Complete sequence of two potentially epidemic KPC-harbouring plasmids in Klebsiella pneumoniae ST11 strains in China

Background: Klebsiella pneumoniae carbapenemase (KPC) has spread across the world. The present study focuses on explore sequence and evolution pathway of two potentially epidemic KPC-harbouring plasmids in Klebsiella pneumonia isolated from Southern China. Methods: Eighteen carbapenemases-producing Klebsiella pneumonia isolates were collected in a tertiary teaching hospital in 2014 in Southern China. The KPC production, antimicrobial susceptibility and MLST were tested for all isolates. Plasmids were purified for all isolates. Plasmids of six isolates were randomly selected to perform Single-molecule real-time sequencing, annotation and bioinformatics analysis. Conjugation and transformation experiment were also performed for the six selected plasmid samples. Furthermore, PCR screening based on the plasmid sequences information were performed for other twelve isolates, to identify the prevalence of the identified plasmids. Results: Two new KPC plasmids, pF5 and pF77, were identified and detected to be highly epidemic in the collected isolates with the rate of 77.8% (14/18), and 92.9% of the plasmid-harbouring isolates were identified in sequence type (ST) 11 group. pF5 belongs to the incompatibility group IncR and IncFIIk, and pF77 belongs to IncFIIk. The two plasmids were non-conjugative. By sequencing and comparing the plasmids sequences in detail, we found these plasmids carrying bla KPC-2 , bla CTX-M-65 , bla SHV-12 , catA2 and fosA3 antimicrobial genes. Additionally, the two plasmids both showed a close relationship with the other two reported KPC-harbouring plasmids pKP1034 and pCT-KPC, which have highly similar backbones and have been considered to evolve from some recombination events of other closely related plasmids pHN7A8, pKPC-LK30 and pKPHS2. We noticed that these previous reported plasmids and our plasmids all have been detected

This pathogen seriously threats public health because of drug resistance against the last clinical resort of carbapenems, thus rendering almost all available treatment options ineffective [1]. One of the primary mechanisms of this pathogen causing resistance is producing the enzyme of Klebsiella pneumoniae carbapenemase (KPC), which is encoded by different variants of bla KPC genes and has spread globally [2].
Plasmids are essential vehicles for the spread of drug-resistance genes such as bla KPC . Among the 22 bla KPC variants, bla KPC-2 is the most dominant one [3]. Different types of plasmids have been identified to carry bla KPC-2 , among which the IncFIIk plasmids are the most frequently detected and widely reported [4,5]. Recently, two such plasmids pKP1034 and pCT-KPC (also known as pCY-KPC334) were identified from the south part of China [6,7]. They both convey high levels of antibiotic resistance ability by carrying bla KPC-2 , bla CTX-M-65 , bla SHV-12 and many other drug-resistant genes [6,7]. For the similarity of the sequences of the two plasmids, they were considered to relate to each other [7].
In this study, we identified two new KPC-harbouring IncFIIk plasmids pF77 and pF5, and they are closely relate to the reported plasmids pKP1034 and pCT-KPC, which are reported to be identified in Southern China. So we investigated characteristics of the plasmids and the isolates carrying them in detail. This information also provides us with deep insights into the diversity as well as the potential evolutionary pathway of the IncFIIk type KPC-harbouring plasmids in K. pneumoniae. In addition, we conducted a survey of this type of plasmid among a collection of KPC-positive K. pneumoniae isolates and uncovered its significant prevalence in a tertiary teaching hospital in Southern China, and its

Multilocus sequence typing (MLST)
Multilocus sequence typing (MLST) analysis of the isolates was performed based on the MLST website (http://bigsdb.pasteur.fr/klebsiella/klebsiella.html). Chromosomal DNA was obtained by the alkaline lysis method using a commercial genomic DNA purification kit (Tiangen Biotech Co., Ltd) according to the manufacturer's instructions. Allele sequences and sequence types (STs) were verified at the website http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html.

Plasmids purification
Plasmid DNA of eighteen isolates was obtained and purified by using a commercial genomic DNA purification kit (Tiangen Biotech Co., Ltd) according to the manufacturer's instructions.

