Plasmid-mediated acquisition and chromosomal integration of blaCTX-M-14 in a subclade of Escherichia coli ST131-H30 clade C1

Kohji Komori; Kotaro Aoki; Sohei Harada; Yoshikazu Ishii; Kazuhiro Tateda

doi:10.1128/aac.00817-24

. 2024 Aug 12;68(9):e00817-24. doi: 10.1128/aac.00817-24

Plasmid-mediated acquisition and chromosomal integration of bla_CTX-M-14 in a subclade of Escherichia coli ST131-H30 clade C1

Kohji Komori ¹, Kotaro Aoki ², Sohei Harada ², Yoshikazu Ishii ^1,^2,^3,^✉, Kazuhiro Tateda ^1,²

Editor: Alessandra Carattoli⁴

PMCID: PMC11373201 PMID: 39133024

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: extended-spectrum β-lactamase (ESBL), Escherichia coli, ST131-H30, chromosomal integration, bla _CTX-M-14

INTRODUCTION

Escherichia coli sequence type (ST)131 is a global multidrug-resistant (MDR) lineage mainly causing bloodstream and urinary tract infections (1 –3). Recent studies using whole-genome sequencing (WGS) have revealed the sublineage structure of ST131, classifying it into clades A, B, and C, each defined by the allele of the type 1 fimbriae fimH adhesin: H41 in clade A, H22 in clade B, and H30 in clade C (4, 5). Molecular clock analysis inferred that mutations in gyrA and/or parC, part of the quinolone resistance-determining region (QRDR), were acquired around 1987 in clade C, leading to its divergence into clades C1 and C2 (6). These clades are strongly associated with CTX-M-type extended-spectrum β-lactamase (ESBL) genes (bla_CTX-M), each possessing a clade-specific bla_CTX-M (7).

Clade C2, also known as H30Rx, commonly harbors bla_CTX-M-15 and is predominant in Europe, North America, and Southeast Asia (3). Conversely, clade C1 strains, also known as H30R, typically possess bla_{CTX-M-9-group} genes, including bla_CTX-M-14, and are prevalent in Japan, Korea, China, Spain, and Canada (3, 8).

The bla_CTX-M genes are originally derived from chromosomal β-lactamase genes of Kluyvera spp. (bla_KLU) and are often carried on plasmids specific to each type of bla_CTX-M (9). The acquisition process of bla_CTX-M by clinically relevant species such as E. coli is proposed to occur as follows (10): an insertion sequence (IS), such as ISEcp1, inserts upstream of bla_KLU. The IS transposes bla_KLU from the chromosome of Kluyvera spp. to a plasmid via transposase activity. This plasmid is subsequently transferred horizontally to other bacterial species, including E. coli.

Plasmids are generally grouped based on incompatibility (11, 12). IncF family plasmids, which possess a toxin-antitoxin system, tend to be stably maintained in bacterial cells (13). Therefore, the transposition of antimicrobial resistance genes into IncF plasmids is particularly concerning for the dissemination of MDR bacteria, as it may lead to the fixation of resistance genes within the host strain lineage. In ST131-H30 clade C2, some IncF plasmids carrying bla_CTX-M-15 have been reported, such as pEC958 with plasmid sequence type (pST) [F2:A1:B-] and pC15-1a with pST [F2:A-:B-], known as the epidemic plasmid in Canada (14 –16). In clade C1 strains, an IncF plasmid carrying bla_CTX-M-27, with pST [F1:A2:B20], is widespread in Japan (17). Although there are few reports of bla_CTX-M-14 being carried on the IncF [F1:A2:B20] plasmid (18), the number of strains carrying it on the chromosome has been increasing recently (19, 20). Chromosomal bla_CTX-M-14 is mostly accompanied by ISEcp1, which is located upstream and possesses high transposase activity (21).

In Japan, ST131 strains harboring bla_CTX-M-14 have been isolated since 2000, with most of them belonging to clade C1 (22). However, investigations on ST131-H30 clade C1 strains isolated during the early emergence period have been limited, primarily using draft WGS data. Consequently, the genetic structure of the plasmids carrying bla_CTX-M-14 has not been fully characterized in many cases, making it unclear how bla_CTX-M-14 was acquired and subsequently stabilized in ST131-H30 clade C1.

This study aimed to characterize the plasmids that contributed to the spread of bla_CTX-M-14 and to elucidate the evolutionary process of E. coli ST131-H30 clade C1 with bla_CTX-M-14. We performed a retrospective analysis using full-length WGS of E. coli strains isolated during the period when the isolation frequency of E. coli with bla_CTX-M-14 increased in Japan. In addition, we compared the full-length genome sequences of ST131-H30 strains analyzed in this study with those in public databases.

MATERIALS AND METHODS

Bacterial strains and genome data

As part of a previous study, we performed a draft WGS of 79 E. coli strains with the bla_{CTX-M-9-group} (23). The strains were selected from an original collection of 329 E. coli strains exhibiting the ESBL phenotype, isolated from humans, companion animals, chicks, and broilers from 1997 to 2019 in Japan. Multiplex PCR for bla_CTX-M genes and PCR-based plasmid Inc grouping were performed on these isolates, and 79 strains harboring bla_{CTX-M-9-group}, with no redundant isolation year and plasmid Inc group, were selected for draft WGS (24, 25) (Fig. S1; Table S1). Full-length genome sequencing was also performed for one of these strains (TUM2805).

