Use of whole genome sequencing of commensal Escherichia coli in pigs for antimicrobial resistance surveillance, United Kingdom, 2018

BackgroundSurveillance of commensal Escherichia coli, a possible reservoir of antimicrobial resistance (AMR) genes, is important as they pose a risk to human and animal health. Most surveillance activities rely on phenotypic characterisation, but whole genome sequencing (WGS) presents an alternative.AimIn this retrospective study, we tested 515 E. coli isolated from pigs to evaluate the use of WGS to predict resistance phenotype.MethodsMinimum inhibitory concentration (MIC) was determined for nine antimicrobials of clinical and veterinary importance. Deviation from wild-type, fully-susceptible MIC was assessed using European Committee on Antimicrobial Susceptibility Testing (EUCAST) epidemiological cut-off (ECOFF) values. Presence of AMR genes and mutations were determined using APHA SeqFinder. Statistical two-by-two table analysis and Cohen's kappa (k) test were applied to assess genotype and phenotype concordance.ResultsOverall, correlation of WGS with susceptibility to the nine antimicrobials was 98.9% for test specificity, and 97.5% for the positive predictive value of a test. The overall kappa score (k = 0.914) indicated AMR gene presence was highly predictive of reduced susceptibility and showed excellent correlation with MIC. However, there was variation for each antimicrobial; five showed excellent correlation; four very good and one moderate. Suggested ECOFF adjustments increased concordance between genotypic data and kappa values for four antimicrobials.ConclusionWGS is a powerful tool for accurately predicting AMR that can be used for national surveillance purposes. Additionally, it can detect resistance genes from a wider panel of antimicrobials whose phenotypes are currently not monitored but may be of importance in the future

The health benefits and risks of growing-your-own produce in an urban environment

The practice of gardening and growing-your-own (GYO) produce in urban areas, has been associated with many potential benefits to health from increased fruit and vegetable consumption and exercise, but also health risks arising from exposure to potentially toxic elements (such as cadmium: Cd, and lead: Pb) in urban soils. However, the potential health benefits of gardening are currently overlooked by authorities during assessments of contaminated land, which may result in access to urban gardens and allotments being incorrectly restricted or removed because of concerns over the impact to human health. The trade-off between health benefits and risks is investigated in this thesis through: the sampling and analysis of the properties of allotment soils (chapter 2); a comparison of plant uptake models (chapter 3) verified using a pot experiment (chapter 4), and a questionnaire survey investigating the effect of gardeners’ routines on benefits and risks (chapter 5). The different areas of study are combined in the creation of a model framework developed to estimate health benefits and risks attributable to urban gardening (chapter 6). A total of 149 allotment plots were sampled across the city of Nottingham and analysed in the laboratory for trace element concentrations by ICP MS. Concentrations of lead (Pb) in top-soils exceeded the category 4 screening level of 84 mg Pb kg-1 dw in most plots (n=129), with a median concentration across all plots of 214 mg Pb kg-1 dw. However, other elemental concentrations (As, Cd, Cr) were below the respective C4SLs for an allotment land-use and unlikely to pose health risks. In comparison, analysis of the results of a questionnaire survey of 120 gardeners’ routines, suggested that gardening more regularly in autumn and winter was positively correlated with categories of increased fruit and vegetable consumption and exercise (one-way ANOVA; p < 0.05). Furthermore, gardeners over the age of 45 scored significantly better in the physical health (but not the mental health) categories of the SF 36 v 2 questionnaire compared to standardised scores when results were adjusted for age and gender (Table 5.9). Health benefits and risks were combined using a prototype Bayesian Network (BN) model developed according to the principles of CLEA (Contaminated Land Exposure Assessment model). Health outcomes from benefits (e.g. fruit and vegetable consumption) and risks (e.g. lead toxicity) were derived using a sub-model; requiring odds and hazard ratio’s for the outcomes of disease. The model predictions suggested that for the study population, whilst there were some benefits attributable to urban gardening, these appeared to be outweighed by health risks from potential exposure to Pb. Further work is required to confirm or refute this finding and to validate and improve the BN model. The study also highlights the importance of considering health benefits of gardening in risk assessment in future for urban gardeners. Future work should also consider weaknesses highlighted in the use of the CLEA model to predict health risks for gardeners, especially in the prediction of plant uptake of toxic elements, and the limited data availability describing the toxic effects of chemicals exceeding a threshold value which may or may not result in SPOSH

Development of a sensitive and rapid method for the measurement of total microbial activity using fluorescein diacetate (FDA) in a range of soils

