Citrobacter freundii carrying blaKPC-2 and blaNDM-1: characterization by whole genome sequencing

A carbapenem-resistant Citrobacter freundii strain WCHCF65 was recovered from hospital sewage and was characterized by genome sequencing and conjugation experiments. The strain carried nine genes encoding β-lactamases including two carbapenemase genes, blaNDM-1 and blaKPC-2. blaNDM-1 was carried on an IncX3 plasmid, which was identical to a plasmid found in a local Escherichia coli, suggesting interspecies horizontal transfer. blaKPC-2 was bracketed by two copies of insertion sequence ISKpn19, which could form a composite transposon with the potential to mobilize blaKPC-2, on a new type of plasmid. The coexistence of blaNDM-1 and blaKPC-2 conferred higher levels of resistance to carbapenems compared with blaNDM-1 or blaKPC-2 alone. The coexistence of these carbapenemase genes, on two different plasmids, in one strain may allow new genetic platforms to be generated to mediate their spread.

Antimicrobial resistance of clinically significant bacteria is a global problem with carbapenem-resistant Enterobacteriaceae (CRE), in particular, emerging as a major threat for medical care. Carbapenem resistance among the Enterobacteriaceae is largely mediated by the production of carbapenemases, which are enzymes able to hydrolyse carbapenems and most other β -lactam antibiotics 1 . There are many types of carbapenemases, among which KPC and NDM were the two major groups produced in the Enterobacteriaceae 1 . bla KPC and bla NDM genes (encoding the KPC or NDM carbapenemases, respectively) are commonly found individually in CRE, but there have been several reports of isolates carrying both bla KPC and bla NDM worldwide [2][3][4][5] . We previously reported an Enterobacter cloacae carrying bla KPC and bla NDM from a blood culture of an ICU patient and demonstrated that the coexistence of both genes in that strain resulted in a slightly higher level of resistance to carbapenems 6 . During a study that specifically screened CRE from hospital sewage, we found a Citrobacter freundii isolate also carrying both bla KPC and bla NDM . Here we report the genomic and plasmid characterization of the isolate.
Both bla NDM-1 and bla KPC-2 were able to be transferred by conjugation, suggesting that they were carried by self-transmissible plasmids. Transconjugants obtained were found harbouring either bla NDM-1 or bla KPC-2 , indicating that the two carbapenemase genes were carried by separate plasmids, designated pNDM1_CF65 and pKPC2_CF65, respectively. An E. coli J53 strain containing both plasmids was obtained by electroporation of a J53:pKPC2_CF65 transconjugant with purified pNDM1_CF65 DNA obtained from a respective transconjugant. A two-or four-fold increase in MIC was observed for imipenem and meropenem when comparing E. coli J53 with both bla NDM-1 and bla KPC-2 compared to J53 having either gene alone (Table 1). This confirmed our previous observation that the coexistence of bla NDM-1 and bla KPC-2 in a single strain could confer slightly elevated levels of carbapenem resistance 6 . Nonetheless, MICs of imipenem and meropenem against J53 containing both carbapenemase genes were still much lower than strain WCHCF65, suggesting that there were additional mechanisms mediating resistance to carbapenems. Previous studies have suggested that impermeability due to alterations of outer membrane porins could lead to resistance to carbapenems 7,8 .
Whole genome sequencing of C. freundii WCHCF65 generated 3,794,588 reads and 474,268,104 clean bases, from which 269 contigs ≥ 500 bp in length (N50, 72,950 bp) were assembled with a 51.9% GC content.
Strain WCHCF65 belonged to a new C. freundii sequence type: ST88. Among the other 48 C. freundii strains with whole genome sequences available in GenBank, strain WCHCF65 was closest to strain ballerup 7851/39 based on phylogenetic analysis. Interestingly, strain ballerup 7851/39 also belonged to a new ST (ST106) and was isolated from a female patient in 1939 in Denmark 9 (Fig. 1). None of the seven alleles of ST88 were identical to those of ST106. The genome of WCHCF65 has the highest average nucleotide identity (ANI; 98.1%) with that of ballerup 7851/39, while the ANI between the genome of WCHCF65 and the remaining 47 C. freundii genomes varied from 90.22% to 92.91%.
