Carbapenemase-producing Enterobacteriaceae in hospital wastewater: a reservoir that may be unrelated to clinical isolates.

BACKGROUND: Carbapenemase-producing Enterobacteriaceae (CPE) are an emerging infection control problem in hospitals worldwide. Identifying carriers may help reduce potential spread and infections. AIM: To assess whether testing hospital wastewater for CPE can supplement patient-based screening for infection prevention purposes in a hospital without a recognized endemic CPE problem. METHODS: Wastewater collected from hospital pipework on 16 occasions during February to March 2014 was screened for CPE using chromID(®) CARBA agar and chromID(®) CPS agar with a 10µg ertapenem disc and combination disc testing. Minimum inhibitory concentrations were determined using British Society for Antimicrobial Chemotherapy methodology and carbapenemase genes detected by polymerase chain reaction or whole-genome sequencing. Selected isolates were typed by pulsed-field gel electrophoresis. FINDINGS: Suspected CPE were recovered from all 16 wastewater samples. Of 17 isolates sent to the Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, six (four Citrobacter freundii and two Enterobacter cloacae complex) were New Delhi metallo-ß-lactamase (NDM) producers and the remaining 11 (six Klebsiella oxytoca and five Enterobacter cloacae complex) were Guiana-Extended-Spectrum-5 (GES-5) producers, the first to be described among Enterobacteriaceae in the UK. The four NDM-producing C. freundii, two NDM-producing E. cloacae complex, and four out of five GES-5-producing E. cloacae complex were each indistinguishable isolates of the same three strains, whereas the six GES-5-producing K. oxytoca overall shared 79% similarity. CONCLUSION: CPE are readily isolated from hospital wastewater using simple culture methods. There are either undetected carriers of CPE excreting into the wastewater, or these CPE represent colonization of the pipework from other sources. Surveillance of hospital wastewater for CPE does not appear helpful for infection control purposes within acute hospitals.

Mycobacterium avium subsp. paratuberculosis in lake catchments, in river water abstracted for domestic use, and in effluent from domestic sewage treatment works: diverse opportunities for environmental cycling and human exposure.

Mycobacterium avium subsp. paratuberculosis from infected animals enters surface waters and rivers in runoff from contaminated pastures. We studied the River Tywi in South Wales, United Kingdom, whose catchment comprises 1,100 km2 containing more than a million dairy and beef cattle and more than 1.3 million sheep. The River Tywi is abstracted for the domestic water supply. Between August 2002 and April 2003, 48 of 70 (68.8%) twice-weekly river water samples tested positive by IS900 PCR. In river water, the organisms were associated with a suspended solid which was depleted by the water treatment process. Disposal of contaminated slurry back onto the land established a cycle of environmental persistence. A concentrate from 100 liters of finished water tested negative, but 1 of 54 domestic cold water tanks tested positive, indicating the potential for these pathogens to access domestic outlets. In the separate English Lake District region, with hills up to 980 m, tests for M. avium subsp. paratuberculosis in the high hill lakes and sediments were usually negative, but streams and sediments became positive lower down the catchment. Sediments from 9 of 10 major lakes receiving inflow from these catchments were positive, with sediment cores indicating deposition over at least 40 to 50 years. Two of 12 monthly 1-liter samples of effluent and a single 100-liter sample from the Ambleside sewage treatment works were positive for M. avium subsp. paratuberculosis. Since Lake Ambleside discharges into Lake Windermere, which is available for domestic supply, there is a potential for these organisms to cycle within human populations.

Mycobacterium avium subsp. paratuberculosis in the catchment area and water of the River Taff in South Wales, United Kingdom, and its potential relationship to clustering of Crohn's disease cases in the city of Cardiff.