Fluorescein diacetate (FDA) hydrolysis is widely accepted as an accurate and simple method for measuring total microbial activity in a range of environmental samples, including soils. Colourless fluorescein diacetate is hydrolysed by both free and membrane bound enzymes, releasing a coloured end product fluorescein which can be measured by spectrophotometry. The current method for measuring FDA hydrolysis in soils is limited in its application. FDA activity was very low in sandy and clayey soils. The low activity observed for these soil types was made difficult to measure by the original authors choice of solvent for terminating the hydrolysis reaction. Acetone (50% v/v) was found to be most efficient at stopping the hydrolysis reaction. During this study acetone (50% v/v) was found to cause a decrease of approximately 37% in the absorbance of fluorescein produced by the soil samples measured. Although this colour loss is independent of initial fluorescein concentration, it makes the measurement of FDA hydrolytic activity extremely difficult in soils with low microbial activity i.e. sandy and/or clayey soils. Chloroform/methanol (2:1 v/v) was found to successfully stop the hydrolysis reaction for up to 50 min in a range of soil samples without causing the loss of colour observed with acetone. By changing the solvent used for terminating the hydrolysis reaction, low activity soils could be measured successfully. Other parameters of the hydrolysis reaction were optimised for the measurement of soil samples including effect of pH. optimum temperature of incubation, amount of soil, time of incubation, amount of substrate and preparation of suitable standards. A new, more sensitive method is proposed adapted from the original method, which provides a more accurate determination of FDA hydrolysis in a wide range of soils

A genomic epidemiological study shows that prevalence of antimicrobial resistance in Enterobacterales is associated with the livestock host, as well as antimicrobial usage

Enterobacterales from livestock are potentially important reservoirs for antimicrobial resistance (AMR) to pass through the food chain to humans, thereby increasing the AMR burden and affecting our ability to tackle infections. In this study 168 isolates from four genera of the order Enterobacterales, primarily Escherichia coli, were purified from livestock (cattle, pigs and sheep) faeces from 14 farms in the United Kingdom. Their genomes were resolved using long- and short-read sequencing to analyse AMR genes and their genetic context, as well as to explore the relationship between AMR burden and on-farm antimicrobial usage (AMU), in the three months prior to sampling. Although E. coli isolates were genomically diverse, phylogenetic analysis using a core-genome SNP tree indicated pig isolates to generally be distinct from sheep isolates, with cattle isolates being intermediates. Approximately 28 % of isolates harboured AMR genes, with the greatest proportion detected in pigs, followed by cattle then sheep; pig isolates also harboured the highest number of AMR genes per isolate. Although 90 % of sequenced isolates harboured diverse plasmids, only 11 % of plasmids (n=58 out of 522) identified contained AMR genes, with 91 % of AMR plasmids being from pig, 9 % from cattle and none from sheep isolates; these results indicated that pigs were a principle reservoir of AMR genes harboured by plasmids and likely to be involved in their horizontal transfer. Significant associations were observed between AMU (mg kg−1) and AMR. As both the total and the numbers of different antimicrobial classes used on-farm increased, the risk of multi-drug resistance (MDR) in isolates rose. However, even when AMU on pig farms was comparatively low, pig isolates had increased likelihood of being MDR; harbouring relatively more resistances than those from other livestock species. Therefore, our results indicate that AMR prevalence in livestock is not only influenced by recent AMU on-farm but also livestock-related factors, which can influence the AMR burden in these reservoirs and its plasmid mediated transmission

The measurement and normalisation of dielectric dissipation factor for diagnostics of transformer insulation

This article describes additional features of the method of Dielectric Dissipation Factor (DDF)/Tangent Delta (tgδ) measurement for a more accurate diagnosis of the condition of the transformer solid insulation. The proposed method is based on determining the DDF weight of solid insulation and oil in the measured value of DDF for the proper insulation zone of the transformer. The article proposes normalisation of DDF values according to the rated voltage of the transformer, and the analysis of the impact of design combining insulation and its condition on recalculation of DDF measurement results at a given temperature to the base temperature

S55746 is a novel orally active BCL-2 selective and potent inhibitor that impairs hematological tumor growth

Escape from apoptosis is one of the major hallmarks of cancer cells. The B-cell Lymphoma 2 (BCL-2) gene family encodes pro-apoptotic and anti-apoptotic proteins that are key regulators of the apoptotic process. Overexpression of the pro-survival member BCL-2 is a well-established mechanism contributing to oncogenesis and chemoresistance in several cancers, including lymphoma and leukemia. Thus, BCL-2 has become an attractive target for therapeutic strategy in cancer, as demonstrated by the recent approval of ABT-199 (Venclexta™) in relapsed or refractory Chronic Lymphocytic Leukemia with 17p deletion. Here, we describe a novel orally bioavailable BCL-2 selective and potent inhibitor called S55746 (also known as BCL201). S55746 occupies the hydrophobic groove of BCL-2. Its selectivity profile demonstrates no significant binding to MCL-1, BFL-1 (BCL2A1/A1) and poor affinity for BCL-XL. Accordingly, S55746 has no cytotoxic activity on BCL-XL-dependent cells, such as platelets. In a panel of hematological cell lines, S55746 induces hallmarks of apoptosis including externalization of phosphatidylserine, caspase-3 activation and PARP cleavage. Ex vivo, S55746 induces apoptosis in the low nanomolar range in primary Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma patient samples. Finally, S55746 administered by oral route daily in mice demonstrated robust anti-tumor efficacy in two hematological xenograft models with no weight lost and no change in behavior. Taken together, these data demonstrate that S55746 is a novel, well-tolerated BH3-mimetic targeting selectively and potently the BCL-2 protein

