Identification of the Causative Bacteria in Musculoskeletal Infections Using 16s rDNA - Denaturing Gradient Gel Electrophoresis Analysis

Musculoskeletal infections are infections of the bone and surrounding tissues. They are currently diagnosed based on culture analysis, which is the gold standard for pathogen identification. However, these clinical laboratory methods are frequently inadequate for the identification of the causative agents, because a large percentage (25-50%) of confirmed musculoskeletal infections are false negatives in which no pathogen is identified in culture. My data supports these results. The goal of this project was to use PCR amplification of a portion of the 16S rRNA gene to test an alternative approach for the identification of these pathogens and to assess the diversity of the bacteria involved. The advantages of this alternative method are that it should increase sample sensitivity and the speed of detection. In addition, bacteria that are non-culturable or in low abundance can be detected using this molecular technique. However, a complication of this approach is that the majority of musculoskeletal infections are polymicrobial, which prohibits direct identification from the infected tissue by DNA sequencing of the initial 16S rDNA amplification products. One way to solve this problem is to use denaturing gradient gel electrophoresis (DGGE) to separate the PCR products before DNA sequencing. Denaturing gradient gel electrophoresis (DGGE) separates DNA molecules based on their melting point, which is determined by their DNA sequence. This analytical technique allows a mixture of PCR products of the same length that electrophoreses through agarose gels as one band, to be separated into different bands and then used for DNA sequence analysis. In this way, the DGGE allows for the identification of individual bacterial species in polymicrobial-infected tissue, which is critical for improving clinical outcomes. By combining the 16S rDNA amplification and the DGGE techniques together, an alternative approach for identification has been used. The 16S rRNA gene PCR-DGGE method includes several critical steps: DNA extraction from tissue biopsies, amplification of the bacterial DNA, PCR product separation by DGGE, amplification of the gel-extracted DNA, and DNA sequencing and analysis. Each step of the method was optimized to increase its sensitivity and for rapid detection of the bacteria present in human tissue samples. The limit of detection for the DNA extraction from tissue was at least 20 Staphylococcus aureus cells and the limit of detection for PCR was at least 0.05 pg of template DNA. The conditions for DGGE electrophoreses were optimized by using a double gradient of acrylamide (6 – 10%) and denaturant (30-70%), which increased the separation between distinct PCR products. The use of GelRed (Biotium) improved the DNA visualization in the DGGE gel. To recover the DNA from the DGGE gels the gel slices were excised, shredded in a bead beater, and the DNA was allowed to diffuse into sterile water overnight. The use of primers containing specific linkers allowed the entire amplified PCR product to be sequenced and then analyzed. The optimized 16S rRNA gene PCR-DGGE method was used to analyze 50 tissue biopsy samples chosen randomly from our collection. The results were compared to those of the Memorial Hermann Hospital Clinical Microbiology Laboratory for the same samples. The molecular method was congruent for 10 of the 17 (59%) culture negative tissue samples. In 7 of the 17 (41%) culture negative the molecular method identified a bacterium. The molecular method was congruent with the culture identification for 7 of the 33 (21%) positive cultured tissue samples. However, in 8 of the 33 (24%) the molecular method identified more organisms. In 13 of the 15 (87%) polymicrobial cultured tissue samples the molecular method identified at least one organism that was also identified by culture techniques. Overall, the DGGE analysis of 16S rDNA is an effective method to identify bacteria not identified by culture analysis.

