Prevalence of blaKPC-2, blaKPC-3 and blaKPC-30—Carrying Plasmids in Klebsiella pneumoniae Isolated in a Brazilian Hospital

Klebsiella pneumoniae carbapenemase (KPC) actively hydrolyzes carbapenems, antibiotics often used a last-line treatment for multidrug-resistant bacteria. KPC clinical relevance resides in its widespread dissemination. In this work, we report the genomic context of KPC coding genes blaKPC-2, blaKPC-3 and blaKPC-30 in multidrug-resistant Klebsiella pneumoniae isolates from Brazil. Plasmids harboring blaKPC-3 and blaKPC-30 were identified. Fifteen additional carbapenem-resistant K. pneumoniae isolates were selected from the same tertiary hospital, collected over a period of 8 years. Their genomes were sequenced in order to evaluate the prevalence and dissemination of blaKPC–harboring plasmids. We found that blaKPC genes were mostly carried by one of two isoforms of transposon Tn4401 (Tn4401a or Tn4401b) that were predominantly located on plasmids highly similar to the previously described plasmid pKPC_FCF3SP (IncN). The identified pKPC_FCF3SP-like plasmids carried either blaKPC-2 or blaKPC-30. Two K. pneumoniae isolates harbored pKpQIL-like (IncFII) plasmids, only recently identified in Brazil; one of them harbored blaKPC-3 in a Tn4401a transposon. Underlining the risk of horizontal spread of KPC coding genes, this study reports the prevalence of blaKPC-2 and the recent spread of blaKPC-3, and blaKPC-30, in association with different isoforms of Tn4401, together with high synteny of plasmid backbones among isolates studied here and in comparison with previous reports.


Introduction
Infections caused by carbapenem-resistant Enterobacteriaceae represent a significant global health threat [1]. Enterobacteriaceae become resistant to carbapenems by three major mechanisms: porin alteration, hyper-expression of efflux pumps, and β-lactamase production [2]. There are three main groups of β-lactamases associated with resistance to carbapenems (carbapenemases): Klebsiella pneumoniae carbapenemase (KPC), metallo-βlactamases, and OXA-48-like β-lactamase. Mobile genetic elements, such as plasmids and transposons, are the main vectors enabling and facilitating the spread of these resistance determinants [3,4].
KPC is a plasmid-encoded enzyme which occurs in several different variants, and its clinical relevance is associated with its worldwide spread [5]. The occurrence of carbapanemases varies according to the geographic region; the KPC-2 variant has been reported as the most frequent carbapenemase in Latin America [6,7]. In Brazil, the first report of KPC-2 occurred in 2006, and soon, numerous reports attested to its widespread dissemination [8][9][10][11]. KPC-2 and KPC-3 differ by only one amino acid (H272Y) and are the most prevalent carbapenemases worldwide [12][13][14][15]. Only KPC-2 is currently considered endemic in Brazil [10,16]. The treatment of infections by carbapenem-resistant Enterobacteriaceae is complicated, with severe clinical and socioeconomic consequences [4,17,18].
Despite the worldwide spread of KPC, genomic studies including KPC-harboring isolates from Latin America, and in particular from Brazil, are underrepresented in the literature. For instance, a recent study evaluated all KPC-harboring plasmids described in the NCBI database, and only 12 out of 257 representative KPC-plasmids evaluated were from Brazil, all harboring bla KPC-2 [19].
Our hospital is one of the Brazilian medical centers enrolled in a longitudinal surveillance study (Study for Monitoring Antimicrobial Resistance Trends, SMART) [20]. Due to our participation, two KPC variants not previously reported in Enterobacteriaceae in Brazil, bla KPC-3 , and bla KPC-30 , were identified in K. pneumoniae isolated in Brazil [21]. Both isolates showed resistance to colistin, a last-resort therapeutic drug against multidrug-resistant Enterobacteriaceae.
In this work, we report the genomic characterization of these two isolates by whole genome sequencing. In addition, 15 K. pneumoniae isolates, collected in the same tertiary hospital over a period of 8 years, were genome-sequenced. We found bla KPC -harboring plasmids in most isolates; these originated from two different plasmids and contained bla KPC-2 , bla KPC-3 , or bla KPC-30 , mostly on one of two isoforms of transposon Tn4401 (Tn4401a or Tn4401b).

