一种肠道硫酸盐还原菌的增菌培养基

目的针对已经分离、纯化的肠道硫酸盐还原菌,建立一种能快速、高效地培养菌体的培养基。方法比较营养丰富的GAM肉汤与常用于培养硫酸盐还原菌的选择性培养基Postgate的培养效果,摸索在GAM肉汤中添加不同浓度的硫酸盐对两种肠道硫酸盐还原菌-Desulfovibrio desulfuricans和Desulfovibrio intestina—zis的培养效果。确定效果最佳的改良GAM培养基配方,并测定在该培养基中D．desulful＇icans的生长曲线。结果与Postgate培养基相比,GAM肉汤能在2d内快速培养D．desulfugicans,但培养至6d时细菌数量大幅降低。在GAM肉汤中添加Na2SO4与FeSO4,在实验浓度范围内,均显著地促进硫酸盐还原菌的生长。在此基础上改良GAM肉汤培养基,培养得到的细菌数量较GAM肉汤显著提高。D．desulfuricans的生长曲线显示,2d时细菌生长达到最高峰,数量可达3．5×10^7 CFU／mL;培养6d,细菌数量为7．3×10^6 CFU／mL。结论基于GAM肉汤改良而得到的增菌培养基,能快速、高效地培养肠道硫酸盐还原菌,为后续进一步研究肠道硫酸盐还原菌的生理功能提供了支持。     

Revealing Sulfate-Reducing Microorganisms in Oilfield Waters (Flysch Carpathians,South-eastern Poland)

Sulphidogenous microorganism communities were isolated from selected oilfield waters in the Flysch Carpathians of south-eastern Poland. Organisms were incubated using the microcosms method with application of two media: minimal medium and modified Postgate C medium with yeast extract or trisodium citrate or monocyclic hydrocarbons from the BTEX group (benzene, toluene, ethylbenzene, and xylene) as the sole carbon source. Activity of sulphidogenic, autochthonous microorganism communities was noted only on the Postgate medium. Beside active sulphate reduction – max. 70%, ca. 74% biodegradation of organic compounds was also observed in the cultures. The highest content of sulphate-reducing bacteria (SRB) in the COD (ca. 83%) was noted in cultures, in which trisodium citrate and yeast extract were applied as the sole carbon source. Molecular analysis indicated not only the presence of SRB such as Desulfobacterium autothrophicum, Desulfovibrio desulfuricans, but also other microorganisms, e.g., Geobacter metallireducens. All these taxa are obligatory or facultative anaerobes, with metabolism linked mostly with elemental sulphur and/or its oxidized forms, as well as iron. Analysis of the mineral composition of the residues confirmed the presence of elemental sulphur, testifying for the active reduction of sulphates by incompletely oxidizing sulphate reducers assigned to the SRB group. Based on the obtained results, it is concluded that the physical and chemical properties of the oilfield waters are favorable for the growth and development of sulphidogenic microorganism assemblages and mineral-forming processes conducted by them.     

Microbiological Reduction of Sulphates in Salty Environments and Mineralogical Characterization of the Transformation Products

This research was focused on the selection, growth and identification of SRB from soils that were subjected to long-term activity of brine, and an evaluation of mineral phases formed during the biodegradation of organic compounds and sulphate reduction. Isolated communities of anaerobic microorganisms were incubated on Postgate C medium with lactate and/or ethanol as the sole carbon source and were adapted for growth at 4% NaCl. Active reduction of sulphates with simultaneous biodegradation of organic compounds was observed in all cultures. The largest reduction of sulphates was noted in cultures with lactate as the sole carbon source; it reached 1438 mg/L, which corresponds to a 43% reduction of sulphates introduced to the medium. SRB activity in the biodegradation of organic compounds varied between 20 and 80% and depended on the level of salinity of the environment in which the SRB communities were isolated, and on the electron donor applied. The presence of biotransformation products in the post-culture deposits in the form of elemental sulphur reflects the activity of the communities. Additionally, the influence of selected communities on the salinity index was analyzed. Active SRB communities decreased the salinity of the environment by as much as 50%. Sulphate-reducing bacteria are an important group of anaerobic microorganisms, especially considering their participation in such geological processes as mineral precipitation and mineralization of organic matter in extreme environmental conditions, including high salinity.     

