Infection and Drug Resistance Dovepress

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ORIGINAL RESEARCH

Genomic Analysis of Carbapenem-Resistant

Hypervirulent Klebsiella pneumoniae in a Chinese
Tertiary Hospital
Lan Chen 1, *, Ying Zhou 2, *, Shanshan Wang 1 , Chunyang Wu 3 , Peiyao Zhou 4 , Bingjie Wang 2 ,
Zhu Chen 5 , Fangyou Yu 2
1
Department of Clinical Laboratory Medicine, The First Affiliated Hospital of Ningbo University, Ningbo, Zhejiang, 315010, People’s Republic of
China; 2Department of Clinical Laboratory Medicine, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, 200082, People’s
Republic of China; 3Department of Respiratory Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, People’s
Republic of China; 4Department of Laboratory Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, People’s Republic of
China; 5Department of Clinical Laboratory Medicine, Ningbo No. 2 Hospital, Ningbo, Zhejiang, 315010, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Zhu Chen; Fangyou Yu, Email [email protected]; [email protected]

Background: Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) has become a clinical crisis and is associated
with significant morbidity and mortality. The prevalence of CR-hvKP has trended upward since 2010. This study aims to describe the
clinical and genomic characteristics of CR-hvKP collected from a tertiary hospital in eastern China, from August 2020 to
October 2021.
Methods: We tested the susceptibility to common antibiotics in these isolates to feature the antibiotic-resistant phenotypes. We also
applied whole-genome sequencing and core-genome phylogenetic to analysis the genetic features of these isolates. Plasmid replicons
were identified by using the PlasmidFinder database, and core-genome phylogenetic analysis by Parsnp database.
Results: All these strains isolated from the patients with serious underlying diseases and poor prognosis. We found all CR-hvKp isolates
exhibited a multidrug-resistant (MDR) phenotype. These results revealed that blaKPC-2 was the predominant carbapenemases gene (n = 53,
84.1%), and ST11-KL64 CR-hvKP strains dominated, forming a single cluster, and differed by an average of 26 core SNPs. We only found
eight ST15 isolates containing KL24 and KL112 type capsules, with the main carbapenem resistance genes being blaOXA-232 and blaKPC-2.
All ST11-KL64 strains had a series of resistance and virulence genes, along with IncHIB-FIB virulence plasmids and IncFII resistance
plasmids, while the prevalence of resistance plasmids like the IncFII plasmid was absence in ST15 isolates.
Conclusion: This suggests that ST11-KL64 CR-hvKP has emerged as the most prevalent hypervirulence and carbapenem-resistant
K. pneumoniae and may contribute to hospital outbreaks of infection, which required most clinical attention.
Keywords: carbapenem-resistant, hypervirulence, Klebsiella pneumoniae, whole-genome sequencing, antimicrobial resistance,
virulence genes

Introduction
Klebsiella pneumoniae (KP) is a common opportunistic pathogen in clinical practice, capable of causing infectious diseases in
the urinary tract, respiratory tract, blood, and soft tissues.1 Hypervirulent Klebsiella pneumoniae (hvKp) has a higher virulence
than KP2 and can cause severe infectious diseases, including pyogenic liver abscess, endophthalmitis, and meningitis.3,4
Although multidrug resistance and hypervirulence were previously thought to follow distinct evolutionary directions with
non-overlapping genomic signatures for each phenotype,5 hvKp has recently garnered more attention due to its increased
likelihood of acquiring antimicrobial resistance (AMR) genes, particularly those that code for carbapenemases.2,6
Carbapenem-resistant hvKp (CR-hvKp) exhibits both hypervirulence and carbapenem resistance phenotypes, making infec­
tions caused by these strains difficult to treat with current antibiotics, and should therefore receive greater attention.7