Molecular characterization of a gene required for entry into S phase of the mitotic cell cycle in the yeast {\it Saccharomyces cerevisiae\/}

A strain of Saccaromyces cerevisiae (SC3B) with a temperature sensitive defect in the synthesis of DNA has been isolated. This defect is due to a single recessive mutation in a gene named INS1 required for the initiation of S phase. Arrested cells carrying the ins1$\sp{ts}$ allele are defective in the completion of G1 to S phase transition events including SPB duplication or separation, initiation of DNA synthesis, normal control of budding, and bud neck stability. The mutation and a gene which complements the mutation were mapped to chromosome IV. The complementing gene was proved to be the wild type allele of the temperature sensitive mutation by genetic linkage of an integrated clone. A very low abundance 4.2 kb RNA message was observed in the strain SC3B which increased greatly in this strain transformed with a multiple copy plasmid carrying the complementing clone. The wild type gene was sequenced and found to encode a 1268 amino acid protein of with a molecular weight of 142,655 Daltons. Computer assisted searches for similar DNA sequences revealed no significant homology matches. However, searches for protein sequence homology revealed a protein (the DIS3 gene product of S. pombe) with a similar sequence over a 534 amino acid stretch to the predicted INS1 gene product. A later search revealed a near identical sequence for a gene (SRK1) also isolated from S. cerevisiae. ^

Palynological richness and evenness: insights from the taxa accumulation curve

Palynology provides the opportunity to make inferences on changes in diversity of terrestrial vegetation over long time scales. The often coarse taxonomic level achievable in pollen analysis, differences in pollen production and dispersal, and the lack of pollen source boundaries hamper the application of diversity indices to palynology. Palynological richness, the number of pollen types at a constant pollen count, is the most robust and widely used diversity indicator for pollen data. However, this index is also influenced by the abundance distribution of pollen types in sediments. In particular, where the index is calculated by rarefaction analysis, information on taxonomic richness at low abundance may be lost. Here we explore information that can be extracted from the accumulation of taxa over consecutive samples. The log-transformed taxa accumulation curve can be broken up into linear sections with different slope and intersect parameters, describing the accumulation of new taxa within the section. The breaking points may indicate changes in the species pool or in the abundance of high versus low pollen producers. Testing this concept on three pollen diagrams from different landscapes, we find that the break points in the taxa accumulation curves provide convenient zones for identifying changes in richness and evenness. The linear regressions over consecutive samples can be used to inter- and extrapolate to low or extremely high pollen counts, indicating evenness and richness in taxonomic composition within these zones. An evenness indicator, based on the rank-order-abundance is used to assist in the evaluation of the results and the interpretation of the fossil records. Two central European pollen diagrams show major changes in the taxa accumulation curves for the Lateglacial period and the time of human induced land-use changes, while they do not indicate strong changes in the species pool with the onset of the Holocene. In contrast, a central Swedish pollen diagram shows comparatively little change, but high richness during the early Holocene forest establishment. Evenness and palynological richness are related for most periods in the three diagrams, however, sections before forest establishment and after forest clearance show high evenness, which is not necessarily accompanied by high palynological richness, encouraging efforts to separate the two.

Isolation of an active gene and of two pseudogenes for mouse U7 small nuclear RNA

Three U7 RNA-related sequences were isolated from mouse genomic DNA libraries. Only one of the sequences completely matches the published mouse U7 RNA sequence, whereas the other two apparently represent pseudogenes. The matching sequence represents a functional gene, as it is expressed after microinjection into Xenopus laevis oocytes. Sequence variations of the conserved cis-acting 5' and 3' elements of U RNA genes may partly explain the low abundance of U7 RNA.

Investigation of Quaternary diatoms in sediment core CRP-1 from the Ross Sea, Antarctica

