Klebsiella pneumoniae is a prevalent nosocomial pathogen responsible for various infections, including pneumonia, urinary tract infection, liver abscess, bacteremia and sepsis (Paczosa and Mecsas, 2016). Although K. pneumoniae has numerous genetic and phenotypic diversities, its successful prevalence in clinical settings can be attributed to antimicrobial resistance and hypervirulence (Lai et al., 2019; Choby et al., 2020). In 1986, seven K. pneumoniae isolates were cultured from a series cases of pyogenic liver abscess complicated by septic endophthalmitis in China Taiwan (Liu et al., 1986). Afterwards, such kind of K. pneumoniae isolates that associated with life-threatening systemic or multiple sites infections were designated as hypervirulent K. pneumoniae (hvKp) to be distinguish from classical K. pneumoniae (cKp) (Russo and Marr, 2019). hvKp represents a distinct and concerning variant of the bacterium that has garnered increasing attention due to its heightened pathogenicity and ability to cause severe infections and poorer patient outcomes with notable clinical impact. hvKp strains exhibit the distinctive characteristic of expressing highly mucoid capsules, resulting in a “hypermucoviscous” colony phenotype (Shon et al., 2013). Their increased virulence is typically ascribed to the carriage of several large, non-conjugative “hypervirulence” plasmids by a few specific clonal lineages and capsule loci (Wyres et al., 2020). The emergence and global spread of hvKp strains pose a significant threat to public health, necessitating a comprehensive understanding of their molecular epidemiological characteristics, mechanisms of virulence, and associated clinical implications.

Though hvKp has been studied for nearly 40 years, its definition remains unclear and controversial (Chen et al., 2023). Many studies summarized the differences between hvKp and CKP, clinical manifestations, capsule typing, hypermucoviscosity, and virulence genes are helpful to identify hvKp (Choby et al., 2020). For example, hvKp isolates often have hypermucoviscous phenotype and carry plasmids encoding virulence genes, and are associated with community-acquired infections in immunocompetent hosts (Struve et al., 2015). Most importantly, hvKp isolates were found more virulent (Lethal dose <10³ CFU) than CKP (Lethal dose >10⁶ CFU) in mouse infection experiments (Russo and MacDonald, 2020; Russo et al., 2021). Notably, many studies have shown that sequence type (ST)23, ST65, ST86 and capsular type K1, K2 were closely associated with hvKp (Liao et al., 2014).

While the majority of hvKp strains exhibit susceptibility to various classes of antimicrobial agents, there have been reports of multidrug-resistant hvKp strains (DeLeo et al., 2023). The emergence of these strains poses significant therapeutic challenges, contributing to a higher morbidity and mortality rates. Addressing the complexities associated with the treatment of multidrug-resistant hvKp infections becomes paramount in mitigating the clinical impact of these formidable strains (Lam et al., 2021). Of particular concern is the emergence of hypervirulent carbapenem-resistant K. pneumoniae, capable of causing challenging-to-treat infections with adverse outcomes (Gorrie et al., 2022). Over the past decade, hv-CRKP has rapidly disseminated, often attributed to the acquisition of pLVPK-like virulence plasmids containing prevalent virulence genes, such as the mucoid regulator gene (rmpA2), distinct mucoid regulator operon (rmpADC), salmochelin receptor gene (iroN), salmochelin biosynthesis genes (iroBCD), aerobactin receptor gene (iutA), and aerobactin biosynthesis genes (iucABCD) (Heng et al., 2023; Liu et al., 2023). Our previous study also highlighted the global evolution and geographic diversity of hypervirulent carbapenem-resistant K. pneumoniae from 1980 to 2022 in 105 countries (Wu et al., 2022). Moreover, the capsule polysaccharide of K. pneumoniae is recognized as a crucial virulence factor, conferring resistance to phagocytosis and serum bactericidal activity (Opoku-Temeng et al., 2019). To date, over 100 capsular (K) serotypes of K. pneumoniae have been characterized. Significantly, several studies highlight the robust association of K1 and K2 serotypes with hypervirulent K. pneumoniae phenotype (Liao et al., 2014). However, it’s noteworthy that the K54 capsular type, especially within representatives of sequence type ST29, has also been linked to hypervirulence recently, expanding the spectrum of capsular serotypes implicated in this enhanced virulence phenotype (Shao et al., 2022).

Although previous studies have extensively explored the molecular epidemiology, virulence factors, and antimicrobial resistance of hvKp, the genomic epidemiological characteristics and population dynamics of hvKp within a single patient across distinct infection episodes remain largely unknown. In this study, we isolated a K2-ST881 and a K54-ST29 K. pneumoniae strain from a single patient suffering multiple site infection in two independent episodes. In addition, we performed a comprehensive genomic epidemiology analysis between hvKp isolates from this study and global ST881 and ST29 K. pneumoniae isolates retrieved from public database to enhance understanding of the potential evolutionary history of hvKp. Finally, the virulence potentials of both isolates were measured by mouse lethality assay. Our study highlights the genomic alterations correlating with clonal switch between two distinct lineages of hvKp that successively replaced each other in vivo.

