Inactivation of murine norovirus on a range of copper alloy surfaces is accompanied by loss of capsid integrity

Norovirus is one of the most common causes of acute viral gastroenteritis. The virus is spread via the fecal-oral route, most commonly from infected food and water, but several outbreaks have originated from contamination of surfaces with infectious virus. In this study, a close surrogate of human norovirus causing gastrointestinal disease in mice, murine norovirus type 1 (MNV-1), retained infectivity for more than 2 weeks following contact with a range of surface materials, including Teflon (polytetrafluoroethylene [PTFE]), polyvinyl chloride (PVC), ceramic tiles, glass, silicone rubber, and stainless steel. Persistence was slightly prolonged on ceramic surfaces. A previous study in our laboratory observed that dry copper and copper alloy surfaces rapidly inactivated MNV-1 and destroyed the viral genome. In this new study, we have observed that a relatively small change in the percentage of copper, between 70 and 80% in copper nickels and 60 and 70% in brasses, had a significant influence on the ability of the alloy to inactivate norovirus. Nickel alone did not affect virus, but zinc did have some antiviral effect, which was synergistic with copper and resulted in an increased efficacy of brasses with lower percentages of copper. Electron microscopy of purified MNV-1 that had been exposed to copper and stainless steel surfaces suggested that a massive breakdown of the viral capsid had occurred on copper. In addition, MNV-1 that had been exposed to copper and treated with RNase demonstrated a reduction in viral gene copy number. This suggests that capsid integrity is compromised upon contact with copper, allowing copper ion access to the viral genome

Identification of diverse antibiotic resistant bacteria in agricultural soil with H218O stable isotope probing combined with high-throughput sequencing.

Background: We aimed to identify bacteria able to grow in the presence of several antibiotics including the ultra-broad-spectrum antibiotic meropenem in a British agricultural soil by combining DNA stable isotope probing (SIP) with high throughput sequencing. Soil was incubated with cefotaxime, meropenem, ciprofloxacin and trimethoprim in 18O-water. Metagenomes and the V4 region of the 16S rRNA gene from the labelled “heavy” and the unlabelled “light” SIP fractions were sequenced. Results: An increase of the 16S rRNA copy numbers in the “heavy” fractions of the treatments with 18O-water compared with their controls was detected. The treatments resulted in differences in the community composition of bacteria. Members of the phyla Acidobacteriota (formally Acidobacteria) were highly abundant after two days of incubation with antibiotics. Pseudomonadota (formally Proteobacteria) including Stenotrophomonas were prominent after four days of incubation. Furthermore, a metagenome-assembled genome (MAG-1) from the genus Stenotrophomonas (90.7% complete) was retrieved from the heavy fraction. Finally, 11 antimicrobial resistance genes (ARGs) were identified in the unbinned-assembled heavy fractions, and 10 ARGs were identified in MAG-1. In comparison, only two ARGs from the unbinned-assembled light fractions were identified. Conclusions: The results indicate that both non-pathogenic soil-dwelling bacteria as well as potential clinical pathogens are present in this agricultural soil and several ARGs were identified from the labelled communities, but it is still unclear if horizontal gene transfer between these groups can occur

QTLs for shelf life in lettuce co-locate with those for leaf biophysical properties but not with those for leaf developmental traits

Developmental and biophysical leaf characteristics that influence post-harvest shelf life in lettuce, an important leafy crop, have been examined. The traits were studied using 60 informative F9 recombinant inbed lines (RILs) derived from a cross between cultivated lettuce (Lactuca sativa cv. Salinas) and wild lettuce (L. serriola acc. UC96US23). Quantitative trait loci (QTLs) for shelf life co-located most closely with those for leaf biophysical properties such as plasticity, elasticity, and breakstrength, suggesting that these are appropriate targets for molecular breeding for improved shelf life. Significant correlations were found between shelf life and leaf size, leaf weight, leaf chlorophyll content, leaf stomatal index, and epidermal cell number per leaf, indicating that these pre-harvest leaf development traits confer post-harvest properties. By studying the population in two contrasting environments in northern and southern Europe, the genotype by environment interaction effects of the QTLs relevant to leaf development and shelf life were assessed. In total, 107 QTLs, distributed on all nine linkage groups, were detected from the 29 traits. Only five QTLs were common in both environments. Several areas where many QTLs co-located (hotspots) on the genome were identified, with relatively little overlap between developmental hotspots and those relating to shelf life. However, QTLs for leaf biophysical properties (breakstrength, plasticity, and elasticity) and cell area correlated well with shelf life, confirming that the ideal ideotype lettuce should have small cells with strong cell walls. The identification of QTLs for leaf development, strength, and longevity will lead to a better understanding of processability at a genetic and cellular level, and allow the improvement of salad leaf quality through marker-assisted breeding

