This study investigated the dissolution-based toxicity mechanism for silver nanoparticles to Escherichia coli K12. The silver nanoparticles, synthesised in the vapour phase, are effective anti-bacterial agents against the Gram-negative bacterium, E. coli K12. The nanoparticles associate with the bacterial cell wall, appearing to interact with the outer and inner membranes, and then dissolve to release Ag(+) into the cell and affect a transcriptional response. The dissolution of these nanoparticles in a modified LB medium was measured by inductively coupled plasma mass spectrometry (ICP-MS) and has been shown to follow a simple first-order dissolution process proportional to the decreasing surface area of the nanoparticles. However, the resulting solution phase concentration of Ag(+), demonstrated by the ICP-MS data, is not sufficient to cause the observed effects, including inhibition of bacterial growth and the differential expression of Cu(+) stress response genes. These data indicate that dissolution at the cell membrane is the primary mechanism of action of silver nanoparticles, and the Ag(+) concentration released into the bulk solution phase has only limited anti-bacterial efficacy.

We report the whole-transcriptome response of Escherichia coli bacteria to acute treatment with silver nanoparticles (AgNPs) or silver ions [Ag(I)] as silver nitrate using gene expression microarrays. In total, 188 genes were regulated by both silver treatments, 161 were up-regulated and 27 were down-regulated. Significant regulation was observed for heat shock response genes in line with protein denaturation associated with protein structure vulnerability indicating Ag(I)-labile -SH bonds. Disruption to iron-sulphur clusters led to the positive regulation of iron-sulphur assembly systems and the expression of genes for iron and sulphate homeostasis. Further, Ag ions induced a redox stress response associated with large (>600-fold) up-regulation of the E. coli soxS transcriptional regulator gene. Ag(I) is isoelectronic with Cu(I), and genes associated with copper homeostasis were positively regulated indicating Ag(I)-activation of copper signalling. Differential gene expression was observed for the silver nitrate and AgNP silver delivery. Nanoparticle delivery of Ag(I) induced the differential regulation of 379 genes; 309 genes were uniquely regulated by silver nanoparticles and 70 genes were uniquely regulated by silver nitrate. The differential silver nanoparticle-silver nitrate response indicates that the toxic effect of labile Ag(I) in the system depends upon the mechanism of delivery to the target cell.

a b s t r a c tThe presence of Escherichia coli in environmental waters is considered as evidence of faecal contamination and is therefore commonly used as an indicator in both water quality and food safety analysis. The long period of time between sample collection and obtaining results from existing culture based methods means that contamination events may already impact public health by the time they are detected. The adoption of molecular based methods for E. coli could significantly reduce the time to detection. A new quantitative real-time PCR (qPCR) assay was developed to detect the ybbW gene sequence, which was found to be 100% exclusive and inclusive (specific and sensitive) for E. coli and directly compared for its ability to quantify E. coli in environmental waters against colony counts, quantitative real-time NASBA (qNASBA) targeting clpB and qPCR targeting uidA. Of the 87 E. coli strains tested, 100% were found to be ybbW positive, 94.2% were culture positive, 100% were clpB positive and 98.9% were uidA positive. The qPCR assays had a linear range of quantification over several orders of magnitude, and had high amplification efficiencies when using single isolates as a template. This compared favourably with qNASBA which showed poor linearity and amplification efficiency. When the assays were applied to environmental water samples, qNASBA was unable to reliably quantify E. coli while both qPCR assays were capable of predicting E. coli concentrations in environmental waters. This study highlights the inability of qNASBA targeting mRNA to quantify E. coli in environmental waters, and presents the first E. coli qPCR assay with 100% target exclusivity. The application of a highly exclusive and inclusive qPCR assay has the potential to allow water quality managers to reliably and rapidly detect and quantify E. coli and therefore take appropriate measures to reduce the risk to public health posed by faecal contamination.Crown

Aquatic microbial communities are central to biogeochemical processes that maintain Earth's habitability. However, there is a significant paucity of data collected from these species in their natural environment. To address this, a suite of ocean-deployable sampling and sensing instrumentation has been developed to retrieve, archive and analyse water samples and their microbial fraction using state of the art genetic assays. Recent deployments have shed new light onto the role microbes play in essential ocean processes and highlight the risks they may pose to coastal populations. Although current designs are generally too large, complex and expensive for widespread use, a host of emerging bio-analytical technologies have the potential to revolutionise this field and open new possibilities in aquatic microbial metrology.

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The selective amplification of E. coli nucleic acid sequences could be used for the early warning of faecal contamination in environmental samples. Modified assays for E. coli DNA and RNA markers are presented with improved integrity and performance over existing methods, and demonstrated using 'ready mixed', preserved reagent mixtures.GeneElute PCR Purification Kit (Sigma, UK) and used directly for RNA synthesis using the Hi Scribe T7 RNA Synthesis Kit (NEB, USA) according to the manufacturers recommended protocol. The synthesised RNA was purified using the RNeasy Mini Kit (Qiagen, UK), and contaminating dsDNA was eliminated using RQ1 RNase-Free DNase (Promega, UK) according to the manufacturers recommended protocol. DNA elimination was confirmed by a null Taqbased PCR. The DNA-free RNA was subsequently purified a second time using the RNeasy Clean-up procedure. The mass of RNA was estimated using a BioAnalyser Instrument, and the RNA 6000 Nano Kit (Agilent, UK). This was used to estimate the number of copies based upon the RNA sequence. A series of RNA copy number standards were prepared by diluting the RNA sample in RT-PCR grade water to between 10 and 10 5 copies per microlitre. Standards were prepared from stock RNA solution immediately prior to use.