Complete Assembly of Escherichia coli Sequence Type 131 Genomes Using Long Reads Demonstrates Antibiotic Resistance Gene Variation within Diverse Plasmid and Chromosomal Contexts

Abstract

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 health care costs. E. coli sequence type 131 (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, bla_CTX-M-15, and bla_CTX-M-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 isolates from six patients. Most long-read assemblies generated entire chromosomes and plasmids as single contigs, in contrast to 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: ISEcp1, IS903B, and Tn2 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 causes a significant number of these infections. ST131 bacteria become resistant to treatments 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 that of 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.

Keywords: MGE; antibiotic resistance; genome assembly; plasmid; sequencing.

FIG 1

The structure of contigs with bla_CTX-M genes. Two of the ST131 bla_CTX-M genes are on chromosomal contigs (VREC0693 and VREC1073). VRES1160 and VREC1013 have IncFIA and IncFII plasmids, respectively, both of which have bla_CTX-M-15 genes. VREC1428 has an IncFIA plasmid with the bla_CTX-M-27 gene. VRES0739 is not shown because it was bla_CTX-M negative and had no large plasmid. The contigs were classified as chromosomal or plasmid derived by mlplasmids so that the bla_CTX-M genes and their genetic flanking context could be examined. Annotation was derived from GalileoAMR based on the Multiple Antibiotic Resistance Annotator (MARA) and database. The bla_CTX-M variants are labeled and circled in red (bla_CTX-M-15), purple (bla_CTX-M-14), or green (bla_CTX-M-27).

FIG 2

Pairwise comparisons of the three bla_CTX-M-positive plasmid-associated contigs show high sequence identity for the two from subclade C2 (VREC1013 and VRES1160) relative to that of one from C1 (VREC1428, top). The BLAST result was visualized with EasyFig v2.2.2 such that the blocks connecting the regions of the contigs represent nucleotide homology: blue for homologous regions in the same direction, and yellow for inversions. Gaps or white spaces denote unique loci or regions present in one contig but not in the other. Gene models are in green, with the direction of transcription shown by arrows. Genes of interest are labeled above each arrow. The bla_CTX-M-27 gene (top) is in mauve, and the two bla_CTX-M-15 genes (middle and bottom) are in red. The table at the bottom shows the contig size, plasmid type, and number of genes per strain. The list and products of the annotated genes are given in Table 5.

FIG 3

The phylogenetic context of the six ST131 genomes (names in large bold font) shows that all except that of VREC1428 are in ST131 subclade C2 (red inner ring, VRES1160, VREC1073, VRES0739, VREC0693, and VREC1013). VREC1428 clustered in subclade C1 (purple inner ring). No new isolate clustered in C0 (green inner ring), B (blue inner ring), or an intermediate cluster (gray inner ring). Clade classification was based on phylogenetic analysis (8) by including the reference NCTC13441, 63 isolates from reference , and 56 isolates from reference with associated classification and bla_CTX-M allele data. VREC1073 and VREC0693 had chromosomal bla_CTX-M genes. The outer ring shows bla_CTX-M-15 (red), bla_CTX-M-14 (purple), and bla_CTX-M-27 alleles (green). The phylogeny was built with RAxML v8.2.11 using 4,457 SNPs from a core genome alignment generated with Roary v3.11.2 and was visualized with iTOL v4.3. Branch support was performed by 100 bootstrap replicates, and the scale bar indicates the number of substitutions per site. This midpoint-rooted phylogeny includes reference genome isolates EC958 and NCTC13441 (both in C2).