Introduction

Klebsiella pneumoniae is one of the most common causes of nosocomial infection. In recent years, the exposure to antibiotics has dramatically increased the drug resistance of K. pneumoniae.1–3K. pneumoniae was first reported to be resistant to penicillin in 1960.4 Carbapenem-resistant Klebsiella pneumoniae (CRKP) has gradually emerged with the introduction of carbapenems and is now a global health concern.3,5 Due to their ease of dissemination and the limited therapeutic options, CRKP infections are usually severe and poorly treated, leading to prolonged hospitalization, increased burden of care, increased risk of multiple infections, and higher mortality rates.6–8 The World Health Organization has identified CRKP as a severe public health threat.9 In China, the China Antimicrobial Surveillance Network (CHINET) has reported a rise in the rates of K. pneumoniae resistance to meropenem from 2.9% in 2005 to 26.3% in 2018, and the resistance rates to imipenem have increased from 3.0% in 2005 to 25% in 2018 (http://www.chinets.com/).

The production of carbapenemases is the chief cause of carbapenem resistance in K. pneumoniae. The three major types of carbapenemases include K. pneumoniae carbapenemase (KPC), metallo-β-lactamases (MBLs) such as New Delhi metallo-β-lactamase (NDM), Imipenemase metallo-β-lactamase (IMP) and Verona integron-encoded metallo-β-lactamase (VIM), and OXA48 enzymes. Among these, IMP exhibits a relatively low prevalence in clinical K. pneumoniae. In China, only a limited number of studies have documented clonal transmission of IMP-4-producing K. pneumoniae among children and newborns,10–12 with only small-scale sample study reporting clonal transmission among adults.13 The specific transmission routes and molecular epidemiological characteristics of these strains remain unclear.

CRKP epidemics exhibit significant regional variations in bacterial characteristics, patients’ baseline characteristics and clinical outcomes, and the distribution of carbapenemases.14 The prevalence of CRKP also varies across different geographical areas in China.15–17 Thus continuous monitoring of CRKP in different areas is worthwhile.

This study investigated the clinical and molecular epidemiological characteristics of CRKP and identified risk factors for the development of CRKP at a large teaching hospital in central China. BlaIMP-4-carrying ST-11 K. pneumoniae discovered as the main prevalent type.

Materials and Methods Bacterial Collection, Identification, and Antimicrobial Susceptibility Testing

Samples from patients infected with CRKP (n = 203) were collected at a tertiary hospital in Wuhan, China, between November 2017 and December 2018. Recurrent infections were excluded, and only the first episode of infections caused by CRKP was included in this analysis. In addition, only patients with complete clinical data were included. All of the specimens were inoculated on a blood agar medium and incubated at a temperature of 37°C for a period of 18 to 24 hours. BD Phoenix™100 Automated Microbiology System (BD Ltd. in Franklin Lakes, NJ, USA) was used to identify the strains and determine their susceptibility to various antimicrobial agents. The modified Hodge test was employed to confirm the presence of carbapenemase-producing strains. All results were interpreted according to the guideline provided by the Clinical and Laboratory Standards Institute (CLSI) M100.18 The disks used for the confirmation test were obtained from Beijing Tiantan Biological Products Corporation in China. K. pneumoniae (ATCC700603) served as the control strain to ensure the accuracy of the experiment. All isolates were stored at −80°C for further study. Patient information, including age, gender, history, diagnosis, and outcome, was obtained from electronic medical records. We also collected the clinical data of a sample of carbapenem-susceptible Klebsiella pneumoniae(CSKP) patients (n = 67) using a systematic sampling method within a list of all CSKP patients in the same hospital between November 2017 and December 2018. A random starting point was chosen, and every 10th identity card number was added to the target CSKP population list.

Detection of Carbapenemase Genes of CRKP Isolates

Bacterial DNA was prepared using the bacteria DNA extraction kit (TIANamp Bacteria DNA kit, China) according to the manufacturer’s instructions. All the samples underwent tests to detect the existence of common carbapenemase genes (blaKPC, blaIMP,blaVIM, blaNDM, and blaOXA) using polymerase chain reaction (PCR) methods. The oligonucleotide primers specific for carbapenemase genes in the PCR assays were designed in this study according to previous studies19–21 and are listed in Table 1. DNA sequencing was performed by Beijing Tsingke Biotech Co., Ltd. (Tianjin, China). The amplification system contained 9.5 μL ddH2O, 12.5 μL multiplex buffer, 1 μL primer1 (10μM), 1 μL primer2 (10μM), and 1 μL template for a final volume of 25 μL. The PCR amplification conditions were as followings: 94°C for 5 minutes (pre-denaturation); 94°C for 30 seconds (denaturation), 56°C for 30 seconds (annealing), and 72°C for 1 minute (extension) (35 cycles); and followed by a final extension at 72°C for 10 minute. Amplified PCR products stained with GelRed were separated by electrophoresis on 1.5% (w/v) agarose (1st base) at 100 V for 30–40 minutes. The molecular size of fragments generated by electrophoresis was determined by comparison to 2-kb DNA ladders (Dalian TaKaRa Corporation), and band patterns were captured under an ultraviolet illuminator. Then, amplified PCR products were sequenced by Sanger sequencing, and the complete sequences were compared with those reported in GenBank using the BLAST algorithm (http://www.ncbi.nlm.nih.gov/blast/).

