New Small Plasmid Harboring bla_KPC-2 in Pseudomonas aeruginosa

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Carbapenem resistance mediated by the production of Klebsiella pneumoniae carbapenemase (KPC) enzymes has been reported worldwide in Enterobacteriaceae, and the bla_KPC-2 gene was found to be associated with different transposons and plasmids (1–3). However, bla_KPC has also emerged in clinical isolates of Pseudomonas aeruginosa. First reported in Colombia in 2006 (4), subsequent worldwide reports described plasmid and chromosomal genetic contexts for this gene. In Brazil, KPC-producing P. aeruginosa was first described in 2012 (5), followed by a second report (6) that highlighted a worrying increase in KPC-producing P. aeruginosa. However, the genetic context of bla_KPC in the Brazilian strains remained unknown. At present, two complete sequences of P. aeruginosa bla_KPC-2-carrying plasmids, pCol-1 (GenBank accession number KC609323) from P. aeruginosa sequence type 308 (ST308) and pPA-2 (GenBank accession number KC609322) from P. aeruginosa ST1006 (7), are available in public databases. The aim of this study was to characterize the genetic context of bla_KPC-2 in a P. aeruginosa isolate from Brazil. Carbapenem-resistant P. aeruginosa (referred to here as BH6) was isolated in Belo Horizonte, Brazil, in 2011. BH6 was recovered from tracheal secretions of a female patient in the intensive care unit of a tertiary medical care center. This isolate was characterized as multidrug resistant, KPC-2 producing, and of ST244 (8). MIC values were ≥32 μg/ml for imipenem, ≥32 μg/ml for meropenem, ≥256 μg/ml for ceftazidime, ≥256 μg/ml for aztreonam, ≥256 μg/ml for cefepime, and 1.0 μg/ml for polymyxin B (9, 10). The bla_KPC-2 gene was isolated by PCR amplification, as previously described (11), followed by Sanger sequencing. The ST was characterized according to the P. aeruginosa MLST database guideline (see http://pubmlst.org/paeruginosa/).

The genomic location of bla_KPC-2 was determined, as previously described, by hybridization on I-CeuI pulsed-field gel electrophoresis (PFGE) using bla_KPC-2 and 16S rRNA genes as probes (to determine whether it was located on the chromosome) and S1 PFGE using bla_KPC-2 as a probe (to determine whether it was plasmid associated) (12). We observed three bands in the gel after S1 PFGE, and all three of them hybridized with the bla_KPC-2 probe. Since it seemed possible that they represented the linear, open-circular, and closed-circular forms of a single plasmid, plasmid DNA was extracted and purified using a PureLink HiPure plasmid filter midiprep kit (Invitrogen) and digested with BamHI (Thermo Fisher Scientific). This treatment generated a single plasmid band that indicated the presence of a single plasmid species. To determine whether this plasmid, pBH6, belonged to a recognized incompatibility (Inc) group, we used a PCR-based replicon typing (PBRT) scheme (13); however, we were unable to identify a particular replicon by using this procedure. Finally, plasmid DNA was sequenced using the Ion PGM system (Life Technologies). De novo assembly was carried out using the CLC Genomics workbench 8.5.0 (CLCbio, Aarhus, Denmark), which generated a single contig that represented the entire circular pBH6 plasmid. This sequence includes a single BamHI site. Gene prediction was carried out with the Prokka pipeline (14). Data files were compiled using Sequin (see http://www.ncbi.nlm.nih.gov/Sequin/), and the SnapGene viewer was used for visualization and analysis. Pairwise alignment was performed by BLASTN and BLASTP homology searches (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (pBH6 GenBank accession number LGVH01000782.1).

