Intestinal Sulfate-reducing Bacteria Research Articles

NADH and NADPH peroxidases as antioxidant defense mechanisms in intestinal sulfate-reducing bacteria

Animal and human feces typically include intestinal sulfate-reducing bacteria (SRB). Hydrogen sulfide and acetate are the end products of their dissimilatory sulfate reduction and may create a synergistic effect. Here, we report NADH and NADPH peroxidase activities from intestinal SRB Desulfomicrobium orale and Desulfovibrio piger. We sought to compare enzymatic activities under the influence of various temperature and pH regimes, as well as to carry out kinetic analyses of enzymatic reaction rates, maximum amounts of the reaction product, reaction times, maximum rates of the enzyme reactions, and Michaelis constants in cell-free extracts of intestinal SRB, D. piger Vib-7, and D. orale Rod-9, collected from exponential and stationary growth phases. The optimal temperature (35 °C) and pH (7.0) for both enzyme’s activity were determined. The difference in trends of Michaelis constants (Km) during exponential and stationary phases are noticeable between D. piger Vib-7 and D. orale Rod-9; D. orale Rod-9 showed much higher Km (the exception is NADH peroxidase of D. piger Vib-7: 1.42 ± 0.11 mM) during the both monitored phases. Studies of the NADH and NADPH peroxidases—as putative antioxidant defense systems of intestinal SRB and detailed data on the kinetic properties of this enzyme, as expressed by the decomposition of hydrogen peroxide—could be important for clarifying evolutionary mechanisms of antioxidant defense systems, their etiological role in the process of dissimilatory sulfate reduction, and their possible role in the development of bowel diseases.

Recent Advances in Metabolic Pathways of Sulfate Reduction in Intestinal Bacteria.

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.

Hydrogen Sulfide Effects on the Survival of Lactobacilli with Emphasis on the Development of Inflammatory Bowel Diseases.

The gut microbiota is a complex component of humans that depends on diet, host genome, and lifestyle. The background: The study purpose is to find relations between nutrition, intestinal lactic acid bacteria (LAB) from various environments (human, animal intestine, and yogurt) and sulfate-reducing microbial communities in the large intestine; to compare kinetic growth parameters of LAB; and to determine their sensitivity to different concentration of hydrogen sulfide produced by intestinal sulfate-reducing bacteria. Methods: Microbiological (isolation and identification), biochemical (electrophoresis), molecular biology methods (DNA isolation and PCR analysis), and statistical processing (average and standard error calculations) of the results were used. The results: The toxicity of hydrogen sulfide produced by sulfate-reducing bacteria, the survival of lactic acid bacteria, and minimal inhibitory concentrations (MIC) were determined. The measured hydrogen sulfide sensitivity values were the same for L. paracasei and L. reuteri (MIC > 1.1 mM). In addition, L. plantarum and L. fermentum showed also a similar sensitivity (MIC > 0.45 mM) but significantly (p < 0.05) lower than L. reuteri and L. paracasei (1.1 > 0.45 mM). L. paracasei and L. reuteri are more sensitive to hydrogen sulfide than L. fermentum and L. plantarum. L. pentosus was sensitive to the extremely low concentration of H2S (MIC > 0.15 mM). Conclusions: The Lactobacillus species were significantly sensitive to hydrogen sulfide, which is a final metabolite of intestinal sulfate-reducing bacteria. The results are definitely helpful for a better understanding of complicated interaction among intestinal microbiota and nutrition.

Toxicity of hydrogen sulfide toward sulfate-reducing bacteria Desulfovibrio piger Vib-7.

