Complete Nucleotide Sequences of Plasmids pEK204, pEK499, and pEK516, Encoding CTX-M Enzymes in Three Major Escherichia coli Lineages from the United Kingdom, All Belonging to the International O25:H4-ST131 Clone

Abstract

We determined the complete nucleotide sequences of three plasmids that encode CTX-M extended-spectrum β-lactamases (ESBLs) in pulsed-field gel electrophoresis-defined United Kingdom variants (strains A, C, and D) of the internationally prevalent Escherichia coli O25:H4-ST131 clone. Plasmid pEK499 (strain A; 117,536 bp) was a fusion of type FII and FIA replicons and harbored the following 10 antibiotic resistance genes conferring resistance to eight antibiotic classes: bla_CTX-M-15, bla_OXA-1, bla_TEM-1, aac6′-Ib-cr, mph(A), catB4, tet(A), and the integron-borne dfrA7, aadA5, and sulI genes. pEK516 (strain D; 64,471 bp) belonged to incompatibility group IncFII and carried seven antibiotic resistance genes: bla_CTX-M-15, bla_OXA-1, bla_TEM-1, aac6′-Ib-cr, catB4, and tet(A), all as in pEK499. It also carried aac3-IIa, conferring gentamicin resistance, and was highly related to pC15-1a, a plasmid encoding the CTX-M-15 enzyme in Canada. By contrast, pEK204 (strain C; 93,732 bp) belonged to incompatibility group IncI1 and carried only two resistance genes, bla_CTX-M-3 and bla_TEM-1. It probably arose by the transposition of Tn3 and ISEcp1-bla_CTX-M-3 elements into a pCOLIb-P9-like plasmid. We conclude that (i) United Kingdom variants of the successful E. coli ST131 clone have acquired different plasmids encoding CTX-M ESBLs on separate occasions, (ii) the bla_CTX-M-3 and bla_CTX-M-15 genes on pEK204 and pEK499/pEK516 represent separate escape events, and (iii) IncFII plasmids harboring bla_CTX-M-15 have played a crucial role in the global spread of CTX-M-15 ESBLs in E. coli.

Escherichia coli strains producing CTX-M extended-spectrum β-lactamases (ESBLs) have emerged as major global pathogens, primarily associated with urinary tract infections, sometimes with contingent bacteremia (3, 25). Internationally disseminated clones have been recognized among these ESBL-producing E. coli strains through the application of multilocus sequence typing. These include E. coli lineages of sequence types ST131 and ST405, which are of particular public health concern (6, 27).

ST131 was first defined in 2008, though it is now known to have been in circulation at least from 2003, and has been reported widely across Europe, North America, and the Far East, often with the CTX-M-15 ESBL encoded by related IncFII plasmids that also encode OXA-1 and TEM-1 β-lactamases and the aminoglycoside/fluoroquinolone acetyltransferase AAC(6′)-Ib-cr (6, 16, 27). It belongs to phylogroup B2, is uropathogenic, and includes many variants with various complements of virulence determinants and divergent pulsed-field gel electrophoresis (PFGE) profiles, albeit generally related at ≥65% banding pattern similarity (15, 21, 23, 27, 27).

Allowing for its PFGE diversity, ST131 E. coli may be more widespread than is currently realized and may be prone to acquire prevalent resistance plasmids that are circulating in a particular area (35). Consistent with this view, ST131 variants in Japan have plasmids that encode group 2 or group 9 CTX-M enzymes, which are the dominant Asian types, rather than CTX-M-15 (31), whereas some United Kingdom members have the bla_CTX-M-3 enzyme with different flanking sequences from the common bla_CTX-M-15 gene. The prevalence of antibiotic-susceptible members of this clone is largely unknown, although one recent study identified carriage in 7% of healthy subjects in the Paris area (23).

In the United Kingdom, E. coli isolates with the CTX-M-15 ESBL have become prevalent since 2003. They include five PFGE-defined strains, A to E, which all belong to ST131 (21, 34), along with PFGE-diverse isolates, some of which also belong to ST131. Strain A, which is the most prevalent variant, is locally dominant (e.g., in Hampshire, Shropshire, and parts of Lancashire); strains B, C, and E are nationally scattered, whereas strain D is local to one center (34). The CTX-M-3 enzyme is associated with strain C isolates from Belfast: producers are less multiresistant than representatives of strain C with the CTX-M-15 enzyme from elsewhere in the United Kingdom.

We report here the complete nucleotide sequences of the bla_CTX-M-15-harboring plasmids pEK499 and pEK516, from representative isolates of United Kingdom strains A and D, respectively, and of the bla_CTX-M-3-harboring plasmid pEK204, from a Belfast representative of strain C.

(Parts of this work were presented as posters at the 17th [17] and 18th [14] European Congress of Clinical Microbiology and Infectious Diseases.).

MATERIALS AND METHODS

Bacterial isolates.

E. coli isolates EO499, H041280204, and EO516 all belong to the O25:H4-ST131 lineage (27, 35) and were selected to represent United Kingdom PFGE-defined strains A, C, and D, respectively (15, 16, 34). Isolates EO499 and EO516 produce CTX-M-15 ESBL, whereas H041280204 produces its close relative, CTX-M-3; their ESBL-encoding plasmids were designated pEK499, pEK516, and pEK204, respectively.

Plasmid sequencing.

Plasmids were transferred by conjugation or electroporation into E. coli strain DH5α or J53 (Table 1) and their sequences were determined by a shotgun cloning method (MWG, Planegg-Martinsried, Germany). Briefly, randomly sheared plasmid fragments of 2 to 3 kb were cloned into the pGEM-T Easy vector and then transformed into E. coli DH10b. Inserts were sequenced by BigDye Terminator chemistry. Sequences were assembled using the Staden package. Combinatorial PCRs, directed PCRs, and walking reads were used to assemble the contigs and to fill in gaps.

TABLE 1.

