Introduction

Klebsiella pneumoniae (K. pneumoniae), a Gram-negative, capsulated bacterium, belongs to the Enterobacteriaceae family.1,2 It is a member of the “ESKAPE” pathogens and can cause various infectious diseases in clinical settings, such as pneumonia, urinary tract infections, bloodstream infections (BSI) and sepsis.3,4 According to the 2024 China Antimicrobial Surveillance Network (CHINET) report, K. pneumoniae ranks second in clinical isolation rates, following Escherichia coli (E. coli). This trend is also observed in BSI,5,6 where K. pneumoniae is even showing a tendency to surpass E. coli.7,8 Moreover, unlike other members of the Enterobacteriaceae family, K. pneumoniae possesses a thick polysaccharide capsule,9 which enhances its resistance to phagocytosis, promoting its survival and increasing its pathogenicity9,10 The rising rates of isolation and the emergence of multidrug-resistant (MDR) strains warrant increased attention.11

β-Lactam antibiotics (including penicillins, cephalosporins, monobactams, among others) form the therapeutic foundation for K. pneumoniae infections. However, with rising incidence and resistance rates of ESBL-producing Enterobacteriaceae (ESBL-E), the efficacy of these agents is increasingly compromised.12,13 Extended-spectrum β-lactamases (ESBL) are bacterial enzymes that hydrolyze broad-spectrum oxyimino-cephalosporins (third- and fourth-generation) and monobactams.14,15 The majority of ESBL are classified as class A enzymes in the Ambler classification, with prominent types including SHV, TEM, and CTX-M.14,16 Since their discovery, ESBL have been reported globally.17,18 In the United States, ESBL-producing bacteria are reported to cause approximately 26,000 antimicrobial-resistant infections and 1,700 associated deaths annually.19 Moreover, ESBL-producing bacteria have been categorized as critical-priority pathogens in WHO’s newly released 2024 Bacterial Priority Pathogens List.20 Relevant studies indicate that ESBL-E are associated with higher patient mortality, prolonged hospital stays, and increased healthcare costs.21 Due to their plasmid-mediated transmissibility, the genetic elements can facilitate the spread of resistance between humans and animals, significantly contributing to the emergence of MDR bacterial phenotypes.22–24 Furthermore, Carbapenems remain the treatment of choice for infections caused by ESBL-producing bacteria,25 but inappropriate empirical treatment can also lead to higher mortality rates.26 Consequently, there is an urgent need for effective strategies to prevent and treat these infections.

Although numerous molecular epidemiological studies on ESBL-Kpn BSI exist in China,27–29 data from Southwest China remain scarce. This study therefore analyzes ESBL-Kpn isolates from BSI patients at the Affiliated Hospital of Southwest Medical University (Luzhou, China), characterizing their molecular profiles and identifying BSI-associated risk factors. Our findings aim to optimize hospital infection control protocols and generate evidence-based strategies for clinical management.

Materials and Methods Bacterial Collection

From January 2020 to June 2023, a total of 371 K. pneumoniae strains were isolated and identified from patients with BSI at the Affiliated Hospital of Southwest Medical University. Identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonik GmbH, Bremen, Germany).

ESBL production was confirmed according to the Clinical and Laboratory Standards Institute (CLSI) 2023-M100.30 Bacterial suspensions were standardized to a 0.5 McFarland turbidity using 0.9% saline, after which they were uniformly inoculated onto Mueller-Hinton (MH) agar plates with a sterile cotton swab. Antimicrobial susceptibility disks for cefotaxime, cefotaxime/clavulanic acid, ceftazidime, and cefotaxime/clavulanic acid were carefully placed on the MH agar plates, which were incubated at 37°C overnight. A test result was considered positive for ESBL production if the zone of inhibition around any of the disks increased by ≥ 5 mm following the addition of clavulanic acid.

Risk Factor Analysis

For the risk factor analysis, patients with ESBL-Kpn BSI between 2020 and 2023 were selected as the case group. During the same period, patients with non-ESBL-Kpn BSI were randomly selected as the control group at a 1:2 ratio. Comprehensive clinical data were collected, including baseline demographics, clinical antibiotic usage, invasive procedures, complications, length of hospital stay, clinical outcomes, and other relevant information.

Antimicrobial Susceptibility Testing (AST)

Antimicrobial susceptibility testing of ESBL-Kpn isolates was performed using the MicroScan WalkAway 96 Plus system (Siemens, Germany) and the microbroth dilution method. The antibiotics tested included amikacin, ampicillin, cefazolin, cefuroxime, ceftriaxone, cefotaxime, ceftazidime, cefepime, ciprofloxacin, gentamicin, levofloxacin, tobramycin, aztreonam, ampicillin/sulbactam, piperacillin/tazobactam, trimethoprim-sulfamethoxazole, cefoperazone/sulbactam, cefoxitin and tigecycline. The results were interpreted according to the CLSI guidelines.30 Tigecycline susceptibility results were interpreted following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. E. coli ATCC25922 served as the control strain, obtained from the China National Health Inspection Center.

Detection of Antimicrobial Resistance Genes

Genomic DNA of the strain was extracted using the boiling method.31 Subsequently, common ESBL resistance genes, including blaSHV, blaTEM, and blaCTX-M, were identified by PCR.32 The primers used are listed in Table S1. Positive samples were then sent to Shenggong Biotechnology Co., Ltd. (Shanghai, China) for Sanger sequencing. The resulting sequencing data were analyzed using the K. pneumoniae database (https://bigsdb.pasteur.fr/klebsiella/).

