Discordance between different bioinformatic methods for identifying resistance genes from short-read genomic data, with a focus on Escherichia coli.

Several bioinformatics genotyping algorithms are now commonly used to characterize 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 simulation tests where 3079 AMR gene variants were inserted into random sequence constructs, KmerResistance was correct for 3076 (99.9 %) simulations, ABRicate for 3054 (99.2 %), ARIBA for 2783 (90.4 %) and SRST2 for 2108 (68.5 %). For simulation tests where two closely related gene variants were inserted into random sequence constructs, KmerResistance identified the correct alleles in 35 338/46 318 (76.3 %) simulations, ABRicate identified them in 11 842/46 318 (25.6 %) simulations, ARIBA identified them in 1679/46 318 (3.6 %) simulations and SRST2 identified them in 2000/46 318 (4.3 %) simulations. In real data, across all methods, 1392/1818 (76 %) isolates had discrepant allele calls for at least 1 gene. In addition to highlighting areas for improvement in challenging scenarios, (e.g. identification of AMR genes at <10× coverage, identifying multiple closely related AMR genes present in the same sample), our evaluations identified some more systematic errors that could be readily soluble, such as repeated misclassification (i.e. naming) of genes as shorter variants of the same gene present within the reference resistance gene database. Such naming errors accounted for at least 2530/4321 (59 %) of the discrepancies seen in real data. Moreover, many of the remaining discrepancies were likely 'artefactual', with reporting of cut-off differences accounting for at least 1430/4321 (33 %) discrepants. Whilst we found that comparing outputs generated by running multiple algorithms on the same dataset could identify and resolve these algorithmic artefacts, the results of our evaluations emphasize the need for developing new and more robust genotyping algorithms to further improve accuracy and performance.

Foxn1 is not essential for T-cell development in teleosts.

In mammals, T-cell development depends on the activity of the Foxn1 transcription factor in the thymic epithelium; mutations in the vertebrate-specific Foxn1 gene are associated with profound T-cell lymphopenia and fatal immunodeficiency. Here, we examined the extent of T-cell development in teleosts lacking a functional foxn1 gene. In zebrafish carrying a deleterious internal deletion of foxn1, reduced but robust lymphopoietic activity is maintained in the mutant thymus. Moreover, pseudogenization or loss of foxn1 in the genomes of deep-sea anglerfishes is independent of the presence or absence of the canonical signatures of the T-cell lineage. Thus, in contrast to the situation in mammals, the teleost thymus can support foxn1-independent lymphopoiesis, most likely through the activity of the Foxn4, an ancient metazoan paralog of Foxn1. Our results imply that during the early stages of vertebrate evolution, genetic control of thymopoiesis was functionally redundant and thus robust; in mammals, the genetic network was reorganized to become uniquely dependent on the FOXN1 transcription factor.

Lymphocyte pathway analysis using naturally lymphocyte-deficient fish.

Comparative phylogenetic analyses are of potential value to establish the essential components of genetic networks underlying physiological traits. For species that naturally lack particular lymphocyte lineages, we show here that this strategy readily distinguishes trait-specific actors from pleiotropic components of the genetic network governing lymphocyte differentiation. Previously, three of the four members of the DNA polymerase X family have been implicated in the junctional diversification process during the somatic assembly of antigen receptors. Our phylogenetic analysis indicates that the presence of terminal deoxynucleotidyl transferase is strictly associated with the facility of V(D)J recombination, whereas PolL and PolM genes are retained even in species lacking Rag-mediated somatic diversification of antigen receptor genes.

A survey of the adaptive immune genes of the polka-dot batfish Ogcocephalus cubifrons.

