Emergence of New Non–Clonal Group 258 High-Risk Clones among Klebsiella pneumoniae Carbapenemase–Producing K. pneumoniae Isolates, France

The worldwide spread of Klebsiella pneumoniae carbapenemase–producing Klebsiella pneumoniae (KPC-Kp) isolates was reported to be caused by dissemination of 1 clonal complex (i.e., clonal group [CG] 258, which includes sequence types [STs] 258 and 512). We conducted whole-genome sequencing and epidemiologic analysis of all KPC-Kp isolates in France in 2018 and found that new successful high-risk clones of ST147, ST307, ST231, and ST383 are now the main drivers of blaKPC genes. The blaKPC genes were mostly carried by Tn4401a and Tn4401d structures and a new non–Tn4401 element. Our epidemiologic investigations showed that the emergence of these non-CG258 KPC-Kp isolates in France was linked to dissemination of these clones from Portugal. Thus, KPC-Kp epidemiology has changed in Europe, at least in several non–KPC-endemic countries of western Europe, such as France and Portugal, where CG258 is not the most prevalent clone.

The worldwide spread of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) isolates was reported to be caused by dissemination of 1 clonal complex (i.e., clonal group [CG] 258, which includes sequence types [STs] 258 and 512). We conducted whole-genome sequencing and epidemiologic analysis of all KPC-Kp isolates in France in 2018 and found that new successful high-risk clones of ST147, ST307, ST231, and ST383 are now the main drivers of bla KPC genes. The bla KPC genes were mostly carried by Tn4401a and Tn4401d structures and a new non-Tn4401 element. Our epidemiologic investigations showed that the emergence of these non-CG258 KPC-Kp isolates in France was linked to dissemination of these clones from Portugal. Thus, KPC-Kp epidemiology has changed in Europe, at least in several non-KPC-endemic countries of western Europe, such as France and Portugal, where CG258 is not the most prevalent clone.
Analysis of the genetic context of bla KPC has revealed that this gene is mostly localized into a class 2 transposon named Tn4401 (12). Several variants of this Tn4401 (Tn4401a through Tn4401i) have been reported with deletions upstream of bla KPC within the promoter region (13,14). Consequently, expression of bla KPC genes is complex and might involve different promoters, depending on the specific genetic environment and bacterial species. The 2 main promoters are named P1, which is in the vicinity of bla KPC , and P2, a hybrid promoter located partly in the inverted repeat right of ISKpn7 (15). In rare cases, bla KPC genes have been described in genetic structure not related to Tn4401 and are named non-Tn4401 elements (NTE) (16). However, in NTE, the expression of bla KPC is mediated by other promoters. Our study aimed to deeply characterize the epidemiology of KPC-Kp circulating in France in 2018.

Strains Collections and Culture Conditions
We included all KPC-Kp sent to France's National Reference Center for Antimicrobial Resistance during January 1-December 31, 2018. As previously described, we used isolates that were recovered from clinical and screening specimens and sent on a voluntary basis by any type of laboratory related to any health facility, such as private and public hospitals, nursing homes, and community laboratories (3,4). These laboratories were located throughout France, including overseas territories. KPC-Kp recovered by the National Reference Center for Antimicrobial Resistance represent ≈85%-90% of the KPC-Kp infection cases reported to the French Public Health Agency (R.A. Bonnin, L. Dortet, unpub. data). The collection used for WGS analysis represents a total of 63 nonduplicate isolates recovered from rectal screening (n = 45), urine (n = 12), blood cultures (n = 1), wound infections (n = 2), and respiratory samples (n = 2) and 1 isolate for which no recovery site information was available. Because the aim of the study was to evaluate the genetic diversity of KPC producers, we discarded from further analysis any duplicate isolates or isolates recovered from the same patient.

WGS and Bioinformatic analysis
We sequenced all KPC-Kp isolates by using Illumina technology as previously described (17). We extracted total DNA from colonies by using the Ultraclean Microbial DNA Isolation Kit (MO BIO Laboratories, https://www.mobio.com) according to the manufacturer's instructions. We prepared the DNA library as previously described (17) and performed de novo assembly and read mappings by using CLC Genomics Workbench 12.0 (QIAGEN, https://www.qiagen. com). We identified the acquired antimicrobial resistance genes by using Resfinder 3.1 (https://cge.cbs. dtu.dk/services/ResFinder/) and the CARD database (https://card.mcmaster.ca). We annotated the genomes by using RAST (18). We performed phylogenic analysis by using CSIphylogeny 1.4 (https:// cge.cbs.dtu.dk/services/CSIPhylogeny) and visualized the genomes by using FigTree 1.4.3 (http:// tree.bio.ed.ac.uk/software/figtree). We performed sequences alignments by using ClustalW (https:// www.genome.jp/tools-bin/clustalw). We analyzed single-nucleotide polymorphisms (SNPs) on the whole genome by using CSIphylogeny V1.4 with parameters as follows: select minimum depth at SNP position at 10×, minimum distance between SNPs at 10 bp, and minimum SNP quality score of 30.
We constructed the genetic contexts by using de novo assembly or by mapping with reference genomes from GenBank and verified by in-house PCR as previously described (17). We analyzed plasmid contents of clinical isolates by using PlasmidFinder 2.1 to search for the replicase gene and by conducting manual searches for genes showing homology with the replicase gene.

