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Potential for water borne and invertebrate transmission of West Nile virus in the Great Salt Lake, Utah

In November and December of 2013, a large mortality event involving 15,000 - 20,000 eared grebes ( Podiceps nigricollis ) occurred at the Great Salt Lake (GSL), UT. The onset of the outbreak in grebes was followed by a mortality event in > 86 bald eagles ( Haliaeetus leucocephalus ). During the die-off, West Nile virus (WNV) was detected by RT-PCR or viral culture in carcasses of grebes and eagles submitted to the National Wildlife Health Center. However, no mosquito activity, the primary vector of WNV, was detected by the State of Utah's WNV monitoring program. Transmission of WNV has rarely been reported during the winter in North America in the absence of known mosquito activity; however, the size of this die-off, the habitat in which it occurred, and the species involved are unique. We experimentally investigated whether WNV could survive in water with a high saline content, as found at the GSL, and whether brine shrimp, the primary food of migrating eared grebes on the GSL, could have played a role in transmission of WNV to feeding birds. We found that WNV can survive up to 72 h at 4°C in water containing 30 — 150 ppt NaCl and brine shrimp, incubated with WNV in 30 ppt NaCl, may adsorb WNV to their cuticle and, through feeding, may infect epithelial cells of their gut. Both mechanisms may have potentiated the WNV die-off in migrating eared grebes on the GSL.

Utah↗

Elucidation of taste- and odor-producing bacteria and toxigenic cyanobacteria in a Midwestern drinking water supply reservoir by shotgun metagenomics analysis

While commonplace in clinical settings, DNA-based assays for identification or enumeration of drinking water pathogens and other biological contaminants remain widely unadopted by the monitoring community. In this study, shotgun metagenomics was used to identify taste-and-odor producers and toxin-producing cyanobacteria over a 2-year period in a drinking water reservoir. The sequencing data implicated several cyanobacteria, including Anabaena spp ., Microcystis spp ., and an unresolved member of the order Oscillatoriales as the likely principal producers of geosmin, microcystin, and 2-methylisoborneol (MIB), respectively. To further demonstrate this, quantitative PCR (qPCR) assays targeting geosmin-producing Anabaena and microcystin-producing Microcystis were utilized, and these data were fitted using generalized linear models and compared with routine monitoring data, including microscopic cell counts, sonde-based physicochemical analyses, and assays of all inorganic and organic nitrogen and phosphorus forms and fractions. The qPCR assays explained the greatest variation in observed geosmin (adjusted R 2 = 0.71) and microcystin (adjusted R 2 = 0.84) concentrations over the study period, highlighting their potential for routine monitoring applications. The origin of the monoterpene cyclase required for MIB biosynthesis was putatively linked to a periphytic cyanobacterial mat attached to the concrete drinking water inflow structure. We conclude that shotgun metagenomics can be used to identify microbial agents involved in water quality deterioration and to guide PCR assay selection or design for routine monitoring purposes. Finally, we offer estimates of microbial diversity and metagenomic coverage of our data sets for reference to others wishing to apply shotgun metagenomics to other lacustrine systems.

Kansas↗

Methane-producing microbial community in a coal bed of the Illinois Basin

A series of molecular and geochemical studies were performed to study microbial, coal bed methane formation in the eastern Illinois Basin. Results suggest that organic matter is biodegraded to simple molecules, such as H 2 and CO2, which fuel methanogenesis and the generation of large coal bed methane reserves. Small-subunit rRNA analysis of both the in situ microbial community and highly purified, methanogenic enrichments indicated that Methanocorpusculum is the dominant genus. Additionally, we characterized this methanogenic microorganism using scanning electron microscopy and distribution of intact polar cell membrane lipids. Phylogenetic studies of coal water samples helped us develop a model of methanogenic biodegradation of macromolecular coal and coal-derived oil by a complex microbial community. Based on enrichments, phylogenetic analyses, and calculated free energies at in situ subsurface conditions for relevant metabolisms (H2-utilizing methanogenesis, acetoclastic methanogenesis, and homoacetogenesis), H 2-utilizing methanogenesis appears to be the dominant terminal process of biodegradation of coal organic matter at this location. Copyright ?? 2008, American Society for Microbiology. All Rights Reserved.