In South Wales, United Kingdom, a populated coastal region lies beneath hill pastures grazed by livestock in which Mycobacterium avium subsp. paratuberculosis is endemic. The Taff is a spate river running off the hills and through the principal city of Cardiff. We sampled Taff water above Cardiff twice weekly from November 2001 to November 2002. M. avium subsp. paratuberculosis was detected by IS900 PCR and culture. Thirty-one of 96 daily samples (32.3%) were IS900 PCR positive, and 12 grew M. avium subsp. paratuberculosis bovine strains. Amplicon sequences from colonies were identical to the sequence with GenBank accession no. X16293, whereas 16 of 19 sequences from river water DNA extracts had a single-nucleotide polymorphism at position 214. This is consistent with a different strain of M. avium subsp. paratuberculosis in the river, which is unculturable by the methods we used. Parallel studies showed that M. avium subsp. paratuberculosis remained culturable in lake water microcosms for 632 days and persisted to 841 days. Of four reservoirs controlling the catchment area of the Taff, M. avium subsp. paratuberculosis was present in surface sediments from three and in sediment cores from two, consistent with deposition over at least 50 years. Previous epidemiological research in Cardiff demonstrated a highly significant increase of Crohn's disease in 11 districts. These bordered the river except for a gap on the windward side. A topographical relief map shows that this gap is directly opposite a valley open to the prevailing southwesterly winds. This would influence the distribution of aerosols carrying M. avium subsp. paratuberculosis from the river.

Analysis of methanogen diversity in a hypereutrophic lake using PCR-RFLP analysis of mcr sequences.

The incidence and diversity of methanogens in Priest Pot, a dynamic and active lake, were monitored by analysing mcrA gene sequences generated from total DNA samples obtained at different times of the year and amplified using the polymerase chain reaction. A number of mcrA clones were analysed by developing an RFLP-based protocol to generate a number of restriction patterns that were assigned to a number of classes. The RFLP patterns for each class were compared with published sequence information for mcrA from cultured methanogens as well as with those from other experimental studies. They could be used to assign tentative identification for some of the Priest Pot clones and also revealed the presence of a number of clones that could not be affiliated to any known methanogens. The limitations of using RFLP profiles of mcrA gene sequences for studying methanogen ecology are discussed.

A comparison of ammonia-oxidiser populations in eutrophic and oligotrophic basins of a large freshwater lake.

A combination of PCR amplification and oligonucleotide probing was used to investigate the populations of ammonia-oxidisers of the beta-Proteobacteria in the eutrophic and oligotrophic basins of Lake Windermere, a large temperate lake in the English Lake District. Numbers of ammonia-oxidisers (MPN) in the Windermere lakewater were low (< 100 cells ml(-1)) throughout the year with the exception of peaks in August, which coincided with stratification, and November in the South Basin where overturn may have introduced ammonia-oxidising bacteria into the water column. Sediment samples contained larger populations of ammonia oxidisers, usually ca. 10(4) per g. dry weight, which remained relatively constant throughout the seasonal cycle in both Basins. DNA was recovered from lakewater and sediment samples and Nitrosospira and N. europaea-eutropha lineage 16S rRNA genes amplified in a nested PCR reaction, with confirmation of identity by oligonucleotide hybridisation. Nitrosospira 16S rDNA was readily detected in all samples and therefore found to be ubiquitous. In contrast, nitrosomonad DNA of the N. europaea-eutropha lineage could only be detected in the oligotrophic North Basin. Enrichment cultures of lakewater samples only exhibited nitrification at low (0.67 mM) and medium (5 mM) ammonium concentrations, whilst sediment enrichments nitrified at all concentrations tested including high (12.5 mM) ammonium medium. These data suggest that ammonia-oxidiser populations may be physiologically distinguished between lakewater and sediment, and that species distribution in a single lake is non-uniform.

Biogeochemical characterisation of a coal tar distillate plume.

The distillation of acidified coal tars for up to 50 years has given rise to a phenol plume approximately 500 m long, 50 m deep and containing up to 15 g l(-1) dissolved organic carbon (DOC) in the Triassic Sandstones aquifer. A conceptual biogeochemical model based on chemical and microbiological analysis of groundwater samples has been developed as a preliminary to more detailed studies of the controls on natural attenuation. While the development of redox zones and the production of methane and carbon dioxide provide evidence of natural attenuation, it appears that degradation is slow. The existence of sulphate in the plume indicates that this electron acceptor has not been depleted and that consequently methanogenesis is probably limited. Based on a simple estimate of sulphate input concentration, a half-life of about 15 years has been estimated for sulphate reduction. Geochemical modelling predicts that increased alkalinity within the plume has not led to carbonate precipitation, and thus within the limits of accuracy of the measurement, alkalinity may reflect the degree of biodegradation. This implies a loss of around 18% of the DOC over a 30-year period. Despite limited degradation, microbial studies show that there are diverse microbial communities in the aquifer with the potential for both anaerobic and aerobic biodegradation. Microbial activity was found to be greatest at the leading edge of the plume where DOC concentrations are 60 mg l(-1) or less, but activity could still be observed in more contaminated samples even though cells could not be cultured. The study suggests that degradation may be limited by the high phenol concentrations within the core of the plume, but that once diluted by dispersion, natural attenuation may proceed. More detailed studies to confirm these initial findings are identified and form the basis of associated papers.

