Eleven Klebsiella pneumoniae strains with different serotypes and/or sequence types were isolated from clinical patients with liver abscess, aimed to investigate their pathogenic potential characteristics and aggressive organ tropism. The K. pneumoniae reference strain NTUH-K2044 (GCA_000009885.1), and the 11 isolates were cultured in YPD (yeast extract peptone dextrose) medium. This study was approved by the medical ethics committee of the Capital Institute of Pediatrics.

For genomic comparison and analysis, 11 K. pneumoniae isolates were subjected to whole-genome sequencing. The sequencing was carried out on the Illumina HiSeq PE150 platform by the Institute of Microbiology, Chinese Academy of Sciences. The genome sequences were assembled using SOAP de novo (version 2.04), and gene annotation was performed with Prokka (version 1.14.6).

To determine the sequence types (STs), multilocus sequence typing (MLST) was applied. The sequences of the housekeeping genes gapA, infB, mdh, pgi, phoE, rpoB, and tonB were submitted to the Institut Pasteur K. pneumoniae MLST database,¹ and the corresponding STs were identified (Diancourt et al., 2005).

Virulence factor identification was performed on 11 K. pneumoniae isolates using VFanalyzer (Virulence Factors of Pathogenic Bacteria)² with NTUH-K2044 designated as the reference strain (Liu et al., 2019). This database catalogs key virulence determinants for Klebsiella, including genes associated with adherence, biofilm formation, efflux pumps, immune evasion, iron acquisition, nutritional factors, regulation, secretion systems, serum resistance, and toxins (Paczosa and Mecsas, 2016).

SNP (single nucleotide polymorphism) mainly refers to the polymorphism of DNA sequences caused by variations in a single nucleotide at the genomic level, including transitions and transversions of a single base, etc. SNPs were identified for 11 K. pneumoniae isolates using the MUMmer (Version 3.23) alignment software, with NTUH-K2044 as the reference genome (Croucher et al., 2015; Gan et al., 2022), and the functions of SNPs were annotated according to the positional relationship and interaction between SNPs and genes. The phylogenetic tree was constructed by the TreeBeST (Version 1.9.2) (Neighbor-Joining, NJ) or PhyML (Maximum likelihood, ML) (Version v3.0).

Antimicrobial susceptibility testing was performed using the Vitek 2 automated analysis system with AST-GN Gram-negative bacterial susceptibility cards, strictly following the manufacturer’s operating procedures. The detection range of minimum inhibitory concentration (MIC) for clinically commonly used antibacterial drugs was set. Bacterial suspensions were prepared using 0.45% normal saline and adjusted to a 0.5 McFarland standard. After loading the bacterial suspensions onto the susceptibility cards, the cards were placed in the Vitek 2 instrument for incubation of 18–24 h, during which the instrument automatically read the MIC values and susceptibility results. Bacterial suspensions were prepared from the same subculture plate, and the concentration and purity of the bacterial suspensions were verified by plating on blood plates free of antibacterial agents. Quality control strain (Klebsiella pneumoniae NTUH-K2044) was included throughout the test to ensure the reliability of the experimental results.

The freshly cultured colony grown on a YPD plate is gently lifted using an inoculation loop by vertically streaking upward three times. If the stretched viscous filament exceeds 5 mm in length upon each pull, the test result is recorded as positive.

The K. pneumoniae strain was cultured with YPD medium and its optical density at 600 nm (OD₆₀₀) was measured at 1 h intervals. For biofilm formation analysis, the strain was diluted (1:50) with fresh medium. The bacterial suspension was then added to a 96-well microplate and incubated for 24 h under 37°C. After incubation, non-adherent bacteria were removed, and the formed biofilms were stained with crystal violet solution. The absorbance of the stained biofims was measured using a spectrophotometer.