Clostridium ramosum, an IgA protease-producing species and its ecology in the human intestinal tract

A bacterial strain isolated from feces of a patient with ulcerative colitis, which had been shown to produce a novel immunoglobulin A (IgA) protease (cleaving both the human IgA1 subclass and IgA2 subclass of A2m(1) allotype) extracellularly, was identified as Clostridium ramosum. By using a selective medium (propionate-rifampicin-gentamicin-colimycin-polymyxin medium) devised for C. ramosum, analysis of the population level of this organism was performed to determine its ecology in the human intestinal tract. C. ramosum was isolated in 20 of 25 fecal samples (80%) from patients with inflammatory bowel disease (I.B.D.) and in 112 of 135 samples (83%) from patients without I.B.D. (control group). C. ramosum was also isolated from 6 of 11 biopsy samples (55%) of the inflamed rectal mucosa from patients with ulcerative colitis and from five of 15 samples (33%) from the intact mucosa of the control group. The population levels of C. ramosum in most of the biopsy samples ranged from 2.3 to 5.0 log10 per gram. The IgA protease-positive C. ramosum was found in only four of 135 fecal samples (3%) and one of 15 biopsy samples (6.7%) from the control group. These results indicate that IgA protease-positive C. ramosum is not likely to play a role in the induction of I.B.D., unless the organism is first isolated from the patient with I.B.D.     

Diversity and distribution of sulphate-reducing bacteria in human faeces from healthy subjects and patients with inflammatory bowel disease

The relative abundance of different groups of sulphate-reducing bacteria (SRB) in faecal DNA collected before and after therapy from patients suffering from Crohn's disease (CD), irritable bowel syndrome (IBS) or ulcerative colitis (UC) has been compared with that from healthy controls. Growth tests revealed that SRB were not more abundant in samples from patients with CD before treatment than in the healthy control group. For most of the 128 samples available, these preliminary results were confirmed using degenerate PCR primers that amplify the dsrAB gene. However, some samples from patients with CD before treatment contained a growth inhibitor that was absent from IBS or UC samples. In-depth sequencing of PCR-generated dsrB fragments revealed that the diversity detected was surprisingly low, with only eight strains of SRB and the sulphite-reducing bacterium, Bilophila wadsworthia, detected above the 0.1% threshold. The proportion of the two major species detected, B. wadsworthia and Desulfovibrio piger, was as high as 93.5% of the total SRB population in the healthy control group and lower in all patient groups. Four previously undescribed species were found: it is impossible to predict whether they are sulphate or sulphite-reducing bacteria.     

On the relationship between sialomucin and sulfomucin expression and hydrogenotrophic microbes in the human colonic mucosa

The colonic mucus layer is comprised primarily of acidomucins, which provide viscous properties and can be broadly classified into sialomucins or sulfomucins based on the presence of terminating sialic acid or sulfate groups. Differences in acidomucin chemotypes have been observed in diseases such as colorectal cancer and inflammatory bowel disease, and variation in sialo- and sulfomucin content may influence microbial colonization. For example, sulfate derived from sulfomucin degradation may promote the colonization of sulfate-reducing bacteria (SRB), which through sulfate respiration generate the genotoxic gas hydrogen sulfide. Here, paired biopsies from right colon, left colon, and rectum of 20 subjects undergoing routine screening colonoscopies were collected to enable parallel histochemical and microbiological studies. Goblet cell sialo- and sulfomucins in each biopsy were distinguished histochemically and quantified. Quantitative PCR and multivariate analyses were used to examine the abundance of hydrogenotrophic microbial groups and SRB genera relative to acidomucin profiles. Regional variation was observed in sialomucins and sulfomucins with the greatest abundance of each found in the rectum. Mucin composition did not appear to influence the abundance of SRB or other hydrogenotrophic microbiota but correlated with the composition of different SRB genera. A higher sulfomucin proportion correlated with higher quantities of Desulfobacter, Desulfobulbus and Desulfotomaculum, relative to the predominant Desulfovibrio genus. Thus, acidomucin composition may influence bacterial sulfate respiration in the human colon, which may in turn impact mucosal homeostasis. These results stress the need to consider mucus characteristics in the context of studies of the microbiome that target intestinal diseases.     

A statistical comparison of two culturing methods for enumerating sulfate-reducing bacteria

The addition of alkaline pyrogallol-soaked cotton wool plugs has been used by other workers to remove oxygen from the headspace gas in culture tubes for the growth of sulfate-reducing bacteria (SRB). This study compares the enumeration fo SRB using the most probable number (MPN) method in tubes with and without the pyrogallol plugs. In both cases, the liquid medium contained two iron finishing nails which help reduce the redox potential of the medium. For each of the 25 samples from oil fields, cooling water systems and natural environments, the time-consuming method using pyrogallol plugs in screw cap tubes yielded virtually the same MPN values as the same method without the plugs and using Kaput® closures on the tubes. However, the pyrogallol plug method, which requires approximately 10-fold more technician time, was superior for pure culture enumeration and in cases where SRB greatly outnumber heterotrophs.     

