Whole-Genome Sequencing Enables Molecular Characterization of Non-Clonal Group 258 High-Risk Clones (ST13, ST17, ST147 and ST307) among Carbapenem-Resistant Klebsiella pneumoniae from a Tertiary University Hospital Centre in Portugal

The carbapenem-resistant Enterobacterales (CRE) strains have been identified by the World Health Organization as critical priority pathogens in research and development of diagnostics, treatments, and vaccines. However, recent molecular information about carbapenem-resistant K. pneumoniae (CRK) epidemiology in Portugal is still scarce. Thus, this study aimed to provide the molecular epidemiology, resistome, and virulome of CRK clinical strains recovered from a tertiary care hospital centre (2019–2021) using polymerase chain reaction (PCR) and the advanced molecular technique whole-genome sequencing (WGS). PCR amplification of carbapenemase genes was performed in 437 carbapenem-resistant K. pneumoniae strains. The most frequent carbapenemases were: KPC-3 (42%), followed by OXA-181 (20%), GES-5 (0.2%), and NDM-1 (0.2%). Additionally, 10 strains (2%) coproduced KPC-3 and OXA-181, and 1 strain coproduced KPC-3 and OXA-48 (0.2%). The genomic population structure of 68 strains characterized by WGS demonstrated the ongoing dissemination of four main high-risk clones: ST13, ST17, ST147, and ST307, while no clones belonging to the European predominant clonal groups (CG15 and CG258) were found. Moreover, we describe one K. pneumoniae ST39-KL62 that coproduced the NDM-1 carbapenemase and the extended-spectrum beta-lactamase CTX-M-15, and one K. pneumoniae ST29-KL54 GES-5 and BEL-1 coproducer. Furthermore, a high prevalence of iron siderophores were present in all CRK strains, with several strains presenting both colibactin and the hypermucoviscosity phenotype. Thus, the data presented here highlight an uncommon molecular epidemiology pattern in Portugal when compared with most European countries, further supporting the emergence and dissemination of nonclonal group 258 hypervirulent multidrug high-risk clones and the need to promote in-depth hospital molecular surveillance studies.


Introduction
Klebsiella pneumoniae is a Gram-negative bacterium with a high public health importance, capable of causing severe organ infections and life-threatening disease in both community-and hospital-acquired infections. K. pneumoniae has emerged as an increasingly resistant pathogen due to its predisposition to acquire multidrug and carbapenem resistance, limiting the therapeutic options [1]. A large variety of carbapenemases have been identified worldwide in K. pneumoniae, namely KPC, GES (Ambler class A), NDM, VIM, IMP (class B), and OXA (Class D), among others [2], positioning K. pneumoniae as one of the top three pathogens of international concern, according to the World Health Organization's Global Priority List of Antibiotic-Resistant Bacteria to Guide Research, Discovery, and Development of New Antibiotics [3].
In Portugal, after the isolation of the first carbapenemase producing a clinical strain of K. pneumoniae at a Lisbon hospital in 2009 [4], an increasing trend of carbapenem resistance among K. pneumoniae has been described, evolving from 3.4% in 2015 to 10.9% in 2019, as reported by the European Centre for Disease Prevention and Control (ECDC) [5]. The last available data in Portugal showed that the most frequent carbapenemase produced K. pneumoniae there were KPC-3 followed by OXA-181 [6][7][8], although other carbapenemases such as GES-5 and OXA-48 were also reported [9,10]. Despite its presence in several countries in Europe, as well as its worldwide spread, the NDM carbapenemase is not frequent in Portuguese clinical strains [11].
K. pneumoniae utilizes a variety of virulence factors such as capsule polysaccharides, adhesins, fimbriae, and iron-binding siderophores (aerobactin, enterobactin, salmochelin, and yersiniabactin) for survival and immune evasion during infection. Although the siderophore content and expression may be variable among different K. pneumoniae genetic lineages, the ability to sequester iron seems to contribute to the pathogenic potential of this organism [1]. Of relevance, it was identified that multidrug-resistant sequence type ST14 KPC-3 carbapenemase K. pneumoniae strains presented a high prevalence of virulent determinants, including the K2 capsular antigen and the siderophore aerobactin [12].
Previous studies indicated that molecular epidemiology in Portugal seems different from the existing molecular epidemiology in Europe. The most common clones detected in the European region are ST11, ST258, and ST512, all belonging to clonal group 258 (CG-258) [13]. In contrast, the most dominant STs reported so far in Portugal are ST13, ST14, ST15, ST17, ST147, ST307 and ST348 [6][7][8][9]12,14,15], all presenting high potential for virulence and dissemination. However, recent data on genomic surveillance of healthcareassociated infections by K. pneumoniae in Portugal are still limited, and the most recent study included strains recovered until 2019 [10]. Moreover, despite the recent interest in the interplay between resistance and virulence determinants in K. pneumoniae clinical strains, their interplay in these uncommon clones remains poorly understood [16]. Therefore, the aim of the present study was to provide updated information on the genomic epidemiology, resistome, and virulome of healthcare-associated infections by K. pneumoniae strains identified at a tertiary university hospital centre in Portugal (2019-2021).