Multilocus Sequence Typing (MLST)

The genetic correlation among all clinical ESBL-Kpn isolates was analyzed using multi-locus sequence typing (MLST).33 The positive PCR products were subsequently sent to Shenggong Biotechnology Co., Ltd. (Shanghai, China) for Sanger sequencing. The resulting sequences were then submitted to the K. pneumoniae MLST database (https://bigsdb.pasteur.fr/cgi-bin/bigsdb/bigsdb.pl?db=pubmlst_klebsiella_seqdef) to determine the STs for each isolated strain. Sequences that could not be accurately matched were uploaded with the necessary information according to the website’s requirements and assigned new STs as needed.

Pulsed-Field Gel Electrophoresis Analysis

The genetic homogeneity among the isolated strains was assessed using XbaI-pulsed-field gel electrophoresis (PFGE), with modifications to previously described protocols.34 Briefly, bacterial DNA was digested with the XbaI restriction endonuclease and subjected to electrophoresis using the CHEF Mapper system (Bio-Rad, USA) for 18 hours, with fragment sizes ranging from 30 to 700 kb. After electrophoresis, the gel was stained with GoldView, and the data were analyzed using BioNumerics software. We acknowledge Shanghai Yibei Technology Co., Ltd. (Shanghai, China) for providing the software trial and technical support.

Serum Resistance Testing

The serum resistance assay was performed following a previously described method with modifications.35 Briefly, 75 µL of healthy human mixed serum was combined with 25 µL of bacterial suspension and incubated on a shaker at 37°C (200 r) for 3 hours. Samples were collected at 0 h, 1 h, 2 h, and 3 h, serially diluted, and plated onto LB agar plates. After overnight incubation at 37°C, colony counts were conducted. Serum resistance of the strains was categorized into six levels based on colony counts.36K. pneumoniae ATCC700603 and K. pneumoniae ATCC2044 served as the control strain.

Capsule Serotyping by PCR and Sequencing

Capsule serotyping was performed as previously described, with minor modifications.37 PCR amplification was conducted using wzi primers, and the positive products were sequenced by Shenggong Biotechnology Co., Ltd. (Shanghai, China). The sequences were then compared on the pasteur website (https://bigsdb.pasteur.fr/) to determine the capsule serotype. The relevant primers are listed in Table S2.

Statistical Analysis

Statistical analyses were performed using SPSS 26.0 (IBM, USA) Chi-square tests or Fisher’s exact tests were used to analyze categorical variables. For continuous variables, independent sample t-tests or non-parametric rank sum tests were employed. Variables with a univariate analysis P < 0.10 and clinical relevance were included in a multivariable binary logistic regression model to identify independent risk factors. Results included calculation of P values, 95% confidence intervals (CIs), and odds ratios (ORs). A significance level of P < 0.05 (two-tailed) was considered statistically significant.

Results Basic Characteristics of the ESBL-Kpn Strains

From January 2020 to June 2023, a total of 45 strains of ESBL-Kpn associated with BSI were collected at the Affiliated Hospital of Southwest Medical University (Luzhou, China). As shown in Figure 1A, the strains were isolated from various departments: the hematology department (n = 7, 15.6%), intensive care unit (n = 5, 11.1%), urology ward (n = 4, 8.9%), neonatology ward (n = 4, 8.9%), vascular surgery (n = 3, 6.7%), and other departments.

Figure 1 Basic characteristics of 45 ESBL-Kpn. (A) Department distribution of 45 isolates; (B) comparison of age distribution in infected patients of different genders; (C) trends in the distribution of antibiotic resistance rates of 45 strains to commonly used antibiotics.

Abbreviation: ns, no statistical difference.

The study population consisted of 26 males (57.8%) and 19 females (42.2%). The ages of the patients ranged from 11 days to 76 years, with a mean age of 45 ± 3.73 years. Specifically, females had a mean age of 41.6 ± 5.5 years, while males averaged 47.5 ± 5.1 years. Although males were generally older than females, the difference was not statistically significant (P > 0.05), as shown in Figure 1B.

Antimicrobial Susceptibility Profiles

Most isolated strains exhibited resistance to most antibiotics. As shown in Table 1, resistance rates were 100% for ampicillin, ceftriaxone, cefotaxime, cefazolin, and cefuroxime, and over 90% for ampicillin/sulbactam. Resistance rates exceeded 80% for trimethoprim-sulfamethoxazole, aztreonam, and cefepime. High resistance was also observed for ciprofloxacin, ceftazidime, levofloxacin, gentamicin, tobramycin, cefoperazone/sulbactam and piperacillin/tazobactam. However, resistance rates were below 10% for cefoxitin, amikacin, and tigecycline.

Table 1 Antimicrobial Susceptibility Profiles of 45 Clinical ESBL-Producing Klebsiella Pneumoniae Strains

Antibiotic resistance rate analysis reveals fluctuating trends as follows: ampicillin/sulbactam (84.6–100%), trimethoprim-sulfamethoxazole (75.0–92.3%), aztreonam (61.5–100%), cefepime (53.8–100%), ceftazidime (53.8–77.8%), and other antibiotics show generally high resistance rates with a tendency to fluctuate and rise. Ciprofloxacin (44.4–91.7%), levofloxacin (11.1–81.8%), gentamicin (22.2–72.7%), tobramycin (11.1–64.6%), piperacillin/tazobactam (18.2%-50.5%) exhibit a slight overall decrease in resistance rates. Furthermore, resistance rates are relatively low for cefoperazone/sulbactam (9.1–25.0%), cefoxitin (9.1–15.4%), and amikacin (0–9.1%). Specific trends in resistance rates are depicted in Figure 1C.

