Search

BackgroundAntimicrobial resistance (AMR) inE. coliis a global problem associated with substantial morbidity and mortality. AMR-associated genes are typically annotated based on similarity to a variants in a curated reference database with an implicit assumption that uncatalogued genetic variation within these is phenotypically unimportant. In this study we evaluated the potential for discovering new AMR-associated gene families and characterising variation within existing ones to improve genotype-to-susceptibility-phenotype prediction inE. coli.MethodsWe assembled a global dataset of 9001E. colisequences of which 8586 had linked antibiotic susceptibility data. Raw reads were assembled using Shovill and AMR genes extracted using the NCBI AMRFinder tool. Mash was used to calculate the similarity between extracted genes using Jaccard distances. We empirically reclustered extracted gene sequences into AMR-associated gene families (70% match) and alleles (ARGs, 100% match).ResultsThe performance of the AMRFinder database for genotype-to-phenotype predictions using strict 100% identity and coverage thresholds did not meet FDA thresholds for any of the eight antibiotics evaluated. Relaxing filters to default settings improved sensitivity with a specificity cost. For all antibiotics, a small number of genes explained most resistance although a proportion could not be explained by known ARGs; this ranged from 75.1% for co-amoxiclav to 3.4% for ciprofloxacin. Only 17,177/36,637 (47%) of ARGs detected had a 100% identity and coverage match in the AMRFinder database. After empirically reclassifying genes at 100% nucleotide sequence identity, we identified 1292 unique ARGs of which 158 (12%) were present ≥10 times, 374 (29%) were present 2-9 times and 760 (59%) only once. Simulated accumulation curves revealed that discovery of new (100%-match) ARGs present more than once in the dataset plateaued relatively quickly whereas new singleton ARGs were discovered even after many thousands of isolates had been included. We identified a strong correlation (Spearman coefficient 0.76 (95% CI 0.72-0.79, pblaTEM-1, we demonstrated that uncatalogued variation, including synonymous variation, is associated with potentially important phenotypic differences (e.g. two common, uncataloguedblaTEM-1alleles with only synonymous mutations compared to the known reference were associated with reduced resistance to co-amoxiclav [aOR 0.57, 95%CI 0.34-0.93, p=0.03] and piperacillin-tazobactam [aOR 0.54, 95%CI 0.32-0.87, p=0.01]).ConclusionsOverall we highlight substantial uncatalogued genetic variation with respect to known ARGs, although a relatively small proportion of these alleles are repeatedly observed in a large international dataset suggesting strong selection pressures. The current approach of using fuzzy matching for ARG detection, ignoring the unknown effects of uncatalogued variation, is unlikely to be acceptable for future clinical deployment. The association of synonymous mutations with potentially important phenotypic differences suggests that relying solely on amino acid-based gene detection to predict resistance is unlikely to be sufficient. Finally, the inability to explain all resistance using existing knowledge highlights the importance of new target gene discovery.

Population-representative estimates of SARS-CoV-2 infection prevalence and antibody levels in specific geographic areas at different time points are needed to optimise policy responses. However, even population-wide surveys are potentially impacted by biases arising from differences in participation rates across key groups. Here, we use spatio-temporal regression and post-stratification models to UK’s national COVID-19 Infection Survey (CIS) to obtain representative estimates of PCR positivity (6,496,052 tests) and antibody prevalence (1,941,333 tests) for different regions, ages and ethnicities (7-December-2020 to 4-May-2022). Not accounting for vaccination status through post-stratification led to small underestimation of PCR positivity, but more substantial overestimations of antibody levels in the population (up to 21%), particularly in groups with low vaccine uptake in the general population. There was marked variation in the relative contribution of different areas and age-groups to each wave. Future analyses of infectious disease surveys should take into account major drivers of outcomes of interest that may also influence participation, with vaccination being an important factor to consider.

The rise of antimicrobial resistance (AMR) is one of the greatest public health challenges, already causing up to 1.2 million deaths annually and rising. Current gold-standard antimicrobial susceptibility tests (ASTs) are low-throughput and can take up to 48 hours, with implications for patient care. We present advances towards a novel, rapid AST, based on the deep-learning of single-cell specific phenotypes directly associated with antimicrobial susceptibility inEscherichia coli. Our models can reliably (80% single-cell accuracy) classify untreated and treated susceptible cells, across a range of antibiotics and phenotypes - including phenotypes not visually distinct to a trained, human observer. Applying models trained on lab-reference susceptible strains to clinical isolates ofE. colitreated with ciprofloxacin, we demonstrate our models reveal significant (p

Following primary SARS-CoV-2 vaccination, understanding the relative extent of protection against SARS-CoV-2 infection from boosters or from breakthrough infections (i.e. infection in the context of previous vaccination) has important implications for vaccine policy. In this study, we investigated correlates of protection against Omicron BA.4/5 infections and anti-spike IgG antibody trajectories after a third/booster vaccination or breakthrough infection following second vaccination in 154,149 adults ≥18y from the United Kingdom general population. We found that higher anti-spike IgG antibody levels were associated with increased protection against Omicron BA.4/5 infection and that breakthrough infections were associated with higher levels of protection at any given antibody level than booster vaccinations. Breakthrough infections generated similar antibody levels to third/booster vaccinations, and the subsequent declines in antibody levels were similar to or slightly slower than those after third/booster vaccinations. Taken together our findings show that breakthrough infection provides longer lasting protection against further infections than booster vaccinations. For example, considering antibody levels associated with 67% protection against infection, a third/booster vaccination did not provide long-lasting protection, while a Delta/Omicron BA.1 breakthrough infection could provide 5-10 months of protection against Omicron BA.4/5 reinfection. 50-60% of the vaccinated UK population with a breakthrough infection would still be protected by the end of 2022, compared to

BackgroundMonitoring infection trends is vital to informing public health strategy. Detecting and quantifying changes in growth rates can inform policymakers’ rationale for implementing or continuing interventions aimed at reducing impact. Substantial changes in SARS-CoV-2 prevalence with emergence of variants provides opportunity to investigate different methods to do this.MethodsWe included PCR results from all participants in the UK’s COVID-19 Infection Survey between 1 August 2020-30 June 2022. Change-points for growth rates were identified using iterative sequential regression (ISR) and second derivatives of generalised additive models (GAMs).Consistency between methods and timeliness of detection were compared.FindingsOf 8,799,079 visits, 147,278 (1·7%) were PCR-positive. Over the time period, change-points associated with emergence of major variants were estimated to occur a median 4 days earlier (IQR 0-8) in GAMs versus ISR, with only 2/48 change-points identified by only one method. Estimating recent change-points using successive data periods, four change-points (4/96) identified by GAMs were not found when adding later data or by ISR; 77% (74/96) of change-points identified by successive GAMs were identified by ISR. Change-points were detected 3-5 weeks after they occurred in both methods but could be detected earlier within specific subgroups.InterpretationChange-points in growth rates of SARS-CoV-2 can be detected in near real-time using ISR and second derivatives of GAMs. To increase certainty about changes in epidemic trajectories both methods could be run in parallel. Running either method in near real-time on different infection surveillance data streams could provide timely warnings of changing underlying epidemiology.FundingUK Health Security Agency, Department of Health and Social Care (UK), Welsh Government, Department of Health (on behalf of the Northern Ireland Government), Scottish Government, National Institute for Health Research.

2.AbstractComplete, accurate, cost-effective, and high-throughput reconstruction of bacterial genomes for large-scale genomic epidemiological studies is currently only possible with hybrid assembly, combining long- (typically using nanopore sequencing) and short-read (Illumina) datasets. Being able to utilise nanopore-only data would be a significant advance. Oxford Nanopore Technologies (ONT) have recently released a new flowcell (R10.4) and chemistry (Kit12), which reportedly generate per-read accuracies rivalling those of Illumina data. To evaluate this, we sequenced DNA extracts from four commonly studied bacterial pathogens, namely Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus, using Illumina and ONT’s R9.4.1/Kit10, R10.3/Kit12, R10.4/Kit12 flowcells/chemistries. We compared raw read accuracy and assembly accuracy for each modality, considering the impact of different nanopore basecalling models, commonly used assemblers, sequencing depth, and the use of duplex versus simplex reads. “Super accuracy” (sup) basecalled R10.4 reads - in particular duplex reads - have high per-read accuracies and could be used to robustly reconstruct bacterial genomes without the use of Illumina data. However, the per-run yield of duplex reads generated in our hands with standard sequencing protocols was low (typically 3.Impact statementOur understanding of microbes has been greatly enhanced by the capacity to evaluate their genetic make-up using a technology known as whole genome sequencing. Sequencers represent microbial genomes as stretches of shorter sequence known as ‘reads’, which are then assembled using computational algorithms. Different types of sequencing approach have advantages and disadvantages with respect to the accuracy and length of the reads they generate; this in turn affects how reliably genomes can be assembled.Currently, to completely reconstruct bacterial genomes in a high-throughput and cost-effective manner, researchers tend to use two different types of sequencing data, namely Illumina (short-read) and nanopore (long-read) data. Illumina data are highly accurate; nanopore data are much longer, and this combination facilitates accurate and complete bacterial genomes in a so-called “hybrid assembly”. However, new developments in nanopore sequencing have reportedly greatly improved the accuracy of nanopore data, hinting at the possibility of requiring only a single sequencing approach for bacterial genomics.Here we evaluate these improvements in nanopore sequencing in the reconstruction of four bacterial reference strains, where the true sequence is already known. We show that although these improvements are extremely promising, for high-throughput, low-cost complete reconstruction of bacterial genomes hybrid assembly currently remains the optimal approach.4.Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article, through supplementary data files, or in publicly accessible repositories.Nanopore fast5 and fastq data are available in the ENA under project accession: PRJEB51164.Assemblies have been made available at: https://figshare.com/articles/online_resource/q20_comparison_genome_assemblies/196838 67.Code and analysis outputs are available at: https://gitlab.com/ModernisingMedicalMicrobiology/assembly_comparison_analysis/-/tree/main (tagged version v0.5.5).

