Dissemination of the high-risk cloneST147 carbapenem-resistant klebsiella pneumoniae from a local tertiary care hospital in the Republic of Korea

Background The emergence of carbapenem-resistant Enterobacterales (CRE) infections is rapidly increasing and represents a serious public threat. In 2020, a total of 16,883 carbapenemase-producing Enterobacterales strains were collected; among these isolates, 21 strains were repeatedly isolated in a local tertiary care hospital. Methods Antimicrobial susceptibility testing was performed using the broth microdilution method. All 21 strains of CRKP were analyzed by PFGE after XbaI digestion. The 21 CRKP strains were sequenced on the Illumina Miseq and Oxford Nanopore GridION platforms. Results These 21 CRKP isolates showed an identical antimicrobial resistance profile, including resistance to ampicillin, carbapenems, cephems, chloramphenicol, fluoroquinolone, macrolides and trimethoprim/sulfamethoxazole. Based on whole-genome analysis, these 21 CRKP isolates shared a common genetic structure (ISAba125-IS630-blaNDM−1-bleMBL) and harbored additional resistance determinants (blaOXA−1, blaCTX−M−15, blaSHV−11, blaSHV−67, aac(6’)-Ib-cr, qnrS1, OqxA, OqxB, catB3, mph(A), sul1, and dfrA12) and mutations in the quinolone resistance-determining regions of gyrA (S83I) and parC (S80I). These isolates belonged to the ST147 and KL64 capsular types, which were carried on IncFIB replicon plasmids. The 21 CRKP strains collected from one hospital were divided into five PFGE patterns, and they were closely related with a minimum similarity value of 95.2%. These isolates were found to be highly related based on the presence of between 2 and 27 SNPs. Conclusions These findings indicate that NDM-1-producing K. pneumoniae ST147 may have been introduced via a common source, implying nosocomial transmission; furthermore, continuous monitoring is necessary to prevent endemic transmission. Supplementary Information The online version contains supplementary material available at 10.1186/s12941-023-00601-2.


Background
The emergence of carbapenem-resistant Enterobacterales (CRE) infections is rapidly accelerating and represents a serious public threat.Recently, among CRE infectious agents, Klebsiella pneumoniae has become one of the most common causative pathogens of hospital-and community-acquired infections in the Republic of Korea [1].Additionally, the predominant carbapenemase genes associated with K. pneumoniae infections are K. pneumoniae carbapenemase (KPC), followed by New Delhi metallo-β-lactamase (NDM), which was reported in a previous study [2].Carbapenem-resistant K. pneumoniae has a significant influence on endemic transmission.
Since the first published records from Hungary and Spain in 2008 and 2009, respectively [3,4].Previous reports have indicated the dissemination of the K. pneumoniae, especially, specific sequence types or clones, including ST147, that are related with dissemination and success of carbapenem-resistant K. pneumoniae; these strains are classified as high-risk STs or clones [5,6].Also, several studies have reported the dissemination of high-risk clones of carbapenem-resistant K. pneumoniae [7][8][9][10].K. pneumoniae ST147 is an emerging high-risk clone because of its successful acquisition of multidrug resistance and high transmissibility, both of which are an important vehicle for dissemination [5].
The Korea Disease Control and Prevention Agency (KDCA) has collected and tested all CRE isolates to monitor antimicrobial resistance profiles and the associated resistance genes by the Korean Antimicrobial Resistance Monitoring System.We confirmed that persistent infections caused by NDM-1-producing K. pneumoniae ST147 occurred in a local tertiary care hospital in the Republic of Korea.In this study, we elucidate the molecular and epidemiological characterization of NDM-1-producing K. pneumoniae ST147 isolates.