Detection and Enumeration of Sulphate-Reducing Bacteria in Estuarine Sediments by Competitive PCR

The distribution of sulphate-reducing bacteria (SRB) in the sediments of the Colne River estuary, Essex, UK covering different saline concentrations of sediment porewater was investigated by the use of quantitative competitive PCR. Here, we show that a new PCR primer set and a new quantitative method using PCR are useful tools for the detection and the enumeration of SRB in natural environments. A PCR primer set selective for the dissimilatory sulphite reductase gene (dsr) of SRB was designed. PCR amplification using the single set of dsr-specific primers resulted in PCR products of the expected size from all 27 SRB strains tested, including Gram-negative and positive species. Sixty clones derived from sediment DNA using the primers were sequenced and all were closely related with the predicted dsr of SRB. These results indicate that PCR using the newly designed primer set are useful for the selective detection of SRB from a natural sample. This primer set was used to estimate cell numbers by dsr selective competitive PCR using a competitor, which was about 20% shorter than the targeted region of dsr. This procedure was applied to sediment samples from the River Colne estuary, Essex, UK together with simultaneous measurement of in situ rates of sulphate reduction. High densities of SRB ranging from 0.2 ? 5.7 × 10⁸ cells ml^{? 1} wet sediment were estimated by the competitive PCR assuming that all SRB have a single copy of dsr. Using these estimates cell specific sulphate reduction rates of 10^{? 17} to 10^{? 15} mol of SO₄ ^{2 ?} cell^{? 1} day^{? 1} were calculated, which is within the range of, or lower than, those previously reported for pure cultures of SRB. Our results show that the newly developed competitive PCR technique targeted to dsr is a powerful tool for rapid and reproducible estimation of SRB numbers in situ and is superior to the use of culture-dependent techniques.     

Examination of thirteen petroliferous formations for hydrocarbon-utilizing sulfate-reducing microorganisms

Virgin cores and production fluids were obtained from seven wells, ranging in depth from 805 ft to 14 492 ft, and examined for the presence of sulfate-reducing bacteria (SRB) using Rosenfeld's sulfate-reducing medium modified by using crude oil in place of lactate. Cores from an additional six wells, ranging in depth from 1160 ft to 13 337 ft were tested for SRB using the modified Rosenfeld medium and API-sulfate-reducing medium. Produced waters from five of the six wells were tested also. All of the eleven produced water samples were positive for SRB while H₂S production was not detected from the core samples.     

Problems of isolating Borrelia burgdorferi from ticks collected in United Kingdom foci of Lyme disease

Abstract. Many isolates of Borrelia burgdorferi have been obtained from ticks and vertebrate tissues collected in North America and continental Europe but only one established culture of United Kingdom Borrelia burgdorferi has been recorded. In this paper we report the isolation of B.burgdorferi from one of 108 tick pools representing 733 ticks and eighty-four tissue samples from twenty-six rodents collected in the U.K., and the subsequent failure to establish the isolate (from ticks collected in Fordingbridge) in culture. In contrast, using identical techniques and culture medium, B.burgdorferi was isolated from one of seven tick pools collected in Switzerland, and from a single pool of ticks collected in Slovakia, and both isolates were successfully passaged. Analysis of questing I.ricinus collected from Fordingbridge by direct immunofluorescence showed 6/32 (19%) of adults and 8/108 (7%) of nymphs were positive for B. burgdorferi , although only one nymph contained ≥ 1000 spirochaetes. To examine further the problem of isolating U.K. B.burgdorferi , twelve Ixodes ricinus tick samples from Fordingbridge, a recognized focus of Lyme disease, were subjected to isolation and culturing techniques, and the procedures monitored by use of the polymerase chain reaction (PCR). Whereas 11/12 samples were PCR positive after 2 weeks in culture, only one was PCR positive after 4 weeks. Motile spirochaetes were not visible by dark-field microscopy in any of the cultures. The results indicate that the standard BSK II medium routinely used to isolate and culture B. burgdorferi does not readily support the replication of the Borrelia species endemic to the U.K.     