Plasmids carry genes conferring antimicrobial resistance (AMR), and other clinically important traits; their ability to move within and between species may provide the machinery for rapid dissemination of such genes. Existing studies using complete plasmid assemblies, which are essential for reliable inference, have been small and/or limited to those carrying particularly antimicrobial resistance genes (ARGs). In this study, we sequenced 1,880 complete plasmids from 738 isolates from bloodstream infections (BSI) in 2009 (194 isolates) and 2018 (368 isolates) in Oxfordshire, UK, plus a stratified selection from intervening years (176 isolates). We demonstrate that plasmids are largely, but not entirely, constrained to 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.

Genome sequencing is pivotal to SARS-CoV-2 surveillance, elucidating the emergence and global dissemination of acquired genetic mutations. Amplicon sequencing has proven very effective for sequencing SARS-CoV-2, but prevalent mutations disrupting primer binding sites have necessitated the revision of sequencing protocols in order to maintain performance for emerging virus lineages. We compared the performance of Oxford Nanopore Technologies (ONT) Midnight and ARTIC tiling amplicon protocols using 196 Delta lineage SARS-CoV-2 clinical specimens, and 71 mostly Omicron lineage samples with S gene target failure (SGTF), reflecting circulating lineages in the United Kingdom during December 2021. 96-plexed nanopore sequencing was used. For Delta lineage samples, ARTIC v4 recovered the greatest proportion of ≥90% complete genomes (81.1%; 159/193), followed by Midnight (71.5%; 138/193) and ARTIC v3 (34.1%; 14/41). Midnight protocol however yielded higher average genome recovery (mean 98.8%) than ARTIC v4 (98.1%) and ARTIC v3 (75.4%), resulting in less ambiguous final consensus assemblies overall. Explaining these observations were ARTIC v4’s superior genome recovery in low viral titre/high cycle threshold (Ct) samples and inferior performance in high titre/low Ct samples, where Midnight excelled. We evaluated Omicron sequencing performance using a revised Midnight primer mix alongside prototype ARTIC v4.1 primers, head-to-head with the existing commercially available Midnight and ARTIC v4 protocols. The revised protocols both improved considerably the recovery of Omicron genomes and exhibited similar overall performance to one another. Revised Midnight protocol recovered ≥90% complete genomes for 85.9% (61/71) of Omicron samples vs. 88.7% (63/71) for ARTIC v4.1. Approximate cost per sample for Midnight (£12) is lower than ARTIC (£16) while hands-on time is considerably lower for Midnight (∼7 hours) than ARTIC protocols (∼9.5 hours).

Given high SARS-CoV-2 incidence, coupled with slow and inequitable vaccine roll-out, there is an urgent need for evidence to underpin optimum vaccine deployment, aiming to maximise global population immunity at speed. We evaluate whether a single vaccination in previously infected individuals generates similar initial and subsequent antibody responses to two vaccinations in those without prior infection. We compared anti-spike IgG antibody responses after a single dose of ChAdOx1, BNT162b2, or mRNA-1273 SARS-CoV-2 vaccines in the COVID-19 Infection Survey in the UK general population. In 100,849 adults who received at least one vaccination, 13,404 (13.3%) had serological and/or PCR evidence of prior infection. Prior infection significantly boosted antibody responses for all three vaccines, producing a higher peak level and longer half-life, and a response comparable to those without prior infection receiving two vaccinations. In those with prior infection, median time above the positivity threshold was estimated to last for >1 year after the first dose. Single-dose vaccination targeted to those previously infected may provide protection in populations with high rates of previous infection faced with limited vaccine supply, as an interim measure while vaccine campaigns are scaled up.

BackgroundHealthcare-associated wastewater reservoirs and asymptomatic gastrointestinal patient colonisation by carbapenemase-producing Enterobacterales (CPE) can be important in nosocomial CPE dissemination and infection. We characterised these niches and within-niche diversity in a blaKPC-associated CPE (KPC-E) endemic healthcare setting, to better understand transmission potential.MethodsWe systematically sampled wastewater sites and patients across three units (six wards) over 6-12 months in 2016 in a KPC-E endemic hospital. We used Illumina sequencing to characterise up to five isolates per sample. Recombination-adjusted phylogenies were used to define genetically related strains; assembly and mapping-based typing approaches were used to characterise antimicrobial resistance gene and insertion sequence profiles, and Tn4401 types/target site sequences. The wider accessory genome was evaluated in a subset of the largest clusters, and those crossing niches.FindingsWastewater site KPC-E-positivity was substantial (101/349 sites [28.9%] positive, 319/4,488 [7.1%] sampling events positive); 183/4,425 (4.1%) of patients were CPE culture-positive over the same timeframe. 13 species and 109 KPC-E strains were observed across niches, and 24% of wastewater and 26% of patient KPC-E-positive samples harboured ≥1 strain. Most diversity was explained by the individual niche, suggesting highly localised factors are important in selection and spread. Tn4401+target site sequence (TSS) diversity was greater in wastewater sites (p4401-associated transposition/evolution and dissemination. Shower/bath and sluice/mop-associated sites were more likely to be KPC-E-positive (Adjusted Odds Ratio [95% CI]: 2.69 [1.44-5.01], p=0.0019 and 2.60 [1.04-6.52], p=0.0410, respectively). Different strains had different transmission and blaKPC dissemination dynamics.InterpretationThere may be substantial KPC-E colonisation of wastewater sites and patients in KPC-E-endemic healthcare settings. Niche-specific factors, and different strains with different transmission dynamics influence carbapenemase gene dissemination. New transmission models incorporating complex, multi-level dynamics are needed to better quantify CPE dissemination to inform interventions and reduce transmission.FundingThis study was supported by the National Institute for Health Research, UK.RESEARCH IN CONTEXTEvidence before this studyWe searched PubMed to identify previous studies using whole genome sequencing (WGS) to characterise in-hospital dissemination and persistence of carbapenemase-producing Enterobacterales (CPEs) or the presence of CPEs in hospital wastewater reservoirs. In our search (01/Jan/1996-30/Sep/2021) we used the terms ((((CPE) OR (KPC)) AND (healthcare-associated OR nosocomial OR hospital-associated)) AND (genom*) AND ((environment OR patient))). We had no restrictions on language. We found sixty-six studies, but these were limited by: (i) evaluating single species or lineages only (n=34), (ii) including patients or environmental outbreaks only, and not both (n=23), (iii) sampling only small numbers (nex vivo or in vivo evolution), two studies did not use WGS, and one study was not primary research.Added value of this studyWe provide a comprehensive assessment of healthcare-associated CPE dissemination and persistence in hospital wastewater reservoirs through systematic sampling of a large number of environmental sites (n=349 sites, n=4,488 samples) and patients (n=4,425) over 6-12 months in a single hospital. For all CPE-positive environmental samples (n=319) and a subset of CPE-positive patient samples (n=97/399 [24.3%] samples from 76/183 [41.5%] CPE-positive patients), we sampled up to five CPE isolates per sample to capture within-niche diversity, and used short-read Illumina WGS (n=1,732 isolates successfully sequenced) to characterise within-niche diversity, changes in colonisation over time, and genetic overlap between patient and environmental niches.Implications of all the available evidenceThrough a detailed and resource-intensive study we captured the dynamics of seeding and dissemination of important carbapenemase genes amongst patients and environmental reservoirs within a hospital. We found differential colonisation and dissemination dynamics for different species and lineages within and between niches. Improved approaches incorporating variable within-niche diversity, accessory distances and horizontal gene transfer in transmission evaluations are required to better understand CPE dissemination and persistence, in order to direct interventions limiting transmission.

