Dissemination of IMP-4-encoding pIMP-HZ1-related plasmids among Klebsiella pneumoniae and Pseudomonas aeruginosa in a Chinese teaching hospital

A total of 26 blaIMP-4-carrying strains of Pseudomonas aeruginosa and Klebsiella pneumoniae were isolated from 2009 to 2013 in a Chinese teaching hospital, and these strains can be assigned into multiple sequence types or allelic profiles as determined by multilocus sequence typing. Of these strains, P. aeruginosa P378 and K. pneumoniae 1220 harbor the IMP-4-encoding plasmids pP378-IMP and p1220-IMP, respectively, whose complete nucleotide sequences are determined to be genetically closely related to the IncN1-type plasmid pIMP-HZ1. pP378-IMP/p1220-IMP-like plasmids are hinted to be present in all the other blaIMP-4-carrying strains, indicating the dissemination of pIMP-HZ1-related plasmids among K. pneumoniae or P. aeruginosa of different genotypes in this hospital. pP378-IMP carries two distinct accessory resistance regions, a blaIMP-4-carrying class 1 integron In823b, and a truncated Tn3-family unit transposon ΔTn6292-3′ harboring the quinolone resistance gene qnrS1. Massive fragmentation and rearrangement of these accessory genetic contents occur among p1220-IMP and IMP-HZ1 relative to pP378-IMP. blaIMP-4 is also present in the In823b remnants from p1220-IMP and IMP-HZ1, while qnrS1 is located in a Tn6292-derive fragment from pIMP-HZ1 but not found in p1220-IMP. pP378-IMP represents the first fully sequenced IncN-type plasmid from P. aeruginosa.

pP378-IMP and p1220-IMP from P. aeruginosa and K. pneumoniae. Two bla IMP-4 -positive strains, P. aeruginosa P378 isolated from the urine specimen of a 36-year-old male with urinary tract infection and consciousness disturbance, and K. pneumoniae 1220 from the blood specimen of a three-month-old baby boy with neonatal septicemia and hyperbilirubinemi, were arbitrarily selected for transferring the bla IMP-4 marker into E. coli EC600 through conjugation, generating the bla IMP-4 -positive E. coli transconjugants P378-IMP-EC600 and 1220-IMP-EC600, respectively. All these four strains had the class B carbapenemase activity and were resistant to piperacillin, piperacillin/tazobactam, cefazolin, cefuroxime, ceftazidime, cefepime, imipenem, and meropenem; moreover, P378 and P378-IMP-EC600, but not 1220 and 1220-IMP-EC600, were resistant to ciprofloxacin and levofloxacin (Table 1). Taken together, either P. aeruginosa P378 or K. pneumoniae 1220 harbors a conjugative bla IMP-4 -carrying plasmid, designated pP378-IMP and p1220-IMP, respectively, which account for the carbapenem resistance phenotype.
Whole-genome sequencing of pP378-IMP and p1220-IMP (with mean coverage > 80), recovered from the P378-IMP-EC600 and 1220-IMP-EC600 strains, respectively, showed that these two plasmids have circularly closed DNA sequences, 51,207 bp and 46,629 bp in length, respectively (Fig. 2). pP378-IMP and p1220-IMP have mean GC contents of 50.5% and 50.7% and contain 64 and 60 predicted open reading frames in total, respectively ( Fig. 2). Backbones of pP378-IMP and p1220-IMP. The entire sequences of pP378-IMP and p1220-IMP are mostly similar to that of pIMP-HZ1 (> 99% query coverage and maximum > 99% nucleotide identity). pP378-IMP, p1220-IMP and pIMP-HZ1 possess the conserved IncN1-type backbone regions, which contain a repA gene and its iterons (RepA-binding sites; regulation of replication) for plasmid replication, the tra genes and kikA-korB for conjugal transfer, the CUP (conserved upstream repeat) -controlled regulon, the stbABC-orfD operon, and resP) for plasmid maintenance (Fig. 2). These backbone regions are highly similar to the IncN1 prototype plasmid R46 from Salmonella enterica serovar Typhimurium.
There are four major genetic differences among the backbones of pP378-IMP, p1220-IMP and IMP-HZ1. First, a total of 7 copies of 37 bp to 40 bp tandem repeats are observed within the repA iterons of pIMP-HZ1, while only 3 copies are found in pP378-IMP and p1220-IMP (Fig. 3). Second, pP378-IMP and IMP-HZ1 contains an intact antirestriction system ecoRII-ecoRIImet (located around 8.5 kb to 11.5 kb nucleotide position of pP378-IMP), while only a truncated ecoRIImet gene is found in p1220-IMP and this truncation likely results from the insertion of ISKpn19 upstream (Fig. 3). Third, the inversion of the conjugal transfer region from orf207 to the 3′ -end remnant of fipA undergone occurs within pP378-IMP and p1220-IMP related to IMP-HZ1 (Fig. 3).