Infection and Drug Resistance 2023:16 6385–6394 6385

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CR-hvKP emerged in the early 2010s and is primarily prevalent in Asia, especially China, but cases have been
reported worldwide.8 In 2016, a lethal outbreak of ST11 CR-hvKP occurred in a Chinese intensive care unit, with 21
ST11 KPC-2-producing CR-hvKP strains isolated from five patients who died during hospitalization.7 CR-hvKP has
spread globally and poses a significant human public health threat.9 Therefore, the early recognition of these hyperviru­
lent strains, including their resistance determinants, is a priority concern.
The aim of our study was to determine the current occurrence of CR-hvKp in a tertiary hospital in eastern China. We
analyzed the clinical outcomes through reviewing medical history and identified different serotypes, virulence-associated
markers, and antimicrobial drug resistance genes among the CR-hvKp isolates by using whole genome sequencing (WGS).

Materials and Methods

Bacterial Strains
To explore the characteristics of the carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) isolates, we collected
63 CR-hvKP isolates from 54 patients with underlying diseases and poor prognosis in a tertiary hospital in Ningbo,
Zhejiang Province, China, from August 2020 to October 2021. The strains from different isolation sites of the same
patient were included in this study. All the information about the patients were listed in Table 1. The speciation was
determined by Matrix-Assisted Laser Flight Desorption/ Ionisation Time of (MALDI-TOF MS). Based on the antibiotic
susceptibility results and the patient’s prognosis, we initially determined whether the infected bacteria were CR-hvKP,
and then further confirmed based on the results of whole-genome sequencing.

Antimicrobial Susceptibility Test

We performed bacterial antimicrobial susceptibility testing using VITEK2 system. MICs were measured for cefoxitin,
ceftriaxone, aztreonam, cefepime, imipenem, tobramycin, gentamicin, amikacin, ciprofloxacin, levofloxacin, piperacillin/
tazobactam, amoxicillin/clavulanic acid and trimethoprim/sulfamethoxazole. For MIC determination, E. coli ATCC
25922 was used as a quality control strain, which was purchased from National Center for Clinical Laboratories and
kept by our laboratory. CLSI2022-M100-ED31was used to determine the interpretative breakpoints.10

Whole-Genome Sequencing and Bioinformatics Analysis

The genomic DNA of these CR-hvKP was extracted using a commercial DNA extraction kit (Qiagen, Germany). The genome
sequencing was then performed by the Illumina NovaSeq 6000 platform, with 2×150 bp paired-end reads. The multilocus
sequence typing (MLST), capsular type, resistance and virulence determinants were determined by the Kleborate (version 0.3.0)
(https://github.com/katholt/Kleborate/). Plasmid replicons were identified using the PlasmidFinder database using the minimum
coverage and minimum identities of 90% (https://cge.cbs.dtu.dk/services/PlasmidFinder/).

Results
Clinical Characteristics of Patients Infecting with CR-hvKp
We collected 63 isolates of carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) from 54 patients at a tertiary
hospital in China. This study included strains from different isolation sites of the same patient, with 9 patients having 2
isolates from different specimens (Table 1). The patients were predominantly males (81.5%, n = 44) with a median age of
66 years. They were mainly from different ICU wards in the hospital (23/54 42.6%), followed by the emergency
intensive care unit (EICU; 6/54, 11.1%), gerontology (5/54; 9.3%), hematology (4/54; 7.4%), neurosurgery (3/54; 5.5%),
emergency ward (3/54; 5.5%) and other wards (10/54; 18.6%). Among the 63 CR-hvKP isolates, a variety of clinical
specimens were involved, including sputum (37/63, 58.7%), blood (11/63, 17.5%), urine (4/63, 6.3%), stool (4/63, 6.3%),
and other specimens (7/63, 11.2%). Notably, some patients had poor prognoses, with 20 patients (37.0%) experiencing
delirium during discharge, 4 patients still requiring treatment, and 2 patients dying during their hospitalization. Due to the
multidrug resistance and higher virulence of infections caused by CR-hvKP isolates, distressing clinical outcomes were
more likely to occur.

