Characterization of a New Transposon, Tn6696, on a blaNDM–1-Carrying Plasmid From Multidrug-Resistant Enterobacter cloacae ssp. dissolvens in China

Background Enterobacter cloacae is an opportunistic pathogen which is responsible for serious nosocomial infections. A gene which plays an important role in resistance to carbapenems is the New Delhi metallo-β-lactamase 1 (NDM-1). Currently, the spread of NDM-1-producing E. cloacae strains is a serious public threat. Methods A multidrug-resistant E. cloacae ssp. dissolvens strain CBG15936 was recovered in 2017 in Guangzhou, China. PCR, S1-pulsed-field gel electrophoresis, and Southern blotting were performed to locate the blaNDM–1 gene. Susceptibility testing and conjugation experiments were also performed. Illumina HiSeq and Nanopore sequencers were used to perform whole-genome sequencing. Results Strain CBG15936 belongs to ST932 and is resistant to carbapenems. The blaNDM–1 gene was found on a ∼62-kb plasmid, which has a conjugation frequency of 1.68 × 10–3 events per donor cell. Genome sequencing and analysis revealed that the NDM-1-carrying IncN1 plasmid contained a new transposon Tn6696, which consists of an intact qnrS1-carrying Tn6292 element, an inverted 8.3-kb Tn3000 remnant, ISkpn19, ΔtnpA, and IS26. Conclusion A new transposon, Tn6696, has been detected on a blaNDM–1-carrying plasmid recovered from multidrug-resistant E. cloacae ssp. dissolvens CBG15936 from China. This finding provides a new perspective regarding the potential for blaNDM–1 to undergo horizontal transfer among drug-resistant bacteria.


INTRODUCTION
Enterobacter cloacae is a gram-negative opportunistic pathogen which belongs to Enterobacteriaceae. Previous studies have reported that E. cloacae are ubiquitous in nature and can be isolated from clinical samples (Davin-Regli et al., 2019). With the extensive use of antibiotics, carbapenem-resistant E. cloacae have become an important nosocomial pathogen which can cause septicemia and lower respiratory tract infections (Mayhall et al., 1979;Daw et al., 1989). In recent years, carbapenem-resistant E. cloacae isolates have been reported worldwide (Davin-Regli and Pagès, 2015). New Delhi metalloβ-lactamase 1 (NDM-1) was first detected in a Swedish patient transferred from India, and it plays a major role in the appearance and dissemination of carbapenem-resistant gramnegative bacteria (Yong et al., 2009;Jamal et al., 2013). NDM-1 has become a burden on the health care system especially in intensive care units (Ahmad et al., 2018;Khalid et al., 2019). The detection rate of NDM-1 in E. cloacae has been increasing globally (Karlowsky et al., 2017). Prevalence of NDM-1-carrying E. cloacae was observed in France (Perry et al., 2011) and Mexico (Torres-Gonzalez et al., 2015). Genome sequencing and analysis also revealed an NDM-1 E. cloacae outbreak in a hospital in the UK (Fairley et al., 2019). In the southwest region of China, 132 carbapenem-resistant E. cloacae isolates were obtained from patients between 2012 and 2016. Twenty (15.2%) of these strains were identified as NDM-1 positive (Jia et al., 2018). It is important to monitor the spread of bla NDM−1 among E. cloacae stains.
Transposons are mobile genetic elements which are able to translocate chromosome or plasmids. Transposons have been shown to carry drug resistance genes and provide antibiotic resistance in pathogenic bacteria (Whittle et al., 2002). Tn6292 is a Tn-family unit transposon which belongs to the Tn3family and has an IS26 at the right end. In addition, Tn6292 contains a quinolone resistance region qnrS1 . Multidrug-resistant (MDR) bacteria containing Tn6292 have been reported many times in China . Tn3000 is bracketed by IS3000 at both ends and contains the bla NDM−1 gene. Tn3000 has been shown to be responsible for transmission of the bla NDM−1 gene in various parts of the world (Campos et al., 2015). Thus, it is important to elucidate the genetic features of transposons in order to explore possible mechanisms of bacterial resistance. Therefore, in this study, we report a carbapenem-resistant E. cloacae ssp. dissolvens strain and the genetic features of the bla NDM−1 -harboring plasmid it carries. In addition, we identify a new transposon, Tn6696, present in this plasmid. These findings provide a new perspective regarding possible mechanisms of gene transmission to mediate drug resistance.

Bacterial Identification
Strain CBG15936 was recovered from the sputum of a patient in Guangzhou, China, in 2017. The strain was identified by using the Vitek 2 Compact System (BioMérieux, France) and confirmed with 16S rRNA gene sequencing. The bla NDM−1 gene was detected by PCR amplification as previously described (Zhang et al., 2013). This isolate was collected through routine surveillance, and verbal consent was obtained as no personally identifiable data were included. The ethics of the study was reviewed and supervised by the Center for Disease Control and Prevention of PLA. All experiments were performed in the biosafety cabinet following the standard procedure.

