Genomic characterization of novel IncFII-type multidrug resistant plasmids p0716-KPC and p12181-KPC from Klebsiella pneumoniae

This study aimed to genetically characterize two fully-sequenced novel IncFII-type multidrug resistant (MDR) plasmids, p0716-KPC and p12181-KPC, recovered from two different clinical Klebsiella pneumoniae isolates. p0716-KPC and p12181-KPC had a very similar genomic content. The backbones of p0716-KPC/p12181-KPC contained two different replicons (belonging to a novel IncFII subtype and the Rep_3 family), the IncFIIK and IncFIIY maintenance regions, and conjugal transfer gene sets from IncFIIK-type plasmids and unknown origins. p0716-KPC and p12181-KPC carried similar three accessory resistance regions, namely ΔTn6209, a MDR region, and the bla KPC-2 region. Resistance genes bla KPC-2, mph(A), strAB, aacC2, qacEΔ1, sul1, sul2, and dfrA25, which are associated with transposons, integrons, and insertion sequence-based mobile units, were located in these accessory regions. p0716-KPC carried two additional resistance genes: aphA1a and bla TEM-1. Together, our analyses showed that p0716-KPC and p12181-KPC belong to a novel IncFII subtype and display a complex chimeric nature, and that the carbapenem resistance gene bla KPC-2 coexists with a lot of additional resistance genes on these two plasmids.

IncFII Y -type plasmid from Klebsiella oxytoca isolated in China 6 . Although highly unusual, these backbone components can function together to promote the replication and stability of pKPC-LK30 in K. pneumoniae.
This work presents the complete sequences of two novel MDR plasmids, p0716-KPC and p12181-KPC, from K. pneumoniae strains isolated from China ( Table 1). The two closely related plasmids belong to a novel IncFII subtype, and displayed a complex chimeric nature with respect to both the plasmid backbone (closely related to pKPHS2 and pKPC-LK30) and the accessory resistance regions. Co-occurrence of bla KPC-2 (carbapenem resistance) with mph(A) (macrolide resistance), strAB and aacC2 (aminoglycoside resistance), qacEΔ1 (quaternary ammonium compound resistance), sul1 and sul2 (sulphonamide resistance), and dfrA25 (trimethoprim resistance) was observed in both plasmids.