K. pneumoniae was streaked on YPD agar; a single colony was activated in YPD medium for 8 h (37°C, 180 r/min). It was then transferred 1:100 to fresh YPD, shaken to logarithmic phase. One milliliter suspension (approximately 3.0 × 10⁷ CFU) was centrifuged, washed 3 times with PBS, fixed in 3% glutaraldehyde at 4°C overnight. 20 μL was loaded on a copper grid, stained with phosphotungstic acid for 1 min, air-dried, and observed via transmission electron microscopy.

To establish a murine acute liver abscess model, male C57BL/6J mice (6–7 weeks old, Charles River Corp., Beijing, China) were randomly divided into 13 groups (n = 10 mice per group). Each group was inoculated intragastrically with K. pneumoniae isolates (10⁷ CFU/200 μL), with the reference strain NTUH-K2044 (at the same dose) and YPD broth (200 μL per mouse) as controls. The survival status of mice in each group was monitored and recorded continuously within 72 h. At the experimental endpoint, mice were anesthetized, and blood samples were collected for routine analysis. Subsequently, liver and lung tissues were harvested for hematoxylin-eosin (H&E) staining and histopathological examination.

Whole-genome sequencing files were submitted to the National Center for Biotechnology Information³ (for specific genome accession numbers, please see Supplementary Table 1).

The survival curves of mice were plotted with GraphPad Prism (version 10.0) and statistically analyzed using the Log-rank test. For assessing differences between the two groups, a two-tailed t-test was performed. All data are shown as mean ± SD. Statistical significance was defined as P < 0.05.

This study characterized 11 clinical K. pneumoniae isolates (Table 1), revealing substantial genotypic diversity through serotyping and sequence typing (ST) analysis. The isolates were classified into six distinct serotypes, with the following distributions: K1 (3 isolates, including ST23, ST700, and one novel ST), K2 (2 isolates, ST65 and ST86), K5 (2 isolates, both ST60), K20 (1 isolate, ST420), K57 (2 isolates, both ST218), and K80 (1 isolate, harboring an additional novel ST). These strains exhibited notable variability in both serotypes and genomic backgrounds.

In virulence gene analysis, the strains showed distinct lineage—specific distribution patterns: K1-ST23 (S1-001) and K1-novel ST (S1-009) carried all 10 target virulence genes and displayed positive string test results; K1-ST700 (S1-003) and K20-ST420 (S20-067) lacked allS, terW, rmpA and magA, allS, respectively, but retained most other virulence genes. K2-ST65 (S2-029) and K57-ST218 (S57-066/077) had unique deletion profiles. The K2-ST65 isolate exhibited deletions of magA, allS, ybtA and kfu, while the two K57-ST218 isolates maintained six intact virulence factors (aerobactin, iroN, terW, iutA, rmpA, and silS). Notably, ybtA was uniquely present in strain S57-066 but absent in S57-077. Consistent with the K2-ST65 strain, both K57-ST218 isolates lacked magA, allS, and kfu virulence determinants. K5-ST60 strains (S5-036/105) maintained only four core virulence genes (aerobactin, kfu, iroN, and ybtA), whereas K2-ST86 (S2-048) and the novel K80 (S80-110) strains showed complete absence of detectable virulence determinants.

Comparative analysis of virulence gene distribution among strains sharing the same serotype but differing in sequence types (STs) revealed distinct patterns: Within the K1 serotype, strains ST23 (S1-001) and novel ST (S1-009) exhibited complete sets of all ten tested virulence genes, whereas strain ST700 (S1-003) lacked allS, terW, and rmpA. Among K2 serotype strains, ST65 (S2-029) tested positive for aerobactin, iroN, terW, iutA, rmpA, and silS but negative for magA, allS, kfu, and ybtA. These findings demonstrate significant intra-serotype heterogeneity in virulence gene profiles across different STs, highlighting the importance of ST-level characterization in virulence assessment.

