Dissemination of the high-risk clone ST147 carbapenem-resistant Klebsiella pneumoniae from a local tertiary care hospital in the Republic of Korea

doi:10.21203/rs.3.rs-2457972/v1

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Dissemination of the high-risk clone ST147 carbapenem-resistant Klebsiella pneumoniae from a local tertiary care hospital in the Republic of Korea

https://doi.org/10.21203/rs.3.rs-2457972/v1

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published 24 Aug, 2023

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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, such as 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-bla_NDM-1-ble_MBL) and harbored additional resistance determinants (bla_OXA-1, bla_CTX-M-15, bla_SHV-11, bla_SHV-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.

Klebsiella pneumoniae

ST147

Carbapenemase

NDM-1

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 [3,4], respectively, several reports have indicated the dissemination of the K. pneumoniae ST147 clone. 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 Korea. In this study, we elucidate the molecular and epidemiological characterization of NDM-1-producing K. pneumoniae ST147 isolates.

Bacterial isolates

In 2020, a total of 16,883 carbapenemase-producing Enterobacterales strains were collected from local public health laboratories, including tertiary care hospitals and long-term care hospitals. Among these isolates, carbapenem-resistant K. pneumoniae (CRKP) isolates were confirmed for 7,808 Enterobacterales strains. In this process, we found that 21 CRKP strains were persistently isolated from a local tertiary care hospital. Here, we characterized these 21 CRKP isolates.

Antimicrobial susceptibility testing

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) [6]. The antimicrobial agents tested were ampicillin, cephems, carbapenems, fluoroquinolone, gentamicin, streptomycin, tetracycline, trimethoprim/sulfamethoxazole and chloramphenicol.

Pulsed-field gel electrophoresis (PFGE)

All 21 strains of CRKP were analyzed by pulsed-field gel electrophoresis (PFGE) after XbaI digestion according to the PulseNet International protocol (https://pulsenetinternational.org/). The genetic relatedness between PFGE patterns was calculated by using BioNumerics v7.6 (Applied Maths, Sint-Martens-Latem, Belgium).

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 MinION 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) [7].

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.

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).

Table 1

Antimicrobial susceptibility profiles of 21 NDM-1-producing *K. pneumoniae* isolates in this study.
Isolates	MIC, µg/L
Isolates	AMI	AMP	FOX	CAZ	CTX	IMI	MER	ETP	DOR	NAL	CIP	SXT	CHL	TET
KR20-0358	< 4	> 64	> 32	> 16	> 32	> 8	16	32	8	> 128	> 16	> 16/304	> 32	4
KR20-0408	< 4	> 64	> 32	> 16	> 32	> 8	8	32	8	> 128	> 16	> 16/304	> 32	64
KR20-0410	< 4	> 64	> 32	> 16	> 32	> 8	8	32	8	> 128	> 16	> 16/304	> 32	4
KR20-0389	< 4	> 64	> 32	> 16	> 32	> 8	8	16	8	> 128	> 16	> 16/304	> 32	4
KR20-0405	8	> 64	> 32	> 16	> 32	> 8	> 32	> 32	> 32	> 128	> 16	> 16/304	> 32	8
KR20-0406	< 4	> 64	> 32	> 16	> 32	> 8	16	32	8	> 128	> 16	> 16/304	> 32	4
KR20-0411	< 4	> 64	> 32	> 16	> 32	> 8	16	32	16	> 128	> 16	> 16/304	> 32	4
KR20-0412	< 4	> 64	> 32	> 16	> 32	> 8	16	32	16	> 128	> 16	> 16/304	> 32	4
KR20-0413	< 4	> 64	> 32	> 16	> 32	> 8	16	32	8	> 128	> 16	> 16/304	> 32	4
KR20-0357	< 4	> 64	> 32	> 16	> 32	> 8	8	32	8	> 128	> 16	> 16/304	> 32	4
KR20-0381	< 4	> 64	> 32	> 16	> 32	> 8	> 32	> 32	> 32	> 128	> 16	> 16/304	> 32	4
KR20-0415	< 4	> 64	> 32	> 16	> 32	> 8	8	32	8	> 128	> 16	> 16/304	> 32	4
KR20-0421	< 4	> 64	> 32	> 16	> 32	> 8	8	32	8	> 128	> 16	> 16/304	> 32	4
KR20-0416	< 4	> 64	> 32	> 16	> 32	> 8	16	32	8	> 128	> 16	> 16/304	> 32	4
KR20-0423	< 4	> 64	> 32	> 16	> 32	> 8	8	32	8	> 128	> 16	> 16/304	> 32	4
KR20-0426	8	> 64	> 32	> 16	> 32	> 8	8	32	8	> 128	> 16	> 16/304	> 32	4
KR20-0427	< 4	> 64	> 32	> 16	> 32	> 8	> 32	> 32	> 32	> 128	> 16	> 16/304	> 32	8
KR20-0428	< 4	> 64	> 32	> 16	> 32	> 8	> 32	> 32	> 32	> 128	> 16	> 16/304	> 32	4
KR20-0430	< 4	> 64	> 32	> 16	> 32	> 8	> 32	> 32	> 32	> 128	> 16	> 16/304	> 32	8
KR20-0434	< 4	> 64	> 32	> 16	> 32	> 8	> 32	> 32	> 32	> 128	> 16	> 16/304	> 32	4
KR20-0369	< 4	> 64	> 32	> 16	> 32	> 8	> 32	> 32	> 32	> 128	> 16	> 16/304	> 32	4

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 KPMX01.568 pattern. Four isolates were divided into four different patterns (KPMX01.347, KPMX01.579, KPMX01.584, and KPMX01.587), differing by only one band.

