Predominant transmission of KPC-2 carbapenemase in Germany by a unique IncN plasmid variant harboring a novel non-transposable element (NTE KPC -Y)

ABSTRACT Carbapenemase-producing Enterobacterales (CPE) pose a major public health threat. Despite active infection prevention efforts, the incidence of KPC-2 carbapenemase-producing Enterobacterales (KPC2-CPE) continues to increase worldwide. In this study, we performed genome sequencing of 135 KPC2-CPE isolates obtained from multiple sources (clinical, hospital environments, and surface water) in Germany between 2013 and 2019 and analyzed them for epidemiological clues regarding transmission. For 92% (124/135) of all isolates, which comprised 14 different species such as Klebsiella, Escherichia, Citrobacter, Enterobacter, Raoultella, and Serratia, KPC-2 was present on an IncN[pMLST15] plasmid. All plasmids carried a novel non-Tn4401-element harboring an aac (3)-IId-bla TEM-1B-bla KPC-2–cassette (designated NTE KPC -Y) that was co-transferred with an adjacent region carrying 12 further antibiotic resistance genes. Identical plasmids were also detected in KPC2-CPE isolates from environmental samples. These plasmids were remarkably stable and were maintained in individual patients colonized with KPC-2 CPEs over a long-term period (>1 year). Thus, a predominant broad host range signature IncN[pMLST15] plasmid mediates transmission of both KPC-2 and associated multiple antimicrobial drug resistance genes in Germany. These data underline the need for in-depth characterization of plasmid carriers of CPE in surveillance and outbreak studies as well as in microbiomes from patients and the environment to identify hidden transmission reservoirs. This information will be essential for the development and implementation of effective infection control and prevention measures to disrupt dissemination of KPC2- CPEs in healthcare and associated environmental settings. IMPORTANCE Current infection control protocols assume that the spread of KPC-2 carbapenemase-producing Enterobacterales (KPC2-CPE) by detected carriers to other in-house patients is through clonal transmission and can be restricted by implementing containment measures. We examined the presence of the bla KPC-2 gene in different genera and species of Enterobacterales isolated from humans at different hospitals and surface waters between 2013 and 2019 in Germany. We found that a single IncN[pMLST15] plasmid carrying the bla KPC-2 gene on a novel non-Tn4401-element (NTEKPC-Y), flanked by an adjacent region encoding 12 other antibiotic resistance genes, was uniquely present in multiple species of KPC2-CPE isolates. These findings demonstrate the selective impact of specific IncN plasmids as major drivers of carbapenemase dissemination and suggest “plasmid-based endemicity” for KPC2-CPE. Studies on the dynamics of plasmid-based KPC2-CPE transmission and its presence in persistent reservoirs need to be urgently considered to implement effective surveillance and prevention measures in healthcare institutions.

the surface water (2017) of a rivulet where the suspected index patient of an outbreak in the Rhine-Main region suffered a near-drowning incident were also included in the study (23).
KPC-2-producing isolates included those from rectal swabs (n = 73), urine samples (n = 12), respiratory samples (n = 12), bloodstream-infections (n = 2), and other sites (n = 21).Isolates from the hospital environment associated with the outbreak (n = 10) and rivulet water samples (n = 5) were included in the study.Detailed information of all KPC2-CPE isolates, including their genomic characteristics, plasmid Inc types, and antimicrobial resistance genes, is presented in Table S1.