Plasmid sequencing, annotation, and bioinformatics analysis
The plasmids of six isolates were randomly selected from the eighteen isolates for sequencing.
Complete sequencing of the plasmids was performed by Single-molecule real-time (SMRT) sequencing. SMRT cell library preparation was conducted by the recommendations provided by the manufacturer and performed using a Pacbio RS II instrument. Each sequence was assembled by the Hierarchical Genome Assembly Process [8,9]. Glimmer and GeneMark were used to predict the coding sequences (CDSs) [10,11]. The CDSs were annotated using BLAST against the NR database.

Conjugation and transformation experiment of the plasmids
The six selected isolates were subjected to conjugation experiments. The conjugation experiments were conducted following a previous report [14]. Rifampin-resistant Escherichia coli EC600 (LacZ -Nal r Rif r ) was used as the recipient strain. Briefly, the donor and recipient strains were cultured overnight and then mixed with fresh Luria-Bertani (LB) broth and incubated for 24 h at 35 °C. Then, the mixture was inoculated on LB agar plates containing rifampin (700 μg/ml) and imipenem (1.5 μg/ml) for 24 h at 35 °C. The colonies that grew on the plates were picked up for antimicrobial susceptibility testing.
All the experiments were repeated three times.
Total plasmids were extracted from the six selected isolates for the transformation experiments via the method mentioned in 2.4. They were then transformed into E. coli DH5α competent cells (Tiangen, China) by the heat shock method, as reported before [15]. Transformants grew overnight on the LB agar containing ceftriaxone (64 mg/L) and then were selected. After testing the existence of the plasmid in the positive transformants via the method mentioned in 2.7, antimicrobial susceptibility of the transformants was determined via the method in 2.1.

PCR screening for the distribution of the pF77-like plasmids
For the similar parts shared between the identified KPC-harbouring plasmids in the selected isolates, we named this type of plasmid as pF77-like plasmid.
The distribution of the pF77-like plasmids in other twelve KPC-harbouring K. pneumoniae isolates was investigated by PCR. PCR primers were designed according to the sequences data of pF77 (Fig 1) [4,16]. The PCR cycling conditions were as follows: an initial denaturation step of 95°C for 5 min, When blasting the backbone sequences of pF77 and pF5, they show both 99% identity to two reported plasmids, pKP1034 and pCT-KPC. However, the query coverage ranges from 48% to 79%, which means pF77 and pF5 are smaller than pKP1034 and pCT-KPC. In addition, although many genes pKP1034 and pCT-KPC were kept in pF77 and pF5, genes such as plasmids stability genes stbA/B, some antibiotic resistance genes (bla TEM-1 and rmtB) and the mercury-resistance clusters are lost in pF77 and/or pF5.
The differences in genetic elements indicate a highly versatile characteristic of this group of the plasmids. They can carry necessary genetic elements and help host cells improve the adaption ability under certain stress conditions, such as the presence of antibiotic and heavy metal toxin compounds [17,18]. They can also help host cells maintain the cost-effectiveness by losing unnecessary elements, for example, the mercury-resistance clusters can be costly for the plasmid to successfully maintained in the strains living without the mercury pressure, which might be the reason for the loss of the clusters in the plasmids [19].

