Increased waterborne blaNDM-1 resistance gene abundances associated with seasonal human pilgrimages to the upper ganges river.

Antibiotic resistance (AR) is often rooted in inappropriate antibiotic use, but poor water quality and inadequate sanitation exacerbate the problem, especially in emerging countries. An example is increasing multi-AR due to mobile carbapenemases, such as NDM-1 protein (coded by blaNDM-1 genes), which can produce extreme drug-resistant phenotypes. In 2010, NDM-1 positive isolates and blaNDM-1 genes were detected in surface waters across Delhi and have since been detected across the urban world. However, little is known about blaNDM-1 levels in more pristine locations, such as the headwaters of the Upper Ganges River. This area is of particular interest because it receives massive numbers of visitors during seasonal pilgrimages in May/June, including visitors from urban India. Here we quantified blaNDM-1 abundances, other AR genes (ARG), and coliform bacteria in sediments and water column samples from seven sites in the Rishikesh-Haridwar region of the Upper Ganges and five sites on the Yamuna River in Delhi to contrast blaNDM-1 levels and water quality conditions between season and region. Water quality in the Yamuna was very poor (e.g., anoxia at all sites), and blaNDM-1 abundances were high across sites in water (5.4 ± 0.4 log(blaNDM-1·mL(-1)); 95% confidence interval) and sediment (6.3 ± 0.7 log(blaNDM-1·mg(-1))) samples from both seasons. In contrast, water column blaNDM-1 abundances were very low across all sites in the Upper Ganges in February (2.1 ± 0.6 log(blaNDM-1·mL(-1))), and water quality was good (e.g., near saturation oxygen). However, per capita blaNDM-1 levels were 20 times greater in June in the Ganges water column relative to February, and blaNDM-1 levels significantly correlated with fecal coliform levels (r = 0.61; p = 0.007). Given that waste management infrastructure is limited in Rishikesh-Haridwar, data imply blaNDM-1 levels are higher in visitor's wastes than local residents, which results in seasonally higher blaNDM-1 levels in the river. Pilgrimage areas without adequate waste treatment are possible "hot spots" for AR transmission, and waste treatment must be improved to reduce broader AR dissemination via exposed returning visitors.

Anaerobic-aerobic sequencing bioreactors improve energy efficiency for treatment of personal care product industry wastes.

Personal care product (PCP) industry liquid wastes contain shampoo residues, which are usually treated by aerobic activated sludge (AS). Unfortunately, AS is expensive for PCP wastes because of high aeration and energy demands, whereas potentially energy-positive anaerobic designs cannot meet effluent targets. Therefore, combined anaerobic-aerobic systems may be the best solution. Seven treatment systems were assessed in terms of energy and treatment performance for shampoo wastes, including one aerobic, three anaerobic (HUASB, AHR and AnCSTR) and three anaerobic-aerobic reactor designs. COD removals were highest in the HUASB-aerobic (87.9 ± 0.4%) and AHR-aerobic (86.8±0.5%) systems, which used 69.2% and 62.5% less energy than aerobic AS. However, actual methane production rates were low relative to theoretical in the UASB and AHR units (∼10% methane/COD removed) compared with the AnCSTR unit (∼70%). Anaerobic-aerobic sequence reactors show promise for treating shampoo wastes, but optimal designs depend upon whether methane production or COD removal is most important to operations.

Nitrification in hybrid bioreactors treating simulated domestic wastewater.

AIM: To provide deeper insights into nitrification process within aerobic bioreactors containing supplemental physical support media (hybrid bioreactors). METHODS AND RESULTS: Three bench-scale hybrid bioreactors with different media size and one control bioreactor were operated to assess how biofilm integrity influences microbial community conditions and bioreactor performance. The systems were operated initially at a 5-day hydraulic retention time (HRT), and all reactors displayed efficient nitrification and chemical oxygen demand (COD) removal (>95%). However, when HRT was reduced to 2.5 days, COD removal rates remained high, but nitrification efficiencies declined in all reactors after 19 days. To explain reduced performance, nitrifying bacterial communities (ammonia-oxidizing bacteria, AOB; nitrite-oxidizing bacteria, NOB) were examined in the liquid phase and also on the beads using qPCR, FISH and DGGE. Overall, the presence of the beads in a reactor promoted bacterial abundances and diversity, but as bead size was increased, biofilms with active coupled AOB-NOB activity were less apparent, resulting in incomplete nitrification. CONCLUSIONS: Hybrid bioreactors have potential to sustain effective nitrification at low HRTs, but support media size and configuration type must be optimized to ensure coupled AOB and NOB activity in nitrification. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that AOB and NOB coupling must be accomplished to minimize nitrification failure.

