A Multidrug Resistance Plasmid pIMP26, Carrying blaIMP-26, fosA5, blaDHA-1, and qnrB4 in Enterobacter cloacae

IMP-26 was a rare IMP variant with more carbapenem-hydrolyzing activities, which was increasingly reported now in China. This study characterized a transferable multidrug resistance plasmid harboring blaIMP-26 from one Enterobacter cloacae bloodstream isolate in Shanghai and investigated the genetic environment of resistance genes. The isolate was subjected to antimicrobial susceptibility testing and multilocus sequence typing using broth microdilution method, Etest and PCR. The plasmid was analyzed through conjugation experiments, S1-nuclease pulsed-field gel electrophoresis and hybridization. Whole genome sequencing and sequence analysis was conducted for further investigation of the plasmid. E. cloacae RJ702, belonging to ST528 and carrying blaIMP-26, blaDHA-1, qnrB4 and fosA5, was resistant to almost all β-lactams, but susceptible to quinolones and tigecycline. The transconjugant inherited the multidrug resistance. The resistance genes were located on a 329,420-bp IncHI2 conjugative plasmid pIMP26 (ST1 subtype), which contained trhK/trhV, tra, parA and stbA family operon. The blaIMP-26 was arranged following intI1. The blaDHA-1 and qnrB4 cluster was the downstream of ISCR1, same as that in p505108-MDR. The fosA5 cassette was mediated by IS4. This was the first report on complete nucleotide of a blaIMP-26-carrying plasmid in E. cloacae in China. Plasmid pIMP26 hosted high phylogenetic mosaicism, transferability and plasticity.

Notoriously, extended and overuse of antibiotics have potentiated globally rapid emergence and spread of carbapenem-resistant Enterobacterales (CRE), posing a serious threat to clinical therapy and infection control [1][2][3] . The major driving force for the diversification and dissemination of CRE has been confirmed as the horizontal transfer of plasmid-mediated carbapenem-hydrolyzing enzymes (i.e., carbapenemase) genes 4 , among which the most prevalent and of particular clinical importance were bla KPC , bla VIM , bla IMP , bla NDM , and bla OXA-48 5 . IMP, one kind of metallo-β-lactamases (MBLs), can efficiently inactivate almost β-lactams except monobactam 5 . IMP-1 was the first transferable MBL detected from Pseudomonas aeruginosa in Japan in 1991 6 ; subsequently, the continuously clinical detection of bla IMP-1 in different species isolates in Japan 7,8 , as well as the discovery of IMP-2 in Italy 9 and IMP-5 in Portugal 10 , marked the beginning of the upcoming flourish of IMP MBLs 11 . IMP-26 was first reported as an IMP-4 variant in Singapore in 2010 from a clinical carbapenem-resistant P. aeruginosa isolate by Koh TH et al. 12 . However, since then, there have been only sporadic reports on the IMP-26-production in Gram-negative bacilli [13][14][15] , especially in Enterobacterales 15 . Notably, isolates expressing IMP-26 were found significantly more resistant to doripenem and meropenem than that expressing IMP-1 13 .