S1-Pulsed-Field Gel Electrophoresis (PFGE) and Conjugation Experiments
Genomic DNA was prepared in agarose plugs and digested with the S1 endonuclease (Takara, Dalian, China). DNA fragments were electrophoresed on a CHEF-DR III system (Bio-Rad, Hercules, United States) for 15 h at 14 • C with run conditions of 6 V/cm and pulse times from 0.22 to 26.29 s. The Salmonella enterica serotype Braenderup H9812 was used as the size marker. To determine the location of the bla NDM−1 gene, DNA was transferred to a positively charged nylon membrane (Roche) and then hybridized with digoxigenin-labeled bla NDM−1 . Conjugation experiments were carried out in LB broth cultures. Azide-resistant Escherichia coli strain J53 [F(-) met pro Azi(r)] (recipient) and strain CBG15936 (donor) were mixed at a 1:3 ratio and then incubated at 37 • C (Yi et al., 2012). After 18 h, transconjugants were selected for 12 h on MacConkey agar plates supplemented with meropenem (4 µg/ml) and sodium azide (150 µg/ml). Horizontal transferability of drug resistance was confirmed with antimicrobial susceptibility testing. Transconjugant-carrying plasmids were subsequently confirmed by PCR amplification and PFGE.

Susceptibility Testing
The minimum inhibitory concentrations (MICs) of amikacin, aztreonam, nitrofurantoin, ciprofloxacin, piperacillin, gentamicin, cefepime, ceftriaxone, ceftazidime, cefotetan, cefazolin, tobramycin, imipenem, and levofloxacin were determined with the Vitek 2 Compact System (Bobenchik et al., 2015). E. coli reference strain ATCC25922 was used as quality control. The MIC value of meropenem was determined using an E-test. The results were interpreted according to Clinical and Laboratory Standards Institute [CLSI] (2018) guidelines.

Whole-Genome Sequencing and Comparative Genome Analysis
Genomic DNA was extracted by using the High Pure PCR Template Preparation Kit (Qiagen, Inc., Valencia, CA, United States). Whole-genome sequencing was subsequently performed by using Illumina HiSeq according to the 350-bp paired-end protocol available from Novogene Company (Beijing, China) and MinION in our lab. The genome was assembled de novo by using a Unicycler (Li et al., 2010). RAST 2.0 was used to annotate the genome sequences obtained (Aziz et al., 2008). ResFinder v3.2 was used to identify acquired antibiotic resistance genes (Jia et al., 2016). Plasmid replicon type was analyzed by using PlasmidFinder (Carattoli et al., 2014). IS sequences were analyzed by using ISfinder (Siguier et al., 2006). Sequences of pNDM1-CBG and three similar plasmids were compared by using BLAST and Easyfig software (Sullivan et al., 2011). Whole-genome sequences of CBG15936 were used to determine multilocus sequence typing (MLST) based on detection of seven housekeeping genes (arcA, aspC, clpX, dnaG, fadD, lysP, and mdh) in pubMLST 1 (Larsen et al., 2012). Twenty-nine sequences of the hsp60 gene of eight Enterobacter clusters were retrieved from NCBI and used for phylogenetic analysis to identify the species of the strain.

Nucleotide Sequence Accession Numbers
The entire sequence of strain, CBG15936, as well as plasmids, pTEM-CBG and pNDM1-CBG, have been deposited in GenBank under accession numbers CP046116, CP046117, and CP046118, respectively.

Genetic Features of the bla NDM−1 -Carrying Plasmid
Plasmid pNDM1-CBG belongs to IncN1, has an average GC content of 52.34%, and has 91 open reading frames. Plasmid pNDM1-CBG also shares 99.95% identity and 100% coverage with plasmid pNDM-BTR, carried by E. coli BTR in Beijing, which was identified in 2013 (Zhao et al., 2017). Meanwhile, pNDM1-CBG shares 100% identity and 83% coverage with plasmid p378-IMP, carried by Pseudomonas aeruginosa in Chongqing, which was identified in 2013 . Both plasmids are composed of a similar ∼40-kb backbone and a variable multidrug resistance region. The backbone is composed of replication (repA), antirestriction (ardA and klcA), stability (stdB), conjugation (tra), and type IV secretion system (virB) genes. Compared with pNDM-BTR, pNDM1-CBG had an inversed Tn3000, an insertion of ISkpn19, a deletion of 144 bp upstream of the MDR region, and deletion of IS26 downstream of the MDR region. Compared with p378-IMP, pNDM1-CBG is missing ardA downstream of the antirestriction region, yet it is present before the stability region. Replacement of In823b (which contains bla IMP−4 downstream of the type IV secretion region) with In191 (which contains dfrA14) is observed in plasmid pNDM1-CBG ( Figure 3A). The MDR region in pNDM1-CBG is composed of a novel transposon designated Tn6696. The latter consists of Tn3000, Tn6292, IS26, ISkpn19, and tnpA. The bla NDM−1 gene FIGURE 2 | S1-PFGE pattern and Southern blotting for strain CBG15936 and E. coli J53 transconjugants. Lanes: Marker, Salmonella serotype Braenderup strain H9812 as a reference size standard. (A) PFGE result for S1-digested plasmid DNA of strain CBG15936. (B) Southern blot hybridization with probes specific to bla NDM−1 ; (C) PFGE result for S1-digested plasmid DNA of strain E. coli J53 transconjugants.