Sulfate-reducing bacteria (SRB) belonging to the intestinal microbiota are the main producers of hydrogen sulfide and their increasing amount due to the accumulation of this compound in the bowel are involved in the initiation and maintenance of inflammatory bowel disease. The purpose of this experiment is to study the relative toxicity of hydrogen sulfide and survival of Desulfovibrio piger Vib-7 through monitoring: sulfate reduction parameters (sulfate consumption, hydrogen sulfide production, lactate consumption and acetate production) and kinetic parameters of these processes. The research is highlighting the survival of intestinal SRB, D. piger Vib-7 under the influence of different hydrogen sulfide concentrations (1-7mM). The highest toxicity of H2S was measured in the presence of concentrations higher than 6mM, where growing was stopped, though metabolic activities were not 100% inhibited. These findings are confirmed by cross correlation and principal component analysis that clearly supported the above mentioned results. The kinetic parameters of bacterial growth and sulfate reduction were inhibited proportionally with increasing H2S concentration. The presence of 5mM H2S resulted in two times longer lag phase and generation time was eight times longer. Maximum rate of growth and hydrogen production was stopped under 4mM, emphasizing the H2S toxicity concentrations to be < 4mM, even for sulfide producing bacteria such as Desulfovibrio. The results are confirming H2S concentrations toxicity toward Desulfovibrio, especially the study novelty should be emphasized where it was found that the exact H2S limits (> 4mM) toward this bacterial strain inhabiting humans and animals intestine.

Analysis of pH Dose-dependent Growth of Sulfate-reducing Bacteria.

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.

Analysis of Physiological Parameters of Desulfovibrio Strains from Individuals with Colitis.

Intestinal sulfate-reducing bacteria are often isolated from patients with inflammatory bowel disease, including ulcerative colitis, and can be involved in the development of gut inflammation. A comparison of the metabolism of intestinal sulfate-reducing bacteria isolated from individuals with colitis and healthy controls using statistical analysis has never been studied and described before. The aim of our research was to evaluate the parameters of dissimilatory sulfate reduction in Desulfovibrio species that were isolated from the feces of healthy objects and individuals with colitis. Principal component analysis indicates that the strains that were isolated from individuals with colitis grouped in the same cluster by biomass accumulation and sulfide production, same as the strains isolated from healthy individuals. Sulfate and lactate consumption measured over time showed negative correlation (Pearson correlations, p<0.01), healthy: -0.760; colitis: -0.770; healthy: -0.828; colitis: -0.847, respectively. The calculated linear regression (R2) was lower in biomass accumulation and hydrogen sulfide production, 0.531; 0.625 respectively. Thus, biomass accumulation and sulfide production, together with measured kinetic parameters play an important factor in bowel inflammation, including ulcerative colitis. Additionally, acetate production can also synergize with H2S, while sulfate consumption and lactate oxidation likely represent minor factors in bowel disease.

Cross-correlation analysis of the Desulfovibrio growth parameters of intestinal species isolated from people with colitis

Sulfate-reducing bacteria can be involved in inflammatory bowel disease. Cross-correlation parameters of their metabolic process in the gut have never been reported before. The aim of the research was to statistically (cross-correlation) evaluate the parameters of growth (biomass) of Desulfovibrio species from colitis people and healthy as well as to investigate the change in dissimilatory sulfate reduction of these bacterial strains. The microbiological, biochemical, chemical, and statistical methods were used in the research. The cross-correlation analysis indicates that the strains isolated from people with colitis shifted to the right side of Y axe by biomass accumulation, sulfate consumption, lactate oxidation as well as hydrogen sulfide and acetate production, compared with the strains isolated from healthy individuals. Different percentages were observed in shifting to the right side of Y axe: biomass accumulation 26%, sulfate consumption 1.5%, sulfide production 5%, lactate oxidation 3% and acetate production 12%. The biomass accumulation of intestinal sulfate-reducing bacteria and their hydrogen sulfide production are the main factors in inflammatory bowel disease development, including ulcerative colitis. Acetate production showed lesser influence while sulfate consumption and lactate oxidation are negligible factors in the inflammatory bowel disease.

Reviewers’ comments. Kushkevych I. V. Intestinal Sulfate-Reducing Bacteria. Masaryk University, Brno, Czech Republic, 2017. – 320 p.

