Evidence of an epidemic spread of KPC-producing Enterobacterales in Czech hospitals

The aim of the present study is to describe the ongoing spread of the KPC-producing strains, which is evolving to an epidemic in Czech hospitals. During the period of 2018–2019, a total of 108 KPC-producing Enterobacterales were recovered from 20 hospitals. Analysis of long-read sequencing data revealed the presence of several types of blaKPC-carrying plasmids; 19 out of 25 blaKPC-carrying plasmids could be assigned to R (n = 12), N (n = 5), C (n = 1) and P6 (n = 1) incompatibility (Inc) groups. Five of the remaining blaKPC-carrying plasmids were multireplicon, while one plasmid couldn’t be typed. Additionally, phylogenetic analysis confirmed the spread of blaKPC-carrying plasmids among different clones of diverse Enterobacterales species. Our findings demonstrated that the increased prevalence of KPC-producing isolates was due to plasmids spreading among different species. In some districts, the local dissemination of IncR and IncN plasmids was observed. Additionally, the ongoing evolution of blaKPC-carrying plasmids, through genetic rearrangements, favours the preservation and further dissemination of these mobile genetic elements. Therefore, the situation should be monitored, and immediate infection control should be implemented in hospitals reporting KPC-producing strains.

Analysis of short-read sequencing results. Forty-nine out of 108 KPC producers, selected as representatives of all different hospitals, bacterial species and susceptibility profiles, were characterized by short-read sequencing using MiSeq (Illumina) platform. Based on short-read data, 44 of the 49 sequenced isolated harboured the bla KPC-2 allele (Table S1), while the five remaining isolates carried the bla KPC-3 gene. The bla KPC-3 allele was identified among 3 K. pneumoniae, 1 K. michiganensis and 1 E. coli isolates. Beside species-specific chromosomal β-lactamases, most of the clinical isolates also carried genes encoding OXA-1/9 oxacillinases (n = 37) and/ or TEM-1 penicillinases (n = 34). The bla CTX-M-15 gene was found among 2 Enterobacter and 5 K. pneumoniae isolates, while 4 out of 5 P. mirabilis harboured the bla CTX-M-14 gene. Additionally, 4 out of 7 Enterobacter isolates cocarried the carbapenemase-encoding gene bla  . All sequenced isolates exhibited a wide variety of resistance genes conferring resistance to aminoglycosides, sulfonamides, trimethoprim, macrolides, streptogramin B, fosfomycin (low-level resistance), fluoroquinolones, chloramphenicol, tetracyclines, and/or rifampicin (Table S1).
WGS data revealed that C. freundii isolates belonged to sequence types ST65 (n = 6), ST580 (n = 3), ST98 (n = 2) and ST8 (n = 1) (Table S1). ST98 C. freundii isolates producing KPC-2 carbapenemase were previously recovered from critically ill patients hospitalized in Germany 17 , while ST8 C. freundii expressing a VIM-4 isoenzyme were identified in Poland 18 , in 2013. On the other hand, the novel ST580 was a single allele variant of ST142, which was previously associated with KPC-2 production in isolates from the University Hospital of Hradec Kralove (Czech Republic) 16 . The isolates belonging to E. cloacae complex were assigned to ST133 (n = 4) and ST421 (n = 3), which haven't been previously associated with the production of KPC-2 carbapenemase. Additionally, in silico hsp60 typing of the genome sequences showed that four Enterobacter isolates belonged to the species Enterobacter hormaechei 19 . The K. pneumoniae isolates included eight STs. Seven KPC-2-producers were distributed in ST101 (n = 4) and ST11 (n = 3). The remaining KPC-2-producing K. pneumoniae isolates belonged to unique STs (ST13, ST17 and ST147), while the K. pneumoniae isolates, which produced the KPC-3 enzyme, were ST307, ST512 and ST846. ST11, ST101, ST147, ST307 and ST512 have been previously associated with the spread of KPC resistance mechanism and have been considered as 'high risk clones' 20,21 . Finally, the E. coli and K. michiganensis (closely related to K. oxytoca) isolates were assigned to diverse STs, as shown in Table S1. Since MLST schemes do not exist for M. morganii and P. mirabilis isolates, phylogenetic clusters for the respective isolates were determined based on core-genome alignment (see below), using the Harvest suite 22 .