Table 1 Primers Designed in This Study for Screening the Carbapenemase Genes of CRKP

Multilocus Sequence Typing (MLST) Analysis and Phylogenetic Tree Construction

Clonal relationships analysis of CRKP strains was performed by MLST. Bacterial DNA extraction was performed as previously described to detect carbapenemase genes.

The housekeeping genes (rpoB, gapA, mdh, pgi, phoE, infB, and tonB) of K. pneumoniae were amplified by PCR, and the alleles and STs were determined using online database tools (http://bigsdb.pasteur.fr/klebsiella/klebsiella.html). The BioNumerics software (version 7.6) was used to construct phylogenetic trees based on MLST sequences using the maximum likelihood method with 1000 bootstrap replicates. To explore patterns of evolutionary descent among isolates, we used global optimal e-BURST (goeBURST) analysis of PHYLOViZ 2.0 software to classify the STs of all strains into clonal complexes (CC), groups, and singletons. The software’s default settings follow the strictest criteria: if two STs differ by only one allele, they are considered single-locus variants (SLVs); if they differ by two alleles, they are considered double-locus variants (DLVs). Only SLVs can form a group. If a group contains enough STs, and one of the STs has the highest number of corresponding SLVs, that ST can be provisionally considered a likely ancestral ST, and the group can be regarded as a CC. STs that do not belong to any group are classified as singletons.

Statistical Analysis

Percentages and frequencies were used to describe categorical variables. The Chi-square test or Fisher’s exact test was used to compare categorical variables. Risk factors for the development of CRKP were analyzed using binary logistic regression. Variables with a P value of <0.05 in the univariate analysis were included in logistic regression models for multivariable analysis. In multivariable logistic regression, risk factors with P-values <0.05 and the odds ratios (ORs) >1 were considered as independent risk factors. A P value of <0.05 was considered to be statistically significant. Analyses were performed using SPSS 26.0 for MacIntosh (SPSS Inc., Chicago, IL, USA).

Ethics Approval and Informed Consent

The study was conducted in accordance with the principles of the Declaration of Helsinki, and the study protocol was approved by the Tongji Medical College Ethics Committee, Huazhong University of Science and Technology (2018-S356). Due to the retrospective nature of the study, patient consent for inclusion was waived. We ensure that the use of anonymized data posed minimal risk to patient privacy.

Results Patient Characteristics

A total of 203 patients with CRKP infection were included in this study, with a median age of 56.0 (interquartile range, [IQR], 38.0–67.0) years, and 136 (67.0%) were men. Of these, 98.0% (199/203) patients had at least one comorbidity, and the common comorbidities were cardiovascular disease (41.4%, 84/205), followed by gastrointestinal disease (36.9%, 75/205) and cerebrovascular disease (31.5%, 64/205). 98.5% (200/203) of the patients were nosocomially infected with CRKP, and 5 (2.5%) were admitted to the relevant department after CRKP infection was detected during outpatient visits. Within 3 months prior to sample collection, 68.5% (139/203) patients had a history of hospitalization, 61.1% (124/203) patients had intensive care unit (ICU) stay, 88.7% (180/203) were exposed to antibiotics, and 89.2% (181/203) patients had undergone invasive procedures. Among those who had undergone invasive procedures, 65.5% (133/203) patients had received venous/arterial catheterization, 63.1% (128/203) had indwelling urinary catheters, 54.2% (110/203) had indwelling gastric catheters, and 43.3% (88/203) underwent invasive mechanical ventilation. The median hospital stay days were 23.0 (14.0–36.0) days. The mortality rate among patients with CRKP was 17.7% (36/205) (Table 2).

Table 2 Demographic and Clinical Characteristics of Patients with CRKP Infection

Drug Resistance Rate and Characteristics of Drug-Resistant Gene in CRKP Strains

All 203 CRKP isolates were confirmed as carbapenemase-producing strains. The highest resistance of all strains was cefazolin (98.5%) excluding intrinsic resistance to ampicillin (100%), followed by piperacillin (97.5%). The highest susceptibility was to tigecycline (100%), followed by minocycline (69.4%) and chloramphenicol (43.9%) (Figure 1).

Figure 1 Resistance profiles of CRKP to common antimicrobials.

