Novel IncR/IncP6 Hybrid Plasmid pCRE3-KPC Recovered from a Clinical KPC-2-Producing Citrobacter braakii Isolate.

Reports of human-pathogenic C. braakii strains, especially of strains showing resistance to carbapenems, are rare. To the best of our knowledge, our results represent the first detection of carbapenemase gene blaKPC-2 in C. braakii strains. In addition, we have studied detailed genetic characteristics of the novel IncR/IncP6 hybrid plasmid pCRE3-KPC, which was isolated from a clinical multidrug-resistant Citrobacter braakii CRE3 strain. Our results may provide further insight into the horizontal transfer of multidrug resistance genes in bacteria and into the genomic diversity and molecular evolution of plasmids.

The IncR replicon was first described in 2009 (13); since then, IncR plasmids have been increasingly reported in Enterobacteriaceae isolates (14). IncR replicons have also been found either as single replicons or as parts of multireplicon plasmids, which includes associations with IncA/C, IncF, IncFIIk, or nontypeable backbones (15). On the basis of prevalence statistics of plasmids containing both the IncR replicon and the bla KPC-2 gene (Table S1), we found that these plasmids usually contain multiple replicons. The bla KPC-2 -carrying plasmid unnamed3 (GenBank accession no. CP027150) contains one IncR replicon from the K. pneumoniae AR_0363 strain, which was that initially reported.
In this work, we have reported the first isolation of a bla KPC-2 -positive C. braakii strain. In addition, we determined the whole genomic sequence of a bla KPC-2 -carrying plasmid that we have named pCRE3-KPC, which was isolated from a clinical multidrugresistant C. braakii CRE3 strain. We compared this plasmid with the following three related plasmids: plasmid unnamed3 (GenBank accession no. CP027150), p10265-KPC (GenBank accession no. KU578314), and pCOL-1 (GenBank accession no. KC609323). Interestingly, we found that plasmid pCRE3-KPC contains both an IncR replicon and an IncP6 replicon belonging to a novel IncR/IncP6 hybrid plasmid. To the best of our knowledge, this is the first report of an IncR/IncP6 hybrid plasmid. Our results may offer insight into the horizontal transfer of resistance genes and provide an overview of plasmid diversity and evolution.
Linear comparisons of plasmid pCRE3-KPC with three related reference plasmids, namely, bla KPC-2 -carrying IncR plasmid unnamed3 (GenBank accession no. CP027150), p10265-KPC (a bla KPC-2 -carrying IncP6 plasmid first reported in China) (16), and pCOL-1 (a bla KPC-2 -carrying IncP6 plasmid, initially identified in Colombia) (18), were conducted. The detailed comparisons revealed that the overall structure of plasmid pCRE3-KPC is highly mosaic and can be divided into the following three distinct modules ( Fig. 1 and  2; see also Fig. S1 in the supplemental material): (i) a first module (ϳ20.5 kb) that is high homologous (Ͼ98.6% identity) to plasmid unnamed3 from the K. pneumoniae AR_0363 strain reported in the United States and extends from the resolution site (res) of ΔTn1722 to gene vagD (virulence-associated gene); (ii) a second module (ϳ27.8 kb) that shares Ͼ99.9% identity with plasmid p10265-KPC (16) from P. aeruginosa strain 10265 isolated in China and extends from the bla KPC-2 gene cluster to ΔTn5563; (iii) a third module comprising the other accessory modules (ϳ13.8 kb) with two novel insertion sequences (ISCfr28 and ISCfr16), the truncated aacC2-tmrB region, ISEc21, and ΔISEc15. On the basis of the study of the hybrid plasmids p675920-1 (20,21) and pKP1034 (22), the majority of the backbone and accessory regions of unnamed3 and p10265-KPC were found to be present in pCRE3-KPC, so pCRE3-KPC may represent a combination resulting from plasmids like these. Compared to the backbone of unnamed3 and p10265-KPC, pCRE3-KPC lost part of its backbone genes (orf711 of unnamed3, Δorf1 and kfrA, and a fragment extending from mobE to orf5 of p10265-KPC) during the recombination process, suggesting that these genes may not be necessary in these plasmids. The gene functions of these plasmids are annotated in detail (see Data Set S1, S2, S3, and S4 in the supplemental material).
Genomic comparison of the backbone regions from pCRE3-KPC and related plasmids. The backbone of each plasmid was further divided into the replication genes and the plasmid maintenance genes, without the conjugal-transfer genes, such that the hybrid pCRE3-KPC plasmid comprised the IncR and IncP6 backbones. The resultant backbone includes two replication genes (IncR replicon repA and IncP6 replicon repB) and two sets of partitioning system parAB genes (Fig. 1).
The IncR backbone from pCRE3-KPC was compared with plasmid unnamed3 (an IncR plasmid; GenBank accession no. CP027150), and their backbones were found to consist of the replication genes (IncR replicon and its iterons) as well as plasmid maintenance genes (parAB, umuCD, and vagDC). However, two differences in their backbones were identified as follows: (i) the orf711 gene (hypothetical protein) is deleted in pCRE3-KPC but complete in plasmid unnamed3 and (ii) the orf258 gene (hypothetical protein) is interrupted into two parts by the insertion of the aac(6=)-Ibcr-related region in plasmid unnamed3 ( Fig. 1 and 2; see also Fig. S1).
Furthermore, p10265-KPC (16) and pCOL-1 (18) can be assigned to the IncP6 incompatibility group, according to replicon-based schemes. The IncP6 backbone of pCRE3-KPC was compared with those of both of the plasmids named above, and the   backbones were found to comprise the replication genes (IncP6 replicon and its iterons) and plasmid maintenance genes (kfrA, parABC, the mob gene cluster, the msrB-msrA-yfcG-corA-orf8 gene cluster, and paeR7IR). Three differences were notable among them ( Fig. 1 and 2; see also Fig. S1): (i) pCRE3-KPC has lost genes (Δorf1 and kfrA) and a fragment extending from mobE (auxiliary protein) to orf5 (hypothetical protein); (ii) the numbers of copies of the 17-bp tandem repeat (GCGCCTGCCTTTGAGTA) within the iterons were 11 in pCRE3-KPC, 6 in p10265-KPC, and 12 in pCOL-1; and (iii) the Δorf8-corA-yfcG-msrA-msrB gene cluster was found to be inverted in pCOL-1. Genomic comparison of the bla KPC-2 gene region from pCRE3-KPC with those from related plasmids. The bla KPC-2 gene is associated with the core bla KPC platform (Tn3-ISKpn27-bla KPC -ΔISKpn6) in most Chinese Enterobacteriaceae strains (23)(24)(25). This core platform is integrated into a ΔISEc33-associated bla KPC-2 cluster, which was initially discovered in the p10265-KPC plasmid from a P. aeruginosa strain (16). In the bla KPC-2 gene cluster of p10265-KPC, the primary genetic structure, Tn3-ISKpn27-bla KPC-2 -ΔISKpn6-korC-orf6-klcA-ΔrepB, may have undergone two evolutionary events (16): (i) insertion of a Δbla TEM-1 gene between ISKpn27 and the Tn3 IRR (right inverted repeat) and (ii) disruption of the tnpA gene (transposase) from Tn3, resulting in its becoming two separate parts, an event caused by insertion of a composite transposon, ISApu1- orf7-ISApu2. The bla KPC-2 -carrying pCRE3-KPC plasmid was detected in an inpatient at a tertiary care hospital in China, and the BLASTN analysis of it showed that the surrounding genetic environment of the bla KPC-2 gene in pCRE3-KPC is highly similar to that in p10265-KPC. The ΔISApu1-orf7-ISApu2 composite transposon is also present in pCOL-1, but it has not been inserted into Tn3 and occurs downstream of ΔTn5403. Furthermore, the bla KPC-2 gene cluster is located downstream of ΔTn1722. Tn1722, a Tn3-family transposon, consists of an IRL (left inverted repeat), tnpA, tnpR (resolvase), res, mcp (methyl-accepting chemotaxis protein), and an IRR (26). ΔTn1722 contains an IRR, tnpA, tnpR, and res in pCRE3-KPC, which is also present in plasmid unnamed3 (GenBank accession no. CP027150) (Fig. 3).