In the present study, we selected 18 additional strains with different combinations of ST, fimH type, and plasmid Inc group based on draft WGS data for full-length genome sequencing using Illumina and nanopore sequencers to obtain complete genome sequences of plasmids carrying bla_CTX-M-14 (Table S2).

As for the IncFII and IncB/O/K/Z plasmids harboring bla_CTX-M-14, a comparative analysis of genetic structural similarity was performed (Fig. S2 and S3), followed by a search for similar plasmids in public databases (Fig. 1 and 2).

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.

In this study, we focused on ST131-H30 strains and obtained genomic data registered in public databases such as GenBank (26). The complete genome sequences of E. coli (n = 3,010, accessed on 29 July 2022) underwent multilocus sequence typing (MLST) and fimH typing to extract the genome sequences of E. coli ST131-H30. Based on the information registered in the BioSample database or relevant literature, we excluded sequences originating from the same strain or lacking information about the isolation year or geographic location. To determine the process by which E. coli ST131-H30 isolated worldwide acquired and stabilized bla_CTX-M-14, we performed plasmid analysis and molecular phylogenetic analysis on the four strains newly sequenced in this study and 141 strains from public databases (Fig. S5). The genetic structure similarity of the IncFII and IncB/O/K/Z plasmids commonly possessed by ST131-H30 clade C strains was compared (Fig. S6). Among the 145 strains, 96 clade C1 strains were analyzed using molecular phylogenetic and molecular clock analysis to characterize the evolutionary process (Fig. 3).

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.

Draft whole-genome sequencing

DNA was extracted from bacterial cells using a boiling method and subsequently purified using Agencourt AMPure XP (Beckman Coulter, Brea, CA, USA). DNA libraries were prepared using the Illumina DNA Prep Kit (Illumina, Inc., CA, USA) and sequenced on a NovaSeq 6000 (Illumina) with 250 bp paired-end reads or a MiSeq (Illumina) with 300 bp paired-end reads. The sequencing read data were trimmed for adapter sequences and reads not passing a quality score of Q30 or higher using Trimmomatic version 0.39 (27). The trimmed reads were then assembled de novo using SPAdes version 3.15.2 (28).

Species identification was performed by average nucleotide identity (ANI) analysis using FastANI version 1.32 (29) against the E. coli type strain DSM 30083 (= JCM 1649 = ATCC 11775) genome (GenBank accession no. CP033092). ANI values above 95% confirmed the strain as E. coli (29, 30).

MLST, fimH typing, identification of acquired antimicrobial resistance genes, detection of QRDR mutations, plasmid Inc grouping, and plasmid MLST (pMLST) were performed using MLST version 2.0, FimTyper version 1.1, ResFinder version 4.1, PointFinder version 2.1, PlasmidFinder version 2.1, and pMLST version 2.0, respectively. We ran those programs with default parameters. These resources are available at the Center for Genomic Epidemiology (https://www.genomicepidemiology.org/).

Long-read sequencing and hybrid assembly with Illumina reads

High molecular weight DNA was extracted from cultured bacterial cells using the NucleoBond AXG 20 column (TaKaRa Bio, Shiga, Japan) combined with the NucleoBond Buffer Set III (TaKaRa Bio). DNA libraries were prepared using the Rapid Barcoding Kit SQK-RBK004 (Oxford Nanopore Technologies: ONT, Oxford, UK) and sequenced using the MinION flow cell R9.4 (ONT) on a MinION Mk1B (ONT). Basecalling and demultiplexing were performed using Guppy v5.0.11 (ONT).

MinION reads were adapter trimmed and quality filtered using NanoFilt version 2.6.0 with a quality score cutoff of Q6 and a minimum length of 5,000 bp (31). The generated reads were subsequently assembled with the Illumina reads, which had been trimmed using Trimmomatic version 0.39 with a quality score cutoff of Q30, using Unicycler version 0.4.8-beta (32). The contigs were polished three times using Pilon version 1.23 (33) based on the Illumina reads. Default parameters were used for all software unless otherwise noted.

Plasmid analysis

Full-length genome sequences were annotated with open reading frames (ORFs) and genes using the DNA Data Bank of Japan Fast Annotation and Submission Tool (34). Insertion sequences and antimicrobial resistance gene locations were confirmed using Prokka version 1.14.6 (35) and ResFinder version 4.1, respectively. Plasmid structure comparisons were performed using EasyFig version 2.2.2 (36). We further targeted the earliest-dated plasmids in each of the detected Inc groups from various genetic lineages and searched public databases for plasmids with similar structures using Nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The obtained plasmid sequences were subsequently compared for similarity.

Core-genome SNP-based phylogenetic analysis of E. coli ST131-H30

Core-genome single-nucleotide polymorphism (SNP)-based phylogenetic analysis was performed on the complete genome sequences of E. coli ST131-H30 strains. First, we simulated short reads based on the contigs generated by the SimSeq simulator (37). These simulated reads were aligned to the genome sequence of a reference strain using the Burrows-Wheeler Aligner with the “SW” option (38). The reference strains used were E. coli EC958 (GenBank accession no. HG941718) for analysis involving the entire clade C, and E. coli CD306 (GenBank accession no. NZ_CP013831) for analysis involving clades C0 and C1.