A predominant IncN[pMLST15] plasmid carrying a novel NTE KPC -Y cassette
For 124 isolates of KPC2-CPEs, the bla KPC-2 gene was located on IncN plasmids that were subtyped as pMLST15.Of these, 121 were single-replicon IncN[pMLST15] plasmids and three were cointegrates of this plasmid with IncF-, IncFII-, and IncQ1 plasmids (Table 1; Table S2).In eight of the remaining 11 KPC2-CPE isolates, the bla KPC-2 gene was located on IncF plasmids.Seven of them were associated with a Tn4401a element, whereas in three isolates, no definite assignment of the location on either the chromosome or plasmid was possible (Table S3).Within the surveillance period, IncN[pMLST15] was detected in 75 of 85 KPC2-CPE from 10 different species and associated with more than 46 MLST types.These isolates were obtained from 45 patients in 19 hospitals distributed across the State of Hesse.Occurrence in species below: no. of isolates and (ST-Types):
The composite structure of the IncN[pMLST15] plasmids consists of a 43-kb IncNcharacteristic backbone (region I), which includes the replication region, a conjugation (tra) system, a stability operon, and an anti-restriction system, similar to that of pNL194, an IncN(pMLST18) plasmid (24).The tra genes are separated into three sections TRA-I, TRA-II, and TRA-III (24).The region between TRA-II and TRA-III (comprising fipA and nuc) is separated by the insertion of an acquired segment of ~35 kb (region II) into the gene nuc, and this region II can in turn be divided into two segments, region IIA and IIB.Region IIA (11 kb) comprises a section carrying three ARGs including bla KPC-2 on a non-Tn4401-element (described in detail as follows), which is commonly present in all IncN[pMLST15] plasmids regardless of size (Fig. 2A).

pCF13141-KPC2
78  Plasmid transfer experiments with the donor strains CF08098 and CP13069 showed bla KPC-2 IncN[pMLST15] transconjugants with plasmids of different sizes (~78 kb and ~60 kb), indicating transfer of resistance to multiple antibiotic classes as well as an inherent instability of the ARGs structure in region II (Fig. S1).

A novel non-Tn4401-KPC2-element (NTE KPC -Y)
The region surrounding bla KPC-2 was unique and differed from both typical Tn4401 structures (tnpR Tn4401 -tnpA Tn4401 -ISkpn7-bla KPC-2 -ISkpn6) as well as from other known non-Tn4401 elements (NTE KPC -I, II, and III) from previous studies (19,25).We provisionally assigned it as NTE KPC -Y, to distinguish it from a newly described NTE KPC -X that is present on an IncN[pMLST15] plasmid in isolates from the United Kingdom (6).Fig. 2B illustrates the differences between the Tn4401-isoforms (a, b, and j) and the non-Tn4401 elements, NTE KPC -Y and NTE KPC -X.In the Tn4401-isoforms, a Tn4401-resolvase, a Tn4401-transpo sase, and an ISKpn7-transposase are present upstream of the bla KPC-2 gene, while for NTE KPC -Y, an IS26 transposase, aac (3)-IId, three CDSs of unknown function, an ISCfr1, and a Tn2-associated bla TEM-1B gene are present.In NTE KPC -X, an IS26 transposase, ISEcp1 element, and a Tn2-associated bla TEM-1B gene are followed by an intact ISKpn6 downstream of the bla KPC-2 gene, whereby the ISKpn6 is split by an IS5403 element.However, the combination of the aac3-IId, bla TEM-1B, and bla KPC-2 resistance genes on NTE KPC -Y is new and has not been hitherto detected.Fig. 3C shows the common insertion site of the bla KPC-2 with its surrounding elements and ARGs of the three KPC-2-bearing variants I, II, and III.The IncN[pMLST15] variant III plasmids in this study also harbored additional ARGs within its dynamic multi-drug resistance region (DMDR [Fig.2C]).We designed specific PCR primer sets based on this unique genetic feature of the IncN[pMLST15]-plasmid and combined it with primer pairs for the pMLST15-back bone (Table S5).This composite PCR assay allowed unambiguous detection of this novel IncN[pMLST15] plasmid in diagnostic reference laboratories.
Several ARG profiles were clustered with respect to the 35-kb inserted segment (Regions IIA + IIB, Tables 2 and 3; Fig. 2).ARG-Profile A with 15 ARGs was the most common type (71%; 89/124) and found in 78% of the 77 KPC2-CPE-patients in 30 hospitals (Table S1).ARG-Profile E containing five ARGs was the next common type (14%; 17/124) and was found in 10 patients in seven hospitals.The remaining minor ARG profiles designated B, C, D, and F and others were detected in 18/124 isolates.
Plasmids carrying ARG profile A were significantly associated with outbreaks.They were predominant in the large multi-species outbreak in 2013/2014 and were recovered six times between 2016 and 2019, generally involving two-three patients colonized with different species at different hospitals, as well as in a single-species outbreak in 2019 (Table S4).Of note, all five IncN[pMLST15] plasmids in the rivulet water sam ples that were collected downstream of a sewage treatment plant had cluster profile A. In contrast, plasmids with ARG profile E were rarely associated with outbreaks but frequently occurred in multiple-species-colonization profiles of individual patients, indicating interspecies spread.The other profiles occurred mostly sporadically, suggest ing that they were occasional minority variants resulting from deletions.