MICs of E. coli recipient strains J53 and DH5α and derivatives containing plasmids pEK499, pEK516, and pEK204

Antibiotic	MIC (μg/ml) for indicated strain
	J53	J53(pEK499)	J53(pEK204)	DH5α	DH5α(pEK516)
Ampicillin	8	>64	>64	16	>64
Co-amoxiclav	8	32	16	8	16
Aztreonam	≤0.125	>64	8	0.25	32
Cefotaxime	≤0.125	256	64	0.25	128
Cefotaxime + clavulanate	≤0.060	0.125	0.125	≤0.060	≤0.060
Ceftazidime	0.25	64	2	0.25	16
Ceftazidime + clavulanate	0.25	0.5	0.25	0.25	0.25
Cefpirome	≤0.125	>64	16	≤0.125	64
Cefpirome + clavulanate	≤0.060	≤0.060	≤0.060	≤0.060	≤0.060
Cefoxitin	8	8	8	8	8
Piperacillin	4	>64	>64	4	64
Piperacillin-tazobactam	4	16	4	4	4
Imipenem	0.25	0.25	0.25	0.5	0.25
Meropenem	≤0.060	≤0.060	≤0.060	≤0.060	≤0.060
Ertapenem	≤0.125	≤0.125	≤0.125	≤0.125	≤0.125
Ciprofloxacin	≤0.125	≤0.125	≤0.125	≤0.125	≤0.125
Amikacin	≤0.5	4	≤0.5	1	2
Gentamicin	0.25	0.25	0.25	0.5	8
Tobramycin	0.25	8	0.5	0.5	8
Streptomycin	1	32	2	1	0.5
Chloramphenicol	2	2	2	2	2
Minocycline	0.25	2	0.25	0.25	4
Tetracycline	1	32	1	1	32
Tigecycline	≤0.250	≤0.250	≤0.250	≤0.250	≤0.250
Sulfamethoxazole	16	>1024	16	2	2
Trimethoprim	0.06	>32	0.06	0.06	0.03
Colistin	≤0.5	≤0.5	≤0.5	≤0.5	≤0.5

pMLST.

The plasmid multilocus sequence typing (pMLST) scheme for IncI1 plasmids (9) was used for pEK204. The relevant fragments of the pilL (254 bp), sogS (254 bp), ardA (343 bp), and repI1 (104 bp) genes and an 812-bp tnbA-pndC region were compared with known allelic variants (http://pubmlst.org/perl/mlstdbnet/mlstdbnet.pl?file=incI1_profiles.xml&page=oneseq).

Bioinformatics.

Open reading frames were predicted and annotated using the Bacterial Annotation System (BASys; http://wishart.biology.ualberta.ca/basys/cgi/submit.pl) (33) and confirmed with DNAMAN 5.2.10 software (Lynnon BioSoft, Lynnon Corporation; http://www.lynnon.com). Each predicted protein was compared against an all-protein database using BlastP (http://blast.ncbi.nlm.nih.gov/Blast.cgi) with a minimum cutoff of 30% identity over 80% length coverage, checking at least two best hits among the COG, KEGG, and nonredundant protein databases. Gene sequences were further compared and aligned with GenBank data using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and CLUSTAL W (http://www.ebi.ac.uk/clustalw) and with reference plasmids by two sequence alignments using the Blastnt-Blast2 algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The IncI1 plasmid R64 (GenBank accession no. NC_005014.1) was used as a reference for annotating pEK204, whereas IncF plasmids pU302L (NC_006816), pIP1206 (AM886293), pRSB107 (AJ851089), and pC15-1a (NC_005327) were used to annotate pEK499 and pEK516. Data files were compiled using Sequin (http://www.ncbi.nlm.nih.gov/Sequin/). The gross structures of whole plasmids were compared with WebACT (http://www.webact.org/WebACT/home) (1), and plasmid maps were prepared using the CGView server (http://stothard.afns.ualberta.ca/cgview_server/) (11).

Nucleotide sequence accession numbers.

The GenBank accession numbers for the three plasmids are EU935738 (pEK516), EU935739 (pEK499), and EU935740 (pEK204).

RESULTS AND DISCUSSION

Analysis of pEK499 (strain A).

Plasmid pEK499 was a circular molecule of 117,536 bp, belonged to incompatibility group F, and represented a fusion of two replicons of types FII and FIA. It harbored 185 predicted genes (Table 2), including 10 conferring resistance to eight antibiotic classes; pEK499 also included various modules that have been identified on other IncF plasmids and which are composed of resistance genes and insertion sequences. With the exception of bla_TEM-1, all the antibiotic resistance genes were clustered in a 25-kb region. They included bla_CTX-M-15 and bla_OXA-1 as well as genes conferring resistance to aminoglycosides and ciprofloxacin (aac6′-Ib-cr), macrolides [mph(A)], chloramphenicol (catB4), and tetracycline [tet(A)]. A 1.8-kb class I integron was present within this multiresistance region and carried dfrA7 and aadA5, encoding trimethoprim and streptomycin resistances, respectively, and sulI, encoding sulfonamide resistance.

TABLE 2.