Funder: Department of Health, Funder: Department of Health & Social Care, BackgroundInformation on SARS-CoV-2 in representative community surveillance is limited, particularly cycle threshold (Ct) values (a proxy for viral load).MethodsWe included all positive nose and throat swabs 26 April 2020 to 13 March 2021 from the UK's national COVID-19 Infection Survey, tested by RT-PCR for the N, S, and ORF1ab genes. We investigated predictors of median Ct value using quantile regression.ResultsOf 3,312,159 nose and throat swabs, 27,902 (0.83%) were RT-PCR-positive, 10,317 (37%), 11,012 (40%), and 6550 (23%) for 3, 2, or 1 of the N, S, and ORF1ab genes, respectively, with median Ct = 29.2 (~215 copies/ml; IQR Ct = 21.9-32.8, 14-56,400 copies/ml). Independent predictors of lower Cts (i.e. higher viral load) included self-reported symptoms and more genes detected, with at most small effects of sex, ethnicity, and age. Single-gene positives almost invariably had Ct > 30, but Cts varied widely in triple-gene positives, including without symptoms. Population-level Cts changed over time, with declining Ct preceding increasing SARS-CoV-2 positivity. Of 6189 participants with IgG S-antibody tests post-first RT-PCR-positive, 4808 (78%) were ever antibody-positive; Cts were significantly higher in those remaining antibody negative.ConclusionsMarked variation in community SARS-CoV-2 Ct values suggests that they could be a useful epidemiological early-warning indicator.FundingDepartment of Health and Social Care, National Institutes of Health Research, Huo Family Foundation, Medical Research Council UK; Wellcome Trust.

2.AbstractSeveral bioinformatics genotyping algorithms are now commonly used to characterise antimicrobial resistance (AMR) gene profiles in whole genome sequencing (WGS) data, with a view to understanding AMR epidemiology and developing resistance prediction workflows using WGS in clinical settings. Accurately evaluating AMR in Enterobacterales, particularly Escherichia coli, is of major importance, because this is a common pathogen. However, robust comparisons of different genotyping approaches on relevant simulated and large real-life WGS datasets are lacking. Here, we used both simulated datasets and a large set of real E. coli WGS data (n=1818 isolates) to systematically investigate genotyping methods in greater detail.Simulated constructs and real sequences were processed using four different bioinformatic programs (ABRicate, ARIBA, KmerResistance, and SRST2, run with the ResFinder database) and their outputs compared. For simulations tests where 3,092 AMR gene variants were inserted into random sequence constructs, KmerResistance was correct for all 3,092 simulations, ABRicate for 3,082 (99.7%), ARIBA for 2,927 (94.7%) and SRST2 for 2,120 (68.6%). For simulations tests where two closely related gene variants were inserted into random sequence constructs, ABRicate identified the correct alleles in 11,382/46,279 (25%) of simulations, ARIBA in 2494/46,279 (5%), SRST in 2539/46,279 (5%) and KmerResistance in 38,826/46,279 (84%). In real data, across all methods, 1392/1818 (76%) isolates had discrepant allele calls for at least one gene.Our evaluations revealed poor performance in scenarios that would be expected to be challenging (e.g. identification of AMR genes at 3.Impact statementWhole-genome sequencing is widely used for studying the epidemiology of antimicrobial resistance (AMR) genes in bacteria; however, there is some concern that outputs are highly dependent on the bioinformatics methods used. This work evaluates these concerns in detail by comparing four different, commonly used AMR gene typing methods using large simulated and real datasets. The results highlight performance issues for most methods in at least one of several simulated and real-life scenarios. However most discrepancies between methods were due to differential labelling of the same sequences related to the assumptions made regarding the underlying structure of the reference resistance gene database (i.e. that resistance genes can be easily classified in well-defined groups). This study represents a major advance in quantifying and evaluating the nature of discrepancies between outputs of different AMR typing algorithms, with relevance for historic and future work using these algorithms. Some of the discrepancies can be resolved by choosing methods with fewer assumptions about the reference AMR gene database and manually resolving outputs generated using multiple programs. However, ideally new and better methods are needed.1.5RepositoriesSequencing data are available at the following NCBI BioProject accession number: PRJNA540750.

We investigated anti-spike IgG antibody responses and correlates of protection following second doses of ChAdOx1 or BNT162b2 SARS-CoV-2 vaccines in the UK general population. In 222,493 individuals, we found significant boosting of anti-spike IgG by second doses of both vaccines in all ages and using different dosing intervals, including the 3-week interval for BNT162b2. After second vaccination, BNT162b2 generated higher peak levels than ChAdOX1. Older individuals and males had lower peak levels with BNT162b2 but not ChAdOx1, while declines were similar across ages and sexes with ChAdOX1 or BNT162b2. Prior infection significantly increased antibody peak level and half-life with both vaccines. Anti-spike IgG levels were associated with protection from infection after vaccination and, to an even greater degree, after prior infection. At least 67% protection against infection was estimated to last for 2-3 months after two ChAdOx1 doses and 5-8 months after two BNT162b2 doses in those without prior infection, and 1-2 years for those unvaccinated after natural infection. A third booster dose may be needed, prioritised to ChAdOx1 recipients and those more clinically vulnerable.

BackgroundThe COVID-19 pandemic is rapidly evolving, with emerging variants and fluctuating control policies. Real-time population screening and identification of groups in whom positivity is highest could help monitor spread and inform public health messaging and strategy.MethodsTo develop a real-time screening process, we included results from nose and throat swabs and questionnaires taken 19 July 2020-17 July 2021 in the UK’s national COVID-19 Infection Survey. Fortnightly, associations between SARS-CoV-2 positivity and 60 demographic and behavioural characteristics were estimated using logistic regression models adjusted for potential confounders, considering multiple testing, collinearity, and reverse causality.FindingsOf 4,091,537 RT-PCR results from 482,677 individuals, 29,903 (0·73%) were positive. As positivity rose September-November 2020, rates were independently higher in younger ages, and those living in Northern England, major urban conurbations, more deprived areas, and larger households. Rates were also higher in those returning from abroad, and working in healthcare or outside of home. When positivity peaked December 2020-January 2021 (Alpha), high positivity shifted to southern geographical regions. With national vaccine roll-out from December 2020, positivity reduced in vaccinated individuals. Associations attenuated as rates decreased between February-May 2021. Rising positivity rates in June-July 2021 (Delta) were independently higher in younger, male, and unvaccinated groups. Few factors were consistently associated with positivity. 25/45 (56%) confirmed associations would have been detected later using 28-day rather than 14-day periods.InterpretationPopulation-level demographic and behavioural surveillance can be a valuable tool in identifying the varying characteristics driving current SARS-CoV-2 positivity, allowing monitoring to inform public health policy.FundingDepartment of Health and Social Care (UK), Welsh Government, Department of Health (on behalf of the Northern Ireland Government), Scottish Government, National Institute for Health Research.

BackgroundSeveral community-based studies have assessed the ability of different symptoms to identify COVID-19 infections, but few have compared symptoms over time (reflecting SARS-CoV-2 variants) and by vaccination status.MethodsUsing data and samples collected by the COVID-19 Infection Survey at regular visits to representative households across the UK, we compared symptoms in new PCR-positives and comparator test-negative controls.ResultsFrom 26/4/2020-7/8/2021, 27,869 SARS-CoV-2 PCR-positive episodes occurred in 27,692 participants (median 42 years (IQR 22-58)); 13,427 (48%) self-reported symptoms (“symptomatic positive episodes”). The comparator group comprised 3,806,692 test-negative visits (457,215 participants); 130,612 (3%) self-reported symptoms (“symptomatic negative visit”). Reporting of any symptoms in positive episodes varied over calendar time, reflecting changes in prevalence of variants, incidental changes (e.g. seasonal pathogens, schools re-opening) and vaccination roll-out. There was a small increase in sore throat reporting in symptomatic positive episodes and negative visits from April-2021. After May-2021 when Delta emerged there were substantial increases in headache and fever in positives, but not in negatives. Although specific symptom reporting in symptomatic positive episodes vs. negative visits varied by age, sex, and ethnicity, only small improvements in symptom-based infection detection were obtained; e.g. adding fatigue/weakness or all eight symptoms to the classic four symptoms (cough, fever, loss of taste/smell) increased sensitivity from 74% to 81% to 90% but tests per positive from 4.6 to 5.3 to 8.7.ConclusionsWhilst SARS-CoV-2-associated symptoms vary by variant, vaccination status and demographics, differences are modest and do not warrant large-scale changes to targeted testing approaches given resource implications.SummaryWithin the COVID-19 Infection Survey, recruiting representative households across the UK general population, SARS-CoV-2-associated symptoms varied by viral variant, vaccination status and demographics. However, differences are modest and do not currently warrant large-scale changes to targeted testing approaches.

The effectiveness of BNT162b2, ChAdOx1, and mRNA-1273 vaccines against new SARS-CoV-2 infections requires continuous re-evaluation, given the increasingly dominant Delta variant. We investigated the effectiveness of the vaccines in a large community-based survey of randomly selected households across the UK. We found that the effectiveness of BNT162b2 and ChAd0x1 against any infections (new PCR positives) and infections with symptoms or high viral burden is reduced with the Delta variant. A single dose of the mRNA-1273 vaccine had similar or greater effectiveness compared to a single dose of BNT162b2 or ChAdOx1. Effectiveness of two doses remains at least as great as protection afforded by prior natural infection. The dynamics of immunity following second doses differed significantly between BNT162b2 and ChAdOx1, with greater initial effectiveness against new PCR-positives but faster declines in protection against high viral burden and symptomatic infection with BNT162b2. There was no evidence that effectiveness varied by dosing interval, but protection was higher among those vaccinated following a prior infection and younger adults. With Delta, infections occurring following two vaccinations had similar peak viral burden to those in unvaccinated individuals. SARS-CoV-2 vaccination still reduces new infections, but effectiveness and attenuation of peak viral burden are reduced with Delta.