Concurrent presence of IncN[pMLST15] plasmids in several species within individual patients or single samples
We observed that the NTE KPC -Y IncN[pMLST15] plasmid was present in multiple bacterial species in 17 individual patients, i.e., in 47 isolates from 10 different species (Fig. 3).Four patients were sampled during the 2013/14 outbreak, and the other 13 were obtained during the surveillance period.The number of the isolates per case was between two and four isolates comprising two to three species, often with a co-occurrence of E. coli and K. pneumoniae.The time span between the first and last isolate ranged between 4 and 167 days, and in two cases, it was even longer than a year (441 and 698 days).In four cases, different species with identical plasmids were detected from single samples such as rectal/stool swab (n = 3) and wound (n = 1).The IncN[pMLST15] plasmids within a patient frequently harbored identical ARG-profiles, mostly profile A.

The IncN[pMLST15] plasmid harboring NTE KPC -Y is unique to Germany
To examine the global distribution of the bla KPC-2 -bearing IncN[pMLST15] plasmid with the NTE KPC -Y-element, we compared it to sequences extracted from the NCBI Genbank and other publicly available databases.We subjected data for 476 wholly sequenced IncN plasmids from public databases (NCBI and PLSDB, 34,513 entries) to further analysis.Of these, 258/476 carry repN_1 (true IncN plasmids) and are typable by pMLST.The most common IncN type was the pMLST7 (n = 64), followed by pMLST5, pMLST1, pMLST6, pMLST9, and pMLST15, with 58, 41, 40, 26, and 16 entries, respectively.We found 17 publicly available IncN[pMLST15] plasmids harboring a bla KPC-2 .These originated mainly from clinical K. pneumoniae or E. coli isolates from Brazil, Israel, the United Kingdom, and France (Table S5).Comparative genomic analysis demonstrated that all plasmids carried bla KPC-2/-3 at a specific site inserted between a truncated nuc (endonuclease) gene and the fipA gene (Fig. 2D).
Surprisingly, all of the previously described IncN[pMLST15] plasmids are carriers of various bla KPC-2 elements, i.e., Tn4401-based or otherwise, regardless of their geograph ical origin (Table S6).We note that three similar plasmids with the novel NTE KPC -Y element were recently found in healthcare institutions in the Czech Republic that borders with Germany (accession no.CP070531, CP070538, and CP070545), suggesting regional dispersion.

