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Antimicrob Agents Chemother. 2016 January; 60(1): 646–649.
Published online 2015 December 31. Prepublished online 2015 November 2. doi:  10.1128/AAC.01886-15
PMCID: PMC4704230

Double Copies of blaKPC-3::Tn4401a on an IncX3 Plasmid in Klebsiella pneumoniae Successful Clone ST512 from Italy


A carbapenem-resistant sequence type 512 (ST512) Klebsiella pneumoniae carbapenemase 3 (KPC-3)-producing K. pneumoniae strain showing a novel variant plasmid content was isolated in Palermo, Italy, in 2014. ST512 is a worldwide successful clone associated with the spread of blaKPC genes located on the IncFIIk pKpQIL plasmid. In our ST512 strain, the blaKPC-3 gene was unusually located on an IncX3 plasmid, whose complete sequence was determined. Two copies of blaKPC-3::Tn4401a caused by intramolecular transposition events were detected in the plasmid.


Extensively drug-resistant (XDR) and pandrug-resistant Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains of the hyperepidemic clonal complex 258 (CC258) are detected worldwide as hospital-acquired pathogens and are frequently responsible for outbreaks. In particular, sequence type 258 (ST258), ST512, ST11, and ST340 are the most frequently detected variants of KPC-producing K. pneumoniae isolates (1, 2).

During May and June 2011, a countrywide Italian survey focusing on the diffusion of carbapenem-nonsusceptible K. pneumoniae isolates showed that the most frequent lineages belonged to CC258 (ST258 or ST512) (3). The epidemiology of KPC-producing K. pneumoniae in Palermo, Italy, also confirmed the emergence of ST258 beginning in 2008 (4). More recently, a 6-month surveillance performed in Sicily suggested that a major epidemiological change is likely ongoing in this geographic area, with ST258 still being prevalent but circulating along with several additional STs, including ST307 and ST273 (4). In particular, only one isolate of ST512 (i.e., K. pneumoniae strain 45) was identified on a total of 94 KPC-producing K. pneumoniae strains (4). This unique isolate of ST512 was further investigated and described in this study.

As shown in Table 1, the ST512 K. pneumoniae 45 strain showed an XDR phenotype (4). It was screened by PCR for the following plasmid-mediated quinolone resistance and β-lactamase genes: qnrA, qnrB, qnrC, qnrD, qnrS, aac(6′)-Ib-cr, qepA, oqxAB, blaKPC, blaVIM, blaNDM, blaOXA-48, blaOXA, blaSHV, blaTEM, blaLAP, blaCTX-M, and blaCMY (5,8). Positive amplicons underwent Sanger DNA sequencing to identify the variant genes. K. pneumoniae 45 was positive for the blaKPC-3, blaOXA-1, blaCTX-M-15, blaTEM-1, aac(6′)-Ib-cr, blaSHV-11, and oqxAB genes. The implementation of the PCR-based replicon typing (PBRT) kit (Diatheva) indicated that plasmids carried by strain 45 were not typeable. Therefore, the blaKPC-3-carrying plasmid was transformed in Escherichia coli DH5α competent cells (Invitrogen) and selected on Luria-Bertani agar plates (Sigma) containing ampicillin (50 μg/ml). The transformants were then screened by PCR for the presence of blaKPC genes. Plasmid DNA of one representative blaKPC-3-positive transformant (named p45) was purified using an Invitrogen plasmid midi kit (Invitrogen) and fully sequenced. A shotgun library was obtained, and sequencing was performed with the 454-GS Junior platform, according to the standard sequencing procedure (Roche Diagnostics). Plasmid coverage was >80×. The reads were aligned and assembled using the Newbler assembler software version (Roche Diagnostics). We found that plasmid p45 was split into three contigs, and the complete sequence was reconstructed by a PCR-based gap closure method. Open reading frames (ORFs) were predicted and annotated using the Artemis software (Wellcome Trust Sanger Institute, United Kingdom). Each predicted protein was compared against the protein database using BLASTP ( The gene sequences were compared and aligned with GenBank data using BLAST ( The IncX3 plasmids pKpS90 and pIncX-SHV (GenBank accession no. JX461340 and JN247852, respectively) were used as references for annotation and comparative analyses.