As shown in Table 1, the string test yielded a distinct distribution across the tested strains, with seven isolates (S1-001/003/009, S2-029, S5-036, S20-067, and S57-066) demonstrating characteristic positive hypermucoviscosity (+), while four strains (S2-048, S5-105, S57-077, and S80-110) were consistently negative (−). Kinetic analysis of 11 clinical K. pneumoniae isolates revealed significant differences in growth performance (Figure 1A). Strain K2-ST65 (S2-029) exhibited the strongest proliferative capacity and an extended logarithmic phase. K1-novel ST (S1-009) and K57-ST218 (S57-077) showed similar growth rates, but the latter displayed a pronounced lag phase before 12 h. Strains with moderate growth capacity included K1-ST23 (S1-001), K1-ST700 (S1-003), K20-ST420 (S20-067), K57-ST218 (S57-066), and K80-novel ST (S80-110); they had a relatively stable peak OD₆₀₀ values, though the timing of entering the stationary phase varied (earlier for S1-003, later for S80-110). Among strains with limited growth, K2-ST86 (S2-048) exhibited the weakest proliferative ability, while K5-ST60 (S5-105) and K5-ST60 (S5-036) displayed phenotypic heterogeneity due to early stagnation and prolonged lag phase, respectively. Notably, though S57-066 and S57-077 belonged to the same lineage (K57-ST218), the former grew faster. In summary, the growth kinetics of the 11 clinical K. pneumoniae isolates showed no direct correlation with virulence gene profiles (e.g., strongly proliferative S2-029 lacked magA/allS, while highly virulent gene-loaded strain S1-001 only exhibited moderate growth).

In biofilm formation assessment (Figure 1B), K1-ST23 (S1-001) demonstrated the strongest biofilm—forming capability, while K57-ST218 (S57-077) displayed the weakest. K5-ST60 (S5-105) and K80-novel ST (S80-110) exhibited relatively strong biofilm formation, other strains showed discontinuous differences—K1-ST700 (S1-003), K1-novel ST (S1-009), K2-ST65 (S2-029), K2-ST86 (S2-048), K20-ST420 (S20-067), whereas K5-ST60 (S5-036), and K57-ST218 (S57-066/077) were much weaker. These observations suggest that K. pneumoniae isolates sharing the same serotype but exhibiting different sequence types (STs) may display divergent biofilm—forming capacities, with K1 serotype isolates demonstrating notably stronger biofilm production. These findings highlight substantial diversity in virulence gene profiles, serotypes, and biofilm formation among K. pneumoniae strains, offering key insights into their distinct pathogenicity mechanisms.

Ultrastructural and colony morphology characteristic analyses (Figure 1C) revealed that all 11 K. pneumoniae isolates maintained a typical rod-shaped morphology and formed grayish-white non-hemolytic colonies, while exhibiting distinct polymorphism at the subcellular level. Eight clinical isolates (S1-001/003/009, S2-029, S20-067, S57-066/077, and S80-110) along with the reference strain NTUH-K2044 consistently exhibited uniform electron-dense bacilli with well-defined capsular structures under electron microscopy. In contrast, three strains (S5-036/105 and S2-048) displayed distinctive intracellular vacuole-like formations. Notably, despite ultrastructural heterogeneity, all strains consistently exhibited a conserved non-hemolysis phenotype on blood agar plates.

The 11 K. pneumoniae strains were subjected to whole-genome sequencing, core single-nucleotide polymorphisms (SNPs) were used to construct the phylogenetic tree (Figure 1D). The clustering analysis results were as follows: S5-105 and S5-036 formed an independent clade, distinctly separated from the other strains. The remaining strains are divided into two main subclades: Subclade 1 contained 7 strains including S1-003, S2-048/029, S80-110, S57-066/077, and S20-067; Subclade 2 includes S1-009/001, and NTUH-K2044. The degree of differentiation revealed that S5-105 and S5-036 exhibited a higher level of differentiation compared to the other strains.