Applied and Environmental Microbiology↗

Formation of tellurium nanocrystals during anaerobic growth of bacteria that use Te oxyanions as respiratory electron acceptors

Certain toxic elements support the metabolism of diverse prokaryotes by serving as respiratory electron acceptors for growth. Here, we demonstrate that two anaerobes previously shown to be capable of respiring oxyanions of selenium also achieve growth by reduction of either tellurate [Te(VI)] or tellurite [Te(IV)] to elemental tellurium [Te(0)]. This reduction achieves a sizeable stable-Te-isotopic fractionation (isotopic enrichment factor [ε] = −0.4 to −1.0 per ml per atomic mass unit) and results in the formation of unique crystalline Te(0) nanoarchitectures as end products. The Te(0) crystals occur internally within but mainly externally from the cells, and each microorganism forms a distinctly different structure. Those formed by Bacillus selenitireducens initially are nanorods (∼10-nm diameter by 200-nm length), which cluster together, forming larger (∼1,000-nm) rosettes composed of numerous individual shards (∼100-nm width by 1,000-nm length). In contrast, Sulfurospirillum barnesii forms extremely small, irregularly shaped nanospheres (diameter < 50 nm) that coalesce into larger composite aggregates. Energy-dispersive X-ray spectroscopy and selected area electron diffraction indicate that both biominerals are composed entirely of Te and are crystalline, while Raman spectroscopy confirms that they are in the elemental state. These Te biominerals have specific spectral signatures (UV-visible light, Raman) that also provide clues to their internal structures. The use of microorganisms to generate Te nanomaterials may be an alternative for bench-scale syntheses. Additionally, they may also generate products with unique properties unattainable by conventional physical/chemical methods.

Applied and Environmental Microbiology↗

Ecophysiology of "halarsenatibacter silvermanii" strain SLAS-1 T , gen. nov., sp. nov., a facultative chemoautotrophic arsenate respirer from salt-saturated Searles Lake, California

Searles Lake occupies a closed basin harboring salt-saturated, alkaline brines that have exceptionally high concentrations of arsenic oxyanions. Strain SLAS-1 T was previously isolated from Searles Lake (R. S. Oremland, T. R. Kulp, J. Switzer Blum, S. E. Hoeft, S. Baesman, L. G. Miller, and J. F. Stolz, Science 308:1305-1308, 2005). We now describe this extremophile with regard to its substrate affinities, its unusual mode of motility, sequenced arrABD gene cluster, cell envelope lipids, and its phylogenetic alignment within the order Halanaero-bacteriales, assigning it the name "Halarsenatibacter silvermanii" strain SLAS-1 T . We also report on the substrate dynamics of an anaerobic enrichment culture obtained from Searles Lake that grows under conditions of salt saturation and whose members include a novel sulfate reducer of the order Desulfovibriales, the archaeon Halorhabdus utahensis, as well as a close homolog of strain SLAS-1 T . Copyright ?? 2009, American Society for Microbiology. All Rights Reserved.

