Joint Position Statement on Degree Requirements for Paramedics.

The National Association of EMS Educators, the National EMS Management Association, and the International Association of Flight and Critical Care Paramedics believe the time has come for paramedics to be trained through a formal education process that culminates with an associate degree. Once implemented a degree requirement will improve the care delivered by paramedics and enhance paramedicine as a heath profession.

IMG/VR: a database of cultured and uncultured DNA Viruses and retroviruses.

Viruses represent the most abundant life forms on the planet. Recent experimental and computational improvements have led to a dramatic increase in the number of viral genome sequences identified primarily from metagenomic samples. As a result of the expanding catalog of metagenomic viral sequences, there exists a need for a comprehensive computational platform integrating all these sequences with associated metadata and analytical tools. Here we present IMG/VR (https://img.jgi.doe.gov/vr/), the largest publicly available database of 3908 isolate reference DNA viruses with 264 413 computationally identified viral contigs from >6000 ecologically diverse metagenomic samples. Approximately half of the viral contigs are grouped into genetically distinct quasi-species clusters. Microbial hosts are predicted for 20 000 viral sequences, revealing nine microbial phyla previously unreported to be infected by viruses. Viral sequences can be queried using a variety of associated metadata, including habitat type and geographic location of the samples, or taxonomic classification according to hallmark viral genes. IMG/VR has a user-friendly interface that allows users to interrogate all integrated data and interact by comparing with external sequences, thus serving as an essential resource in the viral genomics community.

Roles of Thermophiles and Fungi in Bitumen Degradation in Mostly Cold Oil Sands Outcrops.

Oil sands are surface exposed in river valley outcrops in northeastern Alberta, where flat slabs (tablets) of weathered, bitumen-saturated sandstone can be retrieved from outcrop cliffs or from riverbeds. Although the average yearly surface temperature of this region is low (0.7°C), we found that the temperatures of the exposed surfaces of outcrop cliffs reached 55 to 60°C on sunny summer days, with daily maxima being 27 to 31°C. Analysis of the cooccurrence of taxa derived from pyrosequencing of 16S/18S rRNA genes indicated that an aerobic microbial network of fungi and hydrocarbon-, methane-, or acetate-oxidizing heterotrophic bacteria was present in all cliff tablets. Metagenomic analyses indicated an elevated presence of fungal cytochrome P450 monooxygenases in these samples. This network was distinct from the heterotrophic community found in riverbeds, which included fewer fungi. A subset of cliff tablets had a network of anaerobic and/or thermophilic taxa, including methanogens, Firmicutes, and Thermotogae, in the center. Long-term aerobic incubation of outcrop samples at 55°C gave a thermophilic microbial community. Analysis of residual bitumen with a Fourier transform ion cyclotron resonance mass spectrometer indicated that aerobic degradation proceeded at 55°C but not at 4°C. Little anaerobic degradation was observed. These results indicate that bitumen degradation on outcrop surfaces is a largely aerobic process with a minor anaerobic contribution and is catalyzed by a consortium of bacteria and fungi. Bitumen degradation is stimulated by periodic high temperatures on outcrop cliffs, which cause significant decreases in bitumen viscosity.

Paramedic specialization: a strategy for better out-of-hospital care.

Demographic, economic, and political forces are driving significant change in the US health care system. Paramedics are a health profession currently providing advanced emergency care and medical transportation throughout the United States. As the health care system demands more team-based care in nonacute, community, interfacility, and tactical response settings, specialized paramedic practitioners could be a valuable and well-positioned resource to meet these needs. Currently, there is limited support for specialty certifications that demand appropriate education, training, or experience standards before specialized practice by paramedics. A fragmented approach to specialty paramedic practice currently exists across our country in which states, regulators, nonprofit organizations, and other health care professions influence and regulate the practice of paramedicine. Multiple other medical professions, however, have already developed effective systems over the last century that can be easily adapted to the practice of paramedicine. Paramedicine practitioners need to organize a profession-based specialty board to organize and standardize a specialty certification system that can be used on a national level.