Understanding the trajectory, duration, and determinants of antibody responses after SARS-CoV-2 infection can inform subsequent protection and risk of reinfection, however large-scale representative studies are limited. Here we estimated antibody response after SARS-CoV-2 infection in the general population using representative data from 7,256 United Kingdom COVID-19 infection survey participants who had positive swab SARS-CoV-2 PCR tests from 26-April-2020 to 14-June-2021. A latent class model classified 24% of participants as ‘non-responders’ not developing anti-spike antibodies, who were older, had higher SARS-CoV-2 cycle threshold values during infection (i.e. lower viral burden), and less frequently reported any symptoms. Among those who seroconverted, using Bayesian linear mixed models, the estimated anti-spike IgG peak level was 7.3-fold higher than the level previously associated with 50% protection against reinfection, with higher peak levels in older participants and those of non-white ethnicity. The estimated anti-spike IgG half-life was 184 days, being longer in females and those of white ethnicity. We estimated antibody levels associated with protection against reinfection likely last 1.5-2 years on average, with levels associated with protection from severe infection present for several years. These estimates could inform planning for vaccination booster strategies., Most people who are infected with SARS-CoV-2 seroconvert within a few weeks, but the determinants and duration of the antibody response are not known. Here, the authors characterise these features of the immune response using data from a large representative community sample of the UK population.

BackgroundDespite robust efforts, patients and staff acquire SARS-CoV-2 infection in hospitals. In this retrospective cohort study, we investigated whether whole-genome sequencing (WGS) could enhance the epidemiological investigation of healthcare-associated SARS-CoV-2 acquisition.Methods and findingsFrom 17-November-2020 to 5-January-2021, 803 inpatients and 329 staff were diagnosed with SARS-CoV-2 infection across four teaching hospitals in Oxfordshire, UK. We classified cases according to epidemiological definitions, sought epidemiological evidence of a potential source for each nosocomial infection, and evaluated if epidemiologically-linked cases had genomic evidence supporting transmission. We compared epidemiological and genomic outbreak identification.Using national epidemiological definitions, 109/803 (14%) inpatient infections were classified as definite/probable nosocomial, 615 (77%) as community-acquired and 79 (10%) as indeterminate. There was strong epidemiological evidence to support definite/probable cases as nosocomial: 107/109 (98%) had a prior-negative PCR in the same hospital stay before testing positive, and 101(93%) shared time and space with known infected patients/staff. Many indeterminate cases were likely infected in hospital: 53/79 (67%) had a prior-negative PCR and 75 (95%) contact with a potential source. 89/615 (11% of all 803 patients) with apparent community-onset had a recent hospital exposure.WGS highlighted SARS-CoV-2 is mainly imported into hospitals: within 764 samples sequenced 607 genomic clusters were identified (>1 SNP distinct). Only 43/607 (7%) clusters contained evidence of onward transmission (subsequent cases within ≤1 SNP). 20/21 epidemiologically-identified outbreaks contained multiple genomic introductions. Most (80%) nosocomial acquisition occurred in rapid super-spreading events in settings with a mix of COVID-19 and non-COVID-19 patients. Hospitals not routinely admitting COVID-19 patients had low rates of transmission. Undiagnosed/unsequenced individuals prevent genomic data from excluding nosocomial acquisition.ConclusionsOur findings suggest current surveillance definitions underestimate nosocomial acquisition and reveal most nosocomial transmission occurs from a relatively limited number of highly infectious individuals.

ObjectivesGram-negative organisms are common causes of bloodstream infection (BSI) during the neonatal period and early childhood. Whilst several large studies have characterised these isolates in adults, equivalent data (particularly incorporating whole genome sequencing) is lacking in the paediatric population.MethodsWe performed an epidemiological and sequencing based analysis of Gram-negative bloodstream infections in children Results327 isolates (296 successfully sequenced) from 287 patients were included. The burden of infection was predominantly in neonates (124/327[38%]). Most infections were caused by Escherichia coli (149/327[46%])/Klebsiella spp. (69/327[21%]) and Enterobacter hormaechei (34/327[10%]). There was no evidence of an increasing incidence of E. coli BSIs (IRRy 0.96, 95%CI 0.90-1.30, p=0.30) and for Klebsiella spp. there was some evidence that the incidence decreased slightly (IRRy 0.91, 95%CI 0.83-1.00, p=0.06). Similarly the proportion of antimicrobial resistant (across all antimicrobial classes evaluated) isolates did not change over time, though we did identify some evidence of sub-breakpoint increases in gentamicin resistance IRRy 1.86, 95%CI 1.33-2.58, pheterogeneity=0.002. The population structure of E. coli BSI isolates in neonates and children mirrors that in adults with a predominance of STs 131/95/73/69 and the same proportion of O-antigen serotypes covered by the ExPEC-4V vaccine. In most cases there was no evidence of transmission/point-source acquisition and whole genome sequencing was able to refute a previously suspected Serratia marcescens outbreak.ConclusionOur findings support continued use of current empirical treatment guidelines and suggest that O-antigen targeted vaccines may have a role in reducing the incidence of neonatal sepsis.

Real-world data on antibody response post-vaccination in the general population are limited. 45,965 adults in the UK’s national COVID-19 Infection Survey receiving Pfizer-BioNTech or Oxford-AstraZeneca vaccines had 111,360 anti-spike IgG measurements. Without prior infection, seroconversion rates and quantitative antibody levels post single dose were lower in older individuals, especially >60y. Two doses achieved high responses across all ages, particularly increasing seroconversion in older people, to similar levels to those achieved after prior infection followed by a single dose. Antibody levels rose more slowly and to lower levels with Oxford-AstraZeneca vs Pfizer-BioNTech, but waned following a single Pfizer-BioNTech dose. Latent class models identified four responder phenotypes: older people, males, and those having long-term health conditions were more commonly ‘low responders’. Where supplies are limited, vaccines should be prioritised for those not previously infected, and second doses to individuals >60y. Further data on the relationship between vaccine-mediated protection and antibody responses are needed.

Objectives: To assess the effectiveness of COVID-19 vaccination in preventing SARS-CoV-2 infection in the community. Design: Prospective cohort study. Setting: The UK population-representative longitudinal COVID-19 Infection Survey. Participants: 373,402 participants aged ≥16 years contributing 1,610,562 RT-PCR results from nose and throat swabs between 1 December 2020 and 3 April 2021. Main outcome measures: New RT-PCR-positive episodes for SARS-CoV-2 overall, by self-reported symptoms, by cycle threshold (Ct) value (0.9).There was no evidence of a difference in odds of new SARS-CoV-2 infection for individuals having received two vaccine doses and with evidence of prior infection but not vaccinated (P=0.89). Vaccination had a greater impact on reducing SARS-CoV-2 infections with evidence of high viral shedding Ct

ObjectivesWe investigate determinants of SARS-CoV-2 anti-spike IgG responses in healthcare workers (HCWs) following one or two doses of Pfizer-BioNTech or Oxford-AstraZeneca vaccines.MethodsHCWs participating in regular SARS-CoV-2 PCR and antibody testing were invited for serological testing prior to first and second vaccination, and 4 weeks post-vaccination if receiving a 12-week dosing interval. Quantitative post-vaccination anti-spike antibody responses were measured using the Abbott SARS-CoV-2 IgG II Quant assay (detection threshold: ≥50 AU/ml). We used multivariable logistic regression to identify predictors of seropositivity and generalised additive models to track antibody responses over time.ResultsVaccine uptake was 80%, but less in lower-paid roles and Black, south Asian and minority ethnic groups. 3570/3610(98.9%) HCWs were seropositive >14 days post-first vaccination and prior to second vaccination, 2706/2720(99.5%) after Pfizer-BioNTech and 864/890(97.1%) following Oxford-AstraZeneca vaccines. Previously infected and younger HCWs were more likely to test seropositive post-first vaccination, with no evidence of differences by sex or ethnicity. All 470 HCWs tested >14 days after second vaccine were seropositive. Quantitative antibody responses were higher after previous infection: median(IQR) >21 days post-first Pfizer-BioNTech 14,604(7644-22,291) AU/ml vs. 1028(564-1985) AU/ml without prior infection (pConclusionsVaccination leads to detectable anti-spike antibodies in nearly all adult HCWs. Whether differences in response impact vaccine efficacy needs further study.