Enumeration and identification of dominant types of sulfate-reducing bacteria in pulp from a paper-recycling plant: a multiphasic approach

Multiple independent approaches were applied for monitoring the abundance and identity of sulfate-reducing bacteria (SRB) in pulp of a paper-recycling plant suffering from excessive sulfide emission. The methods applied included most-probable-number (MPN) enumeration of cultivable SRB, rate measurements, FISH and PCR-based retrieval of the functional marker genes dsrA and B (encoding the two major subunits of dissimilatory bisulfite reductase) and 16S rRNA genes. The SRB community was composed of phylogenetically highly different lineages all of low abundance relative to the total microbial community in the pulp, which hampered the applicability of FISH. It was also demonstrated that dsrA- or B -targeted PCR primers commonly used for denaturing gradient gel electrophoresis and real-time PCR analyses were biased. However, using a novel approach combining MPN-PCR and terminal restriction fragment length polymorphism analysis of dsrAB amplicons generated from serially diluted DNA extracts allowed the enumeration and identification of the quantitatively most important members of the SRB community. For fast quantification of SRB in the pulp, the dsrAB -MPN-PCR assay and sulfate reduction rate measurements were found to be most suitable.     

Characterization of bacterial community associated to biofilms of corroded oil pipelines from the southeast of Mexico

Microbial communities associated to biofilms promote corrosion of oil pipelines. The community structure of bacteria in the biofilm formed in oil pipelines is the basic knowledge to understand the complexity and mechanisms of metal corrosion. To assess bacterial diversity, biofilm samples were obtained from X52 steel coupons corroded after 40 days of exposure to normal operation and flow conditions. The biofilm samples were directly used to extract metagenomic DNA, which was used as template to amplify 16S ribosomal gene by PCR. The PCR products of 16S ribosomal gene were also employed as template for sulfate-reducing bacteria (SRB) specific nested-PCR and both PCR products were utilized for the construction of gene libraries. The V3 region of the 16S rRNA gene was also amplified to analyse the bacterial diversity by analysis of denaturing gradient gel electrophoresis (DGGE). Ribosomal library and DGGE profiles exhibited limited bacterial diversity, basically including Citrobacter spp., Enterobacter spp. and Halanaerobium spp. while Desulfovibrio alaskensis and a novel clade within the genus Desulfonatronovibrio were detected from the nested PCR library. The biofilm samples were also taken for the isolation of SRB. Desulfovibrio alaskensis and Desulfovibrio capillatus, as well as some strains related to Citrobacter were isolated. SRB consists in a very small proportion of the community and Desulfovibrio spp. were the relatively abundant groups among the SRB. This is the first study directly exploring bacterial diversity in corrosive biofilms associated to steel pipelines subjected to normal operation conditions.     

Microbial community analysis of rectal methanogens and sulfate reducing bacteria in two non-human primate species

Background  Methanogenesis by methanogenic Archaea and sulfate reduction by sulfate reducing bacteria (SRB) are the major hydrogenotrophic pathways in the human colon. Methanogenic status of mammals is suggested to be under evolutionary rather than dietary control. However, information is lacking regarding the dynamics of hydrogenotrophic microbial communities among different primate species.
Methods  Rectal swabs were collected from 10 sooty mangabeys ( Cercocebus atys ) and 10 baboons ( Papio hamadryas ). The diversity and abundance of methanogens and SRB were examined using PCR-denaturing gradient gel electrophoresis (DGGE) and real-time quantitative PCR (qPCR).
Results  The DGGE results revealed that intestinal Archaea and SRB communities differ between mangabeys and baboons. Phylogenetic analyses of Archaea DGGE bands revealed two distinct clusters with one representing a putative novel order of methanogenic Archaea. The qPCR detected a similar abundance of methanogens and SRB.
Conclusions  Intestinal Archaea and SRB coexist in these primates, and the community patterns are host species-specific.     

Case study of the distribution of mucosa-associated Bifidobacterium species, Lactobacillus species, and other lactic acid bacteria in the human colon

The distribution of mucosa-associated bacteria, bifidobacteria and lactobacilli and closely related lactic acid bacteria, in biopsy samples from the ascending, transverse, and descending parts of the colon from four individuals was investigated by denaturing gradient gel electrophoresis (DGGE). Bifidobacterial genus-specific, Lactobacillus group-specific, and universal bacterial primers were used in a nested PCR approach to amplify a fragment of the 16S rRNA gene. DGGE profiles of the bifidobacterial community were relatively simple, with one or two amplicons detected at most sampling sites in the colon. DGGE profiles obtained with Lactobacillus group-specific primers were complex and varied with host and sampling site in the colon. The overall bacterial community varied with host but not sampling site.     