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Table 1 Characteristics of Clinical Cases and Isolates
Patients Years Gender Isolates Source Ward STs KL Virulence Factors Resistance Plasmid Replicons Clinical Outcomes
Old Genes

1 47 Female FK3004 Sputum Neurosurgery ST15 KL24 iucA, rmpA2 KPC-2 IncFIB Improve, discharge
2 22 Male FK3006 Sputum ICU-1 ST15 KL112 iucA, rmpA2 OXA-232 IncFIB, IncHI1B Improve, be
hospitalized
3 72 Male FK3009 Blood Emergency ward ST11 KL64 iucA, rmpA2, rmpA KPC-2 IncFII, IncHI1B, IncR Improve, be
hospitalized
4 51 Male FK3015 Sputum ICU-2 ST65 KL2 iucA, rmpA2, rmpA, iroB KPC-3 IncHI1B, IncX5 Delirious, discharge
51 Male FK3018 Blood ICU-2 ST65 KL2 iucA, rmpA2, rmpA, iroB KPC-3 IncHI1B, IncX5 Delirious, discharge
5 33 Male FK3016 Blood ICU-2 ST11 KL64 iucA, rmpA2 KPC-2 IncFII, IncHI1B, IncR Severe, discharge
6 61 Male FK3021 Sputum ICU-1 ST15 KL24 iucA, rmpA2 KPC-2 IncFIB Cured, discharge
7 60 Male FK3023 Sputum Nephrology ST11 KL64 iucA, rmpA2 KPC-2 IncFII, IncHI1B, IncR, Improve, discharge
IncX3
8 65 Female FK3025 Sputum Gastrointestinal ST11 KL64 iucA, rmpA2 KPC-2 IncFII, IncHI1B, IncR Improve, discharge
surgery
9 88 Male FK3033 Sputum ICU-2 ST11 KL64 iucA, rmpA2 KPC-2 IncFII, IncHI1B, IncR Delirious, discharge
10 59 Male FK3035 Pus Emergency ward ST11 KL64 iucA, rmpA2, rmpA KPC-2 IncFII, IncHI1B, IncR Improve, discharge
11 82 Male FK3040 Sputum EICU ST11 KL64 iucA, rmpA2, rmpA KPC-2 IncFII, IncHI1B, IncR Delirious, discharge
12 62 Male FK3048 Blood Hematology ST65 KL2 iucA, rmpA2, rmpA, iroB NDM-1 IncHI1B, IncX3 Improve, discharge
13 92 Male FK3052 Sputum Gerontology ST11 KL64 iucA, rmpA2, rmpA KPC-2 IncFII, IncHI1B, IncN, Continue treatment
IncR
92 Male FK3121 Sputum Gerontology ST11 KL64 iucA, rmpA2, rmpA KPC-2 IncFII, IncHI1B, IncR Continue treatment
14 55 Male FK3053 Stool ICU-2 ST11 KL64 iucA, rmpA2 KPC-2 IncFII, IncHI1B, IncR Delirious, discharge
15 50 Male FK3054 Pleural fluid ICU-2 ST37 KL25 iucA KPC-2 IncFIB Died
16 76 Female FK3055 Urine Urinary Surgery ST11 KL64 iucA, rmpA2, rmpA KPC-2 IncFII, IncHI1B, IncR Improve, discharge
76 Female FK3056 Blood Urinary Surgery ST11 KL64 iucA, rmpA2, rmpA KPC-2 IncFII, IncHI1B, IncR Improve, discharge
17 51 Female FK3057 Stool Neurosurgery ST11 KL64 iucA, rmpA2 KPC-2 IncFII, IncHI1B, IncR Cured, discharge
18 57 Male FK3058 Sputum EICU ST11 KL64 iucA, rmpA2, rmpA KPC-2, IncFII, IncHI1B, IncR Delirious, discharge
NDM-5
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19 68 Male FK3061 Urine ICU-1 ST412 KL57 iucA, rmpA2, rmpA, iroB KPC-2 - Severe, discharge
68 Male FK3104 Sputum ICU-1 ST412 KL57 iucA, rmpA2, iroB KPC-2 - Severe, discharge
20 70 Male FK3062 Stool ICU-2 ST412 KL57 iucA, rmpA2, rmpA, iroB KPC-2 - Delirious, discharge
70 Male FK3064 Blood ICU-2 ST412 KL57 iucA, rmpA2, rmpA, iroB KPC-2 - Delirious, discharge
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21 87 Male FK3065 Sputum Gastroenterology ST11 KL47 iucA KPC-2 IncFII, IncFIB, IncR Improve
22 91 Male FK3101 Sputum ICU-2 ST37 KL25 iucA KPC-2 IncFIB Died
23 59 Male FK3109 Pipe ICU-2 ST4080 KL64 iucA, rmpA2 KPC-2 IncFII, IncFIB, Delirious, discharge

