The clinical, genomic, and microbiological profile of invasive multi-drug resistant Escherichia coli in a major teaching hospital in the United Kingdom

Escherichia coli is a ubiquitous component of the human gut microbiome, but is also a common pathogen, causing around 40, 000 bloodstream infections (BSI) in the United Kingdom (UK) annually. The number of E. coli BSI has increased over the last decade in the UK, and emerging antimicrobial resistance (AMR) profiles threaten treatment options. Here, we combined clinical, epidemiological, and whole genome sequencing data with high content imaging to characterise over 300 E. coli isolates associated with BSI in a large teaching hospital in the East of England. Overall, only a limited number of sequence types (ST) were responsible for the majority of organisms causing invasive disease. The most abundant (20 % of all isolates) was ST131, of which around 90 % comprised the pandemic O25b:H4 group. ST131-O25b:H4 isolates were frequently multi-drug resistant (MDR), with a high prevalence of extended spectrum β-lactamases (ESBL) and fluoroquinolone resistance. There was no association between AMR phenotypes and the source of E. coli bacteraemia or whether the infection was healthcare-associated. Several clusters of ST131 were genetically similar, potentially suggesting a shared transmission network. However, there was no clear epidemiological associations between these cases, and they included organisms from both healthcare-associated and non-healthcare-associated origins. The majority of ST131 isolates exhibited strong binding with an anti-O25b antibody, raising the possibility of developing rapid diagnostics targeting this pathogen. In summary, our data suggest that a restricted set of MDR E. coli populations can be maintained and spread across both community and healthcare settings in this location, contributing disproportionately to invasive disease and AMR.


Bacterial DNA extraction
All isolates were streaked from frozen stocks onto Iso-Sensitest or Luria Bertani (LB) agar (Oxoid), and incubated overnight at 37°C in a static incubator.Single colonies were picked into a single well of a 96 deep well plate containing Iso-Sensitest broth, before overnight incubation.Bacteria were pelleted and re-suspended in PBS and 20 µL of 100 mg/mL lysozyme solution.DNA extraction was performed using either the MagNAPure small volume nucleic acid extraction kit (Roche) on the FLOW flex platform (Roche), or using the QIAamp 96 DNA QIAcube HT Kit (Qiagen), according to the manufacturer's instructions.Resultant DNA concentration and purity was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific) and a Tapestation 4200 using D1000 ScreenTapes (Agilent Technologies).

Bioinformatics
Command line used for roary core genome alignment: roary -e -n -cd 99 -p 8 ../*.gff Command line used for producing phylogenetic tree from the core genome alignment using iqtree: iqtree -s core_genome_alignment.aln-m GTR+G -bb 1000 Command line for producing SNP distance matrix from the roary core genome alignment: snp-dists core_genome_alignment.aln> core_genome_snp_dists.csv-c Command lines using ariba to prepare the CARD database (Version 3. The 65 genomes identified as ST131 using the srst2 method were mapped against the ST131 reference genome, Escherichia_coli_UPEC_ST131_v0.2.Primers for the pabB gene (TCCAGCAGGTGCTGGATCGT and GCGAAATTTTTCGCCGTACTGT) were used for in silico PCR for O antigen typing, taken from [1] and [2].The 347bp amplicon locus was retrieved from the whole genome alignment (positions 2143355 -2143701 from the reference genome) and inspected in AliView.

Categorisation of E. coli infection class
Classifications were adapted from mandatory reporting surveillance guidance from UKHSA: https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/859759/HCAIDCS_Gram_negative_Submission_Form_2020.pdfHealth-care onset was defined as blood culture positive more than 48 hours after admission.Healthcare associated was defined as blood culture positive less than 48 hours after admission and one of more of the following criteria: hospital admission in previous 28 days, chemotherapy or neutropenia (less than 0.5) in previous 28 days, surgery in previous 28 days, antibiotic prescription in community in previous 28 days, urinary catheter insertion or manipulation in 28 days before admission.
Community onset was defined as blood culture positive less than 48 hours after admission and nil of above risk factors identified from review of clinical note and GP entries in month before admission.
Calculating anti-O25b sensitivity, specificity, positive and negative predictive values for ESBL production and ciprofloxacin susceptibility The following contingency tables were used to calculate the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for samples carrying the O25 antigen in silico and/or being ST131: O25 antigen status vs ESBL production: Bacteria were cultured from storage beads onto LB agar plates.Single colonies were picked and cultured in LB broth overnight at 37⁰C at 200 RPM.Four additional bacterial isolates known from previous work to have different binding patterns were used as controls.Overnight bacterial cultures were diluted 1:200 in LB broth and 50µl of this bacterial suspension was added per well in CellCarrier-96 Ultra plates (Perkin Elmer) and left in a static incubator for 2 hours at 37⁰C.The supernatant was discarded and any remaining bacteria fixed with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) for 15 minutes at room temperature.After washing with PBS, 100µl of the antibody KM467 (diluted to 1µg/ml in PBS + 1% bovine serum albumin) was added for 1 hour at room temperature.After further wash with PBS, 100µl of 2µg/ml Alexa Fluor 647 Goat Anti-Human IgG (ThermoFisher) plus 2µg/ml DAPI (Thermo Fisher) in PBS + 1% bovine serum albumin were added and the plate was incubated for 30 minutes in the dark.After a final wash with PBS, 50µl of PBS was added to each well and the plate was taken for imaging.The antibody binding assay was repeated fivefold.