Abbreviations: AMP, Ampicillin; PIP, Piperacillin; TZP, Piperacillin/tazobactam; AMC, Amoxycillin/clavulanic acid; CEZ, Cefazolin; CAZ, Ceftazidime; CSL, Cefoperazone-sulbactam; CTX, Ceftriaxone; CPE, Cefepime; AZT, Aztreonam; MEM, Meropenem; IPM, Imipenem; GEN, Gentamicin; AMK, Amikacin; CIP, Ciprofloxacin; LVX, Levofloxacin; SXT, Sulfamethoxazole; CHL, Chloramphenicol; MH, Minocycline; TGC, Tigecycline.

The results of drug-resistant gene analysis are shown in Figure 2A. The most common drug-resistant gene was blaIMP-4, identified in 81.0% (166/205) of strains, followed by blaKPC-2 was identified in 24.9% (51/205), and blaNDM-1 was identified in 22.9% (47/205) of strains. Among these, 94 (46.3%) strains had only blaIMP-4 gene, 3 (1.5%) had only blaNDM −1gene, 2 (1.0%) had only blaKPC-2 gene, 28 (13.8%) had both blaKPC-2 and blaIMP-4 genes, 23 (11.3%) had both blaIMP-4 and blaNDM-1 genes, 1 (0.5%) had both blaKPC-2 and blaNDM-1 genes, and 20 (9.9%) had three genes: blaKPC-2, blaIMP-4, and blaNDM-1 three genes. None of the isolates carried blaOXA or blaVIM genes.

Figure 2 (A) Distribution of carbapenemase genes in CRKP strains. (B) Distribution of sequence types in CRKP strains. New STs include 7 types of total 8 strains, others include 33 types of total 37 strains. ST, sequence type.

MLST Analysis

MLST experiments identified a total of 48 distinct STs within the 203 strains. As shown in Figure 2B, the most prevalent STs were ST-11 (59.6%, 121/203), followed by ST-2407 (5.4%, 11/203), ST-15 (3.5%, 7/203), ST-37 (3.0%, 6/203), ST-5254 (2.0%, 4/203), ST-273 (1.5%, 3/203), ST-668 (1.5%, 3/203), and ST-412 (1.5%, 3/203). Additionally, there were also 8 (3.9%) new untitled STs.

Phylogenetic analysis (based on MLST sequences) showed that the 203 CRKP isolates were clustered into different groups which the seven major groups, designated as clades 1 to 7. The phylogenic tree showed that all the ST-11 and ST-15 isolates clustered in clade 1 but belonged to different subclades. The six ST-37 strains all belonged to clade 4. Clade 5 comprised all ST-2407 which was mainly found in pediatric patients, and most of the ST-2407 cases were found carrying blaIMP (Figure S1). From Figure 3, it can be seen that the 48 ST types of the 203 CRKP strains were divided into 6 CCs and 15 singletons, among which 6 CCs contain 174 strains of CRKP, accounting for 85.71% of the total number. CC1 contains 12 ST types of 136 strains of which ST-11 is the ancestral ST, accounting for 70.00% of all strains, and is a dominant CC of which ST-11 located at the center, so it is believed that ST-11 is the primary founder, and the other ST types in CC1 are evolved from ST-11.

Figure 3 Details of the goeBURST clonal complexes detected in this study. The size of each circle, reflects the number of isolates with that MLST genotype. The lengths of lines are not significant.

Comparison Analysis of ST-11 and Non-ST-11 CRKP Infections

This analysis included 183 adult patients, of whom 120 patients were with ST-11 CRKP. There was a higher percentage of patients with non-ST-11 CRKP in the cardiology department (4.8% vs 0.0%, P=0.040) compared to those with ST-11 CRKP. There were no significant differences between the two groups in terms of the distribution across other clinic departments, sites of infection, and comorbidities. Patients with ST-11 CRKP had more ICU stay and underwent more mechanical ventilation (51.7% vs 25.4%, P=0.001) and gastric catheterization (58.3% vs 38.1%, P=0.009) in the three months prior to sample collection than patients with non-ST-11 CRKP (Table 3).

Table 3 Comparison of Patients (≥18 Years) with ST-11 and Non-ST-11 CRKP Infection

Additionally, there were also differences in drug-resistance rates between ST-11 CRKP and non-ST-11 CRKP (Figure 4). Drug-resistance rates of almost all antimicrobial agents, except for minocycline, were higher in the ST-11 group compared to the non-ST-11 group. ST-11 CRKP had a higher rate of resistance and a broader spectrum of resistance compared to non-ST-11 CRKP.

Figure 4 Comparison of resistance rates to common antimicrobials between ST-11 and non ST-11 CRKP strains.

Abbreviations: AMP, Ampicillin; PIP, Piperacillin; TZP, Piperacillin/tazobactam; AMC, Amoxycillin/clavulanic acid; CEZ, Cefazolin; CAZ, Ceftazidime; CSL, Cefoperazone-sulbactam; CTX, Ceftriaxone; CPE, Cefepime; AZT, Aztreonam; MEM, Meropenem; IPM, Imipenem; GEN, Gentamicin; AMK, Amikacin; CIP, Ciprofloxacin; LVX, Levofloxacin; SXT, Sulfamethoxazole; CHL, Chloramphenicol; MH, Minocycline; TGC, Tigecycline.

