Separate F-Type Plasmids Have Shaped the Evolution of the H30 Subclone of Escherichia coli Sequence Type 131

Abstract

The extraintestinal pathogenic Escherichia coli (ExPEC) H30 subclone of sequence type 131 (ST131-H30) has emerged abruptly as a dominant lineage of ExPEC responsible for human disease. The ST131-H30 lineage has been well described phylogenetically, yet its plasmid complement is not fully understood. Here, single-molecule, real-time sequencing was used to generate the complete plasmid sequences of ST131-H30 isolates and those belonging to other ST131 clades. Comparative analyses revealed separate F-type plasmids that have shaped the evolution of the main fluoroquinolone-resistant ST131-H30 clades. Specifically, an F1:A2:B20 plasmid is strongly associated with the H30R/C1 clade, whereas an F2:A1:B- plasmid is associated with the H30Rx/C2 clade. A series of plasmid gene losses, gains, and rearrangements involving IS26 likely led to the current plasmid complements within each ST131-H30 sublineage, which contain several overlapping gene clusters with putative functions in virulence and fitness, suggesting plasmid-mediated convergent evolution. Evidence suggests that the H30Rx/C2-associated F2:A1:B- plasmid type was present in strains ancestral to the acquisition of fluoroquinolone resistance and prior to the introduction of a multidrug resistance-encoding gene cassette harboring bla CTX-M-15. In vitro experiments indicated a host strain-independent low frequency of plasmid transfer, differential levels of plasmid stability even between closely related ST131-H30 strains, and possible epistasis for carriage of these plasmids within the H30R/Rx lineages. IMPORTANCE A clonal lineage of Escherichia coli known as ST131 has emerged as a dominating strain type causing extraintestinal infections in humans. The evolutionary history of ST131 E. coli is now well understood. However, the role of plasmids in ST131's evolutionary history is poorly defined. This study utilized real-time, single-molecule sequencing to compare plasmids from various current and historical lineages of ST131. From this work, it was determined that a series of plasmid gains, losses, and recombinational events has led to the currently circulating plasmids of ST131 strains. These plasmids appear to have evolved to acquire similar gene clusters on multiple occasions, suggesting possible plasmid-mediated convergent evolution leading to evolutionary success. These plasmids also appear to be better suited to exist in specific strains of ST131 due to coadaptive mutations. Overall, a series of events has enabled the evolution of ST131 plasmids, possibly contributing to the lineage's success.

Keywords: Escherichia coli; ST131; genomes; plasmids.

FIG 1

ST131 subclade emergence and designations, based upon fimH allele. FQ_S, fluoroquinolone sensitive; FQ_R, fluoroquinolone resistant; CEPH_R, cephalosporin resistant.

FIG 2

Distribution of F plasmid allele types, island 1, island 2, and the ColIa-containing island among ST131 strains. Strains are ordered by their position in the ST131 phylogenetic tree adapted from reference and colored by fimH allele type according to the figure legend. Strain clades (A, B₁, B₂, C1, and C2) are designated by colored and labeled dashed boxes. Under island 1, island 2, or ColIa, a black-shaded box indicates >99% nucleotide similarity across >90% of the sequence queried.

FIG 3

Linear maps of completed F2:A1 plasmids from H22, H30S, and H30Rx strains. Maps are not to scale. Lines from each map indicate the locations of corresponding isolates in the ST131 phylogenetic tree, with plasmid names colored by fimH allele type. Red numbering below each plasmid indicates IS26 tracer sequences, with those of the same number having the same 8-bp flanking sequence.

FIG 4

Inferred phylogenetic relationships of sequenced ST131 plasmids and closely related database plasmids. Evolutionary history was inferred by the maximum parsimony method, based on 2,414 shared SNP positions among the core regions (32,678 bp) of all plasmids analyzed. Repetitive elements were excluded from the analysis. Isolates are colored by the ST131 sublineage noted in the labels. Numbers at nodes represent bootstrap values based upon 1,000 replicates.

FIG 5

Linear maps of completed ColIa-containing plasmids from H22, H30S, H35, H41, and H30R1 isolates. Maps are not to scale. Lines from each map indicate the location of the corresponding isolate in the ST131 phylogenetic tree, with plasmid names colored by fimH allele type. Red numbering below each plasmid indicates IS26 tracer sequences.

FIG 6

Circular maps displaying nucleotide conservation of plasmid types across ST131 sublineages. (A) pSaT040 (F29:B10 [accession no. CP014496]) used as reference compared to draft assemblies of ColIa-positive isolates. The outer 12 rings are F29:B10-positive isolates, and the inner 27 rings are F1:A2-positive or F29:B10-positive isolates. (B) pMNCRE44_6 (F1:A2:B20 [accession no. CP010882]) used as a reference compared to draft assemblies of ColIa-positive isolates. The outer 21 rings are F1:A2-positive isolates, and the inner 18 rings are F1:A2-positive or F29:B10-positive isolates. (C) pJJ1886-5 (F2:A1 [accession no. CP006789]) used as a reference compared to draft assemblies of all F2:A1-positive isolates. (D) pMNCRE44_6 (F1:A2:B20 [accession no. CP010882]) used as a reference compared to draft assemblies of all F2:A1-positive isolates. The outer two rings are coding regions corresponding to coordinates in GenBank files. The inner two rings represent G+C content and G+C skew, respectively. Asterisks at the zero coordinates designate plasmids of the same replicon allele combination for each respective reference plasmid.

FIG 7

Proposed events leading to currently circulating plasmids in ST131-H30R1 and H30Rx.