Phenotype and Genotype Analysis

All 45 strains were confirmed to produce ESBL through verification experiments following CLSI guidelines. Three types of ESBL resistance genes, blaTEM, blaSHV, and blaCTX-M, were detected. As shown in Figure 2, PCR experiments revealed that 23 strains carried blaTEM, 37 strains carried blaSHV, and 36 strains carried blaCTX-M. Furthermore, 15 strains harbored all three resistance genes simultaneously. The predominant resistance genes identified were blaCTX-M-15 (n = 14).

Figure 2 Molecular characteristics and homology analysis of ESBL-Kpn.

Homology Analysis Results of Clinical Isolates

A total of 26 known STs were identified, with ST15 (n = 5, 11.1%), ST37 (n = 4, 8.9%), ST101 (n = 4, 8.9%), ST23 (n = 3, 6.7%), and ST485 (n = 3, 6.7%) being the most prevalent. Additionally, ST17, ST45, and ST967 were each found in two isolates, while ST14, ST86, ST147, ST193, ST225, ST307, ST340, ST353, ST437, ST502, ST534, ST678, ST1540, ST1798, ST1825, ST1834, ST2004, and ST4065 were each found in one isolate. Two new STs, ST6835 and ST6837, were also discovered.

Further analysis using PFGE experiments revealed that for the majority of strains with the same STs, there was a high similarity in their fingerprint patterns, indicating a high degree of homology and close isolation dates. As shown in Figure 2.

Serum Resistance Test

Nine strains (n = 9, 20%) exhibited high serum resistance, demonstrating unrestricted growth when exposed to healthy human serum, and were categorized as grades 5 and 6. Fifteen strains (n = 15, 33.33%) showed moderate serum resistance, classified as grades 3 and 4. Twenty-one strains (n = 21, 46.67%) were serum-sensitive, being rapidly killed when exposed to healthy human serum, classified as grades 1 and 2, as illustrated in Figure 2.

Capsular Serotyping Profiles

Among the 45 strains, a total of 15 capsular serotypes were identified, with varying distributions. The most prevalent capsular serotypes were K17 (n = 4), K1 (n = 3), K19 (n = 3), K18 (n = 2), K28 (n = 3), and K62 (n = 2). Additionally, 18 strains did not match any known capsular serotype. Among the 27 strains with identified capsular serotypes, an analysis of the relationship between capsular serotypes and STs revealed the following associations: all K17 strains (n = 4) belonged to ST101, all K1 strains (n = 3) belonged to ST23, K19 strains (n = 3) were predominantly associated with ST967 and ST15, K18 strains (n = 2) corresponded to ST6837 and ST15, K28 strains (n = 3) corresponded to ST485 and ST15, and K62 strains (n = 2) all corresponded to ST45. The distribution of capsular serotypes among the 26 ESBL-Kpn strains with identified capsular serotypes and their corresponding STs is shown in Figure 3.

Figure 3 Distribution of capsular serotypes of ESBL-Kpn and their correspondence with STs.

Risk Factors and Multivariate Analysis of ESBL-Kpn BSI

Single-factor statistical analysis identified several key risk factors for BSI caused by ESBL-Kpn, including transferred patient (P = 0.001), gastrointestinal diseases (P = 0.022), history of surgery within the past six months (P = 0.004), use of cephalosporin antibiotics (P = 0.004), and prolonged total hospital stay (P < 0.001). Multivariable analysis further revealed that transferred patient (OR = 2.520, 95% CI 1.071–5.931, P = 0.034), use of cephalosporin antibiotics (OR = 2.761, 95% CI 1.112–6.856, P = 0.029), and prolonged hospital stay (OR = 1.029, 95% CI 1.006–1.052, P = 0.013) were independent risk factors for BSI caused by ESBL-Kpn. The relevant results are shown in Table 2.

Table 2 Clinical Characteristics of ESBL and Non-ESBL Strains

Discussion

K. pneumoniae poses an increasingly serious threat to clinical antimicrobial therapy. Bacteremia caused by ESBL-Kpn is associated with higher mortality.38 This study characterizes the molecular epidemiology and identifies risk factors for 45 ESBL-Kpn isolates from BSI, collected over a three-year period at a teaching hospital in Southwest China. These isolates accounted for 16.7% of all K. pneumoniae BSI, a prevalence lower than reported in other Chinese studies.39 Notably, global prevalence rates vary markedly across regions. A global study by Tsepo Ramatla et al reported an ESBL-Kpn prevalence of 32.7%.40 This significant disparity highlights the urgent need to strengthen infection control measures for preventing ESBL-Kpn BSI.

