Detection of a Novel, and Likely Ancestral, Tn916-Like Element from a Human Saliva Metagenomic Library

Tn916 is a conjugative transposon (CTn) and the first reported and most well characterised of the Tn916/Tn1545 family of CTns. Tn916-like elements have a characteristic modular structure and different members of this family have been identified based on similarities and variations in these modules. In addition to carrying genes encoding proteins required for their conjugation, Tn916-like elements also carry accessory, antimicrobial resistance genes; most commonly the tetracycline resistance gene, tet(M). Our study aimed to identify and characterise tetracycline resistance genes from the human saliva metagenome using a functional metagenomic approach. We identified a tetracycline-resistant clone, TT31, the sequencing of which revealed it to encode both tet(M) and tet(L). Comparison of the TT31 sequence with the accessory, regulation, and recombination modules of other Tn916-like elements indicated that a partial Tn916-like element encoding a truncated orf9 was cloned in TT31. Analysis indicated that a previous insertion within the truncated orf9 created the full length orf9 found in most Tn916-like transposons; demonstrating that orf9 is, in fact, the result of a gene fusion event. Thus, we hypothesise that the Tn916-like element cloned in TT31 likely represents an ancestral Tn916

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

A process for analysis of microarray comparative genomics hybridisation studies for bacterial genomes

<p>Abstract</p> <p>Background</p> <p>Microarray based comparative genomic hybridisation (CGH) experiments have been used to study numerous biological problems including understanding genome plasticity in pathogenic bacteria. Typically such experiments produce large data sets that are difficult for biologists to handle. Although there are some programmes available for interpretation of bacterial transcriptomics data and CGH microarray data for looking at genetic stability in oncogenes, there are none specifically to understand the mosaic nature of bacterial genomes. Consequently a bottle neck still persists in accurate processing and mathematical analysis of these data. To address this shortfall we have produced a simple and robust CGH microarray data analysis process that may be automated in the future to understand bacterial genomic diversity.</p> <p>Results</p> <p>The process involves five steps: cleaning, normalisation, estimating gene presence and absence or divergence, validation, and analysis of data from test against three reference strains simultaneously. Each stage of the process is described and we have compared a number of methods available for characterising bacterial genomic diversity, for calculating the cut-off between gene presence and absence or divergence, and shown that a simple dynamic approach using a kernel density estimator performed better than both established, as well as a more sophisticated mixture modelling technique. We have also shown that current methods commonly used for CGH microarray analysis in tumour and cancer cell lines are not appropriate for analysing our data.</p> <p>Conclusion</p> <p>After carrying out the analysis and validation for three sequenced <it>Escherichia coli </it>strains, CGH microarray data from 19 <it>E. coli </it>O157 pathogenic test strains were used to demonstrate the benefits of applying this simple and robust process to CGH microarray studies using bacterial genomes.</p

Factors associated with plasmid antibiotic resistance gene carriage revealed using large-scale multivariable analysis

Plasmids are major vectors of bacterial antibiotic resistance, but understanding of factors associated with plasmid antibiotic resistance gene (ARG) carriage is limited. We curated > 14,000 publicly available plasmid genomes and associated metadata. Duplicate and replicate plasmids were excluded; where possible, sample metadata was validated externally (BacDive database). Using Generalised Additive Models (GAMs) we assessed the influence of 12 biotic/abiotic factors (e.g. plasmid genetic factors, isolation source, collection date) on ARG carriage, modelled as a binary outcome. Separate GAMs were built for 10 major ARG types. Multivariable analysis indicated that plasmid ARG carriage patterns across time (collection years), isolation sources (human/livestock) and host bacterial taxa were consistent with antibiotic selection pressure as a driver of plasmid-mediated antibiotic resistance. Only 0.42% livestock plasmids carried carbapenem resistance (compared with 12% human plasmids); conversely, tetracycline resistance was enriched in livestock vs human plasmids, reflecting known prescribing practices. Interpreting results using a timeline of ARG type acquisition (determined by literature review) yielded additional novel insights. More recently acquired ARG types (e.g. colistin and carbapenem) showed increases in plasmid carriage during the date range analysed (1994–2019), potentially reflecting recent onset of selection pressure; they also co-occurred less commonly with ARGs of other types, and virulence genes. Overall, this suggests that following acquisition, plasmid ARGs tend to accumulate under antibiotic selection pressure and co-associate with other adaptive genes (other ARG types, virulence genes), potentially re-enforcing plasmid ARG carriage through co-selection

Novel Inducers of the Envelope Stress Response BaeSR in Salmonella Typhimurium: BaeR Is Critically Required for Tungstate Waste Disposal

The RpoE and CpxR regulated envelope stress responses are extremely important for SalmonellaTyphimurium to cause infection in a range of hosts. Until now the role for BaeSR in both the Salmonella Typhimurium response to stress and its contribution to infection have not been fully elucidated. Here we demonstrate stationary phase growth, iron and sodium tungstate as novel inducers of the BaeRregulon, with BaeR critically required for Salmonella resistance to sodium tungstate. We show that functional overlap between the resistance nodulation-cell division (RND) multidrug transporters, MdtA, AcrD and AcrB exists for the waste disposal of tungstate from the cell. We also point to a role for enterobactinsiderophores in the protection of enteric organisms from tungstate, akin to the scenario in nitrogen fixing bacteria. Surprisingly, BaeR is the first envelope stress response pathway investigated in S. Typhimurium that is not required for murine typhoid in either ityS or ityR mouse backgrounds. BaeR is therefore either required for survival in larger mammals such as pigs or calves, an avian host such as chickens, or survival out with the host altogether where Salmonella and related enterics must survive in soil and water