MATERIALS AND METHODS
Bacterial isolates and identification. The clinical C. braakii CRE3 strain was isolated from a drainage sample from a patient at a tertiary care hospital in China on 5 May 2018. Bacterial identification was carried out using a Vitek compact-2 automated system (bioMérieux, France) and was confirmed by 16S rRNA sequencing (35). The genes encoding extended-spectrum ␤-lactamase (36), carbapenemase (37), fluoroquinolone (38), and aminoglycoside (39) were detected by PCR. All the PCR amplicons were sequenced on an ABI 3730 platform (Applied Biosystems, USA).
Plasmid conjugal transfer. The pCRE3-KPC plasmid was recovered from a clinical multidrugresistant C. braakii CRE3 isolate. Conjugation experiments were carried out with cells of rifampin-resistant Escherichia coli strain EC600 as the recipient cells, and the transformation experiments were conducted using cells of E. coli DH5␣ Electro-Cells (TaKaRa, China) as the recipient cells for the plasmid electroporation. Plasmid pCRE3-KPC was extracted from the cells using a Qiagen Plasmid Midi kit (Qiagen, Germany). The plasmid conjugal transfer and electroporation tests were performed as described previously (40,41).
Antimicrobial susceptibility and carbapenemase activity detection. Antimicrobial susceptibility testing was conducted using a Vitek compact-2 automated system (bioMérieux, France). The results were interpreted according to the CLSI (Clinical and Laboratory Standards Institute) 2018 performance standards (42). Carbapenemase activities were detected using mCIM (19).
Sequencing and sequence assembly. The bacterial genomic DNA extracted from the CRE3 isolate using a Wizard Genomic DNA purification kit (Promega, USA) was sequenced on the MiSeq (Illumina, USA) and the MinION (Oxford Nanopore, United Kingdom) platforms. The DNA library was constructed in accordance with a NEB Next Ultra II DNA Library Prep kit for Illumina, and the Illumina sequencing read length used was 300. The library preparations for the MinION platform were performed by the use of a rapid barcoding sequencing kit (SQK-RBK004) according to the protocol of the manufacturer (Oxford Nanopore Technologies), and the results were then loaded into the flow cell (FLO-MIN106D, Oxford Nanopore Technologies) for sequencing. Short Illumina reads were trimmed to remove poor-quality reads using Trimmomatic, and the contigs were assembled using Newbler3.0 (43). The long reads from MinION were combined with the short Illumina reads, which were subjected to hybrid assembly using SPAdesv3.11.1 (44). The hybrid assembly produced several scaffolds, and further bioinformatics analysis verified that the scaffold of the pCRE3-KPC plasmid was successfully cyclized by our in-house script. The correctness was then demonstrated by mapping the Illumina reads to the cyclized scaffold using CLC Genomics Workbench 9.0 (CLC Bio, Denmark), with an average level of read mapping coverage of 817ϫ. The final consensus sequence obtained from CLC Genomics Workbench 9.0 was considered to represent the complete sequence of plasmid pCRE3-KPC.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.