Long-Term Persistence and Leaching of Escherichia coli in Temperate Maritime Soils

Enteropathogen contamination of groundwater, including potable water sources, is a global concern. The spreading on land of animal slurries and manures, which can contain a broad range of pathogenic microorganisms, is considered a major contributor to this contamination. Some of the pathogenic microorganisms applied to soil have been observed to leach through the soil into groundwater, which poses a risk to public health. There is a critical need, therefore, for characterization of pathogen movement through the vadose zone for assessment of the risk to groundwater quality due to agricultural activities. A lysimeter experiment was performed to investigate the effect of soil type and condition on the fate and transport of potential bacterial pathogens, using Escherichia coli as a marker, in four Irish soils (n = 9). Cattle slurry (34 tonnes per ha) was spread on intact soil monoliths (depth, 1 m; diameter, 0.6 m) in the spring and summer. No effect of treatment or the initial soil moisture on the E. coli that leached from the soil was observed. Leaching of E. coli was observed predominantly from one soil type (average, 1.11 ± 0.77 CFU ml⁻¹), a poorly drained Luvic Stagnosol, under natural rainfall conditions, and preferential flow was an important transport mechanism. E. coli was found to have persisted in control soils for more than 9 years, indicating that autochthonous E. coli populations are capable of becoming naturalized in the low-temperature environments of temperate maritime soils and that they can move through soil. This may compromise the use of E. coli as an indicator of fecal pollution of waters in these regions.The contamination of groundwater, including potable water supplies, with microbial pathogens continues to be a global concern (52, 59). Of particular importance in developed countries are the high levels of contamination associated with small-scale and very-small-scale drinking water supplies (5, 19, 57), often groundwater, which serve an estimated 10% of the total population in the European Union (13). The high numbers of these water supplies found to be contaminated with fecal bacteria and thus considered to be unfit for human consumption are worrying because the water from them is often untreated or inadequately treated prior to consumption. Microbial pathogens are known to survive for considerable periods of time in groundwater (29), which increases the health risk due to utilization of contaminated supplies. There are various sources of contamination, but evidence suggests that contamination from the spreading of animal slurries and manures on land can be a significant contributor (3, 33, 53). Spreading of agricultural slurries and manures on land is used by the agricultural sector as a means of nutrient recycling. The health risks associated with the spreading of animal and human wastes containing enteric pathogens have been recognized for a long time (10, 18). Animal manure and wastewaters may contain a broad range of pathogenic microorganisms, including Escherichia coli O157:H7, Campylobacter, Cryptosporidium, Salmonella spp., and pathogenic viruses, which are released into the environment during spreading (15, 22, 55). The levels and incidence of pathogens present in animal manures and slurries are influenced by a number of factors, including herd health, age demographics, stress factors, diet, season, and manure management and storage (37, 39).Soils (and subsoils) often act as a zone for mitigating microbial contamination of groundwater associated with the spreading of animal slurries and manures on land. Some of the pathogenic microorganisms applied to agricultural soils have, however, been observed to leach through the soil into groundwater, which can affect drinking water quality and pose a risk to public health (16, 26, 28, 42, 50), confirming that soil is not always a sufficient obstruction for protection of groundwater (16, 53). Consequently, characterization of the movement of pathogens through the unsaturated soil and subsoil zone (vadose zone) has become critical for assessment of the risk to groundwater posed by agricultural activities (8, 14, 42). The soil and subsoil type is believed to be a major factor influencing the potential transfer of pathogens through soil to groundwater (3, 34, 41, 50). The preapplication moisture status of a soil, which may be influenced by the season, also impacts pathogen survival, fate, and transport (2, 11, 43, 54).E. coli is widely used as an indicator of fecal contamination of water, and certain strains are known to be pathogenic (12). Thus, characterizing this organism''s transport through soil is important because of the health risk posed by the organism itself and with regard to its validity as an indicator of the fate of enteropathogens in the environment. E. coli strains have diverse properties and capabilities that affect their survival and transport in soils (9, 36, 56, 60). Consequently, data obtained by using total E. coli rather than individual surrogate strains can be more representative of the fate and transport of E. coli present in animal slurries. E. coli O157 die-off in soils has been reported to be the same as or quicker than total E. coli die-off, suggesting that data for total E. coli provide a conservative estimate of the survival potential (38, 56). Although many field and laboratory studies have investigated E. coli transport through soil columns (4, 6, 16, 43, 46, 47, 50, 51), most studies have investigated transport through soil to a depth of less than 30 cm. For assessment of the risk of transport to groundwater, such studies may not take into account the variation in soil physical and chemical characteristics with depth (e.g., the frequency and continuity of macropores, organic matter, and moisture contents) that affect bacterial transport. Furthermore, rainfall was often simulated in previous studies, which allows experimental conditions to be controlled but may not be representative of the risk due to variable natural rainfall events over time. In this study, we used intact soil monoliths that were 1 m deep to assess the risk of leaching of total E. coli in four representative Irish soil types under natural rainfall and environmental conditions.The objective of this study was to quantitatively investigate the impact of soil type and season (soil moisture content) on the fate and transport of E. coli spread on four different temperate maritime soil types under natural rainfall conditions. We hypothesized that there would be a greater microbial risk to underlying groundwater with better-drained soil types than with relatively poorly drained soil types following the application of animal slurry. In addition, we hypothesized that E. coli cells spread on wetter spring soils would be transported in greater numbers than E. coli cells spread on drier soils in the summer.     

