Replicative transposition contributes to the evolution and dissemination of KPC-2-producing plasmid in Enterobacterales

ABSTRACT
 Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales are prevalent worldwide and pose an alarming threat to public health. The incidence and transmission of blaKPC-2 gene via horizontal gene transfer (e.g. transposition) have been well documented. However, the dynamics of transposon structure bearing blaKPC-2 and their exact effects on the evolution and dissemination of blaKPC-2 gene are not well characterized. Here, we collected all 161 carbapenem-resistant Enterobacterales (CRE) isolates during the early stage of CRE pandemic. We observed that the prevalence of KPC-2-producing Enterobacterales was mediated by multiple species and sequence types (STs), and that blaKPC-2 gene was located on three diverse variants of Tn1721 in multi-drug resistance (MDR) region of plasmid. Notably, the outbreak of KPC-2-producing plasmid is correlated with the dynamics of transposon structure. Furthermore, we experimentally demonstrated that replicative transposition of Tn1721 and IS26 promotes horizontal transfer of blaKPC-2 and the evolution of KPC-2-producing plasmid. The Tn1721 variants appearing concurrently with the peak of an epidemic (A2- and B-type) showed higher transposition frequencies and a certain superior ability to propagation. Overall, our work suggests replicative transposition contributes to the evolution and transmission of KPC-2-producing plasmid and highlights its important role in the inter- and intra-species dissemination of blaKPC-2 gene in Enterobacterales.


Introduction
Klebsiella pneumoniae carbapenemase (KPC) is a class A serine β-lactamase that efficiently hydrolyzes most β-lactam antimicrobial agents, including carbapenems, limiting treatment options in infected patients seriously [1]. KPC-producing Enterobacterales, particularly K. pneumoniae, have spread worldwide over the last decade, becoming an urgent public health threat [2]. KPC-2, the most commonly identified variant, is a dominant factor leading to carbapenem resistance in Enterobacterales. The bla KPC-2 gene is typically identified in mobile transposon, which is most often situated on conjugative plasmids [2][3][4]. Tn4401 is the major vehicle of bla KPC-2 in most countries and regions, such as Europe [5], the United States [6], and Brazil [7]. In Asia, bla KPC-2 is mostly located on diverse variants of Tn1721 and IS26 [8][9][10].
Horizontal gene transfer (HGT) plays an important role in the evolution of bacteria and the dissemination of antibiotic resistance genes [11]. Tn4401 and Tn1721, typical replicative transposons belonging to Tn3 family, have been proved to mobilize bla KPC-2 at a high transposition frequency, and the latter is capable of transferring bla KPC-2 both internal and external to this element [12,13]. However, few published studies provided comprehensively analysis of epidemic of KPC-2-producing Enterobacterales, the dynamics of genetic structure surrounding bla KPC-2 and transposition mechanism of these elements. Notably, bla KPC-2 is located on diverse variants of Tn1721 that exhibited various transposition frequencies and movement patterns [13], and the mechanism of movement of A2-type (Tn1721-bla KPC-2 -IS26) remains undetermined.
Here, we show that outbreak of KPC-2-producing plasmid is correlated with the dynamics of transposon structure in Enterobacterales. The A2and B-type Tn1721 appearing concurrently with the peak of the epidemic of bla KPC-2 -carrying isolates demonstrated higher transposition frequency. Their specific replicative transposition (the IS26 pattern for A2-type and the Tn1721-bla KPC-2 -IRL2 pattern for B-type) had a certain superior ability to conjugate into another strain. Thus, replicative transposition contributes to the evolution and dissemination of KPC-2-producing plasmid in Enterobacterales, facilitating the inter-and intra-species dissemination of bla KPC-2 .

Clinical isolates
A total of 161 non-duplicated, carbapenem-resistant Enterobacterales (CRE) isolates were collected from August 2006 to December 2010 during routine identification and antimicrobial susceptibility testing by the Microbiology Laboratory, Huashan Hospital, Fudan University (Shanghai, China). This collection comprised all clinical isolates from the first occurrence of KPC-2-producing isolates (K. pneumoniae) to the prevalence of this cabapenemase (Dataset S1). For comparison, a susceptible collection of 112 carbapenem-sensitive K. pneumoniae (CS-KP) were also collected contemporaneously from similar departments (Dataset S2).
PCR screening of bla KPC-2 and IncFII replicon and analysis of genetic environment of bla KPC-2 gene among clinical isolates The bla KPC-2 were identified through the amplification and sequence analysis of a 750-bp polymerase chain reaction (PCR) product [15]. IncFII replicon screen was conducted by PCR-based replicon typing using previously reported primers [16]. The bla KPC-2 -bearing genetic structures were determined by a series of PCR assays as reported previously [15].