Results
Clinical cases. Patient 1 was a 59-year-old male admitted to Hospital 1 in October 2013, where he was diagnosed with a cardiac carcinoma. Nosocomial intra-abdominal infection occurred, and K. pneumoniae 0716 was isolated from ascitic fluid. The patient received intravenous administration of tigecycline, and the patient's acute condition significantly improved.
Patient 2 was an 87-year-old man with chronic obstructive pulmonary disease, chronic bronchitis, pulmonary emphysema, coronary heart disease, and pancreatic carcinoma. Acute pancreatitis and peritonitis developed during treatment in Hospital 2 in September 2013. K. pneumoniae 12181 was isolated from a sputum sample, and the patient was treated with intravenous administration of meropenem plus ciprofloxacin. However, treatment was unsuccessful and the patient died.
Overview of p0716-KPC and p12181-KPC. PCR screening and sequencing indicated the presence of bla KPC-2 , but none of the other carbapenemase genes tested for, in strains 0716 and 12181. The bla KPC markers could be transferred from strains 0716 and 12181 into TOP10 through electroporation, generating the E. coli electroporants 0716-KPC-TOP10 and 12181-KPC-TOP10, respectively. Subsequent high-throughput sequencing indicated that these two electroporants contained the bla KPC-2 -carrying plasmids p0716-KPC and p12181-KPC, respectively. p0716-KPC but not p12181-KPC could be transferred from the respective strains 0716 and 12181 into EC600 through conjugation. In the negative control experiments using only the donor or the recipient, no colonies were observed on the agar plates containing indicated antibiotics, excluding the possibility of antibiotic resistance due to spontaneous mutation.
As expected, the resulting E. coli transformants and transconjugant demonstrated class A carbapenemase activity, and were resistant to ampicillin, amoxicillin/clavulanic acid, piperacillin/tazobactam, cefazolin, ceftriaxone, imipenem, meropenem, and aztreonam, but remained susceptible to macrodantin (data not shown).
Genome sequencing confirmed that p0716-KPC and p12181-KPC were circular DNA molecules, 143,538 bp and 140,089 bp in length, with 192 and 188 predicted open reading frames, respectively ( Figure S1). The modular structure of each plasmid was composed of the backbone region, as well as multiple separate accessory modules inserted within the backbone ( Figure S1). p0716-KPC and p12181-KPC shared 97% query coverage with a maximum nucleotide identity of 99% (Fig. 1).
Backbone regions of p0716-KPC and p12181-KPC. Two representative bla KPC-2 -carrying plasmids pKPHS2 and pKPC-LK30 were included in the genomic comparison with p0716-KPC and p12181-KPC (Fig. 1). p0716-KPC and p12181-KPC were most closely related to pKPHS2 (67% coverage and 99% nucleotide identity; last accessed on August 15, 2015). pKPC-LK30 was selected because it contains a large plasmid maintenance region that is not found in pKPHS2 but was identified in p0716-KPC and p12181-KPC (see below).
The backbones of p0716-KPC and p12181-KPC were almost identical with respect to genetic content. p0716-KPC and p12181-KPC both contained two novel replicons, including a repB Rep_3-family/p0716-KPC gene and a repA IncFII-family gene, both of which were very different from their counterparts in pKPHS2 and pKPC-LK30. The repA IncFII-family gene was most closely related to the IncFII plasmid pEA49-KPC (accession number KU318419) with a nucleotide identity of 94% (last accessed on December 25, 2016). p0716-KPC and p12181-KPC also contained the complete 16-kb IncFII Y -type pKOX_NDM1 maintenance region, and almost the entire non-redundant  Table 1. Major features of p0716-KPC and p12181-KPC and their antibiotic resistance genes and host bacteria.
IncFII K -type maintenance region found in both pKPHS2 and pKPC-LK30 ( Fig. 1). Compared with pKPHS2 and pKPC-LK30, p0716-KPC and p12181-KPC contained two unique backbone regions: a 5.6-kb plasmid maintenance region connected to the repB Rep_3-family/p0716-KPC gene, and a novel 3.2-kb conjugal transfer region contained the pld gene (conjugal transfer endonuclease). This 3.2-kb region was most closely related (93% query coverage and 98% maximum nucleotide identity; last accessed on December 25, 2016) to the MOB F family plasmid pEA49-KPC ( Fig. 1). p0716-KPC and p12181-KPC belonged to a novel IncFII subtype since it contained a novel repA gene belonging to the IncFII family, together with IncFII K /II Y -type maintenance regions. Based on the three key regulatory DNA transfer genes traM, traJ, and finO and the ATPase gene traC, all of which encoded key proteins of a type IV secretion system, p0716-KPC and p12181-KPC were assigned into the subgroup A of the IncF/MOB F12 group 7 .
Accessory regions of p0716-KPC and p12181-KPC. p0716-KPC contained three accessory modules in total, namely ΔTn6029, the MDR region, and the bla KPC-2 region, which were inserted at different sites in the backbone (Fig. 2). ΔTn6029 is a partial fragment of the IS26-based composite transposon Tn6029 8 , and comprises a strAB module flanked by two inverted IS26 elements. As initially characterized in the IncHI1 plasmid pSRC27-H, Tn6029 and Tn4352 are two overlapping transposons likely generated from complex recombination events between IS26, Tn2, and Tn5393c 8 .  The MDR region was further divided into an aacC2/ΔtmrB-related region, In207, and a Tn21-related region. The aacC2/ΔtmrB-related region harbored the aphA1a (aminoglycoside resistance)-carrying Tn4352 8 , which lacked target site duplication signals of transposition. Tn4352 was further connected to a region composed of ΔtmrB, aacC2, and a 135-bp Tn2 remnant containing its inverted repeat right (IRR), resulting in truncation of tmrB (tunicamycin resistance). The association of aacC2-tmrB with Tn2 and IS26, as observed in pCTX-M3 and pU302L, constitutes one example of the multifarious aacC2-harboring structures 9 . In207 was identified in p0716-KPC and p12181-KPC, and is an In4-like integron 10 containing a single dfrA25 cassette. Notably, In207 is connected to the macrolide resistance unit IS26-mph(A)-mrx-mphR(A)-IS6100, a structure frequently associated with class 1 integrons 9 , at its 3ʹ region. This likely occurred through IS6100-mediated recombination, and resulted in deletion of the inverted repeat terminal (IRt). In207 from p0716-KPC and p12181-KPC appeared to be more complete than the prototype In207 (GenBank accession number AB280920), which is only an integron cassette array fragment. The Tn21-related region was organized sequentially as follows: IS26, ∆Tn21, a 288-bp IS903B remnant, and ISKpn14. Tn21, a Tn3-family unit transposon, contained the core transposition module tnpA (transposase)-tnpR (resolvase), tnpM, inserted integron In2, urf2, and the mercuric resistance (mer) operon. The transposon was flanked by 38-bp IRL (inverted repeat left) and IRR sequences, and the In2 insertion was shown to disrupt a presumed ancestral urf2M gene, resulting in urf2 and tnpM 11 . The ∆Tn21 element from p0716-KPC was composed of ΔtniA In2 , the mer operon, and the IS5075-disrupted IRR Tn21 . The IS1111-family element IS5075 targets the terminal inverted repeats of the Tn21-subgroup transposons of the Tn3 family 12 .
p12181-KPC also contained ΔTn6029, the MDR region, and the bla KPC-2 region, which resembled their counterparts in p0716-KPC. Tn4352 in the MDR region and the Tn2-related region in the bla KPC-2 locus were not found in p12181-KPC. Therefore, p0716-KPC contained two additional resistance genes, aphA1a and bla TEM-1 , compared with p12181-KPC. Notably, aphA1a and bla TEM-1 are redundant determinants accounting for resistance to aminoglycosides and β-lactams, respectively, in p0716-KPC. In addition, extensive rearrangement of large fragments was observed not only within, but between the MDR region and the bla KPC-2 region of p12181-KPC relative to p0716-KPC. These rearrangements were likely promoted by IS26-based replicative transposition 16 as multiple copies of IS26 were identified in these two accessory regions. p12181-KPC still maintained three small accessory regions: two IS903D copies and group IIB retro-transposable intron S.ma.I1, none of which were found in p0716-KPC. One copy of IS903D was inserted into a region between trbF and trbB, while traV (an essential gene encoding a core protein of type IV secretion system) was disrupted by a second copy, rendering p12181-KPC non-conjugative.
pKPC-LK30 contained a single accessory resistance region (the bla KPC-2 region) containing two antibiotic resistance genes: bla KPC-2 and bla SHV-11 (Fig. 2). pKPHS2 carried two accessory resistance regions, namely the bla KPC-2 region and the Tn2-related region, each of which contained a single antibiotic resistance gene (bla KPC-2 and bla TEM-1 , respectively) (Fig. 2). All these accessory resistance regions were genetically related to their counterparts from p0716-KPC and p12181-KPC. Compared with pKPC-LK30 and pKPHS2, p0716-KPC and p12181-KPC appear to have acquired many more accessory regions containing several additional resistance genes.