Microbial community and potential functional gene diversity involved in anaerobic hydrocarbon degradation and methanogenesis in an oil sands tailings pond.

Oil sands tailings ponds harbor large amounts of tailings resulting from surface mining of bitumen and consist of water, sand, clays, residual bitumen, and hydrocarbon diluent. Oxygen ingress in these ponds is limited to the surface layers, causing most hydrocarbon degradation to be catalyzed by anaerobic, methanogenic microbial communities. This causes the evolution of large volumes of methane of up to 10(4) m(3)/day. A pyrosequencing survey of 16S rRNA amplicons from 10 samples obtained from different depths indicated the presence of a wide variety of taxa involved in anaerobic hydrocarbon degradation and methanogenesis, including the phyla Proteobacteria, Euryarchaeota, Firmicutes, Actinobacteria, Chloroflexi, and Bacteroidetes. Metagenomic sequencing of DNA isolated from one of these samples indicated a more diverse community than indicated by the 16S rRNA amplicon survey. Both methods indicated the same major phyla to be present. The metagenomic dataset indicated the presence of genes involved in the three stages of anaerobic aromatic hydrocarbon degradation, including genes for enzymes of the peripheral (upper), the central (lower), and the methanogenesis pathways. Upper pathway genes showed broad phylogenetic affiliation (Proteobacteria, Firmicutes, and Actinobacteria), whereas lower pathway genes were mostly affiliated with the Deltaproteobacteria. Genes for both hydrogenotrophic and acetotrophic methanogenesis were also found. The wide variety of taxa involved in initial hydrocarbon degradation through upper pathways may reflect the variety of residual bitumen and diluent components present in the tailings pond.

Metagenomics of hydrocarbon resource environments indicates aerobic taxa and genes to be unexpectedly common.

Oil in subsurface reservoirs is biodegraded by resident microbial communities. Water-mediated, anaerobic conversion of hydrocarbons to methane and CO2, catalyzed by syntrophic bacteria and methanogenic archaea, is thought to be one of the dominant processes. We compared 160 microbial community compositions in ten hydrocarbon resource environments (HREs) and sequenced twelve metagenomes to characterize their metabolic potential. Although anaerobic communities were common, cores from oil sands and coal beds had unexpectedly high proportions of aerobic hydrocarbon-degrading bacteria. Likewise, most metagenomes had high proportions of genes for enzymes involved in aerobic hydrocarbon metabolism. Hence, although HREs may have been strictly anaerobic and typically methanogenic for much of their history, this may not hold today for coal beds and for the Alberta oil sands, one of the largest remaining oil reservoirs in the world. This finding may influence strategies to recover energy or chemicals from these HREs by in situ microbial processes.

Oxygen exposure increases resistance of Desulfovibrio vulgaris Hildenborough to killing by hydrogen peroxide.

Inactivation of PerR by oxidative stress and a corresponding increase in expression of the perR regulon genes is part of the oxidative stress defense in a variety of anaerobic bacteria. Diluted anaerobic, nearly sulfide-free cultures of mutant and wild-type Desulfovibrio vulgaris (10(5)-10(6) colony-forming units/ml) were treated with 0 to 2,500 µM H(2)O(2) for only 5 min to prevent readjustment of gene expression. Survivors were then scored by plating. The wild type and perR mutant had 50% survival at 58 and 269 µM H(2)O(2), respectively, indicating the latter to be 4.6-fold more resistant to killing by H(2)O(2) under these conditions. Significantly increased resistance of the wild type (38-fold; 50% killing at 2188 µM H(2)O(2)) was observed if cells were pretreated with full air for 30 min, conditions that did not affect cell viability. The resistance of the perR mutant increased less (4.6-fold; 50% killing at 1230 µM H(2)O(2)), when similarly pretreated. Interestingly, no increased resistance of either was achieved by exposure with 10.6 µM H(2)O(2) for 30 min, the highest concentration that could be used without killing the cells. Hence, in environments with low D. vulgaris biomass only the presence of external O(2) effectively activates the perR regulon. As a result, mutant strains lacking one of the perR regulon genes ahpC, dvu0772, rbr1 or rbr2 displayed decreased resistance to H(2)O(2) stress only following pretreatment with air.