Note: P value of <0.05 is considered to be statistically significant, **P < 0.01, ***P < 0.001.

Risk Factors Associated with the Development of CRKP Infection

We also used systematic sampling to collect the clinical data of 67 CSKP patients. A comparison of clinical characteristics between CRKP and CSKP patients is shown in Table 4. In bivariate analysis, CRKP patients showed associations with comorbidities, especially gastrointestinal disease, urinary system disease, and cerebrovascular disease, stay in the ICU and cardiology departments, nosocomial infection, lung infection, sputum specimen, previous admission, prior ICU stay, prior antibiotic exposure, prior invasive procedure especially mechanical ventilation, non-invasive ventilation, laryngoscope/bronchoscopy/gastroscope, gastric catheterization, urinary catheterization, venous/arterial catheterization, length of hospital stay, and died outcome. In the multivariate analysis, gastrointestinal disease (OR=6.168, P=0.003), nosocomial infection (OR=5.573, P=0.012), and antibiotic exposure (OR=4.131, P=0.004), as well as urinary catheterization (OR=3.960, P=0.031), and venous/arterial catheterization (OR=2.738, P=0.026) within the prior 3 months before sample collection were independent risk factors significantly associated with CRKP infection.

Table 4 Analysis of Risk Factors of Patients (≥18 Years) with CRKP Infection

Discussion

CRKP has emerged worldwide as a significant challenge for clinical practice and public health. With the increasingly high resistance rate of K. pneumoniae to carbapenems, a comprehensive understanding of CRKP strains in patients is crucial. Our research team has been focusing on the molecular epidemiological characteristics of K. pneumoniae,22,23 and this study we mainly explored the molecular characteristics of CRKP, and identified risk factors for CRKP infection.

This study found that all 203 strains carried carbapenemases, which was consistent with the previous reports in China.24,25 There are mainly three types of carbapenemases: KPC-type enzymes, metallo-β-lactamases (MBLs) which include VIM, IMP, and NDM, and OXA enzymes. In China, it has been reported that KPC is the most prevalent carbapenemase among clinical isolates of CRKP,26–28 but the conclusions reported in different regions of China are not consistent. A study reported that the most common carbapenemase gene was NDM in a hospital of Zunyi, China.25 The present study showed that IMP-4 was the main carbapenemase in the hospital. Studies have shown that IMP is commonly found on the plasmids of IncN, and the horizontal transmission of IncN plasmids leads to sustained outbreaks of IMP in multiple species.29 Moreover, most blaIMP contain type 1 integrons which are usually embedded in transposons and/or plasmids, facilitating horizontal transfer of genetic material.30 These genetic backgrounds play a key role in the acquisition and maintenance of resistance in gram-negative bacteria. Gene site mutations can also occur under the selective pressure of antibiotics selection, which is crucial for the spread and development of resistant bacteria. In addition, studies showed that blaIMP often coexists with other resistance genes, such as aacA, catB, and blaOXA.31 Among the 165 strains carrying the IMP-4 gene in this study, 71 strains carried two or more resistance genes at the same time, indicating the stability and confounding of blaIMP. As a MBL, IMP can be inhibited by ethylenediaminetetraacetic acid (EDTA) but not by clavulanic acid or avibactam. β-Lactam drugs interact with MBLs zinc ions, enabling these enzymes to hydrolyze most cephalosporin and penicillin antibiotics, but not aztreonam. In our research hospital, doctors commonly use ceftazidime avibactam empirically to treat carbapenem-resistant Enterobacteriaceae (CRE) and aztreonam is unavailable in the hospital. This may have contributed to the higher prevalence of IMP-4 and explain the differences between our findings and those of other studies. Our findings may provide valuable insights for guiding clinical antibiotic selection.

ST-11 was identified as the most prevalent ST in this study, consistent with previous studies in China.32–34 ST-11 belongs to the clonal complex group 258 (CC258), which also includes ST-258, ST-512, ST-340 and other subtypes. Globally, ST-258 is the most common type and is widely spread in America and Europe.35 ST-11 is a single-locus variant of ST-258, with 80% of the genes of ST-258 originating from ST-11. However, the difference between the two lies in the tonB housekeeping gene. ST-11 has been reported in many areas of China, such as Zhejiang, Guangdong, Fujian, and Jiangsu, etc.36 Beyond China, ST-11 is also a predominant strain in other Asian countries such as Korea,37 Singapore,38 and Thailand.39 However, the reasons for the predominance of ST-11 strains in Asian countries remain unclear. Li’s study suggested that under similar environmental conditions (24–28°C and 56% relative humidity), ST-11 CRKP isolates survived for a longer period (32–43 days) compared to non-ST-11 CRKP isolates, which survived for less than 30 days.40 Longer survival times favor the transmission of the strain.41 Additionally, hypervirulent ST-11 K. pneumoniae is increasingly being reported,42–44 The ST-11 genome is highly heterogeneous45 and has been found to harbor various resistance genes (eg, KPC, NDM, and VIM) and virulence genes (eg, irp, ybt, and fyu).46 Studies have shown that the highly variable region of bacterial capsule polysaccharide genes may be the main evolutionary region of the ST-11 strain, playing a crucial role in its dissemination and determining its virulence and resistance.47,48 These factors may explain why ST-11 strains can become a dominant type and significantly contribute to spreading resistance genes.