Core-genome alignments were extracted using SAMtools version 1.17 mpileup (39) and VarScan version 2.3.9 mpileup2cns (40). Temporal phylogenetic trees were estimated based on the maximum likelihood method using PhyML version 20120412 (41). These trees were used as starting trees to infer homologous recombination events that imported DNA fragments from strains outside the phylogenetic clade, and a clonal phylogeny with corrected branch lengths was constructed using ClonalFrameML version 1.12 (42). Phylogenetic trees based on SNPs detected in the core genome, excluding homologous recombination regions inferred by ClonalFrameML, were generated by RAxML version 8.2.12 (43) using 1,000 bootstraps and the gamma site substitution model. All trees were visualized using Figtree version 1.4.4.

To further investigate the divergence within clade C1, we performed molecular clock analysis using BEAST version 2.6.7 (44) on 2,401 SNPs in the core genome of 96 strains belonging to clades C0 and C1. We used the Hasegawa, Kishino, and Yano substitution model and a strict clock model. The Markov chain Monte Carlo generations were performed for 30 million steps, with samples taken every 5,000 steps.

Conjugation experiments

In order to verify whether IncFII and IncB/O/K/Z plasmids contribute to the spread of bla_CTX-M-14 in E. coli, conjugation experiments were performed using the filter mating method. The donor strains were E. coli strains harboring bla_CTX-M-14 on a plasmid obtained in this study. The recipient strains used were either E. coli ML4909 (45), which is lactose non-degrading and resistant to sodium azide, or E. coli ML4903 (46), which is lactose degrading and resistant to rifampicin.

Transconjugants were selected on MacConkey agar plates (Eiken Chemical Co., Ltd., Tokyo, Japan) supplemented with cefotaxime at 4 mg/L (Sigma-Aldrich Co. LLC, St. Louis, MO, USA) and sodium azide at 100 mg/L (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) or rifampicin at 100 mg/L (FUJIFILM Wako Pure Chemical Corporation). The presence of the plasmid carrying bla_CTX-M-14 in transconjugants was confirmed by PCR (23). Moreover, we calculated conjugation frequency by dividing the number of transconjugants (CFU/mL) by the number of recipient bacteria (CFU/mL). For strains in which transconjugants were not obtained, we repeated the experiment three times under the same conditions to ensure accuracy.

RESULTS

WGS of E. coli harboring bla_CTX-M-14

We focused on the plasmids carrying bla_CTX-M-14 commonly observed in E. coli strains belonging to ST131-H30 and other STs. Initially, 273 isolates harboring bla_{CTX-M-9-group} were selected from an original collection of 329 ESBL-producing E. coli isolates. From these, 79 strains, with no redundant isolation year and plasmid Inc group, were selected for draft WGS (Table S1). The mean sequence depth of these assembled genomes was 38× (standard deviation: SD, 26×), and the mean N50 was 97,166 bp (SD, 42,921 bp) (Table S1).

Among the 79 strains, 43 carried bla_CTX-M-14 and were classified into 21 STs and 19 fimH types. Eleven of these strains belonged to ST131: seven in H30, three in H41, and one in H27.

From the 43 strains with bla_CTX-M-14, we selected 19 strains with diverse combinations of ST, fimH type, and plasmid Inc group for full-length genome sequencing (Table 1; Table S2). These selected strains belonged to 12 STs and 12 fimH types. The mean sequence depth for long-read sequencing was 134× (SD, 69×).

TABLE 1.

Summary of the genetic lineages and the position of bla_CTX-M-14 in the E. coli strains (n = 19) in this study