Characterization of lipid A from Pseudomonas aeruginosa O-antigenic B band lipopolysaccharide by 1D and 2D NMR and mass spectral analysis.

The Lipid A from the lipopolysaccharide of Pseudomonas aeruginosa was examined by high-field nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). The backbone structure and the position of phosphate substituents were unambiguously established by one- and two-dimensional 1H, 13C, and 31P NMR techniques on a de-O-acylated Lipid A sample. The Lipid A has a beta-(1,6)-glucosamine disaccharide structure which is substituted by phosphomonoesters through glycosidic bonds at C-1 and at C-4'. The configuration of the glycosidically linked phosphate at position C-1 was identified as alpha which is the same as that of Enterobacterial Lipid A. Chemical analysis revealed that the Lipid A contained 2-hydroxydodecanoic, 3-hydroxydodecanoic, dodecanoic, 3-hydroxydecanoic, and hexadecanoic acids in the approximate molar ratios 2.2:2.0:0.2:0.8:0.4. From 1H NMR and fast atom bombardment (FAB) mass spectrometry on the de-O-acylated Lipid A, it was established that both glucosamine residues were N-acylated by 3-hydroxydodecanoic acid. The identity and positions of the ester bound fatty acids in the intact Lipid A were investigated by negative ion FAB-MS. In addition to the hexaacyl and pentaacyl Lipid A species, a tetraacyl species was identified. Heterogeneity due to hydroxylated and nonhydroxylated dodecanoic acid esters could be uniquely localized to the nonreducing beta-glucosamine residue from the fragmentation pattern observed in the negative ion FAB-MS. The complete structure of the Lipid A from P. aeruginosa will be useful in understanding the determinants responsible for the endotoxin activity of this molecule.     

Functional heterogeneity of photosystem II in domain specific regions of the thylakoid membrane of spinach (Spinacia oleracea L.)

A mild sonication and phase fractionation method has been used to isolate five regions of the thylakoid membrane in order to characterize the functional lateral heterogeneity of photosynthetic reaction centers and light harvesting complexes. Low-temperature fluorescence and absorbance spectra, absorbance cross-section measurements, and picosecond time-resolved fluorescence decay kinetics were used to determine the relative amounts of photosystem II (PSII) and photosystem I (PSI), to determine the relative PSII antenna size, and to characterize the excited-state dynamics of PSI and PSII in each fraction. Marked progressive increases in the proportion of PSI complexes were observed in the following sequence: grana core (BS), whole grana (B3), margins (MA), stroma lamellae (T3), and purified stromal fraction (Y100). PSII antenna size was drastically reduced in the margins of the grana stack and stroma lamellae fractions as compared to the grana. Picosecond time-resolved fluorescence decay kinetics of PSII were characterized by three exponential decay components in the grana fractions, and were found to have only two decay components with slower lifetimes in the stroma. Results are discussed in the framework of existing models of chloroplast thylakoid membrane lateral heterogeneity and the PSII repair cycle. Kinetic modeling of the PSII fluorescence decay kinetics revealed that PSII populations in the stroma and grana margin fractions possess much slower primary charge separation rates and decreased photosynthetic efficiency when compared to PSII populations in the grana stack.     