Bacterial antibiotic resistance is mechanistically mediated through four primary pathways: hydrolase production, antibiotic target modification, reduced membrane permeability, and efflux pump overexpression.41 Among these, hydrolase production represents the most prevalent mechanism. Current studies predominantly identify CTX-M as the most prevalent resistance-conferring hydrolase in ESBL-Kpn. First identified in 1990, this β-lactamase demonstrates enhanced cefotaxime-hydrolyzing activity.42,43 CTX-M are primarily categorized into several subfamilies: CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25 groups.44,45 Among the isolates analyzed in this study, three CTX-M-type β-lactamases genes were identified: blaCTX-M-15 (n = 14), blaCTX-M-3 (n = 12), and blaCTX-M-14 (n = 10). Notably, blaCTX-M-15 emerged as the predominant variant among K. pneumoniae isolates from BSI patients at our hospital over the three-year study period. This distribution pattern aligns with global surveillance reports on ESBL genotype epidemiology.31,46,47 Recent reports indicate that K. pneumoniae harboring blaCTX-M-15 is frequently associated with carbapenem-resistant phenotypes (CRKP) and exhibits hypervirulent traits, raising significant concerns for clinical therapy.48–50 Research by Chien-Ming Chao et al revealed blaCTX-M-3 and blaCTX-M-14 as the predominant ESBL genotypes in Taiwan, China, with evidence suggesting their clonal dissemination both within and between healthcare institutions.51 Furthermore, blaCTX-M-3 contributes significantly to distinctive resistance phenotypes in K. pneumoniae, where the IncFII plasmid harboring blaCTX-M-3 serves as the primary transmission vector.52 Furthermore, recent studies identify blaCTX-M-14 and blaCTX-M-55 as the predominant ESBL in Asia, whereas blaCTX-M-1 dominates in Europe,53 demonstrating marked geographical variation in blaCTX-M distribution. These findings highlight the critical necessity for enhanced surveillance of plasmid-mediated clonal transmission in hospital settings.

Analysis of the 45 ESBL-Kpn isolates identified 26 distinct STs; ST15 was the most prevalent. ST15 is recognized as a high-risk K. pneumoniae clone,54,55 with studies confirming its frequent production of ESBL or carbapenemases, often associated with nosocomial outbreaks.56,57 This study found that 80% of ST15 ESBL-Kpn isolates produced blaCTX-M. Similarly, a study by Hang Zhao et al reported significant carriage of blaCTX-M-15 in ST15 CRKP strains.58 Min Wang et al also observed this prevalence of blaCTX-M-15 in ST15 isolates.59 In contrast, Carla Rodrigues et al found that ESBL-encoding genes were primarily observed in clonal group (CG) CG14-I, while CTX-M-15 was particularly prevalent in CG15 and CG15-IIB.60 In addition, the high-risk clone ST101 is associated with enhanced virulence and antimicrobial resistance in K. pneumoniae. Infections caused by ST101 strains show an 11% higher mortality rate compared to non-ST101 infections,61 with most isolates belonging to the K17 capsular serotype.62 Our findings are consistent with these reports, further confirming the clinical significance of this epidemic lineage. However, no statistically significant increase in mortality was observed among ST101-infected patients in our study. This discrepancy may reflect the limited sample size in our cohort. Moreover, ST23 K. pneumoniae belongs to the CG23 clonal group, which is the most virulent clonal group of K. pneumoniae.63 ST23 K. pneumoniae has been widely reported in Asia and is significantly associated with the K1 capsule serotype, which is consistent with the findings of this study.64 This study identified high-level and moderate serum resistance in 24 K. pneumoniae isolates. Serum resistance in K. pneumoniae is mediated by multiple factors, including complement, peptidoglycan-associated lipoprotein and murein lipoprotein.65–67 The prevalence of serum-resistant isolates in our research (53.3%) was lower than rates reported elsewhere.68 This discrepancy may reflect distinct virulence profiles of circulating strains, suggesting that bloodstream isolates in Southwest China exhibit comparatively reduced serum resistance. Notably, ST6385 (18-22-26-59-154-37-49) and ST6387 (25-10-1-1-20-1-929) are novel STs identified in this study. Enhanced vigilance and control measures are warranted to prevent the spread of these emerging clones in Southwest China.

BSI caused by MDR K. pneumoniae in hospital settings are associated with multiple factors, including patient health status, healthcare environment, microbial infections, and treatment-related variables.69 Currently, studies specifically addressing the risk factors for BSI caused by ESBL-producing non-CRKP remain limited. This study identified patient transfer, prior use of cephalosporin antibiotics, and prolonged hospitalization as independent risk factors for BSI caused by ESBL-Kpn. Recently, a study found that recent antimicrobial use was an independent risk factor for BSI caused by ESBL-E, which is consistent with the findings of this study.70 Although carbapenems are widely considered effective for the treatment of ESBL-producing pathogens, their use in specific clinical contexts remains controversial. The indiscriminate application of carbapenems for treating ESBL-Kpn infections, without considering individual circumstances, may exacerbate the ongoing antibiotic resistance crisis.71 Furthermore, ESBL-associated resistance genes are often located on plasmids, which are transferable, facilitating horizontal transfer during patient transitions between healthcare facilities. These findings highlight the importance of rational antibiotic use and the prevention of MDR infections.72 Prognostically, most ESBL-Kpn BSI patients achieved clinical recovery with discharge, while 3 (6.7%) died. This mortality rate is lower than the global average.40 The deceased patients were predominantly elderly individuals and children with severe underlying comorbidities, who are more susceptible to critical illnesses and have a poorer prognosis. These findings provide valuable insights for optimizing clinical patient management, guiding rational antimicrobial use, and preventing the emergence of MDR organisms.

This study has several limitations. First, the sample size of included strains was relatively limited; future studies should expand strain collection and perform phylogenetic analyses of STs using larger cohorts. Second, as a single-center investigation, this study lacks generalizability. Multicenter studies are therefore warranted to enhance molecular epidemiological surveillance of ESBL-Kpn in Southwest China. Such efforts are crucial for comprehensively characterizing resistant strain prevalence and informing evidence-based infection control strategies.