Results
Genome characteristics and MLST types for the K. pneumoniae isolates harboring bla KPC-2 , bla KPC-30 , and bla KPC-3 are summarized in Table 1. All isolates belonged to the Clonal Complex 258 (CC258), except for Kp326 (ST16). Capsule polysaccharide synthesis (cps) locus analysis demonstrated that isolates belonged to five distinct K-loci groups, the most recurrent being KL36, as previously reported [22].
All isolates harbored the bla KPC-2 gene, except for Kp391, harboring bla KPC-30 , and Kp392, harboring bla KPC-3 . We selected three isolates harboring distinct bla KPC variants for a complete genomic characterization: Kp90 (bla KPC-2 ), Kp391 (bla KPC-30 ), and Kp392 (bla KPC-3 ). Major differences between the three genomes are depicted in Figure 1A. We then looked for the genomic loci of the KPC genes; in all strains, the genes were located on contigs that were parts of plasmids ( Figure 1B). This is in agreement with previous observations, that is, the bla KPC gene is mostly carried by a multireplicon IncFIIk-IncFI plasmid [23,24]. In fact, in Kp392, the bla KPC-3 gene was located on a region that is 100% identical to a region of the plasmid pKpQIL (113,637 bp) found in K. pneumoniae ST258 strains [25] and to a region of plasmid pKPC (113,639 bp) found in a ST512 strain [26] ( Figure 1B). Kp392 from this work is also a ST512 strain, and the plasmid in strain Kp392 is a multireplicon IncFIIk-IncFI plasmid ( Figure 1B). Next, we looked into the bla KPC-30 -harboring strain Kp391 that belongs to the well-known ST11 [27][28][29][30]. The bla KPC-30 gene was located on a 49 kb contig that showed high similarity with the 54 kb plasmid pKPC_FCF3SP that carries bla KPC-2 in strain FCF3SP (ST442) isolated from a blood culture of a patient in Brazil [31]. The entire plasmid in strain Kp391 could be reconstructed using pKPC_FCF3SP as template. The assembled plasmid is designated pKPC30_Kp391 ( Figure 1C). All genes present in the lncN plasmid backbone were found in pKPC30_Kp391 ( Figure 1D). The plasmids from strains FCF3SP and Kp391 share 99% nucleotide identity, with a total of 76 SNPs. The strain Kp90 carries a bla KPC-2 -encoding plasmid that shares high similarity with pKPC30_Kp391, pKPC_FCF3SP, as well as pKPC_FCF1305. Figure 1E describes the genomic region of the three plasmids. Fourteen additional K. pneumoniae isolates that were collected in the same tertiary hospital over a period of 8 years were sequenced and analyzed. We searched for the genomic region where the bla KPC genes were located in these 14 strains; in all strains, the genes were located on contigs that were parts of plasmids belonging to two distinct incompatibility groups: lncN or lncFII. Among the 17 sequenced isolates, 15 harbored the lncN backbone, showing high similarity with pKPC_FCF3SP ( Figure 2A). On this plasmid, except for Kp391, harboring bla KPC-30 , all isolates harbored bla KPC-2 . Isolates Kp326 (bla KPC-2 ) and Kp392 (bla KPC-3 ) contained plasmids highly similar to the plasmid pKpQIL (lncFII) ( Figure 2B). pQIL-like (IncFIB) plasmids have just recently been reported in Brazil, in clones belonging to ST16, such as Kp326, and associated with high mortality rates [32]. However, the sequence of the pQIL-like plasmid identified in Brazil is not publicly available, so we were not able to compare ours to it and, to our knowledge, the first sequence made publicly available.
Three isoforms of the Tn4401 transposon were found in the isolates described in this study. Two isoforms are well-known, Tn4401a and Tn4401b. These isoforms differ by a 100 bp deletion in the region upstream of bla KPC in Tn4401a ( Figure 3A). The isoform Tn4401a was carried by pKpQIL-like plasmids (strains Kp326 and Kp392), while the isoform Tn4401b was carried by pKPC_FCF3SP-like plasmids, as previously reported [31]. It has been shown that isolates harboring Tn4401a present higher resistance to carbapenems due to alterations in the promoter region of bla KPC [33,34]. A third isoform of Tn4401 was detected in isolate Kp381. This variant lacks 253 bp downstream from the bla KPC gene, leading to a shorter tnpA/ISKpn6 region (1320 bp/439 aa to 963 bp/320 aa) ( Figure 3B). ity profile of K. pneumoniae, the susceptibility profiles of Kp391 (blaKPC-30), Kp392 (blaKPC-3), and Kp90 (blaKPC-2) are reported in Table 2. All three isolates showed resistance