Within-patient horizontal transfer of pOXA-48 from a hypervirulent Klebsiella pneumoniae SL218 to Serratia marcescens following spread of the K. pneumoniae isolate among hospitalised patients, Denmark, 2021

A hypervirulent Klebsiella pneumoniae SL218 (ST23-KL57), phylogenetically distinct from the classical hypervirulent SL23 (ST23-KL1) lineage, was transmitted between hospitalised patients in Denmark in 2021. The isolate carried a hybrid resistance and virulence plasmid containing blaNDM-1 and a plasmid containing blaOXA-48 (pOXA-48); the latter plasmid was horizontally transferred within-patient to Serratia marcescens. The convergence of drug resistance and virulence factors in single plasmids and in different lineages of K. pneumoniae is concerning and requires surveillance.

A hypervirulent Klebsiella pneumoniae SL218 (ST23-KL57), phylogenetically distinct from the classical hypervirulent SL23 (ST23-KL1) lineage, was transmitted between hospitalised patients in Denmark in 2021. The isolate carried a hybrid resistance and virulence plasmid containing bla NDM-1 and a plasmid containing bla OXA-48 (pOXA-48); the latter plasmid was horizontally transferred within-patient to Serratia marcescens. The convergence of drug resistance and virulence factors in single plasmids and in different lineages of K. pneumoniae is concerning and requires surveillance.
Here, we describe the nosocomial transmission of a novel hypervirulent and multidrug-resistant K. pneumoniae SL218 isolate carrying a bla NDM-1 and a bla OXA-48 β-lactamase among co-patients isolated in a Danish university hospital in 2021. We characterise the plasmid structure of the isolates and describe within-patient horizontal transfer of a plasmid carrying bla OXA-48 from the hypervirulent K. pneumoniae SL218 to Serratia marcescens.

Clinical case description
Patient A with laboratory-confirmed COVID-19 was transferred to a Danish university hospital from a nearby hospital due to severe acute respiratory distress syndrome and need for extracorporeal membrane oxygenation (ECMO). The ECMO was initiated upon admission.
At this hospital, during the COVID-19 pandemic, patients diagnosed with COVID-19 were cohort-isolated in the intensive care unit if isolation in a single room was not possible. Within the isolation cohort, separate staff was allocated to each patient. Patient A was cohortisolated with patients who had been hospitalised outside of Denmark. All patients hospitalised for more than 24 h outside of Denmark within 6 months before admission are screened for CPO carriage and close contact patients are similarly screened. Microbiological cultures of rectal screening swabs from Patient A identified carriage of K. pneumoniae carrying bla OXA-48 and bla NDM-1 on day 8 upon admission. Patient A was screened because a co-patient was found positive for carbapenemase-producing bacteria. The two patients were subsequently isolated in separate rooms as cohort isolation is not used for patients with carbapenemaseproducing organisms.
Patient A was treated with ECMO for overall 46 days during which laboratory testing revealed changing signs of inflammation, including elevated levels of white blood cells, C-reactive protein, and procalcitonin. Klebsiella pneumoniae, S. marcescens, Acinetobacter baumannii, Enterococcus faecium and Candida spp. were cultured repeatedly from different sites, and the patient was treated appropriately with adjusted antibiotic regimens ( Figure 1). Further details on treatment timeline and inflammatory markers can be found in Supplementary Table S1. After 2 months of continued ECMO treatment, the patient developed a cerebral haemorrhage and further treatment was ceased.