DISCUSSION
E. cloacae have a broad range of hosts and have been isolated from wastewater, patients, and soil microcosms (Osborn et al., 2000;Chen et al., 2014;Zhang et al., 2014). Here, we report an NDM-1-producing MDR E. cloacae ssp. dissolvens strain, CBG15936, recovered from China. Our previous study showed that the expression of bla NDM−5 differed in plasmids and that the transconjugants with one plasmid exhibited higherlevel carbapenem resistance than those with two plasmids or FIGURE 3 | A comparative schematic diagram of (A) entire plasmids including VI (39,744 bp), p379-IMP (51,207 bp), pNDM1-CBG (62,663 bp), and pNDM-BTR (59,400 bp) and (B) transposons including Tn6292 in p37-IMP, Tn6696 in pNDM1-CBG, Tn6360 in pNDM-BTR, and Tn3000. Open reading frames are indicated with arrows. Homology regions among different plasmids are denoted with light gray coloring. Gene backbones are shown in dark gray. Black, carmine, cyan, orange, and green arrows represent genes association with replication, antirestriction, stability, conjugation, and the type IV secretion system, respectively. Accessory modules are shown in blue; gene backbones of integron are shown in royal blue; bla NDM−1 is shown in red; qnrS1 is shown in yellow; bla IMP−4 is shown in brown; dfrA14 is shown in purple.
larger plasmids (Yang et al., 2020). Resistance to meropenem of transconjugants and strain CBG15936 may be affected by different NDM expressions and numbers of plasmids. This strain contains a novel transposon, Tn6696, carrying bla NDM−1 and located on the IncN1 plasmid pNDM1-CBG. Considering broad-host-range plasmids generally occurs at a variable frequency from 10 −3 to 10 −6 (Grohmann et al., 2003), pNDM1-CBG exhibited a relatively high transfer frequency. Our results support the potential for cross-species transmission to occur.
The novel transposon, Tn6696, identified in pNDM1-CBG contains an inverted Tn3000 remnant and an intact Tn6292. The latter is a Tn3-family transposon with an IS26 at the right end and has previously been found in a pP378-IMP plasmid obtained from P. aeruginosa recovered in China . Tn3000 was first located in a pEh1A plasmid from Enterobacter hormaechei E0083033-1 and in pEc2A from E. coli E0083033-2 recovered from Brazil (Campos et al., 2015). Therefore, a reorganization event involving fusion of Tn3000 and Tn6292 may have created Tn6696. The NDM-1 gene in Tn6696 is bracketed by two copies of the insertion sequence, ISkpn19. It is hypothesized that the resulting special structure has the potential to mobilize a drug resistance gene (Wu et al., 2016). However, Tn6696 had an additional copy of ISkpn19 and an inverted Tn3000 compared with Tn6360, suggesting that the former may originate from the latter through transposition and inversion of the ISkpn19-Tn3000 structure.
Plasmid pNDM1-CBG also shares high similarity, yet lower coverage, with plasmid VI from E. coli. The main difference between these plasmids is the quite variable MDR regions, which are all inserted in the fipA gene at the same position. In a previous study, fipA was shown to be interrupted into two fragments by MDRs in many different plasmids (Zhao et al., 2017;Yang et al., 2018). Thus, fipA may serve as a "hot spot" for integration of mobile genetic elements.

CONCLUSION
Here, we report a new transposon, Tn6696, in the IncN1 plasmid, pNDM1-CBG, from a ST932 MDR E. cloacae ssp. dissolvens strain recovered in China. We describe the structures of both pNDM1-CBG and Tn6696 in detail. Our work provides a new perspective regarding the potential for a novel horizontal transfer of NDM-1 via Tn6696 to occur among IncN1 plasmids. The latter finding is of great significance for future studies of the dissemination of bla NDM−1 in different species and its monitoring.

DATA AVAILABILITY STATEMENT
The datasets generated for this study can be found in the CP046116, CP046117, and CP046118.

AUTHOR CONTRIBUTIONS
PL, HM, and HS conceived and designed the experiments. QC, LL, YL, and KW performed the experiments. ZL, PHL, and LY analyzed the data. QC and PL wrote the manuscript. All authors read and approved the final manuscript.

FUNDING
The study was supported by the grants from the National Science and Technology Major Project (Grant No. 2018ZX10712001-002-002) and the Beijing Nova Program (Grant No. Z181100006218110).