Conclusion

This study demonstrated a high prevalence of BSI caused by ESBL-Kpn, with ST15 as the predominant STs. Notably, the novel sequence types ST6835 and ST6837 identified in this work represent, to our knowledge, the first reported instances of these STs. Furthermore, the presence of antibiotic resistance genes was a key contributing factor to the observed high-level resistance phenotypes. Hospital transfer, prior cephalosporin use, and prolonged hospitalization were identified as independent risk factors for ESBL-Kpn BSI. These findings highlight the critical need for appropriate antibiotic use and effective infection control measures to prevent ESBL-Kpn BSI and reduce their transmission within healthcare settings.

Data Sharing Statement

The data used or analyzed in this study can be obtained from the corresponding author upon reasonable request.

Ethics Approval and Consent to Participate

The research was approved by the Institutional Ethics Committee at the Affiliated Hospital of Southwest Medical University (KY2022267). This ethical review process strictly followed the fundamental principles established in the Declaration of Helsinki. In addition, all patients participated by providing written informed consent; for minors, written informed consent was obtained from their legal guardians.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

This work was supported by the grants from Sichuan Science and Technology Program (22ZYZF0007, 2023YFQ0096), the Luzhou Science and Technology Program (2024RCM246, 2023SYF138, 2023SYF135), and the Southwest Medical University Science and Technology Program (2024ZKY070).

Disclosure

The authors report no conflicts of interest in this work.

References

1. Hu F, Pan Y, Li H, et al. Carbapenem-resistant Klebsiella pneumoniae capsular types, antibiotic resistance and virulence factors in China: a longitudinal, multi-centre study. Nat Microbiol. 2024;9(3):814–829. doi:10.1038/s41564-024-01612-1

2. Xu L, Li J, Wu W, Wu X, Ren J. Klebsiella pneumoniae capsular polysaccharide: mechanism in regulation of synthesis, virulence, and pathogenicity. Virulence. 2024;15(1):2439509. doi:10.1080/21505594.2024.2439509

3. Denissen J, Reyneke B, Waso-Reyneke M, et al. Prevalence of ESKAPE pathogens in the environment: antibiotic resistance status, community-acquired infection and risk to human health. Int J Hyg Environ Health. 2022;244:114006. doi:10.1016/j.ijheh.2022.114006

4. Li L, Xu X, Cheng P, et al. Klebsiella pneumoniae derived outer membrane vesicles mediated bacterial virulence, antibiotic resistance, host immune responses and clinical applications. Virulence. 2025;16(1):2449722. doi:10.1080/21505594.2025.2449722

5. Saavedra JC, Fonseca D, Abrahamyan A, et al. Bloodstream infections and antibiotic resistance at a regional hospital, Colombia, 2019–2021. Rev Panam Salud Publica. 2023;47:e18. doi:10.26633/rpsp.2023.18

6. Williams A, Coombs GW, Bell JM, et al. Antimicrobial resistance in Enterobacterales, Acinetobacter spp. and Pseudomonas aeruginosa isolates from bloodstream infections in Australian Children, 2013–2021. J Pediatric Infect Dis Soc. 2024;13(12):617–625. doi:10.1093/jpids/piae111

7. Wu H, Li M, Shou C, et al. Pathogenic spectrum and drug resistance of bloodstream infection in patients with acute myeloid leukaemia: a single centre retrospective study. Front Cell Infect Microbiol. 2024;14:1390053. doi:10.3389/fcimb.2024.1390053

8. Foglia F, Della Rocca MT, Melardo C, et al. Bloodstream infections and antibiotic resistance patterns: a six-year surveillance study from southern Italy. Pathog Glob Health. 2023;117(4):381–391. doi:10.1080/20477724.2022.2129161

9. Zierke L, Mourad R, Kohler TP, Müsken M, Hammerschmidt S. Influence of the polysaccharide capsule on virulence and fitness of Klebsiella pneumoniae. Front Microbiol. 2025;16:1450984. doi:10.3389/fmicb.2025.1450984

10. Huang X, Li X, An H, et al. Capsule type defines the capability of Klebsiella pneumoniae in evading Kupffer cell capture in the liver. PLoS Pathog. 2022;18(8):e1010693. doi:10.1371/journal.ppat.1010693

11. Diekema DJ, Hsueh PR, Mendes RE, et al. The microbiology of bloodstream infection: 20-year trends from the SENTRY antimicrobial surveillance program. Antimicrob Agents Chemother. 2019;63(7). doi:10.1128/aac.00355-19

12. Neffe L, Forde TL, Oravcova K, et al. Genomic epidemiology of clinical ESBL-producing Enterobacteriaceae in a German hospital suggests infections are primarily community- and regionally-acquired. Microb Genom. 2022;8(12). doi:10.1099/mgen.0.000901

13. Khadka C, Shyaula M, Syangtan G, et al. Extended-spectrum β-lactamases producing Enterobacteriaceae (ESBL-PE) prevalence in Nepal: a systematic review and meta-analysis. Sci Total Environ. 2023;901:166164. doi:10.1016/j.scitotenv.2023.166164

14. Husna A, Rahman MM, Badruzzaman ATM, et al. Extended-Spectrum β-Lactamases (ESBL): challenges and Opportunities. Biomedicines. 2023;11(11). doi:10.3390/biomedicines11112937