Applied and Environmental Microbiology↗

Denitrification in San Francisco Bay intertidal sediments

The acetylene block technique was employed to study denitrification in intertidal estuarine sediments. Addition of nitrate to sediment slurries stimulated denitrification. During the dry season, sediment-slurry denitrification rates displayed Michaelis-Menten kinetics, and ambient NO 3 &minus; + NO 2 &minus; concentrations (&le;26 &mu;M) were below the apparent K m (50 &mu;M) for nitrate. During the rainy season, when ambient NO 3 &minus; + NO 2 &minus; concentrations were higher (37 to 89 &mu;M), an accurate estimate of the K m could not be obtained. Endogenous denitrification activity was confined to the upper 3 cm of the sediment column. However, the addition of nitrate to deeper sediments demonstrated immediate N 2 O production, and potential activity existed at all depths sampled (the deepest was 15 cm). Loss of N 2 O in the presence of C 2 H 2 was sometimes observed during these short-term sediment incubations. Experiments with sediment slurries and washed cell suspensions of a marine pseudomonad confirmed that this N 2 O loss was caused by incomplete blockage of N 2 O reductase by C 2 H 2 at low nitrate concentrations. Areal estimates of denitrification (in the absence of added nitrate) ranged from 0.8 to 1.2 &mu;mol of N 2 m &minus;2 h &minus;1 (for undisturbed sediments) to 17 to 280 &mu;mol of N 2 m &minus;2 h &minus;1 (for shaken sediment slurries).

California↗

Evaluation of methyl fluoride and dimethyl ether as inhibitors of aerobic methane oxidation

Methyl fluoride (MF) and dimethyl ether (DME) were effective inhibitors of aerobic methanotrophy in a variety of soils. MF and DME blocked consumption of CH 4 as well as the oxidation of 14 CH 4 to 14 CO 2 , but neither MF nor DME affected the oxidation of [ 14 C]methanol or [ 14 C]formate to 14 CO 2 . Cooxidation of ethane and propane by methane-oxidizing soils was also inhibited by MF. Nitrification (ammonia oxidation) in soils was inhibited by both MF and DME. Production of N 2 O via nitrification was inhibited by MF; however, MF did not affect N 2 O production associated with denitrification. Methanogenesis was partially inhibited by MF but not by DME. Methane oxidation was ∼100-fold more sensitive to MF than was methanogenesis, indicating that an optimum concentration could be employed to selectively block methanotrophy. MF inhibited methane oxidation by cell suspensions of Methylococcus capsulatus ; however, DME was a much less effective inhibitor.

California↗

Dissimilatory Fe(III) reduction by the marine microorganism Desulfuromonas acetoxidans

The ability of the marine microorganism Desulfuromonas acetoxidans to reduce Fe(III) was investigated because of its close phylogenetic relationship with the freshwater dissimilatory Fe(III) reducer Geobacter metallireducens. Washed cell suspensions of the type strain of D. acetoxidans reduced soluble Fe(III)-citrate and Fe(III) complexed with nitriloacetic acid. The c -type cytochrome(s) of D. acetoxidans was oxidized by Fe(III)-citrate and Mn(IV)-oxalate, as well as by two electron acceptors known to support growth, colloidal sulfur and malate. D. acetoxidans grew in defined anoxic, bicarbonate-buffered medium with acetate as the sole electron donor and poorly crystalline Fe(III) or Mn(IV) as the sole electron acceptor. Magnetite (Fe 3 O 4 ) and siderite (FeCO 3 ) were the major end products of Fe(III) reduction, whereas rhodochrosite (MnCO 3 ) was the end product of Mn(IV) reduction. Ethanol, propanol, pyruvate, and butanol also served as electron donors for Fe(III) reduction. In contrast to D. acetoxidans, G. metallireducens could only grow in freshwater medium and it did not conserve energy to support growth from colloidal S 0 reduction. D. acetoxidans is the first marine microorganism shown to conserve energy to support growth by coupling the complete oxidation of organic compounds to the reduction of Fe(III) or Mn(IV). Thus, D. acetoxidans provides a model enzymatic mechanism for Fe(III) or Mn(IV) oxidation of organic compounds in marine and estuarine sediments. These findings demonstrate that 16S rRNA phylogenetic analyses can suggest previously unrecognized metabolic capabilities of microorganisms.