Low-energy treatment of colourant wastes using sponge biofilters for the personal care product industry.

Four trickling biofilter designs were assessed as low-energy alternatives to aerobic activated sludge (AS) for the treatment of personal care product industry wastes. The designs included partially submerged packed-media and sponge reactors with and without active aeration. Partial submergence was used to reduce active aeration needs. Simulated colourant wastes (up to COD=12,480 mg/L, TN=128 mg/L) were treated for 201 days, including wastes with elevated oxidant levels. COD and TN removal efficiencies were always >79% and >30% (even without aeration). However, aerated sponge reactors consistently had the highest removal efficiencies, especially for TN (∼60%), and were most tolerant of elevated oxidants. This study shows sponge biofilters have great potential for treating colourant wastes because they achieve high treatment efficiencies and reduce energy use by >40% relative to AS systems.

Mercury levels in sediments and mangrove oysters, Crassostrea rizophorae, from the north coast of Villa Clara, Cuba.

Total mercury levels were quantified in sediments and oyster tissues (Crassostrea rizophorae) from the Sagua la Grande River estuary and offshore mangrove keys 19 km downstream of a chlor-alkali plant (CAP) in Villa Clara, Cuba. Relatively elevated total mercury levels were found in sediments from the estuary itself, ranging from 0.507 to 1.81 µg g(-1) dry weight. However, levels were lower in sediments from the keys farther from the estuary. Oyster mercury levels were always acceptable for human consumption, although levels significantly correlated in sediments and oysters across sampling sites (p < 0.05), which suggests that mercury from the CAP is impacting coastal water quality conditions.

Feasibility tests for treating shampoo and hair colorant wastewaters using anaerobic processes.

Wastes from the personal care product (PCP) industry are often high in biodegradable carbon, which makes them amenable to aerobic biological treatment, although process costs are usually high due to aeration inefficiencies, high electricity demand and production of large amounts of sludge. As such, anaerobic treatment technologies are being considered to lower net energy costs by reducing air use and increasing methane production. To assess the amenability of PCP wastes to anaerobic treatment, methane yields and rates were quantified in different anaerobic reactors treating typical PCP wastes, including wastes from shampoo and hair colorant products. Overall, shampoo wastes were more amenable to methanogenesis with almost double the methane yields compared with colour wastes. To assess relevant microbial guilds, qPCR was performed on reactor biomass samples. Methanosaetaceae abundances were always significantly higher than Methanosarcinaceae and Methanomicrobiales abundances (P < 0.05), and did not differ significantly between waste types. Although colour wastes were less amenable to anaerobic treatment than shampoo wastes, differences cannot be explained by relative microbial abundances and probably result from the presence of inhibiting compounds in hair colorants (e.g., oxidants) at higher levels. Results showed that anaerobic technologies have great potential for treating PCP wastes, but additional work is needed to establish the basis of elevated methane yields and inhibition, especially when colorant wastes are present.

Assessment of total mercury levels in Clarias gariepinus from the Sagua la Grande River, Cuba.

Total mercury levels (Thg) were quantified in Clarias gariepinus captured from the Sagua la Grande River (Cuba) in the vicinity of an active chlor-alkali plant, and relationships among place of capture; fish size, weight, and sex; and THg levels were assessed. THg levels ranged from 67 to 375 ng/g ww in collected fish, never exceeding the Cuban recommended maximum limit for fish consumption of 500 ng/g ww. No significant correlation was observed between mercury levels and fish allometric characteristics (p < 0.05); however, levels were significantly higher in fish captured below the chlor-alkali facility, suggesting a connection between mercury bioaccumulation and plant discharges.

A comparative assessment of molecular biological and direct microscopic techniques for assessing aquatic systems.

Molecular techniques have clear value for community characterization; however, almost all previous datasets are based upon non-molecular measurements and it is hard to compare "old" data with "new" data because few correlations have been made. Therefore, the purpose of this evaluation was to simultaneously use molecular and non-molecular methods within the same sampling program to determine how data compare. Three methods were used for characterizing microbial populations in Lake 260 (L260) at Experimental Lakes Area (Ontario, Canada) during a whole-lake exposure study. Methods included whole-cell microscopic counts (for bacteria, cyanobacteria, algae and zooplankton), chlorophyll a, and small sub-unit (ssu)-rRNA hybridization using EUB-338, EUB-785, CYAN-785-a/b, EUCA-1379 and UNIV-1390 gene probes. Strong correlations were found between the EUB-785 probe signal and "bacteria minus cyanobacteria" direct counts, and the EUB-338 probe signal and "bacteria plus cyanobacteria" counts. Furthermore, the difference in probe signal between EUB-338 and EUB-785 (a presumptive signature for cyanobacteria and plastids) correlated with cyanobacterial direct counts and also with chlorophyll a. However, EUCA-1379 probe signal did not correlate with algae counts, and UNIV-1390 probe signal only correlated with total bacteria counts. Results suggest that, although ssu-rRNA methods are fast, reproducible, and specifically detect "viable" organisms, their use may be limited to non-eukaryotic populations unless new probes are developed that are more specific.

