Complete nucleotide sequence of the pCTX-M3 plasmid and its involvement in spread of the extended-spectrum beta-lactamase gene blaCTX-M-3

Abstract

Here we report the nucleotide sequence of pCTX-M3, a highly conjugative plasmid that is responsible for the extensive spread of the gene coding for the CTX-M-3 extended-spectrum beta-lactamase in clinical populations of the family Enterobacteriaceae in Poland. The plasmid belongs to the IncL/M incompatibility group, is 89,468 bp in size, and carries 103 putative genes. Besides bla(CTX-M-3), it also bears the bla(TEM-1), aacC2, and armA genes, as well as integronic aadA2, dfrA12, and sul1, which altogether confer resistance to the majority of beta-lactams and aminoglycosides and to trimethoprim-sulfamethoxazole. The conjugal transfer genes are organized in two blocks, tra and trb, separated by a spacer sequence where almost all antibiotic resistance genes and multiple mobile genetic elements are located. Only bla(CTX-M-3), accompanied by an ISEcp1 element, is placed separately, in a DNA fragment previously identified as a fragment of the Kluyvera ascorbata chromosome. On the basis of sequence analysis, we speculate that pCTX-M3 might have arisen from plasmid pEL60 from plant pathogen Erwinia amylovora by acquiring mobile elements with resistance genes. This suggests that plasmids of environmental bacterial strains could be the source of those plasmids now observed in bacteria pathogenic for humans.

FIG. 1.

Overview of the pCTX-M3 sequence. ORFs are shown by arrows: pCTX-M3-specific ORFs are in white, ORFs homologous to genes of known function are in black, and ORFs homologous to genes of unknown function are in gray. Functional sequence blocks are highlighted with shades of gray bands and are indicated on the outside: replicon, stabilizing cassettes (PSK and partition), and conjugal transfer system. The middle circle with a thick line indicates the pCTX-M3 sequence identical to that of plasmid pEL60. The inner circle is a schematic representation of blocks of low and high G+C contents. Arrows inside the outer circle indicate the positions in which pEL60 has additional sequences that are absent from pCTX-M3.

FIG. 2.

Comparison of regions coding for the conjugal transfer systems of pCTX-M3 and IncI1 plasmid ColIb-P9. ORFs are presented as arrows: those present in both plasmids are not filled, and ORFs present only in pCTX-M3 or ColIb-P9 are black and gray, respectively, and are shown under or above the scheme of the common structure region.

FIG. 3.

Organization of the 27-kb region of the replicon and the trb genes. The partial pCTX-M3 sequence is drawn schematically out of scale. Arrows, ORFs; black lines, the sites of integration of particular sequence segments; black frames, hypothetical fragments of Tn1 and the integron that are not observed in pCTX-M3, probably due to integration events; vertical bars, IRs of the respective mobile elements and IRi of the integron; gray blocks, the regions of the plasmid backbone.