In the first season of drilling, the Cape Roberts Project (CRP) recovered one drillcore (CRP-l) from Roberts Ridge in western McMurdo Sound, Ross Sea, Antarctica Diatom biostratigraphy places the upper six lithostratigraphic units (Units 1.1, 2.1, 2.2, 2.3, 3.1, and 4.1) of CRP-l (0.0 to 43.15 mbsf) within the Quaternary. Both non-marine and marine Quaternary diatoms occur in variable abundance in the Quaternary interval of CRP- 1 Biostratigraphic data resolve two Quaternary time slices or events within CRP-1. Marine diatom assemblages in Units 4.1 and 3.1 represent sedimentation within the diatom Actinocyclus ingens Zone (1.35 to 0.66 Ma). Further refinement of the age of Unit 3.l places deposition in the interval 1.15 to 0.75 Ma based on the common occurrence of Thalassiosira elliptipora and correlation to the Southern Ocean acme of this taxon The absence of ActiActinocyclus ingens and the presence ot Thalassiosira antarctica in Unit 2.2 require a younger zonal assignment for this interval, within the diatom Thalassiosira lentiginosa Zone (0.66 to 0.0 Ma). A new diatom species. Rouxia leventerae, is described from marine assemblages of Units 2.2, 2.3, 3.1, and 4.l. Lithostratigraphic Unit 3.1 (33.82 to 31.89 mbsf) is a bryozoan-dominated skeletal-carbonate facies. Low abundance of Fragilariopsis curta and Fragilariopsis cylindrus within this unit combined with the relatively high abundance of species associated with open water indicates deposition in waters that remained ice free for much or all of the year Diatom assemblages suggest carbonate deposition in Unit 3.1 is linked to a significant early Pleistocene event in McMurdo Sound, when elevated surface-water temperatures inhibited the formation of sea ice.

(Table S2) Cell concentrations of individual and combined Alexandrium spp. and phylogenetic groups as obtained by qPCR assay and Utermöhl counts, as well as particulate PSP toxin concentrations from discrete water depths

Molecular methods provide promising tools for routine detection and quantification of toxic microalgae in plankton samples. To this end, novel TaqMan minor groove binding probes and primers targeting the small (SSU) or large (LSU) ribosomal subunit (rRNA) were developed for two species of the marine dinoflagellate genus Alexandrium (A. minutum, A. tamutum) and for three groups/ribotypes of the A. tamarense species complex: Group I/North American (NA), Group II/Mediterranean (ME) and Group III/Western European (WE). Primers and probes for real-time quantitative PCR (qPCR) were species-specific and highly efficient when tested in qPCR assays for cross-validation with pure DNA from cultured Alexandrium strains. Suitability of the qPCR assays as molecular tools for the detection and estimation of relative cell abundances of Alexandrium species and groups was evaluated from samples of natural plankton assemblages along the Scottish east coast. The results were compared with inverted microscope cell counts (Utermöhl technique) of Alexandrium spp. and associated paralytic shellfish poisoning (PSP) toxin concentrations. The qPCR assays indicated that A. tamarense (Group I) and A. tamutum were the most abundant Alexandrium taxa and both were highly positively correlated with PSP toxin content of plankton samples. Cells of A. tamarense (Group III) were present at nearly all stations but in low abundance. Alexandrium minutum and A. tamarense (Group II) cells were not detected in any of the samples, thereby arguing for their absence from the specific North Sea region, at least at the time of the survey. The sympatric occurrence of A. tamarense Group I and Group III gives further support to the hypothesis that the groups/ribotypes of the A. tamarense species complex are cryptic species rather than variants belonging to the same species.

Benthic and planktonic foraminifera in the Japan Sea

Biostratigraphy and paleoenvironmental history of deep and surficial waters of the Japan Sea are addressed using sequences recovered from the floor of the backarc basin. The study is divided into two parts: (1) foraminifer biostratigraphy and paleoenvironmental assessment of sedimentary sequences recovered from above igneous basement at the four sites and (2) detailed planktonic foraminifer paleoenvironmental analysis of Quaternary and Pliocene sequences from Sites 794 and 797 in the Yamato Basin. A total of 253 samples were examined for the foraminifer biostratigraphy and 325 samples for the detailed paleoenvironmental study of Quaternary and Pliocene sequences. Low abundance and sporadic occurrence of foraminifers limited interpretation of results. Foraminifer-bearing intervals were correlated where possible to diatom and calcareous nannofossil zonations, and the sequences were successfully assigned to the foraminifer zonation of Matsunaga. Unfortunately, extensive barren intervals and sporadic occurrences of planktonic foraminifers prevented zonation of Quaternary and Pliocene intervals, although some interesting conclusions about paleoenvironment were possible and are listed below. A sequence of Neogene (sensu lato) paleoenvironmental events were identified: (1) deepening of the Yamato basins to middle bathyal depths by the early to middle Miocene, an event contemporaneous with the age of some deep basins known from uplifted sections adjacent to the Japan Basin; (2) cooling of the Japan Sea in the early middle Miocene; (3) oxygenation of deep waters in the late Miocene; (4) further cooling of surficial water masses between the Olduvai Subchron and the Brunhes/Matuyama Boundary; and (5) extermination of lower middle bathyal faunas and replacement by upper middle bathyal faunas near the base of the Quaternary.

