Complete Sequence of a KPC-Producing IncN Multidrug-Resistant Plasmid from an Epidemic Escherichia coli Sequence Type 131 Strain in China

Abstract

We report here the nucleotide sequence of a novel bla_KPC-2-harboring incompatibility group N (IncN) plasmid, pECN580, from a multidrug-resistant Escherichia coli sequence type 131 (ST131) isolate recovered from Beijing, China. pECN580 harbors β-lactam resistance genes bla_KPC-2, bla_CTX-M-3, and bla_TEM-1; aminoglycoside acetyltransferase gene aac(6′)-Ib-cr; quinolone resistance gene qnrS1; rifampin resistance gene arr-3; and trimethoprim resistance gene dfrA14. The emergence of a bla_KPC-2-harboring multidrug-resistant plasmid in an epidemic E. coli ST131 clone poses a significant potential threat in community and hospital settings.

TEXT

The rapid emergence and international spread of Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria are becoming major clinical challenges among high-risk patients in hospitals and long-term acute care facilities (1, 2). KPCs have disseminated among different K. pneumoniae strains, but one predominant sequence type, sequence type 258 (ST258), has successfully spread worldwide (1). In contrast, the dissemination of KPCs in other enterobacterial species, such as Escherichia coli, has not followed the same pattern, and so far, KPC-producing E. coli is not as common as K. pneumoniae. However, a public health concern is that the highly successful uropathogenic E. coli ST131 harboring CTX-M-15 extended-spectrum β-lactamase that has spread in the community will gain further selective advantage as a result of acquiring carbapenem resistance (3, 4). KPCs have been identified in epidemic E. coli ST131 strains (4–6), but the resistance plasmids have not been fully characterized. Here, we report the complete sequence of a novel bla_KPC-2-harboring incompatibility group N (IncN) plasmid from an E. coli ST131 isolate recovered from China.

E. coli strain ECN580 was isolated from a blood culture of a 28-year-old male from a tertiary care hospital in Beijing, China. He was hospitalized due to severe chronic renal failure and was on hemodialysis, with no history of foreign travel in the 6 months before isolation of the organism. The patient was started on empirical treatment with imipenem and cilastatin due to fever at 24 days of hospitalization. Carbapenem-resistant E. coli (ECN580) was identified from blood culture at 28 days of hospitalization, and the patient died 4 days later due to the KPC-associated infection. Multilocus sequence typing (MLST) showed that the isolate belonged to the epidemic E. coli ST131 clone (7). PCR amplification of β-lactamase genes and sequencing of the amplicons demonstrated the presence of bla_KPC-2, bla_CTX-M-3, and bla_TEM-1 (8–10). The bla_KPC-bearing plasmid was successfully transferred by conjugation experiments using ECN580 as donor and E. coli J53AzR as the recipient, as described previously (11). The MICs of the parental isolate and its E. coli J53 transconjugant were determined by broth microdilution in cation-adjusted Mueller-Hinton broth (MHB) using Sensititre GNX2F panels (Thermo Fisher Scientific, Waltham, MA) according to Clinical and Laboratory Standards Institute methods and interpretations (12, 13).