Microbiological analysis of multi-level borehole samples from a contaminated groundwater system.

A range of bacteriological, geochemical process-related and molecular techniques have been used to assess the microbial biodegradative potential in groundwater contaminated with phenol and other tar acids. The contaminant plume has travelled 500 m from the pollutant source over several decades. Samples were obtained from the plume using a multi-level sampler (MLS) positioned in two boreholes (boreholes 59 and 60) which vertically transected two areas of the plume. Activity of the microbial community, as represented by phenol degradation potential and ability to utilise a range of substrates, was found to be influenced by the plume. Phenol degradation potential appeared to be influenced more by the concentration of the contaminants than the total bacterial cell numbers. However, in the areas of highest phenol concentration, the depression of cell numbers clearly had an effect. The types of bacteria present were assessed by culture and DNA amplification by polymerase chain reaction (PCR). Bacterial groups or processes associated with major geochemical processes, such as methanogenesis, sulphate reduction and denitrification, that have the potential to drive contaminant degradation, were detected at various borehole levels. A comparative molecular analysis of the microbial community between samples obtained from the MLS revealed the microbial community was diverse. The examination of microbial activity complemented those results obtained through chemical analysis, and when combined with hydrological data, showed that MLS samples provided a realistic profile of plume effects and could be related to the potential for natural attenuation of the site.

Nucleic acid-based fluorescent probes in microbial ecology: application of flow cytometry.

Microorganisms in natural environments have often been treated as 'black box' systems. Researchers have measured the inputs and outputs of the box, and have made bulk measurements on cell behaviour. However, unravelling the details of the diversity and interactions that exist within these microbial populations has proven exceptionally difficult. The information gained from the black box approach has been invaluable, and has allowed models of global foodwebs to be generated and tested. However, there is still little information about the interactions of individual microbial cells within natural populations. Such studies are essential to fully understand the integrated functioning of ecosystems. To achieve this goal, researchers need to be able to identify individual cells within a population, enumerate them, estimate both viability and activity, and monitor changes in response to relevant parameters. Due to the diversity, heterogeneity and numbers of cells that make up these populations, these measurements require automation and speed. At present, the use of flow cytometry in conjunction with nucleic acid probes provides an excellent method with which to pursue such studies.

Use of combined microautoradiography and fluorescence in situ hybridization to determine carbon metabolism in mixed natural communities of uncultured bacteria from the genus Achromatium.

Combined microautoradiography and fluorescence in situ hybridization (FISH) was used to investigate carbon metabolism in uncultured bacteria from the genus Achromatium. All of the Achromatium species identified in a freshwater sediment from Rydal Water, Cumbria, United Kingdom, which were distinguishable only by FISH, assimilated both [(14)C]bicarbonate and [(14)C]acetate. This extends previous findings that Achromatium spp. present at another location could only utilize organic carbon sources. Achromatium spp., therefore, probably exhibit a range of physiologies, i.e., facultative chemolithoautotrophy, mixotrophy, and chemoorganoheterotrophy, similar to other large sulfur bacteria (e.g., Beggiatoa spp.).

Distribution of oxytetracycline resistance plasmids between aeromonads in hospital and aquaculture environments: implication of Tn1721 in dissemination of the tetracycline resistance determinant tet A.