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IncHI1B, IncR, IncX4
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Table 1 (Continued).

Patients Years Gender Isolates Source Ward STs KL Virulence Factors Resistance Plasmid Replicons Clinical Outcomes
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Old Genes

24 65 Male FK3111 Sputum Hematology ST11 KL64 iucA, rmpA2 KPC-2 IncFII, IncFIB, Delirious, discharge
IncHI1B, IncR
65 Male FK3163 Sputum Hematology ST11 KL64 iucA, rmpA2 KPC-2 IncFII, IncFIB, Delirious, discharge
IncHI1B, IncR
25 93 Male FK3113 Bile Gerontology ST11 KL64 iucA, rmpA2, rmpA KPC-2 IncFII, IncHI1B, IncR Continue treatment
26 68 Female FK3115 Blood ICU-1 ST412 KL57 iucA, rmpA2, rmpA, iroB KPC-2 - Delirious, discharge
27 93 Male FK3118 Sputum Gerontology ST11 KL64 iucA, rmpA2, rmpA KPC-2 IncFII, IncHI1B, IncR Delirious, discharge
28 83 Male FK3119 Blood ICU-1 ST11 KL64 iucA, rmpA2 KPC-2 IncFII, IncHI1B, IncR Delirious, discharge
29 64 Male FK3122 Sputum EICU ST11 KL64 iucA, rmpA2, rmpA KPC-2, IncFII, IncHI1B, IncR Delirious, discharge
NDM-5
30 77 Female FK3124 Sputum EICU ST11 KL64 iucA, rmpA2, rmpA KPC-2 IncFII, IncHI1B, IncR Delirious, discharge
31 93 Male FK3125 Sputum Gerontology ST11 KL64 iucA, rmpA2, rmpA KPC-2 IncFII, IncHI1B, IncR Continue treatment
32 51 Male FK3140 Sputum ICU-1 ST11 KL64 iucA, rmpA2 KPC-2 IncFII, IncHI1B, IncR Improve, discharge
33 92 Male FK3154 Sputum Gerontology ST11 KL64 iucA, rmpA2, rmpA KPC-2 IncFII, IncHI1B, IncR Continue treatment
92 Male FK3159 Secretion Gerontology ST11 KL64 iucA, rmpA2, rmpA KPC-2, IncFII, IncFIB, Continue treatment
OXA-232 IncHI1B, IncR
34 83 Male FK3155 Sputum ICU-1 ST11 KL64 iucA KPC-2 IncFII, IncHI1B, IncR Delirious, discharge
35 73 Male FK3157 Sputum ICU-2 ST412 KL57 iucA, rmpA2, rmpA, iroB KPC-2 - Improve, discharge
36 40 Female FK3160 Stool Hematology ST11 KL64 iucA, rmpA2 KPC-2 IncFII, IncHI1B, IncR Improve, discharge
40 Female FK3165 Blood Hematology ST11 KL64 iucA, rmpA2 KPC-2 IncFII, IncHI1B, IncR Improve, discharge
37 89 Female FK3161 Sputum ICU-1 ST15 KL112 iucA, rmpA2 OXA-232 IncFIB, IncHI1B Delirious, discharge
38 56 Male FK3164 Sputum Neurosurgery ST420 KL20 iucA, rmpA2, rmpA, iroB KPC-2 IncFIA, IncFIB, IncFII, Improve, discharge
IncHI1B
39 58 Female FK3166 Sputum Emergency ward ST11 KL64 iucA, rmpA2 KPC-2 IncFII, IncHI1B, IncR Improve, discharge
40 73 Male FK3168 Urine Cancer ST37 KL25 iucA KPC-2 IncFIB Improve, discharge
Radiochemotherapy
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41 43 Female FK3169 Sputum ICU-2 ST15 KL112 iucA, rmpA2 OXA-232 IncFIB, IncHI1B Uncured
43 Female FK3195 Cerebrospinal ICU-2 ST15 KL112 iucA, rmpA2 OXA-232 IncFIB, IncHI1B Uncured
fluid
42 58 Male FK3170 Sputum ICU-1 ST15 KL112 iucA, rmpA2 OXA-232 IncFIB, IncHI1B Delirious, discharge
43 61 Male FK3171 Blood Hematology ST11 KL64 iucA, rmpA2 KPC-2 IncFII, IncHI1B, IncR Delirious, discharge
44 54 Male FK3174 Blood ICU-2 ST37 KL25 iucA KPC-2 IncFIB, IncR Delirious, discharge

