Gold-iron oxide (Au/Fe3O4) magnetic nanoparticles as the nanoplatform for binding of bioactive molecules through self-assembly.

Nanomedicine plays a crucial role in the development of next-generation therapies. The use of nanoparticles as drug delivery platforms has become a major area of research in nanotechnology. To be effective, these nanoparticles must interact with desired drug molecules and release them at targeted sites. The design of these "nanoplatforms" typically includes a functional core, an organic coating with functional groups for drug binding, and the drugs or bioactive molecules themselves. However, by exploiting the coordination chemistry between organic molecules and transition metal centers, the self-assembly of drugs onto the nanoplatform surfaces can bypass the need for an organic coating, simplifying the materials synthesis process. In this perspective, we use gold-iron oxide nanoplatforms as examples and outline the prospects and challenges of using self-assembly to prepare drug-nanoparticle constructs. Through a case study on the binding of insulin on Au-dotted Fe3O4 nanoparticles, we demonstrate how a self-assembly system can be developed. This method can also be adapted to other combinations of transition metals, with the potential for scaling up. Furthermore, the self-assembly method can also be considered as a greener alternative to traditional methods, reducing the use of chemicals and solvents. In light of the current climate of environmental awareness, this shift towards sustainability in the pharmaceutical industry would be welcomed.

Comparison of In Vitro Approaches to Assess the Antibacterial Effects of Nanomaterials.

The antibacterial properties of nanomaterials (NMs) can be exploited in a range of consumer products (e.g., wound dressings, food packaging, textiles, medicines). There is also interest in the exploitation of NMs as treatments for infectious diseases to help combat antibiotic resistance. Whilst the antibacterial activity of NMs has been assessed in vitro and in vivo in numerous studies, the methodology used is very varied. Indeed, while numerous approaches are available to assess the antibacterial effect of NMs in vitro, they have not yet been systematically assessed for their suitability and sensitivity for testing NMs. It is therefore timely to consider what assays should be prioritised to screen the antibacterial properties of NMs. The majority of existing in vitro studies have focused on investigating the antibacterial effects exhibited by silver (Ag) NMs and have employed a limited range of assays. We therefore compared the antibacterial effects of copper oxide (CuO) NMs to Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis at various concentrations (12.5-200 µg/mL) using a battery of tests (well and disc diffusion, plate counts-time-kill method, optical density measurement-OD, Alamar Blue and live/dead viability assays, and quantitative polymerase chain reaction). CuO NMs were most toxic to B. subtilis and E. coli, while P. aeruginosa was the least sensitive strain. All assays employed detected the antibacterial activity of CuO NMs; however, they varied in their sensitivity, time, cost, technical difficulty and requirement for specialized equipment. In the future, we suggest that a combination of approaches is used to provide a robust assessment of the antibacterial activity of NMs. In particular, we recommend that the time-kill and OD assays are prioritised due to their greater sensitivity. We also suggest that standard operating protocols are developed so that the antibacterial activity of NMs can be assessed using a harmonised approach.

Plasmid Mediated mcr-1.1 Colistin-Resistance in Clinical Extraintestinal Escherichia coli Strains Isolated in Poland.

Objectives: The growing incidence of multidrug-resistant (MDR) bacteria is an inexorable and fatal challenge in modern medicine. Colistin is a cationic polypeptide considered a "last-resort" antimicrobial for treating infections caused by MDR Gram-negative bacterial pathogens. Plasmid-borne mcr colistin resistance emerged recently, and could potentially lead to essentially untreatable infections, particularly in hospital and veterinary (livestock farming) settings. In this study, we sought to establish the molecular basis of colistin-resistance in six extraintestinal Escherichia coli strains. Methods: Molecular investigation of colistin-resistance was performed in six extraintestinal E. coli strains isolated from patients hospitalized in Medical University Hospital, Bialystok, Poland. Complete structures of bacterial chromosomes and plasmids were recovered with use of both short- and long-read sequencing technologies and Unicycler hybrid assembly. Moreover, an electrotransformation assay was performed in order to confirm IncX4 plasmid influence on colistin-resistance phenotype in clinical E. coli strains. Results: Here we report on the emergence of six mcr-1.1-producing extraintestinal E. coli isolates with a number of virulence factors. Mobile pEtN transferase-encoding gene, mcr-1.1, has been proved to be encoded within a type IV secretion system (T4SS)-containing 33.3 kbp IncX4 plasmid pMUB-MCR, next to the PAP2-like membrane-associated lipid phosphatase gene. Conclusion: IncX4 mcr-containing plasmids are reported as increasingly disseminated among E. coli isolates, making it an "epidemic" plasmid, responsible for (i) dissemination of colistin-resistance determinants between different E. coli clones, and (ii) circulation between environmental, industrial, and clinical settings. Great effort needs to be taken to avoid further dissemination of plasmid-mediated colistin resistance among clinically relevant Gram-negative bacterial pathogens.