Oxytetracycline-resistant (OT(r)) mesophilic aeromonads were recovered from untreated hospital effluent (72 isolates) and from fish farm hatchery tanks (91 isolates) at sites within the English Lake District, Cumbria, England. The transfer of OT(r) plasmids from these isolates was investigated. Using Escherichia coli J53-1 as a recipient, 11 isolates from the hospital site and 6 isolates from the fish farm site transferred OT(r) plasmids (designated pFBAOT1 to 17). Original isolates were identified using fatty acid methyl ester and fluorescent amplified fragment length polymorphism comparisons as either Aeromonas hydrophila HG3 (eight isolates), A. veronii b.v. sobria HG8 (six isolates), and A. caviae HGB5 (one isolate). One isolate remained unidentified, and one could not be assigned a taxonomic designation beyond the genus level. Plasmids pFBAOT1 to -17 were screened for the presence of the tetracycline resistance determinants Tet A to E and Tet G. Only determinant Tet A (10 plasmids) was detected in these plasmids, with 7 tet gene determinants remaining unclassified. In all cases, Tet A was located on a 5.5-kb EcoRI restriction fragment. Hybridization with inc-rep probes N, P, Q, W, and U showed pFBAOT3, -4, -5, -6, -7, -9, and -11, from the hospital environment, to be IncU plasmids. Further, restriction fragment length polymorphism (RFLP) analyses and DNA probing demonstrated that pFBAOT plasmids were closely related to IncU OT(r) plasmids pASOT, pASOT2, pASOT3, pRAS1 (originally isolated from A. salmonicida strains from fish farms in Scotland and Norway, respectively), and pIE420 (isolated from a German hospital E. coli strain). In addition, DNA analyses demonstrated that plasmids pRAS1 and pIE420 had identical RFLP profiles and that all fragments hybridized to each other. The presence of tetracycline resistance transposon Tn1721 in its entirety or in a truncated form in these plasmids was demonstrated. These results provided direct evidence that related tetracycline resistance-encoding plasmids have disseminated between different Aeromonas species and E. coli and between the human and aquaculture environments in distinct geographical locations. Collectively, these findings provide evidence to support the hypothesis that the aquaculture and human compartments of the environment behave as a single interactive compartment.

Development and application of molecular tools in the study of IncN-related plasmids from lakewater sediments.

Homology to IncN, P, Q and W inc regions was investigated amongst 114 Hg(2+)-resistant or antibiotic-resistant bacteria isolated from lakewater sediments. No hybridisation signals were found with Inc P, Q and W probes, and only one plasmid, pLV1402, hybridised to the IncN probe. PCR primers designed to conserved regions in the replicon of the IncN plasmid pCU1 and the related beta replicon from pGSH500 were used to amplify a 978-bp fragment from pLV1402, with sequence analysis showing a close relationship (99.2% identity) between their replication genes. A 387-bp region from the pLV1402 rep gene was used to re-screen the isolates and identified another related plasmid, pLV1403. A 3.7-kb probe containing the alpha replicon from pGSH500 hybridised to both pLV1402 and pLV1403, suggesting that both are multi-replicon plasmids. The PCR primers and probes described will be useful in future studies of plasmid diversity.

Phylogenetic relationships of filamentous sulfur bacteria (Thiothrix spp. and Eikelboom type 021N bacteria) isolated from wastewater-treatment plants and description of Thiothrix eikelboomii sp. nov., Thiothrix unzii sp. nov., Thiothrix fructosivorans sp. nov. and Thiothrix defluvii sp. nov.

The relationship of mixotrophic and autotrophic Thiothrix species to morphologically similar chemoorganotrophic bacteria (e.g. Leucothrix species, Eikelboom type 021N bacteria) has been a matter of debate for some years. These bacteria have alternatively been grouped together on the basis of shared morphological features or separated on the basis of their nutrition. Many of these bacteria are difficult to maintain in axenic culture and, until recently, few isolates were available to allow comprehensive phenotypic and genotypic characterization. Several isolates of Thiothrix spp. and Eikelboom type 021N strains were characterized by comparative 16S rRNA sequence analysis. This revealed that the Thiothrix spp. and Eikelboom type 021N isolates formed a monophyletic group. Furthermore, isolates of Eikelboom type 021N bacteria isolated independently from different continents were phylogenetically closely related. The 16S rRNA sequence-based phylogeny was congruent with the morphological similarities between Thiothrix and Eikelboom type 021N. However, one isolate examined in this study (Ben47) shared many morphological features with the Thiothrix spp. and Eikelboom type 021N isolates, but was not closely related to them phylogenetically. Consequently, morphology alone cannot be used to assign bacteria to the Thiothrix/type 021N group. Comparative 16S rRNA sequence analysis supports monophyly of the Thiothrix/type 021N group, and phenotypic differences between the Thiothrix spp. and Eikelboom type 021N bacteria are currently poorly defined. For example, both groups include heterotrophic organisms that deposit intracellular elemental sulfur. It is therefore proposed that the Eikelboom type 021N bacteria should be accommodated within the genus Thiothrix as a new species, Thiothrix eikelboomii sp. nov., and three further new Thiothrix species are described: Thiothrix unzii sp. nov., Thiothrix fructosivorans sp. nov. and Thiothrix defluvii sp. nov.