Genomic analysis

According to the WGS data and MLST scheme of K. pneumoniae, all 21 NDM-1-producing CRKP isolates were identified as ST147 and confirmed to have the same capsular KL64 serotype. Additionally, virulence genes were confirmed to be involved in yersiniabactin (irp1, irp2, ybtAEPQSTUX, and fyuA) and fimbrial adhesion (mrkABCDFHIJ). All 21 ST147-KL64 CRKP isolates coharbored additional resistance determinants, including beta-lactam (bla_OXA−1, bla_CTX−M−15, bla_SHV−11, and bla_SHV−67), quinolone (aac(6')-Ib-cr, qnrS1, OqxA, and OqxB), chloramphenicol (catB3), sulfamethoxazole (sul1), and trimethoprim (dfrA12) resistance genes, as well as mutations in the quinolone resistance-determining regions (QRDRs) of gyrA (S83I) and parC (S80I) (Fig. 1).

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.

Clonal relatedness of bacterial isolates

The phylogenetic analysis of SNPs demonstrated that all strains, including 20 reference strains, differed from each other by 2-2247 SNPs. All K. pneumoniae isolates carrying bla_NDM were typed as ST147. Notably, 21 ST147-KL64 CRKP isolates from this study were shown to be significant phylogroups with limited SNP (2–27) divergences, suggesting an outbreak in one hospital (Fig. 3). Whole-genome comparison showed that 7 reference strains obtained from clinical samples in Italy and the USA (KP-12Pi, KP-26Pi, KP-135LU, KP-1Pi, AR_0145, AR_0152 and MRSN752165) are closely related, differing by 68–109 SNPs

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 [8]. 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 [9]. In this study, we demonstrate that K. pneumoniae ST147-KL64 isolates harboring bla_NDM−1 on IncFIB plasmids have spread in a local tertiary care hospital in the Republic of Korea.

These results support that the possibility of clonal dissemination of K. pneumoniae ST147 among patients. The PFGE results of all strains were different by only one or two bands (> 95.2% similarity). According to SNP analysis, the differences in SNPs among all strains were between 2 and 27 SNPs.

The draft genomes revealed a shared plasmid backbone (> 99% nucleotide identity) among the NDM-1-producing ST147-KL64 isolates from these nosocomial infections (Fig. 2). The bla_NDM−1 gene was encoded on an IncFIB plasmid, although bla_NDM−1 was bracketed by two insertion sequences, ISAba125 and IS630, which belong to the IS30 and IS630 families, respectively. IncFIB plasmids harboring bla_NDM−1 in ST147-KL64 isolates seemed to be horizontally disseminated in the hospital.

IncFIB plasmids harboring bla_NDM−1 were highly homologous with pAR_0145, 7008.20-NDM1 and pM321-NDM1 from K. pneumoniae isolated from the USA, Switzerland and Myanmar (99% nucleotide identity, GenBank accession numbers CP021941, CP082992 and AP018834) [15,16]. These plasmids shared a common plasmid backbone with our isolates and possessed a multidrug resistance region harboring bla_NDM−1.

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 [12–14]. 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 [14,17,18]. 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.

This study identified that persistent infections caused by NDM-1-producing K. pneumoniae ST147 occurred in a local tertiary care hospital in the Republic of Korea. Our findings indicate that NDM-1-producing K. pneumoniae ST147 may have been introduced via a common source, implying nosocomial transmission. Therefore, adequate infection control measure is necessary to prevent the further dissemination in nosocomial and endemic transmission.

CRE : Carbapenem-resistant Enterobacterales

NDM : New Delhi metallo-β-lactamase

CRKP : Carbapenem-resistant Klebsiella pneumoniae

PFGE : Pulsed-field gel electrophoresis

WGS : Whole-genome sequencing

Funding

This work was supported by a grant from the Korea Disease Control and Prevention Agency (grant number 4847-311-210).

Transparency declarations

None to declare.

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No competing interests reported.

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Editorial decision: Major revision
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You are reading this latest preprint version

Dissemination of the high-risk clone ST147 carbapenem-resistant Klebsiella pneumoniae from a local tertiary care hospital in the Republic of Korea

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Materials And Methods

Bacterial isolates

Antimicrobial susceptibility testing

Pulsed-field gel electrophoresis (PFGE)

Whole-genome sequencing (WGS)

WGS analysis

Nucleotide sequence accession numbers

Results

Characteristics of bacterial isolates

PFGE analysis

Genomic analysis

Plasmid analysis

Clonal relatedness of bacterial isolates

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Status:

Journal Publication

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