Strain name	Isolation year	Host ST	Host fimH type	Location of bla_CTX-M-14/plasmid name	Inc group [pST]	Length (bp)	Genes located surrounding bla_CTX-M-14^a	Accession number
TUM1223	1997	405	fimH29	pMTY1223_IncFII	IncFII [F2:A-:B-]	70,302	ISEcp1 - bla_CTX-M-14 - ∆lamB	CP135724
TUM1226	1997	68	fimH49	pMTY1226_IncFII	IncFII [F2:A-:B-]	70,302	ISEcp1 - bla_CTX-M-14 - ∆lamB	CP135721
TUM1586	2002	354	fimH58	pMTY1586_IncFII	IncFII [F2:A-:B-]	72,746	ISEcp1 - bla_CTX-M-14 - ∆lamB	CP135716
TUM1880	2003	297	fimH26	pMTY1880_IncFII	IncFII [F35:A-:B-]	70,737	ISEcp1 - bla_CTX-M-14 - ∆lamB	CP135707
TUM2802	2006	131	fimH30	pMTY2802_IncFII	IncFII [F2:A-:B-]	72,904	ISEcp1 - bla_CTX-M-14 - ∆lamB	CP135693
TUM2830	2006	131	fimH41	pMTY2830_IncFII	IncFII [F2:A-:B-]	70,341	ISEcp1 - bla_CTX-M-14 - ∆lamB	CP135687
TUM3755	2007	752	fimH24	pMTY3755_IncFII_2	IncFII [F35:A-:B-]	70,758	ISEcp1 - bla_CTX-M-14 - ∆lamB	CP143108
TUM9803	2010	48	fimH23	pMTY9803_IncFII	IncFII [F2:A-:B-]	72,265	ISEcp1 - bla_CTX-M-14 - ∆lamB	CP135673
TUM9975	2010	131	fimH30	pMTY9975_IncFII	IncFII [F2:A-:B-]	76,366	ISEcp1 - bla_CTX-M-14 - IS903 - ∆tonB	CP135667
TUM2367	2005	131	fimH30	Chromosome		5,023,218	ISEcp1 - bla_CTX-M-14 - IS903 - ∆tonB	CP135697
				pMTY2367_IncBOKZ	IncB/O/K/Z	104,860	ISEcp1 - bla_CTX-M-14 - IS903 - ∆tonB	CP135699
TUM2805^b	2006	167	Not determined	pMTY2805_IncBOKZ	IncB/O/K/Z	101,720	ISEcp1 - bla_CTX-M-14 - IS903 - ∆tonB	CP128953
TUM2820	2006	1,638	fimH167	pMTY2820_IncBOKZ	IncB/O/K/Z	102,136	ISEcp1 - bla_CTX-M-14 - IS903 - ∆tonB	CP135689
TUM10008	2010	93	fimH30	pMTY10008_IncBOKZ	IncB/O/K/Z	87,747	ISEcp1 - bla_CTX-M-14 - IS903	CP135660
TUM14664	2014	3,075	fimH54	pMTY14664_IncBOKZ	IncB/O/K/Z	128,936	ISEcp1 - bla_CTX-M-14 - IS903	CP135650
TUM17543	2016	12	fimH106	pMTY17543_IncBOKZ	IncB/O/K/Z	90,712	ISEcp1 - bla_CTX-M-14 - IS903	CP135645
TUM1857	2003	131	fimH27	Chromosome		5,199,107	ISEcp1 - bla_CTX-M-14 - IS903 - ∆tonB	CP135709
TUM11356	2012	131	fimH41	Chromosome		4,963,886	ISEcp1 - bla_CTX-M-14 - IS903	CP135656
				pMTY11356_IncF	IncF [F1:A2:B20]	160,382	ISEcp1 - bla_CTX-M-14 - IS903	CP135657
TUM13064	2012	131	fimH30	Chromosome		5,273,163	ISEcp1 - bla_CTX-M-14 - IS903 - ∆tonB	CP135653
TUM14668	2014	131	fimH41	Chromosome		4,829,602	ISEcp1 - bla_CTX-M-14 - IS903 - ∆tonB	CP135646

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^{^a}

The genes located within the ISEcp1-mediated transposable unit flanked by direct repeat sequences were listed.

^{^b}

Data previously published by Ikegaya et al. (23).

Location of bla_CTX-M-14 and structural characteristics of the plasmid determined by full-length WGS

Among the 19 isolates, 9 strains possessed IncFII plasmids harboring bla_CTX-M-14 (Table 1). Six strains carried bla_CTX-M-14 on IncB/O/K/Z plasmids, and one of these strains, TUM2367, also had bla_CTX-M-14 on the chromosome. Another strain carried the gene on both the chromosome and the IncF [F1:A2:B20] plasmid. The remaining three strains carried bla_CTX-M-14 only on the chromosome.

IncFII plasmid

We initially compared the complete genome sequences of the IncFII plasmids carrying bla_CTX-M-14 from nine strains (Fig. S2). In all plasmids, ISEcp1 was located upstream of bla_CTX-M-14. Eight of these plasmids had highly similar structures with 93%–100% similarity in full-length nucleotide sequences, and a partial gene encoding maltoporin LamB (∆lamB) was located downstream of bla_CTX-M-14. Although the remaining plasmid, pMTY9975_IncFII, shared a similar nucleotide sequence in a 53 kb region of its 76 kb total length, the region flanking bla_CTX-M-14 was different. Immediately downstream of bla_CTX-M-14 in pMTY9975_IncFII was IS903 and a partial gene encoding a TonB-dependent receptor (∆tonB).

We searched the public database using BLAST for plasmids with high similarity to pMTY2802_IncFII, which was carried by E. coli ST131-H30 TUM2802 isolated in 2006. We found 24 plasmids with a query cover value over 95%, including pHK01, an epidemic plasmid reported in China in 2004 (47 –49). Among these 24 plasmids, we selected those with high structural similarity to pMTY2802_IncFII and pHK01 and possessed by bacterial species other than E. coli to compare the genetic structure of these plasmids in detail (Fig. 1). The entire structures of pEG356 in Shigella sp. (50) and pKpn1693-CTX-M in Klebsiella sp. (51) were highly similar to pMTY2802_IncFII and pHK01. However, the ESBL gene on pEG356 and pKpn1693-CTX-M was bla_CTX-M-24 not bla_CTX-M-14.

IncB/O/K/Z plasmid

Subsequently, we compared the IncB/O/K/Z plasmids with bla_CTX-M-14 possessed by six strains (Fig. S3). In all these plasmids, bla_CTX-M-14 was flanked by ISEcp1 upstream and IS903 downstream. The sequence similarity of their plasmid backbones ranged from 76% to 96%. These plasmids were categorized into two types based on the location of bla_CTX-M-14 relative to the rep gene: A type, where bla_CTX-M-14 was about 25 kb from rep, and B type, where bla_CTX-M-14 was located 60–100 kb from rep. Additionally, all A-type plasmids had a ΔtonB gene downstream of IS903, whereas B-type plasmids did not.