Open reading frames identified in pEK499

Gene name	Nucleotide positiona	Function encoded
ccdA	408-626	Plasmid maintenance protein, antitoxin component
ccdB	628-933	Plasmid maintenance protein, toxin component
resD	934-1740	Site-specific resolvase that cleaves at the rfsF site
repE	2514-3269	Replication initiation protein of the FIA replicon
sopA	3848-5023	Partitioning protein
sopB	5023-5994	Partitioning protein
yccB	6837-7763	Hypothetical protein
yhdJ	8148-8831	DNA methylase
BASYS001	9067-9501	Hypothetical protein
BASYS002	9501-10328	Hypothetical protein
BASYS003	Compl. 10634-11134	Hypothetical protein
klcA	10745-11170	Putative antirestriction protein
BASYS004	11217-11639	Hypothetical protein
BASYS005	11636-11827	Hypothetical protein
BASYS006	11691-12026	Hypothetical protein
BASYS007	Compl. 12179-12601	Hypothetical protein
BASYS008	12862-13092	Hypothetical protein
BASYS009	13144-14505	Hypothetical protein
BASYS0010	14552-15115	Hypothetical protein
BASYS0011	14564-15220	Hypothetical protein
ltrA	16631-18532	Putative reverse transcriptase
ssb	18727-19167	Single-stranded DNA-binding protein
BASYS0012	Compl. 19022-19498	Hypothetical protein
ykfF	19196-19456	Hypothetical protein
parB	19509-21473	ParB-like partitioning protein
psiB	21528-21962	Plasmid SOS inhibition protein B
psiA	21959-22678	Plasmid SOS inhibition protein A
mok	22900-23108	Modulator of Hok protein, Mok
hok	22958-23116	Postsegregational killing protein Hok
BASYS0013	23882-24010	Hypothetical protein
BASYS0014	Compl. 23990-24262	Hypothetical protein
yubP	24329-25264	Hypothetical protein
gene X (orf39)	Compl. 25561-26208	X-polypeptide; transglycosylation
traM	26494-26877	Mating signal
traJ	27011-27757	Regulation
traY	27851-28078	oriT nicking
traA	28112-28474	F pilin subunit
traL	28479-28790	F pilin assembly
traE	28812-29378	F pilin assembly
traK	29365-30093	F pilin assembly
traB	30093-31520	F pilin assembly
traP	31510-32094	Conjugal transfer protein
trbD	32081-32401	Conjugal transfer protein
trbG	32394-32645	Conjugal transfer protein
traV	32642-33157	F pilus assembly
traR	33292-33513	Conjugal transfer protein
traC	33673-36303	F pilus assembly
tnpA	36350-37054	Transposase of IS26
tnpA	Compl. 37298-38560	Transposase of ISEcp1
tnpA	Compl. 38742-39119	Transposase of Tn3, truncated by the insertion of ISEcp1
tnpR	39088-39681	Resolvase of transposon Tn3
blaTEM-1	39948-40676	β-Lactamase TEM precursor
insA1	40727-41002	Insertion element IS1 protein InsA
insB	41047-41424	Insertion element IS1 protein InsB
yigB	41469-41930	Putative endonuclease precursor
hha	41976-42185	Hemolysin expression-modulating protein
yihA	42223-42813	Hypothetical protein
repA2	43053-43307	Negative regulator of repA1 expression, FII replicon
repA3	43414-43599	Regulator of repA1 expression, FII replicon
repA1	43600-44469	Replication initiation protein RepA1 of FII replicon
repA4	44832-45218	Regulator of repA1 expression, FII replicon
tir	45408-46061	Inner-membrane protein
pemI	46154-46411	Stable plasmid inheritance, antitoxin
pemK	46380-46745	Stable plasmid inheritance, toxin
tnpA	Compl. 46882-49779	Transposase of Tn501
tnpR	49874-50479	Resolvase of Tn501
tnpA	Compl. 50476-51321	Transposase of Tn1721 truncated
pecM	51786-52670	Hypothetical protein
tet(A)	Compl. 52702-53976	Tetracycline resistance protein, class A
tet(R)	53980-54657	Tetracycline repressor protein
tnpA	Compl. 54973-56562	Transposase of Tn1721
tnpA	56553-57257	Transposase of IS26
aac6′-Ib-cr	57263-57955	Aminoglycoside N(6′)-acetyltransferase
blaOXA-1	58041-58916	β-Lactamase OXA-1 precursor
catB4	59054-59602	Chloramphenicol acetyltransferase
tnpA	Compl. 59548-60252	Transposase of IS26
tnpA	Compl. 60299-62314	Transposase of IS26
BASYS0015	62574-62906	Hypothetical protein
blaCTX-M-15	Compl. 62953-63888	Extended-spectrum β-lactamase CTX-M-15
tnpA	63964-64669	Transposase of IS26
tnpM	Compl. 65014-65628	Transposon Tn21 modulator protein
BASYS0016	65116-65580	Hypothetical protein
int1I	Compl. 65567-66580	Class 1 integrase
dhfrVII	66537-67211	Dihydrofolate reductase type VII
aadA5	67342-68130	Aminoglycoside-3′-adenyltransferase
sulI	68590-69516	Dihydropteroate synthase type I
chrA	70003-71208	Putative chromate ion transporter
BASYS000716	71219-71524	Hypothetical protein
tnpA	71676-72497	Putative transposase for insertion sequence
tnpA	71715-72515	Transposase of IS6100
mph(R)	Compl. 73008-73592	Erythromycin resistance repressor protein
mrx	Compl. 73592-74830	Erythromycin resistance regulator protein
mph(A)	Compl. 75732-74827	Macrolide 2-phosphotransferase
tnpA	Compl. 75854-76558	Transposase of IS26
nqrC	76838-77599	Na(+)-translocating NADH-quinone reductase subunit C
ywbL	77886-79832	High-affinity Fe2+/Pb2+ permease
tpd	79870-80400	Hypothetical protein
BASYS00056	80504-81883	Hypothetical protein
ybjZ	81886-83169	Hypothetical protein
lolE	83129-84289	ABC transporter permease protein
ybbA	84294-84989	ABC transporter ATP-binding protein
BASYS00017	Compl. 84600-85265	Hypothetical protein
resA	84958-85461	Thiol-disulfide oxidoreductase
BASYS00018	85486-85971	Hypothetical protein
agp	86093-86647	Glucose-1-phosphatase precursor
tnpA	Compl. 86681-87385	Transposase of IS26
BASYS000046	87386-87718	Putative transposase OrfA of IS629, truncated
BASYS000047	88157-88606	Putative transposase OrfA of IS629, truncated
tnpA	88688-88861	Hypothetical protein TnpA
tnpA	88881-89246	Transposase of IS4
ugpB	Compl. 89305-90669	Putative ABC transporter permease protein
ugpC	Compl. 90608-91732	Putative ABC transporter ATP-binding protein
icc	Compl. 91687-92511	Phosphodiesterase
araQ	Compl. 92522-93409	Putative ABC transporter permease protein
ugpA	Compl. 93399-94277	Putative ABC transporter permease protein
yigB	Compl. 94408-94638	Oxidoreductase
yigB	Compl. 94666-94848	Oxidoreductase
yihH	95249-96139	Dihydrodipicolinate synthase
tdcF	96164-96544	Translation initiation inhibitor
kdgT	96577-97542	Putative 2-keto-3-deoxygluconate permease
yfaX	Compl. 97588-98346	Hypothetical protein
BASYS00018	98945-99229	Hypothetical protein
BASYS00019	99229-99504	Hypothetical protein
BASYS00020	99610-99903	Hypothetical protein
BASYS00021	Compl. 100099-101268	Hypothetical protein
insA1	102357-102632	Insertion sequence IS1 protein InsA
insA1	102677-103054	Insertion sequence IS1 protein InsB
BASYS00016	Compl. 103308-104330	Hypothetical protein
vagD	Compl. 105955-106371	Virulence-associated protein VagD
vagC	Compl. 106368-106598	Virulence-associated protein VagC
pcar	106949-110773	Hypothetical protein
vagD	Compl. 110818-111234	Virulence-associated protein VagD
vagC	Compl. 111231-111461	Virulence-associated protein VagC
BASYS0020	111726-112226	Hypothetical protein
BASYS0021	112239-113012	Hypothetical protein
tnpA	Compl. 113223-114836	Transposase of IS66, ORF3
tnpA	Compl. 114867-115217	Transposase of IS66, ORF2
tnpA	Compl. 115214-115639	Transposase of IS66, ORF1
BASYS0022	115731-116852	Hypothetical protein

