Performance of a Novel Molecular Method in the Diagnosis of Late-Onset Sepsis in Very Low Birth Weight Infants

OBJECTIVE: To compare the use of a generic molecular assay to 'standard' investigations used to assist the diagnosis of late onset bacterial sepsis in very low birth weight infants (VLBW, <1500g).

METHODS: VLBW infants, greater than 48 hours of age, who were clinically suspected to have sepsis were investigated using standard tests (full blood count, C-reactive protein (at presentation) and blood culture), in addition, blood was taken for a universal molecular assay (16S rRNA reverse transcriptase PCR) for comparison. Clinical data were recorded during the suspected infection episode. A validated sepsis score (NEO-KISS) was used to retrospectively determine the presence of sepsis (independent of blood culture). The performance of each of the tests were compared by sensitivity, specificity, positive/negative likihood ratios (+/-LR) and postive/negative predictive values (PPV/NPV).

RESULTS: Sixty-five babies with suspected clinical sepsis were prospectively included. The performance indicators are presented with 95% confidence limits. For the detection of bacteria, blood culture had sensitivity of 0.57 (0.34-0.78), specificity of 0.45 (0.30-0.61); +LR of 1.05 (0.66-1.66) and-LR of 0.94 (0.52-1.7); PPV of 33.3 (18.56-50.97) and NPV of 68.97 (49.17-87.72). Serum CRP had sensitivity of 0.92 (0.64-1) and specificity of 0.36 (0.17-0.59); +LR of 1.45 (1-2.1) and-LR of 0.21 (0.03-1.5); PPV of 44.46 (26.6-66.6) and NPV of 88.9 (51.8-99.7). The universal molecular assay had sensitivity of 0.76 (0.53-0.92), specificity of 0.95 (0.85-0.99); +LR of 16.8 (4.2-66.3) and-LR of 0.25 (0.1-0.5); PPV of 88.9 (65.3-98.6) and NPV of 89.4 (76.9-96.5).

CONCLUSIONS: In VLBW infants this universal molecular assay performed better in the diagnosis of late onset sepsis (LOS) than blood culture and CRP. Further development is required to explore and improve the performance of the assay in real-time diagnosis.

Using microbiological tracers to assess the impact of winter land use restrictions on the quality of stream headwaters in a small catchment.

Diverse land use activities can elevate risk of microbiological contamination entering stream headwaters. Spatially distributed water quality monitoring carried out across a 17km(2) agricultural catchment aimed to characterize microbiological contamination reaching surface water and investigate whether winter agricultural land use restrictions proved effective in addressing water quality degradation. Combined flow and concentration data revealed no significant difference in fecal indicator organism (FIO) fluxes in base flow samples collected during the open and prohibited periods for spreading organic fertilizer, while relative concentrations of Escherichia coli, fecal streptococci and sulfite reducing bacteria indicated consistently fresh fecal pollution reached aquatic receptors during both periods. Microbial source tracking, employing Bacteroides 16S rRNA gene markers, demonstrated a dominance of bovine fecal waste in river water samples upstream of a wastewater treatment plant discharge during open periods. This contrasted with responses during prohibited periods where human-derived signatures dominated. Differences in microbiological signature, when viewed with hydrological data, suggested that increasing groundwater levels restricted vertical infiltration of effluent from on-site wastewater treatment systems and diverted it to drains and surface water. Study results reflect seasonality of contaminant inputs, while suggesting winter land use restrictions can be effective in limiting impacts of agricultural wastes to base flow water quality.

The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability

The interplay between methylation and demethylation of histone lysine residues is an essential component of gene expression regulation and there is considerable interest in elucidating the roles of proteins involved. Here we report that histone demethylase KDM4A/JMJD2A, which is involved in the regulation of cell proliferation and is overexpressed in some cancers, interacts with RNA Polymerase I, associates with active ribosomal RNA genes and is required for serum-induced activation of rDNA transcription. We propose that KDM4A controls the initial stages of transition from 'poised', non-transcribed rDNA chromatin into its active form. We show that PI3K, a major signalling transducer central for cell proliferation and survival, controls cellular localization of KDM4A and consequently its association with ribosomal DNA through the SGK1 downstream kinase. We propose that the interplay between PI3K/SGK1 signalling cascade and KDM4A constitutes a mechanism by which cells adapt ribosome biogenesis level to the availability of growth factors and nutrients.

