Multidrug-resistant Citrobacter freundii ST139 co-producing NDM-1 and CMY-152 from China

The emergence of carbapenemase-producing Citrobacter freundii poses a significant threat to public health worldwide. Here, we reported a C. freundii strain CWH001 which was resistant to all tested antimicrobials except tetracycline. Whole genome sequencing and analysis were performed. The strain, which belonged to a new sequence type ST139, showed close relationship with other foreign C. freundii strains through phylogenetic analysis. A novel variant of the intrinsic blaCMY gene located on the chromosome was identified and designated as blaCMY-152. Coexistence of blaNDM-1 with qnrS1 was found on a conjugative IncN plasmid, which had a backbone appearing in various plasmids. Other class A ESBL genes (blaVEB-3 and blaTEM-1) were also detected on two different novel plasmids. The emergence of multidrug-resistant C. freundii is of major concern, causing great challenges to the treatment of clinical infections. Great efforts need to be taken for the specific surveillance of this opportunistic pathogen.

indicated that the 3.2 kb bla VEB-3 -carrying contig was composed of a novel combination of Klebsiella pneumoniae JM45 plasmid p1 (CP006657, unpublished) and uncultured bacterium plasmid pKAZ5 16 . The presence of the bla NDM-1 and bla VEB-3 genes in the transconjugants was further confirmed by PCR amplification and sequencing. Sulfamethoxazole-trimethoprim ≥320 ≤20 Table 1. Antibiotic susceptibilities of C. freundii strain CWH001 and the E. coli J53 transconjugants. Figure 1. S1-PFGE pattern for strain CWH001 and southern blotting for the bla NDM-1 , bla TEM-1 and bla VEB-3 genes. Lanes: Marker, Salmonella serotype Braenderup strain H9812 as a reference size standard; 1, PFGE result for S1-digested plasmid DNA of strain CWH001; 2-4, southern blot hybridization with the probes specific to bla NDM-1 , bla TEM-1 and bla VEB-3 , respectively. Full length S1-PFGE and southern blotting results are presented in Supplementary Fig. S1. In addition to bla NDM-1 and bla VEB-3 , other resistance genes were also identified in strain CWH001 including bla TEM-1 , qnrS1, dfrA12, armA, fosA3, mphA, sul1, aac(3)-IId and a novel variant of the bla CMY gene. Analysis of the deduced protein sequence of the bla CMY variant revealed a single amino acid substitution at position 22 (Thr → Ala) relative to that of CMY-41. This variant protein was designated CMY-152 (http://www.lahey.org/ Studies/webt.asp). BLAST search and southern blotting revealed the bla CMY-152 gene, together with its regulator gene ampR flanked by the upstream frd genes and the downstream blc gene, was located on the chromosome.