Characterization of bla KPC -carrying genetic units. Based on short-read data, 25 KPC-producing isolates were selected to be sequenced by Sequel I platform, in an attempt to close plasmid sequences. All the 25 isolates showed resistance to cephalosporins and ertapenem while (except for P. mirabilis isolates) remained susceptible to colistin. Some variation in MIC values were noticed, however it is due to the different antibiotic resistance genes content found in each isolate (Table S3). Analysis of long-read sequencing data revealed the presence of several bla KPC -carrying plasmid sequences belonging to different Inc groups and presenting diverse sizes (Table S2). Based on PlasmidFinder analysis of plasmid sequences, 19 out of 25 bla KPC -carrying plasmids could be assigned tο R (n = 12), N (n = 5), C (n = 1) and P6 (n = 1) incompatibility (Inc) groups ( Figure S3). Five of the remaining bla KPC -carrying plasmids were multireplicon, while one plasmid couldn't be typed by the database. All plasmids, except IncN replicons, contained the Tn4401a isoform of the Tn4401 transposon, which is similar to that described in plasmid pNYC, lacking 100 bp upstream of bla KPC gene 11 .
Three out of 12 bla KPC -carrying plasmids, belonging to IncR group, were ⋍54 kb in size, while the nine remaining IncR plasmids sized ⋍89 kb. The IncR plasmids that were ⋍54 kb in size were derivatives of the IncR KPC-2-encoding plasmid pCfr-31816cz (Fig. 1a), which was characterized during an outbreak of KPC-2-producing Enterobacterales in a Czech hospital (Hradec Kralove) 16 . However, they differed from pCfr-31816cz by the presence of an additional 9232-bp sequence (nt 7286 to 16,517; GenBank accession no. CP070521) encoding CcdAB toxin-antitoxin system, and IncFIIA RepA and Ssb proteins. On the other hand, the IncR plasmids that were ⋍89 kb in size showed high degrees of similarity to each other and to the previously described plasmid pCfr-36049cz (Fig. 1b). Plasmid pCfr36049cz was characterized during the KPC-2 outbreak that took place in Hradec Kralove 16  Plasmid p48659_KPC is a fusion derivative of the p52810_KPC and pMmo-37590cz (Fig. 2). pMmo-37590cz is an IncP6 KPC-2-encoding plasmid that was also characterized during the KPC-2 outbreak in Hradec Kralove hospital 16 . Plasmid p48659_KPC contains a 50,603-bp sequence (nt 5917 to 56,519) encoding KPC-2, which is identical to a partial sequence of p52810_KPC. The remaining 11,723-bp sequence of p48659_KPC consists of one segment sharing extensive similarity with sequences carried by pMmo-37590cz. This segment included the IncP6 replication gene repA, the partitioning genes, parA, parB, and parC, and genes encoding a DNA invertase/  www.nature.com/scientificreports/ recombinase (int), a deoxymethyltransferase (dmt), and a DNase (drn) of type II restriction module. Sequence analysis demonstrated that the plasmid p45182_KPC, which was not typed by PlasmidFinder, is 50,582 bp in size and is a derivative of p52810_KPC (Fig. 2). Only two differences between the two plasmids were observed. A 26,069-bp segment (nts 34,454 to 60,522 in p52810_KPC) including IncR plasmidic backbone, a Tn1721-like fragment (consisting of the 38-bp inverted repeat of the transposon, tnpA, tnpR, and tnpM), and intI1 gene of the integron In37, was not present in p45182_KPC. In addition, a second fragment (nts 65,142 to 83,473 in p52810_KPC), being 18,332 bp in size, that contained vir2/3/4/9/10/11 region of IncN3-like plasmids was also absent from p45182_KPC, probably explaining the inability of the plasmid to conjugate. The IncN bla KPC-2 -positive plasmids, which were assigned to ST15 based on the pMLST (https:// cge. cbs. dtu. dk/ servi ces/ pMLST/), comprised the plasmidic backbone and a multidrug resistance (MDR) region inserted downstream the fipA. The IncN plasmidic backbone contained a replication region (repA), a transfer system (traA/B/C/D/N/E/O/F/G), a stability operon (stbA/B/C) and an antirestriction system (ardA/B). The MDR region of IncN plasmids, which ranged from 21,011 to 31,420 bp in size, harboured a Tn4401-derived fragment of 2833-bp, encoding KPC-2 carbapenemase. In comparison to Tn4401b, the Tn4401-derived sequence (designated Tn4401j) had a deletion of 217 bp found upstream of the bla KPC-2 . The Tn4401-derived fragment was disrupted by a Tn3-like sequence, 111 bp upstream of the bla KPC-2 . The Tn3-like sequence was composed of the inverted repeat (IR) of the transposon and the bla TEM-1 resistance gene. A similar bla KPC-2 -carrying genetic environment has been previously identified in the IncN plasmid pCF8698_KPC2, characterized from a C. freundii strain isolated in Germany (GenBank accession no. LN610760) ( Figure S4). The MDR region of the IncN plasmids exhibited additional genes conferring resistance to aminoglycosides, sulfonamides, trimethoprim, macrolides, and/or fluoroquinolones (Table S2, Figure S4).