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45 64 Male FK3175 Sputum ICU-2 ST11 KL64 iucA, rmpA2, rmpA KPC-2 IncFII, IncHI1B, IncR Improve, discharge
46 55 Male FK3181 Sputum Nephrology ST11 KL64 iucA, rmpA2 KPC-2 IncFII, IncHI1B, IncR Improve, discharge

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47 68 Male FK3184 Urine Endocrinology ST11 KL47 iucA, rmpA2 NDM-5 IncFII, IncFIB, Improve, discharge
IncHI1B, IncR, IncX3
48 64 Male FK3186 Sputum ICU-2 ST11 KL64 iucA, rmpA2 KPC-2 IncFII, IncHI1B, IncR Improve, discharge
49 67 Male FK3187 Sputum ICU-1 ST11 KL64 iucA KPC-2 IncFII, IncHI1B, IncR Delirious, discharge
50 43 Male FK3188 Pus EICU ST11 KL64 iucA, rmpA2 KPC-2 IncFII, IncFIB, Uncured
IncHI1B, IncR
51 85 Male FK3191 Sputum EICU ST11 KL64 iucA, rmpA2 KPC-2 IncFII, IncFIB, Delirious, discharge
IncHI1B, IncR
52 63 Male FK3194 Sputum Cardiology ST15 KL24 iucA, rmpA2 KPC-2 IncFIB Improve
53 63 Male FK3196 Sputum Thoracic surgery ST505 KL64 iucA, rmpA, iroB OXA-181 IncFIA, IncFIB, Uncured
IncHI2A, IncHI2,
IncX3
54 63 Male FK3198 Sputum Orthopaedic Sports ST11 KL64 iucA, rmpA2 KPC-2 IncFII, IncFIB, Cured
Medicine IncHI1B, IncR
Abbreviations: EICU, emergency intensive care unit; ICU, intensive care unit.
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Antimicrobial Resistance Phenotype of the CR-hvKp Isolates