Enterobacter cloacae was one member of the normal intestinal microflora of humans and animals, which has also assumed clinical importance and emerged as a major human pathogen causing hospital-acquired bacteremia, nosocomial pneumonia, urinary tract infections and so on 16,17 . In the past decade, the emergence of IMP-producing E. cloacae has been extensively reported as a challenge to clinical therapy because of its rapid worldwide transmission 14,16,18 . And in China, the most common IMP variants found in E. cloacae were IMP-8 and IMP-4 11,19,20 . As for IMP-26-producing E. cloacae, it has been only reported in Chongqing, Shanghai and Beijing worldwide 19,21,22 . Multilocus sequence typing (MLST). A MLST scheme was used to assign E. cloacae to clonal lineages, including seven housekeeping genes (dnaA, fusA, gyrB, leuS, pyrG, rplB, and rpoB) as described by Miyoshi-Akiyama 26 . The combination of seven alleles can define the sequence types (STs) on the MLST website (http://pubmlst.org/ecloacae/). Plasmid conjugation, S1-nuclease pulsed-field gel electrophoresis (S1-PFGE), and southern hybridization. The transferability of the resistance genes was assessed in broth culture using E. coli J53 Azr (sodiumazide-resistant) as the recipient. The transconjugants were selected on MacConkey agar containing sodiumazide (100 mg/L) and meropenem (2 mg/L) or ceftazidime (1 mg/L). PCR was employed to confirm the existence of bla IMP-26 . DNA plugs of the parental and transconjugant digested with S1-nuclease were prepared and separated by PFGE, and then transferred to positively charged nylon membrane (Roche Applied Science, Germany). The membrane was hybridized with digoxigenin-labeled bla IMP-26 specific probes.
DNA sequencing and genomics analysis. Genomic DNA of E. cloacae RJ702 was isolated using ChargeSwitch ® gDNA Mini Bacteria Kit (Life Technologies, Carlsbad, CA, USA) and sequenced by a combination of PacBio RSII (Pacific Biosciences, Menlo Park, CA, USA) and Illumina Hiseq X10 (Illumina, San Diego, CA, USA) sequencing platforms. The assembly was produced firstly using a hybrid de novo assembly solution modified by Koren, in which a de-Bruijn based assembly algorithm and a CLR reads correction algorithm were integrated in "PacBioToCA with Celera Assembler" pipeline 27,28 . The final assembly generated a circular genome sequence with no gap existed. The precise species identification was established based on average nucleotide identity (ANI) between RJ702 and other type strains of E. cloacae subsp. using Orthologous ANI Tool (OAT) recommended by Lee I et al. 29 . Annotation of the genomic sequence and alignment with other similar sequences were carried out using the BLAST Ring Image Generator (BRIG) 30 and SnapGene program v4.3.2. Open reading frames (ORFs) were identified using Glimmer version 3.02 (http://cbcb.umd.edu/software/glimmer/). ORFs less than 300-bp were discarded. Insertion elements and resistance genes were identified using ISFinder (https:// www-is.biotoul.fr) and ResFinder (https://cge.cbs.dtu.dk/services/ResFinder). PlasmidFinder (https://cge.cbs. dtu.dk/services/PlasmidFinder) and pMLST (https://cge.cbs.dtu.dk/services/pMLST/) were employed to detect and type the plasmids. BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to identify related plasmids carrying bla IMP to guide PCR-based gap closure and Sanger sequencing to assemble contigs into complete plasmids.
Nucleotide sequence accession number. The completely annotated sequence of pIMP26 in E. cloacae RJ702 has been deposited in GenBank database (Accession Number: MH399264).