Discussion
The bla KPC genes are largely associated with Tn4401 and Tn6296 14 , which constitute the core bla KPC genetic environments. bla KPC -carrying Tn4401b and its close derivatives are frequently found on plasmids from bacteria isolated in European and American countries [17][18][19] . Tn4401 is rarely found in China 20 , with Tn6296 and its derivatives more frequently identified as the bla KPC platforms, such as those located in pKP048 13 , pKPHS2 4 , pKPC-LK30 5 , and p0716-KPC and p12181-KPC (this study).
bla KPC genes are also commonly identified in plasmids belonging to various incompatibility groups, including IncF, IncI, IncA/C, IncN, IncX, IncR, IncP, IncU, IncW, IncL/M, and ColE, ranging in size from 10-300 kb 21 . The IncF replicons can be classified into the groups FIA, FIB, FIC, and FII 22 . The IncFII plasmids are commonly low copy number plasmids and carry the primary FII replicon, often in association with additional replicons such as FIA and FIB. Moreover, the FII replicons can be further divided into various subtypes, including II K , FII Y , and FII S , generating many compatible variants that can be used to overcome the incompatibility barrier with incoming plasmids 22 .
The IncFII plasmid family can replicate in many different enterobacterial species, and is clearly playing an important role in the dissemination of antimicrobial resistance genes, including bla KPC , amongst Enterobacteriaceae 21 . The bla KPC-3 -carrying IncFII K plasmid pKpQIL and its close derivatives have spread in European and American countries 23 . bla KPC-2 -carrying IncFII K plasmids from China, such as pKP048 and pKPHS2, have very similar core backbone regions but limited overall sequence similarity to pKpQIL. It seems that the pKpQIL-like and pKP048-like plasmids followed distinct evolutionary routes after separating from their common ancestor.
Recovery of the closely related plasmids p0716-KPC and p12181-KPC from two independent cases of nosocomial infection from two different hospitals indicates the potential of trans-regional spread and circulation of these plasmids in hospital settings. p0716-KPC and p12181-KPC belong to a novel IncFII subtype, and display a complex chimeric nature, as observed within the backbone as well as the accessory resistance regions. The replication and stable inheritance of these two plasmids is likely promoted by the coordinated action of the IncFII and Rep_3-family replicons and the IncFII K and IncFII Y maintenance gene sets, respectively.
Production of KPC-2 makes strains containing p0716-KPC or p12181-KPC resistant to almost all β-lactams, including carbapenems. The situation is exacerbated by the presence of five additional classes of antibiotic resistance genes [mph(A), strAB and aacC2, qacEΔ1, sul1 and sul2, and dfrA25] on these two plasmids. The accumulation of various antibiotic resistance genes on a plasmid have resulted from complex horizontal genetic transfer events under selection pressure of multiple antibiotics, and a bacterium will become resistant to multiple antibiotics at once by picking up such a MDR plasmid.
p0716-KPC is conjugative and contains the complete IncFII K conjugal transfer gene content, while p12181-KPC has become non-conjugative likely because of the presence of multiple genetic lesions in the conjugal transfer regions. Non-transmissible plasmids rely largely on vertical transmission to be maintained in populations. Although classical models of plasmid evolution predict that conjugation is necessary for plasmid maintenance, it has been found that compensatory adaptation to ameliorate the cost of plasmid carriage coupled to rare (positive) selection for plasmid-encoded antibiotic resistance is sufficient to stabilize non-transmissible plasmids, explaining why non-conjugative plasmids are common 24 .