Phylogenetic differentiation of two closely related Nitrosomonas spp. That inhabit different sediment environments in an oligotrophic freshwater lake.

The population of ammonia-oxidizing bacteria in a temperate oligotrophic freshwater lake was analyzed by recovering 16S ribosomal DNA (rDNA) from lakewater and sediment samples taken throughout a seasonal cycle. Nitrosospira and Nitrosomonas 16S rRNA genes were amplified in a nested PCR, and the identity of the products was confirmed by oligonucleotide hybridization. Nitrosospira DNA was readily identified in all samples, and nitrosomonad DNA of the Nitrosomonas europaea-Nitrosomonas eutropha lineage was also directly detected, but during the summer months only. Phylogenetic delineation with partial (345 bp) 16S rRNA gene sequences of clones obtained from sediments confirmed the fidelity of the amplified nitrosomonad DNA and identified two sequence clusters closely related to either N. europaea or N. eutropha that were equated with the littoral and profundal sediment sites, respectively. Determination of 701-bp sequences for 16S rDNA clones representing each cluster confirmed this delineation. A PCR-restriction fragment length polymorphism (RFLP) system was developed that enabled identification of clones containing N. europaea and N. eutropha 16S rDNA sequences, including subclasses therein. It proved possible to analyze 16S rDNA amplified directly from sediment samples to determine the relative abundance of each species compared with that of the other. N. europaea and N. eutropha are very closely related, and direct evidence for their presence in lake systems is limited. The correlation of each species with a distinct spatial location in sediment is an unusual example of niche adaptation by two genotypically similar bacteria. Their occurrence and relative distribution can now be routinely monitored in relation to environmental variation by the application of PCR-RFLP analysis.

Natural communities of Achromatium oxaliferum comprise genetically, morphologically, and ecologically distinct subpopulations.

The diversity and ecology of natural communities of the uncultivated bacterium Achromatium oxaliferum were studied by use of culture-independent approaches. 16S rRNA gene sequences were PCR amplified from DNA extracted from highly purified preparations of cells that were morphologically identified as A. oxaliferum present in freshwater sediments from three locations in northern England (Rydal Water, Jenny Dam, Hell Kettles). Cloning and sequence analysis of the PCR-amplified 16S rRNA genes revealed that multiple related but divergent sequences were routinely obtained from the A. oxaliferum communities present in all the sediments examined. Whole-cell in situ hybridization with combinations of fluorescence-labelled oligonucleotide probes revealed that the divergent sequences recovered from purified A. oxaliferum cells corresponded to genetically distinct Achromatium subpopulations. Analysis of the cell size distribution of the genetically distinct subpopulations demonstrated that each was also morphologically distinct. Furthermore, there was a high degree of endemism in the Achromatium sequences recovered from different sediments; identical sequences were never recovered from different sampling locations. In addition to ecological differences that were apparent between Achromatium communities from different freshwater sediments, the distribution of different subpopulations of Achromatium in relation to sediment redox profiles indicated that the genetically and morphologically distinct organisms that coexisted in a single sediment were also ecologically distinct and were adapted to different redox conditions. This result suggests that Achromatium populations have undergone adaptive radiation and that the divergent Achromatium species occupy different niches in the sediments which they inhabit.

Substrate uptake by uncultured bacteria from the genus Achromatium determined by microautoradiography.