Publisher Correction: Genomic content typifying a prevalent clade of bovine mastitis-associated Escherichia coli.

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

Antibacterial Activities of Ga(III) against E. coli Are Substantially Impacted by Fe(III) Uptake Systems and Multidrug Resistance in Combination with Oxygen Levels.

The continued emergence and spread of antimicrobial resistance (AMR), particularly multidrug resistant (MDR) bacteria, are increasing threats driving the search for additional and alternative antimicrobial agents. The World Health Organization (WHO) has categorized bacterial risk levels and includes Escherichia coli among the highest priority, making this both a convenient model bacterium and a clinically highly relevant species on which to base investigations of antimicrobials. Among many compounds examined for use as antimicrobials, Ga(III) complexes have shown promise. Nonetheless, the spectrum of activities, susceptibility of bacterial species, mechanisms of antimicrobial action, and bacterial characteristics influencing antibacterial actions are far from being completely understood; these are important considerations for any implementation of an effective antibacterial agent. In this investigation, we show that an alteration in growth conditions to physiologically relevant lowered oxygen (anaerobic) conditions substantially increases the minimum inhibitory concentrations (MICs) of Ga(III) required to inhibit growth for 46 wild-type E. coli strains. Several studies have implicated a Trojan horse hypothesis wherein bacterial Fe uptake systems have been linked to the promotion of Ga(III) uptake and result in enhanced antibacterial activity. Our studies show that, conversely, the carriage of accessory Fe uptake systems (Fe_acc) significantly increased the concentrations of Ga(III) required for antibacterial action. Similarly, it is shown that MDR strains are more resistant to Ga(III). The increased tolerance of Fe_acc/MDR strains was apparent under anaerobic conditions. This phenomenon of heightened tolerance has not previously been shown although the mechanisms remain to be defined. Nonetheless, this further highlights the significant contributions of bacterial metabolism, fitness, and AMR characteristics and their implications in evaluating novel antimicrobials.

Propionic Acid Promotes the Virulent Phenotype of Crohn's Disease-Associated Adherent-Invasive Escherichia coli.

Propionic acid (PA) is a bacterium-derived intestinal antimicrobial and immune modulator used widely in food production and agriculture. Passage of Crohn's disease-associated adherent-invasive Escherichia coli (AIEC) through a murine model, in which intestinal PA levels are increased to mimic the human intestine, leads to the recovery of AIEC with significantly increased virulence. Similar phenotypic changes are observed outside the murine model when AIEC is grown in culture with PA as the sole carbon source; such PA exposure also results in AIEC that persists at 20-fold higher levels in vivo. RNA sequencing identifies an upregulation of genes involved in biofilm formation, stress response, metabolism, membrane integrity, and alternative carbon source utilization. PA exposure also increases virulence in a number of E. coli isolates from Crohn's disease patients. Removal of PA is sufficient to reverse these phenotypic changes. Our data indicate that exposure to PA results in AIEC resistance and increased virulence in its presence.

Antimicrobial Properties of Gallium(III)- and Iron(III)-Loaded Polysaccharides Affecting the Growth of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, In Vitro.

Antimicrobial resistance (AMR) has become a global concern as many bacterial species have developed resistance to commonly prescribed antibiotics, making them ineffective to treatments. One type of antibiotics, gallium(III) compounds, stands out as possible candidates due to their unique "Trojan horse" mechanism to tackle bacterial growth, by substituting iron(III) in the metabolic cycles of bacteria. In this study, we tested three polysaccharides (carboxymethyl cellulose (CMC), alginate, and pectin) as the binding and delivery agent for gallium on three bacteria (Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus) with a potential bioresponsive delivery mode. Two types of analysis on bacterial growth (minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC)) were carried out while iron(III)-loaded polysaccharide samples were also tested for comparison. The results suggested that gallium showed an improved inhibitory activity on bacterial growth, in particular gallium(III)-loaded carboxymethyl cellulose (Ga-CMC) sample showing an inhibiting effect on growth for all three tested bacteria. At the MIC for all three bacteria, Ga-CMC showed no cytotoxicity effect on human dermal neonatal fibroblasts (HDNF). Therefore, these bioresponsive gallium(III) polysaccharide compounds show significant potential to be developed as the next-generation antibacterial agents with controlled release capability.