E. coli ECN580 exhibited resistance to all tested β-lactams and the inhibitors, including imipenem (MIC > 8 μg/ml), ertapenem (MIC > 8 μg/ml), meropenem (MIC > 8 μg/ml), doripenem (MIC > 4 μg/ml), cefotaxime (MIC > 32 μg/ml), ceftazidime (MIC > 32 μg/ml), cefepime (MIC > 32 μg/ml), aztreonam (MIC > 32 μg/ml), ticarcillin-clavulanate (MIC > 128/2 μg/ml), and piperacillin-tazobactam (MIC > 128/4 μg/ml), as well as co-trimoxazole (MIC > 4/76 μg/ml), amikacin (MIC > 64 μg/ml), gentamicin (MIC > 16 μg/ml), tobramycin (MIC > 16 μg/ml), ciprofloxacin (MIC > 4 μg/ml), and levofloxacin (MIC > 16 μg/ml), but it was susceptible to doxycycline (MIC ≤ 2 μg/ml), minocycline (MIC ≤ 2 μg/ml), tigecycline (MIC ≤ 0.25 μg/ml), colistin (MIC ≤ 1 μg/ml), and polymyxin B (MIC ≤ 1 μg/ml). The E. coli J53 transconjugant had a similar antimicrobial resistance profile as the parental strain but was intermediate against amikacin (MIC = 32 μg/ml) and ciprofloxacin (MIC = 2 μg/ml) and susceptible to gentamicin (MIC = 4 μg/ml) and levofloxacin (MIC = 2 μg/ml).

The bla_KPC-2-harboring plasmid was isolated and purified from E. coli J53 transconjugant using the method described previously (14), and the plasmid genome was sequenced on the Illumina MiSeq platform. The sequencing reads were de novo assembled into consensus contigs using Velvet algorithms (15). The sequence gaps were closed by PCR and standard Sanger sequencing; putative open reading frames (ORFs) were predicted and annotated using the RAST server (http://rast.nmpdr.org) (16).

Plasmid pECN580 is 64,935 bp in size, with an average G+C content of 50.8%, and harbors 93 predicted ORFs (Fig. 1A). pECN is an incompatibility group N (IncN) plasmid, and a query against the plasmid MLST database (http://pubmlst.org/plasmid/) (17) assigns it to plasmid sequence type 7 (ST7), the same sequence type as other carbapenemase-carrying IncN plasmids, including pNDM-BTR (GenBank accession no. KF534788, harboring bla_NDM-1), pK45-67VIM (GenBank accession no. HF955507, harboring bla_VIM-1), and pKo6 (GenBank accession no. KC958437, harboring bla_KPC-2). The IncN plasmid group is one of the most frequently encountered resistance plasmid types in Enterobacteriaceae of human and animal origin, and its members are typically of broad host range and able to transfer by conjugation (17, 18). The core region of pECN580, including a replication module (repA and RepA binding iterons), two transfer (tra) systems, a stability operon (stbAB, ardBR, and umuCD), and an antirestriction system (EcoRII and EcoRII met), is highly similar to those of other IncN plasmids, including the IncN prototype plasmid pR46 (GenBank accession no. AY046276) (Fig. 1A). pECN580 also has two distinct acquired regions harboring a panel of antibiotic resistance genes that are integrated at two previously described “hot spots” (a region downstream of uvp1 and a region between fipA and nuc) on the genome of IncN plasmids (14, 19). The first 8-kb acquired region is located downstream of the uvp1 gene and includes the carbapenemase gene bla_KPC-2 and the trimethoprim resistance gene dfrA14. The second region is located between genes fipA and nuc and carries the β-lactamase genes bla_CTX-M-3 and bla_TEM-1, the quinolone resistance aminoglycoside acetyltransferase gene aac(6′)-Ib-cr, the quinolone resistance gene qnrS1, and the rifampin resistance gene arr-3.

FIG 1.

(A) Plasmid structures of pR46 (GenBank accession no. AY046276), pKo6 (GenBank accession no. KC958437), pECN580 (GenBank accession no. KF914891), and pKPC-LKEc (GenBank accession no. KC788405). Light-blue shading denotes shared regions of homology. ORFs are portrayed by arrows and are colored based on their predicted gene functions. Orange arrows indicate plasmid scaffold regions. The genes associated with the tra locus are indicated by green arrows, and replication-associated genes are indicated by dark-blue arrows. Antimicrobial resistance genes are indicated by red arrows, while arsenic resistance genes are indicated by light-green arrows. Other genes in the accessory region are indicated by yellow arrows. (B) bla_KPC-harboring genetic elements in pBC633 (GenBank accession no. EU176012) (Tn4401b), pKp048 (GenBank accession no. FJ628167), pKo6 (GenBank accession no. KC958437), pECN580 (GenBank accession no. KF914891), and pKPC-LKEc (GenBank accession no. KC788405). Light-blue shading indicates shared regions of homology. Small black arrowheads flanking IS26 indicate the locations of IS26 invert repeats. The plasmid sequenced in this study (pECN580) is indicated with bold type.