^aCompl., gene is the reverse complement of the positions shown.

The structure of pEK499 was compared with the following two fully sequenced plasmids carrying the IncFII-FIA replicons: pRSB107 from a bacterium collected in a sewage treatment plant (32) and pIP1206 from an E. coli clinical isolate in a Belgian hospital (28). Neither of these encodes an ESBL (Fig. 1). pRSB107 encodes an aerobactin iron acquisition system and resistance to penicillins (bla_TEM-1), aminoglycosides [aph(3) and strA-strB], sulfonamides (sulII), macrolides [mph(A)], chloramphenicol (catA), and tetracyclines [tet(A)]; it also carried the class 1 integron-borne trimethoprim resistance gene cassette dhfR (32). In common with pRSB107, pIP1206 also carried catA, tet(A), and bla_TEM-1, but it also harbored (i) two class 1 integrons with the aadA4-dhfr17 and qepA gene cassettes, respectively (the latter gene confers resistance to hydrophilic fluoroquinolones), and (ii) rmtB, which encodes an 16S rRNA m⁷G methyltransferase that confers resistance to all aminoglycosides (28).

FIG. 1.

Major structural features of pEK499 (strain A) and pEK516 (strain D), encoding CTX-M-15 ESBLs, in comparison with IncFII-FIA plasmids pRSB107 (32) and pIP1206 (28) and the IncFII CTX-M-15-encoding plasmid pC15-1a (2). Resistance genes are indicated by colored boxes as follows: green, tetracycline resistance genes [tet(A), tet(R), tet(C), and tet(D)); brown, chloramphenicol resistance genes (catB4 and catA); azure, β-lactamase genes (bla_OXA-1, bla_TEM-1, and bla_CTX-M-15); dark blue, aminoglycoside resistance genes [aacA4, aph(3), aadA1, strA-strB, and rmtB]; yellow, erythromycin, mercuric ion, and quinolone resistance genes [qepA, mer, mph(A), and mph(R)]; pale orange, trimethoprim resistance genes (dfhR, dhfRVII, and dhfr17); and gray, sulfonamide resistance genes (sul1). Transposon-related genes are indicated by black-squared boxes as follows: insertion sequences (IS) are colored in red, and other transposon-associated genes are colored in pale blue. Partitioning-associated genes (parM, ssb, parA, and parB) are indicated by pink-squared white boxes. Iron acquisition, type I DNA restriction, raffinose, arginine deaminase and ABC transporter clusters, and the homocysteine S-methyltransferase (mmu) and the NADH-ubiquinone oxidoreductase genes (nqrC) are indicated by blue-squared white boxes. The locus Tra is indicated by a squared white box with capital letters indicating the respective tra genes (i.e., V, traV; J, traJ; Y, traY; etc.). The antitoxin-toxin genes are indicated by violet (pemI-pemK), pink (ccdA-ccdB), blue (hok-mok), and green (vagC-vagD) double vertical lines, respectively. Repeats 1a, 1b, and 1c are indicated by black vertical lines. Replicons FIA, FIB, and FII are indicated by horizontal black lines. The origin or replication (oriV) is indicated by a circle. The red lines indicate the positions of the Tn3::bla_TEM-1 transposons. Dashed lines represent the plasmid scaffold regions that are in common among plasmids; the black dashed lines indicate the inversion of the common region that occurred in plasmid pEK516 with respect to pC15-1a, the red dashed lines indicate the same inverted region with respect to pEK499; and the green dashed lines indicate the common region among the pEK499, pRSB107, and pIP1206 plasmids. The region of the FIB replicon of pRSB107 that is missing in the pEK499 plasmid is shadowed.

The pEK499 scaffold encoded no fewer than the following five systems to ensure stable plasmid inheritance and postsegregation killing: (i) the postsegregation killing protein Hok and its modulator Mok, located near the parB gene; (ii) the toxin-antitoxin system pemI-pemK, flanking the region of the replicon FII; (iii and iv) two copies of the vagC-vagD virulence-associated genes; and (v) one copy of the toxin-antitoxin system ccdA-ccdB, located in the region of the FIA replicon. This represents the largest number of addiction systems yet described on any IncF plasmid. For comparison, pRSB107 and pIP1206 carry four (two vagC-vagG, ccdA-ccdB, and pemI-pemK) and three (vagC-vagG, ccdA-ccdB, and pemI-pemK) systems, respectively (28, 32). These features seem likely to ensure that pEK499 is maintained in the absence of any antibiotic selective pressure. Plasmids pEK499 and pRSB107 also shared a region with two copies of the module that encodes permeases and ATP binding proteins of the ABC transporter family, and which is also partially present on pIP1206. The contribution of these transporters to virulence and plasmid maintenance has not yet been established.

pEK499 contained an incomplete transfer region composed of the genes traM to traC, but not traW to traX, and thus lacked functional conjugation machinery. Consistent with this observation, pEK499 was not transferable by conjugation in vitro; it was transformed prior to plasmid sequencing. pEK499 also lacked the aerobactin iron acquisition system, and the type I DNA restriction, raffinose and arginine deaminase clusters that have been described in the IncF plasmids pRSK107 and pIP1206, respectively (28, 32).