Microautoradiography was used to investigate substrate uptake by natural communities of uncultured bacteria from the genus Achromatium. Studies of the uptake of (14)C-labelled substrates demonstrated that Achromatium cells from freshwater sediments were able to assimilate (14)C from bicarbonate, acetate, and protein hydrolysate; however, (14)C-labelled glucose was not assimilated. The pattern of substrate uptake by Achromatium spp. was therefore similar to those of a number of other freshwater and marine sulfur-oxidizing bacteria. Different patterns of radiolabelled bicarbonate uptake were noted for Achromatium communities from different geographical locations and indicated that one community (Rydal Water) possessed autotrophic potential, while the other (Hell Kettles) did not. Furthermore, the patterns of organic substrate uptake within a single population suggested that physiological diversity existed in natural communities of Achromatium. These observations are consistent with and may relate to the phylogenetic diversity observed in Achromatium communities. Incubation of Achromatium-bearing sediment cores from Rydal Water with (35)S-labelled sulfate in the presence and absence of sodium molybdate demonstrated that this bacterial population was capable of oxidizing sulfide to intracellular elemental sulfur. This finding supported the role of Achromatium in the oxidative component of a tightly coupled sulfur cycle in Rydal Water sediment. The oxidation of sulfide to sulfur and ultimately to sulfate by Achromatium cells from Rydal Water sediment is consistent with an ability to conserve energy from sulfide oxidation.

Detection, distribution and probable fate of Escherichia coli O157 from asymptomatic cattle on a dairy farm.

The use of commercial anti-Escherichia coli O157-labelled magnetic beads was investigated to improve detection of E. coli O157 by immunomagnetic separation (IMS) from a range of environments on a dairy farm. Immunomagnetic separation proved effective for separation of target cells from laboratory mixtures and during stress in sterile and non-sterile pond water. The IMS procedure was possible with a range of samples (water, faeces, slurry, grass and soil). Non-specific binding of non-target bacterial cells proved problematic in a number of sample types. However, indigenous E. coli O157 cells were detected from samples with a high faecal load, and only with use of IMS. Data on the probable survival and spread of the organism around the farm environment are also discussed.

Ecophysiological Evidence that Achromatium oxaliferum Is Responsible for the Oxidation of Reduced Sulfur Species to Sulfate in a Freshwater Sediment.

Achromatium oxaliferum is a large, morphologically conspicuous, sediment-dwelling bacterium. The organism has yet to be cultured in the laboratory, and very little is known about its physiology. The presence of intracellular inclusions of calcite and sulfur have given rise to speculation that the bacterium is involved in the carbon and sulfur cycles in the sediments where it is found. Depth profiles of oxygen concentration and A. oxaliferum cell numbers in a freshwater sediment revealed that the A. oxaliferum population spanned the oxic-anoxic boundary in the top 3 to 4 cm of sediments. Some of the A. oxaliferum cells resided at depths where no oxygen was detectable, suggesting that these cells may be capable of anaerobic metabolism. The distributions of solid-phase and dissolved inorganic sulfur species in the sediment revealed that A. oxaliferum was most abundant where sulfur cycling was most intense. The sediment was characterized by low concentrations of free sulfide. However, a comparison of sulfate reduction rates in sediment cores incubated with either oxic or anoxic overlying water indicated that the oxidative and reductive components of the sulfur cycle were tightly coupled in the A. oxaliferum-bearing sediment. A positive correlation between pore water sulfate concentration and A. oxaliferum numbers was observed in field data collected over an 18-month period, suggesting a possible link between A. oxaliferum numbers and the oxidation of reduced sulfur species to sulfate. The field data were supported by laboratory incubation experiments in which sodium molybdate-treated sediment cores were augmented with highly purified suspensions of A. oxaliferum cells. Under oxic conditions, rates of sulfate production in the presence of sodium molybdate were found to correlate strongly with the number of cells added to sediment cores, providing further evidence for a role for A. oxaliferum in the oxidation of reduced sulfur.

Survival of cells and DNA of Aeromonas salmonicida released into aquatic microcosms.

The survival of the bacterial fish pathogen Aeromonas salmonicida, and persistence of its DNA, were monitored in aquatic microcosms using selective culture and most probable number PCR. Bacterial cells and naked DNA were released into natural non-sterile microcosms consisting of lake sediment overlayered with lake water. Two different types of surface sediment were used. One was sandy in character, taken from the shoreline whilst the other was a littoral loamy surface mud. Inoculated cells and naked DNA became undetectable from water overlayers within 4 weeks of release. Colony counts of Aer. salmonicida declined below detectable limits after 4 weeks in loamy sediment or 7 weeks in sandy sediment; however, naked DNA and DNA from released cells remained detectable for more than 13 weeks.