There were no chromosomal bla_KPC-2 copies. Sequencing revealed a circular genetic element of 3,652 bp and included the Tn3 transposon resolvase (15), a Tn3 inverted repeat (IR), a carbapenemase-encoding gene (bla_KPC-2), a partial transposase of ISKpn6, and the right end of ISKpn6, IRR (Fig. 1). Positions 3 to 3,027 displayed homology with plasmids pKP13d (82% query coverage, 99% identity [GenBank accession number CP003997]) from a KPC-2-producing Klebsiella pneumoniae Kp13 isolate from Brazil (16) and pFOS18 (89% query coverage, 99% identity [KJ653815]) from a KPC-2-producing K. pneumoniae isolate from China (17) (Fig. 2). Furthermore, P. aeruginosa ST1006 plasmids pCol-1 (56% query coverage, 100% identity [KC609323]) from P. aeruginosa ST308 and pPA-2 (58% query coverage, 99% identity [KC609322]) also show homology covering the bla_KPC-2 gene and the partial ISKpn6 copy.

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FIG 1

Circular plasmid carrying the bla_KPC-2 and origin of replication (ori) genes. The partial copy of ISKpn6 carrying the IRR (black arrow) is shown. A Tn3 IR is located between the bla_KPC-2 and the Tn3 resolvase (longer black arrow).

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FIG 2

Similarities among pBH6 (3,642 bp), pCol-1 (31,529 bp, coordinates 17066 to 19135), pPA2 (7,995 bp, coordinates 1133 to 3283), pFOS18 (23,939 bp, coordinates 4910 to 11956), and pKP13d (45,574 bp, coordinates 4910 to 11956). Red lines indicate the region with 100% similarity among the 5 plasmids. Uncolored lines indicate different regions among the plasmids.

Further analysis found that the region between ∼3200 and 3600 (400 bp) of pBH6 (which includes the DNA outside the known genes and transposon features and may contain the replication origin) is similar to that of several KPC-2-producing plasmids deposited in GenBank. Interestingly, almost all related plasmids bear a rep gene upstream of this region and, as observed in pBH6, a recombinase of the Tn3 family element downstream. A list of related plasmids is shown in Table 1. Only Aeromonas hydrophila pKPC2, K. pneumoniae 565 pKPCAPSS, and Aeromonas hydrophila AH1-pN6 show high similarity to each other over the entire 331-bp region (82% query coverage, 100% identity). The other plasmids, while maintaining high levels of similarity, only span between 100 and 300 bp, which suggests that this region may be related to plasmid replication and recognized as an origin of replication (ori).

It is known that small plasmids, such as pUC19, do not require a plasmid-specific rep gene but can replicate from a plasmid origin using bacterial host enzymes. The genetic environment of bla_KPC-2 here is different from that of those already described. In most Enterobacteriaceae and Pseudomonas spp., bla_KPC-2 appears associated with Tn4401-like transposons (1) carried by different plasmids (13).

To verify whether pBH6 could be established and replicate in other bacteria, purified plasmid DNA was transformed (18, 19) into Escherichia coli DH10B and P. aeruginosa PAO recipient strains followed by selection on MacConkey agar supplemented with ceftazidime (8 μg/ml). Transformants were easily obtained in both recipient strains. To evaluate the stability of pBH6, transformants from each strain isolated on LB solid medium were subcultured for 10 consecutive days in Mueller-Hinton (MH) and LB liquid media with or without 8 μg/ml ceftazidime. After the tenth subculture, both transformants still carried bla_KPC-2 as judged by PCR, suggesting that this small plasmid can replicate and remain stable for several subcultures.

In summary, we have identified and obtained the sequence of a small 3,652-bp plasmid, pBH6, from KPC-producing P. aeruginosa ST244. Plasmid pBH6 carries the bla_KPC-2 gene and remnants of a Tn3 family transposon and of ISKpn6 and may have been derived from the Tn4401 transposon in which ISKpn6 and bla_KPC are juxtaposed (20). When known sequences are taken into account, only 675 bp remain for providing replication functions, which implies that this region carries the origin of plasmid replication. Plasmid pBH6 does not include an identifiable rep gene and can replicate autonomously in E. coli and P. aeruginosa.