to cephalosporins, quinolones, colistin, and carbapenems, and were susceptible to the combination ceftazidime/avibactam. Kp392, harboring the pKpQIL-like plasmid with Tn4401a and blaKPC-3, displayed a MIC of 6 µg/mL for the combination ceftazidime/avibactam (breakpoint for resistance: ≥8 µg/mL); this is higher than for the other two isolates which were clearly susceptible (2 µg/mL) [35]. isolates using strain HS11286 as the reference genome. Genome sizes were similar to the reference genome K. pneumoniae strain HS11286 (5682 kb) and differences are mostly associated with acquired genomic regions. (B) The plasmid in strain Kp392 is highly similar to plasmids pKpQIL and pKPC_D1. The analysis compared the plasmid pKpQIL with the plasmid pKPC_D1 and the genomes of the three strains sequenced here. Only the genome of strain Kp392 contains contigs (including the 21 kb contig that carries blaKPC-3) that match almost entirely the plasmid pKpQIL. The strain Kp391 carries a different plasmid that shares some homology with pKpQIL. The blaKPC-3 gene is highlighted in pink. (C) The blaKPC-30 carrying plasmid pKPC30_Kp391 in strain Kp391 is highly similar to blaKPC-2 carrying plasmids pKPC_FCF3SP and pKPC_FCF1305. The analysis compared the assembled plasmid sequence in strain Kp391 with the plasmids pKPC_FCF3SP and pKPC_FCF1305 and the genomes of the other strains sequenced here (Kp90 and Kp392). pKPC30_Kp391 almost entirely matches the plasmids pKPC_FCF3SP and pKPC_FCF1305. The strain Kp90 also carries a blaKPC-2 encoding plasmid that shares high homology with pKPC30_Kp391, pKPC_FCF3SP and pKPC_FCF1305. The location of blaKPC-2 or blaKPC-30 is highlighted in pink. (D) General structure of pKPC30_Kp391 based on the IncN conserved backbone and the two acquired regions Tn4401b and IS903B. The conserved lncN region carries genes involved in replication and replication regulation (repA, ardB, ardR, ardK, ccgEIII, ccgD, ccgC, ccgAI), genes responsible for plasmid stability (stbA, stbB, stbC, fipA, eex, korA, korB, kikA), in DNA repair (mpr, mucA, mucB), inhibition of type I restriction enzymes isolates using strain HS11286 as the reference genome. Genome sizes were similar to the reference genome K. pneumoniae strain HS11286 (5682 kb) and differences are mostly associated with acquired genomic regions. (B) The plasmid in strain Kp392 is highly similar to plasmids pKpQIL and pKPC_D1. The analysis compared the plasmid pKpQIL with the plasmid pKPC_D1 and the genomes of the three strains sequenced here. Only the genome of strain Kp392 contains contigs (including the 21 kb contig that carries bla KPC-3 ) that match almost entirely the plasmid pKpQIL. The strain Kp391 carries a different plasmid that shares some homology with pKpQIL. The bla KPC-3 gene is highlighted in pink. (C) The bla KPC-30 carrying plasmid pKPC30_Kp391 in strain Kp391 is highly similar to bla KPC-2 carrying plasmids pKPC_FCF3SP and pKPC_FCF1305. The analysis compared the assembled plasmid sequence in strain Kp391 with the plasmids pKPC_FCF3SP and pKPC_FCF1305 and the genomes of the other strains sequenced here (Kp90 and Kp392). pKPC30_Kp391 almost entirely matches the plasmids pKPC_FCF3SP and pKPC_FCF1305. The strain Kp90 also carries a bla KPC-2 encoding plasmid that shares high homology with pKPC30_Kp391, pKPC_FCF3SP and pKPC_FCF1305. The location of bla KPC-2 or bla KPC-30 is highlighted in pink. (D) General structure of pKPC30_Kp391 based on the IncN conserved backbone and the two acquired regions Tn4401b and IS903B. The conserved lncN region carries genes involved in replication and replication regulation (repA, ardB, ardR, ardK, ccgEIII, ccgD, ccgC, ccgAI), genes responsible for plasmid stability (stbA, stbB, stbC, fipA, eex, korA, korB, kikA), in DNA repair (mpr, mucA, mucB), inhibition of type I restriction enzymes (ardA), and conjugative transfer (tra gene region). Transposon Tn4401b comprises tnpR, tnpA, istA, istB, bla KPC , and tnpA. (E) Plasmid gene organization in the vicinity of bla KPC gene in pKPC_Kp90, pKPC_Kp391, pKPC_Kp392, and closely related plasmids. The colors indicate high homology of the genes. 1, bla KPC gene; 2 (tnpA) and 4 (tnpA) (transposase); 3 (istB) and 5 (istA) (mobile element); 6 (tnpR) site-specific recombinase; 7-14 (tra genes) components of a type IV conjugative transfer system; 15-17, hypothetical proteins.