Similarly, 92 M. morganii available genomes in NCBI database were downloaded to compare them with the five isolates sequenced in this study (Fig. 5). Mmo-48659, isolated in HK hospital, clustered alone in a unique node. However, it was closely related to an isolate from South Africa (790 alteration). On the other hand, four isolates (isolated from three different hospitals; Table S1) clustered together, with Mmo-51087 and Mmo-50821 isolated from the same hospital forming a subclade. For the detection of SNPs among the four isolates of this clade, Mmo-51087 was used as a reference. Mmo-50821 and Mmo-46544 showed 22 and 62 alterations, respectively, while Mmo-46903 222 alterations (Table S4).   (Fig. 6). The genomes of three isolates (Cfr-46338, Cfr-49942 and Cfr-48658), which belonged to ST580 and were recovered from HK hospital, clustered together forming a clade. SNPs detection among these isolates showed that Cfr-49942 and Cfr-46338 had 93 and 111 alterations, respectively, compared to Cfr-48658. In a closely related clade, another six genomes from ST65 isolates Cfr-50935, Cfr-48846, Cfr-51238, Cfr-47462, Cfr-48294 and Cfr-47299 clustered together. SNPs detection when compared to Cfr-50935 showed that Cfr-48846 had 25 alterations while Cfr-51238, Cfr-47462, Cfr-47299 and Cfr-48294 had 47, 57, 88 and 99 respectively (Table S4). On the other hand, the genomes of the two ST98 isolates, Cfr-48736 and Cfr-49141, clustered together in a considerable distant clade. These isolates are clustered together with other ST98 C. freundii isolates from the USA and UK. SNPs detection showed that Cfr-48736 had 28 alterations when compared to Cfr-49141. Finally, the isolate Cfr-49969, which was assigned to ST8, resulted in a unique node.
For Enterobacter hormaechei, 126 genomes were downloaded from the NCBI database and were compared with the seven isolates sequenced during this study (Fig. 7). The isolates clustered in two clades. The first clade contained four isolates (Ecl-49142, Ecl-48587, Ecl-48293 and Ecl-49583). Ecl-48293 was used as a reference genome for SNPs detection among these four isolates. Ecl-49142 had 26, Ecl-48587 had 34 and Ecl-49583 had 32 alterations. All ST133 isolates recovered from Czech Republic, South Africa, Japan, Australia and Egypt were grouped in a unique cluster. Additionally, the other cluster contained the three isolates (Ecl-51693, Ecl-51846 and Ecl-52075) which were ST421. For SNPs detection, Ecl-51846 was used as a reference, showing that Ecl-52075 and Ecl-51692 had 12 and 14 alterations, respectively (Table S4).
For K. pneumoniae, 732 genomes were downloaded from the NCBI database to compare them with the 13 sequenced isolates (Fig. 8). Isolates Kpn-51835, Kpn-47158, Kpn-51483, Kpn-53027, Kpn-52813 and Kpn-51069, which were assigned to diverse STs, formed a unique distinct node each. One clade consisting of Kpn-47693, Kpn-O141 and Kpn-A9853 was in close proximity with Kpn-51248 in the neighbouring cluster. Using Kpn-47693 as a reference genome for SNPs detection, Kpn-O141, Kpn-A9853 and Kpn-51248 had 25, 22 and 100 alterations, respectively. The above isolates were grouped together with other ST101 isolates from Italy, USA, Japan, India and South Africa. The last three K. pneumoniae isolates (Kpn-52810, Kpn-A4411 and Kpn-45128) clustered together. Using Kpn-52810 as a reference genome for SNPs detection, Kpn-A4411 had 27 alterations, while Kpn-45128 exhibited 816 alterations (Table S4). Interestingly, the last isolates were clustered with isolates from China, Switzerland, India and the USA, which belonged also to ST11.