Bacterial Strains
A total of 437 carbapenem-resistant K. pneumoniae clinical strains were recovered from a tertiary university hospital centre in Lisbon over a 2-year period, September 2019-2021, using standard clinical operating procedures, and were sent to the Laboratory of Microbiology Research in Environmental Health of the Faculty of Medicine, Universidade de Lisboa (ULisboa), for advanced molecular analysis. The identification was performed by microbiology laboratories using conventional methods or automated systems such as Vitek2 ® (BioMérieux, Marcy, l'Étoile, France) or MicroScan (Snap-on, Kenosha, WI, USA). All strains were maintained frozen in BHI broth (VWR Prolabo, Lisbon, Portugal) plus 15% glycerol at −80 • C. For analysis, the strains were grown in BHI broth for 18 h at 37 • C and seeded in Mueller Hinton agar (VWR Prolabo, Lisbon, Portugal).

Hypermucoviscosity Phenotype
The string test was performed to access the hypermucoviscosity (HMV) phenotype of the strains. A positive string test was defined as the formation of viscous strings of >5 mm in length when a loop was used to stretch the colony on an agar plate.

Molecular Methods of Species Identification and Detection of Carbapenemase Genes
PCR-based screening was performed for confirm the K. pneumoniae identification using the universal 16S rRNA bacterial primers 27F F:5 -AGAGTTTGATCCTGGCTCAG-3 ; 1392R R:5 -GGTTACCTTGTTACGACTT -3 and to identify carbapenemases genes with primers designed for bla OXA-48 F:5 -GGCTGTGTTTTTGGTGGCATC-3 ; R:5 -GCAGCCCTAAACCA TCCGATG-3 , bla KPC [18], bla VIM [19], bla NDM [20], bla GES [21]) using the NZYTaq II 2× Green Master Mix (NZYTech, Lisbon, Portugal) following the manufacturer's instructions. The PCR products were resolved in 1% agarose gel in 10× concentrated Tris-Borate-EDTA (TBE buffer) (NZYTech, Lisbon, Portugal). The PCR assays included positive and negative controls. The positive controls used were positive strains previously sequenced by the Sanger sequencing method. These positive controls were submitted to purification using an ExoCleanUp FAST kit (VWR Prolabo, Lisbon, Portugal) and were sequenced by the Sanger sequencing method at STABVida, Caparica, Portugal. Searches for nucleotide sequences were performed with the BLAST program, which is available at the National Center for Biotechnology Information website (http://www.ncbi.nim.nih.gov; accessed on 20 December 2021). Multiple-sequence alignments were performed with the Clustal Omega program, which is available at https://cge.cbs.dtu.dk/services (accessed on 20 December 2021).

Whole-Genome Sequencing (WGS)
We performed WGS on carbapenem-resistant K. pneumoniae strains with the objective of gaining further insight on the molecular epidemiology, resistome, and virulome of these strains. A total of 68 nonduplicated K. pneumoniae clinical strains were selected based on the carbapenemase gene type identified and within these, by random selection when the number of strains was above one, as previously carried out [6]. The genomic DNA were extracted for WGS from cultures grown overnight in Mueller-Hinton agar, using the NZY Tissue gDNA Isolation kit (NZYTech, Lisbon, Portugal), as per the manufacturer's recom-mendations. The Sequence was done at STABVida Portugal. Sequencing libraries were prepared using the KAPA HyperPrep Library Preparation Kit (Roche, Basel, Switzerland) following the manufacturer's recommended protocol, and sequenced using an Illumina HiSeq Novaseq 6000 platform (Illumina, San Diego, CA, USA) with paired-end reads (2 × 151 bp). The raw data quality control was performed using FASTQC v0.11.9 (Babraham Institute, Cambridge, UK) and the trimming and de novo assembly were performed using CLC Genomics Workbench 12.0.3 (QIAGEN, Aarhus, Denmark). All assemblies were carried out with automatic word size, similarity fraction of 0.95, a length fraction of 0.95, and a minimum contig size of 500 bp.