Since my early investigations with Harry D. Peck, Jr and Jean LeGall, I have been interested in the anaerobic sulfate-reducing bacteria. While my research initially addressed stress response in sulfate reducers, our research group in the past few years has expanded into studying the effect of sul-fate-reducing bacteria on irritable bowel disease. It was through this interest in intestinal sulfate-reducing bacteria that I first became aware of the publications of Dr. Ivan Kushke­ vych. Thus, I have read this monograph with more than casual interest and have concluded that it is an extraordinary contribution which merits publication. In the following, I'll provide comments to support my evaluation. The initial chapter provides background information on the salient characteristics of the sulfate-reducing bacteria including their activities in the intestine. Since this monograph is intended, in part, to be used by students new to the subject, the illustrations and laboratory techniques enable the reader to achieve a deep understanding of the subject.

Activity of ring-substituted 8-hydroxyquinoline-2-carboxanilides against intestinal sulfate-reducing bacteria Desulfovibrio piger

Desulfovibrio genus is dominant among sulfate-reducing bacteria (SRB) in the large intestine of healthy people and animals. It is mostly isolated from patients with inflammatory bowel disease (IBD) and can be involved in the disease initiation. Primary in vitro screening of 8-hydroxyquinoline-2-carboxanilides was performed against Desulfovibrio piger Vib-7 representing SRB. The most effective compounds with MIC90/MBC values in the range of 17–23 µM/20–23 µM, respectively, were substituted in C’(3) by CF3, OCH3, CH3 and in C’(4) by CF3. Their activity was twofold higher than that of ciprofloxacin. These compounds did not express any significant cytotoxic effect on THP-1 cells up to the tested concentration of 30 µM. The antibacterial efficacy of the most active C’(3)-substituted compounds practically did not change with increasing compound lipophilicity, indicating that this position of substitution is favorable for significant antimicrobial effect, while the antibacterial activity of most of C’(2) and C’(4)-substituted derivatives decreased linearly with increasing compound lipophilicity. In addition, the dependence of activity on electronic Hammett’s σ parameter of the substituent R was quasi-parabolic for the most effective C’(3)-substituted compounds.

Molecular Cloning cysK Gene from Escherichia coli Genome, Transferring in the Intestinal Sulfate-Reducing Bacteria and the Expression Analysis of O-acetylserine(thiol)lyase

Sulfate-reducing bacteria produce hydrogen sulfide which is toxic and carcinogenic for intestinal epithelial cells and can cause the development of the inflammatory bowel disease and ulcerative colitis in the humans and animals. Enzyme O-acetylserine(thiol)lyase, localized in Escherichia coli genome, use sulfide as substrate in the cysteine synthesis pathway. In this paper, the molecular cloning cysK gene from E. coli, its genetic transferring in the intestinal sulfate-reducing bacterium Desulfovibrio piger Vib-7 and the expression analysis of the enzyme was studied. Cysteine synthesis from hydrogen sulfide as substrate in the D. piger Vib-7 strain at the first time was demonstrated and characterized. The bacterial growth, sulfate and lactate consumption, accumulation of sulfide, acetate and cysteine synthesis in both D. piger Vib-7 wild-type and mutant-type were tested. The mutant-strain consumed much faster sulfate and lactate producing cysteine in the cultivation medium. The expression of the cysK gene in the mutant-type was studied by the formation of the final reaction product (cysteine) and the activity of O-acetylserine(thiol)lyase enzyme. Cysteine level was directly proportional to consumption of sulfate in the mutant-type and accumulation of sulfide in the wild-type. The D. piger Vib-7 mutant-type completely used sulfate the 48th hour of cultivation, thereafter additional sulfite and sulfide doses from the medium were also consumed and converted to cysteine. The obtained genetically constructed mutant strain bacterium D. piger Vib-7 for therapeutic strategy could be applied as a probiotic substance for subjects with inflammatory bowel disease and ulcerative colitis. This strain can compete with other intestinal sulfate-reducing bacteria, actively growth consuming sulfate and lactate much faster, and converting the toxic sulfide to untoxic cysteine in the gut.Microbes and Health, January 2015. 4(1): 19-24