In the public databases, only two E. coli plasmids, pB16EC0986-2 and pKKo003_1, were structurally similar to the A-type pMTY2367_IncBOKZ with query cover values above 90%. Sequence similarities of pB16EC0986-2 and pKKo003_1 to pMTY2367_IncBOKZ were 97% and 94%, respectively. Although bla_CTX-M-14 was located approximately 25 kb from the rep gene in both plasmids, the upstream ISEcp1 was partly deleted (ΔISEcp1) (Fig. 2A). In pKKo003_1, an inversion of a 2,303 bp region around bla_CTX-M-14 was also observed.

For the B-type plasmid pMTY10008_IncBOKZ, a search resulted in 37 plasmids carrying bla_CTX-M-14 with more than 90% query cover, including the epidemic plasmid pCT, detected in the United Kingdom in 2004 (52, 53). We selected the plasmids with the highest query cover from bacterial species other than E. coli and compared their structures with those of pMTY10008_IncBOKZ and pCT (Fig. 2B). These IncB/O/K/Z plasmids had a conserved backbone structure, and bla_CTX-M-14 was consistently located approximately 63–104 kb from the rep gene.

Conjugation of plasmids carrying bla_CTX-M-14

All the IncFII and IncB/O/K/Z plasmids carrying bla_CTX-M-14 from the 16 strains were successfully transferred via conjugation to the E. coli recipient strains. The conjugation frequency of the IncFII plasmids ranged from 8.0 × 10⁻⁶ to 9.2 × 10⁻³, and those of the IncB/O/K/Z plasmids ranged from 1.9 × 10⁻⁶ to 5.7 × 10⁻³. For the strain TUM11356 possessing bla_CTX-M-14 on the IncF [F1:A2:B20] plasmid, no transconjugants were obtained. Analysis of the transfer (tra) operon of this IncF plasmid revealed an insertion of IS1G and deletions of traA, traF, traN, trbC, and trbE compared to the tra operon of the conjugative IncFII plasmid, pMTY2802_IncFII (Fig. S4).

Relationship between plasmid type and clade of host E. coli ST131-H30 strains

We focused on ST131-H30 strains and conducted core-genome SNP-based phylogenetic analysis using complete genome sequences of the 4 strains in our collection (TUM2367, TUM2802, TUM9975, and TUM13064) and 141 strains from public databases (Fig. S1). The strains from the public database were isolated in 20 countries across Asia, Europe, North America, Africa, and Australia between 2002 and 2021 (Fig. S5). These 145 ST131-H30 strains were classified into three previously reported clades: clade C0 (n = 2), C1 (n = 94), and C2 (n = 49) (Fig. S5). All strains except CCUG 73778 and CD306, regardless of geographic location of isolation, possessed QRDR mutations in gyrA and parC, resulting in amino acid substitutions of Ser-83-Leu and Asp-87-Asn in GyrA, and Ser-80-Ile and Glu-84-Gly/Val in ParC. Of all 145 strains, 55 carried bla_CTX-M-14. Five clade C1 strains and one clade C2 strain possessed bla_CTX-M-14 on IncFII plasmids. Five of these were highly similar to pMTY2802_IncFII with a query cover of 94%–100%, except for pMTY9975_IncFII harbored by the clade C1 strain (Fig. S6A). Additionally, three C1 and four C2 strains carried bla_CTX-M-14 on IncB/O/K/Z plasmids. Six of these IncB/O/K/Z plasmids, except for pMTY2367_IncBOKZ, were similar to B-type pMTY10008_IncBOKZ with a query cover of 73%–86% and also carried bla_CTX-M-14 at the same position on the plasmids (Fig. S6B).

Characterization of subclades in E. coli ST131-H30 clade C1

We conducted a molecular phylogenetic and molecular clock analysis of 96 strains of clades C0 and C1 to determine the molecular characteristics of each subclade and the divergence dates of these subclades (Fig. 3). Moreover, clade C0 strains were included to validate the molecular clock analysis. Clade C0 consisted of two strains, CCUG 73778 and CD306, which did not harbor QRDR mutations. Therefore, it was inferred that the timing of the divergence of clade C1 from clade C0 approximately coincided with that of the emergence of the QRDR mutations.

Clade C1 diverged into eight distinct subclades. The estimated divergence date of clade C1 from clade C0 was around 1983 [95% highest posterior density (HPD), 1977–1988]. Among the C1 subclades, subclades 1, 2, 3, and 8 did not contain clusters of strains with bla_CTX-M genes. In contrast, subclades 4–7 included clusters of strains harboring bla_CTX-M-14 or bla_CTX-M-27. Notably, subclades 4–8 were estimated to have diverged from their common ancestor around 1992 (HPD, 1988–1996). Moreover, all the 34 strains in subclade 5 possessed bla_CTX-M-14 on their chromosomes. This subclade likely emerged approximately in 1999 (HPD, 1995–2002). Meanwhile, 28 out of the 29 strains in subclades 6 and 7 possessed bla_CTX-M-27, which is a single-nucleotide variant of bla_CTX-M-14. Moreover, bla_CTX-M-27 was predominately carried on IncF [F1:A2:B20] plasmids (n = 25) (Fig. S5).

