydrogen and sulfur metabolism. However, information is lacking regarding diversity and community structure of SRB in the gut of piglets. Middle cecum contents were collected from 6 Yorkshire and 6 Meishan piglets at postnatal days (PND) 14, 28 and 49. Piglets were weaned at PND28. Real-time quantitative PCR was performed to detect the number of SRB in the cecum based on dissimilatory sulfite reductase subunit A (dsrA) gene. Prior to real-time PCR, plasmid containing the dsrA gene was constructed and used as external standard to create a standard curve, from which the gene copies of dsrA were calculated. H2S concentration in the cecal contents was measured. Illumina PE250 sequencing of dsrA gene was used to investigate SRB diversity in cecum contents. Results:The qPCR results showed that the number of SRB at PND49 was significantly higher than that at PND28 in Meishan piglets. The concentration of H2S has no significant difference between piglet breeds and between different ages. The Illumina sequencing analysis revealed that the Chao1 richness index was significantly higher at PND49 than that at PND14 and PND28 in Yorkshire piglets. Based on dsrA gene similarities, Proteobacteria, Actinobacteria, and Firmicutes were identified at the phylum level, and most sequences were classified as Proteobacteria. At the genus level, most of sequences were classified as Desulfovibrio. At the species level, Desulfovibrio intestinalis was the predominant SRB in the piglet cecum. The relative abundance and the inferred absolute abundance of Faecalibacterium prausnitzii at PND49 were significantly higher than that at PND14 in Yorkshire piglets. Pig breeds did not affect the dsrA gene copies of SRB, diversity index and community pattern of SRB. Conclusions:Sulfate-reducing bacteria are widely colonized in the cecum of piglets and D. intestinalis is the dominant SRB. The age of piglets, but not the pig breeds affects the diversity and community pattern of SRB.
Project description:Sulfur cycling is primarily driven by sulfate reduction mediated by sulfate-reducing bacteria (SRB) in marine sediments. The dissimilatory sulfate reduction drives the production of enormous quantities of reduced sulfide and thereby the formation of highly insoluble metal sulfides in marine sediments. Here, a novel sulfate-reducing bacterium designated <i>Pseudodesulfovibrio cashew</i> SRB007 was isolated and purified from the deep-sea cold seep and proposed to represent a novel species in the genus of <i>Pseudodesulfovibrio</i>. A detailed description of the phenotypic traits, phylogenetic status and central metabolisms of strain SRB007 allowed the reconstruction of the metabolic potential and lifestyle of a novel member of deep-sea SRB. Notably, <i>P. cashew</i> SRB007 showed a strong ability to resist and remove different heavy metal ions including Co<sup>2+</sup>, Ni<sup>2+</sup>, Cd<sup>2+</sup> and Hg<sup>2+</sup>. The dissimilatory sulfate reduction was demonstrated to contribute to the prominent removal capability of <i>P. cashew</i> SRB007 against different heavy metals via the formation of insoluble metal sulfides.
Project description:BACKGROUND: Arsenic (As) toxicity is primarily based on its chemical speciation. Although inorganic and methylated As species are well characterized in terms of metabolism and formation in the human body, the origin of thiolated methylarsenicals is still unclear. OBJECTIVES: We sought to determine whether sulfate-reducing bacteria (SRB) from the human gut are actively involved in the thiolation of monomethylarsonic acid (MMAV). METHODS: We incubated human fecal and colon microbiota in a batch incubator and in a dynamic gut simulator with a dose of 0.5 mg MMAV in the absence or presence of sodium molybdate, an SRB inhibitor. We monitored the conversion of MMAV into monomethyl monothioarsonate (MMMTAV) and other As species by high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry analysis. We monitored the sulfate-reducing activity of the SRB by measuring hydrogen sulfide (H2S) production. We used molecular analysis to determine the dominant species of SRB responsible for As thiolation. RESULTS: In the absence of sodium molybdate, the SRB activity-primarily derived from Desulfovibrio desulfuricans (piger)-was specifically and proportionally correlated (p < 0.01) to MMAV conversion into MMMTAV. Inactivating the SRB with molybdate did not result in MMAV thiolation; however, we observed that the microbiota from a dynamic gut simulator were capable of demethylating 4% of the incubated MMAV into arsenous acid (iAsIII), the trivalent and more toxic form of arsenic acid (iAsV). CONCLUSION: We found that SRB of human gastrointestinal origin, through their ability to produce H2S, were necessary and sufficient to induce As thiolation. The toxicological consequences of this microbial As speciation change are not yet clear. However, given the efficient epithelial absorption of thiolated methylarsenicals, we conclude that the gut microbiome-and SRB activity in particular-should be incorporated into toxicokinetic analysis carried out after As exposure.
Project description:Sulfate-rich mine water must be treated before it is released into natural water bodies. We tested ethanol as substrate in bioreactors designed for biological sulfate removal from mine water containing up to 9 g L-1 sulfate, using granular sludge from an industrial waste water treatment plant as inoculum. The pH, redox potential, and sulfate and sulfide concentrations were measured twice a week over a maximum of 171 days. The microbial communities in the bioreactors were characterized by qPCR and high throughput amplicon sequencing. The pH in the bioreactors fluctuated between 5.0 and 7.7 with the highest amount of up to 50% sulfate removed measured around pH 6. Dissimilatory sulfate reducing bacteria (SRB) constituted only between 1% and 15% of the bacterial communities. Predicted bacterial metagenomes indicated a high prevalence of assimilatory sulfate reduction proceeding to formation of l-cystein and acetate, assimilatory and dissimilatory nitrate reduction, denitrification, and oxidation of ethanol to acetaldehyde with further conversion to ethanolamine, but not to acetate. Despite efforts to maintain optimal conditions for biological sulfate reduction in the bioreactors, only a small part of the microorganisms were SRB. The microbial communities were highly diverse, containing bacteria, archaea, and fungi, all of which affected the overall microbial processes in the bioreactors. While it is important to monitor specific physicochemical parameters in bioreactors, molecular assessment of the microbial communities may serve as a tool to identify biological factors affecting bioreactor functions and to optimize physicochemical attributes for ideal bioreactor performance.