Molecular typing and phylogenetic analysis.
The NDM-1-producing C. freundii CWH001 did not belong to an existing sequence type and was assigned to a new ST, ST139, using the multi-locus sequence typing (MLST) web server. Phylogenetic analysis revealed a high degree of genetic diversity of 84 available C. freundii genomes with that of CWH001. CWH001 was clustered into clades with overseas strains, and had a close relationship with strain 5-172-05_S1_C1 from Tanzania (Fig. 2). Only 543 SNPs were detected between the chromosomes of strain CWH001 and 5-172-05_S1_C1. However, CWH001 fell into different clades and showing distant phylogenetic relationship to other domestic strains. Sequence alignments revealed the average nucleotide identity (ANI) between CWH001 and other isolates from China ranged from 92.20% to 98.52%, while the ANI between CWH001 and 5-172-05_S1_C1 was 99.50%, indicating a different evolutionary pathway of CWH001 from other domestic strains in China.
Characterization of bla NDM-1 -carrying plasmid pNDM-CWH001. The 59-kb plasmid carrying bla NDM-1 was completely assembled and designated as pNDM-CWH001. pNDM-CWH001 belonged to the incompatibility type IncN. BLAST search against NCBI revealed that pNDM-CWH001 showed 100% coverage and >99% identity to the E. coli plasmid pNDM-BTR 17 from China. Two single nucleotide deletions located within virB4 and virB8, respectively, were identified in pNDM-BTR. pNDM-CWH001 consisted of a bla NDM-1 -containing transposon Tn6360 and a 42.3-kb backbone (Fig. 3a). Tn6360 was composed of an accessory region carrying bla NDM-1 and an intact Tn6292 element carrying qnrS1 (Fig. 3b). The accessory region comprised an IS26, a 427-bp truncated tnpA, and an 8.3-kb Tn3000 remnant (IS3000-ΔISAba125-bla NDM-1 -ble-trpF-tat-ΔcutA1-groES-ΔgroEL). Compared with the prototype Tn3000 18 , the remnant had undergone a deletion of the second copy of IS3000 together with the truncation of groEL in the 3′ extremity, suggesting a possible transposition event. The transposon Tn6292 had a quinolone resistance genetic platform organized as IS26-qnrS1-ISkpn19, which has been repeatedly reported in previous plasmids 19,20 and was likely introduced due to the inter-plasmid transfer as a transposable element 21 .
The backbone of pNDM-CWH001 also presented >98% identity to those of pMR3-OXA181 22 (100% coverage) and pIMP-GZ1058 23 (92% coverage). The backbone contained a set of core genes for plasmid replication (repA), conjugation (tra genes), stability (stdB), antirestriction (ardA and klcA) and type IV secretion system (virB genes). However, there existed an inversion of a 1-kb region in plasmid pNDM-CWH001 and pNDM-BTR, which encoded aldehyde dehydrogenase and transcriptional regulator. An additional IS26 was inserted following this inversion region. The bla NDM-1 -carrying transposon Tn6360 was integrated into the fipA gene, which was interrupted into two fragments in pNDM-CWH001 compared to plasmid R46 24 and may serve as a "hotspot" for insertion of transposable elements.
Genetic features of plasmid pTEM-CWH001. The bla TEM-1 gene was located on a novel plasmid designated as pTEM-CWH001, which had the length of 107,391 bp and comprised a combination of C. freundii plasmid p112298-KPC 9 and Salmonella enterica plasmid pF8475 25 . pTEM-CWH001 could not be assigned to any known incompatibility group. The deduced replication initiator RepA presented >98% amino acid similarity with various IncFII family RepA proteins from Citrobacter. An insertion of ISEc42 between conjugal transfer genes tra and trb were observed, which was likely to impair the expression of the trbABC operon and may result in a non-transferable plasmid. pTEM-CWH001 harbored a Tn21-like structure bound by the transposition genes (tnpAR) and the mer operon in the 5′ and 3′ portion, respectively. The bla TEM-1 gene and an insertion sequence ISCfr1 were located upstream of the Tn21-like structure. Compared with the prototype Tn21, this structure had undergone the replacement of aadA1 by dfrA12 and an insertion of a macrolide resistance operon organized as mphA-mrx-mphR in the class 1 integron In2, suggesting possible frequent transposition events.

Discussion
The ability to produce NDM-1 carbapenemases has been acquired by diverse Enterobacteriaceae species and posed a significant threat to public health. Our study identified a bla NDM-1 -positive C. freundii isolate with coexistence of other multiple resistant determinants (bla VEB-3 , bla TEM-1 and bla CMY-152 ) and provided detailed genetic characteristics of the NDM-1-carrying IncN plasmid pNDM-CWH001. Plasmids belonging to the IncN group are typically broad-host-range and self-conjugative 26 . The high transfer frequency of pNDM-CWH001 demonstrated its great potential to transfer across species. The resistance-determining region in those pNDM-CWH001-like plasmids was all inserted within the fipA gene. Interestingly, the fipA-encoded protein was reported to inhibit the conjugal transfer of some plasmids 27 . The interruption of the fipA gene could facilitate the ability of the plasmids of the plasmids to accumulate in diverse hosts and may serve as a "hotspot" for integration of mobile elements. Comparative analysis revealed that the acquisition of Tn6292 and the Tn3000 remnant might be subsequently integrated into pNDM-CWH001-like plasmids, highlighting the urgency of further surveillance and genetic analysis of such flexible mobile units for better understanding of extensive resistance dissemination. Recent studies have reported the simultaneous presence of multiple resistance genes in C. freundii strains isolated in China 8,9,11 . However, CWH001 showed long-distance dispersals from other C. freundii isolates in China and gained some resistance determinants (bla VEB-3 and fosA3) that were rarely identified in other C. freundii isolates. Previous study has reported C. freundii strain WCHCF65 from China clustered with strains from Denmark 8 . Phylogenetic analysis revealed that domestic C. freundii isolates showed close relationship with overseas ones but fell into distinct clusters, indicating different evolution and dissemination route. Plasmid pNDM-BTR was isolated from Beijing in 2013. Though lacking of epidemiological association, the close spatial and temporal proximity between pNDM-CWH001 and pNDM-BTR in China suggested possible dissemination of this novel plasmid, and more attention should be devoted to monitoring the epidemic spread of such bla NDM-1 -carrying IncN plasmids among Enterobacteriaceae.
In summary, our study characterized a multidrug-resistant C. freundii isolate harboring multiple ESBL-encoding genes. Strain CWH001 belonged to a novel sequence type ST139 with a self-transferable plasmid pNDM-CWH001, which may facilitate the bla NDM-1 gene dissemination. Phylogenetic analysis revealed that CWH001 had different origin from domestic isolates but gained multidrug resistance. Our findings further emphasize the threat of NDM-1 carbapenemase circulation among diverse species, and urgent actions should be taken to control the potential rapid spread of such plasmids.