Studies have shown a predominance of ST-11 blaKPC-2K. pneumoniae in China,49 where this study identified a predominance of ST-11 blaIMP-4 in our research. Zheng’s study showed that blaIMP-4-positive isolates were resistant to all β-lactam antimicrobial drugs (namely ampicillin, cefotaxime, meropenem, imipenem, and amoxicillin/clavulanate) and trimethoprim/sulfamethoxazole, classifying them as multidrug-resistant strains.50 The ST-11 clone is highly transmissible.48 When an ST-11 clone carries blaIMP-4, its resistance capabilities and transmissibility may be enhanced, potentially contributing to more K. pneumoniae infections. While no study has reported on ST-11 K. pneumoniae carrying blaIMP-4, however, sporadic studies on other STs of K. pneumoniae carrying blaIMP-4 have shown multiple resistance genes and high transmission characteristics.13,51 Therefore, blaIMP-4-carrying ST-11 K. pneumoniae could be a continually evolving threat and warrants prospective monitoring.

The phylogenic tree showed that ST-11 and ST-15 isolates belonged to two subclades in clade 1. One study revealed that most carbapenemase-positive K. pneumoniae isolates belong to four clonal lineages: ST-11, ST-15, ST-101, and ST-258/512, and their derivatives. These lineages are considered “high-risk” clones, which commonly share a recent ancestor, epidemic success, and a defined geographic distribution.5 In the present study, all 8 ST-15 cases included 3 were ICU isolates and 2 were hematology isolates, implying that ST-15 may always associate with more severe conditions. Besides, ST-2407 was assigned to another distantly related clades in which 10 out of 11 cases were paediatric isolates and most of the ST-2407 cases harbored blaIMP. A Chinese study on paediatric CRKP also found that ST-2407 was the most frequent MLST, but it also suggested that all CRKP isolates mainly carried the carbapenemases resistance genes blaNDM-1 and blaKPC-2, which is inconsistent with our findings.52 In addition to ST-11, we also identified 7 new STs of CRKP isolates disseminated with the hospital. Although their proportion was extremely small, STs that undergo within-hospital transmission are more difficult to control and are more likely to develop into high-risk clones that warrant monitoring.

In this study, gastrointestinal disease, nosocomial infection, prior antibiotic exposure, urinary catheterization, and venous/arterial catheterization were identified as independent risk factors for CRKP infection. With the advancement of medical technology, invasive operations are increasingly being applied to clinical practice. While these procedures have significant benefits for the diagnosis and treatment of diseases, they also increase the risk of bacterial colonization. Invasive operations lead to congestion, edema, and decreased local immunity of the surrounding tissues, weakening the body’s intrinsic barrier against bacteria, and creating conditions for bacterial invasion.53 A meta-analysis study also suggested that invasive ventilation, central venous catheterization, and prior use of antimicrobials were all independent risk factors for CRKP infection.54 Therefore, invasive operations should be minimized, whenever clinically feasible, to reduce the risk of CRKP infection when condition permitting.55

This study had several limitations, including being a single-center retrospective analysis, which may limit the generalizability of the results to other institutions or geographical areas. Moreover, further studies are needed to investigate the specific mechanisms underlying the transmission of drug-resistant genes and strains.

Conclusions

This study documented the distribution of drug-resistant genes and MLST typing of CRKP and investigated the risk factors of CRKP development. In our findings, identified blaIMP-4-carrying ST-11 K. pneumoniae as the most prevalent type, which could represent a continually evolving threat and warrants prospective monitoring. Gastrointestinal disease, nosocomial infection, antibiotic exposure, urinary catheterization, and venous/arterial catheterization within 3 months before sample collection were independent risk factors for CRKP infection. Strict policies for antibiotic use, cautious decisions regarding the implementation of invasive procedures, and careful management of patients with catheters are necessary to prevent CRKP infections.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No.82270021). We thank all study participants and all staff who provided help for our program in our hospital. The abstract of this paper was presented at the Annual Conference of Respiratory Diseases of Chinese Medical Association – 2023 (24 National Academic Conference on Respiratory Diseases).

Disclosure

The authors report no conflicts of interest in this work.