Analysis of pEK516 (strain D).

Plasmid pEK516 was a circular molecule of 64,471 bp, harboring 103 predicted genes (Table 3) and belonging to incompatibility group IncFII. It carried the following seven genes encoding antibiotic resistance clustered in a 22-kb region: bla_CTX-M-15, bla_OXA-1, bla_TEM-1, aac6′-Ib-cr, aac3-IIa, catB4, and tet(A). An ISEcp1 element was located 48 bp upstream of bla_CTX-M-15. pEK516 shared 75% of its DNA sequence with pEK499, albeit with considerable rearrangements (Fig. 1); notably, both plasmids carried the region containing the FII replicon and the hok-mok and parB genes. However, pEK516 was 53 kb (45%) smaller than pEK499 but carried the type I partitioning locus (parM and stbB), ensuring stable plasmid inheritance. Moreover, its Tn3::bla_TEM-1 module flanked the FII replicon, whereas this module was located close to the deleted transfer region on pEK499 (Fig. 1, red line). The resistance region of pEK516 encoded an AAC(3) enzyme, absent from pEK499 and conferring resistance to gentamicin, but lacked the class 1 integron with genes conferring resistance to trimethoprim, streptomycin, and sulfonamides, also the macrolide resistance cluster. Consequently, it was similar to pC15-1a (2), from a widespread Canadian strain of E. coli with the CTX-M-15 ESBL and probably also belonging to the ST131 clone. In fact, three genetic events potentially accounted for all of the differences observed between pC15-1a and pEK516, namely, (i) the partial deletion of the tra region, (ii) the inversion of the region between the FII replicon and the parM gene, and (iii) the acquisition of catB4 close to bla_OXA-1 within the resistance region of pEK516.

TABLE 3.

Open reading frames identified in pEK516

Gene name	Nucleotide positiona	Function encoded
tnpA	Compl. 34-2931	Transposase of Tn501
tnpR	3023-3630	Resolvase of Tn21
tnpA	3627-4473	Transposase of Tn1721
pecM	4938-5823	Hypothetical protein
tet(A)	Compl. 5853-7128	Tetracycline resistance protein, class A
tet(R)	7132-7809	Tetracycline resistance regulator protein
tnpA	Compl. 8124-9714	Transposase of Tn1721
tnpA	9705-9980	NH-2 terminal of the transposase of IS26, pseudogene
tnpA	9980-10663	COOH− terminal of the transposase of IS26, pseudogene
aac6′-Ib-cr	10669-11361	Aminoglycoside N(6′)- acetyltransferase
blaOXA-1	11447-12322	β-Lactamase OXA-1 precursor
catB4	12460-13008	Chloramphenicol acetyltransferase
tnpA	Compl. 12954-13658	Transposase of IS26
aacC3	13765-14625	Aminoglycoside N(3′)-acetyltransferase III
BASYS0001	14638-15180	Hypothetical protein
BASYS000006	15251-15583	Hypothetical protein
insFI	15580-16428	Putative transposase of IS986/IS6110
tnpA	Compl. 16374-17078	Transposase of IS26
tnpA	Compl. 17125-19140	Transposase of Tn3, truncated
amb	19400-19732	Hypothetical protein
blaCTX-M-15	Compl. 19779-20654	Extended-spectrum β-lactamase CTX-M-15
tnpA	Compl. 20910-22172	Transposase ISEcp1
tnpA	Compl. 22354-22731	Transposase of Tn3
tnpR	22700-23293	Resolvase of Tn3
blaTEM-1	23476-24336	β-Lactamase TEM precursor
tnpA	Compl. 2447-26954	Transposase of Tn21
tnpA	Compl. 27097-27801	Transposase of IS26
repA1	Compl. 28926-29795	Replication initiation protein RepA1 of the FII replicon
repA4	Compl. 30088-30342	Regulator of repA1 expression, FII replicon
yihA	Compl. 30582-31172	Hypothetical protein
hha	Compl. 31210-31419	Hemolysin expression-modulating protein
yigB	Compl. 31465-31938	Putative endonuclease precursor
insB2	Compl. 31971-32474	Insertion element IS1 protein InsB
yfhA	Compl. 32697-33119	Hypothetical protein
traR	Compl. 33112-33333	Conjugal transfer protein
traV	Compl. 33468-33983	F pilus assembly
trbD	Compl. 33980-34300	Conjugal transfer protein
traP	Compl. 34287-34874	Conjugal transfer protein
traB	Compl. 34843-36294	F pilin assembly
traK	Compl. 36294-37022	F pilin assembly
traE	Compl. 37009-37575	F pilin assembly
traL	Compl. 37597-37908	F pilin assembly
traA	Compl. 37923-38282	F pilin subunit
traY	Compl. 38315-38542	oriT nicking
traJ	Compl. 38679-39350	Regulation
traM	Compl. 39544-40029	Mating signal
gene X (orf39)	40250-40852	X− polypeptide; transglycosylation
yubP	Compl. 41149-41970	Hypothetical protein
yeiA	Compl. 42081-42377	Hypothetical protein
yehA	42677-43054	Hypothetical protein
tnpA	Compl. 43058-44461	Transposase of ISPsy5
ECs1338	Compl. 44702-45052	Hypothetical protein
ECs1337	Compl. 45049-45474	Hypothetical protein
hok	Compl. 45832-45990	Postsegregational killing protein Hok
mok	Compl. 45836-46048	Modulator of Hok protein, Mok
psiA	Compl. 46270-46989	Plasmid SOS inhibition protein A
psiB	Compl. 46986-47423	Plasmid SOS inhibition protein B
yefA	Compl. 47489-48064	ParB-like nuclease
parB	Compl. 48091-49512	ParB-like partitioning protein
BASYS00066	49537-49884	Hypothetical protein
ydeA	Compl. 49573-49812	Hypothetical protein
ssb	Compl. 49864-50430	Single-strand binding protein
yddA	50693-51154	Hypothetical protein
ydcA	Compl. 51240-51803	Hypothetical protein
ydbA	Compl. 51850-53211	Hypothetical protein
SC	Compl. 54531-54722	Hypothetical protein
ycjA	Compl. 54719-55141	Hypothetical protein
yicB	Compl. 55188-55613	Antirestriction protein KlcA
ychA	Compl. 56031-56858	Hypothetical protein
ycgB	Compl. 56858-57292	Hypothetical protein
P030	Compl. 57306-57527	Hypothetical protein
ycgA	Compl. 57528-58175	Hypothetical protein
parM	58728-59690	Plasmid segregation protein
stbB	59690-60043	Stable plasmid inheritance protein
ycdB	Compl. 60166-60447	Hypothetical protein
ycdA	60476-60712	Hypothetical protein
tnpA	62389-63093	Transposase of IS26
tir	63133-63684	Inner-membrane protein
pemI	63777-64034	Stable plasmid inheritance, antitoxin
pemK	63967-64368	Stable plasmid inheritance, toxin