Category
Antibiotics MIC (mg/L)/antimicrobial susceptibility 1220 1220-IMP-EC600 P378 P378-IMP-EC600 EC600 Table 1. Antimicrobial drug susceptibility profiles. S = sensitive; R = resistant; I = intermediate. The fourth major genetic difference ( Fig. 4) is found within the CUP-controlled regulon 10 . A total of four putative operons, namely the CUPA operon, the CUPB operon, the CUP5 operon, the CUP4 operon, the CUP3 operon, the CUP2 operon and the CUP1 operon, are arranged within this regulon; each of these operons contains a putative ArdK-binding site and a promoter, which are responsible for ArdK-dependent expression of corresponding genes 10 . Compared with pIMP-HZ1, the translocation of the CUP2 operon occurs within pP378-IMP and p1220-IMP, which most likely results from the homologous recombination mediated by CUP1, CUP2 and CUP4 (Fig. 4).
Multiple copies of IS26 are present in the In823b-and Tn6292-related regions of pP378-IMP, p1220-IMP and IMP-HZ1, and the common component IS26 would act as an adaptor 11,12 to mediate massive fragmentation and rearrangements of In823b-and Tn6292-related regions in p1220-IMP and IMP-HZ1 relative to pP378-IMP (Fig. 5), leaving different mosaic assemblies from the remnants of In823b and Tn6292 in p1220-IMP and IMP-HZ1. Nevertheless, all these accessory genetic contents are integrated at two "hotspots" (Fig. 1), namely a region downstream of resP (resolvase) and a region within fipA (fertility inhibition protein), which has been previously described in IncN1 plasmids 2,4 .
Compare with the fragmentary In823b-related regions in pIMP-HZ1 and p1220-IMP, the In823b integron from pP378-IMP looks like a primitive form flanked by a complete set of inverted repeats (IRs, 25 bp in length) and direct repeats (DRs, 5 bp in length: target site duplication signals of transposition) (Fig. 5) 13 . The 5′ -conserved segment [5′ -CS: IRi (inverted repeat initial)-intl1 (integrase)-attI] of In823b is disrupted by the insertion of IS26 into intl1. In823b contains a single resistance gene cassette bla IMP-4 -attC blaIMP-4 , and a group IIc intron Kl.pn.I3 disrupts an unusual attC site that appears to be a chimera between attC blaIMP-4 and attC dfrA14GC . Downstream of attC dfrA14GC is a structure mobC (Mobilization protein)-IRi-IS6100-IRt (inverted repeat terminal), but the typical 3′ -conserved segment [3′ -CS: qacED1 (quaternary ammonium compound resistance)-sulI (sulfonamide resistance)-IRt] in not found. The expression of bla IMP-4 is driven by a single promoter PcW  . which is a derivate of the weak promoter PcW and much stronger than PcW due to the C to G mutation 2 bp upstream of the − 10 element 14 .
Compared to In823b and Tn6292-3′ from pP378-IMP, massive fragmentation of these two accessory regions, followed by further inversion and translocation of the resulting In823b-and Tn6292-derived fragments, occurs in p1220-IMP and IMP-HZ1, leaving the assembly of different combinations of accessory regions with a very complex mosaic nature in these two plasmids (Fig. 5). pP378-IMP contains a total of two resistance genes bla IMP-4 and qnrS1, which are captured by In823b and Δ Tn6292-3′ , respectively. bla IMP-4 is also present in the In823b-derived elements In823b-1 and In823b-2 (which can be discriminated as the partial regions of In823b) from p1220-IMP and IMP-HZ1, respectively. qnrS1 is also present in Δ Tn6292-5′ from pIMP-HZ1, but it not found in p1220-IMP.
Prevalence of pP378-IMP/p1220-IMP-related plasmids. A total of 12 backbone genes repA, mrr, kikA, traL, traB, traF, traJ, stdB, ccgAII, ardA, mucB, and ardK as well as the accessory quinolone-resistance gene qnrS1 were arbitrarily selected for PCR detection, followed by amplicon sequencing (data not shown). It was found that all these 12 backbone genes were present in all the bla IMP-4 -carrying 19 K. pneumoniae strains and 7 P. aeruginosa strains (Table S1). The above results indicated that pP378-IMP/p1220-IMP-like plasmids were harbored in all these bla IMP-4 -carrying K. pneumoniae and P. aeruginosa strains. The qnrS1 gene was detected in 3 bla IMP-4 -carrying K. pneumoniae strains and in 4 bla IMP-4 -carrying P. aeruginosa strains (Table S1), denoting the probable coexistence of the In823-derived bla IMP-4 regions and the Tn6296-derived qnrS1 regions in these strains.

Methods
Bacterial strains and identification. Bacterial species was identified by 16S rRNA gene sequencing 17 and by PCR detection of K. pneumoniae-specific gene khe 18 , P. aeruginosa-specific oafA 19 and A. baumannii-specific bla OXA-51 20 . The major plasmid-borne carbapenemase and extended-spectrum β -lactamase genes were screened for by PCR 21 , followed by amplicon sequencing on ABI 3730 Sequencer (LifeTechnologies, CA, USA). The MLST schemes for K. pneumoniae and P. aeruginosa were derived from the PubMLST database (http://pubmlst.org/).

Plasmid conjugal transfer.
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 P378 or 1220 as donor. 3 ml of overnight culture of each of donor and recipient bacteria were mixed together, harvested and resuspended in 80 μ l of Brain Heart Infusion (BHI) broth (BD Biosciences). The mixture was spotted on a 1 cm 2 filter membrane that was placed on BHI agar (BD Biosciences) plate, and then incubated for mating at 37 °C for 12 to 18 h. Bacteria were washed from filter membrane and spotted on Muller-Hinton (MH) agar (BD Biosciences) plate containing 1000 μ g/ml rifampin and 2 μ g/ml imipenem for selection of bla IMP -positive E. coli transconjugants.
Bacterial antimicrobial susceptibility test. Bacterial  Plasmid sequencing and sequence assembly. Plasmid DNA was isolated from E. coli transconjugant using Qiagen large construct kit (Qiagen, Hilden, Germany), and sequenced by whole-genome shotgun strategy in combination with Illumina HiSeq 2500 (Illumina, San Diego, CA, USA) sequencing technology. Reads from each sample were trimmed to remove poor quality sequences, and then the contigs were assembled with Velvet. The gaps were filled through combinatorial PCR and Sanger sequencing on ABI 3730 Sequencer.