Phenotypic and molecular characterisation of Klebsiella pneumoniae and Serratia marscesens isolates
Antimicrobial susceptibility testing was performed using European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines and expert rules [3]. Susceptibility testing was performed by disk diffusion and minimal inhibitory concentrations (MIC) using gradient tests, except for colistin where microbroth MIC determination was done. Phenotypic susceptibility is summarised in Table 1. In addition to the agents shown in Table 1, the K. pneumoniae isolate was tested for trimethoprim/sulfametoxazol (MIC: 32 mg/L), tobramycin (R: 6 mm) and tigecycline (MIC: 2 mg/L). The initial K. pneumoniae isolate was susceptible to colistin (MIC: 0.25 mg/L) but isolates after day 27 had developed resistance (MIC: 16 mg/L). Synergy was observed for the combination of ceftazidime/avibactam and aztreonam using gradient tests.
For elaborated laboratory methods, please see the supplementary material. We isolated K. pneumoniae from two patients during their hospitalisation. Both K. pneumoniae isolates displayed a hypermucoid phenotype (positive string test). A picture demonstrating this test result is appended to this publication in Supplementary Figure S1.
Whole genome sequencing revealed that the isolates belonged to SL218 (scgST-34342), the capsule type inferred from Kleborate (https://github.com/klebgenomics/Kleborate; [4]) was KL57, and SNP analysis performed with BacDist (https://github.com/MigleSur/ BacDist; [5]), reference genome: GCF_006364295.1_ ASM636429v1) disclosed that the two isolates were identical, indicating transmission between the patients during their cohort isolation. The K. pneumoniae SL218 isolate from patient A was subjected to Oxford Nanopore Technology sequencing, and UniCycler hybrid assembly resulted in six circular contigs: a     diameter. a Phenotypic susceptibility testing was performed according to EUCAST guidelines [3]. b Synergy was observed for the combination of ceftazidime/avibactam and aztreonam using gradient tests. c Not used due to inducible AmpC.

Hybrid virulence and resistance plasmid of Klebsiella pneumoniae
The large repB-type plasmid of 247,143 bp was closely related to the 202,175 bp virulence plasmid from Kp_ Goe_154414 (CP018338). Both plasmids had regions homologous to the classical hypervirulence plasmid pLVPK (219,385 bp (NC005249) [10]) but compared with this, both plasmids contained a 21,363 bp deletion that included the salmochelin (iroBCDN) locus present in pLVPK (Figure 3). Compared with pLVPK, the repB plasmid of the K. pneumoniae SL218 isolate also contained a 48,549 bp insertion containing multiple antimicrobial resistance genes ( Figure 3; Table 2), among which bla NDM-1 was identified in a Tn125 remnant. This multidrug resistance region is highly homologous to similar regions described in hybrid virulence and resistance plasmids that occur for example in K. pneumoniae SL147 (MW911671; 99% query coverage and 100% nucleotide identity) and SL395 (MW911666; 100% query coverage and 100% nucleotide identity) reported from Russia [11] (Figure 3).
The bla OXA-48 gene was located within a Tn1999 element on a separate IncL plasmid of 62,722bp (pOXA-48). This plasmid is highly homologous to other OXA-48 encoding plasmids prevalent in Europe (JN626286) [12].

In vivo horizontal transfer of pOXA-48 to Serratia marcescens
In addition, two S. marcescens strains were consecutively isolated from patient A. The second S. marcescens isolate was recovered 5 days after the first isolate and, unlike the first isolate, exhibited resistance to piperacillin/tazobactam and diminished susceptibility to meropenem ( Table 1). Comparison of the genomes with BacDist considering 88% of the genome (reference genome GCF_000513215.1_DB11), uncovered only a single SNP difference between the two isolates. However, the second S. marcescens isolate had acquired two plasmids, a 63,589 bp IncL plasmid containing bla OXA-48 and a 2,058 bp ColpVC plasmid. The 63,589 bp IncL plasmid was identical to the IncL pOXA-48 plasmid from K. pneumoniae except that it had acquired an additional insertion sequence element. The 2,058 bp ColpVC plasmids of S. marcescens and K. pneumoniae were identical and appeared to have been mobilised with the pOXA-48 plasmid. We make available a detailed illustration of which genes were associated to the two plasmids in Supplementary Figure S2. IncL pOXA-48 plasmids are promiscuously transmitted among Enterobacterales, as exemplified in an outbreak of bla OXA-48 -carrying K. pneumoniae in a Dutch hospital involving 118 patients [8]. In 55 of the patients, more than one species of Enterobacterales harboured the pOXA-48, including S. marcescens.