Substantial Extracellular Metabolic Differences Found Between Phylogenetically Closely Related Probiotic and Pathogenic Strains of Escherichia coli.

Since its first isolation a century ago, the gut inhabitant Escherichia coli strain Nissle 1917 has been shown to have probiotic activities; however, it is yet not fully elucidated which differential factors play key roles in its beneficial interactions with the host. To date, no metabolomics studies have been reported investigating the potential role of small molecules in functional strain differentiation of Nissle from its genetically close neighbors. Here, we present results of liquid chromatography coupled to high-resolution mass spectrometry characterization of extracellular metabolomes of E. coli strains as a proxy of their bioactivity potential. We found that phylogroup B2 strains exported a more diverse arsenal of metabolites than strains of other phylogroups. Zooming into the phylogroup B2 metabolome identified consistent substantial differences between metabolic output of E. coli Nissle and other strains, particularly in metabolites associated to the Argimine biosynthesis pathway. Nissle was found to release higher levels of Ornithine and Citrulline whilst depleting greater amounts of Arginine from the medium. Moreover, a novel Nissle-specific metabolite not reported before in bacteria, 5-(Carbamoylamino)-2-hydroxypentanoic acid (Citrulline/Arginic Acid related) was observed. Finally, Nissle, CFT073 and NCTC12241/ATCC25922 shared the excretion of N5-Acetylornithine, whereas other strains released N2-Acetylornithine or no N-Acetylornithine at all. Thus, we found substantial metabolic differences in phylogenetically very similar E. coli strains, an observation which suggests that it is justified to further investigate roles of small molecules as potential modulators of the gut environment by probiotic, commensal, and pathogenic strains, including E. coli Nissle 1917.

Author Correction: Host-associated niche metabolism controls enteric infection through fine-tuning the regulation of type 3 secretion.

The original version of this Article contained an error in the spelling of the author David Ruano-Gallego, which was incorrectly given as David R. Gallego. This has now been corrected in both the PDF and HTML versions of the Article.

Host-associated niche metabolism controls enteric infection through fine-tuning the regulation of type 3 secretion.

Niche-adaptation of a bacterial pathogen hinges on the ability to recognize the complexity of signals from the environment and integrate that information with the regulation of genes critical for infection. Here we report the transcriptome of the attaching and effacing pathogen Citrobacter rodentium during infection of its natural murine host. Pathogen gene expression in vivo was heavily biased towards the virulence factor repertoire and was found to be co-ordinated uniquely in response to the host. Concordantly, we identified the host-specific induction of a metabolic pathway that overlapped with the regulation of virulence. The essential type 3 secretion system and an associated suite of distinct effectors were found to be modulated co-ordinately through a unique mechanism involving metabolism of microbiota-derived 1,2-propanediol, which dictated the ability to colonize the host effectively. This study provides novel insights into how host-specific metabolic adaptation acts as a cue to fine-tune virulence.

Variant O89 O-Antigen of E. coli Is Associated With Group 1 Capsule Loci and Multidrug Resistance.

