The use of human specimens and all related experimental protocols were approved by the Committee on Human Research of the 307th Hospital of the People's Liberation Army and that of the First Affiliated Hospital of Anhui Medicial University, and carried out in accordance with the approved guidelines. The research involving biohazards and all related procedures were approved by the Biosafety Committee of the Beijing Institute of Microbiology and Epidemiology. Bacterial species was identified by 16S rRNA gene sequencing (Frank et al., 2008). The major plasmid-borne carbapenemase genes were screened for by PCR (Chen et al., 2015), followed by amplicon sequencing on ABI 3730 Sequencer.

Plasmid conjugal transfer experiments were carried out with the rifampin-resistant Escherichia coli EC600 (LacZ⁻, Nal^R, Rif^R) being used as recipient and strain 0801 or 7121 or 17285 or as donor (Chen et al., 2015). 3 ml of overnight culture of each of donor and recipient bacteria were mixed together, harvested and resuspended in 80 μl of Brain Heart Infusion (BHI) broth (BD Biosciences). The mixture was spotted on a 1 cm² filter membrane that was placed on BHI agar (BD Biosciences) plate, and then incubated for mating at 37°C for 12–18 h. Bacteria were washed from filter membrane and spotted on Muller-Hinton (MH) agar (BD Biosciences) plate containing 1,000 μg/ml rifampin and 2 μg/ml imipenem for selection of bla_IMP-positive E. coli transconjugants.

Activity of class A/B/D carbapenemases in bacterial cell extracts was determined via a modified CarbaNP test (Chen et al., 2015). Overnight bacterial cell culture in MH broth was diluted 1:100 into 3 ml of fresh MH broth, and bacteria were allowed to grow at 37°C with shaking at 200 rpm to reach an OD₆₀₀ of 1.0 to 1.4. If required, ampicillin was used at 200 μg/ml. Bacterial cells were harvested from 2 ml of the above culture, and washed twice with 20 mM Tris-HCl (pH 7.8). Cell pellets were resuspended in 500 μl of 20 mM Tris-HCl (pH 7.8), and lysed by soniation, followed by centrifugation at 10,000 × g at 4°C for 5 min. 50 μl of the supernatant (the enzymatic bacterial suspension) were mixed with 50 μl of substrate I to V, respectively, followed by incubation at 37°C for a maximum of 2 h. Substrate I: 0.054% phenol red plus 0.1 mM ZnSO₄ (pH7.8). Substrate II: 0.054% phenol red plus 0.1 mM ZnSO₄ (pH7.8), and 0.6 mg/μl imipenem. Substrate III: 0.054% phenol red plus 0.1 mM ZnSO₄ (pH7.8), 0.6 mg/μl mg imipenem, and 0.8 mg/μl tazobactam. Substrate IV: 0.054% phenol red plus 0.1 mM ZnSO₄ (pH7.8), 0.6 mg/μl mg imipenem, and 3 mM EDTA (pH7.8). Substrate V: 0.054% phenol red plus 0.1 mM ZnSO₄ (pH7.8), 0.6 mg/μl mg imipenem, 0.8 mg/μl tazobactam, and 3 mM EDTA (pH7.8).

Bacterial antimicrobial susceptibility was tested by BioMérieux VITEK 2 and interpreted as per Clinical and Laboratory Standards Institute (CLSI) guidelines (ClSI, 2015).

Plasmid DNA was isolated from E. coli transconjugant using a Qiagen Large Construct Kit, and then sequenced with 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 (Illumina). Reads from each sample were trimmed to remove poor quality sequences, and then the contigs were assembled with Newbler 2.6 (Nederbragt, 2014).

Open reading frames and pseudogenes were predicted using RAST 2.0 (Brettin et al., 2015) combined with BLASTP/BLASTN searches (Boratyn et al., 2013) against the UniProtKB/Swiss-Prot (Boutet et al., 2016) and RefSeq (O'leary et al., 2016) databases. Annotation of resistance genes, mobile elements, and other features was carried out using the online databases including CARD (Jia et al., 2016), ResFinder (Zankari et al., 2012), BacMet (Pal et al., 2014), ISfinder (Siguier et al., 2006), INTEGRALL (Moura et al., 2009), and the Tn Number Registry (Roberts et al., 2008). Multiple and pairwise sequence comparisons were performed using MUSCLE 3.8.31 (Edgar, 2004) and BLASTN, respectively. Gene organization diagrams were drawn in Inkscape 0.48.1.

The complete sequences of p0801-IMP, p7121-IMP, and p17285-IMP were submitted to GenBank under accession numbers KT345947, KX784502, and KX784503, respectively.