Water hyacinths (Eichhornia crassipes) as indicators of heavy metal impact of a large landfill on the Almendares River near Havana, Cuba.

The Almendares River is central to recreational and other activities in Havana, Cuba. However, monitoring indicated significant heavy metal contamination in river sediments, especially below Calle 100, the largest landfill in Havana. This work extended previous sediment studies by determining complementary Cu, Pb, Ni, Cr, Cd, and Zn levels in indigenous water hyacinths (Eichhornia crassipes; EC) above and below the landfill. Pb, Cu, and Zn were significantly elevated in EC roots below the landfill and also correlated with sediment data (p < 0.05), implying elevated levels likely result from landfill activity and might be useful biomonitors as river remediation proceeds.

Cryptic striations in the upper mantle revealed by hafnium isotopes in southeast Indian ridge basalts.

The Earth's mantle is isotopically heterogeneous on length scales ranging from centimetres to more than 10(4) kilometres. This heterogeneity originates from partial melt extraction and plate tectonic recycling, whereas stirring during mantle convection tends to reduce it. Here we show that mid-ocean ridge basalts from 2,000 km along the southeast Indian ridge (SEIR) display a bimodal hafnium isotopic distribution. This bimodality reveals the presence of ancient compositional striations (streaks) in the Indian Ocean upper mantle. The number density of the streaks is described by a Poisson distribution, with an average thickness of approximately 40 km. Such a distribution is anticipated for a well-stirred upper mantle, in which heterogeneity is continually introduced by plate tectonic recycling, and redistributed by viscous stretching and convective refolding.

Field assessment of oxytetracycline exposure to the freshwater macrophytes Egeria densa Planch. and Ceratophyllum demersum L.

In a microcosm study, two aquatic macrophytes, Egeria densa and Ceratophyllum demersum were exposed to nominal concentrations of 0, 5, 20, 50, and 250 microg/L oxytetracycline (n=3), plus 20 microg/L oxytetracycline amended with additional nitrogen (N) and phosphorus (P). Responses were monitored bi-weekly over a six-week exposure period. Both plant species exhibited a significant decline in growth in the 250 microg/L oxytetracycline and the N- and P-amended units. Decreased light penetration resulting from accumulating oxytetracycline by-products appears to be the primary modifier in the growth of these plants. Increased susceptibility to oxytetracycline exposure was noted in some paired plantings (e.g., E. densa root development), relative to individual plants in these treatments, however, no clear explanation for this response is available. Based on the toxicity data generated in this study, we estimate that current concentrations of oxytetracycline in freshwater environments do not pose a direct risk to E. densa and C. demersum.

Fate and effects of enrofloxacin in aquatic systems under different light conditions.

The fate and effects of fluoroquinolone antibacterials (FQ) in the environment is of significance because of apparent increased FQ resistance in environmental and clinical organisms. Here we simultaneously assessed the fate and effects of enrofloxacin (enro), an FQ often used in agriculture, on the chemistry and in situ microbial communities in receiving waters. We added enro to 25 microg/L in nine outdoor mesocosms maintained under three light conditions (in triplicate): full sunlight typical of the upper epilimnion (100% full-light exposure, FLE), partial shading typical of the lower epilimnion (28% FLE), and near-complete shading typical of the hypolimnion (0.5% FLE). Enro disappearance and ciprofloxacin (cipro) formation were monitored over time using LC/MS, and water chemistry and ambient microbial communities (using denaturing gradient gel electrophoresis; DGGE) were characterized. Enro half-lives were 0.8, 3.7, and 72 days for the 100%, 28%, and 0.5% FLE treatments, respectively, creating three distinct FQ exposure scenarios. Although FQ exposures ranged from approximately 6 microg/L for 24 h to approximately 21 microg/L for 30 days, no statistically significant exposure effects were noted in water quality or microbial communities (as indicated by whole-community 16S rDNA DGGE analysis and specific amplification of the QRDR region of gyrase A). Small changes in water chemistry were noted over time; however, changes could not be specifically attributed to FQs. In general, enro addition had minimal effect on water column conditions at the levels and durations used here; however, further investigation is needed to assess effects in aquatic sediments.