Antimicrobial MIC µg/mL Profile MIC µg/mL Profile MIC µg/mL Profile
MIC: minimum inhibitory concentration. Breakpoints for Tigecycline are not available for K.
pneumoniae [36]. As a means to understand the impact of the different bla KPC genes in the susceptibility profile of K. pneumoniae, the susceptibility profiles of Kp391 (bla KPC-30 ), Kp392 (bla KPC-3 ), and Kp90 (bla KPC-2 ) are reported in Table 2. All three isolates showed resistance to cephalosporins, quinolones, colistin, and carbapenems, and were susceptible to the combination ceftazidime/avibactam. Kp392, harboring the pKpQIL-like plasmid with Tn4401a and bla KPC-3 , displayed a MIC of 6 µg/mL for the combination ceftazidime/avibactam (breakpoint for resistance: ≥8 µg/mL); this is higher than for the other two isolates which were clearly susceptible (2 µg/mL) [35].
MIC: minimum inhibitory concentration. Breakpoints for Tigecycline are not available for K. pneumoniae [36].

Discussion
KPC-producing Enterobacteriaceae have been considered a pandemic in the history of Gram-negative bacteria [37]. According to gene sequences deposited in Genbank (https://www.ncbi.nlm.nih.gov/pathogens/refgene/#kpc, accessed on 4 February 2021) at the time of this submission (date: 4 February 2021), a total of 66 blaKPC gene variants had been reported.
As a result of the antibiotic resistance surveillance study, SMART, two new KPC variants were detected in K. pneumoniae in Brazil: blaKPC-3 and blaKPC-30 [21]. This work was conducted to gain an understanding of the genetic context surrounding blaKPC genes in the tertiary hospital where the new variants were detected, using this information to

Discussion
KPC-producing Enterobacteriaceae have been considered a pandemic in the history of Gram-negative bacteria [37]. According to gene sequences deposited in Genbank (https://www.ncbi.nlm.nih.gov/pathogens/refgene/#kpc, accessed on 4 February 2021) at the time of this submission (date: 4 February 2021), a total of 66 bla KPC gene variants had been reported.
As a result of the antibiotic resistance surveillance study, SMART, two new KPC variants were detected in K. pneumoniae in Brazil: bla KPC-3 and bla KPC-30 [21]. This work Pathogens 2021, 10, 332 7 of 11 was conducted to gain an understanding of the genetic context surrounding bla KPC genes in the tertiary hospital where the new variants were detected, using this information to discuss the scenario in Brazil.
KPC-3, still not endemic in Brazil, differs by one amino acid substitution (H272Y) from KPC-2 [38]. This single amino acid change has been reported to increase the catalytic efficiency of the enzyme by up to nine times when degrading ceftazidime [14], and, most recently, it has been described to increase the MIC for ceftazidime/avibactam [14,15,39]. This is particularly worrisome since, in Brazil, this combination has only recently been approved as a treatment option for multidrug-resistant bacteria (June 2018). Strains Kp90 (harboring bla KPC-2 ), Kp391 (harboring bla KPC-30 ), and Kp392 (harboring bla KPC-3 ) showed very similar susceptibility profiles for carbapenems and ceftazidime. However, Kp392 displayed a MIC of 6 µg/mL for ceftazidime/avibactam (breakpoint for resistance: ≥8 µg/mL) [35], compared to Kp90 and Kp391, which were clearly susceptible (2 µg/mL). We suggest that the increased resistance to ceftazidime/avibactam found for Kp392 (MIC of 6 mg/mL) is mostly associated with the presence of KPC-3, in agreement with previous studies [14,40]. Additionally, in Kp392, the pKpQIL-like plasmid harbored the bla KPC-3 gene in a Tn4401a transposon; this transposon has been associated with increased resistance to carbapenems due to modifications in the promoter region that increase the expression of the bla KPC-3 gene (a 100 bp deletion upstream of this gene) [33,34].
Plasmid pKpQIL belongs to the lncFII incompatibility group and has often been reported in association with antibiotic resistance and epidemic isolates. It was first reported in Israel in 2006, but a retrospective study revealed that it had been carried by a K. pneumoniae isolate from a patient in New York in 2004 [41][42][43][44]. This plasmid was the first bla KPC -bearing plasmid identified in epidemic ST258 strains, becoming well-known for the early dissemination of KPC-encoding genes [25,45,46]. Despite its worldwide spread, pKpQIL-like plasmids were only recently reported in Brazil in K. pneumoniae ST16 [25,32,41,43,44,47,48]. In this work, isolates harboring this plasmid were collected in 2016 and 2017 in strains belonging to ST16 and ST512, suggesting a recent, and possibly local, spread.
Plasmids belonging to IncN, such as pKPC_FCF3SP, and highly similar plasmids identified in this study (in strains isolated since 2011), have been reported in Brazil since 2005; the earliest report of an IncN plasmid carrying bla KPC-2 was in 1997 [19,31,49]. Even though pKPC_FCF3SP is associated with the bla KPC-2 gene, here we report an isolate carrying a pKPC_FCF3SP-like plasmid with bla KPC-30 . The bla KPC-30 gene sequence has been previously reported in one strain from Brazil, but without any associated publication (strain 1472816, GenBank accession number KY646302.1). The bla KPC-30 variant shows a single-point mutation compared to bla KPC-2 , leading to one amino acid change (R6H) in the region coding for the signal peptide. In this regard, a high degree of synteny between bla KPC-2 and bla KPC-30 -carrying plasmids was noted and, as matter of fact, highly similar plasmids have often been identified in epidemic strains, such as those belonging to CC258 [45,50,51].
In conclusion, in this work we reported the genetic background for bla KPC found in carbapenem-resistant K. pneumoniae isolates from Brazil. Plasmids highly similar to pKpQIL and pKPC_FCF3SP harbored bla KPC-2, bla KPC-3 , bla KPC-30 , in association with different isoforms of Tn4401, an active transposon encoding insertion sequences (ISKpn7 and ISKpn6) capable of efficiently mobilizing the bla KPC gene to random targets. The diversity and structural complexity of genetic elements carrying bla KPC genes suggest that they play major roles in actively promoting transposition of these genes to various genetic locations in the bacterial genome, but also suggest that they may increase the genetic plasticity of plasmids, leading to improved ability to coexist with bacterial hosts [45]. However, we also observed high synteny of plasmid backbones among isolates studied here and in comparison with previous reports from Brazil and the rest of the world. This highlights the importance of surveillance for early detection and implementation of control measures to prevent the rapid dissemination of bla KPC in the clinical environment.

