Genetic structures at the origin of acquisition of the beta-lactamase bla KPC gene

Abstract

Genetic structures surrounding the carbapenem-hydrolyzing Ambler class A bla KPC gene were characterized in several KPC-positive Klebsiella pneumoniae and Pseudomonas aeruginosa strains isolated from the United States, Colombia, and Greece. The bla KPC genes were associated in all cases with transposon-related structures. In the K. pneumoniae YC isolate from the United States, the beta-lactamase bla KPC-2 gene was located on a novel Tn3-based transposon, Tn4401. Tn4401 was 10 kb in size, was delimited by two 39-bp imperfect inverted repeat sequences, and harbored, in addition to the beta-lactamase bla KPC-2 gene, a transposase gene, a resolvase gene, and two novel insertion sequences, ISKpn6 and ISKpn7. Tn4401 has been identified in all isolates. However, two isoforms of this transposon were found: Tn4401a was found in K. pneumoniae YC and K. pneumoniae GR from the United States and Greece, respectively, and differed by a 100-bp deletion, located just upstream of the bla KPC-2 gene, compared to the sequence of Tn4401b, which was found in the Colombian isolates. In all isolates tested, Tn4401 was flanked by a 5-bp target site duplication, the signature of a recent transposition event, and was inserted in different open reading frames located on plasmids that varied in size and nature. Tn4401 is likely at the origin of carbapenem-hydrolyzing beta-lactamase KPC mobilization to plasmids and its further insertion into various-sized plasmids identified in nonclonally related K. pneumoniae and P. aeruginosa isolates.

FIG. 1.

Plasmid extractions from cultures of the different isolates and their transconjugants or transformants (A) and Southern hybridization carried out with an internal probe for bla_KPC-2 (B). Lanes 1, K. pneumoniae YC; lanes 2, E. coli J53 transconjugant harboring plasmid pNYC-1; lanes 3, P. aeruginosa 2404; lanes 4, P. aeruginosa KG2505 transformant harboring plasmid pCOL; lanes 5, E. coli J53 transformant harboring plasmid pCOL; lanes 6, K. pneumoniae KN2303; lanes 7, E. coli J53 transconjugant harboring plasmid pBC2303; lanes 8, K. pneumoniae KPC-negative strain; lanes 9, K. pneumoniae KN633; lanes 10, E. coli J53 electroporant harboring plasmid pBC633; lanes 11, K. pneumoniae GR; lanes 12, E. coli J53 transconjugant harboring plasmid pNGR-1; lanes 13, E. coli 50192 harboring four plasmids (7, 48, 66, and 154 kb); lanes 14, E. coli J53 reference strain (only the chromosomal band is visible). C, chromosome.

FIG. 2.

Schematic representation of bla_KPC-positive structures identified in enterobacterial isolates. (a) Salmonella enterica serovar Cubana (Salmonella cubana) (14); (b) Enterobacter cloacae (unpublished data; GenBank accession no. AM774409); (c) K. pneumoniae KN2303 (28); (d) K. pneumoniae pYW (30); (e) recombinant clone pRYC-1 containing the bla_KPC-2-coding region from K. pneumoniae YC (16). The vertical dotted lines indicate the largest known structure of the bla_KPC genetic environment. Genes and their corresponding transcription orientations are indicated by horizontal arrows. Grey triangles represent IRL and IRR of Tn4401. IRR1 represents another IRR (black triangle) that is disrupted by the ISKpn7 insertion. Small and empty triangles represent the inverted repeats of ISKpn6 and ISKpn7. TSDs are indicated above the sequence.

FIG. 3.

Schematic representation of Tn4401 structures identified on naturally occurring plasmids pCOL from P. aeruginosa 2404 (29), pBC2303 from K. pneumoniae (28), pBC633 from K. pneumoniae (28), pNGR from K. pneumoniae (6), and pNYC-1 from K. pneumoniae (16). Genes and their corresponding transcription orientations are indicated by horizontal arrows. Tn4401 is delimited by two IR sequences (grey triangles). Small and empty triangles represent the IRs of ISKpn6 and ISKpn7. Tn4401 target site duplications are indicated. The location of a 100-bp deletion in pNGR and pNYC-1 is indicated by vertical lines. The disrupted ORFs resulting from the Tn4401 insertion are indicated. In the case of pBC2303, Tn4401 inserted into an ORF that is located at the left end of another transposon. This ORF was also disrupted by a 220-bp miniature IR element. Small arrowheads with numbers indicate the primers listed in Table 1 and used for PCR mapping.

FIG. 4.

Alignment of the 39-bp Tn4401 IRs. (A) IRL and IRR; (B) IRL with a reconstructed IRR1. The underlined nucleotides correspond to the nucleotides duplicated after ISKpn7 insertion. Identical positions are indicated by asterisks.

FIG. 5.

Genesis of Tn4401 and origin of bla_KPC mobilization. Three steps might have been necessary for the genesis of Tn4401. (A) Insertion of a Tn3-based transposon delimited by IRL and IRR1, upstream of bla_KPC; (B) insertion of ISKpn6 and ISKpn7, which disrupted IRR1; (C) another IRR located just downstream of bla_KPC and IRL are recognized by the transposase, leading to the excision of Tn4401, which can then insert into a novel target sequence.