BackgroundNatural and vaccine-induced immunity will play a key role in controlling the SARS-CoV-2 pandemic. SARS-CoV-2 variants have the potential to evade natural and vaccine-induced immunity.MethodsIn a longitudinal cohort study of healthcare workers (HCWs) in Oxfordshire, UK, we investigated the protection from symptomatic and asymptomatic PCR-confirmed SARS-CoV-2 infection conferred by vaccination (Pfizer-BioNTech BNT162b2, Oxford-AstraZeneca ChAdOx1 nCOV-19) and prior infection (determined using anti-spike antibody status), using Poisson regression adjusted for age, sex, temporal changes in incidence and role. We estimated protection conferred after one versus two vaccinations and from infections with the B.1.1.7 variant identified using whole genome sequencing.Results13,109 HCWs participated; 8285 received the Pfizer-BioNTech vaccine (1407 two doses) and 2738 the Oxford-AstraZeneca vaccine (49 two doses). Compared to unvaccinated seronegative HCWs, natural immunity and two vaccination doses provided similar protection against symptomatic infection: no HCW vaccinated twice had symptomatic infection, and incidence was 98% lower in seropositive HCWs (adjusted incidence rate ratio 0.02 [95%CI ConclusionNatural infection resulting in detectable anti-spike antibodies and two vaccine doses both provide robust protection against SARS-CoV-2 infection, including against the B.1.1.7 variant.SummaryNatural infection resulting in detectable anti-spike antibodies and two vaccine doses both provided ≥ 85% protection against symptomatic and asymptomatic SARS-CoV-2 infection in healthcare workers, including against the B.1.1.7 variant. Single dose vaccination reduced symptomatic infection by 67%.

Analysing the flanking sequences surrounding genes of interest is often highly relevant to understanding the role of mobile genetic elements (MGEs) in horizontal gene transfer, particular for antimicrobial resistance genes. Here, we present Flanker, a Python package which performs alignment-free clustering of gene flanking sequences in a consistent format, allowing investigation of MGEs without prior knowledge of their structure. These clusters, known as ‘flank patterns’, are based on Mash distances, allowing for easy comparison of similarity across sequences. Additionally, Flanker can be flexibly parameterised to finetune outputs by characterising upstream and downstream regions separately and investigating variable lengths of flanking sequence. We apply Flanker to two recent datasets describing plasmid-associated carriage of important carbapenemase genes (blaOXA-48 and blaKPC-2/3) and show that it successfully identifies distinct clusters of flank patterns, including both known and previously uncharacterised structural variants. For example, Flanker identified four Tn4401 profiles that could not be sufficiently characterised using TETyper or MobileElementFinder, demonstrating the utility of Flanker for flanking gene characterisation. Similarly, using a large (n=226) European isolate dataset, we confirm findings from a previous smaller study demonstrating association between Tn1999.2 andblaOXA-48upregulation and demonstrate 17 flank patterns (compared to the 5 previously identified). More generally the demonstration in this study that flank patterns are associated with to geographical regions and antibiotic susceptibility phenotypes suggests that they may be useful as epidemiological markers. Flanker is freely available under an MIT license athttps://github.com/wtmatlock/flanker.Data SummaryNCBI accession numbers for all sequencing data used in this study is provided in Supplementary Table 1. The analysis performed in this manuscript can be reproduced in a binder environment provided on the Flanker Github page (https://github.com/wtmatlock/flanker).

BackgroundA new variant of SARS-CoV-2, B.1.1.7/VOC202012/01, was identified in the UK in December-2020. Direct estimates of its potential to enhance transmission are limited.MethodsNose and throat swabs from 28-September-2020 to 2-January-2021 in the UK’s nationally representative surveillance study were tested by RT-PCR for three genes (N, S and ORF1ab). Those positive only on ORF1ab+N, S-gene target failures (SGTF), are compatible with B.1.1.7/VOC202012/01. We investigated cycle threshold (Ct) values (a proxy for viral load), percentage of positives, population positivity and growth rates in SGTF vs non-SGTF positives.Results15,166(0.98%) of 1,553,687 swabs were PCR-positive, 8,545(56%) with three genes detected and 3,531(23%) SGTF. SGTF comprised an increasing, and triple-gene positives a decreasing, percentage of infections from late-November in most UK regions/countries, e.g. from 15% to 38% to 81% over 1.5 months in London. SGTF Ct values correspondingly declined substantially to similar levels to triple-gene positives. Population-level SGTF positivity remained low (ConclusionsDirect population-representative estimates show that the B.1.1.7/VOC202012/01 SARS-CoV-2 variant leads to higher infection rates, but does not seem particularly adapted to any age group.

BackgroundIt is critical to understand whether infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) protects from subsequent reinfection.MethodsWe investigated the incidence of SARS-CoV-2 PCR-positive results in seropositive and seronegative healthcare workers (HCWs) attending asymptomatic and symptomatic staff testing at Oxford University Hospitals, UK. Baseline antibody status was determined using anti-spike and/or anti-nucleocapsid IgG assays and staff followed for up to 30 weeks. We used Poisson regression to estimate the relative incidence of PCR-positive results and new symptomatic infection by antibody status, accounting for age, gender and changes in incidence over time.ResultsA total of 12219 HCWs participated and had anti-spike IgG measured, 11052 were followed up after negative and 1246 after positive antibody results including 79 who seroconverted during follow up. 89 PCR-confirmed symptomatic infections occurred in seronegative individuals (0.46 cases per 10,000 days at risk) and no symptomatic infections in those with anti-spike antibodies. Additionally, 76 (0.40/10,000 days at risk) anti-spike IgG seronegative individuals had PCR-positive tests in asymptomatic screening, compared to 3 (0.21/10,000 days at risk) seropositive individuals. Overall, positive baseline anti-spike antibodies were associated with lower rates of PCR-positivity (with or without symptoms) (adjusted rate ratio 0.24 [95%CI 0.08-0.76, p=0.015]). Rate ratios were similar using anti-nucleocapsid IgG alone or combined with anti-spike IgG to determine baseline status.ConclusionsPrior SARS-CoV-2 infection that generated antibody responses offered protection from reinfection for most people in the six months following infection. Further work is required to determine the long-term duration and correlates of post-infection immunity.

SUMMARYThe prevalence and proliferation of antimicrobial resistant bacteria is considered one of the critical issues of our time. Wastewater is a habitat for complex microbial communities where bacteria share genes of antimicrobial resistance through horizontal gene transfer. Hospital wastewater drainage systems are an ideal reservoir for environmental and pathogenic bacteria to interface and exchange genes of antimicrobial resistance1. Highly resistant Enterobacterales are of the greatest concern because they not only cause human infections but can also thrive in the environment2,3 and have shown to transmit from sink drains to patients4. Replacement of contaminated plumbing may be the most intuitive and widely deployed response once highly resistant potentially pathogenic bacteria are identified in a sink drain. Here we show using shotgun metagenomic sequencing of sink drain biofilms (in 6 intensive care patient rooms, 36 subsamples) paired with microbial culture analysis, an evident shift of biofilm community structure towards increased abundance of Enterobacteriaceae in the newly replaced sink plumbing. Significant increase in resistome load and abundance of clinically relevant resistance and typically mobile genes in the newly replaced plumbing was also observed. Findings from this study demonstrate that exchanging contaminated plumbing for new plumbing may actually have the unexpected consequence of increased abundance of Enterobacterales and genes of antimicrobial resistance in the sink drains. Disruption of preexisting complex environmental biofilms may result in an unintended increase in the amount of antimicrobial resistant Enterobacterales which are targeted for elimination. Temporal variability is an important attribute of ecosystems affecting colonization dynamics and biotic interactions (composition of introduced and resident communities) are key to functional outcomes. Absence of competition within an ecosystem favors invasion.

Background Serological tests are widely used in various medical disciplines for diagnostic and monitoring purposes. Unfortunately, the sensitivity and specificity of test systems is often poor, leaving room for false positive and false negative results. However, conventional methods used to increase specificity decrease sensitivity and vice versa. Using SARS-CoV-2 serology as an example, we propose here a novel testing strategy: the "Sensitivity Improved Two-Test" or "SIT2" algorithm. Methods SIT2 involves confirmatory re-testing of samples with results falling in a predefined retesting-zone of an initial screening test, with adjusted cut-offs to increase sensitivity. We verified and compared the performance of SIT2 to single tests and orthogonal testing (OTA) in an Austrian cohort (1,117 negative, 64 post-COVID positive samples) and validated the algorithm in an independent British cohort (976 negatives, 536 positives). Results The specificity of SIT2 was superior to single tests and non-inferior to OTA. The sensitivity was maintained or even improved using SIT2 when compared to single tests or OTA. SIT2 allowed correct identification of infected individuals even when a live virus neutralization assay could not detect antibodies. Compared to single testing or OTA, SIT2 significantly reduced total test errors to 0.46% (0.24-0.65) or 1.60% (0.94-2.38) at both 5% or 20% seroprevalence. Conclusion For SARS-CoV-2 serology, SIT2 proved to be the best diagnostic choice at both 5% and 20% seroprevalence in all tested scenarios. It is an easy to apply algorithm and can potentially be helpful for the serology of other infectious diseases.