Plasmid pKPC2_CF65 was 40.9 kb in size and contained genes for conjugation and partitioning but genes encoding the replicon are yet to be determined. The backbone of pKPC2_CF65 had only 67% nucleotide identity (65% coverage) with the closest plasmid matches: p34399-43.500 kb (GenBank accession number CP010387) and p35734-109.753 kb (CP012163) and pCAV1669-34 (CP011648) from E. cloacae, pKPC_CAV1741 (CP011656) from C. freundii, and pCAV1374-34 (CP011629) from Klebsiella oxytoca. Both the PlasmidFinder tool and PCR-based replicon typing could not assign pKPC2_CF to a known incompatibility group. The above findings suggest that pKPC2_CF65 is a novel type of plasmid. On pKPC2_CF65, bla KPC-2 was the only known and complete antimicrobial resistance gene with no known acquired virulence factors being detected. Two copies of ISKpn19 flank bla KPC-2 on pKPC2_CF65 and potentially represent a composite transposon, which has not seen before. However, bla KPC-2 is seen in a similar immediate genetic context on pKPC2_EC14653, a plasmid from an E. cloacae isolate recovered in the same hospital 6 (Fig. 2). On both plasmids, a Tn3-like transposon interrupted by the insertion of insertion sequence ISKpn27 can be found upstream of bla KPC-2 (Fig. 2). Downstream of bla KPC-2 there was a copy of ISKpn6 and several open reading frames including korC (function unknown) and klcA (encoding antirestriction protein) that are often seen with bla KPC-2 in other genetic contexts such as in pKPC2_ EC14653. However, the repB gene (encoding a putative replication initiation protein), which was present on pKPC2_EC14653 6 , was truncated by the second copy of ISKpn19 in pKPC2_CF65. Given the similarity between the genetic contexts of bla KPC-2 between these two plasmids from the same hospital, it can be suggested that the insertion of two copies of ISKpn19 into a pKPC2_EC14653-like plasmid prior to the movement of bla KPC-2 via an ISKpn19-mediated composite transposon or homologous recombination to a pKPC2_CF65 progenitor.
The coexistence of bla NDM-1 and bla KPC-2 has been previously identified in five enterobacterial isolates of different species (i.e. C. freundii, E. cloacae, Enterobacter hormaechei and Klebsiella pneumoniae) 2-6 . In all previous isolates and strain WCHCF65, the two carbapenemase genes were not located on a common plasmid. However, the coexistence of two or more plasmids in a single strain may allow new platforms for spreading carbapenemase genes to be generated. Of note, during this study, another C. freundii isolate (strain 112298) was reported to carry both bla NDM-1 and bla KPC-2 , which was recovered from a patient in Guangzhou, southern China in 2013 5 . There was no obvious epidemiological link between strain 112298 and strain WCHCF65 that were recovered from these two hospitals (approximately 1,200 km apart in distance). pNDM_CF65 and the plasmid carrying bla NDM-1 in C. freundii strain 112298 were almost identical except a 3.5 kb inversion and 546 bp deletion in the latter plasmid. Both plasmids were also highly similar to other IncX3 plasmids from strains recovered in China 11 . In contrast, the plasmids carrying bla KPC-2 in the two C. freundii isolates had completely different backbones.
In conclusion, the study characterized a C. freundii isolate carrying both bla KPC-2 and bla NDM-1 that were carried by different plasmids. The coexistence of two carbapenemase genes in a single strain is of significance as it confers a higher level of resistance to carbapenems and may allow new platforms to be generated for mediating the further spread of the carbapenemase genes. Two copies of ISKpn19, which bracketed bla KPC-2 , could form a composite transposon, representing a new mechanism for mediating the mobilization of bla KPC-2 .

Material and Methods
The isolate, in vitro susceptibility and screening for carbapenemase genes. Hospital sewage (1 mL) was obtained from the influx of the wastewater treatment plant in West China Hospital of Sichuan University, Chengdu, western China, in January 2015. The sewage was diluted 1:10 and an aliquot (100 μ L) was spread onto a CHROMAgar Orientation (CHROMAgar, Paris, France) agar plate containing 2 μ g/mL meropenem and then incubated at 35 °C overnight under aerobic conditions. Colonies were patched on CHROMAgar plates containing 2 μ g/mL meropenem to ensure they were resistant to carbapenems and were then screened for acquired carbapenemase-encoding genes bla GES , bla KPC , bla IMP , bla IMI , bla NDM , bla OXA-48 and bla VIM using PCR as described previously [14][15][16][17] . Amplicons containing the complete coding sequence of bla NDM 14 or bla KPC 17 were sequenced in both directions using an ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA) at the Beijing Genomics Institute (Beijing, China).
Preliminary species identification was performed by partially sequencing the gyrB gene as described previously 18 . MICs of amikacin, ceftazidime, ciprofloxacin, colistin, imipenem, meropenem and tigecycline were determined using the microdilution broth method and breakpoints following recommendations of the Clinical Laboratory Standards Institute (CLSI) 19 . As no CLSI-defined breakpoints for tigecycline and colistin were available, those defined by the Food and Drug Administration (FDA) were used for tigecycline and those by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) 20 were used for colistin.