Hypervirulent K. pneumoniae (hvKp) has emerged as a significant global pathogen capable of causing both community-acquired and hospital-acquired infections, even in individuals without underlying health issues. This pathogen is harbored in the gastrointestinal tract, contributing to its dissemination in both community and healthcare settings (Russo and Marr, 2019). Initially identified in East Asia, hvKp gained prominence as a leading cause of pyogenic liver abscesses. Over subsequent decades, it has extended its reach globally, causing a spectrum of infections (Choby et al., 2020). An alarming trend is the emergence of multidrug-resistant hypervirulent strains, presenting a new and formidable challenge in the battle against this already perilous pathogen. Of particular concern is the convergence of virulence and carbapenem resistance (e.g., the bla_KPC-2 gene carried by a pLVPK-like virulence plasmid) in a single K. pneumoniae hybrid plasmid, which is the most worrisome (Kong et al., 2021; Wu et al., 2023b).

HvKp is often found as part of the gut microbiome, contributing to its dissemination in both community and hospital settings. Previous study has identified K. pneumoniae in the stool of 10 to 35% of individuals (Choby et al., 2020). In hospitalized patients, the rates of K. pneumoniae intestinal colonization are notably higher, ranging between 19 and 38% (Martin et al., 2016; Gorrie et al., 2017). Furthermore, there is compelling evidence suggesting a direct link between gut colonization by hvKp and subsequent infection in the same individual. For instance, approximately half of ICU K. pneumoniae infections were found to be caused by the patient’s own gastrointestinal strain, emphasizing the role of gut colonization in the pathogenesis of hvKp infections (Gorrie et al., 2017). Another study also disclosed a moderate carriage rate of hvKp in the healthy population, ranging from 4 to 5.19% (Yang et al., 2022). There is a phylogenetically close relationship between intestinal colonization of hvKp and strains known to be implicated in clinical infections (Teo et al., 2024). These findings underscore the potential role of human gut as a reservoir for hvKp and suggest it may reside in the gut that contribute to infections in other body sites. In this case, the patient had a history of gastrointestinal hemorrhage and had kept drinking alcohol for several years. In the first episode, his headache started on the day after drinking and aggravated during the following week till admission. Hence, even though no K. pneumoniae strain was successfully isolated from fecal sample of the patient, may partially owing to antibiotic usage, it is reasonable to speculate that these life-threatening infections may result from translocations of intestinal colonized hvKp isolates under the circumstance of intestinal mucosal barrier impairment induced by alcohol. Therefore, active screening and antibiotic-mediated decolonization of K. pneumoniae from the gut of high-risk patients might be able to reduce the chance of hvKp infections.

The emergence of ST881 and ST29 hvKp strains reported in this study has presented unique challenges and implications for clinical management. The clinical implications of clonal replacement of hvKp strains underscore the adaptability and persistence of these pathogens within the host environment (Tang et al., 2023). This phenomenon may be influenced by a range of factors, including selective pressures, host immune responses, and the interplay between the bacterial strains and the host microenvironment. Taking the history of disease and drinking habit of this patient into consideration, his immune function of intestinal mucosal barrier was likely to be declined, leading to vulnerability to intestinal bacterial colonization and translocation, which may be responsible for his reinfection of different hvKp isolates.

Clinical examination of this patient suggested that multiple sites infections were developed in both episodes, indicating high virulence and pathogenicity of these two strains. In addition to intracranial and blood steam infection, the ST881 isolate EDhvKp-1 (EDhvKp-2) was also associated with a suspicious liver abscess, which disappeared on CT scan after antibiotic therapy. The ST29 isolate EDhvKp-3 caused blood steam infection, accompanied by prostatitis and infective cardiac endocarditis. Furthermore, the metastatically spreads of infection lesions were also observed in mouse lethality assay, while bacterial suspensions were inoculated into abdominal cavity, multiple micro abscesses were formed inside the livers. These findings were consistent with the clinical and microbiological features of hvKp reported previously. The virulence scoring system has been structured for evaluating the virulence level in K. pneumoniae assigned by a series of virulence-related factors (ybt, clb and iuc) in genome loci (Lam et al., 2021). In our study, the virulence score indicated that the ST881-KL2 isolate EDhvKp-1 was less virulent than the ST29-KL54 isolate EDhvKp-3. This result was consistent with our mouse lethality assay, suggesting that the complete virulence plasmid may enhance the virulence of isolate EDhvKp-3. Interestingly, the rmpA2 gene was truncated in the EDhvKp-3, which suggested that the hypermucoviscosity in EDhvKp-3 was mainly mediated by other virulence genes rather than rmpA2.