Vitek 2 automated analysis system was used to evaluate the MICs of common antibiotics of 11 clinical K. pneumoniae strains. As shown in Table 2, overall resistance phenotype showed that 9 of 11 strains were pan-susceptible, with an overall low drug resistance rate. All strains were 100% sensitive to carbapenems (imipenem, meropenem), cephalosporins (ceftazidime, cefepime), aminoglycosides (amikacin, tobramycin), and monobactam (aztreonam). Among quinolones, S5-105 exhibited resistance to ciprofloxacin (MIC = 1 μg/mL) and intermediate susceptibility to levofloxacin (MIC = 1 μg/mL). Two isolates showed non-susceptible phenotypes to penicillins and β-lactamase inhibitors: S5-036 displayed intermediate susceptibility to sulbactam (MIC = 16 μg/mL), while S57-066 exhibited intermediate susceptibility to piperacillin (MIC = 32 μg/mL) and resistance to sulbactam (MIC ≥ 32 μg/mL). All other tested antimicrobial agents remained fully susceptible. This suggests that the studied K. pneumoniae population exhibits favorable susceptibility to most antimicrobial agents, with low resistance pressure.

We systematically evaluated the pathogenicity of 11 K. pneumoniae isolates in mice, revealing significant heterogeneity in virulence, manifested as a complex correlation between pathological damage severity and survival rates. The standard virulent strain NTUH-K2044 (72-h survival rate: 80%, Figure 2A) and highly pathogenic strains (e.g., S1-001/003, S2-029/048) induced typical suppurative liver damage, characterized by multifocal scattered abscess formation in hepatic lobules, accompanied by neutrophil infiltration, deposition of eosinophilic necrotic debris, and hepatocyte degeneration (ranging from swelling to coagulative necrosis) (Figure 2B). Among these, S1-009 and S2-029 exhibited the strongest pathogenicity (survival rate: 50%). In contrast, low—pathogenicity strains (S5-036/105, S57-066/077) caused only mild pathological damage; the novel strain S80-110 performed more different, with a 100% survival rate and no significant lesions. Notably, the pathology-survival correlation displayed specificity: while most strains showed a negative correlation between damage severity and survival rate (e.g., S1/S2 series), S20-067 triggered severe abscesses yet maintained a 100% survival rate, and S5-036/105 caused mild pathological damage but had only an 80% survival rate.

Next, we evaluated lung injury in infected mice, and the results revealed significant differences in pulmonary pathological damage caused by different K. pneumoniae isolates (Figure 2C). The highly pathogenic NTUH-K2044 strain exhibited near-normal alveolar architecture, with no detectable inflammatory infiltration. In contrast, most clinical strains induced varying degrees of lung injury: S1-001 and S80-110 led to severe alveolar destruction accompanied by extensive inflammatory consolidation; S2-029 and S2-048 displayed diffuse inflammatory infiltration and disruption of alveolar structure; S1-003 and S20-067 presented with alveolar septal thickened or focal tissue damage; and S5-036 showed only mild alveolar injury. Notably, S5-105 and S57-066 maintained intact alveolar structures with minimal inflammatory responses, which closely resembled the pathological features of uninfected control lung tissue. Of particular interest, strain K80-novel ST (S80-110), triggered severe pulmonary inflammatory responses. These findings highlight the complexity of tissue tropism and pathogenic mechanisms among different K. pneumoniae strains.

Blood test results revealed characteristic trends in mice infected with different K. pneumoniae isolates: highly pathogenic strains (NTUH-K2044, S1-001/003/009, S2-029) all induced significant decreases in white blood cells (WBC) (Figure 2D) and percentage of lymphocytes (LYM%) (Figure 2F), accompanied by increased percentage of neutrophils (NEU) (Figure 2E). Among them, S2-029 and K1-novel ST (S1-009) were particularly notable. This hematological pattern (WBC↓, LYM%↓, NEU%↑) was associated with severe inflammatory liver damage and significantly reduced survival rates. In contrast, strains including S2-048, S5-036/105, and S20-067 primarily caused selective decreases in WBC or LYM%, with no concurrent increase in NEU%. Some strains (S57-066/077, S80-110) showed essentially normal blood indicators. Notably, although the changes in blood tests generally correlated with pathogenicity, certain strains S20-067 exhibited inconsistencies between the severity of blood test abnormalities and liver damage, suggesting the potential existence of independent pathogenic mechanisms.