Materials and Methods
Bacterial isolation and identification. The bla NDM-1 -positive C. freundii strain CWH001 was recovered from the blood sample of a 63-year-old male patient through routine surveillance in Wuhan, China, in 2014. The species level identification was performed by using Vitek 2 compact system (bioMérieux, France) and confirmed by 16S rDNA sequencing 28 . The presence of genes encoding carbapenemases and ESBLs was determined by PCR and sequencing [29][30][31] . The entire bla NDM , bla TEM , bla VEB and bla CMY genes were amplified with previously described primers [32][33][34][35]  Southern blotting and Conjugation experiment. Genomic DNA from strain CWH001 was prepared in agarose plugs and digested with the S1 endonuclease (Takara, Dalian, China). DNA fragments were separated by PFGE through a CHEF-DR III system (Bio-Rad, Hercules, USA). The plasmid DNA was transferred to a positively charged nylon membrane (Roche) and hybridized with the digoxigenin-labeled probes specific to bla NDM-1 , bla TEM-1 , bla VEB-3 and bla CMY-152 .
Conjugation experiment was carried out by broth and filter mating using the clinical strain CWH001 as donors and azide-resistant E. coli strain J53 as the recipient. The donor and recipient cultures were mixed at a ratio of 1:3 in LB broth and incubated at 37 °C for 18 hours. The mixture was inoculated into MacConkey agar plates containing 4 μg/ml meropenem and 150 μg/ml sodium azide. The transconjugants were selected after 12 h of incubation. Horizontal transferability of drug resistance was assessed by antimicrobial susceptibility testing and the transconjugants carrying resistant markers (bla NDM-1 , bla VEB-3 ) were confirmed by PCR amplification.
Whole genome sequencing and phylogenetic analysis. Total DNA was extracted from cultured bacterium using the QIAamp DNA minikit (Qiagen, Inc., Valencia, CA). Sequencing was carried out using an Illumina HiSeq. 2500 platform with a 350-bp insert size at Novogene Company (Beijing, China). The genome was assembled de novo using SOAPdenovo (v2.04) 37 with an average 110-fold coverage. Scaffolding and gap filling were performed using SSPACE and GapFiller 38,39 . Plasmids pNDM-BTR, p112298-KPC and pF8475 were selected as reference. Gaps were closed using reference-guided assembly and manually checked by re-mapping raw reads against the plasmids. Genome sequences were annotated using RAST 40 . Plasmid replicon type was identified using PlasmidFinder 41 (Enterobacteriaceae).
Seven housekeeping genes (arca-aspc-clpx-dnag-fadd-lysp-mdh) were extracted from the genome of CWH001 and used in MLST typing through the MLST web server 42 . Genome sequences of 84 currently available C. freundii isolates were downloaded from the NCBI database for phylogenetic analysis (accessed 12th June 2017). C. freundii strain B38 (GenBank accession number CP016762) was used as the reference for comparison. Reads mapping was performed using BWA (v0.7.12) 43 . SNPs were identified using SAMtools (v1.3) 44 . The resulting 27366 SNPs were concatenated and aligned to construct the Maximum-Likelihood phylogenetic tree using RAxML (v8.2.4) with the general time reversible (GTR) model and a gamma distribution 45 . ANIs between CWH001 and other genomes were calculated using JSpeciesWS 46 to evaluate the genome similarity.
Nucleotide sequence accession number. The shotgun whole genome sequence of strain CWH001 and complete sequence of plasmids pNDM-CWH001 and pTEM-CWH001 have been deposited in NCBI GenBank under accession number PEHH00000000.