Through cgSNP-based phylogenetic analysis, we found that 24 of 32 (75%) ST29-KL54 hvKp were originated from China, while the remaining were isolated from India (n = 2), Japan (n = 2), Madagascar (n = 1) and Thailand (n = 1). We hypothesized that the clonal transmission of ST29-KL54 occurred in China, which is consistent with the report of its dissemination in a tertiary hospital in China, 2018 (Qiu et al., 2023). A previous study reported a case of sepsis and brain abscess patient caused by an infection of the ST29-KL54 strain TAKPN-1 (Shao et al., 2022). This strain is most closely related to strain EDhvKp-3 in this study. However, strain TAKPN-1 lacks the pLVPK-like virulence plasmid, implying that the acquisition of the virulence genes by EDhvKp-3 may have occurred through horizontal transmission. EDhvKp-1 showed the highest degree of similarity to strain 502, which harbored extra virulence genes iroBCDN, rmpA, rmpC and rmpD and antimicrobial resistance genes aadA1, dfrA12 and mph (A), in comparison to EDhvKp-1. We identified the KL2 in all K. pneumoniae lineages ST881, this particular serotype combination has been broadly associated with hvKp (Sanchez-Lopez et al., 2019). Of note, this was the first report of KL2-ST881 hvKp recovered from China.

We also confirmed the genetic contexts of rmpA, rmpA2, rmpC, rmpD, iroBCDN, iucABCD and iutA genes in pEDhvKp-3 plasmid shared high homology with pLVPK-like virulence plasmid. Diverse insertion sequences were discovered in the upstream and downstream regions of virulence genes, including iroBCDN with an upstream IS3 and IS66 transposase and iucABCD with an upstream IS630 transposase, both of which were facilitated by horizontal transfer. Out of 180 ST29 K. pneumoniae strains, 30 (16.7%) were not of human origin. Previous studies have revealed the presence of hvKp in several sources including clinical settings, the environment and animals (Furlan et al., 2020; Mario et al., 2023). This presence contributed to a shared reservoir of antimicrobial resistance genes and virulence genes, posing a significant public health risk. Therefore, it is imperative to enhance the range of surveillance and implement the One Health strategy to prevent its further dissemination among different settings.

While this study represents the first investigation into the in vivo clonal replacement of two hypervirulent K. pneumoniae lineages ST881 and ST29, it is essential to acknowledge its limitations. A notable constraint is the relatively small sample size within a single patient, which may have an impact on the generalizability and robustness of the findings. In the future, it is imperative to conduct large-scale clinical studies involving patient cohorts and comprehensive investigations into the factors influencing clonal replacement of hvKp. This includes examining selection pressures, host immunological responses, and environmental factors, all of which are essential for advancing our understanding of hvKp infections. Furthermore, although the virulence traits of each strain were assessed through the identification of key virulence genes and animal lethality experiment, the origin and transmission dynamics of these hvKp strains are still unknown. Future research should not only concentrate on the genomic characteristics of hypervirulent strains but also delve into the gut colonization of hvKp directly precedes infection from the gut microbiome of health individuals.

In conclusion, the clonal replacement of hypervirulent K. pneumoniae ST881 and ST29 lineages within the same patient emphasizes the complexity of hypervirulent K. pneumoniae infections, laying the groundwork for developing strategies to impede the persistence of these formidable bacterial pathogens. Our study provided a dynamic insight into clinical bacterial colonization and infection, highlighting the potential merit of microbiological screening in relatively healthy people, especially for those patients who have had systemic infection episodes or chronic impairments in their intestinal tracts. Further research into the genetic determinants, clinical outcomes, and factors influencing the dynamics of clonal replacement is essential for refining our understanding of the pathogenesis of hypervirulent K. pneumoniae and informing strategies for effective clinical management and infection control.

The genome sequences of the three K. pneumoniae isolates recovered in this study were deposited in the NCBI GenBank database under the BioProject accession number PRJNA553055.

The studies involving humans were approved by Ethics Committee of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, China. The human samples used in this study were acquired from a by-product of routine care. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements. The animal study was approved by Ethics Committee of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, China. The study was conducted in accordance with the local legislation and institutional requirements.

NLi: Data curation, Writing – original draft. NLo: Formal analysis, Investigation, Writing – original draft. JH: Data curation, Writing – original draft. ZC: Data curation, Writing – original draft. BL: Data curation, Writing – original draft. ZZ: Formal analysis, Visualization, Writing – review & editing. YH: Conceptualization, Project administration, Supervision, Validation, Writing – review & editing. LC: Project administration, Supervision, Writing – review & editing. WX: Conceptualization, Investigation, Methodology, Supervision, Visualization, Writing – original draft, Writing – review & editing.