Magmatic and amagmatic seafloor generation at the ultraslow-spreading Gakkel ridge, Arctic Ocean.

A high-resolution mapping and sampling study of the Gakkel ridge was accomplished during an international ice-breaker expedition to the high Arctic and North Pole in summer 2001. For this slowest-spreading endmember of the global mid-ocean-ridge system, predictions were that magmatism should progressively diminish as the spreading rate decreases along the ridge, and that hydrothermal activity should be rare. Instead, it was found that magmatic variations are irregular, and that hydrothermal activity is abundant. A 300-kilometre-long central amagmatic zone, where mantle peridotites are emplaced directly in the ridge axis, lies between abundant, continuous volcanism in the west, and large, widely spaced volcanic centres in the east. These observations demonstrate that the extent of mantle melting is not a simple function of spreading rate: mantle temperatures at depth or mantle chemistry (or both) must vary significantly along-axis. Highly punctuated volcanism in the absence of ridge offsets suggests that first-order ridge segmentation is controlled by mantle processes of melting and melt segregation. The strong focusing of magmatic activity coupled with faulting may account for the unexpectedly high levels of hydrothermal activity observed.

Discovery of abundant hydrothermal venting on the ultraslow-spreading Gakkel ridge in the Arctic Ocean.

Submarine hydrothermal venting along mid-ocean ridges is an important contributor to ridge thermal structure, and the global distribution of such vents has implications for heat and mass fluxes from the Earth's crust and mantle and for the biogeography of vent-endemic organisms. Previous studies have predicted that the incidence of hydrothermal venting would be extremely low on ultraslow-spreading ridges (ridges with full spreading rates <2 cm x yr(-1)-which make up 25 per cent of the global ridge length), and that such vent systems would be hosted in ultramafic in addition to volcanic rocks. Here we present evidence for active hydrothermal venting on the Gakkel ridge, which is the slowest spreading (0.6-1.3 cm x yr(-1)) and least explored mid-ocean ridge. On the basis of water column profiles of light scattering, temperature and manganese concentration along 1,100 km of the rift valley, we identify hydrothermal plumes dispersing from at least nine to twelve discrete vent sites. Our discovery of such abundant venting, and its apparent localization near volcanic centres, requires a reassessment of the geologic conditions that control hydrothermal circulation on ultraslow-spreading ridges.

Design and synthesis of novel antibacterial agents with inhibitory activity against DNA polymerase III.

4-Substituted 2-amino-6-(anilino)pyrimidines have been found to be selective inhibitors of DNA polymerase III, a replicative enzyme known to be essential in the DNA synthesis of Gram-positive bacteria. Among the analogues, 18 displayed an IC(50) of 10 microM against DNA polymerase III from Staphylococcus aureus.

Effect of oxygen level on simultaneous nitrogenase and sMMO expression and activity in Methylosinus trichosporium OB3b and its sMMO(C) mutant, PP319: aerotolerant N2 fixation in PP319.

Soluble methane monooxygenase (sMMO) expression and activity were monitored under conditions that either promoted or suppressed the expression of nitrogenase in Methylosinus trichosporium OB3b wild-type (WT) and in its sMMO-constitutive mutant, PP319. Both WT and mutant cultures had reduced sMMO activity and protein levels under elevated O2 conditions (188 microM) compared with low O2 conditions (24 microM). Simultaneous N2 fixation also reduced sMMO activity in both cultures when O2 was low. However, when O2 levels were increased, nitrogenase expression ceased and sMMO activity was reduced by approximately 77% in the WT, whereas sMMO and nitrogenase expression and activity in PP319 were relatively unaffected by the higher O2 levels. Western immunoblot analysis showed that the nitrogenase Fe protein resolved as two components (apparent molecular mass of 30.5 and 32 kDa) in both the WT and PP319 when O2 levels were low. When O2 levels were high, only the 32-kDa form of the Fe protein was present in PP319, whereas neither form was detectable in the WT. Aerotolerant N2 fixation appears to be associated with the 32-kDa Fe protein in M. trichosporium OB3b.

Upper-mantle dynamics revealed by helium isotope variations along the southeast Indian ridge.

