Further Spread of a blaKPC-Harboring Untypeable Plasmid in Enterobacteriaceae in China

The wide spread of Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae is great threat to public health in China. Plasmids are among the major factors mediating blaKPC gene dissemination. A total of 156 carbapenem-resistant Enterobacteriaceae (CRE) isolates were identified in a tertiary hospital in China. Six KPC-producing isolates, namely, E. coli (n = 2), E. asburiae (n = 1), C. freundii (n = 1), C. portucalensis (n = 1), and C. koseri (n = 1), tested positive for the pCKPC18-1-like untypeable plasmid, which was described recently in C. freundii. All 6 plasmids could be easily transferred into E. coli by chemical transformation or conjugation and were confirmed by sequencing to harbor blaKPC−2. Multilocus PCRs and EcoRI-RFLP revealed that the 6 untypeable plasmids belonged to 2 isoforms. High-throughput sequencing of representative plasmids (pCP40 and pEC86) led to the identification of 2 plasmids that shared the common backbone genes repA, DnaJ, StpA, and yafB, which were characteristic of the untypeable plasmid, and had similar blaKPC−2 genetic contexts of the Tn3-Tn4401 chimera. Nucleotide comparison revealed high sequence identity of the 2 plasmids with previously reported blaKPC−2-carrying untypeable plasmids. In particular, the pCP40 plasmid from C. portucalensis and the pHS062105-3 plasmid from K. pneumoniae differed by only 20 single-nucleotide polymorphisms (SNPs). To the best of our knowledge, this is the first report of a blaKPC-harboring untypeable plasmid spread into E. coli, E. asburiae, and C. koseri strains in China.


INTRODUCTION
Carbapenems are often used as the last effective agents in the treatment of severe infections caused by multidrug-resistant gram-negative bacteria, especially strains expressing high-level AmpC cephalosporinase or extended spectrum β-lactamases (ESBLs). However, the emergence of carbapenem-hydrolyzing enzymes has greatly limited the effectiveness of these agents (Nordmann et al., 2009;Zhang et al., 2017).
Plasmids play an important role in the dissemination of the bla KPC−2 gene. Only one identified bla KPC−18 gene has been determined to be located on the chromosome (Thomson et al., 2016), while nearly all of the other identified bla KPC genes were harbored on plasmids. Plasmids of the incompatibility groups F (IncF), N (IncN), L/M (IncL/M), and X (IncX) have been reported to mediate bla KPC−2 gene transfer in Enterobacteriaceae (Cuzon et al., 2010;Jiang et al., 2010;Chen et al., 2013;Chen Y. T. et al., 2014).
Recently, a novel bla KPC -harboring untypeable plasmid (pCKPC18-1) encoding a replication protein that could not be assigned to any known incompatibility group was described in China (Zheng et al., 2018). To date, the likely plasmid has only been detected in Citrobacter freundii, Klebsiella pneumonia, and Enterobacter cloacae strains (Jiang et al., 2015;Shen et al., 2016;Zheng et al., 2018). In this study, we provided evidence for the further spread of the bla KPC -carrying untypeable plasmid in Enterobacteriaceae in the southwestern Zhejiang province of China.

Antimicrobial Susceptibility Testing
Antimicrobial susceptibilities were first determined by the disc diffusion (Kirby-Bauer, K-B) method. The minimum inhibitory concentrations (MICs) of imipenem, ertapenem, ceftazidime, ceftriaxone, cefepime, ampicillin, aztreonam, ciprofloxacin, levofloxacin, trimethoprim/sulfamethoxazole, tobramycin, gentamicin, and amikacin were detected by the VITEK2 Compact system with AST-GN13 cards or E-test (bioMérieux, France) according to the manufacturer's instructions. The results were interpreted according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (Wayne, 2017). E. coli ATCC 25922 was used for quality control.

Multilocus PCR and RFLP Analysis of Plasmids
Transformation and conjugation experiments were performed to acquire purified single plasmids as described previously (Jiang et al., 2010(Jiang et al., , 2015Shen et al., 2016;Zheng et al., 2018). The bla KPC−2 -carrying plasmids were extracted with a Plasmid Miniprep Kit (Transgen Biotech, China) from the transconjugants. Multilocus PCR was performed to evaluate the relationships of the 6 untypeable plasmids discovered in this study. In addition to the repA and bla KPC genes, primers ( Table 1) were designed to target the backbone genes taxA, virB5, virB11, and repB and the mobile elements Tn1721-TnpA and Tn1721-TnpR. Amplicons were analyzed by electrophoresis and sequencing. Meanwhile, plasmids were digested with EcoRI and subjected to restriction fragment length polymorphism (RFLP) by electrophoresis on 1% agarose (Sangon, China) gels in 1×TAE buffer as reported before (Ho et al., 2012).

