Ten Years of Population-Level Genomic Escherichia coli and Klebsiella pneumoniae Serotype Surveillance Informs Vaccine Development for Invasive Infections.

BACKGROUND: The incidence of bloodstream infections (BSIs) caused by Escherichia coli and Klebsiella pneumoniae is increasing, with substantial associated morbidity, mortality, and antimicrobial resistance. Unbiased serotyping studies to guide vaccine target selection are limited. METHODS: We conducted unselected, population-level genomic surveillance of bloodstream E. coli and Klebsiella pneumoniae isolates from 2008 to 2018 in Oxfordshire, United Kingdom. We supplemented this with an analysis of publicly available global sequencing data (n = 3678). RESULTS: We sequenced 3478 E. coli isolates (3278 passed quality control) and 556 K. pneumoniae isolates (535 [K-antigen] and 549 [O-antigen] passed quality control). The 4 most common E. coli O-antigens (O1/O2/O6/O25) were identified in 1499/3278 isolates; the incidence of these O-types increased over time (incidence rate ratio per year [IRRy] = 1.14, 95% confidence interval [CI]: 1.11-1.16). These O-types accounted for 616/1434 multidrug-resistant (MDR) and 173/256 extended-spectrum beta-lactamase (ESBL)-resistant isolates in Oxfordshire but only 19/90 carbapenem-resistant isolates across all studies. For Klebsiella pneumoniae, the most common O-antigens (O2v2/O1v1/O3b/O1v2) accounted for 410/549 isolates; the incidence of BSIs caused by these also increased annually (IRRy = 1.09; 95% CI: 1.05-1.12). These O-types accounted for 122/148 MDR and 106/123 ESBL isolates in Oxfordshire and 557/734 carbapenem-resistant isolates across all studies. Conversely we observed substantial capsular antigen diversity. Analysis of 3678 isolates from global studies demonstrated the generalizability of these findings. For E. coli, based on serotyping, the ExPEC4V and ExPEC10V vaccines under investigation would cover 46% and 72% of Oxfordshire isolates respectively, and 47% and 71% of MDR isolates. CONCLUSIONS: O-antigen targeted vaccines may be useful in reducing the morbidity, mortality, and antimicrobial resistance associated with E. coli and K. pneumoniae BSIs.

A Candida auris Outbreak and Its Control in an Intensive Care Setting.

BACKGROUND: Candida auris is an emerging and multidrug-resistant pathogen. Here we report the epidemiology of a hospital outbreak of C. auris colonization and infection. METHODS: After identification of a cluster of C. auris infections in the neurosciences intensive care unit (ICU) of the Oxford University Hospitals, United Kingdom, we instituted an intensive patient and environmental screening program and package of interventions. Multivariable logistic regression was used to identify predictors of C. auris colonization and infection. Isolates from patients and from the environment were analyzed by whole-genome sequencing. RESULTS: A total of 70 patients were identified as being colonized or infected with C. auris between February 2, 2015, and August 31, 2017; of these patients, 66 (94%) had been admitted to the neurosciences ICU before diagnosis. Invasive C. auris infections developed in 7 patients. When length of stay in the neurosciences ICU and patient vital signs and laboratory results were controlled for, the predictors of C. auris colonization or infection included the use of reusable skin-surface axillary temperature probes (multivariable odds ratio, 6.80; 95% confidence interval [CI], 2.96 to 15.63; P<0.001) and systemic fluconazole exposure (multivariable odds ratio, 10.34; 95% CI, 1.64 to 65.18; P=0.01). C. auris was rarely detected in the general environment. However, it was detected in isolates from reusable equipment, including multiple axillary skin-surface temperature probes. Despite a bundle of infection-control interventions, the incidence of new cases was reduced only after removal of the temperature probes. All outbreak sequences formed a single genetic cluster within the C. auris South African clade. The sequenced isolates from reusable equipment were genetically related to isolates from the patients. CONCLUSIONS: The transmission of C. auris in this hospital outbreak was found to be linked to reusable axillary temperature probes, indicating that this emerging pathogen can persist in the environment and be transmitted in health care settings. (Funded by the National Institute for Health Research Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance at Oxford University and others.).

Nanopore metagenomic sequencing of influenza virus directly from respiratory samples: diagnosis, drug resistance and nosocomial transmission, United Kingdom, 2018/19 influenza season.