Bacterial surface polysaccharides play significant roles in fitness and virulence. In Gram-negative bacteria such as Escherichia coli, major surface polysaccharides are lipopolysaccharide (LPS) and capsule, representing O- and K-antigens, respectively. There are multiple combinations of O:K types, many of which are well-characterized and can be related to ecotype or pathotype. In this investigation, we have identified a novel O:K permutation resulting through a process of major genome reorganization in a clade of E. coli. A multidrug-resistant, extended-spectrum ß-lactamase (ESBL)-producing strain - E. coli 26561 - represented a prototype of strains combining a locus variant of O89 and group 1 capsular polysaccharide. Specifically, the variant O89 locus in this strain was truncated at gnd, flanked by insertion sequences and located between nfsB and ybdK and we apply the term O89m for this variant. The prototype lacked colanic acid and O-antigen loci between yegH and hisI with this tandem polysaccharide locus being replaced with a group 1 capsule (G1C) which, rather than being a recognized E. coli capsule type, this locus matched to Klebsiella K10 capsule type. A genomic survey identified more than 200 E. coli strains which possessed the O89m locus variant with one of a variety of G1C types. Isolates from our collection with the combination of O89m and G1C all displayed a mucoid phenotype and E. coli 26561 was unusual in exhibiting a mucoviscous phenotype more recognized as a characteristic among Klebsiella strains. Despite the locus truncation and novel location, all O89m:G1C strains examined showed a ladder pattern typifying smooth LPS and also showed high molecular weight, alcian blue-staining polysaccharide in cellular and/or extra-cellular fractions. Expression of both O-antigen and capsule biosynthesis loci were confirmed in prototype strain 26561 through quantitative proteome analysis. Further in silico exploration of more than 200 E. coli strains possessing the O89m:G1C combination identified a very high prevalence of multidrug resistance (MDR) - 85% possessed resistance to three or more antibiotic classes and a high proportion (58%) of these carried ESBL and/or carbapenemase. The increasing isolation of O89m:G1C isolates from extra-intestinal infection sites suggests that these represents an emergent clade of invasive, MDR E. coli.

Immunomodulation of Host Chitinase 3-Like 1 During a Mammary Pathogenic Escherichia coli Infection.

Chitin is a N-acetyl-d-glucosamine biopolymer that can be recognized by chitin-binding proteins. Although mammals lack chitin synthase, they induce proteins responsible for detecting chitin in response to bacterial infections. Our aim was to investigate whether chitinase 3-like 1 (CHI3L1) has a potential role in the innate immunity of the Escherichia coli (E. coli) infected mammary gland. CHI3L1 protein was found to be secreted in whey of naturally coliform-affected quarters compared to whey samples isolated from healthy udders. In addition, gene expression of CHI3L1 was confirmed in udder tissue of cows experimentally infected with a mammary pathogenic E. coli (MPEC) strain. Despite the known anatomical differences, the bovine udders' innate immune response was mimicked by applying an experimental mouse model using MPEC or non-MPEC isolates. The effect of CHI3L1 expression in the murine mammary gland in response to coliform bacteria was investigated through the use of CHI3L1-/- mice as well as through treatment with either a pan-caspase inhibitor or chitin particles in wild-type mice. The local induction of CHI3L1 postinfection with different E. coli strains was demonstrated to be independent of both bacterial growth and mammary interleukin (IL)-8 levels. Indeed, CHI3L1 emerged as a regulator impacting on the transcytosis of Ly6G-positive cells from the interstitial space into the alveolar lumen of the mammary tissue. Furthermore, CHI3L1 was found to be upstream regulated by caspase activity and had a major downstream effect on the local pro-inflammatory cytokine profile, including IL-1beta, IL-6, and RANTES/CCL5. In conclusion, CHI3L1 was demonstrated to play a key role in the cytokine and caspase signaling during E. coli triggered inflammation of the mammary gland.

Postgenomics Characterization of an Essential Genetic Determinant of Mammary Pathogenic Escherichia coli.

Escherichia coli are major bacterial pathogens causing bovine mastitis, a disease of great economic impact on dairy production worldwide. This work aimed to study the virulence determinants of mammary pathogenic E. coli (MPEC). By whole-genome sequencing analysis of 40 MPEC and 22 environmental ("dairy-farm" E. coli [DFEC]) strains, we found that only the fec locus (fecIRABCDE) for ferric dicitrate uptake was present in the core genome of MPEC and that it was absent in DFEC genomes (P < 0.05). Expression of the FecA receptor in the outer membrane was shown to be citrate dependent by mass spectrometry. FecA was overexpressed when bacteria were grown in milk. Transcription of the fecA gene and of the inner membrane transport component fecB gene was upregulated in bacteria recovered from experimental intramammary infection. The presence of the fec system was shown to affect the ability of E. coli to grow in milk. While the rate of growth in milk of fec-positive (fec+) DFEC was similar to that of MPEC, it was significantly lower in DFEC lacking fec Furthermore, deletion of fec reduced the rate of growth in milk of MPEC strain P4, whereas fec-transformed non-mammary gland-pathogenic DFEC strain K71 gained the phenotype of the level of growth in milk observed in MPEC. The role of fec in E. coli intramammary pathogenicity was investigated in vivo in cows, with results showing that an MPEC P4 mutant lacking fec lost its ability to induce mastitis, whereas the fec+ DFEC K71 mutant was able to trigger intramammary inflammation. For the first time, a single molecular locus was shown to be crucial in MPEC pathogenicity.IMPORTANCE Bovine mastitis is the major infectious disease in dairy cows and the leading cause of economic loss to the global dairy industry, directly contributing to the price of dairy products on supermarket shelves and the financial hardships suffered by dairy farmers. Mastitis is also the leading reason for the use of antibiotics in dairy farms. Good farm management practices in many countries have dramatically reduced the incidence of contagious mastitis; however, the problems associated with the incidence of environmental mastitis caused by bacteria such as Escherichia coli have proven intractable. E. coli bacteria cause acute mastitis, which affects the health and welfare of cows and in extreme cases may be fatal. Here we show for the first time that the pathogenicity of E. coli causing mastitis in cows is highly dependent on the fecIRABCDE ferric citrate uptake system that allows the bacterium to capture iron from citrate. The Fec system is highly expressed during infection in the bovine udder and is ubiquitous in and necessary for the E. coli bacteria that cause mammary infections in cattle. These results have far-reaching implications, raising the possibility that mastitis may be controllable by targeting this system.