Antimicrobial effect of salicylamide derivatives against intestinal sulfate-reducing bacteria

Sulfate-reducing bacteria (SRB) are most likely involved in both the initiation and maintenance of inflammatory bowel disease (IBD); unfortunately present antibacterial chemotherapeutics used in the treatment of IBD have been ineffective. Thus, the antimicrobial activity of salicylamide derivatives against two different genera of intestinal SRB, Desulfovibrio and Desulfomicrobium, was investigated. Six 2-(phenylcarbamoyl)phenyl N-[(benzyloxy)carbonyl]alkanoates and three 2-hydroxy-N-[(2S)-1-oxo-1-(phenylamino)alkan-2-yl]benzamides showed MIC values in the range from 0.22 to 0.35μM against Desulfovibrio piger Vib-7 and in the range from 0.27 to 8.52μM against Desulfomicrobium sp. Rod-9, while MIC values of ciprofloxacin were 41.2μM and 39.3μM. The highest potency against the two strains was observed for 4-chloro-N-{(2S)-1-[(3,4-dichlorophenyl)amino]-3-methyl-1-oxobutan-2-yl}-2-hydroxybenzamide (MIC 0.22μM and 0.27μM). 4-Chloro-2-[(4-nitrophenyl)carbamoyl]phenyl (2S)-2-{[(benzyloxy)carbonyl]amino}-3-methylbutanoate showed high activity against D. piger Vib-7 (MIC=0.26μM), while 4-chloro-2-[(4-methylphenyl)carbamoyl]phenyl (2S)-2-[(tert-butoxycarbonyl)amino]-3-(1H-indol-2-yl)propanoate expressed high activity against Desulfomicrobium sp. Rod-9 (MIC=0.31μM). Structure–activity relationships are discussed.

Dissimilatory sulfate reduction in the intestinal sulfate-reducing bacteria

The study of the intestinal sulfate-reducing bacteria, the process of dissimilatory sulfate reduction and accumulation of hydrogen sulfide, as well as their role in the inflammatory bowel diseases, including ulcerative colitis, in animals and human have increa­singly attracted the attention of scientists. New opportunities for studying inflammatory bowel disease and the assessment of the effectiveness of its treatment is an urgent problem of modern biology and medicine. In this review, brief characteristics of these bacteria and their mechanism of dissimilatory sulfate reduction were described based on modern literature data and own research. The characteristics of substrates for intestinal sulfate-reducing bacteria and the thermodynamic properties of their electron donors were also described. Special attention was paid to the mechanism and stages of sulfate dissimilation including role of enzymes involved in this process. Based on our results, general scheme of dissimilatory sulfate reduction sho­wing the activity of each enzyme of the process was demonstrated. The described physiological and biochemical parameters are important for a more detailed understanding of sulfate dissimilation in the human and animal bowel, as well as studying the mechanisms of action of the antimicrobial prophylactics and the therapy against specific components involved in the pathogenesis of the disease. It is also essential for understanding the mechanisms of bowel diseases and for evaluating the effectiveness of its therapy.

Kinetic properties of pyruvate ferredoxin oxidoreductase of intestinal sulfate-reducing bacteria Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9

Intestinal sulfate-reducing bacteria reduce sulfate ions to hydrogen sulfide causing inflammatory bowel diseases of humans and animals. The bacteria consume lactate as electron donor which is oxidized to acetate via pyruvate in process of the dissimilatory sulfate reduction. Pyruvate-ferredoxin oxidoreductase activity and the kinetic properties of the enzyme from intestinal sulfate-reducing bacteria Desulfovibrio piger and Desulfomicrobium sp. have never been well-characterized and have not been yet studied. In this paper we present for the first time the specific activity of pyruvate-ferredoxin oxidoreductase and the kinetic properties of the enzyme in cell-free extracts of both D. piger Vib-7 and Desulfomicrobium sp. Rod-9 intestinal bacterial strains. Microbiological, biochemical, biophysical and statistical methods were used in this work. The optimal temperature (+35°C) and pH 8.5 for enzyme reaction were determined. The spectral analysis of the puri- fied pyruvate-ferredoxin oxidoreductase from the cell-free extracts was demonstrated. Analysis of the kinetic properties of the studied enzyme was carried out. Initial (instantaneous) reaction velocity (V0), maximum amount of the product of reaction (Pmax), the reaction time (half saturation period) and maximum velocity of the pyruvate-ferredoxin oxidoreductase reaction (V ) were defined. Michaelis constants (Km) of the enzyme reaction were calculated for both intestinal bacterial strains. The studies of the kinetic enzyme properties in the intestinal sulfate-reducing bacteria strains in detail can be prospects for clarifying the etiological role of these bacteria in the development of inflammatory bowel diseases.