To clarify the antibiotic-resistant phenotype of these CR-hvKp isolates, we tested their susceptibility to 13 antibiotics
(Table 2). We found that CR-hvKp was resistant to multiple antibiotic classes, with most isolates showing high-level
resistance to carbapenems and all β-lactam antibiotics, including ceftriaxone, cefepime, cefoxitin, and aztreonam
(Table 2). Resistance to fluoroquinolones was also frequent, with 92.1% and 88.9% of isolates resistant to ciprofloxacin
and levofloxacin, respectively. However, the isolates were relatively susceptible to three antimicrobials: gentamicin,
tobramycin, and amikacin, with resistance rates of 23.8%, 22.2%, and 15.9%, respectively. All CR-hvKp exhibited
a multidrug-resistant (MDR) phenotype showing resistant to three or more antibiotic classes and 27.0% were resistant to
all six antimicrobial classes.

Genomic Phylogeny of CR-hvKp Isolates

To gain a deeper understanding of the molecular mechanisms behind these strains, we conducted whole-genome
sequencing and core-genome phylogenetic analysis (Figure 1). The results showed that the ST11 CR-hvKP strains
dominated, forming a single cluster, and differed by an average of 26 core SNPs, indicating clonal expansion. These
ST11 strains contained the KL64 capsule type and had a series of resistance and virulence genes, along with IncHI1B-
FIB virulence plasmids and IncFII resistance plasmids (Figure 1). This suggests that ST11-KL64 CR-hvKP has emerged
as the most prevalent hypervirulence and carbapenem-resistant K. pneumoniae and may contribute to hospital outbreaks
of infection.
While ST15 K. pneumoniae was identified as the second most frequent CRKP clone in hospital infections after ST11
K. pneumoniae, hypervirulent ST15 CRKP was uncommon. We found eight ST15 isolates containing KL24 and KL112
type capsules, with the main carbapenem resistance genes being blaOXA-232 and blaKPC-2. The prevalence of resistance
plasmids like the IncFII plasmid did not observed in ST15 isolates, but only the IncHI1B-FIB virulence plasmid in these
ST15 clusters.
In addition to the ST11 and ST15 CR-hvKP prevalence clones, we found several hypervirulent clones like ST412 and
ST65 that obtained resistance elements to generate hypervirulent CRKP (Figure 1). Although the classical hypervirulent
clone ST412 isolates contained the blaKPC-2 gene, we observed no plasmids, indicating that the gene may have been
obtained through other mobile elements. For the ST65 strains, the resistance phenotype mainly attributed to another
resistance plasmid, the IncX plasmid, harboring blaNDM-1 and blaKPC-3 genes. The co-existence of different plasmids and
the diversity of plasmids simultaneously contributed to the transmission of both resistant and hypervirulent phenotypes in
CR-hvKP isolates.

Table 2 Antibiotic Susceptibilities of 63 Carbapenem-Resistant K. pneumoniae

Antibiotics Sensitivity (%) Intermediate (%) Resistance (%)

Aztreonam 1.6 0.00 98.4

Ciprofloxacin 6.3 1.6 92.1
Levofloxacin 6.3 4.8 88.9
Tobramycin 69.8 8.0 22.2
Gentamicin 73.0 3.2 23.8
Amikacin 80.9 3.2 15.9
Cefoxitin 4.8 9.5 85.7
Ceftriaxone 0.00 0.00 100.0
Cefepime 3.2 1.6 95.2
Imipenem 1.6 1.6 96.8
Piperacillin/Tazobactam 0.0 0.00 100.0
Trimethoprim/Sulfamethoxazole 42.9 - 57.1
Amoxicillin/Clavulanic acid 0.00 1.6 98.4

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Figure 1 Phylogenetic tree based on core genome alignment of 63 Carbapenem-Resistant Hypervirulent Klebsiella pneumoniae in a tertiary hospital in East China. The
different STs and KLs is color-coded and illustrated at the tips. The clusters also differed with several colors. The occurrence of resistance and virulence genes, and plasmid
replicons were also color-coded. The tree was rooted in the midpoint. Scale bar represents 0.01 mutations per nucleotide position.