BACKGROUND: The anglerfish, belonging to the teleost order Lophiiformes, are a diverse and species-rich group of fish that are known to exhibit a number of unique morphological, reproductive and immunological adaptations. Work to date has identified the loss of specific adaptive immune components in two of the five Lophiiformes sub-orders (Lophioidei and Ceratioidei), while no anomalies have been identified to date in two other sub-orders, Antennaroidei and Chaunacoidei. The immunogenome of the fifth sub-order, Ogcocephaloidei has not yet been investigated, and we have therefore used whole genome shotgun sequencing, combined with RNA-seq, to survey the adaptive immune capabilities of the polka-dot batfish, O. cubifrons, as a representative of this as yet unexplored sub-order. RESULTS: We find that the O. cubifrons genome encodes the core genes needed to mount adaptive T and B cell responses. These genes include those necessary for rearranging and editing antigen receptors, the antigen receptors themselves; as well as the co-receptors, signalling molecules, and antigen presenting molecules (both class I and class II) needed for B cell and T cell development and activation. CONCLUSIONS: From an immune perspective, the polka-dot batfish has a canonical complement of adaptive immune genes, and does not exhibit any of the adaptive immune changes previously identified in monkfish and oceanic anglerfish.

Penicillin Binding Protein Substitutions Cooccur with Fluoroquinolone Resistance in Epidemic Lineages of Multidrug-Resistant Clostridioides difficile.

Clostridioides difficile remains a key cause of healthcare-associated infection, with multidrug-resistant (MDR) lineages causing high-mortality (≥20%) outbreaks. Cephalosporin treatment is a long-established risk factor, and antimicrobial stewardship is a key control. A mechanism underlying raised cephalosporin MICs has not been identified in C. difficile, but among other species, this is often acquired via amino acid substitutions in cell wall transpeptidases (penicillin binding proteins [PBPs]). Here, we investigated five C. difficile transpeptidases (PBP1 to PBP5) for recent substitutions, associated cephalosporin MICs, and co-occurrence with fluoroquinolone resistance. Previously published genome assemblies (n = 7,096) were obtained, representing 16 geographically widespread lineages, including healthcare-associated ST1(027). Recent amino acid substitutions were found within PBP1 (n = 50) and PBP3 (n = 48), ranging from 1 to 10 substitutions per genome. ß-Lactam MICs were measured for closely related pairs of wild-type and PBP-substituted isolates separated by 20 to 273 single nucleotide polymorphisms (SNPs). Recombination-corrected phylogenies were constructed to date substitution acquisition. Key substitutions such as PBP3 V497L and PBP1 T674I/N/V emerged independently across multiple lineages. They were associated with extremely high cephalosporin MICs; 1 to 4 doubling dilutions >wild-type, up to 1,506 µg/mL. Substitution patterns varied by lineage and clade, showed geographic structure, and occurred post-1990, coincident with the gyrA and/or gyrB substitutions conferring fluoroquinolone resistance. In conclusion, recent PBP1 and PBP3 substitutions are associated with raised cephalosporin MICs in C. difficile. Their co-occurrence with fluoroquinolone resistance hinders attempts to understand the relative importance of these drugs in the dissemination of epidemic lineages. Further controlled studies of cephalosporin and fluoroquinolone stewardship are needed to determine their relative effectiveness in outbreak control. IMPORTANCE Fluoroquinolone and cephalosporin use in healthcare settings has triggered outbreaks of high-mortality, multidrug-resistant C. difficile infection. Here, we identify a mechanism associated with raised cephalosporin MICs in C. difficile comprising amino acid substitutions in two cell wall transpeptidase enzymes (penicillin binding proteins). The higher the number of substitutions, the greater the impact on phenotype. Dated phylogenies revealed that substitutions associated with raised cephalosporin and fluoroquinolone MICs were co-acquired immediately before clinically important outbreak strains emerged. PBP substitutions were geographically structured within genetic lineages, suggesting adaptation to local antimicrobial prescribing. Antimicrobial stewardship of cephalosporins and fluoroquinolones is an effective means of C. difficile outbreak control. Genetic changes associated with raised MIC may impart a "fitness cost" after antibiotic withdrawal. Our study therefore identifies a mechanism that may explain the contribution of cephalosporin stewardship to resolving outbreak conditions. However, due to the co-occurrence of raised cephalosporin MICs and fluoroquinolone resistance, further work is needed to determine the relative importance of each.

ReadItAndKeep: rapid decontamination of SARS-CoV-2 sequencing reads.