Desk studies on feasibility of horizontal standard rapid methods for detection of E. coli (including E. coli O157) and Salmonella

The emerging methods becoming available for the rapid detection and enumeration of E. coli (including E. coli O157) and Salmonella in sludges, soil and treated biowastes have been evaluated with a view to possible future standardisation. The main methods that are available for the detection and enumeration of E. coli (including E. coli O157) and Salmonella have been developed largely for analysis of food and water and can be broadly divided into four groups. Proprietary Quantitray® technology, equivalent to the 5-tube most probable number (MPN) technique, employing disposable plastic trays for enumeration of E. coli and Salmonella. Immunological, involving a short or overnight pre-enrichment of the target organism followed by specific detection of cellular antigen in either a lateral flow device or following immunomagnetic capture. Molecular, involving PCR amplification of target DNA sequences from low numbers of cells, or preferably following a short pre-enrichment of the organism to amplify numbers and demonstrate viability prior to molecular detection. Physico-chemical, involving techniques such as measurement of impedance changes during enrichment and growth in appropriate media. The merits of each are described, in relation to their suitability for use with sludge, soil and biowastes. Since the majority of agar and MPN broth techniques take between 24-96 hours for identification and enumeration, we define “rapid” as any technique that detects, and if possible, enumerates the target organism in under 24 hours.All of the methods described have strengths and weaknesses, dependent on not only the Regulators’ types of requirements for sludge, soil and biowaste analysis but also their sensitivity, specificity, speed and cost. It is unlikely therefore that there can be only one methodology applicable to both E. coli (and E. coli O157) and Salmonella detection. Nevertheless, it is considered feasible to formulate horizontal standards to cover rapid analysis of E. coli and Salmonella in sludge, soil, soil improvers, growing media, and biowaste. None of the methods have been extensively evaluated for sewage sludge, soils or biowastes. As such, there is an urgent need for their modification and evaluation as part of the next phase of the Project Horizonta

Desk studies on feasibility of horizontal standard rapid methods for detection of Clostridium perfringens and enterococci in sludges, soil, soil improvers, growing media and biowastes

The existing methods currently available for the detection and enumeration of clostridia and enterococci in sludges and treated biowastes have been evaluated with a view to possible standardisation. The main methods used for the detection and enumeration of Clostridium and Enterococcus spp. have been developed largely for analysis of food and water and can be broadly divided into three groups. Quantification of colonies on agar media; most probable number (MPN) quantification in indicator broth using conventional test tube technology; and proprietary Quantitray® technology equivalent to the 5-tube MPN technique employing disposable plastic trays for enumeration of enterococci. The merits of each are described. At least one report has suggested that the use of m-CP agar medium, which is used in the reference method in the European Union, is not suitable for recovering C. perfringens spores from groundwater. This questions its possible use as a method for detecting C. perfringens in sludge, soil, soil improvers, growing media, and biowaste.Indeed, all of the methods described for detection of C. perfringens and enterococci have strengths and weaknesses, dependent on not only the Regulators’ types of requirements for sludge, soil and biowaste analysis but also their sensitivity, specificity, speed and cost. Nevertheless, it is considered feasible to formulate horizontal standards to cover analysis of C. perfringens and enterococci in sludge, soil, soil improvers, growing media, and biowaste. However, none of the methods have been extensively evaluated for these waste types. As such, there is an urgent need for their modification and evaluation as part of the next phase of the Project Horizonta