References

1. Yang Y, Chen J, Lin D, et al. Prevalence and drug resistance characteristics of carbapenem-resistant Enterobacteriaceae in Hangzhou, China. Front Med. 2018;12(2):182–188. doi:10.1007/s11684-017-0529-4

2. Wang G, Zhao G, Chao X, et al. The characteristic of virulence, biofilm and antibiotic resistance of Klebsiella pneumoniae. Int J Environ Res Public Health. 2020;18:17. doi:10.3390/ijerph18010017

3. Miller WR, Arias CA. ESKAPE pathogens: antimicrobial resistance, epidemiology, clinical impact and therapeutics. Nat Rev Microbiol. 2024;22:598–616. doi:10.1038/s41579-024-01054-w

4. Munoz-Price LS, Poirel L, Bonomo RA, et al. Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases. Lancet Infect Dis. 2013;13:785–796. doi:10.1016/S1473-3099(13)70190-7

5. David S, Reuter S, Harris SR, et al. Epidemic of carbapenem-resistant Klebsiella pneumoniae in Europe is driven by nosocomial spread. Nat Microbiol. 2019;4:1919–1929. doi:10.1038/s41564-019-0492-8

6. Xu L, Sun X, Ma X. Systematic review and meta-analysis of mortality of patients infected with carbapenem-resistant Klebsiella pneumoniae. Ann Clin Microbiol Antimicrob. 2017;16:18. doi:10.1186/s12941-017-0191-3

7. Xiao T, Zhu Y, Zhang S, et al. A retrospective analysis of risk factors and outcomes of carbapenem-resistant Klebsiella pneumoniae bacteremia in nontransplant patients. J Infect Dis. 2020;221:S174–s183. doi:10.1093/infdis/jiz559

8. Lou T, Du X, Zhang P, et al. Risk factors for infection and mortality caused by carbapenem-resistant Klebsiella pneumoniae: a large multicentre case-control and cohort study. J Infect. 2022;84:637–647. doi:10.1016/j.jinf.2022.03.010

9. Guidelines for the prevention and control of carbapenem-resistant Enterobacteriaceae, Acinetobacter baumannii and Pseudomonas aeruginosa in health care facilities. Geneva: World Health Organization. 2017. Available from: http://www.who.int/csr/publications/i/item/9789241550178. Accessed November1, 2017.

10. Bai Y, Shao C, Hao Y, et al. Using whole genome sequencing to trace, control and characterize a hospital infection of IMP-4-producing Klebsiella pneumoniae ST2253 in a Neonatal unit in a tertiary hospital, China. Front Public Health. 2021;9:755252. doi:10.3389/fpubh.2021.755252

11. Wang X, Qin J, Xiang G, et al. Nosocomial dissemination of blaIMP-4 among Klebsiella pneumoniae by horizontal gene transfer and clonal spread: the epidemic IncN plasmids and the emerging high-risk IMP-4-producing ST101 clone. J Antimicrob Chemother. 2023;78:2890–2894. doi:10.1093/jac/dkad326

12. Wu W, Wei S, Xue CX, et al. An IncN-ST7 epidemic plasmid mediates the dissemination of carbapenem-resistant Klebsiella pneumoniae in a neonatal intensive care unit in China over 10 years. Int J Antimicrob Agents. 2023;62:106921. doi:10.1016/j.ijantimicag.2023.106921

13. Liu Z, Li J, Wang H, et al. Clonal transmission of bla(IMP-4)-carrying ST196 Klebsiella pneumoniae isolates mediated by the IncN plasmid in China. J Glob Antimicrob Resist. 2024;38:116–122. doi:10.1016/j.jgar.2024.05.002

14. Wang M, Earley M, Chen L, et al. Clinical outcomes and bacterial characteristics of carbapenem-resistant Klebsiella pneumoniae complex among patients from different global regions (CRACKLE-2): a prospective, multicentre, cohort study. Lancet Infect Dis. 2022;22:401–412. doi:10.1016/S1473-3099(21)00399-6

15. Hu Y, Liu C, Shen Z, et al. Prevalence, risk factors and molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae in patients from Zhejiang, China, 2008-2018. Emerg Microbes Infect. 2020;9:1771–1779. doi:10.1080/22221751.2020.1799721

16. Wang F, Zou X, Zhou B, et al. Clinical characteristics of carbapenem-resistant Klebsiella pneumoniae infection/colonisation in the intensive care unit: a 9-year retrospective study. BMJ Open. 2023;13:e065786. doi:10.1136/bmjopen-2022-065786

17. Luo Q, Lu P, Chen Y, et al. ESKAPE in China: epidemiology and characteristics of antibiotic resistance. Emerging Microbes Infect. 2024;13:2317915. doi:10.1080/22221751.2024.2317915

18. CLSI. Performance Standards for Antimicrobial Susceptibility Testing: 34th Edition. Clinical and Laboratory Standards Institute (CLSI); 2024.