SUMMARY: Viral sequence data from clinical samples frequently contain contaminating human reads, which must be removed prior to sharing for legal and ethical reasons. To enable host read removal for SARS-CoV-2 sequencing data on low-specification laptops, we developed ReadItAndKeep, a fast lightweight tool for Illumina and nanopore data that only keeps reads matching the SARS-CoV-2 genome. Peak RAM usage is typically below 10 MB, and runtime less than 1 min. We show that by excluding the polyA tail from the viral reference, ReadItAndKeep prevents bleed-through of human reads, whereas mapping to the human genome lets some reads escape. We believe our test approach (including all possible reads from the human genome, human samples from each of the 26 populations in the 1000 genomes data and a diverse set of SARS-CoV-2 genomes) will also be useful for others. AVAILABILITY AND IMPLEMENTATION: ReadItAndKeep is implemented in C++, released under the MIT license, and available from https://github.com/GenomePathogenAnalysisService/read-it-and-keep. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Developmental dynamics of two bipotent thymic epithelial progenitor types.

T cell development in the thymus is essential for cellular immunity and depends on the organotypic thymic epithelial microenvironment. In comparison with other organs, the size and cellular composition of the thymus are unusually dynamic, as exemplified by rapid growth and high T cell output during early stages of development, followed by a gradual loss of functional thymic epithelial cells and diminished naive T cell production with age1-10. Single-cell RNA sequencing (scRNA-seq) has uncovered an unexpected heterogeneity of cell types in the thymic epithelium of young and aged adult mice11-18; however, the identities and developmental dynamics of putative pre- and postnatal epithelial progenitors have remained unresolved1,12,16,17,19-27. Here we combine scRNA-seq and a new CRISPR-Cas9-based cellular barcoding system in mice to determine qualitative and quantitative changes in the thymic epithelium over time. This dual approach enabled us to identify two principal progenitor populations: an early bipotent progenitor type biased towards cortical epithelium and a postnatal bipotent progenitor population biased towards medullary epithelium. We further demonstrate that continuous autocrine provision of Fgf7 leads to sustained expansion of thymic microenvironments without exhausting the epithelial progenitor pools, suggesting a strategy to modulate the extent of thymopoietic activity.

Epidemiological data and genome sequencing reveals that nosocomial transmission of SARS-CoV-2 is underestimated and mostly mediated by a small number of highly infectious individuals.

OBJECTIVES: Despite robust efforts, patients and staff acquire SARS-CoV-2 infection in hospitals. We investigated whether whole-genome sequencing enhanced the epidemiological investigation of healthcare-associated SARS-CoV-2 acquisition. METHODS: From 17-November-2020 to 5-January-2021, 803 inpatients and 329 staff were diagnosed with SARS-CoV-2 infection at four Oxfordshire hospitals. We classified cases using epidemiological definitions, looked for a potential source for each nosocomial infection, and evaluated genomic evidence supporting transmission. RESULTS: 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. 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. 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. CONCLUSIONS: Current surveillance definitions underestimate nosocomial acquisition. Most nosocomial transmission occurs from a relatively limited number of highly infectious individuals.

Population-level faecal metagenomic profiling as a tool to predict antimicrobial resistance in Enterobacterales isolates causing invasive infections: An exploratory study across Cambodia, Kenya, and the UK.