The Distribution of Antibiotic Resistance Genes and Virulence Factors

The presence of carbapenem-resistant genes in all 63 isolates is concerning, as it limits the options for effective antimicrobial
treatment. Most of the isolates carried the blaKPC gene, particularly blaKPC-2, but other carbapenemase genes such as blaOXA
and blaNDM were also found. A small proportion of isolates carried multiple carbapenemase genes, which further complicates
treatment options. Additionally, a significant proportion of isolates carried beta-lactamase genes.
The hypervirulence of hvKp is due to its unique characteristics, including capsule, lipopolysaccharide, siderophores,
and allantoin metabolism. Genetic biomarkers such as iucA, iroB, rpmA, and rmpA2 can be used to identify hvKp strains.
All isolates in the study carried the iucA gene, which is a critical factor contributing to the high pathogenicity of hvKP.
The iroB gene was detected in 11 isolates, and the rpmA (26/63, 41.3%) and rmpA2 (55/63, 87.3%) genes were present in
a significant proportion of isolates. The combination of these virulence determinants varied among different ST types,
leading to different hypervirulence phenotypes.
Overall, the emergence of CR-hvKP strains with limited treatment options and high virulence is a significant public
health concern, and further studies are needed to understand the molecular mechanisms behind these strains and develop
effective prevention and treatment strategies.

Discussion
HvKp is an emerging pathogen that has been reported in the community setting and has recently caused infections in
healthcare settings, with higher virulence and mortality rates.11,12 In this study, we used whole-genome sequencing
(WGS) in conjunction with phenotypic surveillance to characterize CR-hvKP isolates collected from a large tertiary
public health hospital in eastern China. This study aims to complement and update the growing body of epidemiological
findings on this pathogen.