Location and surrounding structures of bla_CTX-M-14 on the chromosome in ST131-H30 clade C1 strains

To investigate whether C1-subclade 5 strains had acquired the chromosomal bla_CTX-M-14 genes in a single acquisition event and subsequently inherited them by progeny strains, we analyzed the chromosomal location and surrounding genetic structure of bla_CTX-M-14 in these strains. We also investigated the genetic environment of chromosomal bla_CTX-M-14 in strains from other subclades for comparison. Forty strains from subclades 2, 4, or 5 possessed one to five copies of chromosomal bla_CTX-M-14 with all these genes (Fig. S5), except in STO_Bone8 (which had ∆ISEcp1), containing intact ISEcp1 sequences upstream and conserved right and left inverted repeat sequences for the ISEcp1-mediated transposable units (Table S3). Using the genome sequence of strain CD306 (GenBank accession number NZ_CP013831) as a reference, we analyzed the regions flanked by direct repeat (DR) sequences for unit length and chromosomal location as ISEcp1-mediated transposable units. The lengths of these transposable units ranged from 3,001 to 4,801 bp. For those missing downstream DRs (such as TUM2367, F16EC0593-1, and E17EC0383-2), the unit and chromosome boundaries were analyzed, revealing unit lengths from 3,633 to 30,154 bp. The ISEcp1-mediated transposable units were located at 24 different positions on the chromosome, with 34 subclade 5 isolates consistently having an integration at position bp 927155 (Fig. 4). The transposable units of 31 out of these 34 subclade C5 isolates shared a common 4,798 bp structure comprising ISEcp1-bla_CTX-M-14-IS903-∆tonB.

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.

The remaining three strains (STO_Bone8, E17EC0383-1, and F16EC0593-1) had IS insertions or inversions in the transposable unit but otherwise shared a common structure. Additionally, 14 out of the 34 subclade 5 isolates harbored one to four additional ISEcp1-mediated transposable units containing bla_CTX-M-14 at various other positions, although the insertion positions differed in all but one pair of strains with high overall genetic similarity (E16EC0458-5 and E16EC0945-1). The positions of insertion of ISEcp1-mediated transposable units with bla_CTX-M-14 were also diverse among subclade 2 and 4 strains.

DISCUSSION

In this study, we detected the IncFII and IncB/O/K/Z plasmids carrying bla_CTX-M-14 in diverse genetic lineages of E. coli from the 2000s. Molecular phylogenetic analysis indicated that ST131-H30 clade C1 strains acquired a plasmid carrying bla_CTX-M-14, followed by the integration of bla_CTX-M-14 into the chromosome, resulting in the emergence of C1-subclade 5, which universally harbors chromosomal bla_CTX-M-14.

bla_CTX-M-14 originated from the chromosomal β-lactamase gene of Kluyvera georgiana (bla_KLUY) (54) and is hypothesized to have spread horizontally to other Enterobacterales mediated by IS elements such as ISEcp1 and conjugative plasmids (10). Previous studies have reported that plasmids in various Inc groups carried bla_CTX-M-14 using PCR typing methods (55, 56). Notably, IncF plasmids, such as pHK01, were prevalent in China and Korea (47 –49, 57), while IncK plasmids, such as pCT, were prevalent in Spain and the UK (53, 58 –60). In this study, IncFII plasmids similar to pHK01 and IncB/O/K/Z plasmids similar to pCT were detected in E. coli belonging to diverse genetic lineages isolated between 1997 and 2016 (Fig. S2 and S3). Therefore, these plasmids likely contributed to the spread of bla_CTX-M-14 in Japan as well. We confirmed that IncFII and IncB/O/K/Z plasmids carrying bla_CTX-M-14 can transfer horizontally into E. coli, consistent with previous reports (61). Additionally, genomic analysis identified plasmids with high similarity to pMTY2802_IncFII or pMTY10008_IncBOKZ, found in E. coli strains in Japan in this study, within E. coli, Shigella sonnei, and Klebsiella pneumoniae isolates from various countries (Fig. 1 and 2). These plasmids are thus expected to spread by conjugation not only to E. coli but also to other Enterobacteriaceae. In Japan, Korea, and Spain, E. coli strains harboring bla_CTX-M isolated from patients in the 2000s belonged to highly diverse genetic lineages, including ST131 (57, 62, 63), likely due to the horizontal transfer of the gene via these plasmids. In contrast, transconjugants for the IncF plasmid with bla_CTX-M-14, pMTY11356_IncF, were not obtained, and conjugal transfer-related genes traA, traF, traN, trbE, and trbC were deleted (Fig. S4). Notably, TraF is a 26-kDa periplasmic protein required for the F-pilus extension (64), and it has been reported that the traF knockout results in the failure to obtain a transconjugant (65). We hypothesize that the deletion of traF is responsible for the failure of conjugative transmission in pMTY11356_IncF.