Genomic network analysis of environmental and livestock F-type 1 plasmid populations

F-type plasmids are diverse and of great clinical significance, often carrying genes conferring antimicrobial resistance (AMR) such as extended-spectrum β-lactamases, particularly in Enterobacterales. Organising this plasmid diversity is challenging, and current knowledge is largely based on plasmids from clinical settings. Here, we present a network community analysis of a large survey of F-type plasmids from environmental (influent, effluent and upstream/downstream waterways surrounding wastewater treatment works) and livestock settings. We use a tractable and scalable methodology to examine the relationship between plasmid metadata and network communities. This reveals how niche (sampling compartment and host genera) partition and shape plasmid diversity. We also perform pangenome-style analyses on network communities. We show that such communities define unique combinations of core genes, with limited overlap. Building plasmid phylogenies based on alignments of these core genes, we demonstrate that plasmid accessory function is closely linked to core gene content. Taken together, our results suggest that stable F-type plasmid backbone structures can persist in environmental settings while allowing dramatic variation in accessory gene content that may be linked to niche adaptation. The association of F-type plasmids with AMR may reflect their suitability for rapid niche adaptation

Harmonisation of European tests for serological diagnosis of Brucella infection in bovines

Summary The principal methods for the serological diagnosis of bovine brucellosis are the complement fixation test (CFT), serum agglutination test (SAT), Rose-Bengal test (RBT), indirect enzyme-linked immunosorbent assay (iELISA) and more recently the competitive ELISA (cELISA) and the fluorescent polarisation assay (FPA). Guidelines set by the World Organisation for Animal Health (OIE) describe methods and diagnostic thresholds for each of these tests. Many countries have adopted these methods for the purposes of eradication of brucellosis and have legislated for the use of these tests (the CFT and SAT in particular) for the prevention of the spread of the disease through international trade. Within the European Union (EU) each member state has a National Reference Laboratory which regulates the quality of brucellosis diagnosis and works to the recommendations set by the OIE. This article describes the results from the first three EU ring trials assessing the harmonisation of diagnostic tests between each member state. The general level of harmony for SAT, CFT, and iELISA was found to be good, but issues of standardisation of the RBT, cELISA and FPA remain. The cELISA and FPA in particular need further work to create European harmony. The ring trials also proved successful at providing specific evidence of poor performance in some areas. The decision on whether or not to take action on the basis of these results rested with the individual laboratories concerned. The increase in the number of participants in these trials over time reflected the enlargement of the EU and increased the need for quality assurance

Identification of a New Antimicrobial Resistance Gene Provides Fresh Insights Into Pleuromutilin Resistance in Brachyspira hyodysenteriae, Aetiological Agent of Swine Dysentery

Brachyspira hyodysenteriae is the aetiological agent of swine dysentery, a globally distributed disease that causes profound economic loss, impedes the free trade and movement of animals, and has significant impact on pig health. Infection is generally treated with antibiotics of which pleuromutilins, such as tiamulin, are widely used for this purpose, but reports of resistance worldwide threaten continued effective control. In Brachyspira hyodysenteriae pleuromutilin resistance has been associated with mutations in chromosomal genes encoding ribosome-associated functions, however the dynamics of resistance acquisition are poorly understood, compromising stewardship efforts to preserve pleuromutilin effectiveness. In this study we undertook whole genome sequencing (WGS) and phenotypic susceptibility testing of 34 UK field isolates and 3 control strains to investigate pleuromutilin resistance in Brachyspira hyodysenteriae. Genome-wide association studies identified a new pleuromutilin resistance gene, tva(A) (tiamulin valnemulin antibiotic resistance), encoding a predicted ABC-F transporter. In vitro culture of isolates in the presence of inhibitory or sub-inhibitory concentrations of tiamulin showed that tva(A) confers reduced pleuromutilin susceptibility that does not lead to clinical resistance but facilitates the development of higher-level resistance via mutations in genes encoding ribosome-associated functions. Genome sequencing of antibiotic-exposed isolates identified both new and previously described mutations in chromosomal genes associated with reduced pleuromutilin susceptibility, including the 23S rRNA gene and rplC, which encodes the L3 ribosomal protein. Interesting three antibiotic-exposed isolates harboured mutations in fusA, encoding Elongation Factor G, a gene not previously associated with pleuromutilin resistance. A longitudinal molecular epidemiological examination of two episodes of swine dysentery at the same farm indicated that tva(A) contributed to development of tiamulin resistance in vivo in a manner consistent with that seen experimentally in vitro. The in vitro studies further showed that tva(A) broadened the mutant selection window and raised the mutant prevention concentration above reported in vivo antibiotic concentrations obtained when administered at certain doses. We show how the identification and characterisation of tva(A), a new marker for pleuromutilin resistance, provides evidence to inform treatment regimes and reduce the development of resistance to this class of highly important antimicrobial agents