BackgroundSARS-CoV-2 IgG antibody measurements can be used to estimate the proportion of a population exposed or infected and may be informative about the risk of future infection. Previous estimates of the duration of antibody responses vary.MethodsWe present 6 months of data from a longitudinal seroprevalence study of 3217 UK healthcare workers (HCWs). Serial measurements of IgG antibodies to SARS-CoV-2 nucleocapsid were obtained. Bayesian mixed linear models were used to investigate antibody waning and associations with age, gender, ethnicity, previous symptoms and PCR results.ResultsIn this cohort of working age HCWs, antibody levels rose to a peak at 24 (95% credibility interval, CrI 19-31) days post-first positive PCR test, before beginning to fall. Considering 452 IgG seropositive HCWs over a median of 121 days (maximum 171 days) from their maximum positive IgG titre, the mean estimated antibody half-life was 85 (95%CrI, 81-90) days. The estimated mean time to loss of a positive antibody result was 137 (95%CrI 127-148) days. We observed variation between individuals; higher maximum observed IgG titres were associated with longer estimated antibody half-lives. Increasing age, Asian ethnicity and prior self-reported symptoms were independently associated with higher maximum antibody levels, and increasing age and a positive PCR test undertaken for symptoms with longer antibody half-lives.ConclusionIgG antibody levels to SARS-CoV-2 nucleocapsid wane within months, and faster in younger adults and those without symptoms. Ongoing longitudinal studies are required to track the long-term duration of antibody levels and their association with immunity to SARS-CoV-2 reinfection.SummarySerially measured SARS-CoV-2 anti-nucleocapsid IgG titres from 452 seropositive healthcare workers demonstrate levels fall by half in 85 days. From a peak result, detectable antibodies last a mean 137 days. Levels fall faster in younger adults and following asymptomatic infection.

SynopsisBackgroundAntimicrobial resistance (AMR) surveillance of bloodstream infections is challenging in low- and middle-income countries (LMICs), limited laboratory capacity preventing routine patient-level susceptibility testing. Other specimen types could provide an effective approach to surveillance.ObjectivesOur study aims to systematically evaluate the relationship between resistance prevalence in non-sterile sites and bloodstream infections.MethodsAssociations between resistance rates in Escherichia coli and Staphylococcus aureus isolates from blood and other specimens were estimated in Oxfordshire, UK, 1998-2018, comparing proportions resistant in each calendar year using time series cross-correlations and across drug-years. We repeated analysis across publicly-available data from four high-income and 12 middle-income countries, and in three hospitals/programmes in LMICs.Results8102 E. coli bloodstream infections, 322087 E. coli urinary tract infections, 6952 S. aureus bloodstream infections and 112074 S. aureus non-sterile site cultures were included from Oxfordshire. Resistance trends over time in blood versus other specimens were strongly correlated (maximum cross-correlation 0.51-0.99, strongest associations in the same year for 18/27 pathogen-drug combinations). Resistance prevalence was broadly congruent across drug-years for each species. 276/312 (88%) species-drug-years had resistance prevalence in other specimen types within ±10% of that blood isolates. Results were similar across multiple countries and hospitals/programmes in high/middle/low income-settings.ConclusionsResistance in bloodstream and less invasive infections are strongly related over time, suggesting the latter could be a surveillance tool for AMR in LMICs. These infection sites are easier to sample and cheaper to obtain the necessary numbers of susceptibility tests, providing more cost-effective evidence for decisions including empiric antibiotic recommendations.

IncF plasmids are diverse and of great clinical significance, often carrying genes conferring antimicrobial resistance (AMR) such as extended-spectrum β-lactamases, particularly inEnterobacteriaceae. 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 IncF 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 IncF plasmid backbone structures can persist in environmental settings while allowing dramatic variation in accessory gene content that may be linked to niche adaptation. The recent association of IncF plasmids with AMR likely reflects their suitability for rapid niche adaptation.

Escherichia coli and other Enterobacteriaceae are highly diverse species with ‘open’ pangenomes1,2, where genes move intra- and inter-species via horizontal gene transfer3. These species can cause clinical infections4,5 as well as persist environmentally6,7. Environmental populations have been suggested as important reservoirs of antimicrobial resistance (AMR) genes. However, as most analyses focus on clinical isolates8,9, the pangenome dynamics of natural populations remain understudied, particularly the role of plasmids. Here, we reconstructed near-complete genomes for 828 Enterobacteriaceae, including 553 Escherichia spp. and 275 non-Escherichia species with 2,293 circularised plasmids in total, collected from nineteen locations (livestock farms and wastewater treatment works in the United Kingdom) within a 30km radius at three timepoints over the course of a year. We find different dynamics for the chromosomal and plasmid-borne components of the pangenome, showing that plasmids have a higher burden of both AMR genes and insertion sequences, and AMR plasmids show evidence of being under stronger selective pressure. Focusing on E. coli, we observe that plasmid dynamics are more strongly dominated by niche and local geography, rather than phylogeny or season. Our results highlight the diversity of the AMR reservoir in these species and niches, and the importance of local strategies for controlling the emergence and spread of AMR.

The incidence of bloodstream infections (BSIs) caused by Enterobacteriaceae (e.g. Escherichia coli, Klebsiella pneumoniae) continues to increase globally and the threat of untreatable disease is substantial1. Prophylactic vaccines represent an alternative approach to combating antimicrobial resistance (AMR) by reducing antibiotic usage and preventing infections caused by AMR-associated strains. To investigate their potential utility, we performed in silico serotyping on 4035 E. coli/K. pneumoniae BSI from population-level surveillance in Oxfordshire (2008-2018) in addition to 3678 isolates from previous studies. Most infections, including those associated with AMR, were caused by isolates with a small subset of O-antigens, with no evidence that the proportion of BSIs caused by these changed significantly over time. O-antigen targeted vaccines might therefore be useful in reducing the significant morbidity and mortality2 associated with BSIs. Vaccines may also have a role in preventing the spread of carbapenem resistance genes into common serotypes associated with community-onset disease.

A hospital outbreak of carbapenem-resistant Enterobacteriales was detected by routine surveillance. Whole genome sequencing and subsequent analysis revealed a conserved promiscuous OXA-48 carrying plasmid as the defining factor within this outbreak. Four different species of Enterobacteriales were involved in the outbreak.Escherichia coliST399 accounted for 20/55 of all the isolates. Comparative genomics with publicly availableE. coliST399 sequence data showed that the outbreak isolates formed a unique clade. The OXA-48 plasmid identified in the outbreak differed from other known plasmids by an estimated five homologous recombination events. We estimated a lower bound to the plasmid conjugation rate to be 0.23 conjugation events per lineage per year. Our analysis suggests co-evolution between the plasmid and its main bacterial host to be a key driver of the outbreak. This is the first study to report carbapenem-resistantE. coliST399 carrying OXA48 as the main cause of a plasmid-borne outbreak within a hospital setting. This study supports complementary roles for both plasmid conjugation and clonal expansion in the emergence of this outbreak.

BackgroundLaboratory diagnosis of SARS-CoV-2 infection (the cause of COVID-19) uses PCR to detect viral RNA (vRNA) in respiratory samples. SARS-CoV-2 RNA has also been detected in other sample types, but there is limited understanding of the clinical or laboratory significance of its detection in blood.MethodsWe undertook a systematic literature review to assimilate the evidence for the frequency of vRNA in blood, and to identify associated clinical characteristics. We performed RT-PCR in serum samples from a UK clinical cohort of acute and convalescent COVID-19 cases (n=212), together with convalescent plasma samples collected by NHS Blood and Transplant (NHSBT) (n=111 additional samples). To determine whether PCR-positive blood samples could pose an infection risk, we attempted virus isolation from a subset of RNA-positive samples.ResultsWe identified 28 relevant studies, reporting SARS-CoV-2 RNA in 0-76% of blood samples; pooled estimate 10% (95%CI 5-18%). Among serum samples from our clinical cohort, 27/212 (12.7%) had SARS-CoV-2 RNA detected by RT-PCR. RNA detection occurred in samples up to day 20 post symptom onset, and was associated with more severe disease (multivariable odds ratio 7.5). Across all samples collected ≥28 days post symptom onset, 0/143 (0%, 95%CI 0.0-2.5%) had vRNA detected. Among our PCR-positive samples, cycle threshold (ct) values were high (range 33.5-44.8), suggesting low vRNA copy numbers. PCR-positive sera inoculated into cell culture did not produce any cytopathic effect or yield an increase in detectable SARS-CoV-2 RNA.ConclusionsvRNA was detectable at low viral loads in a minority of serum samples collected in acute infection, but was not associated with infectious SARS-CoV-2 (within the limitations of the assays used). This work helps to inform biosafety precautions for handling blood products from patients with current or previous COVID-19.