Effect of sodium bisulfite injection on the microbial community composition in a brackish-water-transporting pipeline.

Pipelines transporting brackish subsurface water, used in the production of bitumen by steam-assisted gravity drainage, are subject to frequent corrosion failures despite the addition of the oxygen scavenger sodium bisulfite (SBS). Pyrosequencing of 16S rRNA genes was used to determine the microbial community composition for planktonic samples of transported water and for sessile samples of pipe-associated solids (PAS) scraped from pipeline cutouts representing corrosion failures. These were obtained from upstream (PAS-616P) and downstream (PAS-821TP and PAS-821LP, collected under rapid-flow and stagnant conditions, respectively) of the SBS injection point. Most transported water samples had a large fraction (1.8% to 97% of pyrosequencing reads) of Pseudomonas not found in sessile pipe samples. The sessile population of PAS-616P had methanogens (Methanobacteriaceae) as the main (56%) community component, whereas Deltaproteobacteria of the genera Desulfomicrobium and Desulfocapsa were not detected. In contrast, PAS-821TP and PAS-821LP had lower fractions (41% and 0.6%) of Methanobacteriaceae archaea but increased fractions of sulfate-reducing Desulfomicrobium (18% and 48%) and of bisulfite-disproportionating Desulfocapsa (35% and 22%) bacteria. Hence, SBS injection strongly changed the sessile microbial community populations. X-ray diffraction analysis of pipeline scale indicated that iron carbonate was present both upstream and downstream, whereas iron sulfide and sulfur were found only downstream of the SBS injection point, suggesting a contribution of the bisulfite-disproportionating and sulfate-reducing bacteria in the scale to iron corrosion. Incubation of iron coupons with pipeline waters indicated iron corrosion coupled to the formation of methane. Hence, both methanogenic and sulfidogenic microbial communities contributed to corrosion of pipelines transporting these brackish waters.

Microbial community succession in a bioreactor modeling a souring low-temperature oil reservoir subjected to nitrate injection.

Injection of up-flow packed-bed bioreactors with excess volatile fatty acids and limiting concentrations of nitrate and sulfate gave complete reduction of nitrate from 0 to 5.5 cm and complete or near-complete reduction of sulfate from 3.2 to 11.5 cm along the bioreactor flow path. Most of the biomass (85%) and most of the genes for nitrate reduction (narG, 96%; napA, 99%) and for sulfate reduction (dsrB, 91%) were present near the inlet (0-5.5 cm) of the 37-cm-long bioreactor. Microbial community analysis by a combination of denaturing gradient gel electrophoresis and pyrosequencing of 16S rRNA amplicons indicated that nitrate-reducing Arcobacter and Pseudomonas species were located from 0 to 3.2 cm and throughout, respectively. Desulfobulbus species were the main sulfate reducers present and acetotrophic methanogens of the genus Methanosaeta predominated at 20-37 cm. Overall, the results indicated a succession of microbial communities along the bioreactor flow path. In the absence of nitrate, the sulfate reduction zone moved nearer to the bioreactor inlet. The sulfide concentration in the bioreactor effluent was temporarily lowered after nitrate injection was re-started. Hence, the bioreactor sulfide output could be disrupted by pulsed, not by constant nitrate injection, as demonstrated also previously in a low-temperature oil field.

Effects of biocides on gene expression in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough.