The phylogenetic position and ultrastructure of the uncultured bacterium Achromatium oxaliferum.

Achromatium oxaliferum is a large, morphologically conspicuous, sediment-dwelling bacterium. Nothing is known concerning its phylogeny and it has eluded all attempts at laboratory cultivation. The limited physiological description of A. oxaliferum has been based on morphological features of the bacterium such as the presence of intracellular sulphur inclusions. A. oxaliferum cells were purified from a wetland region close to Rydal Water (Cumbria, UK). Scanning and transmission electron microscopy revealed that a number of morphologically distinct A. oxaliferum cell-types, based on cell surface features and the size and abundance of calcite and sulphur inclusions within the cells, were present in a single sample of purified cells. PCR was used to amplify almost full-length 16S rRNA gene sequences from DNA extracted from A. oxaliferum cells directly purified from sediments. The PCR products were cloned and partial sequences (approx. 400 bp) were determined for seven of the clones. Three different sequence clusters were recovered from the clone libraries. A near full-length (1489 bp) 16S rRNA gene sequence was determined for a representative clone of the most dominant sequence-type (52% of the sequences). Comparative sequence analysis showed A. oxaliferum to form a deep branching lineage within the gamma-subdivision of the Proteobacteria. A. oxaliferum was related most closely to the Chromatium assemblage that includes sulphur-oxidizing symbiotic bacteria, purple sulphur bacteria, and sulphur- and iron-oxidizing thiobacilli. Phylogenetic inferences made using distance, parsimony and maximum likelihood methods all placed A. oxaliferum with this group of bacteria. Bootstrap support for a relationship with any particular lineage within the assemblage was weak. The seven clone sequences recovered from the A. oxaliferum cells however formed a monophyletic group well supported by bootstrap analysis (85-100% support depending on the analysis done). It was concluded that A. oxaliferum was related to organisms of the Chromatium assemblage but constituted a novel lineage within this group of bacteria. A. oxaliferum cells were confirmed as the source of the 16S rRNA sequence obtained, by the use of a fluorescently-labelled 16S rRNA-targeted oligonucleotide specific for the A. oxaliferum rRNA sequence.

Isolation and identification of methanogen-specific DNA from blanket bog peat by PCR amplification and sequence analysis.

The presence of methanogenic bacteria was assessed in peat and soil cores taken from upland moors. The sampling area was largely covered by blanket bog peat together with small areas of red-brown limestone and peaty gley. A 30-cm-deep core of each soil type was taken, and DNA was extracted from 5-cm transverse sections. Purified DNA was subjected to PCR amplification with primers IAf and 1100Ar, which specifically amplify 1.1 kb of the archaeal 16S rRNA gene, and ME1 and ME2, which were designed to amplify a 0.75-kb region of the alpha-subunit gene for methyl coenzyme M reductase (MCR). Amplification with both primer pairs was obtained only with DNA extracted from the two deepest sections of the blanket bog peat core. This is consistent with the notion that anaerobiosis is required for activity and survival of the methanogen population. PCR products from both amplifications were cloned, and the resulting transformants were screened with specific oligonucleotide probes internal to the MCR or archaeal 16S rRNA PCR product. Plasmid DNA was extracted from probe-positive clones of both types and the insert was sequenced. The DNA sequences of 8 MCR clones were identical, as were those of 16 of the 17 16S rRNA clones. One clone showed marked variation from the remainder in specific regions of the sequence. From a comparison of these two different 16S rRNA sequences, an oligonucleotide was synthesized that was 100% homologous to a sequence region of the first 16 clones but had six mismatches with the variant. This probe was used to screen primary populations of PCR clones, and all of those that were probe negative were checked for the presence of inserts, which were then sequenced. By using this strategy, further novel methanogen 16S rRNA variants were identified and analyzed. The sequences recovered from the peat formed two clusters on the end of long branches within the methanogen radiation that are distinct from each other. These cannot be placed directly with sequences from any cultured taxa for which sequence information is available.