Transformation and conjugation experiments of bla KPC-2 -bearing plasmids
Thirty-five bla KPC-2 -bearing plasmids were obtained by transformation or conjugation (Dataset S3). Plasmids of clinical isolates were extracted with a Qiagen Plasmid Midi kit (Qiagen, Germany) and examined by agarose gel electrophoresis, and then transformed into E. coli DH5α Electrocompetent cells by electroporation (Micro-Pulser electroporator; Bio-Rad, USA). Conjugation experiment was performed with E. coli J53 (AZ r ) as the recipient. Transformants and conjugants were selected on MacConkey agar containing IPM (AZ was also used in conjugants selection) and identified by VITEK 2 system (bioMérieux, France) and were further subjected to PCR amplification of IncFII replicons, bla KPC-2 and genetic environment of bla KPC-2 according to our previous operations. For plasmids originated from clinical isolates of E. coli, each transformant was also identified by detecting the deletion of lacY gene as E. coli DH5α lacks the lac operon. Primers are listed in Table S1.

Bioinformatics analysis
All complete genome sequences of Enterobacterales publicly available (5152 in total) and all of the plasmid sequences harbored in these strains (10,507 in total) were downloaded from NCBI database in August 2021 (Chromosomes in Dataset S4 and plasmids in Dataset S5). The bla KPC-2 gene was identified by using nucleotide BLAST. Plasmid incompatibility type was determined by comparing with information in the Plasmid MLST locus/sequence definitions database (https://pubmlst.org/bigsdb?db=pubmlst_ plasmid_seqdef& page = sequenceQuery).

Plasmids construction
The primers and plasmids used in this study are listed in Table S1 and Table S2, respectively. pHS10842 (GenBank accession no. KP125892), a vector favorable for use in the exploration of the transposition mechanism of Tn1721-like transposons, has been described previously [13].
For pHS10842-ΔtnpA Tn1721 , IRR and tnpR fragments were amplified with primers JP934/JP935 and JP936/JP937, respectively. The IRR fragment share 20-bp sequences with AhdI restriction sites of pHS10842 and the tnpR fragments, respectively. The tnpR fragments also share 20-bp sequences with the IRR fragment and AflII restriction sites of pHS10842, respectively. The two fragments were subcloned into AhdI and AflII restriction sites of pHS10842 by use of the NEBuilder HiFi DNA assembly master mix (New England BioLabs, USA), generating the derivative with the tnpA deletion of Tn1721, pHS10842-ΔtnpA Tn1721 .
The transposase deletion was introduced into IS26 by digesting plasmid pHS10842 with SwaI and XmnI to removal a 560-bp fragment (base 11,366 to base 11,925 in GenBank accession no. KP125892) and generate blunt ends. The DNA was ligated and transformed into competent cells of E. coli DH5α. pHS10842-ΔtnpA Tn1721 Δtnp26 was constructed in a similar strategy with pHS10842-ΔtnpA Tn1721 .

Transposition assays and molecular characterization of transposition events
Transposition assays were performed as described previously [13,17]. Transposition frequency is calculated as the number of IPM r TMP r STR r transconjugants per TMP r STR r transconjugant. For each transposition event, the movement patterns were determined by agarose gel electrophoresis and Southern hybridization [13]. The exact insertion site and target site duplication of Tn1721 and IS26 were determined by using primes specific for regions internal to Tn1721 and IS26 and primers specific for R388 as described previously [13]. Primers are listed in Table S1.

Analysis of the target site consensus sequence
The relative frequencies of the AT and GC contents of the region extending from 50 bp upstream to 50 bp downstream of the duplicated target site for IS26 (8 bp) were calculated and plotted on a line graph. The pictures of the relative frequencies of the bases at each position were generated with the Pictogram program (http://genes.mit.edu/pictogram. html).

Statistics
Statistical significance was assessed by Fisher's exact test or Chi-square test with Yates' correction using GraphPad Prism8 software (https://www.graphpad. com/). P < 0.05 was considered statistically significant.