Novel insights into the Proteus mirabilis crystalline biofilm using real-time imaging

The long-term use of indwelling catheters results in a high risk from urinary tract infections (UTI) and blockage. Blockages often occur from crystalline deposits, formed as the pH rises due to the action of urease-producing bacteria; the most commonly found species being Proteus mirabilis. These crystalline biofilms have been found to develop on all catheter materials with P. mirabilis attaching to all surfaces and forming encrustations. Previous studies have mainly relied on electron microscopy to describe this process but there remains a lack of understanding into the stages of biofilm formation. Using an advanced light microscopy technique, episcopic differential interference contrast (EDIC) microscopy combined with epifluorescence (EF), we describe a non-destructive, non-contact, real-time imaging method used to track all stages of biofilm development from initial single cell attachment to complex crystalline biofilm formation. Using a simple six-well plate system, attachment of P. mirabilis (in artificial urine) to sections of silicone and hydrogel latex catheters was tracked over time (up to 24 days). Using EDIC and EF we show how initial attachment occurred in less than 1 h following exposure to P. mirabilis. This was rapidly followed by an accumulation of an additional material (indicated to be carbohydrate based using lectin staining) and the presence of highly elongated, motile cells. After 24 h exposure, a layer developed above this conditioning film and within 4 days the entire surface (of both catheter materials) was covered with diffuse crystalline deposits with defined crystals embedded. Using three-dimensional image reconstruction software, cells of P. mirabilis were seen covering the crystal surfaces. EDIC microscopy could resolve these four components of the complex crystalline biofilm and the close relationship between P. mirabilis and the crystals. This real-time imaging technique permits study of this complex biofilm development with no risk of artefacts due to sample manipulation. A full understanding of the stages and components involved in crystalline encrustation formation will aid in the development of new protocols to manage and ultimately prevent catheter blockage

Comparison of polyvinyl chloride membrane electrodes sensitive to alkylphosphonium ions for the determination of the electrical difference (ΔΨ) of Streptococcus mutans and Lactobacillus casei

Polyvinyl chloride membrane electrodes sensitive to tetraphenyl phosphonium (TPP+), butyltriphenyl phosphonium (bTPP+), and methyltriphenyl phosphonium (mTPP+) ions have been compared for the determination of the electrical potential difference (ΔΨ) of the oral bacteria, Streptococcus mutans DR0001 6 and Lactobacillus casei RB1014. All three types of electrode proved suitable for determining Δ,gY, although the TPP+-sensitive electrode was particularly susceptible to interference by protonmotive force (Δp) dissipators known to inhibit sugar uptake by the bacteria. The mTPP+-sensitive electrode was the least affected. Similarly, both strains had high nonspecific binding capacity for TPP+ and bTPP+ ions, and this increased for all three ions when the bacteria were heated to 80°C for 1 h to abolish glucose uptake and metabolism. This heat-treatment procedure is therefore not a suitable control for determination of nonspecific binding to cells. However, 1% ( v v) toluene, 20 μm gramicidin, or 10 μm valinomycin effectively depolarized the bacteria without interfering with nonspecific binding. The ionophores were therefore used subsequently for the determination of nonspecific binding of the lipid-soluble cations. The mTPP+ ion and corresponding electrode proved the most effective system, and ΔΨ values of -89 and -107 mV were obtained for S. mutans and L. casei, respectively, harvested from glucose-limited continuous cultures and incubated in 100 mm Hepes-KOH buffer (pH 7.0), containing 1 mm dithiothreitol and 10 mm glucose. Although the ΔΨ of S. mutans decreased significantly in the presence of Mes-KOH and potassium phosphate buffers at pH 7.0, it increased to -119 mV in Tris-HCl buffer (pH 7.0). Addition of 100 mm KCl to the Tris buffer showed that the inhibiting effects of the former buffers resulted from their high K+ content. Glucose uptake and acid production by both strains was also markedly inhibited by high concentrations of Na+. The measuring system revealed that the ΔΨ of both strains decreased by up to 50% in the presence of 100 mm Na+, supporting the proposed role for Na+ in the deenergization of Δp in oral bacteria.</p