BACKGROUND: Antimicrobial resistance (AMR) in Enterobacterales is a global health threat. Capacity for individual-level surveillance remains limited in many countries, whilst population-level surveillance approaches could inform empiric antibiotic treatment guidelines. METHODS: In this exploratory study, a novel approach to population-level prediction of AMR in Enterobacterales clinical isolates using metagenomic (Illumina) profiling of pooled DNA extracts from human faecal samples was developed and tested. Taxonomic and AMR gene profiles were used to derive taxonomy-adjusted population-level AMR metrics. Bayesian modelling, and model comparison based on cross-validation, were used to evaluate the capacity of each metric to predict the number of resistant Enterobacterales invasive infections at a population-level, using available bloodstream/cerebrospinal fluid infection data. FINDINGS: Population metagenomes comprised samples from 177, 157, and 156 individuals in Kenya, the UK, and Cambodia, respectively, collected between September 2014 and April 2016. Clinical data from independent populations included 910, 3356 and 197 bacterial isolates from blood/cerebrospinal fluid infections in Kenya, the UK and Cambodia, respectively (samples collected between January 2010 and May 2017). Enterobacterales were common colonisers and pathogens, and faecal taxonomic/AMR gene distributions and proportions of antimicrobial-resistant Enterobacterales infections differed by setting. A model including terms reflecting the metagenomic abundance of the commonest clinical Enterobacterales species, and of AMR genes known to either increase the minimum inhibitory concentration (MIC) or confer clinically-relevant resistance, had a higher predictive performance in determining population-level resistance in clinical Enterobacterales isolates compared to models considering only AMR gene information, only taxonomic information, or an intercept-only baseline model (difference in expected log predictive density compared to best model, estimated using leave-one-out cross-validation: intercept-only model = -223 [95% credible interval (CI): -330,-116]; model considering only AMR gene information = -186 [95% CI: -281,-91]; model considering only taxonomic information = -151 [95% CI: -232,-69]). INTERPRETATION: Whilst our findings are exploratory and require validation, intermittent metagenomics of pooled samples could represent an effective approach for AMR surveillance and to predict population-level AMR in clinical isolates, complementary to ongoing development of laboratory infrastructures processing individual samples.

Niche and local geography shape the pangenome of wastewater- and livestock-associated Enterobacteriaceae.

Escherichia coli and other Enterobacteriaceae are diverse species with "open" pangenomes, where genes move intra- and interspecies via horizontal gene transfer. However, most analyses focus on clinical isolates. The pangenome dynamics of natural populations remain understudied, despite their suggested role as reservoirs for antimicrobial resistance (AMR) genes. Here, we analyze near-complete genomes for 827 Enterobacteriaceae (553 Escherichia and 274 non-Escherichia spp.) with 2292 circularized plasmids in total, collected from 19 locations (livestock farms and wastewater treatment works in the United Kingdom) within a 30-km radius at three time points over a year. We find different dynamics for chromosomal and plasmid-borne genes. Plasmids have a higher burden of AMR genes and insertion sequences, and AMR-gene-carrying plasmids show evidence of being under stronger selective pressure. Environmental niche and local geography both play a role in shaping plasmid dynamics. Our results highlight the importance of local strategies for controlling the spread of AMR.

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

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

Evolution of thymopoietic microenvironments.

In vertebrates, the development of lymphocytes from undifferentiated haematopoietic precursors takes place in so-called primary lymphoid organs, such as the thymus. Therein, lymphocytes undergo a complex differentiation and selection process that culminates in the generation of a pool of mature T cells that collectively express a self-tolerant repertoire of somatically diversified antigen receptors. Throughout this entire process, the microenvironment of the thymus in large parts dictates the sequence and outcome of the lymphopoietic activity. In vertebrates, direct genetic evidence in some species and circumstantial evidence in others suggest that the formation of a functional thymic microenvironment is controlled by members of the Foxn1/4 family of transcription factors. In teleost fishes, both Foxn1 and Foxn4 contribute to thymopoietic activity, whereas Foxn1 is both necessary and sufficient in the mammalian thymus. The evolutionary history of Foxn1/4 genes suggests that an ancient Foxn4 gene lineage gave rise to the Foxn1 genes in early vertebrates, raising the question of the thymopoietic capacity of the ancestor common to all vertebrates. Recent attempts to reconstruct the early events in the evolution of thymopoietic tissues by replacement of the mouse Foxn1 gene by Foxn1-like genes isolated from various chordate species suggest a plausible scenario. It appears that the primordial thymus was a bi-potent lymphoid organ, supporting both B cell and T cell development; however, during the course of vertebrate, evolution B cell development was gradually diminished converting the thymus into a site specialized in T cell development.

Retracing the evolutionary emergence of thymopoiesis.

