ColE1-like plasmid pIP843 of Klebsiella pneumoniae encoding extended-spectrum beta-lactamase CTX-M-17

Abstract

The resistance of Klebsiella pneumoniae BM4493, isolated in Ho Chi Minh City, Vietnam, to cefotaxime and aztreonam was due to production of a novel beta-lactamase, CTX-M-17. The bla(CTX-M-17) gene was borne by 7,086-bp plasmid pIP843, which was entirely sequenced and which was found to belong to the ColE1 family. The 876-bp bla(CTX-M-17) gene differed from bla(CTX-M-14) by 2 nucleotides, which led to the single amino acid substitution Glu289-->Lys. bla(CTX-M-17) was flanked upstream by an ISEcp1-like element and downstream by an insertion sequence (IS) IS903 variant designated IS903-C. The transcriptional start site of bla(CTX-M-17) was located 109 nucleotides upstream from the initiation codon in the ISEcp1-like element, which also provided the promoter sequences. Plasmid pIP843, which was non-self-transferable and nonmobilizable, contained five open reading frames transcribed in the same orientation. Regions homologous to sequences coding for putative RNA II and RNA I transcripts, a rom gene, which is involved in initiation of replication, and a cer-like gene, which is responsible for the stability of ColE1-like plasmids, were identified. Consensus sequences for putative replication (oriV) and transfer (oriT) origins were present. Results of primer extension experiments indicated that ISEcp1 provides the promoter for expression of bla(CTX-M-17) and may contribute to dissemination of this gene.

FIG. 1.

Genetic organization of plasmid pIP843. Heavy bar, regulatory region for replication; thin line, noncoding region; bar consisting of multiple rectangles, AT-rich region. The putative functional regions are as follows: RNA II transcript (nt 6024 to 6676); RNA I transcript (complementary to nt 6031 to 6128); oriV replication origin (nt 6527 to 6529); rom-like genes (ORF1; nt 6717 to 6908); oriT transfer origin (nt 6934 to 300); cer-like region (nt 346 to 611); ISEcp1-like element (nt 1138 to 2973); ISEcp1-like ORF2 transposase (nt 1324 to 2586); CTX-M-17 ORF3 (nt 2836 to 3711); IS903 (nt 3714 to 4770); IS903-C ORF4 transposase (nt 1324 to 2586); and outer membrane lipoprotein ORF5 (nt 4918 to 5706).

FIG. 2.

Identification of the transcriptional start site for bla_CTX-M-17 in strain BM4493 by primer extension analysis. (A) Lane 1, primer elongation product obtained with oligodeoxynucleotide PE and 50 μg of total RNA of BM4493 (arrow); lanes T, G, C, and A, results of sequencing reactions performed with pIP843 DNA as a template and the PE primer. (B) Sequence from nt 2676 to 2950 (the numbering is that for the sequence of the strain with GenBank accession no. AY033516); +1, transcriptional start site for bla_CTX-M-17 in BM4493, indicated by an arrow; the −35 and −10 promoter sequences upstream from the transcriptional start site are underlined with a thin line; the right terminal repeat of the ISEcp1-like element is underlined with a thick line; the ATG start codon of bla_CTX-M-17 is boxed; and the ribosome-binding site (rbs) is underlined with dots. The location of the PE primer is indicated with an arrow at the bottom.