Plasmid-mediated acquisition and chromosomal integration of blaCTX-M-14 in a subclade of Escherichia coli ST131- H 30 clade C1

Abstract

Escherichia coli ST131 is a multidrug-resistant lineage associated with the global spread of extended-spectrum β-lactamase-producing organisms. Particularly, ST131 clade C1 is the most predominant clade in Japan, harboring bla_CTX-M-14 at a high frequency. However, the process of resistance gene acquisition and spread remains unclear. Here, we performed whole-genome sequencing of 19 E. coli strains belonging to 12 STs and 12 fimH types collected between 1997 and 2016. Additionally, we analyzed the full-length genome sequences of 96 ST131-H30 clade C0 and C1 strains, including those obtained from this study and those registered in public databases, to understand how ST131 clade C1 acquired and spread bla_CTX-M-14. We detected conjugative IncFII plasmids and IncB/O/K/Z plasmids carrying bla_CTX-M-14 in diverse genetic lineages of E. coli strains from the 1990s to the 2010s, suggesting that these plasmids played an important role in the spread of bla_CTX-M-14. Molecular phylogenetic and molecular clock analyses of the 96 ST131-H30 clade C0 and C1 strains identified 8 subclades. Strains harboring bla_CTX-M-14 were clustered in subclades 4 and 5, and it was inferred that clade C1 acquired bla_CTX-M-14 around 1993. All 34 strains belonging to subclade 5 possessed bla_CTX-M-14 with ISEcp1 upstream at the same chromosomal position, indicating their common ancestor acquired bla_CTX-M-14 in a single ISEcp1-mediated transposition event during the early formation of the subclade around 1999. Therefore, both the horizontal transfer of plasmids carrying bla_CTX-M-14 to diverse genetic lineages and chromosomal integration in the predominant genetic lineage have contributed to the spread of bla_CTX-M-14.

Keywords: Escherichia coli; ST131-H30; blaCTX-M-14; chromosomal integration; extended-spectrum β-lactamase (ESBL).

Fig 1

Structural comparison of IncFII plasmids carrying bla_CTX-M-14 in this study and those in public databases. The gray scale indicates similarity of the genome sequence, with closer to black indicating higher similarity. Block arrows indicate confirmed or putative open reading frames (ORFs) and their orientations. Arrow size is proportional to the predicted ORF length. The meanings of the arrow colors are as follows: red, bla_CTX-M-14; orange, bla_CTX-M-24; yellow, ∆lamB; cyan, transposase genes; blue, conjugal transfer genes; green, replication initiation protein gene; fuchsia, other antimicrobial resistance genes. Putative, hypothetical, and unknown genes are represented by gray arrows. Square brackets indicate host species, isolation year, and country, and parentheses indicate the accession number of the plasmid.

Fig 2

Structural comparison of IncB/O/K/Z plasmids carrying bla_CTX-M-14 in this study and those in public databases. The gray scale indicates similarity of the genome sequence, with closer to black indicating higher similarity. Block arrows indicate confirmed or putative open reading frames (ORFs) and their orientations. Arrow size is proportional to the predicted ORF length. The meanings of the arrow colors are as follows: red, bla_CTX-M-14; black, ∆tonB; cyan, transposase genes; blue, conjugal transfer genes; green, replication initiation protein gene. Putative, hypothetical, and unknown genes are represented by gray arrows. The A-type pMTY2367_IncBOKZ is shown in (A), and the B-type pMTY10008_IncBOKZ is shown in (B). Square brackets indicate host species, isolation year, and country, and parentheses indicate the accession number of the plasmid.

Fig 3

A time-calibrated phylogeny of E. coli ST131-H30 clade C0 (n = 2) and C1 (n = 94). The phylogenetic tree was constructed using BEAST v.2.6.7 based on 2,401 bp single-nucleotide polymorphisms in the core genome, excluding homologous recombination regions, for 96 strains in this study and in the public database. The core-genome region comprised 83.6% (4,241,105/5,073,822 bp) of the genome of the reference strain, E. coli strain CD306 (GenBank accession number NZ_CP013831). We used the Hasegawa, Kishino, and Yano substitution model and strict clock model. The Markov chain Monte Carlo generations were performed for 30 million steps and sampled every 5,000 steps. The x-axis indicates the time estimates for the emergence of the corresponding subclade. CH indicates the chromosome. Isolated countries were classified by geographic region according to the following colors: fuchsia, Asia; blue, North America; green, Europe.

Fig 4

Chromosomal location of the ISEcp1-mediated transposable units with bla_CTX-M-14 in E. coli ST131-H30 clade C1 strains using the genome sequence of strain CD306 (GenBank accession number NZ_CP013831) as the reference and the surrounding structure of bla_CTX-M-14 in the transposable units. The ISEcp1-mediated transposable unit is defined as the region inside direct repeats adjacent to inverted repeat left or inverted repeat right. For strains in which ISEcp1 was disrupted or the downstream DR was not intact, the boundary was defined as the site where a sequence matching the chromosomal nucleotide sequence of the reference genome appeared. Block arrows indicate confirmed or putative ORFs and their orientations. Arrow size is proportional to the predicted ORF length. The meanings of the arrow colors are as follows: red, bla_CTX-M-14; yellow, ∆lamB; black, ∆tonB; cyan, transposase genes; blue, conjugal transfer genes; green, replication initiation protein genes. Putative, hypothetical, and unknown genes are represented by gray arrows.

Fig 5

The dissemination and adaptive evolution scenario of bla_CTX-M-14. The illustration was created with BioRender.com.