Helium isotope variations in igneous rocks are important for relating isotopic heterogeneity to convective mixing in the Earth's mantle. High 3He/4He ratios at many ocean islands, along with lower and relatively uniform values in mid-ocean-ridge basalts (MORBs), are thought to result from a well mixed upper-mantle source for MORB and a distinct deeper-mantle source for ocean island basalts. At finer scales, 3He/4He variations along mid-ocean ridges have been related to underlying mantle heterogeneity, but relationships between the scales of geochemical segmentation and mantle convection remain enigmatic. Here we present helium isotope data for MORB glasses recovered along approximately 5,800 km of the southeast Indian ridge, and develop an approach to quantitatively relate spatial variations in geochemical and geophysical parameters at the Earth's surface. A point-to-point correlation analysis reveals structure in the helium isotope data at length scales of approximately 150 and approximately 400 km that appears to be related to secondary convection in the underlying mantle.

Copper-binding compounds from Methylosinus trichosporium OB3b.

Two copper-binding compounds/cofactors (CBCs) were isolated from the spent media of both the wild type and a constitutive soluble methane monooxygenase (sMMOC) mutant, PP319 (P. A. Phelps et al., Appl. Environ. Microbiol. 58:3701-3708, 1992), of Methylosinus trichosporium OB3b. Both CBCs are small polypeptides with molecular masses of 1,218 and 779 Da for CBC-L1 and CBC-L2, respectively. The amino acid sequence of CBC-L1 is S?MYPGS?M, and that of CBC-L2 is SPMP?S. Copper-free CBCs showed absorption maxima at 204, 275, 333, and 356 with shoulders at 222 and 400 nm. Copper-containing CBCs showed a broad absorption maximum at 245 nm. The low-temperature electron paramagnetic resonance (EPR) spectra of copper-containing CBC-L1 showed the presence of a copper center with an EPR splitting constant between those of type 1 and type 2 copper centers (g = 2.087, g = 2.42 G, A = 128 G). The EPR spectrum of CBC-L2 was more complex and showed two spectrally distinct copper centers. One signal can be attributed to a type 2 Cu2+ center (g = 2.073, g = 2.324 G, A = 144 G) which could be saturated at higher powers, while the second shows a broad, nearly isotropic signal near g = 2.063. In wild-type strains, the concentrations of CBCs in the spent media were highest in cells expressing the pMMO and stressed for copper. In contrast to wild-type strains, high concentrations of CBCs were observed in the extracellular fraction of the sMMOC mutants PP319 and PP359 regardless of the copper concentration in the culture medium.

Isolation of copper biochelates from Methylosinus trichosporium OB3b and soluble methane monooxygenase mutants.

Methylosinus trichosporium OB3b produces an extracellular copper-binding ligand (CBL) with high affinity for copper. Wild-type cells and mutants that express soluble methane monooxygenase (sMMO) in the presence and absence of copper (sMMOc) were used to obtain cell exudates that were separated and analyzed by size exclusion high-performance liquid chromatography. A single chromatographic peak, when present, contained most of the aqueous-phase Cu(II) present in the culture medium. In mutant cultures that were unable to acquire copper, extracellular CBL accumulated to high levels both in the presence and in the absence of copper. Conversely, in wild-type cultures containing 5 microM Cu(II), extracellular CBL was maintained at a low, steady level during exponential growth, after which the external ligand was rapidly consumed. When Cu(II) was omitted from the growth medium, the wild-type organism produced the CBL at a rate that was proportional to cell density. After copper was added to this previously Cu-deprived culture, the CBL and copper concentrations in the medium decreased at approximately the same rate. Apparently, the extracellular CBL was produced throughout the period of cell growth, in the presence and absence of Cu(II), by both the mutant and wild-type cultures and was reinternalized or otherwise utilized by the wild-type cultures when it was bound to copper. CBL produced by the mutant strain facilitated copper uptake by wild-type cells, indicating that the extracellular CBLs produced by the mutant and wild-type organisms are functionally indistinguishable. CBL from the wild-type strain did not promote copper uptake by the mutant. The molecular weight of the CBL was estimated to be 500, and its association constant with copper was 1.4 x 10(16) M-1. CBL exhibited a preference for copper, even in the presence of 20-fold higher concentrations of nickel. External complexation may play a role in normal copper acquisition by M. trichosporium OB3b. The sMMOc phenotype is probably related to the mutant's inability to take up CBL-complexed copper, not to a defective CBL structure.

Molecular typing of Helicobacter pylori isolates from a multicenter U.S. clinical trial by ureC restriction fragment length polymorphism.

The molecular typing of 81 pretreatment Helicobacter pylori isolates and the comparison of 18 pretreatment-posttreatment pairs is described by restriction fragment length polymorphism (RFLP) of the ureC gene. The results of our study show the extreme genomic diversity of H. pylori and indicate that infection by H. pylori in the United States does not appear to be limited to a small number of RFLP types.