Genetic Diversity of KPC-Kp
Phylogenetic analysis revealed that the 63 KPC-Kp belonged to 15 different clones (STs) circulating in France (Figure 2; Appendix 1). Although many studies have asserted that CG258 is responsible for the spread of bla KPC (6,(8)(9)(10)(11), in our collection, only 8 isolates (12.7%) belonged to CG258 (4 each for ST258 and ST512). Furthermore, epidemiologic investigations revealed no link between these isolates (Figure 2). Because KPC is not widely disseminated in France, we did not expect to observe such clonal diversity. Indeed, the epidemiology of KPC in France is not comparable to what was reported in nearby countries in Europe where KPC is endemic, such as Greece and Italy, and where the spread of bla KPC-2/-3 is clearly linked to CG258 (24,25). Among the 8 isolates we identified that belonged to CG258, 3 were recovered from patients with travel history in Greece (isolate 175C3 and isolate 177H5) and Italy (isolate 160C2). In France, the 3 most prevalent clones are ST307 (with 15 isolates), ST147 (12 isolates), and ST13 (7 isolates). By using the 21 SNP cutoff value proposed by David et al. to identity a single hospital outbreak caused by a ST258 and ST512 cluster (8), we identified that the ST307 clone was overrepresented because of an outbreak that included 11 isolates (Figure 2; Appendix 2, https://wwwnc.cdc.gov/EID/article/26/6/19-1517-App2.pdf). However, this ST307 also included 4 isolates that were not related to this outbreak, such as the 195I4 strain, which was isolated from a patient who traveled in Crete (Greece) and possesses an additional carbapenemase-encoding gene (bla NDM-4 ). The second most prevalent clone, ST147, seemed to have disseminated upon distinct events (Appendix 2). Most of the ST147 isolates have been recovered from different areas with no epidemiologic link between the patients (Figure 2). A link with Portugal has been identified for most (9/11) patients infected or colonized with a KPC-3-producing K. pneumoniae of ST147 (Figure 2). The same link with Portugal was observed for 4 patients infected or colonized with a KPC-3-producing K. pneumoniae of ST231. Strains from ST383 represented a small outbreak for which cross contamination was evidenced (<20 SNPs between 4 isolates [Appendix 2]). One isolate (171J7) was distantly related to other clones and corresponded to K. variicola. KPC-2-producing K. pneumoniae isolates of ST11 were predominantly linked to patients who had a history of travel in Asia (China and Vietnam), where this ST is known to be the main vector of bla KPC dissemination.

Diversity of Genetic Vehicle Involved in Spread of bla KPC
Analysis of the close genetic context of bla KPC highlighted diversity in the genetic structures at the origin of the acquisition of the carbapenemase-encoding gene. The well-known Tn4401a (in 29 isolates) and Tn4401d (in 26 isolates) were the most prevalent structures identified (Figure 2 and 3; Appendix 1). The KPC-Kp of the 2 main clones ST307 and ST147, bla KPC , is carried on Tn4401a in ST307 and Tn4401d in ST147. Two unrelated isolates (ST11-167I9 and the K. variicola 171J7 isolate) harbored bla KPC in the Tn4401b isoform. In the remaining 6 isolates (ST273-171J9, ST147-199D1, ST1788-189B3, ST11-171G8, ST11-190F6, and ST11-171J10), bla KPC-2 is localized in an NTE element (Figures 2, 3). Although 3 isolates belonged to ST11, they displayed 200-800 SNPs of differences along their core genome, indicating that they were unrelated (Appendix 2). The links with 3 different countries (Portugal for ST11-171G8, China for ST11-190F6, and Vietnam for ST11-171J10) are consistent with this unrelatedness. Analysis of NTE elements revealed 4 different structures even if common features were observed (Figure 3). For instance, the presence of a fragment of ISKpn6 downstream of bla KPC and a copy of ISKpn27 upstream were always present (Figure 3). DRs of TATAGG bracketing ISKpn27 indicated a transposition process that occurred inside the resolvase gene of Tn3. Immediately upstream of bla KPC-2 (74 bp), the presence of the inverted repeat right of Tn3 is present in all NTE, indicating that all these structures were related. However, the NTE differed by the size of the deletions that are present between ISKpn27 and bla KPC-2 (from 280 bp in NTE-190F6 to 940 bp in NTE-199D1). We could observe a remnant of bla TEM-1 in longer structures, but it was not functional anymore. Analysis of the 4 NTE revealed that in NTE-199D1, several copies of IS26 bracketed the whole structure, indicating that this IS might be involved in its acquisition by transposition or a recombination event. IS26 has been recently demonstrated to be able to transpose and thus create a class I transposon by targeting another copy of IS26 (26). NTE-171J10 is inserted in the fip gene of IncNtype plasmids with the presence of DRs surrounding the NTE-171J0 (Figure 3). The fip gene has already been demonstrated to be an integration hot spot in IncN-type plasmids (27,28). DRs as well as putative inverted repeats of Tn3-family transposon are present at the integration site ( Figure 3). Moreover, the presence of the complete Tn3 transposase gene indicated that NTE-171J10 might be functional. In NTE-189B3, a new class I transposon carrying a protein of unknown function has been identified. DRs bracketed ISApu1 and ISApu2, indicating a transposition process mediated by these close insertion sequences (Figure 3).