Discussion
KPC-producing Enterobacterales represent a major threat of global dimensions for public health. The current study described the change of the epidemiological situation in Czech hospitals, from the sporadic cases or outbreaks to the epidemic spread of KPC-producing isolates ( Figure S1). During 2018-2019, 108 KPC producers were recovered from 22 different Czech hospitals located throughout the country. Additionally, the bla KPC gene was found among diverse species and clones of Enterobacterales family ( Figure S2). Phylogenetic analysis indicated that P. mirabilis and M. morganii isolates, carrying the bla KPC -like gene, didn't exhibit close relationship with isolates characterized previously from other geographical areas. Additionally, phylogenetic analysis showed that the KPC-2-producing E. hormaechei isolates belonged to two distinct clones (Fig. 7), assigned as ST133 and ST421 based on MLST. The ST421 isolates weren't closely related with other isolates analysed, using parsnp software, while ST133 isolates clustered together with isolates from South Africa, Japan, Australia and Egypt. However, ST133 isolates, recovered from other geographical areas, weren't associated with the production of KPC-2 carbapenemase. Regarding C. freundii isolates, phylogenetic analysis revealed two main clones, which were assigned to ST65 and ST580 based on MLST. These two clones were distinct to each other and to the isolates included in the analysis. Isolates Cfr-48736 and Cfr-49141 grouped together with other ST98 isolates from the UK and USA, while the isolate Cfr-49969 was clustered with other ST8 isolates from Australia and the USA. However, the later clones have been associated with the production of KPC-2 and VIM-4 carbapenemases in Germany and Poland 17,18 , respectively. On the other hand, 6 out of 13 K. pneumoniae isolates characterized by WGS belonged to unique STs. Interestingly, parsnp phylogenetic analysis clustered these isolates with other K. pneumoniae isolates, belonging to the same STs, from worldwide. Additionally, the 7 remaining K. pneumoniae isolates belonged to two distinct clades. The later clades included ST11 and ST101 isolates from different geographical origins. Among K. pneumoniae, the 'high risk' clones, ST11, ST101, ST147 and ST512, that have been previously associated with the spread of KPC resistance mechanism were found 20,26 . In agreement with recent reports, those data confirm that high-risk clones, other than CC258, currently contribute to spread of KPC resistance mechanism in Europe 24,25 . Finally, the KPC-producing E. coli and K. michiganensis isolates belonged to unique STs. These findings underline the ongoing spread of the KPC resistance mechanism among different species and clones.
The analysis of the genetic units carrying the bla KPC -like genes revealed the presence of a wide variety of plasmids involved in the spread of the KPC resistance mechanism. Some of the observed plasmid-types, like IncFII K2 pKpQIL, IncFII K2 -IncR pIT-12C73, and IncR-IncN 3 pCfr-36049cz, have been previously described to www.nature.com/scientificreports/ be responsible for the spread of the bla KPC -like genes 16,20,25 . Additionally, some novel emerging plasmid-types, as the IncN pCF8698_KPC2 originally described from Germany (GenBank accession no. CP070521), the IncFIB K / FII K pRIVM_C008981_1 firstly characterized from a Dutch collection 24 , and the hybrid IncFII/FIB/C 2 /N plasmid p49969_KPC characterized during this study, were identified to disseminate the bla KPC -like genes. A few fusion derivatives of the bla KPC -carrying plasmids described above were observed. These data verify the presence of some successful plasmid lineages spreading the KPC resistance mechanism, but also highlight the ongoing evolution of the mobile genetic elements involved in the dissemination of clinically important resistance mechanisms. For example, IncR plasmids carrying bla KPC genes have played a significant role in the spread of the specific resistance mechanism, in the Czech Republic. But, IncR plasmids have also been involved with the spread of other important carbapenemases, like NDM and VIM 27,28 . Additionally, in agreement with previous studies 20,25,29 , IncF plasmids are one of the major factors contributing to the worldwide spread of KPC carbapenemases. Moreover, the distribution of the different plasmid types detected suggests local dissemination with IncR plasmid spreading in middle part of the map especially in Hradec Kralove and Nymburk ( Figure S3), while the IncN plasmid spreading in the North West of the Bohemian region. However, Prague seems like the melting pot, in which all plasmid families were detected, indicating the transient admission of patients from surrounding districts to Prague for specialized treatment. Moreover, this is confirmed by the fact that most of the isolated strains in Prague comes from private labs which provides services for many hospitals and long-term care facilities in and outside Prague. This route of dissemination could be explained by the spread of specific plasmid families within the same region, like IncR plasmids, or crossing the borders via travelling, like IncN plasmid from Germany.