Activity and kinetic properties of phosphotransacetylase from intestinal sulfate-reducing bacteria

Phosphotransacetylase activity and the kinetic properties of the enzyme from intestinal sulfate-reducing bacteria Desulfovibrio piger and Desulfomicrobium sp. has never been well-characterized and has not been studied yet. In this paper, the specific activity of phosphotransacetylase and the kinetic properties of the enzyme in cell-free extracts of both D. piger Vib-7 and Desulfomicrobium sp. Rod-9 intestinal bacterial strains were presented at the first time. The microbiological, biochemical, biophysical and statistical methods in this work were used. The optimal temperature and pH for enzyme reaction was determined. Analysis of the kinetic properties of the studied enzyme was carried out. Initial (instantaneous) reaction velocity (V0), maximum amount of the product of reaction (Pmax), the reaction time (half saturation period, τ) and maximum velocity of the phosphotransacetylase reaction (Vmax) were defined. Michaelis constants (Km) of the enzyme reaction (3.36 ± 0.35 mM for D. piger Vib-7, 5.97 ± 0.62 mM for Desulfomicrobium sp. Rod-9) were calculated. The studies of the phosphotransacetylase in the process of dissimilatory sulfate reduction and kinetic properties of this enzyme in intestinal sulfate-reducing bacteria, their production of acetate in detail can be perspective for clarification of their etiological role in the development of the humans and animals bowel diseases. These studies might help in predicting the development of diseases of the gastrointestinal tract, by providing further details on the etiology of bowel diseases which are very important for the clinical diagnosis of these disease types.

ACTIVITY AND KINETIC PROPERTIES OF ADENOSINE 5′-PHOSPHOSULFATE REDUCTASE IN THE INTESTINAL SULFATE-REDUCING BACTERIA

Adenosine 5′-phosphosulfate (APS) reductase activity and the kinetic properties of the enzyme from intestinal sulfate-reducing bacteria Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9 has never been well-characterized and has not been studied yet. The aim and background of this work was to investigate the dissimilatory APS reductase activity in cell-free extract of intestinal sulfate-reducing bacteria D. piger Vib-7 and Desulfomicrobium sp. Rod-9 and to carry out the kinetic analysis of enzymatic reaction. Methods. Microbiological, biochemical, and biophysical methods of the studies, and statistical processing of the results were used; the obtained data were compared with those from literature. Results. Dissimilatory APS reductase activity in the sulfate-reducing bacteria isolated from human intestine was studied. The highest activity of the enzyme (0.34±0.029 U×mg-1 protein) was measured in the cell-free extract prepared from D. piger Vib-7 cells then from Desulfomicrobium sp. Rod-9 (0.22±0.018 U×mg-1 protein). The optimal temperature (+35 oC) and pH (8.0) for APS reductase reaction were determined. The analysis of the kinetic properties of the bacterial APS reductase was carried out. The APS reductase activity, initial (instantaneous) reaction rate (V0) and maximum rate of the APS reductase reaction (Vmax) in both D. piger Vib-7 and Desulfomicrobium sp. Rod-9 bacterial strains were defined. Michaelis constants (Km) of the enzyme reaction (4.33±0.47 and 3.57±0.32 mM for D. piger Vib-7 and Desulfomicrobium sp. Rod-9, respectively) were determined. Conclusion. The described results of these studies can be the prospects to clarify the etiological role of these bacteria in the development of inflammatory bowel diseases humans and animals.