BackgroundInfluenza virus presents a considerable challenge to public health by causing seasonal epidemics and occasional pandemics. Nanopore metagenomic sequencing has the potential to be deployed for near-patient testing, providing rapid infection diagnosis, rationalising antimicrobial therapy, and supporting infection-control interventions.AimTo evaluate the applicability of this sequencing approach as a routine laboratory test for influenza in clinical settings.MethodsWe conducted Oxford Nanopore Technologies (Oxford, United Kingdom (UK)) metagenomic sequencing for 180 respiratory samples from a UK hospital during the 2018/19 influenza season, and compared results to routine molecular diagnostic standards (Xpert Xpress Flu/RSV assay; BioFire FilmArray Respiratory Panel 2 assay). We investigated drug resistance, genetic diversity, and nosocomial transmission using influenza sequence data.ResultsCompared to standard testing, Nanopore metagenomic sequencing was 83% (75/90) sensitive and 93% (84/90) specific for detecting influenza A viruses. Of 59 samples with haemagglutinin subtype determined, 40 were H1 and 19 H3. We identified an influenza A(H3N2) genome encoding the oseltamivir resistance S331R mutation in neuraminidase, potentially associated with an emerging distinct intra-subtype reassortant. Whole genome phylogeny refuted suspicions of a transmission cluster in a ward, but identified two other clusters that likely reflected nosocomial transmission, associated with a predominant community-circulating strain. We also detected other potentially pathogenic viruses and bacteria from the metagenome.ConclusionNanopore metagenomic sequencing can detect the emergence of novel variants and drug resistance, providing timely insights into antimicrobial stewardship and vaccine design. Full genome generation can help investigate and manage nosocomial outbreaks.

Reconciling the Potentially Irreconcilable? Genotypic and Phenotypic Amoxicillin-Clavulanate Resistance in Escherichia coli.

Resistance to amoxicillin-clavulanate, a widely used beta-lactam/beta-lactamase inhibitor combination antibiotic, is rising globally, and yet susceptibility testing remains challenging. To test whether whole-genome sequencing (WGS) could provide a more reliable assessment of susceptibility than traditional methods, we predicted resistance from WGS for 976 Escherichia coli bloodstream infection isolates from Oxfordshire, United Kingdom, comparing against phenotypes from the BD Phoenix (calibrated against EUCAST guidelines). A total of 339/976 (35%) isolates were amoxicillin-clavulanate resistant. Predictions based solely on beta-lactamase presence/absence performed poorly (sensitivity, 23% [78/339]) but improved when genetic features associated with penicillinase hyperproduction (e.g., promoter mutations and copy number estimates) were considered (sensitivity, 82% [277/339]; P < 0.0001). Most discrepancies occurred in isolates with MICs within ±1 doubling dilution of the breakpoint. We investigated two potential causes: the phenotypic reference and the binary resistant/susceptible classification. We performed reference standard, replicated phenotyping in a random stratified subsample of 261/976 (27%) isolates using agar dilution, following both EUCAST and CLSI guidelines, which use different clavulanate concentrations. As well as disagreeing with each other, neither agar dilution phenotype aligned perfectly with genetic features. A random-effects model investigating associations between genetic features and MICs showed that some genetic features had small, variable and additive effects, resulting in variable resistance classification. Using model fixed-effects to predict MICs for the non-agar dilution isolates, predicted MICs were in essential agreement (±1 doubling dilution) with observed (BD Phoenix) MICs for 691/715 (97%) isolates. This suggests amoxicillin-clavulanate resistance in E. coli is quantitative, rather than qualitative, explaining the poorly reproducible binary (resistant/susceptible) phenotypes and suboptimal concordance between different phenotypic methods and with WGS-based predictions.

DNA Thermo-Protection Facilitates Whole-Genome Sequencing of Mycobacteria Direct from Clinical Samples.