In conclusion, our results show that the increased prevalence of KPC-producing isolates was due to plasmids being conjugative and spreading among different species and clones. Additionally, the ongoing evolution through genetic rearrangements, observed in bla KPC -carrying plasmids, favour the preservation and further dissemination of these mobile genetic elements. Therefore, the situation should be monitored, and immediate infection control should be implemented in hospitals reported.

Material and methods
Bacterial isolates, carbapenemase production and susceptibility testing.   Short-read whole genome sequencing. Forty-nine KPC-producing Enterobacterales were selected for complete sequencing, using the Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA). These isolates were selected as representatives of all different hospitals, bacterial species and susceptibility profiles. The genomic DNAs of the clinical isolates were extracted using the DNA-Sorb-B kit (Sacace Biotechnologies S.r.l., Como, Italy). Multiplexed DNA libraries were prepared using the Nextera XT library preparation kit, and 300-bp paired-end sequencing was performed on the Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA) using the MiSeq v3 600-cycle reagent kit. Initial paired-end reads were quality trimmed using the Trimmomatic tool v0.33 34 and then, assembled by use of the de Bruijn graph-based de novo assembler SPAdes v3.14.0 35 . Map. Maps of the Czech Republic was created using the Leaflet package 36 in R-studio 37 from R-project 38 .
Long-read whole genome sequencing. Based on the results of short-read sequencing (see below), twenty-five KPC producers were selected to be sequenced using long-read sequencing technology, to help close the whole plasmid sequences. These isolates were selected as representatives of all different hospitals, bacterial species, STs, replicon profiles and KPC alleles.
Genomic DNA was extracted from the clinical isolates using NucleoSpin Microbial DNA kit (Macherey-Nagel, Germany). Whole genome sequencing (WGS) was performed on the Sequel I platform (Pacific biosciences, Menlo Park, CA, United States). Microbial multiplexing protocol was used for the library preparation according to the manufacturer instructions for Sheared DNA. DNA shearing was performed using the Megaruptor 2 (Diagenode, Liege, Belgium) using long hydropores producing 10 kb long inserts. No size selection was performed during the library preparation. The Microbial Assembly pipeline offered by the SMRT Link v9.0 software was used to perform the assembly and circularization with minimum seed coverage of 30X. Assembled sequences were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP).  Transfer of bla KPC -like genes. Conjugal transfer of bla KPC -like genes from the clinical strains was carried out in mixed broth cultures 45 , using the rifampicin-resistant E. coli A15 laboratory strain as a recipient. Transconjugants were selected on MacConkey agar plates supplemented with rifampicin (150 mg/l) and ampicillin (50 mg/l). Transconjugants were confirmed to be KPC producers by PCR 11 and the MALDI-TOF MS meropenem hydrolysis assay 30 . Phylogenetic analysis. Genetic diversity and phylogenetic relationship between the sequenced samples and global genomes were studied. All phylogenies were created using core genome, recombination and single nucleotide polymorphisms (SNPs) using parsnp v1.2, available in the harvest suite 22 using a corresponding reference genome. SNPs identified in local collinear blocks were subsequently used for reconstructing an approximate maximum-likelihood tree using FastTree2 46 while including the general time reversible (GTR) model of nucleotide substitution. The Shimodaira-Hasegawa test implemented in FastTree2 was used to assess the support for significant clustering in the observed phylogeny. The interactive tree of life or iTOL (https:// itol. embl. de/). 47 was used for the graphic illustration of the trees along with relative annotations.
For the construction of the SNPs-based phylogenies, 582 Proteus mirabilis genomes were downloaded from NCBI assembly database including complete and draft genomes, using ASM6996v1 as reference. Similarly, 92 genomes for Morganella morganii (ASM1428397v1 as reference), 118 genomes for Citrobacter freundii (Cfr-49,969 as reference), 126 genomes for Enterobacter hormaechei (Ecl-48,293 as reference) and 732 genomes for K. pneumoniae using Kpn-48,293 as reference.
Moreover, isolates from the study that clustered together forming a clade or/and subclade were investigated further. SNPs among the isolates within the clade/subclade were compared to a reference genome within the selected set using snippy v. 4.4.3 (https:// github. com/ tseem ann/ snippy).
Nucleotide sequence accession numbers. The nucleotide sequence of the genomes and plasmids were deposited and available in GenBank under the BioProject number PRJNA700516; all accession numbers can be retrieved from Table S5.