Plasmid sequences comparison and analysis
The backbone of pF77 can be divided into three modules, and all of them are flanked by IS26. The first module (from 26065 to 39750) contains bla CTX-M-65 and the second module (from 39751 to 54956) contains bla SHV-12 and bla KPC-2 . These two modules are similar to the multi-resistant region (MRR) of pKP1034 or pCT-KPC, and they were considered to origin from the same sequence of plasmid pHN7A8 [6]. The third module contains (from 1 to 26064) catA2, which was only similar to the sequence of pKP1034 (Fig. 1). pF77 is identified to belong to the incompatibility group IncFIIk.
Despite containing the same three modules with pF77, pF5 extends with the other one module between the first and second modules. This extra module is also flanked by IS26. The resistant gene fosA3 keeps in this module. By comparing the sequences in detail, this module is similar with the sequence of pCT-KPC via deleting the mercury-resistance clusters and a reversion event of part of the sequences (Fig. 1). This is a conserved region among most IncR plasmids such as pKPHS2 [6]. Thus pF5 is a multi-replicon plasmid and belongs to both IncR and IncFIIkgroup. Such a presence of multiple replicons can facilitate the plasmid to interact with the broader range of hosts [4].
Additionally, an unreported genetic element structure of the bla KPC-2 gene has been identified in plasmids pF77 and pF5. Unlike the classic non-Tn4401 mobile element structure of bla KPC in pKP1034 and pCT-KPC, which is a typical core element structure of ISKpn8-bla KPC-2 -ΔISKpn6 [20], the element structure upstream the bla KPC-2 in pF77 and pF5 consisted of insB and insA (Fig. 2), which together constitute IS1 instead of ISKpn8 [21]. As far as we know, this type of core element structure of IS1-bla KPC-2 -ΔISKpn6 has not been reported yet.This structure derives from the IS1-mediated homologous recombination (HR) event of deletion of the sequences between bla KPC-2 and insB in pCT-KPC and pKP1034 ( Fig. 1 and 2).
We also noticed that these previously reported plasmids and our plasmids were all detected in the period of 2013-2014 in Southern China. They share similar backbone and were carried by the clonal ST11 K. pneumonia strains. In addition to their sequence comparison, a hypothesised evolutionary pathway was considered for these plasmids. pF77 is supposed to derive from plasmids pHN7A8 and pKPC-LK30, and pF5 is supposed to derive from plasmids pHN7A8 and pKPHS2 (Fig 1). While pKP1034 also being hypothesised to evolve from plasmids pHN7A8 and pKPC-LK30 [6], and pCT-KPC has been hypothesised to evolve from plasmids pHN7A8 and pKPHS2 [7, 22,23], it seems there is a highly diversity of the group of plasmids generated from pHN7A8, pKPC-LK30 and pKPHS2, via IS26mediated HR events including deletion and reversion.

Microbiological characterisation of the isolates
Antimicrobial susceptibility tests showed that the isolates were all resistant to most of the antimicrobial agents. However, two isolates harbouring pF77 and one harbouring pF5 were susceptible to amikacin (≤ 2 µg/ml), gentamicin (4 µg/ml) and tobramycin (2 µg/ml), while the other two isolates harbouring pF77 were resistant to all of the three antimicrobilas. For the other one isolate harbouring pF5, it was susceptible to amikacin (≤ 2 µg/ml) and was resistant to gentamicin and tobramycin. The detailed results are presented in Table 3.
No Escherichia coli EC600 transconjugant recovered by conjugation of both two plasmids in all experiments, which is the same result with that of pKP1034 and pCT-KPC. But the E. coli DH5α transformants positive with plasmids pF5 and pF77 confer significantly higher resistance to β-lactams than their parental strain E. coli DH5α component cells (Table 3), which confirm the antimicrobial resistance ability of the plasmids.
MLST analysis revealed that the six isolates all belonged to the epidemic ST11 K. pneumoniae (Table   2). For other non-sequenced isolates, all of them belonged to ST11, except one isolate belonged to ST524.

Prevalence and dissemination of the pF77-like plasmids
By PCR the conserved locations of the sequences, eight out of twelve non-sequenced isolates were tested to carry the pF77-like plasmids. Therefore, this type of plasmids were epidemic in our collected isolates (14/18, 77.8%). Moreover, the majority (13/14, 92.9%) of the plasmid were harboured in ST11 isolates and one of the isolates belonged to ST524, which is very close to ST11 with only a single nucleotide mutation at 278 position of infB (C→T). Interestingly, pKP1034 and pCT-KPC were also identified in ST11 K. pneumoniae strains in Southern China (Table 2). Therefore, this type of plasmids seems to disseminate mainly in ST11 clonal strains in K. pneumoniae. Considering the three modules of pF77 discussed in 3.2 are highly conserved in the identified plasmids, the genes and structures in the modules may contribute to their successful dissemination in the strains [24].

Conclusions
In this study, we identified two newly reported KPC-harbouring plasmids pF5 and pF77. They were grouped as pF77-like plasmids and share three conservative modules in their sequences. The pF77like plasmids were found to be highly prevalent in the collected KPC-harbouring K. pneumoniae isolates. Plasmid sequences comparison suggested a potential evolutionary pathway of pHN7A8-, pKPC-LK30-and pKPHS2-derived plasmids in Klebsiella pneumoniae strains in China. Through this pathway, a diversity of plasmids has emerged from frequent HR events between variable integration sites. This mechanism can facilitate them to rapidly adapt to a different environment and be able to confer a high level of antibiotic resistance. Furthermore, clonal dissemination and a regional epidemic of pF77-like plasmids in the ST11 K. pneumoniae isolates have been identified in our study, which highlights a broad concern of their further evolvement to be a growing clinical and public health