Mycobacterium tuberculosis is the leading cause of death from bacterial infection. Improved rapid diagnosis and antimicrobial resistance determination, such as by whole-genome sequencing, are required. Our aim was to develop a simple, low-cost method of preparing DNA for sequencing direct from M. tuberculosis-positive clinical samples (without culture). Simultaneous sputum liquefaction, bacteria heat inactivation (99°C/30 min), and enrichment for mycobacteria DNA were achieved using an equal volume of thermo-protection buffer (4 M KCl, 0.05 M HEPES buffer, pH 7.5, 0.1% dithiothreitol [DTT]). The buffer emulated intracellular conditions found in hyperthermophiles, thus protecting DNA from rapid thermodegradation, which renders it a poor template for sequencing. Initial validation experiments employed mycobacteria DNA, either extracted or intracellular. Next, mock clinical samples (infection-negative human sputum spiked with 0 to 105Mycobacterium bovis BCG cells/ml) underwent liquefaction in thermo-protection buffer and heat inactivation. DNA was extracted and sequenced. Human DNA degraded faster than mycobacteria DNA, resulting in target enrichment. Four replicate experiments achieved M. tuberculosis detection at 101 BCG cells/ml, with 31 to 59 M. tuberculosis complex reads. Maximal genome coverage (>97% at 5× depth) occurred at 104 BCG cells/ml; >91% coverage (1× depth) occurred at 103 BCG cells/ml. Final validation employed M. tuberculosis-positive clinical samples (n = 20), revealing that initial sample volumes of ≥1 ml typically yielded higher mean depths of M. tuberculosis genome coverage, with an overall range of 0.55 to 81.02. A mean depth of 3 gave >96% 1-fold tuberculosis (TB) genome coverage (in 15/20 clinical samples). A mean depth of 15 achieved >99% 5-fold genome coverage (in 9/20 clinical samples). In summary, direct-from-sample sequencing of M. tuberculosis genomes was facilitated by a low-cost thermo-protection buffer.

The Impact of Vaccination and Prior Exposure on Stool Shedding of Salmonella Typhi and Salmonella Paratyphi in 6 Controlled Human Infection Studies.

BACKGROUND: Shedding of Salmonella Typhi or Paratyphi in the stool or urine leads to contamination of food or water, which is a prerequisite for transmission of enteric fever. Currently, there are limited data on the effect of vaccination or prior exposure on stool shedding. METHODS: Six Salmonella Typhi or Paratyphi human challenge studies were conducted between 2011 and 2017. Participants were either unvaccinated or vaccinated with 1 of 4 vaccines: Vi-polysaccharide (Vi-PS), Vi-tetanus-toxoid conjugate vaccine (Vi-TT), live oral Ty21a vaccine, or an experimental vaccine (M01ZH09). Daily stool cultures were collected for 14 days after challenge. RESULTS: There were 4934 stool samples collected from 430 volunteers. Participants who received Vi-PS or Vi-TT shed less than unvaccinated participants (odds ratio [OR], 0.34; 95% confidence interval [CI], 0.15-0.77; P = .010 and OR, 0.41; 95% CI, 0.19-0.91, P = .029 for Vi-PS and Vi-TT, respectively). Higher anti-Vi immunoglobulin G titers were associated with less shedding of S. Typhi (P < .0001). A nonsignificant reduction in shedding was associated with Ty21a vaccine (OR, 0.57; 95% CI, 0.27-1.20; P = .140). Individuals previously exposed to S. Typhi shed less than previously unexposed individuals (OR, 0.30; 95% CI, 0.1-0.8; P = .016). Shedding of S. Typhi was more common than S. Paratyphi. CONCLUSIONS: Prior vaccination with Vi vaccines, or natural infection, reduces onward transmission of S. Typhi. Field trials of Vi-TT should be designed to detect indirect protection, reflecting the consequence of reduced stool shedding observed in the human challenge model.

Whole-Genome Sequencing for Predicting Clarithromycin Resistance in Mycobacterium abscessus.

Mycobacterium abscessus is emerging as an important pathogen in chronic lung diseases, with concern regarding patient-to-patient transmission. The recent introduction of routine whole-genome sequencing (WGS) as a replacement for existing reference techniques in England provides an opportunity to characterize the genetic determinants of resistance. We conducted a systematic review to catalogue all known resistance-determining mutations. This knowledge was used to construct a predictive algorithm based on mutations in the erm(41) and rrl genes which was tested on a collection of 203 sequentially acquired clinical isolates for which there were paired genotype/phenotype data. A search for novel resistance-determining mutations was conducted using a heuristic algorithm. The sensitivity of existing knowledge for predicting resistance in clarithromycin was 95% (95% confidence interval [CI], 89 to 98%), and the specificity was 66% (95% CI, 54 to 76%). The subspecies alone was a poor predictor of resistance to clarithromycin. Eight potential new resistance-conferring single nucleotide polymorphisms (SNPs) were identified. WGS demonstrated probable resistance-determining SNPs in regions that the NTM-DR line probe cannot detect. These mutations are potentially clinically important, as they all occurred in samples that were predicted to be inducibly resistant and for which a macrolide would therefore currently be indicated. We were unable to explain all resistance, raising the possibility of the involvement of other as yet unidentified genes.