^aCompl., gene is the reverse complement of the positions shown.

Both pEK516 and pC15-1a carried only two addiction systems, pemI-pemK and hok-mok, and resembled the IncFII portion of pEK499. pEK516 showed a greater deletion of the transfer region than pEK499, since the latter contained traC, which was missing in pEK516. Despite this, pEK516 was transferred by conjugation in vitro (16); so it seems likely that the tra deletion(s) occurred during subsequent storage and prior to plasmid sequencing.

In summary, pEK516 represented a highly related variant of the previously identified plasmid, pC15-1a (2). Plasmids of the FII group harboring bla_CTX-M-15 have wide geographic scatter (4, 6) and have played a crucial role in the global spread of CTX-M-15 ESBLs in E. coli.

Analysis of pEK204 (strain C).

Plasmid pEK204 was a circular molecule of 93,732 bp, harboring 112 predicted genes (Table 4), and could be transferred by conjugation in vitro. It belonged to incompatibility group IncI1, which is characterized by the presence of a gene cluster encoding a thin, type IV pilus required for liquid matings (18) and the RepZ replicase gene. As such, it was distinct from two other fully sequenced plasmids encoding the CTX-M-3 enzyme, pCTX-M3, which is widespread in Poland (10, 26), and pK29 (5). pEK204 was assigned to a new IncI1 pMLST type (9), ST16 (I1, A5, S8, P6, and T10), with four unique alleles (pil6, sogS8, ardA5, and trbA-pndC10). However, its gross structure revealed strong similarity to IncI1 plasmid pCOLIb-P9 (GenBank AB021078), although the region that includes the colicin 1b gene (pCOLIb-P9 nucleotides 8310 to 20275) was absent, and to R64, which is the reference plasmid for the IncI1 group (GenBank accession no. AP005147; Fig. 2). In comparison with those of R64, pEK204 lacked the arsenic, tetracycline, and streptomycin resistance genes and the addiction systems mck-kor and parA-parB; this deleted region was substituted by its own resistance region. The colinearity between the pEK204 and R64 plasmid scaffolds was therefore well maintained in the transfer region, with the exception of the shufflons—a characteristic feature of IncI1 plasmids that may act as a biological switch (12, 19, 20)—which appeared to be rearranged in pEK204 with respect to R64 and also by the insertion of an IS66 element.

TABLE 4.