In E. coli ST131-H30, the IncF plasmid is linked to bla_CTX-M, such as IncF [F2:A1:B-] with bla_CTX-M-15 and IncF [F1:A2:B2] with bla_CTX-M-27 (16, 66, 67), although literature about a plasmid carrying bla_CTX-M-14 has been limited. Our plasmid analysis of 145 E. coli ST131-H30 strains suggested that IncFII plasmids similar to pMTY2802_IncFII and IncB/O/K/Z plasmids similar to pMTY10008_IncBOKZ likely played important roles in the acquisition of bla_CTX-M-14 in ST131-H30.

The evolutionary process leading to the creation of the E. coli ST131-H30 lineage harboring bla_CTX-M has been investigated in several previous studies using molecular clock analysis. It was predicted that E. coli ST131-H30 emerged around 1980 (95% HPD, 1973–1986), acquired fluoroquinolone resistance, and later diverged into clades C1 and C2 around 1987 (95% HPD, 1983–1992), subsequently acquiring bla_CTX-M (6, 7). To investigate the further evolution within ST131-H30 clade C1, we performed molecular phylogenetic and molecular clock analysis using complete genomes and estimated the divergence date of the subclades (Fig. 3). Initially, we estimated that QRDR mutations occurred around 1989 in clade C1. These mutations conferred fluoroquinolone resistance and provided a selective advantage under antimicrobial use for ST131-H30 strains worldwide. As this estimated date was consistent with previous reports (6), the analysis method used in this study seems valid. Among the eight subclades of clade C1, most strains in subclades 4–8 harbored bla_CTX-M-14 or bla_CTX-M-27. Assuming that bla_CTX-M-27 evolved from bla_CTX-M-14, it was inferred that the common ancestor of these subclades acquired bla_CTX-M-14 around 1993 (1989–1996). This estimation appears reasonable, as bla_CTX-M-14 was first reported in 1995 from Shigella spp. isolated from a patient in South Korea (68).

We also focused on subclade 5, which harbored chromosomal bla_CTX-M-14. All 34 strains of subclade 5 had a transposable unit containing the ISEcp1-bla_CTX-M-14-IS903-∆tonB structure at the same position on the chromosome (bp 927155 of the genomic sequence of strain CD306). Therefore, this ISEcp1-mediated transposable unit was acquired by a single transposition event in the common ancestral strain of these isolates. Considering the period when subclade 5 was formed, it was estimated that this event took place no later than 1999.

Furthermore, we focused on the genetic structure of IS903-∆tonB, situated downstream of bla_CTX-M-14, to identify the plasmid from which the chromosomal bla_CTX-M-14 in subclade 5 originated. Among the plasmids analyzed in this study, only one each of the A-type IncB/O/K/Z plasmid (pMTY2367_IncBOKZ) and the IncFII plasmid (pMTY9975_IncFII) possessed intact ISEcp1 and IS903-∆tonB. However, drawing a definitive conclusion regarding their origin proved challenging, as only one of the strains within ST131 (TUM2367 and TUM9975) carried each of these plasmids.

Fourteen of the 34 isolates harbored similar transposable units at different chromosomal positions in addition to the bp 927155 position. However, the insertion positions were diverse, and the length downstream of bla_CTX-M-14 was equal to or less than that of the transposable unit at the bp 927155 position. Therefore, multiple copies of bla_CTX-M-14 on the chromosome may have arisen by replicative transposition of the ISEcp1-mediated unit following an integration at the bp 927155 position. Moreover, chromosomal bla_CTX-M-14 was located in the vicinity of dnaA, and the copy number of genes was amplified using either copy-and-paste or tandem amplification methods to adjust the transcript level of bla_CTX-M-14 and extend the resistance spectrum (19). Additionally, transposition of bla_CTX-M-14 to the chromosome among subclade 5 strains may allow these strains to stably possess the gene. Moreover, strains with chromosomal bla_NDM-5 were more stable in gene inheritance for at least 30 days than those with the gene on a plasmid (69).

In a subclade of E. coli ST38 with bla_OXA-48, transfer of the composite transposon containing bla_OXA-48 from the plasmid to the chromosome resulted in stable maintenance of the resistance gene with a reduced fitness cost (70). About a quarter of the 115 ESBL-producing K. pneumoniae bloodstream isolates belonging to ST307 or ST48 from six hospitals in Korea had bla_CTX-M on their chromosomes, suggesting the advantage of chromosomal location of the resistance genes for stable transmission in clinical settings (71).

Overall, we conceptualized a bla_CTX-M-14 spreading process consisting of the following three phases (Fig. 5). In the first phase, bla_KLUY on the chromosome of K. georgiana was transposed to a plasmid and horizontally transferred to other Enterobacterales via conjugation. In the second phase, bla_CTX-M-14 was horizontally transferred to E. coli of diverse genetic lineages, including ST131-H30, mediated by plasmids, such as IncFII and IncB/O/K/Z plasmids, around 1993. In the third phase, E. coli ST131-H30 strains with bla_CTX-M-14 disseminated while retaining these plasmids. During this process, bla_CTX-M-14 in an E. coli ST131-H30 C1 strain has been integrated into the chromosome by ISEcp1-mediated transposition. This process may facilitate the spread of bla_CTX-M-14 with stable retention, followed by the creation of C1-subclade 5 around 1999. In this study, there are five limitations. First, it is a retrospective study using stored E. coli strains with bla_CTX-M-14 isolated before 2016. The collection criteria were not standardized. Therefore, there is a potential for bias regarding the geographic regions and sources of the isolates. Second, only 19 out of the 329 strains underwent full-length WGS. Consequently, the findings may not fully represent the characteristics of all plasmids harboring bla_CTX-M-14. Third, this study did not include the most recent isolates from Japan, which may affect the relevance of the findings to current conditions. Fourth, the origin and mechanism of bla_CTX-M-14 acquisition by IncFII and IncB/O/K/Z plasmids remain unclear. Fifth, 32 of the 34 subclade 5 isolates in the ST131-H30 clade C1 were from South Korea. Despite this, we used all full-length genome sequences of E. coli ST131-H30 in public databases.