Two class 1 integron-like elements were identified in pECN580. The first element is an IntI1-aac(6′)-Ib-cr-aar-3-Tn3-bla_TEM-1-bla_CTX-M-3-ISEcp1 gene cassette, located upstream of gene fipA. The second one harbors dfrA14 with its 3′-conserved sequence truncated by the insertion of an IS6100 element, located downstream of bla_KPC. The quinolone resistance gene, qnrS1, is flanked by a Tn3-like transposase gene and a truncated IS2 element disrupted by the insertion of an IS26 element. This qnrS1-containing element is highly similar to the qnrS1-harboring region (>99.9% nucleotide identities) identified in pKOX_R1 in a Klebsiella oxytoca isolate from Taiwan (20).

The bla_KPC-2 gene in pECN580 is carried on a 5-kb composite transposon-like element flanked by two IS26 elements, which is distinct from the bla_KPC-bearing Tn4401 transposon and the genetic element described in plasmid pKp048 (21, 22) (Fig. 1B). The transposon-like element in pECN580 was initially described in plasmid pKPC-LKEc (GenBank accession no. KC788405) from a carbapenem-resistant ST410 E. coli isolated in Taiwan (Fig. 1B) (23). pKPC-LKEc is a chimeric plasmid composed of three regions that showed homology to IncI, IncN, and RepFIC plasmids (23). A comparison between pECN580 and pKPC-LKEc indicated that they share a highly similar 28.5-kb element (>99.9% nucleotide identities), flanked by an ISEcp1 and an IS26 element (Fig. 1A). This plasmid structure leads to speculation that the IS elements may facilitate recombination between the plasmids and explain the movement of the IncN region.

A further BLAST search of the pECN580 sequence against the GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) reveals that it is highly similar to plasmid pKo6, a multidrug-resistant plasmid from a Klebsiella pneumoniae subsp. ozaenae isolate in China (GenBank accession no. KC958437), with 99% query coverage and maximum 100% identities (Fig. 1A). There are only two differences between these closely related plasmids. The region between repA and uvp1 in pECN580, which harbors the RepA binding iterons repeats involved in the control of plasmid replication and copy numbers (24), is 780 bp shorter than that in pKo6 (Fig. 1A). The second major difference relates to the insertion sites of IS26-d (located downstream of bla_KPC-2) on the composite transposon-like bla_KPC-harboring element (Fig. 1B). IS26-d is inserted 224 bp upstream of the stop codon of ΔISKpn6 in pKo6, while it is integrated 389 bp upstream of that in pECN580 and pKPC-LKEc (Fig. 1B). Taken together, the high sequence similarity between pKo6 and pECN580 suggests they evolved from a common plasmid and that they have independently spread to different species (E. coli and K. pneumoniae subsp. ozaenae, respectively).

In summary, we describe here the complete sequence of a novel bla_KPC-2-harboring IncN plasmid from a multidrug-resistant E. coli ST131 isolate. The novel bla_KPC-2-harboring composite transposon-like element found in pECN580 and related plasmids (pKPC-LKEc and pKo6) highlights the plasticity and complexity of these highly promiscuous plasmids. The spread of bla_KPC-2-harboring multidrug-resistant plasmids, e.g., pECN580, into E. coli ST131, which is associated with community-acquired infections, poses a new public health threat.

Nucleotide sequence accession number.

The complete nucleotide sequence of pECN580 was deposited as GenBank accession no. KF914891.