19. Poirel L, Walsh TR, Cuvillier V, et al. Multiplex PCR for detection of acquired carbapenemase genes. Diagn Microbiol Infect Dis. 2011;70:119–123. doi:10.1016/j.diagmicrobio.2010.12.002

20. Doyle D, Peirano G, Lascols C, et al. Laboratory detection of Enterobacteriaceae that produce carbapenemases. J Clin Microbiol. 2012;50:3877–3880. doi:10.1128/JCM.02117-12

21. Mlynarcik P, Roderova M, Kolar M. Primer evaluation for pcr and its application for detection of carbapenemases in Enterobacteriaceae. Jundishapur J Microbiol. 2016;9:e29314. doi:10.5812/jjm.29314

22. Wang XR, Chen JC, Kang Y, et al. Prevalence and characterization of plasmid-mediated blaESBL with their genetic environment in Escherichia coli and Klebsiella pneumoniae in patients with pneumonia. Chin Med J. 2012;125:894–900.

23. Liu J, Du SX, Zhang JN, et al. Spreading of extended-spectrum β-lactamase-producing Escherichia coli ST131 and Klebsiella pneumoniae ST11 in patients with pneumonia: a molecular epidemiological study. Chin Med J. 2019;132:1894–1902. doi:10.1097/CM9.0000000000000368

24. Wyres KL, Lam MMC, Holt KE. Population genomics of Klebsiella pneumoniae. Nat Rev Microbiol. 2020;18:344–359. doi:10.1038/s41579-019-0315-1

25. Shen M, Chen X, He J, et al. Antimicrobial resistance patterns, sequence types, virulence and carbapenemase genes of carbapenem-resistant Klebsiella pneumoniae clinical isolates from a tertiary care teaching hospital in Zunyi, China. Infect Drug Resist. 2023;16:637–649. doi:10.2147/IDR.S398304

26. hang V, Wang Q, Yin Y, et al. Epidemiology of carbapenem-resistant Enterobacteriaceae infections: report from the China CRE network. Antimicrob Agents Chemother. 2018;62:10–128.

27. Chen D, Li H, Zhao Y, et al. Characterization of carbapenem-resistant Klebsiella pneumoniae in a tertiary hospital in Fuzhou, China. J Appl Microbiol. 2020;129:1220–1226. doi:10.1111/jam.14700

28. Jin Z, Wang Z, Gong L, et al. Molecular epidemiological characteristics of carbapenem-resistant Klebsiella pneumoniae among children in China. AMB Express. 2022;12:89. doi:10.1186/s13568-022-01437-3

29. Yamagishi T, Matsui M, Sekizuka T, et al. A prolonged multispecies outbreak of IMP-6 carbapenemase-producing Enterobacterales due to horizontal transmission of the IncN plasmid. Sci Rep. 2020;10:4139. doi:10.1038/s41598-020-60659-2

30. Zhang Y, Wang X, Wang Q, et al. Emergence of tigecycline nonsusceptible and imp-4 carbapenemase-producing K2-ST65 hypervirulent Klebsiella pneumoniae in China. Microbiol Spectr. 2021;9:e0130521. doi:10.1128/Spectrum.01305-21

31. Kizny Gordon A, Phan HTT, Lipworth SI, et al. Genomic dynamics of species and mobile genetic elements in a prolonged blaIMP-4-associated carbapenemase outbreak in an Australian hospital. J Antimicrob Chemother. 2020;75:873–882. doi:10.1093/jac/dkz526

32. Hu F, Zhu D, Wang F, et al. Current status and trends of antibacterial resistance in China. Clin Infect Dis. 2018;67:S128–s134. doi:10.1093/cid/ciy657

33. Shao C, Wang W, Liu S, et al. molecular epidemiology and drug resistant mechanism of carbapenem-resistant Klebsiella pneumoniae in elderly patients with lower respiratory tract infection. Front Public Health. 2021;9:669173. doi:10.3389/fpubh.2021.669173

34. Zeng L, Yang C, Zhang J, et al. An outbreak of carbapenem-resistant Klebsiella pneumoniae in an intensive care unit of a major teaching hospital in Chongqing, China. Front Cell Infect Microbiol. 2021;11:656070. doi:10.3389/fcimb.2021.656070

35. Peirano G, Bradford PA, Kazmierczak KM, et al. Importance of clonal complex 258 and IncF(K2-like) plasmids among a global collection of Klebsiella pneumoniae with bla(KPC). Antimicrob Agents Chemother. 2017;61:10–128.