Phylogenetic Analysis
A minimum-spanning tree (MST) was constructed using the Phyloviz V2.0 software (available at https://bitbucket.org/phyloviz/phyloviz-main/downloads; accessed on 6 October 2021) and the goeBURST algorithm therein implemented. The MLST data used in the construction of the MST was retrieved from the downloadable scheme MLST profiles of K. pneumoniae available at the Pasteur MLST website (http://www.pasteur.fr/mlst; accessed on 6 October 2021).

Ethical Approval
The strains were obtained as part of routine diagnostic testing and were analysed anonymously. The study proposal was analysed and dismissed from evaluation by the Ethics Committee of the Lisbon Academic Medical Centre of the Faculty of Medicine, Universidade de Lisboa, Portugal (Nr. 248/21, 22 September 2021).

Discussion
Our study provided updated information on the genomic epidemiology, resistome, and virulome of healthcare-associated infections by K. pneumoniae strains in Portugal, and revealed a different molecular epidemiology there (ST13, ST17, ST307, ST147) compared to most countries in Europe (CG 258). Moreover, we reported the dominance of KPC-3 and OXA-181 carbapenemases.
One K. pneumoniae strain coproduced the Guiana extended-spectrum carbapenemase GES-5 and the extended-spectrum beta-lactamase BEL-1. Both GES-5 and BEL-1 genes seem to be commonly identified in Pseudomonas aeruginosa, rather than Enterobacterales [22,23]. The first GES-5-producing Enterobacterales in Portugal (2014) was identified in a K. pneumoniae recovered from aquatic environmental samples [22]. The association of bla GES-5 and bla BEL-1 genes located on a class 3 integron (In1144) was described in a K. pneumoniae ST252 clinical strain isolated in 2009 from northern Portugal. In this class 3 integron, the first gene cassette was the bla IMP-8 gene [24]. Recently, a study described a K. pneumoniae ST147 isolated in 2016 at a tertiary care hospital in Lisbon that produced the bla GES-5 and bla BEL-1 genes in combination with the carbapenemase KPC-3, and showed a similar class 3 integron (In4885). Although this integron only harbored the bla GES-5 and bla BEL-1 genes, and lacked the bla IMP-8 gene, both integrons were also located in a ColRNAI plasmid [25]. Another study in Portugal described the clinical isolate K. pneumoniae ST29, identified in 2017, coproducing both bla GES-5 and bla BEL-1 genes but located in a ColE1 plasmid [9]. Interestingly, our K. pneumoniae strain, which was isolated from a bloodstream infection in 2020, also belonged to ST29 and showed decreased susceptibility to imipenem and meropenem, but was resistant to ertapenem and also had a class 3 integron (In257) with three gene cassettes (bla GES-5 , bla BEL-1 , and aac(6 )-Ib4 genes) located in a ColRNAI plasmid. The data herein support that for the successful emergence and dissemination of bla GES-5 genes, in combina-tion or not with other carbapenemases such as IMP-8 and KPC-3, they are integrated in a class 3 integron and located in a ColRNAI plasmid.
The strain resistant to ceftazidime/avibactam identified was a New Delhi metallo-βlactamase (NDM) producer that is known to be resistant to ceftazidime-avibactam due to the lack of inhibitory effect of avibactam towards such metallo-β-lactamase producers [26], and is not related to the emergence and dissemination of KPC-3 variants [27].
To date, only few cases of members of Enterobacterales producing NDM-1 have been previously reported in Portugal. Two subsequent NDM-1-producing strains were found in Morganella morganii and Proteus mirabilis from a single patient [28], one NDM-1producing Providencia stuartii [29], and Enterobacter spp. producing NDM-1 strains were also found in water samples in Lis River, in the central region of Portugal [23]. NDM carbapenemases are commonly found in K. pneumoniae and Escherichia coli and are increasing worldwide, particularly in several European countries, including Spain [30]. Nevertheless, the reports in Portugal are scarce. Recently, an outbreak caused by NDM-1-producing Klebsiella pneumoniae ST11-KL105 lineage (in clinical and environmental strains) during the COVID-19 pandemic in Portugal was described [31]. However, herein we report a NDM-1-producing Klebsiella pneumoniae ST39 clone, which has been previously reported in other countries harboring the bla NDM-1 gene [32,33]. Our strain coproduced NDM-1 and CTX-M-15 enzymes, and although NDM producers seemed to be very resistant to most therapeutic regimens [2], the fact that this strain was resistant to all antibiotics tested (including tigecycline) is worrisome and requires further attention.