Although sulfate-reducing bacteria (SRB), such as Desulfovibrio vulgaris Hildenborough (DvH) are often eradicated in oil and gas operations with biocides, such as glutaraldehyde (Glut), tetrakis (hydroxymethyl) phosphonium sulfate (THPS), and benzalkonium chloride (BAC), their response to these agents is not well known. Whole genome microarrays of D. vulgaris treated with biocides well below the minimum inhibitory concentration showed that 256, 96, and 198 genes were responsive to Glut, THPS, and BAC, respectively, and that these three commonly used biocides affect the physiology of the cell quite differently. Glut induces expression of genes required to degrade or refold proteins inactivated by either chemical modification or heat shock, whereas BAC appears to target ribosomal structure. THPS appears to primarily affect energy metabolism of SRB. Mutants constructed for genes strongly up-regulated by Glut, were killed by Glut to a similar degree as the wild type. Hence, it is difficult to achieve increased sensitivity to this biocide by single gene mutations, because Glut affects so many targets. Our results increase understanding of the biocide's mode of action, allowing a more intelligent combination of mechanistically different agents. This can reduce stress on budgets for chemicals and on the environment.

Effect of sulfide on growth physiology and gene expression of Desulfovibrio vulgaris Hildenborough.

Hydrogen sulfide, the metabolic end product of sulfate-reducing bacteria (SRB), is toxic to most life forms. This includes the SRB themselves. Although many of these are probably among the most sulfide resistant life forms, the presence of sulfide nevertheless presents a stress, which SRB must overcome. Although the response of SRB, especially the genus Desulfovibrio, to numerous stressors has been studied, their response to sulfide stress is unknown. We determined the effect of sulfide stress by comparing cells of Desulfovibrio vulgaris Hildenborough grown under conditions in which sulfide accumulated (high sulfide, 10 mM) with cells grown under conditions in which sulfide was removed by continuous gassing (low sulfide, 1 mM). High sulfide decreased the instantaneous growth rate constant and the final cell density of the culture by 52 and 33%, respectively, indicating a decreased bioenergetic fitness. Changes in gene expression caused by exposure to high sulfide were determined using full-genome D. vulgaris microarrays. The transcription of ribosomal protein-encoding genes was decreased, in agreement with the lower growth rate of D. vulgaris under high sulfide conditions. Interestingly, expression of the gene for DsrD, located downstream of the genes for dissimilatory sulfite reductase was also strongly down-regulated. In contrast, the expression of many genes involved in iron accumulation, stress response and proteolysis were increased. This indicates that high sulfide represents a significant stress condition, in which the bioavailability of metals like iron may be lowered. Overall this leads to a reduced growth rate and less efficient biomass production.

A genomic island of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough promotes survival under stress conditions while decreasing the efficiency of anaerobic growth.

A 47 kb genomic island (GEI) bracketed by 50 bp direct repeats, containing 52 annotated genes, was found to delete spontaneously from the genome of Desulfovibrio vulgaris Hildenborough. The island contains genes for site-specific recombinases and transposases, rubredoxin:oxygen oxidoreductase-1 (Roo1) and hybrid cluster protein-1 (Hcp1), which promote survival in air and nitrite stress. The numbering distinguishes these from the Roo2 and Hcp2 homologues for which the genes are located elsewhere in the genome. Cells with and without the island (GEI(+) and GEI(-) cells respectively) were obtained by colony purification. GEI(-) cells arise in anaerobic cultures of colony-purified GEI(+) cells, indicating that the site-specific recombinases encoded by the island actively delete this region. GEI(+) cells survive better in microaerophilic conditions due to the presence of Roo1, whereas the Hcps appear to prevent inhibition by sulfur and polysulfide, which are formed by chemical reaction of sulfide and nitrite. Hence, the island confers resistance to oxygen and nitrite stress. However, GEI(-) cells have a higher growth rate in anaerobic media. Microarrays and enzyme activity stains indicated that the GEI(-) cells have increased expression of genes, which promote anaerobic energy conservation, explaining the higher growth rate. Hence, while lowering the efficiency of anaerobic metabolism, the GEI increases the fitness of D. vulgaris under stress conditions, a feature reminiscent of pathogenicity islands which allow more effective colonization of environments provided by the targeted hosts.

Gene expression by the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough grown on an iron electrode under cathodic protection conditions.