Dissimilatory sulfite reductase in cell-free extracts of intestinal sulfate-reducing bacteria

Dissimilatory sulfite reductase activity in different fractions of the sulfate-reducing bacteria Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9 isolated from human intestine was studied. Sulfite reductase, an important enzyme in the process of sulfur metabolism in these bacteria, was solubilized from the membrane fraction. The highest activity of the enzyme in the cell-free extract of the bacterial strains was measured (0.032±0.0026 and 0.028±0.0022 U×mg -1 protein for D. piger Vib-7 and Desulfomicrobium sp. Rod-9, respectively) compared to other fractions. The optimal temperature (+30…35 °C) and pH 7.0 for sulfite reductase reaction in the extracts of both bacterial strains was determined. The spectral analysis of purified sulfite reductase from cell-free extracts was carried out. The absorption maxima were 284, 391, 412, 583, and 630 nm, as well as 287, 393, 545, and 581 nm for sulfite reductase of D. piger Vib-7 and Desulfomicrobium sp. Rod-9, respectively. Analysis of the kinetic properties of the bacterial sulfite reductase has been carried out. The sulfite reductase activity, initial (instantaneous) reaction rate ( V 0 ) and maximum rate of the sulfite reductase reaction ( V max ) were higher in the D. piger Vib-7 cells than in the Desulfomicrobium sp. Rod-9. However, Michaelis constants ( K m ) of the enzyme activity were similar for both bacterial strains. The studies of the sulfite reductase activity, the kinetic properties of this enzyme in the intestinal sulfate-reducing bacteria strains, and their production of hydrogen sulfide in detail can be useful for clarification of the etiological role of these bacteria in the development of inflammatory bowel diseases in humans and animals. Keywords : sulfate-reducing bacteria, sulfite reductase, intestinal microbiocenosis, inflammatory bowel diseases, ulcerative colitis.

Biotic methylation of mercury by intestinal and sulfate-reducing bacteria and their potential role in mercury accumulation in the tissue of the soil-living Eisenia foetida

Monomethylmercury as one of the most toxic mercury species influences the health and development of higher organisms and tends to accumulate in the tissue of animals and humans. The aim of this study was to explore the mercury methylating capability of (1) intestinal microbiota of the soil-living earthworm Eisenia foetida (E. foetida) and (2) intestinal sulfate reducing-bacteria in pure cultures. After exposing animals to inorganic mercury chloride (4 mg kg−1 Hg2+) in soil and sterile soil for ten days, the amount of methylmercury in tissue was measured. Despite sterilization of soil, the accumulation of the organic mercury species in tissue was 51 ng g−1. To elucidate the potential of mercury methylation by the intestinal bacterial community, the methylation of inorganic mercury (0.1 mg L−1 Hg2+) in growth media inoculated with soil, gut content and gut epithelia was compared. The media with microorganisms of gut content and gut epithelia reached 710–980 pg mL−1 methylmercury while the methylmercury amount in samples inoculated with soil was below the detection limit. This lead to the assumption that, in this experiment, the soil microbiota is not the main source for methylmercury. Furthermore, we investigated the distribution of mercury in tissue thin-sections of worms kept in soil containing mercury chloride using laser ablation inductively coupled plasma mass spectrometry. Thereby, a mercury gradient with highest signal intensities in the intestines and lowest intensities in the epidermis was measured. These results indicated that the biotic production of methylmercury in the gut of the soil-living earthworm E. foetida is mainly related to the intestinal and, to a much lesser extent, to the soil microbiota in their habitat. Moreover, we demonstrated the capability of intestinal sulfate-reducing bacteria in pure cultures to be a source for methylmercury. Ten of 14 intestinal sulfate-reducing bacteria were found to be able to produce methylmercury in growth medium. In the presence of 0.1 mg L−1 inorganic HgCl2 bacterial cultures produced 100–1200 pg mL−1 methylmercury in 12 h. The mercury methylating species were Desulfovibrio spp., Desulfovibrio piger, Desulfovibrio giganteus, Desulfovibrio termitidis, Desulfotomaculum ruminis and Desulfobulbus propionicus, and two enrichment cultures, Zy1 and EF4, originating from the gut of a damselfly and E. foetida, respectively. To the best of our knowledge, this is the first study concerning biotic methylation including several pure and enrichment strains of intestinal microorganisms and it is a new insight that mercury methylating capability was found within the genus Desulfotomaculum.