Antimicrobial susceptibility testing and conjugation assay
Antimicrobial susceptibility testing was performed using the broth microdilution method with customized Sensititre KRCDC2F and KORN panels (TREK Diagnostic Systems, United Kingdom) in accordance with the guidelines established by the Clinical and Laboratory Standards Institute (CLSI) [12].The antimicrobial agents tested were ampicillin, cephems, carbapenems, fluoroquinolone, gentamicin, streptomycin, tetracycline, trimethoprim/sulfamethoxazole and chloramphenicol.Conjugal transfer of carbapenem resistance genes were examined using azide-resistant E. coli J53 as the recipient strain.21 bla NDM−1 positive CRKP isolates were selected for this assay.Donor and recipient cells from Luria-Bertani broth cultures were mixed in a ratio of 1:5 and transconjugants were selected on MacConkey agar plates (Difco, USA) supplemented with imipenem (1 mg/L) and sodium azide (200 mg/L).Carriage of bla NDM-1 in the transconjugant was confirmed by PCR and MICs.

Whole-genome sequencing (WGS)
Genomic DNA of the 21 isolates was isolated using a Blood and Tissue Kit (Qiagen, Stockach, Germany) according to the manufacturer's protocol.Short-read DNA libraries were prepared using an Illumina Nextera Flex library preparation kit and sequenced on a MiSeq sequencer (Illumina, San Diego, CA, USA).A long-read GridION sequencing library was prepared by using a ligation sequencing kit (SQK-LSK109), and sequencing was carried out using a version R9.4.1 flow cell (FLO-MIN 106D).

WGS analysis
The contigs of genomic sequences were assembled with a minimum contig size threshold of 200 bp using the de novo assembly tool within the CLC genomic workbench 21.0.3.Assembled sequences were analyzed for resistance genes (ResFinder 4.1), sequence type (MLST 2.0) and plasmid replicon types (PlasmidFinder 2.1) using bioinformatics web tools available from the Center for Genomic Epidemiology (CGE) website (https://www.genomicepidemiology.org/).The capsular type and virulence genes of these isolates were confirmed using the Bacterial Isolate Genome Sequence Database (https:// bigsdb.pasteur.fr/klebsiella/).Single-nucleotide polymorphisms (SNPs) of the 21 CRKP isolates were identified using CSI phylogeny 1.4 (https://cge.food.dtu.dk/services/CSIPhylogeny/) by comparison with the reference strain K. pneumoniae KP5 (GenBank accession no.CP012426) [13].The phylogenetic analysis was performed by aligning the whole genome of K. pneumoniae KP5 with the whole genomes of other representative NDM-producing K. pneumoniae strains available in the GenBank database (Table S1).

Nucleotide sequence accession numbers
The whole-genome sequences of these strains were deposited with the National Center for Biotechnology Information (NCBI) under the Bio-Project PRJNA813961.The two plasmids sequences were submitted to the GenBank database and can be found under accession numbers OQ785270-OQ785271.

Characteristics of bacterial isolates
Among the isolates, 21 CRKP strains were continuously isolated from June to December in a local tertiary care hospital; 10 were from sputum samples, 4 from urine samples, 3 from wound swabs, 2 from abscess samples and 1 each from a rectal swab and a bronchial washing fluid.Antimicrobial susceptibility testing showed that the 21 CRKP isolates were resistant to ampicillin, carbapenems, cephems, chloramphenicol, fluoroquinolone, macrolides and trimethoprim/sulfamethoxazole, whereas all isolates were susceptible to amikacin, colistin, gentamicin and tetracycline, except for the KR20-0408 isolate, which was resistant to tetracycline (Table 1).

PFGE analysis
All isolates were divided into five different PFGE patterns and were closely related at 95.2% of the minimum similarity value (Fig. 1).Seventeen isolates were clustered together into the KPMX01.568pattern.Four isolates were divided into four different patterns (KPMX01.347,KPMX01.579,KPMX01.584, and KPMX01.587),differing by only one band.