Plasmid Sequencing and Annotation
Representative plasmids were fragmented by the whole-genome shotgun (WGS) approach and libraries were constructed. Genomic DNA were completely sequenced by next-generation sequencing (NGS) on an Illumina MiSeq platform with 2 × 400 bp paired-end reads. Adapters were removed using AdapterRemoval (ver. 2.1.7), and the high quality reads were screened through SOAPec (v2.0) with a Kmer frequency of 17. Sequences were then assembled with the A5-miseq (v20160825) and SPAdes (v3.9.0) programs. Protein-coding genes were predicted using GeneMarkS (v4.28) software, and coding sequence (CDS) annotations were performed using the BLASTP program with the NR database, followed by manual inspection.

Bacterial Strains and Antimicrobial Susceptibility Testing
Among all the 156 strains, 6 bla KPC−2 producers [2 E. coli strains (EC84 and EC86), 1 E. asburiae strain (EAK7), 1 C. freundii strain (CF111), 1 C. portucalensis strain (CP40), and 1 C. koseri strain (CK61)] isolated from different patients were positive for the untypeable plasmid (Supplementary S1). EAK7, which was isolated from hematology wards in December 2010, was the first untypeable plasmid carrier identified in our hospital. Then it was CP40 in January 2012, CK61 in September 2012, EC84 in May 2013, EC86 in June 2013 and CF111 in October 2014. However, except for the 2 E. coli strains isolated from the ICU, all the strains were isolated from different wards. No apparent contact among the patients was validated. None of the other CRE isolates were positive for the untypeable plasmid. All 6 isolates were resistant to carbapenems, third-generation cephalosporins and aztreonam, but were susceptible to colistin ( Table 2). Notably, C. portucalensis CP40 was resistant to sulfonamides and aminoglycosides but was susceptible to quinolones; however, C. freundii CF111 was exhibited the opposite resistance and susceptibility. Both E. coli EC84 and EC86 were resistant to levofloxacin, ciprofloxacin, trimethoprim/sulfamethoxazole, gentamicin and tobramycin and were susceptible to amikacin. Nevertheless, all the transconjugants showed similar susceptibility profiles that were resistant to the β-lactams but susceptible to quinolones, aminoglycosides and sulfonamides.

Analysis of the Genetic Relatedness
According to the MLST results, both E. coli EC84 and EC86 belonged to ST648 and showed identical PFGE patterns (Supplementary S2), suggesting the clonal relatedness of the 2 strains. The C. freundii CF111 belonged to sequence type ST116.

Multilocus PCR and RFLP Analysis of Plasmids
Single plasmids were successfully obtained via transformation (pCP40, pCF111, pEC84, and pEC86) and conjugation (pEAK7 and pCK61). All the plasmids tested positive for repA and bla KPC . Upon amplification of the TaxA, virB5, virB11, Tn1721-TnpA, and Tn1721-TnpR sequences, positive results, which exhibited 100% sequence identity with pCKPC18-1, were obtained with pEAK7, pCP40, pCK61, and pCF111; however, none of these genes were detected in pEC84 and pEC86 ( Figure 1A). Interestingly, fragments of different lengths were obtained from all the plasmids with the forward and reverse primers CH4F and CH5R, targeting Tn1721-TnpA and repB, respectively. The fragment lengths were approximately 2,500 bp from pEAK7, pCP40, pCK61 and pCF111 and 1,000 bp from pEC84 and pEC86. Sequencing analysis revealed that the large and small fragments shared 100% identity with pCKPC18-1 (CP022276) and pKPC-ECN49 (KP726894), respectively. Consistent with the multilocus PCR results, 2 different patterns were observed in the RFLP analysis ( Figure 1B). The plasmids pEAK7, pCP40, pCK61, and pCF111 yielded identical fragments, which differed from the fragments from pEC84 and pEC86.

Characteristics of the bla KPC-2 -Carrying Untypeable Plasmids
The plasmids pCP40 and pEC86, isolated from C. portucalensis and E. coli strains respectively, were chosen as representative plasmids for complete genome sequencing. The plasmid pCP40 is a 42,848-bp closed circular DNA with an average G+C content of 50.1%. Annotation of the final sequence of pCP40 revealed 50 open reading frames (ORFs), 32 of which encoded homologous proteins with known functions. In the backbone structure, there were genes encoding an untypeable replication protein, the molecular chaperone DnaJ, the type IV secretory pathway   Frontiers in Microbiology | www.frontiersin.org (VirB1-10), the DNA-binding protein StpA, the antirestriction protein Klca, and the transcriptional repressor protein KorC.

Nucleotide Accession Numbers
The complete sequences of pCP40 and pEC86 have been deposited in GenBank under the accession numbers MH328006 and MH328007, respectively.

