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 beta-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.

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.

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).

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.