Results
The prevalence of KPC-2-producing Enterobacterales was mediated by multiple species and STs All of the 161 CRE were identified as bla KPC-2 -positive, including K. pneumoniae (112, 69.56%), E. coli (15, 9.32%), Citro. freundii (15, 9.32%), K. aerogenes (12,7.45%) and other species (7, 4.35%) ( Figure 1A). Among the 112 K. pneumoniae isolates, ST11 was the most prevalent ST, followed by ST423, ST65 and ST977, and PFGE of ST11 K. pneumoniae indicated five diverse subtypes with a criterion of 75% identity ( Figure 1B). For comparison, we collected CS-KP as susceptible controls that were matched by time and ward. As expected, all CS-KP isolates were bla KPC-2negative and the STs of the susceptible collection were scattered without any dominant STs ( Figure  S1). E. coli, Citro. freundii and K. aerogenes isolates were comprised of several STs. Together, these results suggested that bla KPC-2 is the chief culprit leading to carbapenem resistance and that the prevalence of bla KPC-2 in Enterobacterales was mediated by multiple species and STs, rather than clonal spread.
bla KPC-2 genes were usually located on three diverse variants of Tn1721 in MDR region of plasmid The bla KPC-2 genes were usually reported to be located on MDR region of plasmid, and IncFII plasmids contributed significantly to the global prevalence of bla KPC among K. pneumoniae [4,9]. To determine the correlation between IncFII plasmids and bla KPC-2 gene, we conducted a bioinformatic analysis of 5152 complete genome sequences of Enterobacterales publicly available (including 5152 chromosomes and 10,506 plasmids in total, Dataset S4 and Dataset S5). Among these, 311 strains were identified to be bla KPC-2 -positive, and the overwhelming majority of bla KPC-2 genes were found on plasmids (300/311, 96.46%). Remarkably, most of the bla KPC-2 -bearing plasmids belonged to IncFII group, but this group was less common in bla KPC-2 -negative plasmids [174/300 (58%) versus 2195/10206 (21.51%), P < 0.0001; Table 1]. Moreover, IncFII plasmids were significantly over-represented in bla KPC-2 -positive K. pneumoniae compared with that in bla KPC-2 -negative group [151/204 (74.02%) versus 657/2701 (24.32%), P < 0.0001; Table 1]. These findings suggest that bla KPC-2 genes are mostly located on plasmids in Enterobacterales, and that IncFII is the most common Incompatibility group, especially in K. pneumoniae.
To evaluate this correlation in clinical isolates, we first performed IncFII replicon screening on all clinical isolates. Significantly, the detection rate of IncFII replicon was 84.82% in K. pneumoniae and the rate was slightly lower for E. coli (73.33%) and K. aerogenes (66.67%) ( Table S3). None of Citro. freundii isolates was detected to IncFII-positive. However, the rate was much lower in carbapenem-susceptible collection in comparison with either CR-KP or CRE collection (49.11% vs. 84.82% for CR-KP, P < 0.0001; 49.11% vs. 72.05% for CRE, P = 0.0001, Figure 2A). Next, the linkage between IncFII plasmids and bla KPC-2 gene was determined in thirty-five transformants and conjugants containing bla KPC-2 -positive plasmid by PCR amplification of IncFII replicons and genetic environment of bla KPC-2 (see Dataset S3 for details).
Interestingly, 80% of plasmids carrying bla KPC-2 gene (28/35) belong to IncFII group. Finally, five plasmids in various sizes (two IncFII-negative and four IncFII-positive, Dataset S3) were selected for complete sequence analysis to furtherly confirm the correlation between IncFII plasmids and bla KPC-2 gene.