Metagenomic Nanopore Sequencing of Influenza Virus Direct from Clinical Respiratory Samples.

Influenza is a major global public health threat as a result of its highly pathogenic variants, large zoonotic reservoir, and pandemic potential. Metagenomic viral sequencing offers the potential for a diagnostic test for influenza virus which also provides insights on transmission, evolution, and drug resistance and simultaneously detects other viruses. We therefore set out to apply the Oxford Nanopore Technologies sequencing method to metagenomic sequencing of respiratory samples. We generated influenza virus reads down to a limit of detection of 102 to 103 genome copies/ml in pooled samples, observing a strong relationship between the viral titer and the proportion of influenza virus reads (P = 4.7 × 10-5). Applying our methods to clinical throat swabs, we generated influenza virus reads for 27/27 samples with mid-to-high viral titers (cycle threshold [CT ] values, <30) and 6/13 samples with low viral titers (CT values, 30 to 40). No false-positive reads were generated from 10 influenza virus-negative samples. Thus, Nanopore sequencing operated with 83% sensitivity (95% confidence interval [CI], 67 to 93%) and 100% specificity (95% CI, 69 to 100%) compared to the current diagnostic standard. Coverage of full-length virus was dependent on sample composition, being negatively influenced by increased host and bacterial reads. However, at high influenza virus titers, we were able to reconstruct >99% complete sequences for all eight gene segments. We also detected a human coronavirus coinfection in one clinical sample. While further optimization is required to improve sensitivity, this approach shows promise for the Nanopore platform to be used in the diagnosis and genetic analysis of influenza virus and other respiratory viruses.

Same-Day Diagnostic and Surveillance Data for Tuberculosis via Whole-Genome Sequencing of Direct Respiratory Samples.

Routine full characterization of Mycobacterium tuberculosis is culture based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near the point of care. We demonstrate a low-cost method of DNA extraction directly from patient samples for M. tuberculosis WGS. We initially evaluated the method by using the Illumina MiSeq sequencer (40 smear-positive respiratory samples obtained after routine clinical testing and 27 matched liquid cultures). M. tuberculosis was identified in all 39 samples from which DNA was successfully extracted. Sufficient data for antibiotic susceptibility prediction were obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. With Illumina MiSeq/MiniSeq, the workflow from patient sample to results can be completed in 44/16 h at a reagent cost of £96/£198 per sample. We then employed a nonspecific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to cultured Mycobacterium bovis strain BCG DNA and to combined culture-negative sputum DNA and BCG DNA. For flow cell version R9.4, the estimated turnaround time from patient to identification of BCG, detection of pyrazinamide resistance, and phylogenetic placement was 7.5 h, with full susceptibility results 5 h later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of MinION is the ability to continue sequencing until sufficient coverage is obtained, providing a potential solution to the problem of variable amounts of M. tuberculosis DNA in direct samples.

Mycobacterial DNA extraction for whole-genome sequencing from early positive liquid (MGIT) cultures.

We developed a low-cost and reliable method of DNA extraction from as little as 1 ml of early positive mycobacterial growth indicator tube (MGIT) cultures that is suitable for whole-genome sequencing to identify mycobacterial species and predict antibiotic resistance in clinical samples. The DNA extraction method is based on ethanol precipitation supplemented by pretreatment steps with a MolYsis kit or saline wash for the removal of human DNA and a final DNA cleanup step with solid-phase reversible immobilization beads. The protocol yielded ≥0.2 ng/µl of DNA for 90% (MolYsis kit) and 83% (saline wash) of positive MGIT cultures. A total of 144 (94%) of the 154 samples sequenced on the MiSeq platform (Illumina) achieved the target of 1 million reads, with <5% of reads derived from human or nasopharyngeal flora for 88% and 91% of samples, respectively. A total of 59 (98%) of 60 samples that were identified by the national mycobacterial reference laboratory (NMRL) as Mycobacterium tuberculosis were successfully mapped to the H37Rv reference, with >90% coverage achieved. The DNA extraction protocol, therefore, will facilitate fast and accurate identification of mycobacterial species and resistance using a range of bioinformatics tools.