Escherichia coli and Klebsiella spp. are important human pathogens that cause a wide spectrum of clinical disease. In healthcare settings, sinks and other wastewater sites have been shown to be reservoirs of antimicrobial-resistant E. coli and Klebsiella spp., particularly in the context of outbreaks of resistant strains amongst patients. Without focusing exclusively on resistance markers or a clinical outbreak, we demonstrate that many hospital sink drains are abundantly and persistently colonised with diverse populations of E. coli, Klebsiella pneumoniae and Klebsiella oxytoca, including both antimicrobial-resistant and susceptible strains. Using whole genome sequencing (WGS) of 439 isolates, we show that environmental bacterial populations are largely structured by ward and sink, with only a handful of lineages, such as E. coli ST635, being widely distributed, suggesting different prevailing ecologies which may vary as a result of different inputs and selection pressures. WGS of 46 contemporaneous patient isolates identified one (2%; 95% CI 0.05-11%) E. coli urine infection-associated isolate with high similarity to a prior sink isolate, suggesting that sinks may contribute to up to 10% of infections caused by these organisms in patients on the ward over the same timeframe. Using metagenomics from 20 sink-timepoints, we show that sinks also harbour many clinically relevant antimicrobial resistance genes including blaCTX-M, blaSHV and mcr, and may act as niches for the exchange and amplification of these genes. Our study reinforces the potential role of sinks in contributing to Enterobacterales infection and antimicrobial resistance in hospital patients, something that could be amenable to intervention.IMPORTANCEEscherichia coli and Klebsiella spp. cause a wide range of bacterial infections, including bloodstream, urine and lung infections. Previous studies have shown that sink drains in hospitals may be part of transmission chains in outbreaks of antimicrobial-resistant E. coli and Klebsiella spp., leading to colonisation and clinical disease in patients. We show that even in non-outbreak settings, contamination of sink drains by these bacteria is common across hospital wards, and that many antimicrobial resistance genes can be found and potentially exchanged in these sink drain sites. Our findings demonstrate that the colonisation of handwashing sink drains by these bacteria in hospitals is likely contributing to some infections in patients, and that additional work is needed to further quantify this risk, and to consider appropriate mitigating interventions.

Antimicrobial resistance (AMR) is a global health threat, especially in low-/middle-income countries (LMICs), where there is limited surveillance to inform empiric antibiotic treatment guidelines. Enterobacterales are amongst the most important causes of drug-resistant bacterial infections. We developed a novel AMR surveillance approach for Enterobacterales by profiling pooled human faecal metagenomes from three sites (n=563 individuals; Cambodia, Kenya, UK) to derive a taxonomy-adjusted AMR metric (“resistance potential”) which could be used to predict the aggregate percentage of resistant invasive Enterobacterales infections within each setting. Samples were sequenced (Illumina); taxonomic and resistance gene profiling was performed using ResPipe. Data on organisms causing bacteraemia and meningitis and antibiotic susceptibility test results from 2010-2017 were collated for each site. Bayesian generalised linear models with a binomial likelihood were fitted to determine the capacity of the resistance potential to predict AMR in Enterobacterales infections in each setting. The most informative model accurately predicted the numbers of resistant infections in the target populations for 14/14 of antibiotics in the UK, 12/12 in Kenya, and 9/12 in Cambodia. Intermittent metagenomics of pooled human samples could represent a powerful pragmatic and economical approach for determining and monitoring AMR in clinical infections, especially in resource-limited settings.

BackgroundThe global dissemination of colistin resistance encoded by mcr-1 has been attributed to extensive use of colistin in livestock, threatening colistin efficacy in medicine. The emergence of mcr-1 in common pathogens, such as Escherichia coli, is of particular concern. Therefore, China banned the use of colistin in animal feed from May 1ST 2017. We investigated subsequent changes in mcr-1 prevalence, and the genomic epidemiology of mcr-1-positive Escherichia coli (MCRPEC).MethodsSampling was conducted pre- (October-December 2016) and post-colistin ban (October-December, 2017 and 2018, respectively). 3675 non-duplicate pig fecal samples were collected from 14 provinces (66 farms) in China to determine intervention-related changes in mcr-1 prevalence. 15193 samples were collected from pigs, healthy human volunteers, colonized and infected hospital inpatients, food and the environment in Guangzhou, to characterize source-specific mcr-1 prevalence and the wider ecological impact of the ban. From these samples, 688 MCRPEC were analyzed with whole genome sequencing (WGS), plasmid conjugation and S1-PFGE/Southern blots to characterize associated genomic changes.FindingsAfter the ban, mcr-1 prevalence decreased significantly in national pig farms, from 45·0% (308/684 samples) in 2016, to 19·4% (274/1416) in 2018 (pmcr-1 was predominantly found on plasmids (632/688 [92%]). Common mcr-1 plasmid types included IncX4, IncI2 and IncHI2 (502/656 [76.5%]); significant increases in IncI2-associated mcr-1 and a distinct lineage of mcr-1-associated IncHI2 were observed post-ban. Changes in the frequency of mcr-1-associated flanking sequences (ISApl1-negative MCRPEC), 63 core genome SNPs and 30 accessory genes were also significantly different after the ban, consistent with rapid genetic adaptation in response to changing selection pressures.InterpretationA rapid, ecosystem-wide, decline in mcr-1 was observed after banning the use of colistin in animal feed, with associated genetic changes in MCRPEC. Genomic surveillance is key to assessing and monitoring stewardship interventions.FundingNational Natural Science Foundation of China

With multidrug resistant (MDR) Enterobacteriales on the rise, a non-toxic agent with a unique mechanism of action such as fosfomycin seems attractive. However, establishing accurate fosfomycin susceptibility testing for non-E. coliin a clinical microbiology laboratory remains problematic. We evaluated fosfomycin susceptibility by multiple methods with multiple strains and species of KPC-producing clinical isolates collected at a single center between 2008 and 2016. In addition, we assessed the presence of fosfomycin resistance genes from whole genome sequencing (WGS) data using NCBI’s AMRFinder and custom HMM search. Susceptibility testing was performed using glucose-6-phosphate supplemented fosfomycin E-Test and Kirby-Bauer disk diffusion (DD) assays, and compared to agar dilution. Clinical Laboratory and Standards Institute (CLSI) breakpoints forE. coliwere applied for interpretation. Overall, 63% (60/96) of isolates were susceptible by E-Test, 70% (67/96) by DD, and 88% (84/96) by agar dilution.FosAwas detected in 80% (70/88) of previously sequenced isolates, with species-specific associations and alleles, andfosA-positive isolates were associated with higher MIC distributions. Disk potentiation testing was performed using sodium phosphonoformate to inhibitfosAand showed significant increases in the zone diameter of DD testing for isolates that werefosA-positive compared tofosA-negative. The addition of sodium phosphonoformate (PPF) corrected 10/14 (71%) major errors in categorical agreement with agar dilution. Our results indicate thatfosAinfluences the inaccuracy of susceptibility testing by methods readily available in a clinical laboratory when compared to agar dilution. Further research is needed to determine the impact offosAon clinical outcomes.

Several emerging pathogens have arisen as a result of selection pressures exerted by modern healthcare. Klebsiella quasipneumoniae was recently defined as a new species, yet its prevalence, niche, and propensity to acquire antimicrobial resistance genes are not fully described. We have been tracking inter- and intra-species transmission of the Klebsiella quasipneumoniae carbapenemase (KPC) gene, blaKPC, between bacteria isolated from a single institution. We applied a combination of Illumina and PacBio whole-genome sequencing to identify and compare K. quasipneumoniae from patients and the hospital environment over 10 and five-year periods respectively. There were 32 blaKPC-positive K. quasipneumoniae isolates, all of which were identified as K. pneumoniae in the clinical microbiology laboratory, from eight patients and 11 sink drains, with evidence for seven separate blaKPC plasmid acquisitions. Analysis of a single subclade of K. quasipneumoniae subspecies quasipneumoniae (n=23 isolates) from three patients and six rooms demonstrated seeding of a sink by a patient, subsequent persistence of the strain in the hospital environment, and then probable transmission to another patient. Longitudinal analysis of this strain demonstrated the acquisition of two unique blaKPC plasmids and then subsequent within-strain genetic rearrangement through transposition and homologous recombination. Our analysis highlights the apparent molecular propensity of K. quasipneumoniae to persist in the environment as well as acquire carbapenemase plasmids from other species and enabled an assessment of the genetic rearrangements which may facilitate horizontal transmission of carbapenemases.

Widespread resistance to antibiotics is among the gravest threats to modern medicine, and controlling the spread of multi-drug resistant Enterobacteriaceae has been given priority status by the World Health Organization. Interventions to reduce transmission within hospital wards may be informed by modifiable patient-level risk factors for becoming colonised, however understanding of factors that influence a patient’s risk of acquisition is limited. We analyse data from a one year prospective carriage study in a neonatal intensive care unit in Cambodia using Bayesian hierarchical models to estimate the daily probability of acquiring multi-drug resistant organisms, while accounting for patient-level time-varying covariates, including interactions between species, and interval-censoring of transmission events. We estimate the baseline daily probability for becoming colonised with third generation cephalosporin resistant (3GC-R) Klebsiella pneumoniae as 0.142 (95% credible interval [CrI] 0.066, 0.27), nearly ten times higher than the daily probability of acquiring 3GC-R Escherichia coli (0.016 [95% CrI 0.0038, 0.049]). Prior colonization with 3GC-R K. pneumoniae was associated with a greatly increased risk of a patient acquiring 3GC-R E. coli (odds ratio [OR] 6.4 [95% CrI 2.8, 20.9]). Breast feeding was associated with a reduced risk of colonization with both 3GC-R K. pneumoniae (OR 0.73 [95% CrI 0.38, 1.5]) and E. coli (OR 0.62 [95% CrI 0.28, 1.6]). The use of an oral probiotic (Lactobacillus acidophilus) did not show clear evidence of protection against colonization with either 3GC-R K. pneumoniae (OR 0.83 [95% CrI 0.51, 1.3]) or 3GC-R E. coli (OR 1.3 [95% CrI 0.77, 2.1]). Antibiotic consumption within the past 48 hours did not strongly influence the risk of acquiring 3GC-R K. pneumoniae. For 3GC-R E. coli, ceftriaxone showed the strongest effect for increasing the risk of acquisition (OR 2.2 [95% CrI 0.66, 6.2]) and imipenem was associated with a decreased risk (OR 0.31 [95% CrI 0.099, 0.76). Using 317 whole-genome assemblies of K. pneumoniae, we determined putatively related clusters and used a range of models to infer transmission rates. Model comparison strongly favored models with a time-varying force of infection term that increased in proportion with the number of colonized patients, providing evidence of patient-to-patient transmission, including among a cluster of Klebsiella quasipneumoniae. Our findings provide support for the hypothesis that K. pneumoniae can be spread person-to-person within ward settings. Subsequent horizontal gene transfer within patients from K. pneumoniae provides the most parsimonious explanation for the strong association between colonization with 3GC-R K. pneumoniae and acquisition of 3GC-R E. coli.