Outbreak of KPC-2-producing plasmids is correlated with the dynamics of transposon structure
In order to clarify the progression of bla KPC-2 outbreak, we comprehensively analyzed all data obtained in molecular epidemiological investigation. Figure 3 provides an overview of the evolution of bla KPC-2 bearing structure in Enterobacterales. Samples are plotted by month of isolation, wards, species, STs of most common resistant specie (K. pneumoniae) and transposable elements carrying bla KPC-2 gene. As shown in Figure 3, bla KPC-2 genes that were located in three distinct Tn1721-like transposons on plasmid, originated from K. pneumoniae, then became increasingly prevalent in this specie and spread in Enterobacterales further. The epidemic of bla KPC-2 -carrying plasmid reported  At the second epidemic stage, KPC-2-producing plasmid were prevalent in K. pneumoniae ST11 that consisted of several different subtypes, and spread into other Enterobacterales. A2-type Tn1721 was the dominant structure carrying bla KPC-2 . In the mixed epidemic period, KPC-2-producing isolates increased furtherly. This stage involved three distinct Tn1721 variants, and the number of emerging B-type was almost equal to that for A2-type during the same period.
Altogether, outbreak of KPC-2-producing plasmid was correlated with the dynamics of bla KPC-2 -bearing transposon structure, and A2-and B-type Tn1721 appeared concurrently with the peak of bla KPC-2 epidemic.
Replicative transposition promotes horizontal transfer of bla  A critical step in the dissemination process of bla KPC-2 is the HGT of mobile genetic elements (MGEs) surrounding this determinant. According to our comprehensive analysis of epidemiological data, it was presumed that replicative transposons, Tn1721 and IS26, can mobilize bla KPC-2 through transposition, promoting the dissemination of bla KPC-2 in Enterobacterales. In our previous study, A1-and B-type Tn1721 have been shown to transfer bla KPC-2 both internal and external to this element, and target transposition into 5-bp region that gradually exhibits a degenerated degree of AT-rich regions from both sides to the middle and that is immediately flanked by GC-rich regions [13]. Here, we characterized the movement and target site of A2-type Tn1721 (Tn1721-bla KPC-2 -IS26).
Two distinct patterns of movement mediated by Tn1721 and IS26 existed in this chimera. Tn1721 pattern was the same as the one detected in A1-and Btype Tn1721 previously, including cointegrate  forming and resolving steps [13]. In addition to having the Tn1721 pattern, a different IS26 pattern via replicative transposition was also detected in several cases of A2-type Tn1721 ( Figure 4B). Only one plasmid (P3) was obtained from the transconjugant. The donor (pHS10842) and target (R388) plasmids generated this cointegrate (P3) in which both plasmids fused together by directly repeated copies of IS26. This result was supported by a series of data ( Figure  4A), as follows. (i) the size of P3 was larger than that of R388 and P2. (ii) Southern blot analysis showed that P3 contained bla KPC-2 , Tn1721, sul1, IS26. (iii) A series of PCRs confirmed that P3 had the same bla KPC-2 -bearing genetic structure (Tn1721-bla KPC-2 -IS26) as that of pHS10842, and the junctions between donor and target in each case were amplified and sequenced with primers specific for regions internal to IS26 and primers specific for R388. Consistent with previous reports [18], 8-bp target site duplication was evidenced for each transposition events (Figure 5). At the target sites, the AT content for regions from t2 to t7 stabilize at 60-70%, while the AT content at t1 and t8 was slightly lower (46%). Nucleotide composition analysis revealed the t2 and t3 positions were found to be predominantly T residues (54% and 38%, respectively), while t6 and t7 positions were mostly A residues with the same percentage as that for t2 and t3, respectively. It is noteworthy that the insertion sites mostly carried one or even more AA or/and TT nucleotide tandems. These data suggested that IS26 preferentially targets AT-rich regions with AA and TT nucleotide tandems.
The transposition frequency of A2-type Tn1721 was measured to be 3.8 × 10 −6 ( Table 3). This represented a 4-fold increase in efficiency compared to that of A1-type, a bla KPC-2 -bearing structure lacking IS26, and 30% of that for B-type Tn1721 containing an additional left inverted repeat (IRL2). The tnpA deletion of either Tn1721 or IS26 decreased half of the frequency, and both tnpA deletions led to a functional inability to mobilize bla KPC-2 . Hence, bla KPC-2 embedded between Tn1721 and IS26 was transferred at an apparently higher frequency owing to the existence of both elements.
Taken together, our findings indicate that all three transposons were capable of transferring bla KPC-2 through replicative transposition, and that A2-and B-type Tn1721 showed higher transposition frequencies and had a certain superior ability to propagation.