All the strains coproducing both KPC-3 and OXA-181/OXA-48 showed resistance to carbapenems but susceptibility to ceftazidime/avibactam. OXA-48 K. pneumoniae producers are not common in Portugal [6,8,12,25,34], despite its emergence in several Mediterranean countries [35]. The first OXA-48 K. pneumoniae-producing strains were found in 10 strains in the northern region of Portugal in 2018 [10] and after that, only one other study reported a K. pneumoniae OXA-48-producing strain in the same region of Portugal [8]. This study reported a coproduction of KPC-3 and OXA-48 K. pneumoniae strain in a different region of the country, suggesting that although it is not a frequent carbapenemase gene in Portugal, the presence of OXA-48 in K. pneumoniae is not limited to a certain region.
The emergence and dissemination of K. pneumoniae strains that possess both MDR and hypervirulence is a major concern worldwide. The association between genotypes and hypervirulence phenotype is rather complex, and the differences between hypervirulent and classical strains can often be difficult to access. With the help of whole-genome sequencing (WGS) of K. pneumoniae strains, it is now clear that some differences exist in the accessory genome between hypervirulent and classical strains, and that the former contain genes related to increased virulence, such as genes that confer HMV, siderophores, and the genotoxin colibactin, among others [36]. Limited data are available regarding virulence determinants in K. pneumoniae in Portugal. One study conducted by Caneiras et al. identified higher accumulation of virulence factors as type 1 and type 3 fimbrial adhesins (fimH and mrkD), iron siderophore aerobactin (iucC), the capsule polysaccharide K2 serotype, and haemolysin (khe) in hospital-acquired urinary tract infections by MDR K. pneumoniae clinical strains compared with strains recovered from a community setting, which showed lower resistance and virulence [6].
In this study, among the 68 K. pneumoniae strains studied by WGS, 63 (93%) presented yersiniabactin harbored in distinct integrative and conjugative element (ICEs), and 16 (25%) of the strains with yersiniabactin also had the genotoxin colibactin (ICEKp10 encodes colibactin synthetic genes) [37]. This is of relevance, since ICEs are extremely prevalent in hypervirulent K. pneumoniae lineages, including in nearly 90% of CG23 and close to 75% of all hypervirulent K. pneumoniae (hvKp) strains [37]. Furthermore, the HMV phenotype contributes to a large majority of hvKp strains [37], and our data revealed 11 strains with positive HMV phenotype, all of which coded the yersiniabactin virulence gene.
A prevalence of O1/O2 (47/68; 69%) serotypes was present across the studied strains and was congruent with previous reports in Portugal [8,25]. In contrast, a wide variety of K locus serotypes was found herein, where the KL25 (15/68; 22.1%) associated with O5 serotype was the most dominant. Of relevance, no K1/K2 serotypes were found, contrasting with previous findings by Caneiras et al., which included strains recovered until 2013 [6]. On the other hand, a study conducted in Portugal that included strains recovered after 2013 also found no K1/K2 [8], which corroborated the findings of this study, suggesting the emergence of different serotypes in the country. It is known that K1 and K2 serotypes are the most common capsular types in K. pneumoniae, which commonly exhibit an HMV phenotype and are usually associated with hypervirulent strains. However, serotyping alone is not an entirely accurate indicator of hypervirulence, as other serotypes aside from K1 and K2 have also been identified among hvKp strains [36]. In fact, among the most common K locus serotypes related to hypervirulent strains is K1, followed by K2, K5, and K57 [37]. Moreover, in contrast to MDR infections, hypervirulent K. pneumoniae infections worldwide are dominated by the same subset of lineages, namely CG23, followed by CG65 (including ST65 and ST375) and CG86, among others to a lesser extent [37]. Noteworthy, one strain belonging to the novel ST5840 clone found in this study was associated with the KL5;O3b serotype, ybt 10, ICEKp4, and KPC-3 enzyme. Although ST5840 did not seem to be related to the most common clonal groups associated with hvKp infections, nonetheless, the KL5 serotype with yersiniabactin production suggested a possible potential for hypervirulence in this strain.