Nanopore sequencing of influenza A and B in Oxfordshire and the United Kingdom, 2022-23.

OBJECTIVES: We evaluated Nanopore sequencing for influenza surveillance. METHODS: Influenza A and B PCR-positive samples from hospital patients in Oxfordshire, UK, and a UK-wide population survey from winter 2022-23 underwent Nanopore sequencing following targeted rt-PCR amplification. RESULTS: From 941 infections, successful sequencing was achieved in 292/388 (75 %) available Oxfordshire samples: 231 (79 %) A/H3N2, 53 (18 %) A/H1N1, and 8 (3 %) B/Victoria and in 53/113 (47 %) UK-wide samples. Sequencing was more successful at lower Ct values. Most same-sample replicate sequences had identical haemagglutinin segments (124/141, 88 %); 36/39 (92 %) Illumina vs. Nanopore comparisons were identical, and 3 (8 %) differed by 1 variant. Comparison of Oxfordshire and UK-wide sequences showed frequent inter-regional transmission. Infections were closely-related to 2022-23 vaccine strains. Only one sample had a neuraminidase inhibitor resistance mutation. 849/941 (90 %) Oxfordshire infections were community-acquired. 63/88 (72 %) potentially healthcare-associated cases shared a hospital ward with ≥ 1 known infectious case. 33 epidemiologically-plausible transmission links had sequencing data for both source and recipient: 8 were within ≤ 5 SNPs, of these, 5 (63 %) involved potential sources that were also hospital-acquired. CONCLUSIONS: Nanopore influenza sequencing was reproducible and antiviral resistance rare. Inter-regional transmission was common; most infections were genomically similar. Hospital-acquired infections are likely an important source of nosocomial transmission and should be prioritised for infection prevention and control.

The plasmidome associated with Gram-negative bloodstream infections: A large-scale observational study using complete plasmid assemblies.

Plasmids carry genes conferring antimicrobial resistance and other clinically important traits, and contribute to the rapid dissemination of such genes. Previous studies using complete plasmid assemblies, which are essential for reliable inference, have been small and/or limited to plasmids carrying antimicrobial resistance genes (ARGs). In this study, we sequenced 1,880 complete plasmids from 738 isolates from bloodstream infections in Oxfordshire, UK. The bacteria had been originally isolated in 2009 (194 isolates) and 2018 (368 isolates), plus a stratified selection from intervening years (176 isolates). We demonstrate that plasmids are largely, but not entirely, constrained to a single host species, although there is substantial overlap between species of plasmid gene-repertoire. Most ARGs are carried by a relatively small number of plasmid groups with biological features that are predictable. Plasmids carrying ARGs (including those encoding carbapenemases) share a putative 'backbone' of core genes with those carrying no such genes. These findings suggest that future surveillance should, in addition to tracking plasmids currently associated with clinically important genes, focus on identifying and monitoring the dissemination of high-risk plasmid groups with the potential to rapidly acquire and disseminate these genes.

Enterobacterales plasmid sharing amongst human bloodstream infections, livestock, wastewater, and waterway niches in Oxfordshire, UK.

Plasmids enable the dissemination of antimicrobial resistance (AMR) in common Enterobacterales pathogens, representing a major public health challenge. However, the extent of plasmid sharing and evolution between Enterobacterales causing human infections and other niches remains unclear, including the emergence of resistance plasmids. Dense, unselected sampling is essential to developing our understanding of plasmid epidemiology and designing appropriate interventions to limit the emergence and dissemination of plasmid-associated AMR. We established a geographically and temporally restricted collection of human bloodstream infection (BSI)-associated, livestock-associated (cattle, pig, poultry, and sheep faeces, farm soils) and wastewater treatment work (WwTW)-associated (influent, effluent, waterways upstream/downstream of effluent outlets) Enterobacterales. Isolates were collected between 2008 and 2020 from sites <60 km apart in Oxfordshire, UK. Pangenome analysis of plasmid clusters revealed shared 'backbones', with phylogenies suggesting an intertwined ecology where well-conserved plasmid backbones carry diverse accessory functions, including AMR genes. Many plasmid 'backbones' were seen across species and niches, raising the possibility that plasmid movement between these followed by rapid accessory gene change could be relatively common. Overall, the signature of identical plasmid sharing is likely to be a highly transient one, implying that plasmid movement might be occurring at greater rates than previously estimated, raising a challenge for future genomic One Health studies.