15. Karaiskos I, Giamarellou H. Carbapenem-Sparing Strategies for ESBL producers: when and how. Antibiotics. 2020;9(2). doi:10.3390/antibiotics9020061

16. Castanheira M, Simner PJ, Bradford PA. Extended-spectrum β-lactamases: an update on their characteristics, epidemiology and detection. JAC Antimicrob Resist. 2021;3(3):dlab092. doi:10.1093/jacamr/dlab092

17. Patil S, Wagh K, Lopes B, Liu S, Wen F. ESBL encoding third-generation cephalosporin resistance observed in bloodstream infection in India. J Hosp Infect. 2023;133:98–99. doi:10.1016/j.jhin.2022.12.019

18. Wang C, Zhang H, Zhao R, Tsui CK, Deng S. Genomic characterization and antimicrobial resistance of ESBL-producing, Escherichia coli isolates in Suzhou, China. Infect Drug Resist. 2025;18:1049–1057. doi:10.2147/idr.S488794

19. Li R, Xu H, Tang H, Shen J, Xu Y. The characteristics of extended-spectrum β-lactamases (ESBLs)-producing Escherichia coli in bloodstream infection. Infect Drug Resist. 2023;16:2043–2060. doi:10.2147/idr.S400170

20. Sati H, Carrara E, Savoldi A, et al. The WHO bacterial priority pathogens list 2024: a prioritisation study to guide research, development, and public health strategies against antimicrobial resistance. Lancet Infect Dis. 2025. doi:10.1016/s1473-3099(25)00118-5

21. Kayinamura MP, Muhirwa A, Kamaliza AC, et al. Prevalence of extended-spectrum beta-lactamase-producing enterobacteriaceae and associated clinical implications at the university teaching hospital of Kigali in Rwanda. Am J Trop Med Hyg. 2024;111(3):565–568. doi:10.4269/ajtmh.23-0605

22. Menezes J, Frosini SM, Weese S, et al. Transmission dynamics of ESBL/AmpC and carbapenemase-producing Enterobacterales between companion animals and humans. Front Microbiol. 2024;15:1432240. doi:10.3389/fmicb.2024.1432240

23. Lü Y, Kang H, Fan J. A novel bla (CTX-M-65)-harboring IncHI2 plasmid pE648CTX-M-65 isolated from a clinical extensively-drug-resistant Escherichia coli ST648. Infect Drug Resist. 2020;13:3383–3391. doi:10.2147/idr.S269766

24. Hawkey J, Wyres KL, Judd LM, et al. ESBL plasmids in Klebsiella pneumoniae: diversity, transmission and contribution to infection burden in the hospital setting. Genome Med. 2022;14(1):97. doi:10.1186/s13073-022-01103-0

25. Tamma PD, Heil EL, Justo JA, Mathers AJ, Satlin MJ, Bonomo RA. Infectious Diseases Society of America 2024 Guidance on the Treatment of Antimicrobial-Resistant Gram-Negative Infections. Clin Infect Dis. 2024. doi:10.1093/cid/ciae403

26. Mancuso G, De Gaetano S, Midiri A, Zummo S, Biondo C. The challenge of overcoming antibiotic resistance in Carbapenem-resistant gram-negative bacteria: “Attack on Titan”. Microorganisms. 2023;11(8). doi:10.3390/microorganisms11081912

27. Tang Y, Xu C, Xiao H, Wang L, Cheng Q, Li X. Gram-negative bacteria bloodstream infections in patients with hematological malignancies - the impact of pathogen type and patterns of antibiotic resistance: a retrospective cohort study. Infect Drug Resist. 2021;14:3115–3124. doi:10.2147/idr.S322812

28. Liang T, Xu C, Cheng Q, Tang Y, Zeng H, Li X. Epidemiology, risk factors, and clinical outcomes of bloodstream infection due to extended-spectrum Beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in hematologic malignancy: a retrospective study from Central South China. Microb Drug Resist. 2021;27(6):800–808. doi:10.1089/mdr.2020.0033

29. Xiao S, Chen T, Wang H, et al. Drug susceptibility and molecular epidemiology of Klebsiella pneumoniae bloodstream infection in ICU patients in Shanghai, China. Front Med Lausanne. 2021;8:754944. doi:10.3389/fmed.2021.754944

30. CLSI. Performance Standards for Antimicrobial Susceptibility Testing. 33th ed. CLSI M100. Clinical and Laboratory Standards Institute; 2023.

31. Zeng Z, Wei Y, Ye C, et al. Carbapenem-resistant Enterobacter cloacae complex in Southwest China: molecular characteristics and risk factors caused by NDM producers. Infect Drug Resist. 2024;17:1643–1652. doi:10.2147/idr.S447857

32. Li Z, Ding Z, Yang J, et al. Carbapenem-resistant Klebsiella pneumoniae in Southwest China: molecular characteristics and risk factors caused by KPC and NDM producers. Infect Drug Resist. 2021;14:3145–3158. doi:10.2147/idr.S324244

33. Diancourt L, Passet V, Verhoef J, Grimont PA, Brisse S. Multilocus sequence typing of Klebsiella pneumoniae nosocomial isolates. J Clin Microbiol. 2005;43(8):4178–4182. doi:10.1128/jcm.43.8.4178-4182.2005

34. Jiménez-Rojas V, Villanueva-García D, Miranda-Vega AL, et al. Gut colonization and subsequent infection of neonates caused by extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae. Front Cell Infect Microbiol. 2023;13:1322874. doi:10.3389/fcimb.2023.1322874