A vast array of STs (17 in total) were detected. ST13 was the most prevalent, followed by ST17, ST307, and ST147, accounting for more than two-thirds of all studied strains. All ST13 strains were KPC-3 carbapenemase producers, and moreover, coproduced bla KPC-3 and bla OXA-181 /bla OXA-48 genes. ST13 clones have been described in Portugal in KPC-3producing strains [9]; nevertheless, the coproduction of both bla KPC-3 and bla OXA-181/OXA-48 genes in K. pneumoniae has not yet been reported in this country. ST17 has already been described harboring KPC [10] and OXA-181 [9] in other studies in Portugal. ST147 clones have also been previously reported, but harboring the bla KPC gene [8,14,25], opposite to what was found in this study, in which all ST147 strains produced OXA-181. Still, the strains belonging to the ST147 clone had the same KL64/O2v1 serotype, as previously reported [8,14,25]. All ST307 clones presented the KL102/O2v2 serotype, but only one strain produced carbapenemases genes (bla KPC-3 ). However, previous reports described ST307 clones with no carbapenemase produced and with the presence of KL102/O2v2 serotypes. Interestingly, a French report described an outbreak with K. pneumoniae KPC-producing ST307 clones, as well as KPC-producing ST147 and ST13 clones from patients who had recently established a link with Portugal [38]. This study also supported an emergent molecular shift in K. pneumoniae epidemiology in Portugal, as in Europe, particularly for the appearance of ST147 and ST307 high-risk clones [39], a cause of concern [40], and recently associated with high mortality in COVID-19 patients [41].
Indeed, previous national reports of carbapenemase-producing K. pneumoniae strains in Portuguese healthcare facilities already revealed a different molecular epidemiology compared to most countries in Europe. To date, it has been reported that Portugal presented more clones belonging to CG15 than to CG258 [6,7,9,12,15]. However, strains belonging to CG15 that were previously frequently found were not present in this study. In fact, the molecular epidemiological characterization of clinical strains resistant to carbapenems demonstrated a different epidemiological scenario in the last 2 years: ST13, ST17, ST147, and ST307 remained among the most frequently found MLST profiles. Additionally, clones belonging to the CG258 were also not present, which highlighted the different molecular epidemiology in Portugal compared the most of the countries in Europe.
Our study had some limitations, given that the WGS analysis was performed on 68 selected K. pneumoniae strains and not on the complete collection of strains. Moreover, we included strains from a single tertiary university hospital centre, which cannot represent the complete epidemiological situation in the country. However, it is important to highlight that is one of the major hospital centres in Portugal, and considering that it is a tertiary care centre, it receives patients from other hospitals in the country. Moreover, herein a comprehensive microbiological and molecular characterization of carbapenem-resistant K. pneumoniae clinical strains was performed, contributing updated data on the genomic epidemiology, resistome, and virulome. Future studies should expand both the collection of strains analysed by WGS and the number of hospitals involved, in order to provide a more complete picture of the molecular patterns of K. pneumoniae in Portugal.

Conclusions
In conclusion, we reported a molecular epidemiology shift in Portugal and an uncommon clone detection compared to Europe (where CG15 and CG258 are still dominant). The high-risk clones ST13, ST17, ST147, and ST307 were the most predominant among the carbapenem-resistant K. pneumoniae strains studied. Furthermore, we described K. pneumoniae strains with a high prevalence of siderophores, suggesting the emergence of hypervirulent strains in Portugal. These results emphasized the relevance of performing continuous molecular surveillance, especially through WGS.