Open reading frames identified in pEK204

Gene name	Nucleotide positiona	Function encoded
repY	378-467	Regulatory protein of RepZ
repZ	455-1486	Replication protein of the I1 replicon
yafA	Compl. 2556-3065	Hypothetical protein
yafB	Compl. 3121-3681	Hypothetical protein
yagA	3966-5423	Hypothetical protein
tnpA	Compl. 5711-5806	Transposase of Tn3, truncated
tnpA	5997-7259	Transposase of ISEcp1
blaCTX-M-3	7594-8469	β-Lactamase CTX-M-3 precursor
tnpA	Compl. 8772-11930	Transposase of Tn3, truncated
tnpR	11899-12492	Resolvase of Tn3
blaTEM-1	12675-13535	β-Lactamase TEM-1 precursor
impB	Compl. 15037-16308	UV protection and mutation protein
impA	Compl. 16308-16745	UV protection and mutation protein
impC	Compl. 16742-16990	UV protection and mutation protein
yccA	17098-17370	Hypothetical protein
yccB	17381-18310	Hypothetical protein
ycdA	18307-18628	Hypothetical protein
parM	19078-20058	Plasmid segregation protein ParM
stbB	20055-20483	Stable plasmid inheritance protein
ycdB	20874-21557	Putative methylase
yceA	21558-21779	Hypothetical protein
yceB	21793-22227	Hypothetical protein
ycfA	22272-23042	Hypothetical protein
ycgB	23455-23880	Hypothetical protein
ycgC	23927-24349	Hypothetical protein
ychA	24401-24703	Hypothetical protein
ssb	25322-25849	Single-stranded DNA-binding protein
ykfF	25880-26140	Hypothetical protein
ycjA	26193-28157	Hypothetical protein
psiB	28117-28644	Plasmid SOS inhibition protein B
psiA	28641-29360	Plasmid SOS inhibition protein A
ygaA	29357-29953	Hypothetical protein
ygbA	30025-30915	Hypothetical protein
ardA	30415-30915	Antirestriction protein
ydfA	31644-32078	Hypothetical protein
ydfB	32172-32438	Hypothetical protein
BASYS00078	32615-33004	Hypothetical protein
ygbB	33001-33903	Hypothetical protein
ygeA	Compl. 34202-34453	Hypothetical protein
BASYS00079	Compl. 34487-34798	Hypothetical protein
ydiA	35032-35880	Hypothetical protein
yggA	Compl. 35966-36301	Hypothetical protein
nikA	36532-36867	Relaxosome component protein
nikB	36878-39577	Relaxase
trbC	Compl. 39614-41905	Hypothetical protein
trbB	Compl. 39614-41905	Hypothetical protein
trbA	Compl. 42987-44195	Hypothetical protein
finQ	Compl. 44502-45431	Hypothetical protein
pndC	45769-46053	Postsegregation killing system, counter protein for PndA
pndA	45908-46060	Postsegregation killing protein
exc	Compl. 47893-48555	Surface exclusion protein
traY	Compl. 48626-50863	Integral membrane protein
traX	Compl. 50891-51475	F pilin acetylation
traW	Compl. 51504-52706	F pilus assembly
traV	Compl. 52673-53287	F pilus assembly
traU	Compl. 53287-56331	F pilus assembly
traT	Compl. 56421-57221	Surface exclusion
traS	Compl. 57205-57393	Surface exclusion
traR	Compl. 57457-57861	Hypothetical protein
traQ	Compl. 58439-59143	Conjugal transfer protein
traP	Compl. 58439-59143	Conjugal transfer protein
traO	Compl. 59143-60432	Hypothetical protein
traN	Compl. 60435-61418	Aggregate stability
traM	Compl. 661429-62121	Mating signal
traL	Compl. 62118-62465	F pilus assembly
sogL	Compl. 62483-66286	DNA primase
sogS	Compl. 62483-65020	Regulative protein
nuc	Compl. 66340-66894	EDTA-resistant nuclease
traK	Compl. 66906-67196	F pilus assembly
traJ	Compl. 67193-68341	ATP-binding protein
traI	Compl. 68338-69156	DNA helicase
traH	Compl. 69153-69611	F pilus assembly
traG	Compl. 70005-70589	F pilus assembly
traF	Compl. 70649-71851	F pilus assembly
traE	Compl. 71937-72761	F pilus assembly
rci	Compl. 72912-74066	Shufflon-specific DNA recombinase
tnpA	75447-77060	Transposase of IS66
pilV	Compl. 77739-79163	Type IV prepilin cluster
pilU	Compl. 79162-79819	Type IV prepilin cluster, prepilin peptidase
pilT	Compl. 79804-80364	Type IV prepilin cluster
pilS	Compl. 80374-80988	Type IV prepilin cluster, prepilin
pilP	Compl. 80988-84132	Type IV prepilin cluster
pilR	Compl. 81005-82103	Type IV prepilin cluster, integral membrane protein
pilQ	Compl. 82115-83669	TypeIV prepilin cluster, ATP-binding protein
pilP	Compl. 83680-84132	Type IV prepilin cluster
pilO	Compl. 84179-85414	Type IV prepilin cluster
pilN	Compl. 85407-87089	Type IV prepilin cluster, secretin protein
pilM	Compl. 87103-87540	Type IV prepilin cluster
pilL	Compl. 87540-88607	Type IV prepilin cluster, lipoprotein
pilK	Compl. 88641-89234	Type IV prepilin cluster
pilJ	Compl. 89231-89527	Type IV prepilin cluster
pilI	Compl. 89694-89948	Type IV prepilin cluster
BASYS00080	Compl. 90108-90661	Hypothetical protein
traC	Compl. 90834-91517	F pilus assembly
traB	Compl. 91771-92304	F pilus assembly
traA	Compl. 92309-92432	F pilin subunit

^aCompl., gene is the reverse complement of the positions shown.

FIG. 2.

Major structural features of pEK204 (strain C), encoding CTX-M-3 ESBL, in comparison with IncI1 plasmid R64. Resistance genes are indicated by colored boxes as follows: green, tetracycline resistance genes (tet(A), tet(R), tet(C), and tet(D)); azure, β-lactamase genes (bla_TEM-1 and bla_CTX-M-15); dark blue, aminoglycoside resistance genes (strA-strB); and yellow, arsenic resistance genes (arsA and arsB). Transposon-related genes are indicated by black-squared boxes as follows: insertion sequences (IS) are colored in red, and other transposon-associated genes are colored in pale blue. Partitioning-associated genes (parA, parB, and parM) are indicated by pink-squared white boxes. Blue-squared white boxes indicate characteristic IncI1 clusters (impABC, psiAB, pndAC, and pilV-IV). The Tra locus is indicated by a squared white box with capital letters indicating the respective tra genes (i.e., Y, traY; X, traX; W, traW; etc.). The antitoxin-toxin genes (mck-kor) are indicated by green double vertical lines. The shufflons are indicated by azure and dark blue vertical lines. The I1 replicon is indicated by a horizontal black line. The origin of transfer (oriT) is indicated by a circle. Dashed lines represent the plasmid scaffold regions that are common to both plasmids. Pale orange-colored boxes indicate the IncI1-pMLST target sites (repI1, ardA, trbA-pndC, SogS, and pilIV).

In contrast to multiresistance plasmids pEK499 and pEK516, pEK204 carried only two known resistance genes, bla_CTX-M-3 and bla_TEM-1 (Fig. 3). The bla_CTX-M-3 gene, which encodes an ESBL that differs from CTX-M-15 by only a Asp240→Gly substitution, has previously been detected on plasmids belonging to different incompatibility groups and with broad host ranges (4), including IncL/M (pCTX-M-3; ca. 90 kb) in Poland (10, 26) and IncHI2 (pK29; ca. 270 kb) in Taiwan (5), as well as the IncA/C, IncFII, and IncN types (4). Large (>90-kb) IncI1 plasmids encoding the CTX-M-3 enzyme have been reported previously in diverse members of the Enterobacteriaceae in a university hospital in Taiwan (24). Moreover, IncI1 plasmids have been reported to encode myriad other β-lactamases besides the CTX-M-3 enzyme, including CTX-M-1, CTX-M-2, CTX-M-9, CTX-M-14, CTX-M-15, and CTX-M-24, as well as TEM-1 (as here), TEM-52, SHV-12, several CMY (acquired AmpC) enzymes, and the metallocarbapenemase VIM-1 (4, 5, 9).