Applied and Environmental Microbiology↗

Autotrophic, hydrogen-oxidizing, denitrifying bacteria in groundwater, potential agents for bioremediation of nitrate contamination

Addition of hydrogen or formate significantly enhanced the rate of consumption of nitrate in slurried core samples obtained from an active zone of denitrification in a nitrate-contaminated sand and gravel aquifer (Cape Cod, Mass.). Hydrogen uptake by the core material was immediate and rapid, with an apparent K m of 0.45 to 0.60 μM and a V max of 18.7 nmol cm -3 h -1 at 30°C. Nine strains of hydrogen-oxidizing denitrifying bacteria were subsequently isolated from the aquifer. Eight of the strains grew autotrophically on hydrogen with either oxygen or nitrate as the electron acceptor. One strain grew mixotrophically. All of the isolates were capable of heterotrophic growth, but none were similar to Paracoccus denitrificans , a well-characterized hydrogen-oxidizing denitrifier. The kinetics for hydrogen uptake during denitrification were determined for each isolate with substrate depletion progress curves; the K m s ranged from 0.30 to 3.32 μM, with V max s of 1.85 to 13.29 fmol cell -1 h -1 . Because these organisms appear to be common constituents of the in situ population of the aquifer, produce innocuous end products, and could be manipulated to sequentially consume oxygen and then nitrate when both were present, these results suggest that these organisms may have significant potential for in situ bioremediation of nitrate contamination in groundwater.

Massachusetts↗

Novel processes for anaerobic sulfate production from elemental sulfur by sulfate-reducing bacteria

Sulfate reducers and related organisms which had previously been found to reduce Fe(III) with H 2 or organic electron donors oxidized S 0 to sulfate when Mn(IV) was provided as an electron acceptor. Organisms catalyzing this reaction in washed cell suspensions included Desulfovibrio desulfuricans, Desulfomicrobium baculatum, Desulfobacterium autotrophicum, Desulfuromonas acetoxidans , and Geobacter metallireducens . These organisms produced little or no sulfate from S 0 with Fe(III) as a potential electron acceptor or in the absence of an electron acceptor. In detailed studies with Desulfovibrio desulfuricans , the stoichiometry of sulfate and Mn(II) production was consistent with the reaction S 0 + 3 MnO 2 + 4H + →SO 4 2- + 3Mn(II) + 2H 2 O. None of the organisms evaluated could be grown with S 0 as the sole electron donor and Mn(IV) as the electron acceptor. In contrast to the other sulfate reducers evaluated, Desulfobulbus propionicus produced sulfate from S 0 in the absence of an electron acceptor and Fe(III) oxide stimulated sulfate production. Sulfide also accumulated in the absence of Mn(IV) or Fe(III). The stoichiometry of sulfate and sulfide production indicated that Desulfobulbus propionicus disproportionates S 0 as follows: 4S 0 + 4H 2 O→SO 4 2- + 3HS - + 5 H + . Growth of Desulfobulbus propionicus with S 0 as the electron donor and Fe(III) as a sulfide sink and/or electron acceptor was very slow. The S 0 oxidation coupled to Mn(IV) reduction described here provides a potential explanation for the Mn(IV)-dependent sulfate production that previous studies have observed in anoxic marine sediments. Desulfobulbus propionicus is the first example of a pure culture known to disproportionate S 0 .

Applied and Environmental Microbiology↗

Growth of strain SES-3 with arsenate and other diverse electron acceptors

The selenate-respiring bacterial strain SES-3 was able to use a variety of inorganic electron acceptors to sustain growth. SES-3 grew with the reduction of arsenate to arsenite, Fe(III) to Fe(II), or thiosulfate to sulfide. It also grew in medium in which elemental sulfur, Mn(IV), nitrite, trimethylamine N -oxide, or fumarate was provided as an electron acceptor. Growth on oxygen was microaerophilic. There was no growth with arsenite or chromate. Washed suspensions of cells grown on selenate or nitrate had a constitutive ability to reduce arsenate but were unable to reduce arsenite. These results suggest that strain SES-3 may occupy a niche as an environmental opportunist by being able to take advantage of a diversity of electron acceptors.