Assaying the polyadenylation state of mRNAs

The poly(A) tail present at the 3' end of most eukaryotic mRNAs can play a critical role in message translation and stability. Therefore, identifying alterations in poly(A) tail length can yield important insights into an mRNA's function and subsequent physiological impact. Here, we present three methods for assaying polyadenylation of a specific mRNA in the context of total cellular RNA. The first method described, oligo(dT)/RNase H-Northern analysis, is the classic labor-intensive assay for polyadenylation and is included for historical reference and as a potential experimental control for the poly(A) test (PAT) assays described subsequently. The PAT methods-rapid amplification of cDNA ends-PAT (RACE-PAT), and ligase-mediated PAT (LM-PAT)-are polymerase chain reaction-driven assays that allow speed, sensitivity, and length quantitation. The PAT assays can be conducted in a single day and can readily detect the poly(A) status of an mRNA present in subnanogram quantities of total cellular RNA.     

Sequencing of the Dutch Elm Disease Fungus Genome Using the Roche/454 GS-FLX Titanium System in a Comparison of Multiple Genomics Core Facilities

As part of the DNA Sequencing Research Group of the Association of Biomolecular Resource Facilities, we have tested the reproducibility of the Roche/454 GS-FLX Titanium System at five core facilities. Experience with the Roche/454 system ranged from <10 to >340 sequencing runs performed. All participating sites were supplied with an aliquot of a common DNA preparation and were requested to conduct sequencing at a common loading condition. The evaluation of sequencing yield and accuracy metrics was assessed at a single site. The study was conducted using a laboratory strain of the Dutch elm disease fungus Ophiostoma novo-ulmi strain H327, an ascomycete, vegetatively haploid fungus with an estimated genome size of 30–50 Mb. We show that the Titanium System is reproducible, with some variation detected in loading conditions, sequencing yield, and homopolymer length accuracy. We demonstrate that reads shorter than the theoretical minimum length are of lower overall quality and not simply truncated reads. The O. novo-ulmi H327 genome assembly is 31.8 Mb and is comprised of eight chromosome-length linear scaffolds, a circular mitochondrial conti of 66.4 kb, and a putative 4.2-kb linear plasmid. We estimate that the nuclear genome encodes 8613 protein coding genes, and the mitochondrion encodes 15 genes and 26 tRNAs.     

A microaliquoting technique for precise histological annotation and optimization of cell content in frozen tissue specimens

Knowledge of the exact cell content of frozen tissue samples is of growing importance in genomic research. We developed a microaliquoting technique to measure and optimize the cell composition of frozen tumor specimens for molecular studies. Frozen samples of 31 mesothelioma cases were cut in alternating thin and thick sections. Thin sections were stained and evaluated visually. Thick sections, i.e., microaliquots, were annotated using bordering stained sections. A range of cellular heterogeneity was observed among and within samples. Precise annotation of samples was obtained by integration and compared to conventional single face and “front and back” section estimates of cell content. Front and back estimates were more highly correlated with block annotation by microaliquoting than were single face estimates. Both methods yielded discrepant estimates, however, and for some studies may not adequately account for the heterogeneity of mesothelioma or other malignancies with variable cellular composition. High yield and quality RNA was extracted from precision annotated, tumor-enriched subsamples prepared by combining individual microaliquots with the highest tumor cellularity estimates. Microaliquoting provides accurate cell content annotation and permits genomic analysis of enriched subpopulations of cells without fixation or amplification.     

Breeding ecology of the Seychelles Black Parrot Coracopsis barklyi

Knowledge of breeding ecology is required for many conservation interventions. The Seychelles Black Parrot Coracopsis barklyi, endemic to the island of Praslin, is vulnerable to extinction. We aimed to improve understanding of C. barklyi breeding ecology to aid conservation planning. We present the results of four years of research, including nesting cavity characteristics and availability, reproductive success, breeding parameters, parental behaviour and reproductive strategy. Thirty-six breeding attempts were studied over the four seasons. Nests were mainly located in Coco de Mer palms Lodoicea maldivica. Deeper cavities with more canopy cover were preferred. There may be a shortage of high-quality nesting cavities in intensive breeding seasons. Average clutch size was 2.2 eggs, incubation period was c. 15 d and egg fertility was 71%. Rats were key nest predators, causing the failure of up to 33% of breeding attempts. The probability of nest success was 53%. At least 57% of fledglings survived their first year. This species breeds cooperatively and practices a highly unusual side-by-side copulation. We discuss the implications of the results in the context of former, ongoing and potential conservation measures for C. barklyi including translocation, invasive species management, nest box provisioning, habitat restoration and further research.