Materials and Methods
Bacterial strains and identification. The use of human specimens and all related experimental protocols was approved by the Committee on Human Research of all the institutions (Beijing Institute of Microbiology and Epidemiology, the 307th Hospital of the People's Liberation Army, and Navy General Hospital), and was carried out in accordance with the approved guidelines. Informed consent was obtained from patients where indicated. Our research was carried out in accordance with the Declaration of Helsinki.
Imipenem-non-susceptible K. pneumoniae strains 0716 and 12181 were isolated from two inpatients with hospital-acquired infections from two different public hospitals, and there was no epidemiological link between the two patients. Bacterial species identification was performed by 16 S rRNA gene sequencing 25 . The major plasmid-borne carbapenemase genes were screened by PCR 26 . All PCR amplicons were sequenced on an ABI 3730 Sequencer using the PCR primers.

Plasmid conjugal transfer. Plasmid conjugal transfer experiments were carried out using rifampin-resistant
Escherichia coli strain EC600 as the recipient, and K. pneumoniae strains 0716 and 12181 as donors. Aliquots (3 ml) of overnight culture of each donor and recipient strain were mixed, harvested, and resuspended in 80 μl of Brain Heart Infusion (BHI) broth (BD Biosciences). The mixtures were spotted on 1 cm 2 hydrophilic nylon membrane filters with a 0.45-µm pore size (Millipore), which were then placed on BHI agar (BD Biosciences) plates and incubated at 37 °C for 12-18 h. Bacteria were washed from the filter membranes and spotted on Muller-Hinton (MH) agar (BD Biosciences) plates containing 1 mg/ml rifampin and 2 μg/ml imipenem for selection of bla KPC -positive E. coli transconjugants.

Plasmid electroporation.
To prepare competent E. coli TOP10 cells for plasmid electroporation, 200 ml of overnight culture in Super Optimal Broth (SOB) at an optical density (OD 600 ) of 0.4-0.6 were washed three times with electroporation buffer (0.5 M mannitol and 10% glycerol), and then concentrated into a final volume of 2 ml. A 1-µg aliquot of plasmid DNA, isolated from strain 0716 or 12181 using a Qiagen Plasmid Midi Kit, was mixed with 100 μl of competent cells for electroporation at 25 μF, 200 Ω, and 2.5 kV. Immediately following electroporation, cells were suspended in 500 μl of SOB, and an appropriate aliquot was spotted on an SOB agar plate containing 2 μg/ml imipenem for selection of bla KPC -positive E. coli transformants.
Antimicrobial susceptibility testing. Antimicrobial susceptibility testing was conducted using the VITEK 2 system (bioMérieux) according to the manufacturer's instructions, and interpreted as per the Clinical and Laboratory Standards Institute guidelines 27 . Plasmid sequencing and annotation. Plasmid DNA was isolated from E. coli transformants using a Qiagen Large Construct Kit, and then sequenced from a paired-end library with an average insert size of 500 bp, and a mate-pair library with average insert size of 5,000 bp, using an Illumina MiSeq sequencer. The circled DNA contigs were assembled using Newbler 2.6 28 . Open reading frames and pseudogenes were predicted using RAST 2.0 29 combined with BLASTP/BLASTN 30 searches against the UniProtKB/Swiss-Prot 31 and RefSeq 32 databases. Annotation of resistance genes, mobile elements, and other features was carried out using CARD 33 36 , and the Tn Number Registry 37 . Multiple and pairwise sequence comparisons were performed using MUSCLE 3.8.31 38 and BLASTN, respectively. Gene organization diagrams were drawn in Inkscape 0.48.1.
Nucleotide sequence accession numbers. The complete nucleotide sequences of p0716-KPC and p12181-KPC were submitted to GenBank under accession numbers KY270849 and KY270850, respectively.