35. Park S, Kim H, Ko KS. Reduced virulence in tigecycline-resistant Klebsiella pneumoniae caused by overexpression of ompR and down-regulation of ompK35. J Biomed Sci. 2023;30(1):22. doi:10.1186/s12929-023-00910-w

36. Liu Y, Liu PP, Wang LH, Wei DD, Wan LG, Zhang W. Capsular polysaccharide types and virulence-related traits of epidemic KPC-producing Klebsiella pneumoniae isolates in a Chinese University Hospital. Microb Drug Resist. 2017;23(7):901–907. doi:10.1089/mdr.2016.0222

37. Brisse S, Passet V, Haugaard AB, et al. wzi Gene sequencing, a rapid method for determination of capsular type for Klebsiella strains. J Clin Microbiol. 2013;51(12):4073–4078. doi:10.1128/jcm.01924-13

38. Li D, Huang X, Rao H, et al. Klebsiella pneumoniae bacteremia mortality: a systematic review and meta-analysis. Front Cell Infect Microbiol. 2023;13:1157010. doi:10.3389/fcimb.2023.1157010

39. Zhang F, Li Y, Lv Y, Zheng B, Xue F. Bacterial susceptibility in bloodstream infections: results from China Antimicrobial Resistance Surveillance Trial (CARST) Program, 2015–2016. J Glob Antimicrob Resist. 2019;17:276–282. doi:10.1016/j.jgar.2018.12.016

40. Ramatla T, Mafokwane T, Lekota K, et al. “One Health” perspective on prevalence of co-existing extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae: a comprehensive systematic review and meta-analysis. Ann Clin Microbiol Antimicrob. 2023;22(1):88. doi:10.1186/s12941-023-00638-3

41. Urban-Chmiel R, Marek A, Stępień-Pyśniak D, et al. Antibiotic resistance in bacteria–a review. Antibiotics. 2022;11(8). doi:10.3390/antibiotics11081079

42. Romyasamit C, Sornsenee P, Kawila S, Saengsuwan P. Extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae: insights from a tertiary hospital in Southern Thailand. Microbiol Spectr. 2024;12(7):e0021324. doi:10.1128/spectrum.00213-24

43. Watanabe N, Watari T, Otsuka Y, Ito M, Yamagata K, Fujioka M. Antimicrobial resistance and AmpC production in ESBL-producing Klebsiella pneumoniae and Klebsiella quasipneumoniae: a retrospective study in Japanese clinical isolates. PLoS One. 2024;19(5):e0303353. doi:10.1371/journal.pone.0303353

44. Walther-Rasmussen J, Høiby N. Cefotaximases (CTX-M-ases), an expanding family of extended-spectrum beta-lactamases. Can J Microbiol. 2004;50(3):137–165. doi:10.1139/w03-111

45. D’Andrea MM, Arena F, Pallecchi L, Rossolini GM. CTX-M-type β-lactamases: a successful story of antibiotic resistance. Int J Med Microbiol. 2013;303(6–7):305–317. doi:10.1016/j.ijmm.2013.02.008

46. Akenten CW, Khan NA, Mbwana J, et al. Carriage of ESBL-producing Klebsiella pneumoniae and Escherichia coli among children in rural Ghana: a cross-sectional study. Antimicrob Resist Infect Control. 2023;12(1):60. doi:10.1186/s13756-023-01263-7

47. Kakuta N, Nakano R, Nakano A, et al. Molecular characteristics of extended-spectrum β-lactamase-producing Klebsiella pneumoniae in Japan: predominance of CTX-M-15 and emergence of hypervirulent clones. Int J Infect Dis. 2020;98:281–286. doi:10.1016/j.ijid.2020.06.083

48. Howard-Jones AR, Sandaradura I, Robinson R, et al. Multidrug-resistant OXA-48/CTX-M-15 Klebsiella pneumoniae cluster in a COVID-19 intensive care unit: salient lessons for infection prevention and control during the COVID-19 pandemic. J Hosp Infect. 2022;126:64–69. doi:10.1016/j.jhin.2022.05.001

49. Hou B, Zhou Y, Wang W, et al. Characterization of ST15-KL112 Klebsiella pneumoniae Co-Harboring Bla (oxa-232) and rmtF in China. Infect Drug Resist. 2024;17:2719–2732. doi:10.2147/idr.S462158

50. Fang Y, Li X, Wu Z, et al. Emergence of an XDR Klebsiella pneumoniae ST5491 strain co-harboring NDM-5, MCR-1.1, tmexCD1-toprJ1, and a novel plasmid carrying CTX-M-15. Front Microbiol. 2025;16:1581851. doi:10.3389/fmicb.2025.1581851

51. Chao CM, Lai CC, Yu WL. Epidemiology of extended-spectrum β-lactamases in Enterobacterales in Taiwan for over two decades. Front Microbiol. 2022;13:1060050. doi:10.3389/fmicb.2022.1060050

52. Li P, Yan L, Song J, Lin C, Zeng F, Zeng S. Involvement of the bla (CTX-M-3) gene in emergence of a peculiar resistance phenotype in Klebsiella pneumoniae. Front Cell Infect Microbiol. 2025;15:1545157. doi:10.3389/fcimb.2025.1545157