Abundance and diversity of sedimentary bacterial communities in a coastal productive setting in the Western Irish Sea

The bacterial community composition and biomass abundance from a depositional mud belt in the western Irish Sea and regional sands were investigated by phospholipid ester-linked fatty acid profiling, denaturing gradient gel electrophoresis and barcoded pyrosequencing of 16S rRNA genes. The study area varied by water depth (12-111 m), organic carbon content (0.09-1.57% TOC), grain size, hydrographic regime (well-mixed vs. stratified), and water column phytodetrital input (represented by algal polyunsaturated PLFA). The relative abundance of bacterial-derived PLFA (sum of methyl-branched, cyclopropyl and odd-carbon number PLFA) was positively correlated with fine-grained sediment, and was highest in the depositional mud belt. A strong association between bacterial biomass and eukaryote primary production was suggested based on observed positive correlations with total nitrogen and algal polyunsaturated fatty acids. In addition, 16S rRNA genes affiliated to the classes Clostridia and Flavobacteria represented a major proportion of total 16S rRNA gene sequences. This suggests that benthic bacterial communities are also important degraders of phytodetrital organic matter and closely coupled to water column productivity in the western Irish Sea.

Biological phosphorus removal during high-rate, low-temperature, anaerobic digestion of wastewater.

We report, for the first time, extensive biologically-mediated phosphate removal from wastewater during high-rate anaerobic digestion (AD). A hybrid sludge bed/fixed-film (packed pumice stone) reactor was employed for low-temperature (12°C) anaerobic treatment of synthetic sewage wastewater. Successful phosphate removal from the wastewater (up to 78% of influent phosphate) was observed, mediated by biofilms in the reactor. Scanning electron microscopy and energy dispersive X-ray analysis revealed the accumulation of elemental phosphorus (~2%) within the sludge bed and fixed-film biofilms. 4’, 6-diamidino-2-phenylindole (DAPI) staining indicated phosphorus accumulation was biological in nature and mediated through the formation of intracellular inorganic polyphosphate (polyP) granules within these biofilms. DAPI staining further indicated that polyP accumulation was rarely associated with free cells. Efficient and consistent chemical oxygen demand (COD) removal was recorded, throughout the 732-day trial, at applied organic loading rates between 0.4-1.5 kg COD m-3 d-1 and hydraulic retention times of 8-24 hours, while phosphate removal efficiency ranged from 28-78% on average per phase. Analysis of protein hydrolysis kinetics and the methanogenic activity profiles of the biomass revealed the development, at 12˚C, of active hydrolytic and methanogenic populations. Temporal microbial changes were monitored using Illumina Miseq analysis of bacterial and archaeal 16S rRNA gene sequences. The dominant bacterial phyla present in the biomass at the conclusion of the trial were the Proteobacteria and Firmicutes and the dominant archaeal genus was Methanosaeta. Trichococcus and Flavobacterium populations, previously associated with low temperature protein degradation, developed in the reactor biomass. The presence of previously characterised polyphosphate accumulating organisms (PAOs) such as Rhodocyclus, Chromatiales, Actinobacter and Acinetobacter was recorded at low numbers. However, it is unknown as yet if these were responsible for the luxury polyP uptake observed in this system. The possibility of efficient phosphate removal and recovery from wastewater during AD would represent a major advance in the scope for widespread application of anaerobic wastewater treatment technologies.

Metagenomic characterisation of the viral community of lough neagh, the largest freshwater lake in Ireland

Lough Neagh is the largest and the most economically important lake in Ireland. It is also one of the most nutrient rich amongst the world's major lakes. In this study, 16S rRNA analysis of total metagenomic DNA from the water column of Lough Neagh has revealed a high proportion of Cyanobacteria and low levels of Actinobacteria, Acidobacteria, Chloroflexi, and Firmicutes. The planktonic virome of Lough Neagh has been sequenced and 2,298,791 2×300 bp Illumina reads analysed. Comparison with previously characterised lakes demonstrates that the Lough Neagh viral community has the highest level of sequence diversity. Only about 15% of reads had homologs in the RefSeq database and tailed bacteriophages (Caudovirales) were identified as a major grouping. Within the Caudovirales, the Podoviridae and Siphoviridae were the two most dominant families (34.3% and 32.8% of the reads with sequence homology to the RefSeq database), while ssDNA bacteriophages constituted less than 1% of the virome. Putative cyanophages were found to be abundant. 66,450 viral contigs were assembled with the largest one being 58,805 bp; its existence, and that of another 34,467 bp contig, in the water column was confirmed. Analysis of the contigs confirmed the high abundance of cyanophages in the water column.