Complete assembly ofEscherichia coliST131 genomes using long reads demonstrates antibiotic resistance gene variation within diverse plasmid and chromosomal contexts

The incidence of infections caused by extraintestinal Escherichia coli (ExPEC) is rising globally, which is a major public health concern. ExPEC strains that are resistant to antimicrobials have been associated with excess mortality, prolonged hospital stays and higher healthcare costs. E. coli ST131 is a major ExPEC clonal group worldwide with variable plasmid composition, and has an array of genes enabling antimicrobial resistance (AMR). ST131 isolates frequently encode the AMR genes bla CTX-M-14/15/27 , which are often rearranged, amplified and translocated by mobile genetic elements (MGEs). Short DNA reads do not fully resolve the architecture of repetitive elements on plasmids to allow MGE structures encoding bla CTX-M genes to be fully determined. Here, we performed long read sequencing to decipher the genome structures of six E. coli ST131 isolated from six patients. Most long read assemblies generated entire chromosomes and plasmids as single contigs, contrasting with more fragmented assemblies created with short reads alone. The long read assemblies highlighted diverse accessory genomes with bla CTX-M-15 , bla CTX-M-14 and bla CTX-M-27 genes identified in three, one and one isolates, respectively. One sample had no bla CTX-M gene. Two samples had chromosomal bla CTX-M-14 and bla CTX-M-15 genes, and the latter was at three distinct locations, likely transposed by the adjacent MGEs: IS Ecp1 , IS 903B and Tn 2 . This study showed that AMR genes exist in multiple different chromosomal and plasmid contexts even between closely-related isolates within a clonal group such as E. coli ST131. Importance Drug-resistant bacteria are a major cause of illness worldwide and a specific subtype called Escherichia coli ST131 cause a significant amount of these infections. ST131 become resistant to treatment by modifying their DNA and by transferring genes among one another via large packages of genes called plasmids, like a game of pass-the-parcel. Tackling infections more effectively requires a better understanding of what plasmids are being exchanged and their exact contents. To achieve this, we applied new high-resolution DNA sequencing technology to six ST131 samples from infected patients and compared the output to an existing approach. A combination of methods shows that drug-resistance genes on plasmids are highly mobile because they can jump into ST131’s chromosomes. We found that the plasmids are very elastic and undergo extensive rearrangements even in closely related samples. This application of DNA sequencing technologies illustrates at a new level the highly dynamic nature of ST131 genomes.