Pyomyositis is a severe bacterial infection of skeletal muscle, commonly affecting children in tropical regions and predominantly caused by Staphylococcus aureus. To understand the contribution of bacterial genomic factors to pyomyositis, we conducted a genome-wide association study of S. aureus cultured from 101 children with pyomyositis and 417 children with asymptomatic nasal carriage attending the Angkor Hospital for Children in Cambodia. We found a strong relationship between bacterial genetic variation and pyomyositis, with estimated heritability 63.8% (95% CI 49.2-78.4%). The presence of the Panton-Valentine leucocidin (PVL) locus increased the odds of pyomyositis 130-fold (p =10-17.9). The signal of association mapped both to the PVL-coding sequence and the sequence immediately upstream. Together these regions explained > 99.9% of heritability. Our results establish staphylococcal pyomyositis, like tetanus and diphtheria, as critically dependent on expression of a single toxin and demonstrate the potential for association studies to identify specific bacterial genes promoting severe human disease.

Farm animals have been identified as reservoirs of Clostridium difficile PCR-ribotype 078 (RT078). Since 2005, the incidence of human clinical cases (frequently severe), with this genotype has increased. We aimed to understand this change, by studying the recent evolutionary history of RT078. Phylogenetic analysis of international genomes (isolates from 2006–2014) revealed several recent clonal expansions. A common ancestor of each expansion had independently acquired different alleles of the tetracycline resistance gene tetM. Consequently, an unusually high proportion of RT078 genomes were tetM positive (76.5%). Additional tetracycline resistance determinants were also identified, some for the first time in C. difficile (efflux pump tet40). Each tetM-clonal expansion lacked geographic structure, indicating rapid international spread. Resistance determinants for C. difficile-infection-triggering antimicrobials including fluoroquinolones and clindamycin were comparatively rare in RT078. Tetracyclines are used intensively in agriculture; this selective pressure, plus rapid spread via the food-chain may explain the increased RT078 prevalence in humans.

BackgroundKlebsiella pneumoniae is an important and increasing cause of life-threatening disease in hospitalised neonates. Third generation cephalosporin resistance (3GC-R) is frequently a marker of multi-drug resistance, and can complicate management of infections. 3GC-R K. pneumoniae is hyper-endemic in many developing country settings, but its epidemiology is poorly understood and prospective studies of endemic transmission are lacking. We aimed to determine the transmission dynamics of 3GC-R K. pneumoniae in a newly opened neonatal unit (NU) in Cambodia.MethodsWe performed a prospective longitudinal study between September and November 2013. Rectal swabs from 37 consented patients were collected upon NU admission and every three days thereafter. Morphologically different colonies from swabs growing cefpodoxime-resistant K. pneumoniae were selected for whole-genome sequencing (WGS).Results32/37 (86%) patients screened positive for 3GC-R K. pneumoniae and 93 colonies from 119 swabs were sequenced. Isolates were resistant to a median of six (range 3-9) antimicrobials. WGS revealed high diversity; pairwise distances between isolates from the same patient were either 0-1 SNV or >1,000 SNVs; 19/32 colonized patients harboured K. pneumoniae colonies differing by >1000 SNVs. Diverse lineages accounted for 18 probable importations to the NU and nine probable transmission clusters involving 19/37 (51%) of screened patients. Median cluster size was 5 patients (range 3-9).ConclusionsThe epidemiology of 3GC-R K. pneumoniae was characterised by multiple introductions and a dense network of cross-infection, with half of screened neonates part of a transmission cluster. Efforts to reduce the 3GC-R K. pneumoniae disease burden should consider targeting both processes.

SynopsisBackgroundmcr-1-mediated transmissible colistin resistance in Enterobacteriaceae is concerning, given colistin is frequently used as a treatment of last resort in multidrug-resistant Enterobacteriaceae infections. Reported rates of human mcr-1 gastrointestinal carriage have historically been low.ObjectivesTo identify trends in human gastrointestinal carriage of mcr-1 positive and mcr-1-positive/cefotaxime-resistant Enterobacteriaceae in Guangzhou, China, 2011-2016, and investigate the genetic contexts of mcr-1 in a subset of mcr-1-positive/cefotaxime-resistant strains using whole genome sequencing (WGS).MethodsOf 8,022 faecal samples collected, 497 (6.2%) were mcr-1- positive, and 182 (2.3%) mcr-1-positive/cefotaxime-resistant. Trends in carriage were assessed using iterative sequential regression. A subset of mcr-1-positive isolates was sequenced (Illumina), and genetic contexts of mcr-1 were characterised.ResultsWe observed marked increases in mcr-1 (now ~30% prevalence) and more recent (since January 2014) increases in mcr-1-positive/third-generation cephalosporin-resistant Enterobacteriaceae human colonisation (pmcr-1-positive/third-generation cephalosporin-resistant isolates were commonly multi-drug resistant.WGS of 50 mcr-1/third-generation cephalosporin-resistant isolates (49 Escherichia coli; 1 Klebsiella pneumoniae) demonstrated bacterial strain diversity (39 E. coli sequence types); mcr-1 in association with common plasmid backbones (IncI, IncHI2/HI2A, IncX4) and sometimes in multiple plasmids; frequent mcr-1 chromosomal integration; and loss of the mcr-1-associated insertion sequence ISApl1 in some plasmids. Significant sequence similarity with published mcr-1 plasmid sequences was consistent with spread amongst pig, chicken and human reservoirs.ConclusionsThe high positivity rate (~10%) of mcr-1 in multidrug-resistant E. coli colonising humans is a clinical threat; the diverse genetic mechanisms (strains/plasmids/insertion sequences) associated with mcr-1 have likely contributed to its dissemination, and will facilitate its persistence.

The recent widespread emergence of carbapenem resistance in Enterobacteriaceae is a major public health concern, as carbapenems are a therapy of last resort in this family of common bacterial pathogens. Resistance genes can mobilize via various mechanisms including conjugation and transposition, however the importance of this mobility in short-term evolution, such as within nosocomial outbreaks, is currently unknown. Using a combination of short- and long-read whole genome sequencing of 281 blaKPC-positive Enterobacteriaceae isolated from a single hospital over five years, we demonstrate rapid dissemination of this carbapenem resistance gene to multiple species, strains, and plasmids. Mobility of blaKPC occurs at multiple nested genetic levels, with transmission of blaKPC strains between individuals, frequent transfer of blaKPC plasmids between strains/species, and frequent transposition of the blaKPC transposon Tn4401 between plasmids. We also identify a common insertion site for Tn4401 within various Tn2-like elements, suggesting that homologous recombination between Tn2-like elements has enhanced the spread of Tn4401 between different plasmid vectors. Furthermore, while short-read sequencing has known limitations for plasmid assembly, various studies have attempted to overcome this with the use of reference-based methods. We also demonstrate that as a consequence of the genetic mobility observed herein, plasmid structures can be extremely dynamic, and therefore these reference-based methods, as well as traditional partial typing methods, can produce very misleading conclusions. Overall, our findings demonstrate that non-clonal resistance gene dissemination can be extremely rapid, presenting significant challenges for public health surveillance and achieving effective control of antibiotic resistance.ImportanceIncreasing antibiotic resistance is a major threat to human health, as highlighted by the recent emergence of multi-drug resistant “superbugs”. Here, we tracked how one important multi-drug resistance gene spread in a single hospital over five years. This revealed high levels of resistance gene mobility to multiple bacterial species, which was facilitated by various different genetic mechanisms. The mobility occurred at multiple nested genetic levels, analogous to a Russian doll set where smaller dolls may be carried along inside larger dolls. Our results challenge traditional views that drug-resistance outbreaks are due to transmission of a single pathogenic strain. Instead, outbreaks can be “gene-based”, and we must therefore focus on tracking specific resistance genes and their context rather than only specific bacteria.