Ethics approval and informed consent. The study was approved by Ruijin Hospital Ethics Committee
(Shanghai Jiao Tong University School of Medicine), and the Review Board exempted the requirements for informed consent as this study only focused on bacteria.
Genome sequencing of RJ702. Whole genome sequencing generated 1,458,457 single reads and 4.70 Gb clean data total bases, which were de novo assembled to 184 contigs (75 > 1,000 bp; N50: 269,528 bp; N90: 45,440 bp). The bases in all contigs of RJ702 was 5.01 Mb with a 54.69% G + C content. The size of chromosome was 4,303,224 bp, and the bases in all contigs of two plasmids in RJ702 were 329,420 bp (pIMP26) and 78,322 bp respectively. PlasmidFinder presented that plasmid pIMP26 hosted two replicons, of which IncHI2 was 327 bp and IncHI2A was 630 bp; while the other plasmid hosted none.

Discussion
The undesirable antibiotic resistance (especially carbapenem-resistance) has appeared and disseminated rapidly in Gram-negative bacilli, which was attributed largely to the acquisition of multiple resistance genes by horizontal plasmid-mediated genes transfer 4,5 . Our study was to map the genetic environment of a novel multi-drug-resistance plasmid pIMP26, in order to provide a new insight for the potential spread of bla IMP-26 and fosA5 or correlations between genetic diagnosis and clinical treatment.
Firstly, the backbone of pIMP26 was blasted with different plasmids in BLAST. The origins of functional modules in pIMP26, such as multiple antibiotic resistance determinants, stably conjugal transfer (tra and trh family), mobile elements and plasmid maintenance (stb family) (Fig. 2), represented a strong transferability, stability and plasticity of this plasmid 31 . IncHI2 was one of the most prevalent broad-host-range plasmid families carrying different resistance determinants simultaneously in Enterobacterales 4,31,32 . As previously reported on E. cloacae, most β-lactamase-encoding genes (bla SHV-12 , bla CTX-M-15 , bla NDM-1 , bla IMP-4 , etc.) were also located on IncHI2 plasmids (subtype ST1) of 290~340-kb in size 18,[31][32][33][34][35] , and our study also fit it. It should be noted that the similar backbone shared by pIMP26 and other plasmids (Fig. 2) in clinical isolates of E. cloacae, K. pneumoniae and S. enterica from different areas strongly suggested that inter-species genetic exchange also occurred, thus broadening the host range and dissemination of combined cargo genes. Besides, pIMP26 contained a wide variety of transposable elements carrying known antibiotic resistance genes. Tn3 family transposon was the medium of TEM genes and fosA5 was also located in Tn3 in pIMP26 (Figs 2 and 3). The archetype of Tn3 was known as some of the earliest unit transposons identified in Gram-negative bacilli. Tn3 family members demonstrated transposition immunity, but homologous and/or res-mediated recombination between related elements can occur, creating hybrid elements 31 . And this would explain multiple Tn3-mediated resistance elements in pIMP26 in this study. However, further study is definitely needed to characterize the mechanisms behind the transfer or recombination of Tn3.
IMP-26, firstly found in P. aeruginosa in Singapore, was differed from IMP-4 at position 145 (G to T change); the translated amino acid sequence differed from IMP-4 at residue 49 (phenylalanine for valine) 12 . Blast searches indicated that the genetic structure surrounding bla IMP-26 has only revealed in a study from Vietnam up to now, containing intI1-bla IMP-26 -qacG-aac(6′)-Ib-orf3-orf4 (Fig. 2) 13 , and our study was the first time focusing on the complete nucleotide of the plasmid carrying bla IMP-26 . Interesting was the bla IMP-26 region in pIMP26 different from that found in Vietnam (though both located on intI1) 13 ; but same as the bla IMP-4 cluster of pIMP-4 in www.nature.com/scientificreports www.nature.com/scientificreports/ Shanghai (Genbank ID: FJ384365) (Fig. 3). It prompted that the bla IMP-26 detected in our study maybe originated from bla IMP-4 or the genetic mutation may occur during transfer of bla IMP-26 cassette.
The prevalence and dissemination of fosA5 have probably been underestimated 36 . Previous study once found IS10 playing an important role in the mobilization of fosA5 37 . However, the upper half consistent with pHKU1 in  www.nature.com/scientificreports www.nature.com/scientificreports/ pIMP26 indicated that IS4 might also related to its mobilization 36 (Fig. 3). Plasmid carrying bla DHA was usually reported also carrying qnrB4, bla SHV-12 38 . This suggested that the cassette in common of qnrB4 and bla DHA-1 (Fig. 3) (including that in pIMP26) was derived from the same immediate ancestor. The qnrB4-bla DHA -containing region of pIMP26 was located after the 3′ conserved sequence (3′-CS) of intI1 (Fig. 3), containing aac(6′)-IIc, qacEΔ1 and sul1. Besides, an insertion sequence common region 1 (ISCR1) was identified downstream of sul1. ISCR1 could mobilize the nearby sequence and a truncated 3′-CS from one integron to the 3′-CS of another integron through rolling-circle transposition, and provide a promoter for the expression of nearby genes 39 ; this may lead to the co-carriage of multiple resistant genes in one plasmid and the multi-drug resistance of clinical isolates.
Interestingly, our study showed the qnrB4-and aac(6′)-Ib3-harboring RJ702 susceptible to quinolones (MIC = 0.5 or 0.25). We speculated that it was due to the absence of other mechanisms of chromosomal resistance (e.g. alterations in type II topoisomerases) in RJ702 other than plasmid-mediated quinolone resistant (PMQR) genes. Researchers found that PMQR mechanism caused only low-level quinolone-resistance on its own, which may not exceed the clinical breakpoints of susceptibility for quinolones but facilitated selections of higher-level resistance and posed threats to the treatment of infections by microorganisms hosting PMQR genes 40 , which could validate our speculation and underline the necessity of monitoring on PMQR genes. This is the first report on the entire structure of bla IMP-26 -carring plasmid. To some extent, our study evidenced the increasing clinical significance of IncHI2 replicons as resistance genes' reservoirs and provided insights on the possibilities of further spread in China and highlighted the needs for intensive surveillance and precautions.

Data Availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.