36. Wang Q, Wang X, Wang J, et al. Phenotypic and genotypic characterization of carbapenem-resistant Enterobacteriaceae: data from a longitudinal large-scale CRE study in China (2012-2016). Clin Infect Dis. 2018;67(suppl_2):S196–s205. doi:10.1093/cid/ciy660

37. Kim JH, Cho YY, Choi JY, et al. Two distinct genotypes of KPC-2-producing Klebsiella pneumoniae isolates from South Korea. Antibiotics. 2021;11:10. doi:10.3390/antibiotics11010010

38. Balm MN, Ngan G, Jureen R, et al. Molecular characterization of newly emerged blaKPC-2-producing bla KPC-2 -producing Klebsiella pneumoniae in Singapore. J Clin Microbiol. 2012;50:475–476. doi:10.1128/JCM.05914-11

39. Kim SY, Ko KS. Diverse plasmids harboring bla(CTX-M-15) in Klebsiella pneumoniae ST11 isolates from several Asian countries. Microb Drug Resist. 2019;25:227–232. doi:10.1089/mdr.2018.0020

40. Wei L, Wu L, Wen H, et al. Spread of carbapenem-resistant Klebsiella pneumoniae in an intensive care unit: a whole-genome sequence-based prospective observational study. Microbiol Spectr. 2021;9:e0005821. doi:10.1128/Spectrum.00058-21

41. iu Y, Zhang X, Cai L, et al. Enhanced survival of ST-11 carbapenem-resistant Klebsiella pneumoniae in the intensive care unit. Infect Control Hosp Epidemiol. 2020;41:740–742. doi:10.1017/ice.2020.68

42. Yang X, Sun Q, Li J, et al. Molecular epidemiology of carbapenem-resistant hypervirulent Klebsiella pneumoniae in China. Emerg Microbes Infect. 2022;11:841–849. doi:10.1080/22221751.2022.2049458

43. Lan P, Jiang Y, Zhou J, et al. A global perspective on the convergence of hypervirulence and carbapenem resistance in Klebsiella pneumoniae. J Glob Antimicrob Resist. 2021;25:26–34. doi:10.1016/j.jgar.2021.02.020

44. Zhang Y, Jin L, Ouyang P, et al. Evolution of hypervirulence in carbapenem-resistant Klebsiella pneumoniae in China: a multicentre, molecular epidemiological analysis. J Antimicrob Chemother. 2020;75:327–336. doi:10.1093/jac/dkz446

45. Li DX, Zhai Y, Zhang Z, et al. Genetic factors related to the widespread dissemination of ST11 extensively drug-resistant carbapenemase-producing Klebsiella pneumoniae strains within hospital. Chin Med J. 2020;133:2573–2585. doi:10.1097/CM9.0000000000001101

46. Dong N, Zhang R, Liu L, et al. Genome analysis of clinical multilocus sequence type 11 Klebsiella pneumoniae from China. Microb Genom. 2018;4:e000149.

47. Andrade LN, Vitali L, Gaspar GG, et al. Expansion and evolution of a virulent, extensively drug-resistant (polymyxin B-resistant), QnrS1-, CTX-M-2-, and KPC-2-producing Klebsiella pneumoniae ST11 international high-risk clone. J Clin Microbiol. 2014;52:2530–2535. doi:10.1128/JCM.00088-14

48. Zhao J, Liu C, Liu Y, et al. Genomic characteristics of clinically important ST11 Klebsiella pneumoniae strains worldwide. J Glob Antimicrob Resist. 2020;22:519–526. doi:10.1016/j.jgar.2020.03.023

49. Ding L, Shen S, Chen J, et al. Klebsiella pneumoniae carbapenemase variants: the new threat to global public health. Clin Microbiol Rev. 2023;36:e0000823. doi:10.1128/cmr.00008-23

50. Zheng Z, Liu L, Ye L, et al. Genomic insight into the distribution and genetic environment of bla(IMP-4) in clinical carbapenem-resistant Klebsiella pneumoniae strains in China. Microbiol Res. 2023;275:127468. doi:10.1016/j.micres.2023.127468

51. Fang L, Shen Y, Chen R, et al. The characterization of an IncN-IncR fusion plasmid co-harboring bla(TEM-40), bla(KPC-2), and bla(IMP-4) derived from ST1393 Klebsiella pneumoniae. Sci Rep. 2024;14:26723. doi:10.1038/s41598-024-78205-9

52. Zhang X, Xue J, Shen MJ, et al. Molecular typing and drug resistance analysis of carbapenem-resistant Klebsiella pneumoniae from paediatric patients in China. J Infect Dev Ctries. 2022;16:1726–1731. doi:10.3855/jidc.17003

53. Li Y, Li J, Hu T, et al. Five-year change of prevalence and risk factors for infection and mortality of carbapenem-resistant Klebsiella pneumoniae bloodstream infection in a tertiary hospital in North China. Antimicrob Resist Infect Control. 2020;9:79. doi:10.1186/s13756-020-00728-3

54. Liu P, Li X, Luo M, et al. Risk factors for carbapenem-resistant Klebsiella pneumoniae infection: a meta-analysis. Microb Drug Resist. 2018;24:190–198. doi:10.1089/mdr.2017.0061

55. Dai G, Xu Y, Kong H, et al. Risk factors for carbapenem-resistant Klebsiella pneumoniae infection and associated clinical outcomes. Am J Transl Res. 2021;13:7276–7281.