53. Yu K, Huang Z, Xiao Y, Gao H, Bai X, Wang D. Global spread characteristics of CTX-M-type extended-spectrum β-lactamases: a genomic epidemiology analysis. Drug Resist Updat. 2024;73:101036. doi:10.1016/j.drup.2023.101036

54. Hu Y, Yang Y, Feng Y, et al. Prevalence and clonal diversity of carbapenem-resistant Klebsiella pneumoniae causing neonatal infections: a systematic review of 128 articles across 30 countries. PLoS Med. 2023;20(6):e1004233. doi:10.1371/journal.pmed.1004233

55. Song S, Zhao S, Wang W, et al. Characterization of ST11 and ST15 Carbapenem-resistant hypervirulent Klebsiella pneumoniae from patients with ventilator-associated pneumonia. Infect Drug Resist. 2023;16:6017–6028. doi:10.2147/idr.S426901

56. 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(11):1919–1929. doi:10.1038/s41564-019-0492-8

57. Lam MMC, Wick RR, Watts SC, Cerdeira LT, Wyres KL, Holt KE. A genomic surveillance framework and genotyping tool for Klebsiella pneumoniae and its related species complex. Nat Commun. 2021;12(1):4188. doi:10.1038/s41467-021-24448-3

58. Zhao H, He Z, Li Y, Sun B. Epidemiology of carbapenem-resistant Klebsiella pneumoniae ST15 of producing KPC-2, SHV-106 and CTX-M-15 in Anhui, China. BMC Microbiol. 2022;22(1):262. doi:10.1186/s12866-022-02672-1

59. Wang M, Guo H, He F, Xu J. Genomic and phylogenetic analysis of a multidrug-resistant Klebsiella pneumoniae ST15 strain co-carrying bla(OXA-232) and bla(CTX-M-15) recovered from a gallbladder infection in China. J Glob Antimicrob Resist. 2022;30:228–230. doi:10.1016/j.jgar.2022.06.023

60. Rodrigues C, Lanza VF, Peixe L, Coque TM, Novais Â. Phylogenomics of globally spread clonal Groups 14 and 15 of Klebsiella pneumoniae. Microbiol Spectr. 2023;11(3):e0339522. doi:10.1128/spectrum.03395-22

61. Roe CC, Vazquez AJ, Esposito EP, Zarrilli R, Sahl JW. Diversity, virulence, and antimicrobial resistance in isolates from the newly emerging Klebsiella pneumoniae ST101 lineage. Front Microbiol. 2019;10:542. doi:10.3389/fmicb.2019.00542

62. Pristas I, Ujevic J, Bodulić K, et al. The association between resistance and virulence of Klebsiella pneumoniae in high-risk clonal lineages ST86 and ST101. Microorganisms. 2024;12(10). doi:10.3390/microorganisms12101997

63. Liu Y, Jian Z, Wang Z, et al. Clinical characteristics and molecular epidemiology of ST23 Klebsiella pneumoniae in China. Infect Drug Resist. 2023;16:7597–7611. doi:10.2147/idr.S428067

64. Wyres KL, Nguyen TNT, Lam MMC, et al. Genomic surveillance for hypervirulence and multi-drug resistance in invasive Klebsiella pneumoniae from South and Southeast Asia. Genome Med. 2020;12(1):11. doi:10.1186/s13073-019-0706-y

65. Opstrup KV, Bennike TB, Christiansen G, Birkelund S. Complement killing of clinical Klebsiella pneumoniae isolates is serum concentration dependent. Microbes Infect. 2023;25(4):105074. doi:10.1016/j.micinf.2022.105074

66. Hsieh PF, Liu JY, Pan YJ, et al. Klebsiella pneumoniae peptidoglycan-associated lipoprotein and murein lipoprotein contribute to serum resistance, antiphagocytosis, and proinflammatory cytokine stimulation. J Infect Dis. 2013;208(10):1580–1589. doi:10.1093/infdis/jit384

67. Walker KA, Miller VL. The intersection of capsule gene expression, hypermucoviscosity and hypervirulence in Klebsiella pneumoniae. Curr Opin Microbiol. 2020;54:95–102. doi:10.1016/j.mib.2020.01.006

68. Ballén V, Gabasa Y, Ratia C, Ortega R, Tejero M, Soto S. Antibiotic resistance and virulence profiles of Klebsiella pneumoniae strains isolated from different clinical sources. Front Cell Infect Microbiol. 2021;11:738223. doi:10.3389/fcimb.2021.738223

69. Yoon EJ, Gwon B, Liu C, et al. Beneficial chromosomal integration of the genes for CTX-M extended-spectrum β-lactamase in Klebsiella pneumoniae for stable propagation. mSystems. 2020;5(5). doi:10.1128/mSystems.00459-20

70. Vance MK, Cretella DA, Ward LM, Vijayvargiya P, Garrigos ZE, Wingler MJB. Risk factors for bloodstream infections due to ESBL-Producing Escherichia coli, Klebsiella spp. and Proteus mirabilis. Pharmacy. 2023;11(2). doi:10.3390/pharmacy11020074

71. Tamma PD, Mathers AJ. Navigating treatment approaches for presumed ESBL-producing infections. JAC Antimicrob Resist. 2021;3(1):dlaa111. doi:10.1093/jacamr/dlaa111

72. Alav I, Pordelkhaki P, de Resende PE, et al. Cobalt complexes modulate plasmid conjugation in Escherichia coli and Klebsiella pneumoniae. Sci Rep. 2024;14(1):8103. doi:10.1038/s41598-024-58895-x