K. pneumoniae 0801, K. oxytoca 7121, and C. freundii 17285 were isolated from three different inpatients designated Patient 1 to Patient 3, respectively, with nosocomial infections. Patient 1 was a 35-year-old woman admitted to Hospital 1 in May 2013 and diagnosed to have acute lymphoblastic leukemia, and she received chemotherapy for 1 week. Pulmonary infection, septicemia and recurrent fever occurred during chemotherapy, and she received empirical intravenous administration with moxifloxacin. K. pneumoniae 0801was isolated from the blood specimens on the next day after chemotherapy. The patient was discharged 3 days later upon request from her family members.

Patient 2 was a 43-year-old woman admitted to Hospital 1 in January 2014 and diagnosed to have acute myeloid leukemia, and she received hematopoietic stem cell transplantation. Pulmonary infection occurred in the convalescent period, and K. oxytoca 7121 was then isolated from the sputum specimens. The patient received intravenous administration with flucloxacillin empirically at first, which was switched into levofloxacin based on antimicrobial susceptibility test results. Her symptoms associated with pulmonary infections progressively improved. The patient was discharged at 2 weeks after transplantation.

Patient 3 was a 66-year-old woman admitted to Hospital 2 in July 2013 and diagnosed to have adult onset Still's disease, and she received antianaphylactic treatment. Urinary tract infection occurred at 1 week after hospitalization, and C. freundii 17285 was then isolated from the voided midstream urine specimens. The patient received intravenous administration with teicoplanin. Her symptoms associated with infection and adult onset Still's disease progressively improved. The patient was discharged at 2 weeks after hospitalization.

PCR screening assay indicated the presence of bla_IMP but not any of the other carbapenemase genes tested in strains 0801, 7121, and 17285, and the bla_IMP-carrying plasmids were designated p0801-IMP, p7121-IMP, and p17285-IMP, respectively. Each of these plasmids could be transferred into strain EC600 through conjugation, generating E. coli transconjugants 0801-IMP-EC600, 7121-IMP-EC600, and 17285-IMP-EC600, respectively, indicating that all these three plasmids are conjugative. All the above wild-type and transconjugant strains have the class B carbapenemase activity (data not shown), and are resistant to ceftriaxone, ceftazidime, imipenem and meropenem (Table 1). Strains 0801, 7121, and 17285 are resistant to gentamicin but the other strains remain susceptible to this drug, and all of them are susceptible to amikacin.

Genome sequencing shows that p0801-IMP, p7121-IMP, and p17285-IMP have 42,580-bp, 42,461-bp and 43797-bp circularly closed DNA sequences, respectively, all of which carry 54 predicted open reading frames in total (Figure S1). These three plasmids belong to the IncN2 group because each of them contains an IncN2-type repA (plasmid replication initiation) gene. Further comparative genomics analysis is applied to all the five integron-carrying IncN2 plasmids p0801-IMP, p7121-IMP and p17285-IMP (this study), pJIE137 (Partridge et al., 2012), and p34983-59.134kb (accession number CP010378), together with the IncN2 reference plasmid pYNKP001-NDM (Sun et al., 2015), and the modular structure of each plasmid is divided into the IncN2 backbone as well as one or more accessory modules inserted at different sites of the backbone (Figure S1, Figure 1). Although p271A is the first fully sequenced IncN2 plasmid, pYNKP001-NDM is more appropriate as the IncN2 reference, because a 5.2-kb backbone region within the CUP-controlled regulon is absent from p271A relative to pYNKP001-NDM (Poirel et al., 2011; Sun et al., 2015).

The six plasmids involved in genomic comparison possess conserved IncN2 backbones, each of which can be further divided into the regions responsible for plasmid replication (repA and its iterons), maintenance [the CUP (conserved upstream repeat)-controlled regulon, the stbABC-orfD operon, and resD), and conjugal transfer (tra and kikA-korB) (Figure S1, Figure 1). There are two major differences among the backbones of these six plasmids: (i) a region between repA and its iterons from pYNKP001-NDM differs from all the other counterparts, and (ii) insertion, deletion, and rearrangement occur within the CUP-controlled regulons.

Gene organization and function of the IncN1 CUP-controlled regulon (Delver and Belogurov, 1997) have been described in the IncN1 reference plasmid R46 (accession number AY046276). Similarly, four putative operons (i.e., the ardK operon, the CUP-4 operon, the CUP-3 operon, the CUP-2 operon and the CUP-1 operon) arranged in the same orientation are annotated within the IncN2 CUP-controlled regulon, and CUP-4, CUP-3, CUP-2 and CUP-1 are located at the 5′-ends of the last four operons, respectively (Figure 2). Each of these operons contains a putative ArdK-binding site and a promoter, accounting for ArdK-dependent expression of operon-borne genes. A 40-bp deletion is found within CUP-3 of p7121-IMP. An ISEcp1-bla_CTX-M-62-Δorf477-orfRA1-14 transposition unit is inserted between CUP-4 and ardB in pJIE137 (Partridge et al., 2012), which would impair the gene expression of the CUP-4 operon. In p0801-IMP, homologous recombination medicated by CUP-3 and CUP-1 likely leads to an inversion of the orf792 to ros region as well as the disruption of CUP-3 and CUP-1.

p0801-IMP and p7121-IMP carry a novel class 1 integron In1223, containing 5′-CS, the cassette array bla_IMP-1-gcu162-aacA4-aadA6, and the complete Tn402 tni module (tniABQ-res-tniR), which is bordered by IRi (inverted repeat at the integrase end) and IRt (inverted repeat at the tni end) (Figure 3A). bla_IMP-1 and aacA4/aadA6 account for resistance to carbapenems and aminoglycosides, respectively, while gcu162 is a novel gene cassette of unknown function.