Discussion
Recombination contributes considerably to evolution and diversification of K. pneumoniae [1,13,14], and classical 7-locus MLST lacks discriminatory power to Phylogenetic comparison with Parsnp and Harvest suite to illustrate genetic distance between K. pneumoniae of this study in the context of SL218 isolates identified in Russia and later France and Finland from a risk assessment by the European Centre for Disease Prevention and Control [23] and available in GenBank (Accession numbers SRR7181964 and SRS7484649) and isolates belonging to SL218 and carrying OXA-48, NDM-1 or both, described in recent work [9]. The SL218 isolates of the present study are most closely related to NMI1734 (48-50 SNPs) isolated in Poland and carrying bla OXA-48. The scalebar represents substitutions per site, the maximum single nucleotide polymorphism (SNP) distance across tree is 159 SNPs.
accurately capture phylogenetic lineages. As a result, a substantial fraction of classical 7-locus sequence types (ST) are polyphyletic, i.e. a single ST comprises separate phylogenetic lineages [13]. Using a 629-locus core genome MLST (scgMLST) scheme, it is possible to define sublineages (SL) that more accurately reflect phylogeny, i.e. most SL are monophyletic, containing exclusively a single phylogenetic lineage [13]. Certain sublineages, such as SL11, SL15, SL101 and SL147 are globally distributed, often associated with horizontally acquired antimicrobial resistance genes and frequently detected in nosocomial outbreaks [15,16]. Other sublineages (e.g. SL23 or SL65) are associated with hypervirulence but not resistance [17] and are the causative agents of community-acquired invasive infections such as liver abscess, endophthalmitis and meningitis. They are characterised by the expression of virulence factors, most which are encoded on virulence plasmids (e.g. pK2044 and pLVPK) [18].
Classical 7-loci MLST classifies both SL23 and SL218 to ST23, however, SL218 (ST23/KL57) isolates are progeny of a recombination event between ST218 and SL395 (ST395) lineages and are phylogenetically not closely related to SL23 isolates [19]. Although hypervirulence and multidrug resistance seemingly appeared in distinct clonal lineages [1], convergent evolution occurs when multidrug-resistant lineages acquire virulence factors or, conversely, hypervirulent lineages acquire antimicrobial resistance genes. Examples of the former are KPC-2-producing K. pneumoniae SL11 isolates identified in China, which had additionally acquired a pLVPK-like virulence plasmid [20], and NDM-1producing K. pneumoniae SL147 isolates carrying a hybrid multidrug resistance-virulence plasmid detected in Tuscany, Italy [21]. Examples of the latter include hypervirulent K. pneumoniae SL23 isolates, whose genomes encode a bla OXA-48 observed in Ireland and K. pneumoniae SL218 isolates producing NDM-1 or OXA-48, respectively observed in Finland and France [22,23]. The hypermucoid SL218 isolate described here is an additional example of convergence of virulence factors and antimicrobial resistance genes within the same sublineage.

Conclusion
The carriage of a hybrid resistance and virulence plasmid containing key virulence factors aerobactin (iut) and rmpAC and a multidrug resistance region carrying bla NDM1 may be especially concerning. might be established and endemic in at least parts of Europe. The detection of hybrid virulence and multidrug resistance plasmids in K. pneumoniae SL218 is concerning and requires continued surveillance.

Ethical statement
The spouse of Patient A provided written informed consent for the publication of the paper.

Funding statement
The study was supported by a grant from the Novo-Nordisk Foundation (Reference Number NNF16OC0023482). Role of funders: All authors are employed by public hospitals in the Capital Region, Denmark, which are public institutions. Foundations had no role in designing and conducting the study, analysis, and interpretation the data, or the writing, review, and approval of the manuscript.

Data availability statement
Data are presented in full in the manuscript or in the Supplementary data. Raw Illumina reads and genome assemblies have been uploaded to NCBI under Bioproject PRJNA944929.