Contusotruncana contusa abundances and morphotypes in the cretaceous mid-latitude South Atlantic

Spatial and temporal patterns in test size and shape (test conicity and spiral roundness) and absolute abundance (accumulation rate) of the planktonic foraminifer Contusotruncana contusa were studied in the South Atlantic Ocean (DSDP sites 356, 516, 525 and 527) during an interval corresponding to the last 800 kyr of the Cretaceous. The variation in absolute abundance of C. contusa was characterised by alternating periods of high and low abundance; some of these periods were traceable across the entire mid-latitude South Atlantic Ocean. While the mean spiral roundness did not show any interpretable patterns, a sudden increase of the mean test size and mean test conicity occurred between 65.3 and 65.2 Ma (based on linear interpolation within the Cretaceous part of Subchron C29R) at all sites studied, indicating a poleward migration followed by rapid withdrawal of the low-latitude C. contusa morphotypes from the mid-latitude South Atlantic Ocean. We suggest that this event was caused by a short period of surface-water warming in the southern mid-latitudes corresponding to the brief high-latitude warming event and associated faunal migrations in the Boreal and Austral realms.

Lower to middle Miocene calcareous nannofossil events from the Ontong Java Plateau

Uppermost Oligocene through middle Miocene calcareous nannofossil events that were considered potentially useful from a biostratigraphic point of view have been investigated from Ocean Drilling Program Sites 806 and 807 in the western equatorial Pacific Ocean. Comparisons have been made to the corresponding events from other equatorial regions and the mid-latitude North Atlantic. In terms of biostratigraphic reliability, defined by the ability of the pertinent species to provide distinctive marker events and synchroneity over geographic distance, the investigated events can be classified into four general categories: The good markers: last occurrence (LO) Sphenolithus ciperoensis, first occurrence (FO) S. delphix, LO S. delphix, FO S. belemnos, LO S. belemnos, FO S. heteromorphus, termination acme (TA) Discoaster deflandrei, and LO Sphenolithus heteromorphus. The poor markers: LO Helicosphaera recta, TA Cyclicargolithus abisectus, LO Triquetrorhabdulus carinatus, and FO Calcidiscus macintyrei. Ecologically controlled markers with regional value: LO Dictyococcites bisectus, LO Helicosphaera ampliaperta, FO Reticulofenestra pseudoumbilica, LO Cyclicargolithus floridanus, and LO Coronocyclus nitescens. The low abundance markers: FO Discoaster druggii, gradational form of Sphenoliths dissimilis/Sphenolithus belemnos, FO Triquetrorhabdulus rugosus, and FO T. rioensis.

Diatom assamblage in samples from sediment traps across the Pacific Sector of the Southern Ocean