Applied and Environmental Microbiology↗

Inhibition of methane oxidation by Methylococcus capsulatus with hydrochlorofluorocarbons and fluorinated methanes

The inhibition of methane oxidation by cell suspensions of Methylococcus capsulatus (Bath) exposed to hydrochlorofluorocarbon 21 (HCFC-21; difluorochloromethane [CHF 2 Cl]), HCFC-22 (fluorodichloromethane [CHFCl 2 ]), and various fluorinated methanes was investigated. HCFC-21 inhibited methane oxidation to a greater extent than HCFC-22, for both the particulate and soluble methane monooxygenases. Among the fluorinated methanes, both methyl fluoride (CH 3 F) and difluoromethane (CH 2 F 2 ) were inhibitory while fluoroform (CHF 3 ) and carbon tetrafluoride (CF 4 ) were not. The inhibition of methane oxidation by HCFC-21 and HCFC-22 was irreversible, while that by methyl fluoride was reversible. The HCFCs also proved inhibitory to methanol dehydrogenase, which suggests that they disrupt other aspects of C1 catabolism in addition to methane monooxygenase activity.

Applied and Environmental Microbiology↗

Size-selective predation on groundwater bacteria by nanoflagellates in an organic-contaminated aquifer

Time series incubations were conducted to provide estimates for the size selectivities and rates of protistan grazing that may be occurring in a sandy, contaminated aquifer. The experiments involved four size classes of fluorescently labeled groundwater bacteria (FLB) and 2- to 3-μm-long nanoflagellates, primarily Spumella guttula (Ehrenberg) Kent, that were isolated from contaminated aquifer sediments (Cape Cod, Mass.). The greatest uptake and clearance rates (0.77 bacteria · flagellate −1 · h −1 and 1.4 nl · flagellate −1 · h −1 , respectively) were observed for 0.8- to 1.5-μm-long FLB (0.21-μm 3 average cell volume), which represent the fastest growing bacteria within the pore fluids of the contaminated aquifer sediments. The 19:1 to 67:1 volume ratios of nanoflagellate predators to preferred bacterial prey were in the lower end of the range commonly reported for other aquatic habitats. The grazing data suggest that the aquifer nanoflagellates can consume as much as 12 to 74% of the unattached bacterial community in 1 day and are likely to have a substantive effect upon bacterial degradation of organic groundwater contaminants.

Massachusetts↗

A single Alal 39-to-Glu substitution in the Renibacterium salmoninarum virulence-associated protein p57 results in antigenic variation and is associated with enhanced p57 binding to Chinook salmon leukocytes

The gram-positive bacterium Renibacterium salmoninarum produces relatively large amounts of a 57-kDa protein (p57) implicated in the pathogenesis of salmonid bacterial kidney disease. Antigenic variation in p57 was identified by using monoclonal antibody 4C11, which exhibited severely decreased binding to R. salmoninarum strain 684 p57 and bound robustly to the p57 proteins of seven other R. salmoninarum strains. This difference in binding was not due to alterations in p57 synthesis, secretion, or bacterial cell association. The molecular basis of the 4C11 epitope loss was determined by amplifying and sequencing the two identical genes encoding p57, msa1 and msa2 . The 5&prime; and coding sequences of the 684 msa1 and msa2 genes were identical to those of the ATCC 33209 msa1 and msa2 genes except for a single C-to-A nucleotide mutation. This mutation was identified in both the msa1 and msa2 genes of strain 684 and resulted in an Ala 139 -to-Glu substitution in the amino-terminal region of p57. We examined whether this mutation in p57 altered salmonid leukocyte and rabbit erythrocyte binding activities. R. salmoninarum strain 684 extracellular protein exhibited a twofold increase in agglutinating activity for chinook salmon leukocytes and rabbit erythrocytes compared to the activity of the ATCC 33209 extracellular protein. A specific and quantitative p57 binding assay confirmed the increased binding activity of 684 p57. Monoclonal antibody 4C11 blocked the agglutinating activity of the ATCC 33209 extracellular protein but not the agglutinating activity of the 684 extracellular protein. These results indicate that the Ala 139 -to-Glu substitution altered immune recognition and was associated with enhanced biological activity of R. salmoninarum 684 p57.