The genome sequence of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough was reanalyzed to design unique 70-mer oligonucleotide probes against 2,824 probable protein-coding regions. These included three genes not previously annotated, including one that encodes a c-type cytochrome. Using microarrays printed with these 70-mer probes, we analyzed the gene expression profile of wild-type D. vulgaris grown on cathodic hydrogen, generated at an iron electrode surface with an imposed negative potential of -1.1 V (cathodic protection conditions). The gene expression profile of cells grown on cathodic hydrogen was compared to that of cells grown with gaseous hydrogen bubbling through the culture. Relative to the latter, the electrode-grown cells overexpressed two hydrogenases, the hyn-1 genes for [NiFe] hydrogenase 1 and the hyd genes, encoding [Fe] hydrogenase. The hmc genes for the high-molecular-weight cytochrome complex, which allows electron flow from the hydrogenases across the cytoplasmic membrane, were also overexpressed. In contrast, cells grown on gaseous hydrogen overexpressed the hys genes for [NiFeSe] hydrogenase. Cells growing on the electrode also overexpressed genes encoding proteins which promote biofilm formation. Although the gene expression profiles for these two modes of growth were distinct, they were more closely related to each other than to that for cells grown in a lactate- and sulfate-containing medium. Electrochemically measured corrosion rates were lower for iron electrodes covered with hyn-1, hyd, and hmc mutant biofilms than for wild-type biofilms. This confirms the importance, suggested by the gene expression studies, of the corresponding gene products in D. vulgaris-mediated iron corrosion.

Phoenix 2: a locally installable large-scale 16S rRNA gene sequence analysis pipeline with Web interface.

We have developed Phoenix 2, a ribosomal RNA gene sequence analysis pipeline, which can be used to process large-scale datasets consisting of more than one hundred environmental samples and containing more than one million reads collectively. Rapid handling of large datasets is made possible by the removal of redundant sequences, pre-partitioning of sequences, parallelized clustering per partition, and subsequent merging of clusters. To build the pipeline, we have used a combination of open-source software tools and custom-developed Perl scripts. For our project we utilize hardware-accelerated searches, but it is possible to reconfigure the analysis pipeline for use with generic computing infrastructure only, with a considerable reduction in speed. The set of analysis results produced by Phoenix 2 is comprehensive, including taxonomic annotations using multiple methods, alpha diversity indices, beta diversity measurements, and a number of visualizations. To date, the pipeline has been used to analyze more than 1500 environmental samples from a wide variety of microbial communities, which are part of our Hydrocarbon Metagenomics Project (http://www.hydrocarbonmetagenomics.com). The software package can be installed as a local software suite with a Web interface. Phoenix 2 is freely available from http://sourceforge.net/projects/phoenix2.

Function of periplasmic hydrogenases in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough.

The sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough possesses four periplasmic hydrogenases to facilitate the oxidation of molecular hydrogen. These include an [Fe] hydrogenase, an [NiFeSe] hydrogenase, and two [NiFe] hydrogenases encoded by the hyd, hys, hyn1, and hyn2 genes, respectively. In order to understand their cellular functions, we have compared the growth rates of existing (hyd and hyn1) and newly constructed (hys and hyn-1 hyd) mutants to those of the wild type in defined media in which lactate or hydrogen at either 5 or 50% (vol/vol) was used as the sole electron donor for sulfate reduction. Only strains missing the [Fe] hydrogenase were significantly affected during growth with lactate or with 50% (vol/vol) hydrogen as the sole electron donor. When the cells were grown at low (5% [vol/vol]) hydrogen concentrations, those missing the [NiFeSe] hydrogenase suffered the greatest impairment. The growth rate data correlated strongly with gene expression results obtained from microarray hybridizations and real-time PCR using mRNA extracted from cells grown under the three conditions. Expression of the hys genes followed the order 5% hydrogen>50% hydrogen>lactate, whereas expression of the hyd genes followed the reverse order. These results suggest that growth with lactate and 50% hydrogen is associated with high intracellular hydrogen concentrations, which are best captured by the higher activity, lower affinity [Fe] hydrogenase. In contrast, growth with 5% hydrogen is associated with a low intracellular hydrogen concentration, requiring the lower activity, higher affinity [NiFeSe] hydrogenase.