Symbolic action and constraint: the cultural logic of the 2017 UK General Election.

This paper examines the influence of both the agential and structural aspects of culture on the 2017 UK General Election. The empirical section of the paper is organised around three aspects of the Labour campaign narrative: its promise to provide a national 'alternative', its mobilisation of Corbyn as simultaneously an individual and an icon, and its use of rallies and social media as alternative stages from which to project its meanings. It identifies the material conditioning, coding and counter-coding, narrativisation and counter-narrativisation of key events, issues, and figures, demonstrating how the substantive contestation of the campaign occurred within a consensual formal grammar that constrained symbolic action and often produced consequences unintended by cultural workers themselves. It, therefore, demonstrates how culture retained a relative autonomy in influencing meaning outcomes in part due to its formal semiotic logic. It introduces the notion of 'code-rerouting' to cultural sociology's stock of conceptual tools and highlights how failure to conform to the structuring norms of civil rituals, such as presenting oneself to public scrutiny during a campaign, or failure to project coherence between surface and depth, individual and icon, past and present, or between private and public can prove fatal to performative felicity.

Changes in paediatric respiratory infections at a UK teaching hospital 2016-2021; impact of the SARS-CoV-2 pandemic.

Objective To describe the impact of the SARS-CoV-2 pandemic on the incidence of paediatric viral respiratory tract infection in Oxfordshire, UK. Methods Data on paediatric Emergency Department (ED) attendances (0-15 years inclusive), respiratory virus testing, vital signs and mortality at Oxford University Hospitals were summarised using descriptive statistics. Results Between 1-March-2016 and 30-July-2021, 155,056 ED attendances occurred and 7,195 respiratory virus PCRs were performed. Detection of all pathogens was suppressed during the first national lockdown. Rhinovirus and adenovirus rates increased when schools reopened September-December 2020, then fell, before rising in March-May 2021. The usual winter RSV peak did not occur in 2020/21, with an inter-seasonal rise (32/1,000 attendances in 0-3 yr olds) in July 2021. Influenza remained suppressed throughout. A higher paediatric early warning score (PEWS) was seen for attendees with adenovirus during the pandemic compared to pre-pandemic (p = 0.04, Mann-Witney U test), no other differences in PEWS were seen. Conclusions SARS-CoV-2 caused major changes in the incidence of paediatric respiratory viral infection in Oxfordshire, with implications for clinical service demand, testing strategies, timing of palivizumab RSV prophylaxis, and highlighting the need to understand which public health interventions are most effective for preventing respiratory virus infections.

Ten-year longitudinal molecular epidemiology study of Escherichia coli and Klebsiella species bloodstream infections in Oxfordshire, UK.

BACKGROUND: The incidence of Gram-negative bloodstream infections (BSIs), predominantly caused by Escherichia coli and Klebsiella species, continues to increase; however, the causes of this are unclear and effective interventions are therefore hard to design. METHODS: In this study, we sequenced 3468 unselected isolates over a decade in Oxfordshire (UK) and linked this data to routinely collected electronic healthcare records and mandatory surveillance reports. We annotated genomes for clinically relevant genes, contrasting the distribution of these within and between species, and compared incidence trends over time using stacked negative binomial regression. RESULTS: We demonstrate that the observed increases in E. coli incidence were not driven by the success of one or more sequence types (STs); instead, four STs continue to dominate a stable population structure, with no evidence of adaptation to hospital/community settings. Conversely in Klebsiella spp., most infections are caused by sporadic STs with the exception of a local drug-resistant outbreak strain (ST490). Virulence elements are highly structured by ST in E. coli but not Klebsiella spp. where they occur in a diverse spectrum of STs and equally across healthcare and community settings. Most clinically hypervirulent (i.e. community-onset) Klebsiella BSIs have no known acquired virulence loci. Finally, we demonstrate a diverse but largely genus-restricted mobilome with close associations between antimicrobial resistance (AMR) genes and insertion sequences but not typically specific plasmid replicon types, consistent with the dissemination of AMR genes being highly contingent on smaller mobile genetic elements (MGEs). CONCLUSIONS: Our large genomic study highlights distinct differences in the molecular epidemiology of E. coli and Klebsiella BSIs and suggests that no single specific pathogen genetic factors (e.g. AMR/virulence genes/sequence type) are likely contributing to the increasing incidence of BSI overall, that association with AMR genes in E. coli is a contributor to the increasing number of E. coli BSIs, and that more attention should be given to AMR gene associations with non-plasmid MGEs to try and understand horizontal gene transfer networks.