Conclusion

Our study revealed the significant role of IncFII and IncB/O/K/Z plasmids in the global spread of bla_CTX-M-14 across diverse bacterial species, including E. coli ST131-H30. Furthermore, the ST131-H30 clade C1 strains likely disseminated while carrying plasmids containing bla_CTX-M-14, leading to the emergence of a strain with chromosomal integration of bla_CTX-M-14, thereby contributing to its spread. Our findings highlight the critical influence of horizontal plasmid transmission and chromosomal integration, especially in pandemic lineages, on the dissemination and evolution of antimicrobial-resistant strains.

ACKNOWLEDGMENTS

This work was supported by the Ministry of Health, Labor, and Welfare of Japan (grant numbers: H30-Shokuhin-Ippan-006 and 21KA1004), the Japan Society for the Promotion of Science KAKENHI Grant-in-Aid for Scientific Research (C) to Y.I. (grant number JP18K08452), and the Project Research Grant No. 22-7 of Toho University School of Medicine. K.K. is a recipient of the BIKEN Taniguchi Scholarship.

The authors thank Enago for the English language review.

Contributor Information

Yoshikazu Ishii, Email: ishiiyo@hiroshima-u.ac.jp.

Alessandra Carattoli, Universita degli studi di roma La Sapienza, Rome, Italy.

DATA AVAILABILITY

WGS data have been deposited in the GenBank database under the BioProject accession numbers PRJNA984451, PRJNA984747, and PRJNA1019817. The GenBank accession numbers assigned to each WGS data are provided in Tables S1 and S2.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/aac.00817-24.

Figure S1. aac.00817-24-s0001.tiff.

The strain selection process for comparative analysis of plasmids carrying bla_CTX-M-14 and molecular phylogenetic analysis of ST131-H30 used in this study.

aac.00817-24-s0001.tiff^{(11.9MB, tiff)}

DOI: 10.1128/aac.00817-24.SuF1

Figure S2. aac.00817-24-s0002.tiff.

Structural comparison of IncFII plasmids carrying bla_CTX-M-14 possessed by E. coli in diverse genetic lineages in this study.

aac.00817-24-s0002.tiff^{(11.9MB, tiff)}

DOI: 10.1128/aac.00817-24.SuF2

Figure S3. aac.00817-24-s0003.tiff.

Structural comparison of IncB/O/K/Z plasmids carrying bla_CTX-M-14 possessed by E. coli in diverse genetic lineages in this study.

aac.00817-24-s0003.tiff^{(11.9MB, tiff)}

DOI: 10.1128/aac.00817-24.SuF3

Figure S4. aac.00817-24-s0004.tiff.

Structural comparison of the IncF plasmid carrying bla_CTX-M-14 and the conjugative IncFII plasmid.

aac.00817-24-s0004.tiff^{(11.9MB, tiff)}

DOI: 10.1128/aac.00817-24.SuF4

Figure S5. aac.00817-24-s0005.xlsx.

Strain information and genetic sublineages of E. coli ST131-H30 in this study and public databases used for molecular phylogenetic analysis.

aac.00817-24-s0005.xlsx^{(99.3KB, xlsx)}

DOI: 10.1128/aac.00817-24.SuF5

Figure S6. aac.00817-24-s0006.tiff.

Structure comparison of IncFII and IncB/O/K/Z plasmids carrying bla_CTX-M-14 harbored by ST131-H30 strains.

aac.00817-24-s0006.tiff^{(11.9MB, tiff)}

DOI: 10.1128/aac.00817-24.SuF6

Table S1. aac.00817-24-s0007.xlsx.

Whole genome sequencing-analyzed E. coli strains with bla_{CTX-M-9 group} in our strain collection.

aac.00817-24-s0007.xlsx^{(29KB, xlsx)}

DOI: 10.1128/aac.00817-24.SuF7

Table S2. aac.00817-24-s0008.xlsx.

Full-length genome sequencing-analyzed E. coli strains with bla_CTX-M-14 in our strain collection.

aac.00817-24-s0008.xlsx^{(16.9KB, xlsx)}

DOI: 10.1128/aac.00817-24.SuF8

Table S3. aac.00817-24-s0009.xlsx.

Insertion location and surrounding structure of bla_CTX-M-14 on the chromosome of a reference strain.

aac.00817-24-s0009.xlsx^{(18.4KB, xlsx)}

DOI: 10.1128/aac.00817-24.SuF9

ASM does not own the copyrights to Supplemental Material that may be linked to, or accessed through, an article. The authors have granted ASM a non-exclusive, world-wide license to publish the Supplemental Material files. Please contact the corresponding author directly for reuse.

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