Replicative transposition of Tn1721 contributes to the evolution and dissemination of KPC-2producing plasmid
Given that Tn1721 variants have been shown various capacities of transferring bla KPC-2 via replicative transposition, an attractive hypothesis is that the A2and B-type Tn1721 occasionally appearing in the late-stage stood out from the rest through selection for their ability to facilitate dissemination of bla KPC-2 . As discussed above, the outbreak of KPC-2-producing plasmid was correlated with the dynamics of bla KPC-2 -bearing Tn1721 structure, and A2-and Btype Tn1721 arising concurrently with the peak of bla KPC-2 epidemic showed apparently higher transposition frequencies. Such an association and different movement capabilities imply that replicative transposition of Tn1721 variants contributes to the evolution and dissemination of KPC-2-producing plasmid in Enterobacterales. This mechanism could enable bla KPC-2 gene to search for suitable host spontaneously, dealing with the antibiotic pressure from the environment.

Discussions
KPC-producing Enterobacterales that have spread extensively throughout the world, are an important cause of nosocomial infections, especially urinary tract infections, respiratory tract infections, and bloodstream-associated infections [1]. During the last decade, several studies have described of diverse transposable elements surrounding bla KPC-2 gene [9,10,12]. However, little is known about the exact impacts of these genetic environment change and its correlation with epidemic of bla KPC-2 gene. Our investigation of clinical CRE provided a detailed description of the whole process from bla KPC-2 gene first occurrence, progress to final prevalence. We observed that the prevalence of KPC-2-producing Enterobacterales was mediated by multiple species and STs, and that bla KPC-2 gene was located on three diverse variants of Tn1721 in multi-drug resistance (MDR) region of plasmid. IncFII was the most common Incompatibility group. Further study indicated that replicative transposition contributed to the evolution and dissemination of KPC-2-producing plasmid.
Transposable elements play an important role in the genetic variation and evolution of bacteria. The bla KPC-2 gene is mostly located on transposable elements, such as Tn4401, Tn1721, and IS26 [8,13]. To date, eight variants of Tn4401 (Tn4401a to Tn4401h) have been identified, with Tn4401a and Tn4401b being the most widespread [1,12,19,20]. Notably, these isoforms demonstrate lacking tnpA or/and tnpR, or have deletions in the noncoding region upstream of bla KPC leading to enhanced or reduced expression of this carbapenemase [21]. It is widely reported that Tn1721 variants are the dominant structures in Asia, particularly in China [8][9][10]15,22]. Given that the structure of Tn1721 variants in current bla KPC-2 epidemic is too complicated and diverse to track the dissemination process of this gene, we focus on the early stage of bla KPC-2 epidemic. In addition to the dynamics of bla KPC-2 -bearing transposon structure raised by this study, host and plasmid factors as well as antibiotic pressure from environment were also worth further study.
Our findings indicated that all three Tn1721 variants were capable of mobilizing bla KPC-2 via replicative transposition, and A2-and B-type Tn1721 exhibited higher transposition frequencies than A1type. Such various capacities are presumably associated with cointegrate resolution of Tn1721 in transposition assay. For the transconjugants with the Tn1721 pattern, only the cointegrate could be conjugated into the recipient (E. coli HB101, STR resistant [STR r ]) and screened on LB agar containing IPM, TMP, and STR in the transposition assay. Once the cointegrate resolved in donor strain, neither plasmid generated here could survive. Because P1 lacked the TMP resistance (TMP r ) and the ability to conjugate into recipient (STR r ), even though it possessed IPM resistance (IPM r ), while P2 was an opposite case. In contrast, both the cointegrate and plasmid resolved from the cointegrate with Tn1721-bla KPC-2 -IRL2 pattern possessed IPM r and TMP r , as well as had the ability to conjugate to recipient (STR r ). For the transposition via IS26, there is no resolution process. It means that the cointegrate (P3) with IS26 pattern possessed IPM r and TMP r , as well as had the ability of conjugal transfer. Consequently, it survived on LB agar containing IPM, TMP, and STR. In other words, the plasmid with Tn1721-bla KPC-2 -IRL2 pattern (in B-type) or IS26 pattern (in A2-type) had a certain superior ability of conjugal transfer and became more widespread. These results strongly supported our molecular epidemiological findings that the two peaks of bla KPC-2 epidemic followed the appearance of A2-and B-type Tn1721, reflecting an important role for replicative transposition of Tn1721 and IS26 in the evolution and transmission of bla KPC-2 -carrying plasmid in Enterobacterales.
In conclusion, our work demonstrates replicative transposition facilitates the evolution and transmission of KPC-2-producing plasmid in Enterobacterales, and highlights its important role in the dissemination of antibiotic resistance genes between pathogenic bacterial species.