Livestock-Associated Methicillin-Resistant Staphylococcus aureus From Animals and Animal Products in the UK

[EN] Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is an emerging problem in many parts of the world. Although animal-adapted LA-MRSA has been known for many years, recent reports suggest a possible increasing trend in the zoonotic transmission of LA-MRSA in Europe. Since its emergence in the early 2000¿s, several investigations have indicated that persons in prolonged, repeated contact with affected livestock are at a higher risk of becoming colonized with LA-MRSA. LA-MRSA monitoring in livestock is voluntary under current EU legislation, and not all member states, including the UK, participate. UK LA-MRSA isolates have been detected through scanning surveillance, where samples are submitted from clinically diseased livestock for diagnostic investigation, and research studies. Surveys conducted on retail beef, pig and poultry meat on sale in the UK have also detected LA-MRSA. Taken together these results suggest that LA-MRSA is present in the UK, possibly at low prevalence level, as suggested by available evidence. In this review, we examine the data available from UK livestock and animal products, and make recommendations for future. We also review the findings from whole genome sequencing (WGS) of the possible lineage of some UK livestock isolates.We are grateful to the Veterinary Medicines Directorate in the UK for funding this work through VMD0533. FM-J contributed during a sabbatical to the APHA with a grant from Consellería de Educación y Ciencia of Generalitat Valenciana (BEST/2017/050). CM contributed during a sabbatical to the APHA which was supported by a Lecturer research grant from the Santander bank (programme XIII Convocatoria de ayudas a la movilidad investigadora CEU-Banco Santander).Anjum, MF.; Marco-Jiménez, F.; Duncan, D.; Marin-Orenga, C.; Smith, RP.; Evans, SJ. (2019). Livestock-Associated Methicillin-Resistant Staphylococcus aureus From Animals and Animal Products in the UK. Frontiers in Microbiology. 10:1-7. https://doi.org/10.3389/fmicb.2019.02136S171

Monitoring Antimicrobial Resistance and Drug Usage in the Human and Livestock Sector and Foodborne Antimicrobial Resistance in Six European Countries

Introduction: Antimicrobial resistance (AMR), associated with antimicrobial use (AMU), is a major public concern. Surveillance and monitoring systems are essential to assess and control the trends in AMU and AMR. However, differences in the surveillance and monitoring systems between countries and sectors make comparisons challenging. The purpose of this article is to describe all surveillance and monitoring systems for AMU and AMR in the human and livestock sectors, as well as national surveillance and monitoring systems for AMR in food, in six European countries (Spain, Germany, France, the Netherlands, the United Kingdom and Norway) as a baseline for developing suggestions to overcome current limitations in comparing AMU and AMR data. Methods: A literature search in 2018 was performed to identify relevant peer-reviewed articles and national and European grey reports as well as AMU/AMR databases. Results: Comparison of AMU and AMR systems across the six countries showed a lack of standardization and harmonization with different AMU data sources (prescription vs sales data) and units of AMU and AMR being used. The AMR data varied by sample type (clinical/non-clinical), laboratory method (disk diffusion, microdilution, and VITEK, among others), data type, ie quantitative (minimum inhibition concentration (MIC) in mg/L/inhibition zone (IZ) in mm) vs qualitative data (susceptible-intermediate-resistant (SIR)), the standards used (EUCAST/CLSI among others), and/or the evaluation criteria adopted (epidemiological or clinical). Discussion: A One Health approach for AMU and AMR requires harmonization in various aspects between human, animal and food systems at national and international levels. Additionally, some overlap between systems of AMU and AMR has been encountered. Efforts should be made to improve standardization and harmonization and allow more meaningful analyses of AMR and AMU surveillance data under a One Health approach.Peer Reviewe

Factors associated with plasmid antibiotic resistance gene carriage revealed using large-scale multivariable analysis

Plasmids are major vectors of bacterial antibiotic resistance, but understanding of factors associated with plasmid antibiotic resistance gene (ARG) carriage is limited. We curated > 14,000 publicly available plasmid genomes and associated metadata. Duplicate and replicate plasmids were excluded; where possible, sample metadata was validated externally (BacDive database). Using Generalised Additive Models (GAMs) we assessed the influence of 12 biotic/abiotic factors (e.g. plasmid genetic factors, isolation source, collection date) on ARG carriage, modelled as a binary outcome. Separate GAMs were built for 10 major ARG types. Multivariable analysis indicated that plasmid ARG carriage patterns across time (collection years), isolation sources (human/livestock) and host bacterial taxa were consistent with antibiotic selection pressure as a driver of plasmid-mediated antibiotic resistance. Only 0.42% livestock plasmids carried carbapenem resistance (compared with 12% human plasmids); conversely, tetracycline resistance was enriched in livestock vs human plasmids, reflecting known prescribing practices. Interpreting results using a timeline of ARG type acquisition (determined by literature review) yielded additional novel insights. More recently acquired ARG types (e.g. colistin and carbapenem) showed increases in plasmid carriage during the date range analysed (1994–2019), potentially reflecting recent onset of selection pressure; they also co-occurred less commonly with ARGs of other types, and virulence genes. Overall, this suggests that following acquisition, plasmid ARGs tend to accumulate under antibiotic selection pressure and co-associate with other adaptive genes (other ARG types, virulence genes), potentially re-enforcing plasmid ARG carriage through co-selection