There are two accessory modules in each of pJIE137 (Partridge et al., 2012) and p17285-IMP. As shown in Figures 3A,B, p17285-IMP contains In655 (inserted into resD) and a 2554-bp ISEcp1-orfRA1-14 transposition unit (inserted between orf333 and orf648), while pJIE137 harbors ISEcp1-bla_CTX-M-62-Δorf477-orfRA1-14 (inserted within the CUP-4 operon) and In27 (inserted between resD and orf333). In655 differs from In1223 by presence of a distinct cassette array bla_IMP-8-aacA4 and, notably, its Tn402-family tni module is maximally only 95% identical to the others at nucleotide level. ISEcp1 captures and arranges orfRA1-14 and bla_CTX-M-62-Δorf477 at its immediately downstream, which generates the transposition units ISEcp1-orfRA1-14 and ISEcp1-bla_CTX-M-62-Δorf477, respectively, bordered by terminal inverted repeats IRL_ISEcp1 and IRR_ISEcp1. Similar ISEcp1-bla_CTX-M-Δorf477 structures (containing different variants of the bla_CTX-M-1 group) are found on plasmids from various bacterial hosts, while the ISEcp1-orfRA1-14 elements are found on plasmids from only enterobacterial species. ISEcp1-bla_CTX-M-62-Δorf477-orfRA1-14 is a hybrid of ISEcp1-bla_CTX-M-62-Δorf477 and an ISEcp1-orfRA1-14-related element that originates from splitting of ISEcp1-orfRA1-14 into the 5′- and 3′-parts, followed by inversion of the 5′-part (Zong et al., 2010).

Tn6325 (Figure 3A) from p34983-59.134kb is a novel derivative of Tn1696 belonging to the Tn21 subgroup of the Tn3 transposon family. Tn1696, located in the IncP1 plasmid R1033 from clinical P. aeruginosa, is generated from insertion of In4 within the resolution (res) site of a transposon backbone structure IRL-tnpA (transposase)-tnpR (resolvase)-res-mer (mercury resistance locus)-IRR, interrupting res into two separate parts (Partridge et al., 2001). Close Tn1696 relatives, which contain different In4-type integrons inserted at exactly the same position as In4, have been found on plasmids such as pHCM1, pSRC125 and pSRC26 (Cain et al., 2010). Tn6325 differs from Tn1696 by (i) insertion a distinct integron In1130 at the same position as In4 within res (Figure 3C), and (ii) disruption of IRL_Tn6325 into two parts by IS4321 (Figure 3A) that is a hunter of terminal inverted repeats of Tn21 subgroup transposons (Partridge and Hall, 2003).

The modular structure of a typical complex class 1 integron is organized sequentially as IRi, 5′-CS, variable region 1 (VR1), the first copy of 3′-CS [3′-CS1: qacED1 (quaternary ammonium compound resistance)-sul1 (sulfonamide resistance)], the common region ISCR1, VR2, the second copy of 3′-CS (3′-CS2; qacED1-sul1-orf5-orf6), tni, and IRt (Partridge et al., 2009). In27 from pJIE137 and In1130 from p34983-59.134kb (Figure 3A) belong to complex class 1 integrons because they contain all of the above core components with modifications of 3′-CS2. For both In27 and In1130, the connection of VR2 [ΔsapA-orf462-qnrB2 (quinolone resistance)] with 3′-CS2 leads to the truncation of qacED1 at the 5′-terminus of 3′-CS2. In addition, the tni module within the 3′-CS2 has been replaced by an IS6100 element in In1130, while the 3′-CS2 of In27 is interrupted into two separate parts ΔqacED1-sul1-Δorf5 and ΔtniA due to the insertion of a 6.8-kb region [composed of the chromate-resistance unit chrA-orf98 and the macrolide-resistance unit IS26-mph(A)-mrx-mphR(A)-IS6100], which is highly similar to the 3′-region of In37 from p112298-KPC (Feng et al., 2015). In In1130, ISCR1 is interrupted by the insertion of a cryptic Tn3-family unit transposon Tn5403.

The insertion of each of In1223, In655, ISEcp1-orfRA1-14, In27, ISEcp1-bla_CTX-M-62- Δorf477-orfRA1-14, In1130 and Tn6325 into the relevant plasmids leaves target site duplication signals of transposition, manifesting as various types of 5-bp direct repeat at the sites of insertion.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.