An array of four sediment trap moorings recorded the particulate flux across the Antarctic Circumpolar Current (ACC) at 170 °W, between November 1996 and January 1998, as part of the US JGOFS-Antarctic Environment and Southern Ocean Process Study (AESOPS) program. The trap locations represent sampling within the Polar Frontal Zone, the Antarctic Polar Front, the Antarctic Zone and the Southern Antarctic Zone. Here we report observations from 1000 m below the sea-surface compared to seafloor and surface water distributions. Sub-sample splits from each trap were obtained and total diatom flux and species composition were determined. The diatom fluxes were quantified using both a dilution and a 'spike' method to allow for the rapid repeatability of measurements. Diatom flux was found to be highly seasonal across the ACC particularly at higher latitudes. Marine snow aggregates of intact diatom cells and chains were the major components of the biogenic flux. Siliceous particle size was noted to decrease with increasing latitude, which could be aligned with a shift of the diatom assemblage to small-size species/sea-ice affiliated species. A 'double-structured' diatom flux was recorded at the location of the Antarctic Polar Front trap, with a shift in the diatom assemblage from larger to smaller diatoms in the second flux episode. The sediment trap assemblage shows deviations from the surface water assemblage, while surface sediment samples indicate that significant dissolution occurs after 1000 m and at the sediment-water interface. Estimation of diatom biovolumes across the ACC shows that large diatoms have the potential to greatly impact biogenic fluxes to the ocean interior despite their low fluxes. Small species of the genus Fragilariopsis could potentially export as much Corg as Fragilariopsis kerguelensis near the retreating ice edge. However, their low abundance in the surface sediments also suggests that these diatoms are a shallow export species.

Environment and habitat characteristics of wintering lemmings in the Arctic

Snow cover has dramatic effects on the structure and functioning of Arctic ecosystems in winter. In the tundra, the subnivean space is the primary habitat of wintering small mammals and may be critical for their survival and reproduction. We have investigated the effects of snow cover and habitat features on the distributions of collared lemming (Dicrostonyx groenlandicus) and brown lemming (Lemmus trimucronatus) winter nests, as well as on their probabilities of reproduction and predation by stoats (Mustela erminea) and arctic foxes (Vulpes lagopus). We sampled 193 lemming winter nests and measured habitat features at all of these nests and at random sites at two spatial scales. We also monitored overwinter ground temperature at a subsample of nest and random sites. Our results demonstrate that nests were primarily located in areas with high micro-topography heterogeneity, steep slopes, deep snow cover providing thermal protection (reduced daily temperature fluctuations) and a high abundance of mosses. The probability of reproduction increased in collared lemming nests at low elevation and in brown lemming nests with high availability of some graminoid species. The probability of predation by stoats was density dependent and was higher in nests used by collared lemmings. Snow cover did not affect the probability of predation of lemming nests by stoats, but deep snow cover limited predation attempts by arctic foxes. We conclude that snow cover plays a key role in the spatial structure of wintering lemming populations and potentially in their population dynamics in the Arctic.

Miocene radiolarians of the Sulu Sea

Radiolarians are sporadic in sediments collected in the Sulu Sea during ODP Leg 124. Due to the generally poor preservation and low abundance of radiolarians in Sulu Sea sediments, no biostratigraphic datums are well defined, although three radiolarian zones are identified. Most samples containing radiolarians are pelagic or hemipelagic clays with varying proportions of volcanic ash. Detailed analysis of Sulu Sea radiolarians was limited to Miocene successions. Pliocene and Quaternary occurrences of radiolarians were noted but have not been zoned. The late middle Miocene of Sites 769 and 771 is represented by an assemblage of radiolarians (Diartus petterssoni Zone) that is entirely replaced by massive pyrite. This type of preservation develops only under anoxic conditions. The development of widespread anoxia in Sulu Sea waters in the late middle Miocene was probably the result of hydrologic isolation of basin waters, and may be associated with eustatic sea level fall over the silled basin. Upper lower Miocene pelagic and hemipelagic sediments that overlie pyroclastics and basalt flows in the Sulu Sea sites contain moderately to very poorly preserved radiolarians of the Calocycletta costata Zone. A thin unit of marine claystone was recovered from between the thick pyroclastics and basement rocks at Site 768. Radiolarians present in these claystones are rare and very poorly preserved. This radiolarian assemblage probably represents the C. costata Zone, although very poor preservation and low abundance make this interpretation equivocal. The radiolarian zones identified constrain the age of basin formation to late early Miocene or earlier.