Gastrointestinal and microbial responses to sulfate-supplemented drinking water in mice.

There is increasing evidence that hydrogen sulfide (H2S), produced by intestinal sulfate-reducing bacteria (SRB), may be involved in the etiopathogenesis of chronic diseases such as ulcerative colitis and colorectal cancer. The activity of SRB, and thus H2S production, is likely determined by the availability of sulfur-containing compounds in the intestine. However, little is known about the impact of dietary or inorganic sulfate on intestinal sulfate and SRB-derived H2S concentrations. In this study, the effects of short-term (7 day) and long-term (1 year) inorganic sulfate supplementation of the drinking water on gastrointestinal (GI) sulfate and H2S concentrations (and thus activity of resident SRBs), and the density of large intestinal sulfomucin-containing goblet cells, were examined in C3H/HeJBir mice. Additionally, a PCR-denaturing gradient gel electrophoresis (DGGE)-based molecular ecology technique was used to examine the impact of sulfate-amended drinking water on microbial community structure throughout the GI tract. Average H2S concentrations ranged from 0.1 mM (stomach) to 1 mM (cecum). A sulfate reduction assay demonstrated in situ production of H2S throughout the GI tract, confirming the presence of SRB. However, H2S generation and concentrations were greatest in the cecum and colon. Sulfate supplementation of drinking water did not significantly increase intestinal sulfate or H2S concentrations, suggesting that inorganic sulfate is not an important modulator of intestinal H2S concentrations, although it altered the bacterial profiles of the stomach and distal colon of 1-year-old mice. This change in colonic bacterial profiles may reflect a corresponding increase in the density of sulfomucin-containing goblet cells in sulfate-supplemented compared with control mice.

Molecular ecological analysis of the succession and diversity of sulfate-reducing bacteria in the mouse gastrointestinal tract.

Intestinal sulfate-reducing bacteria (SRB) growth and resultant hydrogen sulfide production may damage the gastrointestinal epithelium and thereby contribute to chronic intestinal disorders. However, the ecology and phylogenetic diversity of intestinal dissimilatory SRB populations are poorly understood, and endogenous or exogenous sources of available sulfate are not well defined. The succession of intestinal SRB was therefore compared in inbred C57BL/6J mice using a PCR-based metabolic molecular ecology (MME) approach that targets a conserved region of subunit A of the adenosine-5'-phosphosulfate (APS) reductase gene. The APS reductase-based MME strategy revealed intestinal SRB in the stomach and small intestine of 1-, 4-, and 7-day-old mice and throughout the gastrointestinal tract of 14-, 21-, 30-, 60-, and 90-day-old mice. Phylogenetic analysis of APS reductase amplicons obtained from the stomach, middle small intestine, and cecum of neonatal mice revealed that Desulfotomaculum spp. may be a predominant SRB group in the neonatal mouse intestine. Dot blot hybridizations with SRB-specific 16S ribosomal DNA (rDNA) probes demonstrated SRB colonization of the cecum and colon pre- and postweaning and colonization of the stomach and small intestine of mature mice only. The 16S rDNA hybridization data further demonstrated that SRB populations were most numerous in intestinal regions harboring sulfomucin-containing goblet cells, regardless of age. Reverse transcriptase PCR analysis demonstrated APS reductase mRNA expression in all intestinal segments of 30-day-old mice, including the stomach. These results demonstrate for the first time widespread colonization of the mouse intestine by dissimilatory SRB and evidence of spatial-specific SRB populations and sulfomucin patterns along the gastrointestinal tract.