Evaluation of the sulfate-reducing bacterial population associated with stored swine slurry

Hydrogen sulfide, produced by sulfate-reducing bacteria (SRB), is one of the most potent malodors emitted from anaerobic swine waste storage systems. However, little is known about the prevalence and diversity of SRB in those systems. The goals of this study were to evaluate the SRB population in swine manure storage systems and to develop quantitative, real-time PCR (QRT-PCR) assays to target four of the SRB groups. Dissimilatory sulfite reductase (DSR) gene sequences were obtained from swine slurry stored in underground pits (43 clones) or in lagoons (34 clones). QRT-PCR assays were designed to target the dsrA gene of four novel groups of SRB. Sequences of dsrA clones from slurry samples grouped with those from three different cultured SRB: Desulfobulbus sp. (46 clones), Desulfovibrio sp. (24 clones and 5 isolates), and Desulfobacterium sp. (7 clones). However, DsrA sequences from swine slurry clones were generally less than 85% similar to those of cultured organisms. SRB from all four targeted SRB groups were detected in underground waste storage pits (6.6 x 10(3)-8.5 x 10(7) dsrA copies mL(-1) slurry), while only two groups of SRB were detected in lagoons (3.2 x 10(5)-2.5 x 10(6) dsrA copies mL(-1) slurry). To date, this is the only study to evaluate the phylogeny and concentration of SRB in any livestock waste storage system. The new QRT-PCR assays should facilitate sensitive, specific detection of the four novel groups of SRB in livestock waste storage systems.     

Occurrence of sulphate-reducing bacteria in human faeces and the relationship of dissimilatory sulphate reduction to methanogenesis in the large gut

Sulphate-reducing bacteria (SRB) were enumerated in 40 faecal samples obtained from two different human populations in the United Kingdom and rural South Africa. Species able to metabolize acetate, lactate, propionate, butyrate, H₂/CO₂, succinate, pyruvate, valerate, ethanol and a glutamate/serine/alanine mixture were found in faeces from both populations. Although a variety of nutritionally and morphologically distinct species of SRB belonging to the genera Desulfotomaculum, Desulfobacter, Desulfomonas and Desulfobulbus were identified, Desulfovibrio types always predominated. Significant numbers of SRB were present only in faecal samples from subjects whose breath methane excretion was low or undetectable. Reduced or absent methanogenesis in the presence of SRB was confirmed in fermentation studies with faecal slurrries. Fourteen of 20 (70%) British faecal samples contained SRB and the remainder produced methane. The reverse was the case with 20 rural black South Africans, where only three (15%) of the samples had significant levels of SRB; the remaining 85% produced methane. These results suggest that to a large extent, dissimilatory sulphate reduction and methanogenesis are mutually exclusive in the human large gut.     

Occurrence of sulphate-reducing bacteria in human faeces and the relationship of dissimilatory sulphate reduction to methanogenesis in the large gut

Sulphate-reducing bacteria (SRB) were enumerated in 40 faecal samples obtained from two different human populations in the United Kingdom and rural South Africa. Species able to metabolize acetate, lactate, propionate, butyrate, H2/CO2, succinate, pyruvate, valerate, ethanol and a glutamate/serine/alanine mixture were found in faeces from both populations. Although a variety of nutritionally and morphologically distinct species of SRB belonging to the genera Desulfotomaculum, Desulfobacter, Desulfomonas and Desulfobulbus were identified, Desulfovibrio types always predominated. Significant numbers of SRB were present only in faecal samples from subjects whose breath methane excretion was low or undetectable. Reduced or absent methanogenesis in the presence of SRB was confirmed in fermentation studies with faecal slurries. Fourteen of 20 (70%) British faecal samples contained SRB and the remainder produced methane. The reverse was the case with 20 rural black South Africans, where only three (15%) of the samples had significant levels of SRB; the remaining 85% produced methane. These results suggest that to a large extent, dissimilatory sulphate reduction and methanogenesis are mutually exclusive in the human large gut.     

Case Study of the Distribution of Mucosa-Associated Bifidobacterium Species, Lactobacillus Species, and Other Lactic Acid Bacteria in the Human Colon

The distribution of mucosa-associated bacteria, bifidobacteria and lactobacilli and closely related lactic acid bacteria, in biopsy samples from the ascending, transverse, and descending parts of the colon from four individuals was investigated by denaturing gradient gel electrophoresis (DGGE). Bifidobacterial genus-specific, Lactobacillus group-specific, and universal bacterial primers were used in a nested PCR approach to amplify a fragment of the 16S rRNA gene. DGGE profiles of the bifidobacterial community were relatively simple, with one or two amplicons detected at most sampling sites in the colon. DGGE profiles obtained with Lactobacillus group-specific primers were complex and varied with host and sampling site in the colon. The overall bacterial community varied with host but not sampling site.