DISCUSSION
In this study, we detected the presence of bla KPC-2 in different genera and species of Enterobacterales isolated from human and inanimate environments between 2013 and 2019 in Germany.This study analyzed isolates from 61 hospitals in the Federal State of Hesse serving a population of ~6.3 million inhabitants together with additional isolates from other regions in Germany including samples obtained from surface waters.We detected broad species-based dissemination of the bla KPC-2 gene facilitated by intraand inter-species horizontal gene transfer (HGT) of a single IncN  plasmid.This transfer also led to the emergence of highly antibiotic-resistant bacteria that carry up to 14 other ARGs encoded on the same plasmid (Table 2).Previously, an association of KPC-2 with plasmids of different incompatibility (Inc) groups, such as FII, N, R, A/C2, P, L/M, Q1, FIB, U, FIA, X3, Q2, and N3, was reported, with FII and N being the most common (10).However, the proportion of the previously mentioned plasmids reported that are in fact cointegrates with an IncN plasmid, as detected in this study, is not known.
Currently, 36% of all reported plasmids worldwide carry the bla KPC-2 gene on the transposon element Tn4401 (10), indicating the importance of transposons in transmis sion of this resistance.In our study, 94% were associated with NTE KPC -Y, a novel non-Tn4401-KPC2-element carried on IncN[pMLST15] plasmids, while only 6% of all isolates carry KPC-2 on a Tn4401a transposable element.The NTE KPC -Y element is unlikely to be mobile because it is always coupled to the presence of the IncN[MLST15] plasmid, that is, present even in cointegrates.Thus, the NTE KPC -Y element relies on the replication and maintenance properties of the broad host range IncN plasmid for effective dissemination and persistence in a range of different environments (Fig. 4).
In the 3-year SurvCARE surveillance study, IncN[pMLST15] comprised 88% of all KPC2-CPEs detected (Table 1).Plasmids carrying the ARG profile A significantly con tributed to outbreaks, causing a large multi-species outbreak in southern Hesse in 2013/2014 (Table 1), six smaller multi-species outbreaks in several hospitals between 2016 and 2019, as well as a single-species outbreak in 2019 (Table S4).A largely identical NTE KPC -Y IncN[pMLST15] plasmid was previously reported from isolates obtained during outbreaks from hospitals in Berlin in 2016 (26) and more recently in Cologne (2020-2021) (Xanthopoulou K et al.ECCMID 2022, oral presentation O0234).Cumulatively, these data suggest that hitherto undetected transmission events may have resulted in the spread and persistence of this plasmid over large distances and time periods.The presence of the plasmid in environmental KPC2-CPE from surface water is deeply concerning as it emphasizes the presence of reservoirs in diverse ecological habitats.More recently, the detection of similar IncN[pMLST15] plasmids with NTE KPC -Y in C. freundii and E. hormaechei isolates from Ostrov, Koli, and Prague in the Czech Republic suggests multi-species regional, transboundary spread.
While the NTE KPC -Y element is clearly unique to Germany, similar NTE KPC-2 elements with unique compositions have been described in other countries such as Argentina, Brazil, China, Russia, and the United Kingdom (6,17,19).Their frequent association with broad host range IncN plasmids, which provide a versatile platform for the capture of these genes and its genetic plasticity, would facilitate transfer of large sets of ARGs even when antibiotics unrelated to carbapenems are used during therapy.An additional layer of complexity introduced by multi-species outbreaks exploits the ability of these plasmids to find appropriate bacterial hosts, e.g., Citrobacter, Enterobacter, and Raoultella, which are present in a wide variety of near-patient and community environments.This distribution is concerning as there are currently no effective strategies to force the elimination of these plasmids from their various hosts.
Our study has several limitations.The isolates submitted were part of mandatory reporting to regional healthcare authorities as well as voluntary contributions to the National Reference Center, and the study was limited in terms of access to clinical data of patients in the individual hospitals and healthcare institutions based on data protection considerations.The systematic surveillance study was geographically restricted to the state of Hesse and covers a catchment area of 21.115 km².In addition, environmental sampling at the individual institutions was not performed.
We provide evidence that the HGT properties of a single plasmid rather than clonal expansion of successful genetic lineages dominated the dissemination of bla KPC-2 in Germany.Its location on a novel non-transposable Tn4401 element (NTE KPC -Y) suggests that plasmid-encoded factors contribute to the endemicity and long-term persistence of KPC-2 in community-based, hospital, and environmental reservoirs.Data from studies that examine for the presence of plasmids of KPC2-CPE in community and hospital microbiomes, both in patients and the environment, are urgently needed to inform and complement current containment measures to enable the development of effective transmission-control protocols.These results emphasize the need, already now, to be aware of a prominent role of transmissible plasmids during CPE surveillance and their contribution to transmission events.

Sample size
To estimate the statistical power of our study, we used data on CPE prevalence from the National Reference Center for Enterobacterales at the Robert-Koch Institute in Germany between 2017 and 2019 (27)(28)(29).Based on these data, we expected to achieve a statistical power of 95% with a significance level (α) of 0.05 if we had a sample size of 145 isolates.Here, we performed genome sequencing of all 346 CPE samples obtained in this study, which increased the statistical power of our study to more than 99% with the same significance level.

Phenotypic characterization of the bacterial isolates and plasmid conjuga tion assay
All bacterial isolates were identified using mass spectrometry MALDI-TOF MS (VITEK MS, Biomerieux, Nürtingen Germany), characterized for their phenotypical antimicrobial susceptibility using the automated VITEK2 system (Biomerieux, Nürtingen, Germany), and interpreted following EUCAST guidelines.For plasmid conjugation assays, the E. coli strain J53 was used as a recipient, as previously described (30).