Antimicrobial susceptibility profiles of the original KPC-producing K. pneumoniae isolates and transformantsa

As shown in Fig. 1, our results indicated that p45 is 63,203 bp in size and shows the typical IncX3 scaffold, including the replicase gene, tax, and pilX gene clusters (9). The blaKPC-3 gene is located in the Tn3-like element Tn4401a (10). However, we observed that two copies of Tn4401a were present at a distance of 8,658 bp within the IncX3 plasmid scaffold of p45.

Major structural features of plasmids p45, pKpS90, and pIncX-SHV. Predicted open reading frames (ORFs) on plasmids are represented by white arrows. The ORFs of p45 were identified in this study; the ORFs of pKpS90 and pIncX-SHV were deduced from GenBank ...

One copy of Tn4401a was integrated within an IS3000-a1 element, which was interrupted by the blaKPC-3::Tn4401a insertion. The duplication of a 5-bp sequence was identified at the site of integration, immediately flanking the inverted repeat right (IRR) and inverted repeat left (IRL) of Tn4401a. The blaSHV-11 gene was identified 4 kb from the blaKPC-3::Tn4401a integration site, followed by an IS26 element. The same structure carrying IS26-blaSHV-11-IS3000 was previously detected in plasmid pIncX-SHV (11; GenBank accession no. JN247852). In a similar plasmid (i.e., pKpS90), blaKPC-2::Tn4401a was integrated in the same region as p45 but within a different site, causing the interruption of the ygbK gene (12). The progenitor of p45 was therefore a pIncX-SHV-like plasmid carrying the blaSHV-11 gene (11).

The second copy of Tn4401a in p45 was integrated in topB (a gene constantly present in all IncX3 plasmids) and in opposite orientation compared to that of the first transposon (Fig. 1). The target site duplication was also identified in this site, adjacent to the inverted repeats of the transposon. It is plausible that the acquisition of the second Tn4401a in p45 occurred by intramolecular transposition. In the literature, the simultaneous presence of two blaKPC copies located in trans on different plasmids in the same bacterial host was previously reported in several collections (13,16). However, only two examples of in cis blaKPC genes were previously described and differed from the arrangement described for p45. Two blaKPC-3::Tn4401a copies were identified in MNCRE44, an extraintestinal pathogenic E. coli strain belonging to the ST131 H30R subclade and found in the United States (13). Its plasmid (pMNCRE44_5) was a 116-kb hybrid of IncX3 and IncFIA(HI1) plasmids, and the two transposon copies were located as one copy for each of the two fused plasmid portions (13). The duplication of the similar transposon blaKPC-2::Tn4401b was described for the IncN plasmid S9 from K. pneumoniae in the United States (17).

The MICs for several antibiotics for KPC-producing K. pneumoniae strains of ST512 and ST258 carrying blaKPC-3 on pKpQIL (18), those of KPC-producing K. pneumoniae 45 (ST512 and with IncX3) and those of their corresponding E. coli DH5α transformants, were determined implementing the microdilution ESB1F and GNX2F plates (Trek Diagnostics) (Table 1). As result, we noted that the E. coli DH5α transformant carrying the IncX3 plasmid with two copies of blaKPC-3::Tn4401a showed significantly increased MICs for carbapenems, cephalosporins, and β-lactam–β-lactamase inhibitor combinations compared to those of the transformant carrying the classical pKpQIL plasmid. This phenomenon was not observed for the original K. pneumoniae strains of different STs and possessing different blaKPC genetic backgrounds. The difference in MICs may be due not only to the double copy of the blaKPC genes and their levels of gene expression (14) but also to different copy numbers of IncX3 and pKpQIL.

In conclusion, our study describes the change in the typical plasmid content of ST512 K. pneumoniae: the worldwide-described pKpQIL plasmid carrying blaKPC (19; GenBank accession no. GU595196) was replaced by an IncX3 plasmid carrying two copies of blaKPC-3::Tn4401a. The change in plasmid type in K. pneumoniae strain 45 might represent an important evolution of the ST512 lineage.

Nucleotide sequence accession number. The GenBank file for K. pneumoniae strain ST512 plasmid p45-IncX3 was compiled using Sequin ( and deposited at the NCBI GenBank under accession no. KT362706.

Funding Statement

The Italian Flagship “InterOmics” project (PB.P05) funded by MIUR and coordinated by the Italian Council of National Research (CNR) (A.C.) provided funding.


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