Rapid and robust analytical protocol for E. coli STEC bacteria subspecies differentiation using whole cell MALDI mass spectrometry.

Whole cell MALDI is regularly used for the identification of bacteria to species level in clinical Microbiology laboratories. However, there remains a need to rapidly characterize and differentiate isolates below the species level to support outbreak management. We describe the implementation of a modified preparative approach for MALDI-MS combined with a custom analytical computational pipeline as a rapid procedure for subtyping Shigatoxigenic E. coli (STEC) and accurately identifying strain-specifying biomarkers. The technique was able to differentiate E. coli O157:H7 from other STEC. Within O157 serotype O157:H7 isolates were readily distinguishable from Sorbitol Fermenting O157 isolates. Overall, nine homogeneous groups of isolates were distinguished, each exhibiting distinct profiles of defining mass spectra features. This offers a robust analytical tool useable in reference/diagnostic public health scenarios.

Unexpected differential metabolic responses of Campylobacter jejuni to the abundant presence of glutamate and fucose.

INTRODUCTION: Campylobacter jejuni is the leading cause of foodborne bacterial enteritis in humans, and yet little is known in regard to how genetic diversity and metabolic capabilities among isolates affect their metabolic phenotype and pathogenicity. OBJECTIVES: For instance, the C. jejuni 11168 strain can utilize both L-fucose and L-glutamate as a carbon source, which provides the strain with a competitive advantage in some environments and in this study we set out to assess the metabolic response of C. jejuni 11168 to the presence of L-fucose and L-glutamate in the growth medium. METHODS: To achieve this, untargeted hydrophilic liquid chromatography coupled to mass spectrometry was used to obtain metabolite profiles of supernatant extracts obtained at three different time points up to 24 h. RESULTS: This study identified both the depletion and the production and subsequent release of a multitude of expected and unexpected metabolites during the growth of C. jejuni 11168 under three different conditions. A large set of standards allowed identification of a number of metabolites. Further mass spectrometry fragmentation analysis allowed the additional annotation of substrate-specific metabolites. The results show that C. jejuni 11168 upon L-fucose addition indeed produces degradation products of the fucose pathway. Furthermore, methionine was faster depleted from the medium, consistent with previously-observed methionine auxotrophy. CONCLUSIONS: Moreover, a multitude of not previously annotated metabolites in C. jejuni were found to be increased specifically upon L-fucose addition. These metabolites may well play a role in the pathogenicity of this C. jejuni strain.

MALDIrppa: quality control and robust analysis for mass spectrometry data.

Summary: This R package helps to implement a robust approach to deal with mass spectrometry (MS) data. It is aimed at alleviating reproducibility issues and pernicious effects of deviating signals on both data pre-processing and downstream data analysis. Based on robust statistical methods, it facilitates the identification and filtering of low-quality mass spectra and atypical peak profiles as well as monitoring and data handling through pre-processing, which extends existing computational tools for high-throughput data. Availability and implementation: MALDIrppa is implemented as a package for the R environment for data analysis and it is freely available to download from the CRAN repository at https://CRAN.R-project.org/package=MALDIrppa. Contact: javier.palarea@bioss.ac.uk.

The bile salt sodium taurocholate induces Campylobacter jejuni outer membrane vesicle production and increases OMV-associated proteolytic activity.