Whole-genome sequencing (WGS)
All isolates underwent WGS (Illumina MiSeq or NextSeq 500).A subset of the isolates was re-sequenced by long-read sequencing using the PacBio Single-Molecular-Real-Time (SMRT) or Nanopore technology to complete the genome.The Illumina library prep aration and sequencing were carried out as previously described (31).Briefly, DNA sequencing libraries were prepared using the Nextera XT kit (Illumina MiSeq system (Illumina, Netherlands BV, Eindhoven, the Netherlands) according to the manufacturer's introductions and sequenced either on a MiSeq instrument with 2 × 300 cycles or on a NextSeq instrument with 2 × 150 cycles.The SMRT-sequencing was carried out on a PacBio RSII machine (Pacific Biosciences, MenloPark, CA, USA), as described earlier (31).Sequencing using Nanopore technology with a MinION sequencer was performed as described previously (32).

Genome assembly and plasmid-sequence completion
The Illumina-sequenced reads were assembled de novo using CLC Genomics Workbench version 8.0.1 (Qiagen, Aarhus A/S, Denmark) and/or SPAdes genome Assembler (33).For PacBio SMRT sequencing, the assembly was performed either using RS HGAP Assembly 3 or SMRT-Link Microbial Assembly v.10.1.0,using default parameters.The validity of each assembly was cross-checked using the RS_Bridgemapper.1 protocol, and each replicon was circularized independently.Finally, the circulated genome sequence was error-corrected by mapping of Illumina reads to the finished genomes using BWA (34), with subsequent variant calling using VarScan (35).A consensus concordance of QV60 was obtained for all of the genomes.For Nanopore sequencing, hybrid assembly was carried out using Unicycler v0.4.6.
For sequence finishing (completion) of the bla KPC-2 -bearing plasmids of the isolates sequenced only by Illumina, contig-mapping and read-mapping against closed bla KPC-2bearing plasmid-genomes from the SMRT-sequencing as references, by using SeqManPro (10.0), MAUVE (2.3.1), and CLC-Workbenches (8.0.1), were performed to complete the bla KPC-2 -encoding plasmids of the remaining isolates that were only sequenced by short-read-sequencing.
To determine the phylogenetic variation of the IncN plasmids, a maximum likeli hood phylogeny was constructed using the Neighbor Joining Method based on the whole sequence alignment (CLC Genomics Workbench v.10.1.1)using default parameter settings.The phylogenetic tree was generated by MEGA6 (41).
Plasmid sequence comparison with the sequence of pCP13069-KPC2 as the reference was generated by using BLAST Ring Image Generator (BRIG) (42).
The NCBI Genbank database and other available complete sequence data were used to search the global distribution of the bla KPC-2 -bearing IncN[pMLST15] plasmid with the specific non-Tn4401-KPC-element.IncN plasmid sequences (n = 476) and information from the plasmid database (PLSDB, https://ccb-microbe.cs.uni-saarland.de/plsdb/, as of 15 August 2022) were subjected to further analysis.

TABLE 1
Contribution of the bla KPC-2 containing NTE KPC -Y-carrying IncN[pMLST15] plasmids based on antimicrobial resistance gene (ARG) profiles in Germany from 2013 to 2019 (n = 124) c,d

Table 2 .
c SurvCARE Hesse: a regional genome-based CRE surveillance study (State of Hesse).d ND: not determined.

TABLE 3
Genomic characteristics of the bla KPC-2 -bearing plasmids based on long-read sequencing Plasmid

TABLE 3
Genomic characteristics of the bla KPC-2 -bearing plasmids based on long-read sequencing (Continued) a these both plasmids carried only one copy of sul1.b pEC_Surv190-KPC2-2 harbored an E-like profile without strA,, but sul2.c pKA36387-KPC2 harbored neither a NTE KPC -Y nor Tn4401.d bla KPC-2 and bla TEM-1 are not adjacent to each other.e pKP11394-KPC2 harbored two Tn4401a with bla KPC-2 .