Clinical Isolates
K. pneumoniae isolates harboring bla KPC genes were identified as K. pneumoniae by MALDI-TOF MS (Bruker Daltonics, Billerica, MA, USA) between 2011 and 2017. All patients had hospital-acquired infections, with a history of previous hospitalization and antimicrobial use (carbapenem, quinolone, cephalosporin, or piperacillin-tazobactam) during the 30 days prior to K. pneumoniae isolation.

Antibiotic Susceptibility Testing
Antibiotic susceptibility was determined using the Vitek 2 XL System (bioMérieux, Craponne, France). Additionally, susceptibility to ceftazidime/avibactam, imipenem, and meropenem was also carried out by the epsilometric (Etest ® ) method. Broth microdilution was performed for MIC testing of polymyxin B with Pseudomonas aeruginosa ATCC 27853 used as a reference for susceptibility. Antimicrobial susceptibility results were interpreted according to BrCAST/EUCAST guidelines [52].

Genome Sequencing
Genomic DNA isolation of K. pneumoniae isolates was performed as previously described [53]. Concentration and purity of the isolated DNA was first checked with a NanoDrop ND-1000 (Peqlab, Erlangen, Germany), and the exact concentration was determined using the Qubit ® dsDNA HS Assay Kit, as recommended by the manufacturer (Life Technologies GmbH, Darmstadt, Germany). Illumina shotgun libraries were prepared using the Nextera XT DNA Sample Preparation Kit and subsequently sequenced on a MiSeq system using the reagent kit v3 with 600 cycles (Illumina, San Diego, CA, USA), as recommended by the manufacturer. Quality filtering was done with version 0.36 of Trimmomatic [54]. Assembly was performed using the SPAdes genome assembler software version 3.13.0 [55], using an average of 2,009,581 paired-end reads (range: 1,716,540-2,544,588). Qualimap version 2.2.1 [56] was used to validate the genome assembly and determine the sequence coverage. The average coverage was 81-fold (range: 69-103). Comparative genome and plasmid analyses were done using RAST and visualized with BRIG [57]. Insertion sequences were identified by IS finder [58].