Campylobacter jejuni, the leading cause of bacterial acute gastroenteritis worldwide, secretes an arsenal of virulence-associated proteins within outer membrane vesicles (OMVs). C. jejuni OMVs contain three serine proteases (HtrA, Cj0511, and Cj1365c) that cleave the intestinal epithelial cell (IEC) tight and adherens junction proteins occludin and E-cadherin, promoting enhanced C. jejuni adhesion to and invasion of IECs. C. jejuni OMVs also induce IECs innate immune responses. The bile salt sodium taurocholate (ST) is sensed as a host signal to coordinate the activation of virulence-associated genes in the enteric pathogen Vibrio cholerae. In this study, the effect of ST on C. jejuni OMVs was investigated. Physiological concentrations of ST do not have an inhibitory effect on C. jejuni growth until the early stationary phase. Coculture of C. jejuni with 0.1% or 0.2% (w/v) ST stimulates OMV production, increasing both lipid and protein concentrations. C. jejuni ST-OMVs possess increased proteolytic activity and exhibit a different protein profile compared to OMVs isolated in the absence of ST. ST-OMVs exhibit enhanced cytotoxicity and immunogenicity to T84 IECs and enhanced killing of Galleria mellonella larvae. ST increases the level of mRNA transcripts of the OMVs-associated serine protease genes and the cdtABC operon that encodes the cytolethal distending toxin. Coculture with ST significantly enhances the OMVs-induced cleavage of E-cadherin and occludin. C. jejuni OMVs also cleave the major endoplasmic reticulum chaperone protein BiP/GRP78 and this activity is associated with the Cj1365c protease. These data suggest that C. jejuni responds to the presence of physiological concentrations of the bile salt ST that increases OMV production and the synthesis of virulence-associated factors that are secreted within the OMVs. We propose that these events contribute to pathogenesis.

Host species adaptation of TLR5 signalling and flagellin recognition.

Toll-like receptor 5 (TLR5) recognition of flagellin instigates inflammatory signalling. Significant sequence variation in TLR5 exists between animal species but its impact on activity is less well understood. Building on our previous research that bovine TLR5 (bTLR5) is functional, we compared human and bovine TLR5 activity and signalling in cognate cell lines. bTLR5 induced higher levels of CXCL8 when expressed in bovine cells and reciprocal results were found for human TLR5 (hTLR5) in human cells, indicative of host cell specificity in this response. Analysis of Toll/interleukin-1 receptor (TIR) sequences indicated that these differential responses involve cognate MyD88 recognition. siRNA knockdowns and inhibitor experiments demonstrated that there are some host differences in signalling. Although, PI3K activation is required for bTLR5 signalling, mutating bTLR5 F798 to hTLR5 Y798 within a putative PI3K motif resulted in a significantly reduced response. All ruminants have F798 in contrast to most other species, suggesting that TLR5 signalling has evolved differently in ruminants. Evolutionary divergence between bovine and human TLR5 was also apparent in relation to responses measured to diverse bacterial flagellins. Our results underscore the importance of species specific studies and how differences may alter efficacy of TLR-based vaccine adjuvants.

A population genomics approach to exploiting the accessory 'resistome' of Escherichia coli.

The emergence of antibiotic resistance is a defining challenge, and Escherichia coli is recognized as one of the leading species resistant to the antimicrobials used in human or veterinary medicine. Here, we analyse the distribution of 2172 antimicrobial-resistance (AMR) genes in 4022 E. coli to provide a population-level view of resistance in this species. By separating the resistance determinants into 'core' (those found in all strains) and 'accessory' (those variably present) determinants, we have found that, surprisingly, almost half of all E. coli do not encode any accessory resistance determinants. However, those strains that do encode accessory resistance are significantly more likely to be resistant to multiple antibiotic classes than would be expected by chance. Furthermore, by studying the available date of isolation for the E. coli genomes, we have visualized an expanding, highly interconnected network that describes how resistances to antimicrobials have co-associated within genomes over time. These data can be exploited to reveal antimicrobial combinations that are less likely to be found together, and so if used in combination may present an increased chance of suppressing the growth of bacteria and reduce the rate at which resistance factors are spread. Our study provides a complex picture of AMR in the E. coli population. Although the incidence of resistance to all studied antibiotic classes has increased dramatically over time, there exist combinations of antibiotics that could, in theory, attack the entirety of E. coli, effectively removing the possibility that discrete AMR genes will increase in frequency in the population.