DISCUSSION
Plasmids are one of the major causes responsible for the rapid dissemination of the bla KPC gene (Cuzon et al., 2010;Jiang et al., 2010;Chen et al., 2013;Chen Y. T. et al., 2014). The novel plasmid pFOS18, characterized by backbone genes encoding a replication protein (RepA) that could not be assigned to any known incompatibility group, the molecular chaperone DnaJ, the DNA-binding protein StpA, the antirestriction protein Klca, and the transcriptional repressor protein KorC, was first described in 2015 from a fosfomycin-resistant KPCproducing K. pneumoniae strain in Zhejiang province (Jiang et al., 2015). Recently, plasmids containing identical repA genes were detected in Citrobacter freundii (pCKPC18-1) and Enterobacter cloacae (pKPC-ECN49) stains in the Zhejiang and Jiangsu provinces, respectively (Zheng et al., 2018).
In this study, we identified the untypeable plasmids in 6 Enterobacteriaceae. Besides C. freundii strain, the untypeable plasmid was detected in 2 E. coli, 1 E. asburiae, 1 C. portucalensis and 1 C. koseri strains. Although E. asburiae was the first strain carrying the untypeable plasmid isolated in our hospital, clonal spread of this strain was not detected. The 2 E. coli strains exhibited identical ST types, PFGE profiles and susceptibility patterns, suggesting clonal spread of the E. coli strains. However, due to limited information, it is not clear whether the CF111 (ST116) is related to the C. freundii strains carrying pCKPC18-1 in Hangzhou or pP10159-3 in Chongqing (Zheng et al., 2018).
Consistent with the previous studies (Jiang et al., 2015;Shen et al., 2016;Zheng et al., 2018), 2 different subtypes of the untypeable plasmids were characterized in the 6 strains according to multilocus-PCR and RFLP analysis. The plasmids pEAK7, pCP40, pCK61, and pCF111 yielded identical multilocus-PCR results matching pCKPC18-1, while the other 2 (pEC84 and pEC86) were more related to pFOS18. Complete sequencing of the 2 representative plasmids, pCP40 and pEC86, showed that they were 42,848 and 24,228-bp in size, respectively. Although they differed in size, identical replication initiators and similar backbone genes and bla KPC−2 genetic contexts were observed in the 2 plasmids. Comparative analysis revealed that pCP40 shared high query coverage and identity with untypeable plasmids isolated from K. pneumoniae, C. freundii and E. cloacae in different areas of China. In particular, pCP40 present in the C. portucalensis isolate and pHS062105-3 from a K. pneumoniae strain differed by only 20 SNPs. Taken together, the high sequence similarity among pCP40, pKPC-ECN49, pHS062105-3, pP10159-3, and pCKPC18-1 suggested that these plasmids evolved from a common plasmid and spread independently (Zheng et al., 2018).
A previous study showed that the K. pneumoniae strains could transfer their bla KPC−2 -carrying untypeable plasmids to the azide-resistant E. coli strain J53, implying the possibility of horizontal transfer of the untypeable plasmid (Shen et al., 2016). Moreover, the transfer regions of these untypeable plasmids also share high identity with the transferable IncN plasmid p1 (CP006657) (Zheng et al., 2018). In our study, 2 strains (EAK7 and CK61) successfully transferred untypeable plasmids to the recipient, thereby reconfirming the horizontal transfer capacity. Take account of the high similarity of pEAK7, pCP40, pCK61, and pCF111, the untypeable plasmid may transfer among Enterobacteriaceae, mediating bla KPC dissemination. However, after 5 attempts, the E. coli strains (EC84 and EC86) failed in conjugation experiments. Compared with pCP40, complete sequencing revealed the main difference of lacking the type IV secretory system in pEC86. The type IV secretory system has been reported to deliver DNA and protein substrates from donor to target bacterial cells by conjugation (Christie, 2004), implying that it is the factor responsible for the horizontal transferability of the untypeable plasmid.
In summary, this study characterized the epidemiological contexts of 6 CRE isolates carrying the untypeable plasmids in our hospital and identified two plasmids, namely, pCP40 and pEC86. To the best of our knowledge, this is the first description of a bla KPC -harboring untypeable plasmid in E. coli, E. asburiae, and C. koseri strains. This work provided evidence of the spread of this bla KPC -harboring untypeable plasmid in Enterobacteriaceae in China and highlights the FIGURE 1 | Relatedness of the 6 untypeable plasmids. (A) Mutilocus-PCR results. Primers targeting the genes repA, bla KPC , TaxA, virB5, virB11, Tn1721-TnpA, Tn1721-TnpR, and repB were designed. Two profiles were obtained after evaluation of the amplicons by electrophoresis. The plasmids pEAK7, pCP40, pCK61, and pCF111 exhibited one pattern, while pEC84 and pEC86 exhibited a different pattern. (B) RFLP results. Fragments were separated by electrophoresis on a 1% agarose gel in 1 × TAE buffer. Two different patterns, one for the plasmids pEAK7, pCP40, pCK61, and pCF111 and another for pEC84 and pEC86, were obtained. EA, E. asburiae; CP, C. portucalensis; CF, C. freundii; CK, C. koseri; EC, E. coli; M, DNA marker.

AUTHOR CONTRIBUTIONS
JH and ZZ were responsible for the study design and data interpretation. HD, YZ, XH, and JR collected all the clinical isolates and performed susceptibility tests. YS, GH, and RW carried out PCRs, transformation and conjugation experiments. JH performed RFLP and wrote the report. All authors revised, reviewed, and approved the final report.