Discussion
In France, KPC producers (84.1% of K. pneumoniae) represent only 6.8% of all carbapenemase producers, far away from the global 72.7% found in Europe in 2013 (8). This relatively low prevalence of KPC producers in France compared with OXA-48-like and NDM producers has been reported since 2012 (3,4,29). We demonstrated unexpected clonal diversity among KPC-Kp isolated in France. A few overrepresented clones were identified (i.e., ST307, ST147, ST231, and ST13). However, ST307 was involved in a regional outbreak, whereas ST147 and ST13 were identified in different parts of France. Most of the patients colonized or infected with KPC-Kp had a clear link with Portugal, where these 4 STs were recently described to be the more prevalent (30,31). The KPC-2-producing K. pneumoniae isolates identified in France were predominantly recovered from patients with a history of travel in Greece (ST258) or Asia (ST11).
Regarding antimicrobial susceptibility of KPC-Kp in France, the relative high susceptibility to imipenem (30.3%) and meropenem to a lesser extent (18.2%) are in agreement with previous reports from Italy, where ST512 is highly prevalent (26.6% susceptibility to meropenem) (32). Conversely, data from the United States and Taiwan indicated that KPC-Kp are more resistant to carbapenems in those parts of the world, where ST258 is more prevalent (33,34).
Altogether, our results indicate that the KPC-Kp epidemiology has changed in Europe during the past 5 years. In 2018, ST258 and ST512 K. pneumoniae were no longer the main drivers of KPC resistance, at least in several non-KPC-endemic countries of western Europe, such as France and Portugal (30,31). KPC-Kp epidemiology also appears to have begun changing in some countries, such as Italy and Colombia, where CG-258 KPC-Kp was previously known to be endemic. This change is indicated by the reported emergence of ST307 and ST273 KPC-Kp in Sicilia (Italy) (35) and ST307 and ST14 KPC-Kp in Colombia (36). This change in the global epidemiology of KPC-Kp might have an effect on the identification of these carbapenemase producers with the molecular methods dedicated to the identification of GC258 K. pneumoniae (37,38).
In addition, our study highlights the dissemination of bla KPC genes in high-risk clones of K. pneumoniae (ST307 and ST147), genetic features that might provide an advantage in adaptation to the hospital environment and the human host (39). These clones already convey several antimicrobial-resistance genes, including genes encoding other carbapenemases of NDM and OXA-48-like types (40,41). Accordingly, we might now fear the emergence of ST307 and ST147 high-risk clones of K. pneumoniae that can co-produce multiple carbapenemases. A recent study demonstrated the importance of ST307 in the dissemination of bla OXA-181 in South Africa (42). In that study, >600 isolates belonging to ST307 were recovered and analyzed, and the results demonstrated the importance of this clone as a carrier of carbapenemase genes in all continents. Another study used Bayesian analysis to demonstrate that ST307 emerged in the mid-1990s (43). ST307 had been strongly associated with the diffusion of bla CTX-M-15 (43) and now is associated with the dissemination of carbapenemase genes (42).
In conclusion, we found that the epidemiology of KPC-Kp has changed in Europe, in particular, with emergence of non-CG258 KPC-Kp isolates in France, linked to dissemination from Portugal. This change in epidemiology has to be considered by microbiologists because a few diagnostic assays specifically designed for the identification of ST-258 KPC-Kp isolates will not be able to detect non-CG258 KPC-Kp isolates.
This work was funded in part by the University Paris-Sud, France. L.D., T.N., and R.A.B. are members of the Laboratory of Excellence in Research on Medication and Innovative Therapeutics, which is supported by a grant from France's National Research Agency (grant no. ANR-10-LABX-33).

About the Author
Dr. Bonnin is an associate professor in Medical Microbiology at the University of Paris-Sud Medical School and a member of the Associated National Reference Center for Carbapenemase-Producing Enterobacteriaceae. His research focuses on bacterial genomes and transcriptomes with a special interest in mobile genetic elements and evolution.