The onset of lymphocyte development in the vertebrate primordial thymus, about 500 million years ago, represents one of the foundational events of the emerging adaptive immune system. Here, we retrace the evolutionary trajectory of thymopoiesis, from early vertebrates to mammals, guided by members of the Foxn1/4 transcription factor gene family, which direct the differentiation of the thymic microenvironment. Molecular engineering in transgenic mice recapitulated a gene duplication event, exon replacements, and altered expression patterns. These changes predictably modified the lymphopoietic characteristics of the thymus, identifying molecular features contributing to conversion of a primordial bipotent lymphoid organ to a tissue specializing in T cell development. The phylogenetic reconstruction associates increasing efficiency of T cell generation with diminishing B cell-generating capacity of the thymus during jawed vertebrate evolution.

Author Correction: Elephant shark genome provides unique insights into gnathostome evolution.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

High precision Neisseria gonorrhoeae variant and antimicrobial resistance calling from metagenomic Nanopore sequencing.

The rise of antimicrobial-resistant Neisseria gonorrhoeae is a significant public health concern. Against this background, rapid culture-independent diagnostics may allow targeted treatment and prevent onward transmission. We have previously shown metagenomic sequencing of urine samples from men with urethral gonorrhea can recover near-complete N. gonorrhoeae genomes. However, disentangling the N. gonorrhoeae genome from metagenomic samples and robustly identifying antimicrobial resistance determinants from error-prone Nanopore sequencing is a substantial bioinformatics challenge. Here, we show an N. gonorrhoeae diagnostic workflow for analysis of metagenomic sequencing data obtained from clinical samples using R9.4.1 Nanopore sequencing. We compared results from simulated and clinical infections with data from known reference strains and Illumina sequencing of isolates cultured from the same patients. We evaluated three Nanopore variant callers and developed a random forest classifier to filter called SNPs. Clair was the most suitable variant caller after SNP filtering. A minimum depth of 20× reads was required to confidently identify resistant determinants over the entire genome. Our findings show that metagenomic Nanopore sequencing can provide reliable diagnostic information in N. gonorrhoeae infection.

Differential occupational risks to healthcare workers from SARS-CoV-2 observed during a prospective observational study.

We conducted voluntary Covid-19 testing programmes for symptomatic and asymptomatic staff at a UK teaching hospital using naso-/oro-pharyngeal PCR testing and immunoassays for IgG antibodies. 1128/10,034 (11.2%) staff had evidence of Covid-19 at some time. Using questionnaire data provided on potential risk-factors, staff with a confirmed household contact were at greatest risk (adjusted odds ratio [aOR] 4.82 [95%CI 3.45-6.72]). Higher rates of Covid-19 were seen in staff working in Covid-19-facing areas (22.6% vs. 8.6% elsewhere) (aOR 2.47 [1.99-3.08]). Controlling for Covid-19-facing status, risks were heterogenous across the hospital, with higher rates in acute medicine (1.52 [1.07-2.16]) and sporadic outbreaks in areas with few or no Covid-19 patients. Covid-19 intensive care unit staff were relatively protected (0.44 [0.28-0.69]), likely by a bundle of PPE-related measures. Positive results were more likely in Black (1.66 [1.25-2.21]) and Asian (1.51 [1.28-1.77]) staff, independent of role or working location, and in porters and cleaners (2.06 [1.34-3.15]).

The immunogenetics of sexual parasitism.

Sexual parasitism has evolved as a distinctive mode of reproduction among deep-sea anglerfishes. The permanent attachment of males to host females observed in these species represents a form of anatomical joining, which is otherwise unknown in nature. Pronounced modifications to immune facilities are associated with this reproductive trait. The genomes of species with temporarily attaching males lack functional aicda genes that underpin affinity maturation of antibodies. Permanent attachment is associated with additional alterations, culminating in the loss of functional rag genes in some species, abolishing somatic diversification of antigen receptor genes, the hallmark of canonical adaptive immunity. In anglerfishes, coevolution of innate and adaptive immunity has been disentangled, implying that an alternative form of immunity supported the emergence of this evolutionarily successful group of vertebrates.

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.