Cloning of bla CMY-137 . The complete coding sequence of bla CMY-137 was amplified with primers 65_N92-1EcoRI (AGAATTCAGTCGCTCAACAGAGGGAAA, the EcoRI restriction site was underlined; binding to ampR, upstream of bla CMY-137 ) and CMY137-dowEcoRI (AGAATTCTCGCCAGTGAAGTAGGCTTT; binding to a gene downstream of bla CMY-137 ). The amplicon was 2 kb in size and contained the 1,146-bp complete coding sequence of bla CMY-137 . The amplicon was restricted using EcoRI (NEB, Ipswich, MA, USA) and then was ligated to EcoRI-restricted pBC SK vector (Agilent, Santa Clara, CA, USA) using T4 ligase (NEB). The ligated fragments were electroporated into E. coli DH5α and the bla CMY-137 -containing transformants were selected on LB agar plates containing 100 μ g/mL ampicillin. The presence of bla CMY-137 in transformants was confirmed by PCR and sequencing. MICs of ampicillin, ceftazidime, cefotaxime, cefoxitin, imipenem, piperacillin and piperacillin-tazobactam were determined using the broth microdilution method following recommendations of CLSI.
Whole genome sequencing and analysis. Genomic DNA of strain WCHCF65 was prepared using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) and was subjected to paired-end (2 × 125 bp) whole genome sequencing with approximately 100× coverage using the HiSeq 2500 Sequencer (Illumina, San Diego, CA, USA) following the manufacturer's protocol at the Beijing Genomics Institute. The Spades program 21 was used to de novo assemble reads into contigs and annotation of the genomic sequence was carried out using the Prokka program 22 . ST was determined by using the genomic sequence to query the multi-locus sequence typing (MLST) database of C. freundii (http://pubmlst.org/cfreundii/) and the MLST tool from the Center for Genomic Epidemiology (http://genomicepidemiology.org/). Antimicrobial resistance genes were predicted using the ResFinder tool (80%, minimum threshold for identity; 60%, minimum coverage) from the Center for Genomic Epidemiology. To confirm the chromosomal location of bla CMY-137 , the contig containing this gene was linked to a contig containing genes encoding ribosomal proteins by PCRs using primers designed based on available contig sequences and Sanger dideoxy sequencing. Acquired virulence factors were predicted using the VirulenceFinder tool (85%, minimum threshold for identity; 60%, minimum coverage) from the Center for Genomic Epidemiology to query the known virulence factors of E. coli.
Phylogenetic relatedness of WCHCF65 to the 48 C. freundii genomes available in GenBank (http://www.ncbi.nlm. nih.gov/genome/2850, accessed November 24, 2015) was investigated using the Harvest suite 23 . The ANI between the genome sequence of WCHCF65 and the 48 C. freundii genomes was calculated using the web-based JSpeciesWS tool 24 .
Plasmid replicons carried by WCHCF65 were determined using the PlasmidFinder tool (80%, minimum threshold for identity) from the Center for Genomic Epidemiology. The sequence of plasmids carrying bla NDM-1 or bla KPC-2 were completely circularised with gaps between contigs closed by Sanger dideoxy sequencing of amplicons from PCRs using primers designed based on available contig sequences. For pKPC2_CF65, PCR-based replicon typing was performed as described previously 25 .

Plasmid manipulation experiments.
Conjugation experiments of WCHCF65 were carried out in broth using azide-resistant E. coli strain J53 as the recipient; potential transconjugants were selected on LB agar plates containing 4 μ g/mL meropenem and 150 μ g/mL sodium azide. To obtain a J53 strain containing both bla NDM -and bla KPC -carrying plasmids (pNDM1_CF65 and pKPC2_CF65, respectively), plasmid DNA was prepared from J53 containing pNDM1_CF65 by alkaline lysis and was then electroporated into J53 containing pKPC2_CF65. Potential transformants were selected on LB agar plates with 250 μ g/mL zeocin (Invitrogen, Waltham, MA, USA), a bleomycin antibiotic. As the recipient carried bla KPC-2 and was resistant to carbapenems, the use of carbapenems was therefore no longer able to distinguish the recipient and the transformant. Nonetheless, the recipient did not carry any bleomycin-resistant genes but the bleomycin-resistant gene ble was located downstream of bla NDM-1 . Therefore, bleomycin could be used to screen the transformants. The presence of bla NDM and bla KPC in transconjugants and transformants were confirmed by PCR.
Nucleotide sequence accession number. Reads and the Whole Genome Shotgun Sequencing project of C. freundii WCHCF65 have been deposited into DDBJ/EMBL/GenBank under the accession numbers SRR2153225 and LIDR01000000, respectively. The sequence of pKPC2_CF65 has been deposited into DDBJ/EMBL/GenBank under accession number KU176944.