Diatom stratigraphy of ODP Leg 150 holes

Diatoms are present in middle to lower upper Miocene sections of all holes examined during Leg 150, but are generally absent or in low abundance in Pleistocene to middle upper Miocene sediments. An exception is the alternating diatom-rich, diatom-poor intervals in upper Quaternary sediments. Five new diatom zones, covering an interval from near the lower/middle Miocene boundary to the lower upper Miocene, are proposed. Some of the taxon used to define these zones are also used in zonal schemes for the East Coast of the United States, and allow for correlations to be drawn between this region and Leg 150 sites. Lower Miocene and older levels are not included in this study. Although older Tertiary diatoms are present at some of the sites, dissolution has largely compromised their usefulness as zonal markers.

Pigments, phytoplankton and its production parameters in the Obskaya Guba and adjacent Kara Sea in the latest September 2007

Material was collected in the Ob River estuary and the adjacent shallow Kara Sea shelf between 71°14.0'N and 75°33.0'N at the end of September 2007. Latitudinal zonation in phytoplankton distribution was demonstrated; this zonation was determined by changes in salinity and concentration of nutrients. Characteristic of the phytocenosis in the southern desalinated zone composed of freshwater diatom and green algae species were high population density (1500000 cells/l), biomass (210 ?g C/l), chlorophyll concentration (4.5 ?g/l), and uniform distribution in the water column. High primary production (~40 ?g C/l/day) was recorded in the upper 1.5 m layer. The estuarine frontal zone located to the north had a halocline at depth 3-5 m. Freshwater species with low abundance (250000 cells/l), biomass (24 ?g C/l), and chlorophyll concentration (1.5 ?g/l) dominated above the halocline. Marine diatom algae, dinoflagellates, and autotrophic flagellates formed a considerable part of the phytocenosis below the halocline; community characteristics were two-fold lower as compared with the upper layer. Maximal values of primary production (~10 ?g C/l/day) were recorded in the upper 1.5 m layer. The phytocenosis in the seaward zone was formed by marine alga species and was considerably poorer as compared with the frontal zone. Assimilation rates of carbon per chlorophyll a at the end of the vegetation season within the studied area were low, average 0.4-1.0 ?g C/?g Chl/hour in the upper layer and 0.03-0.1 ?g C/?g Chl/hour below the pycnocline.

Miocene through Pleistocene dinoflagellates from ODP Site 170-1039

Palynological data from offshore Costa Rica, allow us to investigate the relationship between dinoflagellate cyst assemblages and changes in regional oceanic primary productivity. From Miocene to Pleistocene, productivity at ODP Site 1039 was influenced by tectonic drift, as Site 1039 approached the continent, from the Equator to its current position at ~10°N. In addition, dinoflagellate abundance is modulated by regional productivity events, which modified primary productivity, as also indicated by available data on calcareous nannofossils, diatoms, TOC, and CaCO3 content. Five palynomorph intervals are defined. The early-late Miocene one, dominated by Batiacasphaera, represents relatively stable, productive oceanic conditions before the closure of the Indonesian and Panama Seaways. The late Miocene decrease in palynomorph recovery is related to the Carbonate Crash Event. The high abundance and diversity of the assemblages at the end of the late Miocene to early Pliocene indicate increased productivity related to the Global Biogenic Bloom, and a change in dominance from Batiacasphaera to Impagidinium to Nematosphaeropsis. The low abundance of the late Pliocene interval is related to El Niño-like conditions, and there is another change related to the disappearance of Batiacasphaera and dominance of Impagidinium, Nematosphaeropsis, and Operculodinium. The abundant Pleistocene assemblages represent increased marine productivity, and a high influx of continental palynomorphs and bissacate pollen, associated with the proximity of the Costa Rica Dome. Pleistocene dinoflagellates are characterized by Spiniferites and Selenopemphix, together with rare Impagidinium and Nematosphaeropsis.