Why meaning-making matters: the case of the UK Government's COVID-19 response.

Through analysis of the UK government's management of the COVID-19 outbreak, this paper offers an empirical demonstration of the principle of culture's relative autonomy. It does so by showing how the outcome of meaning-making struggles had impacts on political legitimacy, public behaviour, and control over the spread of the virus. Ultimately, these impacts contributed to the avoidable deaths of tens of thousands of UK citizens. Dividing the crisis into phases within a secular ritual passage or 'social drama', it shows how each phase was defined by struggles between the government and other actors to code the unfolding events in an appropriate moral way, to cast actors in their proper roles, and to plot them together in a storied fashion under a suitable narrative genre. Taken together, these processes constituted a conflictual effort to define the meaning of what was occurring. The paper also offers more specific contributions to cultural sociology by showing why social performance theory needs to consider the effects of casting non-human actors in social dramas, how metaphor forms a powerful tool of political action through simplifying and shaping complex realities, and how casting can shift responsibility and redefine the meaning of emotionally charged events such as human death.

Case Report: Disseminated, rifampicin resistant Mycobacterium bovis (BCG) infection in an immunocompromised child.

Background: Bacillus Calmette-Guérin (BCG) is a live-attenuated vaccine used world-wide for prevention of tuberculosis disease. In some immunocompromised hosts it has the potential to cause disease. As with other members of the M. tuberculosis complex it has the potential for acquiring drug resistance. Methods: We reviewed 10 years of paediatric clinical BCG strains referred to our clinical microbiology laboratory in Oxford where they underwent whole genome sequencing. We present a case series comparing clinical, pathogen genetic and pathogen phenotypic data, and consider the clinical implications. Results: We identified 15 BCG isolates from 8 children under 16 years old. Only one child had clinical disease with the other seven reported as local inoculation-site reactions. Case 1 suffered disseminated disease secondary to an undiagnosed IL-12/IFNγ receptor defect and the BCG isolates evolved two different rifampicin resistance mutations. Across all 15 isolates, phenotypic resistance to each first line drug was seen.  Conclusions: BCG is a safe and effective vaccine in children. Most clinical specimens in our series were not related to disease. However, in the context of rare pathogen-specific immunocompromise, BCG can cause pathology and acquire drug resistance under selection from therapy.

Optimized use of Oxford Nanopore flowcells for hybrid assemblies.

Hybrid assemblies are highly valuable for studies of Enterobacteriaceae due to their ability to fully resolve the structure of mobile genetic elements, such as plasmids, which are involved in the carriage of clinically important genes (e.g. those involved in antimicrobial resistance/virulence). The widespread application of this technique is currently primarily limited by cost. Recent data have suggested that non-inferior, and even superior, hybrid assemblies can be produced using a fraction of the total output from a multiplexed nanopore [Oxford Nanopore Technologies (ONT)] flowcell run. In this study we sought to determine the optimal minimal running time for flowcells when acquiring reads for hybrid assembly. We then evaluated whether the ONT wash kit might allow users to exploit shorter running times by sequencing multiple libraries per flowcell. After 24 h of sequencing, most chromosomes and plasmids had circularized and there was no benefit associated with longer running times. Quality was similar at 12 h, suggesting that shorter running times are likely to be acceptable for certain applications (e.g. plasmid genomics). The ONT wash kit was highly effective in removing DNA between libraries. Contamination between libraries did not appear to affect subsequent hybrid assemblies, even when the same barcodes were used successively on a single flowcell. Utilizing shorter run times in combination with between-library nuclease washes allows at least 36 Enterobacteriaceae isolates to be sequenced per flowcell, significantly reducing the per-isolate sequencing cost. Ultimately this will facilitate large-scale studies utilizing hybrid assembly, advancing our understanding of the genomics of key human pathogens.