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The majority of CR-hvKP isolates in our study were recovered from patients hospitalized in the ICU, EICU, and
hematology departments. Patients hospitalized in these departments are at a greater risk of CR-hvKP infections, mainly
due to immune status alterations, the use of broad-spectrum antibiotics, frequent comorbidities, and the use of invasive
devices.13–16 These CR-hvKP isolates showed 27.0% resistance to all six antimicrobial classes. Due to their multidrug
resistance and higher virulence, the prognosis of some patients was poor, with 37.0% of patients experiencing delirium at
discharge. Therefore, to prevent transmission, we should screen for CRE carriage to enable early detection and
implementation of eradication measures.
Our study revealed a high diversity of STs (n=8), with ST11-KL64 K. pneumoniae being the predominant blaKPC
clone in China. ST11-KL64 CR-hvKP has been identified as a cause of bacterial liver abscess,17 bacteremia, and other
infections, and it is a high-risk clinical pathogen that has gained worldwide attention.18 Notably, a Chinese report linked
pneumonia with high mortality to ST11-KL64 CR-hvKP.19 Our study results are consistent with these findings, with
ST11-KL64 (n=37, 58.7%) accounting for most of the strains. In a multicenter study, ST11 also accounted for most
infections (66.6%), followed by ST45 (8.2%), ST15, and ST290 (5.4% each).20 KPC-producing ST15 K. pneumoniae
have caused outbreaks.21 However, a recent Chinese surveillance study reported a lower frequency of ST15 clones,20 In
our study, ST15 was the second most frequent CR-hvKP clone in hospital infections after ST11. Interestingly, we found
that ST15-KL112 only carried OXA-232 genes, instead of blaKPC-2. All in all, ST11-KL64 CR-hvKP poses a significant
challenge for clinicians in various clinical settings and warrants further attention.
The 63 CR-hvKP isolates posed multi-drug-resistant features that exhibited high-level resistance to all β-lactam
antibiotics and carbapenems, but remained susceptible to aminoglycosides. All the isolates harboring carbapenemases
gene, which make the major contribution to the MDR phenotype. In this study, blaKPC-2 genes were predominant among
CR-hvKP isolates (84.1%, n = 53), similar finding was noted in Asia and America where blaKPC-2 is class A enzymes
highly prevalent in CR-hvKP isolates.22 Moreover, the blaKPC-2 genes were distributed across almost all STs, while the
diversity of strains carrying OXA-48-like genes was lower. In other countries, such as India, Iran, Russia, and Italy, the
majority of CR-hvKP isolates were OXA-48-positive strains.22 It is important to note that global immigration may lead to
changes in the linkages between bacterial resistance mechanisms and regions or cities.23 Therefore, surveillance of AMR
trends should be maintained, and areas with low prevalence cannot be ignored. Furthermore, CR-hvKP strains simulta­
neously producing two or more carbapenemases can cause serious infectious diseases with high mortality.22 In our study,
two CR-hvKP strains producing blaKPC-2 and blaNDM-5 carbapenem resistance genes were reported.
All these 63 strains were found to carry virulence genes, including aerobactin (iucA), salmochelin (iroB), and
regulators of mucoid phenotype (rmpA and rmpA2). Siderophore systems are crucial for bacterial pathogenicity, enabling
them to scavenge iron from host transport proteins, allowing them to survive and proliferate in the host.24 Aerobactin
plays a crucial role in both in vivo and vivo survival of hvKp, compared to other siderophores.2 Aerobactin has been
identified as the most prevalent siderophore in hvKp.2 These investigations indicate that aerobactin (iucA) is the primary
determinant of the virulence of hvKP. Our study showed the presence of iucA universally among all isolates, and all 63
isolates harbored carbapenemase-encoding genes, indicating the convergence of hypervirulence and multidrug resistance.
While salmochelin (iroB) was detected in only 5.4% of bacteremia isolates,20 it was present in 17.5% of isolates in
our study. We also observed a higher frequency of rmpA in isolates (41.3%) compared to bacteremia isolates in
a previous study (25.2%).20 The gene encoding aerobactin (iucA) was detected in 10.3% of isolates in Singapore,
while the remaining virulence genes (iro, rmpA, and rmpA2) were far less prevalent, occurring in only 2.4% to 4.5% of
the isolates.25 The regulators of mucoid phenotype genes, rmpA/A2, were associated with hypermucoviscosity.26 Strains
with rmpA/A2 were mainly enriched in KL1/KL2 and ST23/ST86/ST65 hypervirulent clones.26 However, rmpA/A2 was
distributed across most strains in our study, except for the ST37-KL25 strains. The most pandemic linage observed in our
study was ST11-KL64, which harbored iucA, rpmA, and rmpA2.
The copresence of iucA, iroB, rmpA, and rmpA2 was observed in most isolates of ST412-KL57 and ST65-KL2 in our
study, except for FK3104. Twenty-seven of the 63 CR-hvKP isolates (42.9%) harbored three or more hypervirulence
genes. K. pneumoniae isolates carrying hypervirulence genes pose a risk of transmission and constitute a significant
public health threat once they exhibit hypervirulence in vitro and vivo.

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Conclusion
ST11-KL64 CR-hvKP has emerged as the most prevalent hypervirulence and carbapenem-resistant K. pneumoniae and
contribute to the transmission of both resistance and hypervirulence phenotype, which required most clinical attention.

Ethics Statement
The research protocol was approved by the Ethics Committee of The First Affiliated Hospital of Ningbo University
(2023019A). We confirm that all adult participants gave their informed consent. Guidelines outlined in the Declaration of
Helsinki were followed.

Funding
This research is supported by Medical Scientific Research Foundation of Zhejiang Province, Grant No.2023KY1072.
This research is also supported by Medical Scientific Research Foundation of Zhejiang Province, Grant No.2022KY1124.

Disclosure
The authors report that there are no competing interests to declare for this work.

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