FIG. 3.

Detail of the resistance region of pEK204, which arose through the acquisition of Tn3 and ISEcp1-bla_CTX-M-3 by a pCOL1b-P9-like plasmid. The 5-bp direct repeats consistent with transposition events are shown. The disrupted tnpA gene of the Tn3 element is shaded black.

The sequence data suggest that pEK204 arose by the transposition of Tn3 and ISEcp1-bla_CTX-M-3 elements into a pCOLIb-P9-like plasmid. This Tn3 element inserted after the position equivalent to nucleotide 8269 of pCOLIb-P9 and was flanked by 5-bp direct repeats of TTTTC. Both of the Tn3 terminal inverted repeats, IR_L and IR_R, were intact, but the tnpA gene (encoding the transposase) was disrupted by ISEcp1-bla_CTX-M-3 (Fig. 3). The ISEcp1 element was located 128 bp upstream of bla_CTX-M-3, and the linking sequence was identical to that of pCTX-M-3 (10), though different from that (48 bp) between ISEcp1 and bla_CTX-M-15 on pEK516 or the remnant of this IS element on pEK499. This underscores the point that, although they differ only by one nucleotide, the bla_CTX-M-3 and bla_CTX-M-15 genes in the United Kingdom plasmids represent separate escape events from Kluyvera spp.

The ISEcp1-bla_CTX-M-3 element of pEK204 was flanked by 5-bp direct repeats of TATTG, consistent with ISEcp1-mediated transposition, which prefers AT-rich target sequences (29). Despite disruption, the Tn3 transposase (tnpA) gene on pEK204 was still predicted to encode a protein of 999 amino acids, comprising 970 (ca. 97%) “genuine” amino acids plus 29 “new” C-terminal amino acids resulting from the read-through of the in-frame ISEcp1-bla_CTX-M-3 insertion. The genuine C-terminal residues of the Tn3 transposase were encoded by a ΔtnpA remnant located between IR_L and ISEcp1 (Fig. 3). The disruption of transposons by other transposable elements is a feature common to many bacterial genomes and plasmids, including all of those reported here. The resistance region of pEK204 represents a simple example of this but raises the question of whether this plasmid evolved via one or two transposition events. If the predicted transposase of Tn3 on pEK204 remains functional, it would be expected to mediate the transposition of a new element, still defined by the Tn3 terminal inverted repeats IR_L and IR_R but encoding CTX-M-3 ESBL in addition to TEM-1 penicillinase (Fig. 3). The potential for the simultaneous transposition of two distinct β-lactamase genes from pEK204-like plasmids has public health importance and will be investigated further. It is relevant in context that Tn3 played a major role in the huge dissemination of TEM-1 β-lactamase in the 1960s and 1970s.

Concluding remarks.

We have determined the complete sequences of the CTX-M ESBL-encoding plasmids found in three United Kingdom variants (A, C, and D) of the pathogenic, multiresistant ST131 E. coli lineage. Strain A (pEK499), which is widespread and locally dominant in the United Kingdom, has been found in Austria (8) and, more recently, in Bolzano, northern Italy (R. Aschbacher, D. M. Livermore, and N. Woodford, unpublished data). Strain C is also widely found in the United Kingdom: some Belfast isolates of the strain have the CTX-M-3 enzyme encoded by pEK204, as here, but others, including many from the United Kingdom mainland, have CTX-M-15, possibly encoded by a different plasmid. In contrast to these widely disseminated strains, the third ST131 variant studied, strain D (pEK516), is prevalent only in Shropshire, a county on the English-Welsh border.

Clearly, differences exist among variants of the ST131 clone and these probably have significant impact on their relative success and prevalence. Success is likely to reflect a complex combination of characteristics, both intrinsic to the PFGE-defined variant or acquired on plasmids and mobile genetic elements, interacting with local pressures and opportunities relating to antibiotic usage and patient types. It is clear, however, that these strains and their multiresistance plasmids have been moving beyond the hospital environment in the United Kingdom for some years (34). A recent study identified ESBL-producing E. coli in the bowel flora of 40% of residents of long-term-care facilities in Belfast; almost half were colonized by strain A (30) and most of the remainder by diverse ST131 variants harboring pEK204-like plasmids (7). The multitude of addiction systems present on pEK499 will, in particular, ensure that it is maintained even in the absence of antibiotic selection.

Many patients with E. coli producing CTX-M ESBLs, most of them elderly, present with community-onset infections in increasing numbers, providing evidence of true community acquisition (25). This age distribution may change over time, leading to new problems. A study from Hong Kong found significant rates of ESBL production (particularly of the CTX-M-14 enzyme) among urinary isolates of E. coli from women of all ages; the prevalence was 7.3% among community-onset infections in the 18-to-35 age group (13). Another study, from Canada, of community-onset infections caused by ESBL-producing E. coli recorded a much broader age distribution among patients deemed to have community-acquired infections versus those considered to have health care-associated infections (22). Rising rates of E. coli with CTX-M ESBLs in the genitourinary tracts of sexually active women raise the alarming possibility that these enzymes might “escape” into sexually transmitted bacterial pathogens, specifically Neisseria gonorrhoeae. Oral and intramuscular oxyimino-cephalosporins, such as cefixime and ceftriaxone, are widely used as a first-line treatment for uncomplicated gonorrhea, and any evolution of ESBL-producing gonococci would be a catastrophic development.

In summary, United Kingdom E. coli strains A, C, and D belonging to the internationally disseminated O25:H4-ST131 clone (21, 27, 34) have acquired different plasmids encoding CTX-M ESBLs on separate occasions.