Applied and Environmental Microbiology↗

Infectious Cryptosporidium parvum oocysts in final reclaimed effluent

Water samples collected throughout several reclamation facilities were analyzed for the presence of infectious Cryptosporidium parvum by the focus detection method-most-probable-number cell culture technique. Results revealed the presence of infectious C. parvum oocysts in 40% of the final disinfected effluent samples. Sampled effluent contained on average seven infectious oocysts per 100 liters. Thus, reclaimed water is not pathogen free but contains infectious C. parvum.

Applied and Environmental Microbiology↗

Vulnerability of drinking-water wells in La Crosse, Wisconsin, to enteric-virus contamination from surface water contributions

Human enteric viruses can contaminate municipal drinking-water wells, but few studies have examined the routes by which viruses enter these wells. In the present study, the objective was to monitor the municipal wells of La Crosse, Wisconsin, for enteric viruses and determine whether the amount of Mississippi River water infiltrating the wells was related to the frequency of virus detection. From March 2001 to February 2002, one river water site and four wells predicted by hydrogeological modeling to have variable degrees of surface water contributions were sampled monthly for enteric viruses, microbial indicators of sanitary quality, and oxygen and hydrogen isotopes. 18O/ 16O and 2H/1H ratios were used to determine the level of surface water contributions. All samples were collected prior to chlorination at the wellhead. By reverse transcription-PCR (RT-PCR), 24 of 48 municipal well water samples (50%) were positive for enteric viruses, including enteroviruses, rotavirus, hepatitis A virus (HAV), and noroviruses. Of 12 river water samples, 10 (83%) were virus positive by RT-PCR. Viable enteroviruses were not detected by cell culture in the well samples, although three well samples were positive for culturable HAV. Enteroviruses detected in the wells by RT-PCR were identified as several serotypes of echoviruses and group A and group B coxsackieviruses. None of the well water samples was positive for indicators of sanitary quality, namely male-specific and somatic coliphages, total coliform bacteria, Escherichia coli, and fecal enterococci. Contrary to expectations, viruses were found in all wells regardless of the level of surface water contributions. This result suggests that there were other unidentified sources, in addition to surface water, responsible for the contamination.

Applied and Environmental Microbiology↗

Redox transformations of arsenic oxyanions in periphyton communities

Periphyton ( Cladophora sp.) samples from a suburban stream lacking detectable dissolved As were able to reduce added As(V) to As(III) when incubated under anoxic conditions and, conversely, oxidized added As(III) to As(V) with aerobic incubation. Both types of activity were abolished in autoclaved controls, thereby demonstrating its biological nature. The reduction of As(V) was inhibited by chloramphenicol, indicating that it required the synthesis of new protein. Nitrate also inhibited As(V) reduction, primarily because it served as a preferred electron acceptor to which the periphyton community was already adapted. However, part of the inhibition was also caused by microbial reoxidation of As(III) linked to nitrate. Addition of [ 14 C]glucose to anoxic samples resulted in the production of 14 CO 2 , suggesting that the observed As(V) reduction was a respiratory process coupled to the oxidation of organic matter. The population density of As(V)-reducing bacteria within the periphyton increased with time and with the amount of As(V) added, reaching values as high as ∼10 6 cells ml −1 at the end of the incubation. This indicated that dissimilatory As(V) reduction in these populations was linked to growth. However, As(V)-respiring bacteria were found to be present, albeit at lower numbers (∼10 2 ml −1 ), in freshly sampled periphyton. These results demonstrate the presence of a bacterial population within the periphyton communities that is capable of two key arsenic redox transformations that were previously studied in As-contaminated environments, which suggests that these processes are widely distributed in nature. This assumption was reinforced by experiments with estuarine samples of Cladophora sericea in which we detected a similar capacity for anaerobic As(V) reduction and aerobic As(III) oxidation.

Applied and Environmental Microbiology↗