Plasmid analysis
According to whole-genome sequence analysis, 21 ST147-KL64 CRKP isolates shared a common genetic structure.The plasmid incompatibility group of the 21 NDM-1-producing ST147 K. pneumoniae included an IncFIB replicon plasmid approximately 54 kb in size (Fig. 2).The representative ST147-KL64 CRKP genome showed that the flanking regions of the bla NDM−1 gene surrounded the insertion sequences ISAba125 and IS630 upstream and downstream of the bleomycin resistance gene (ISAba125-IS630-bla NDM−1 -ble MBL ).The other resistance region was carried by IS26 and orf477 on the transposon unit orf477-bla CTX−M−15 -IS26 (Fig. 2).This plasmid carried other beta-lactamase genes and those conferring resistance to quinolone, chloramphenicol, and sulfamethoxazole.Attempts to transfer these NDM-1-harboring plasmids by conjugation were unsuccessful.However, the long-and short-read sequencing results confirmed that the bla NDM−1 gene was located on the IncFIB replicon plasmid.

Discussion
From 2017 to 2020, the Korean Antimicrobial Resistance Monitoring System for CRE infections reported that K. pneumoniae was the most frequent pathogen (64.3%), followed by Escherichia coli (18%) and Enterobacter cloacae (3.8%); among these pathogens, bla KPC (75.4%) and bla NDM (18%) were the predominant carbapenemase genotypes [2].The first identification of nosocomial infections associated with NDM-1 producing K. pneumoniae were reported in the Republic of Korea in 2010 [14].K. pneumoniae was known as one of the causes of nosocomial infections, and it transferred rapidly due to the efficiency of colonization and acquired resistance to antibiotics [15].In this study, we demonstrate that K. pneumoniae ST147-KL64 isolates harboring bla NDM−1 have spread in a local tertiary care hospital in the Republic of Korea.
Attempts to transfer these NDM-1-harboring IncFIB plasmids by conjugation were unsuccessful, in agreement with the absence of four conjugation modules (oriT, relaxase, type IV coupling protein [T4CP], and T4SS) from their backbone.However, the results showed that both PFGE and SNP analyses results identified genetically highly related isolates (> 95.2% similarity and 2-27 SNPs differences).Therefore, we confirmed that this nosocomial infection was related to the clonal spread of NDM-1 producing K. pneumoniae rather than the horizontal transmission of plasmids.
Incorporating public bla NDM -producing K. pneumoniae ST147 data into the analysis revealed that these isolates formed a single cluster, suggesting the acquisition of bla NDM by a clonally spreading K. pneumoniae ST147-KL64 strain in one hospital.In addition, these isolates have an identical capsular type, plasmid replicon type, resistance determinant set and mobile genetic element set, which may have been introduced from common sources, implying nosocomial transmission.However, the limited acquisition of NDM-1-producing K. pneumoniae ST147-KL64 could not be explained by the spread from humans to humans or contamination of the environment.
An NDM-producing ST147-KL64 clone has been reported annually in other countries, such as the UK, the USA and Italy [18][19][20].Recently, several cases have demonstrated the prevalence of NDM-producing K. pneumoniae associated with nosocomial infections, including cases in the USA, Pakistan and the Netherlands [9,20,21].A previous study showed that the K. pneumoniae ST147 clone has a global distribution and that it has been responsible for several nosocomial outbreaks worldwide [5].
The emergence of the NDM-1-producing K. pneumoniae ST147-KL64 clone in a local tertiary care hospital is a concern not only for transmission in hospitals but also for the spread of community-acquired transmission.Thus, increased monitoring is necessary to prevent the dissemination of high-risk clones, and adequate infection control measures against the spread of national and transnational infections, especially those targeting underlying mechanisms, are needed.

Fig. 2 Fig. 1
Fig. 2 Sequence alignment analysis of IncFIBbla NDM−1 plasmids Linear alignment of the sequences of two representative bla NDM−1 harboring K. pneumoniae strains in this study.The gray shaded area indicates the nucleotide similarity between the corresponding genetic loci in each plasmid

Table 1
Antimicrobial susceptibility profiles of 21 NDM-1-producing K. pneumoniae isolates in this study